A2AR SIGNALING IN TREG CELLS IS CRITICAL FOR MAINTAINING IMMUNE HOMEOSTASIS AND THEIR SUPPRESSIVE FUNCTION

Introduction: Regulatory T (Treg) cells play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses. Treg cells can generate extracellular adenosine, which has been implicated in their immunosuppressive activity via activation of adenosine receptor A2a (A2aR) in effector T cells. However, it was not clear how A2aR activation affects the biology of Treg cells. Objective: To investigate the intrinsic function of A2aR in Treg cells. Methods: Treg cells from A2aRΔTreg (Foxp3CreAdora2af/f) and Foxp3Cre mice were used for the analysis of their molecular phenotype, function, and stability ex vivo. A2aRΔTreg mice were also investigated for the aging-associated spontaneous systemic lymphoproliferative disease development and experimental autoimmune encephalomyelitis (EAE) model (protocol number of ethics committee: 251/2019). Results: A2aR-ΔTreg mice showed normal Treg and T cell development in multiple tissues at young ages. However, aged (1.5 years old) A2aR-ΔTreg mice developed a spontaneous lupus-like disease phenotype, with systemic lymphoproliferative response (CD4+CD44hiCD62LloCD69+ T cells) and production of anti-nuclear autoantibodies. Aged A2aRΔTreg mice also showed increase number of Foxp3+ Treg cells, but with impaired immunosuppressive function. Interestingly, young A2aRΔTreg mice develop a severe form of EAE. Moreover, progression of EAE symptoms in A2aR-ΔTreg mice occurred despite the mice maintaining high numbers of Treg cells in spleen and peripheral lymph nodes, and disease severity was accompanied by increased frequency of IFN-γ- and IL-17-producing CD4 T cells in the spinal cord and brain. Conclusion: Our results suggest that control of A2aR signaling in Treg cells is critical to maintaining their homeostasis and function in autoimmune diseases and aging.

A BIOINFORMATIC APPROACH TO DISCOVER GUT MICROBIOTA GENE PRODUCTS RESPONSIBLE FOR INDUCING ROR(γt)+ TREG CELLS

Regulatory T cells (Treg) have a vital role in controlling immunological homeostasis and responses. A unique Treg cell population found within the colonic mucosa is characterized by expressing the RAR-related orphan receptor gamma t (ROR(γt)) transcription factor. These ROR(γt)+ Treg cells are only induced upon specific microbial colonization of the intestinal tract, being involved in keeping immune tolerance in the gastrointestinal tract. The ROR(γt)+ Treg cells have also been involved in food antigen tolerance, with a role in controlling food allergy development. Although some commensal bacteria can induce the proliferation of ROR(γt)+ Treg cells, the mechanisms by which they are induced in the intestine are still unknown. Here, we analyze the genomes of 26 human microbiome bacteria members reported as inducers of distinct Treg levels in a murine model. These genomes were grouped in two categories based on their level of ROR(γt)+ Treg induction: high inducers (n=7) or low inducers (n= 11). Bacteria with intermediate ROR(γt)+ Treg induction levels were removed from further analysis. The two groups remaining were compared using Fisher's exact test (p value <0.05). We obtained 27 genes associated with the high induction group and 1 gene enriched in the low induction group. Differential genes were annotated for their function and metabolic pathway memberships using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Pathway signals associated with a high induction profile included Lipopolysaccharide biosynthesis, Peptidoglycan biosynthesis. Specific genes associated with a high ROR(γt)+ Treg induction include: alpha-2-macroglobulin (αMs), UDP-N-acetyl-L-fucosamine (UDP-l-FucNAc) synthase, D-sedoheptulose 7-phosphate isomerase (GmhA), chemotaxis protein MotB. The αMs are reported to act as a proteinase inhibitor, with a possible role in cell defense following host attack, suggesting an accord with the idea of these bacteria inducing host tolerance through ROR(γt)+ Treg cells. UDP-l-FucNAc synthase, GmhA and MotB are cell surface proteins that could be involved in host recognition and tolerance, as already reported for some bacterial cell wall derived polysaccharides that promote Treg cell induction in the intestine. The genomic signals detected here can be further tested experimentally to confirm possible avenues by which the microbiome controls immune homeostasis, particularly the induction of ROR(γt)+ Treg cells.

A BRIEF ANALYSIS ABOUT GUT MICROBIOME DIVERSITY IN ASTHMATICS

Introduction: The gut microbiota can influence immune responses at distant sites, such as the lung, via multiple mechanisms and plays several important roles in the development, regulation, and maintenance of healthy immune response. Several studies have linked unbalanced of the gut microbiota with an altered risk of asthma later in life. Objectives: To assess microbial diversity from the microbiota in stool samples from asthmatic individuals. Methods: This project is part of a multicenter study conducted in five countries. A total of 57 stool samples were collected from each subject (29 samples from asthmatics and 28 samples from non-asthmatics individuals) the bacterial 16S rDNA targeting the V4 region was amplified using PCR and sequenced by Illumina MiSeq high-throughput sequencing. The bioinformatics analysis was conducted using QIIME2 (version 2021.4) and data visualization and analysis using R (version 4.1.0). Results and Conclusions: The richness and diversity of the microbial community, i.e. alpha diversity, in asthmatics was lower than that in non-asthmatics, although no significant difference in ASVs and Shannon index was observed. Beta diversity analysis was performed by Bray–Curtis dissimilarity analysis, indicated a significant difference in beta diversity between the two groups. This study supports information of the microbial diversity and richness of species among asthmatics and non-asthmatics individuals.

Acetate increases the frequency of regulatory T cells and ameliorates the type 1 diabetes in experimental model

Introduction: Type 1 diabetes mellitus (T1D) is the most common chronic autoinflammatory disease among children and adolescents worldwide. It is characterized by the progressive destruction of pancreatic beta cells induced by inflammatory responsemediated by self-reactive T and B cells. Regulatory T cells (Tregs) have immunoregulatory mechanisms over T cell activation, proliferation and their defect in number and funtion is related to the onset of autoimmune diseases such as T1D. In recent years, the composition of the intestinal microbiome has gained prominence over the maturation of the immune system. Short chain fatty acids (SCFA), mainly acetate, propionate and butyrate are molecules derived from the metabolism of dietary fiber by bacteria of the intestinal microbiota. It is possible to observe the increased frequency of Treg in T helper (Th) naïve cell cultures containing high concentrations of butyrate. However, little is known about the properties of other SCFA on the development and function of Tregs. Objective: To evaluate the influence of acetate on the differentiation and stability of Tregs during the immunomodulation of T1D. Methods: Acetate (150 mM) was provided in the water of C57BL6 mice ad libitum. From the fifth day of acetate supply in the mice drinking water, 40mg/kg of streptozotocin were inoculated intraperitoneally daily for five consecutive days. On the 21st day of acetate supply, mice were euthanized and clinical samples were collected. Results: Mice given acetate had lower fasting blood glucose and decrease incidence of diabetes than diabetic mice given no acetate in their drinking water. Acetate consumption increased the number of regulatory T cells (FOXP3+ CD4+) as well as LAPTGF-β1+ Tregs in pancreatic and cecal lymph nodes. The frequency and number of follicular Tregs (CD4+ FOXP3+ CXCR5+ PD1+) in the cecal lymph node was also increased in the acetate group. Despite we did not observe the effect of acetate (1µM and 10µM) on the differentiation of Tregs in vitro, acetate was able to increase the stability of FOXP3 expression in Tregs in vitro within two days of culture. Conclusion: This study observed that acetate confers protection against the development of T1D, increasing the frequency and number of Tregs in lymph nodes draining the inflammatory site in a mechanism that suggests a sustained stability of the FOXP3 gene.

A COMPLEX NETWORK OF MIRNA AND HOST GENE SHIFT MACROPHAGE RESPONSE TO LPS AND LEISHMANIA INFECTION

Introduction: Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family is composed of a large number of intracellular pathogen recognition receptors that function as sensors for microbial-derived and danger-associated molecules in the cytoplasm of host cells. The member of NLR family, NLRP12 (NALP12) contributes to suppressing the production of proinflammatory cytokines and chemokines by interfering with canonical and noncanonical NF-κB signaling pathways. The microRNA (miRNAs) family miR-290/295 in murine and miR-371/373 in human are encoded in the antisense nearly to NLRP12. MiRNAs work downstream of TFs regulating post-transcriptional levels of mRNAs. The Leishmania parasite can subvert macrophage’s inflammatory response to survive in this hostile environment, interfering in the mRNAs and miRNAs host-expression. Thus, we aimed to investigate the participation of NLRP12 and host miRNAs in murine and human macrophages infected with L. amazonensis or stimulated with LPS. Materials and Methods: We analyzed the NLPR12 and miRNA expression in Bone-marrow-derived macrophages of BALB/c mice and human THP-1 derived macrophages infected with L. amazonensis (MOI 5:1) or stimulated with LPS (100ng/mL) for 4 and 24 h. Results: We observed a distinct expression of NLRP12 in infected BALB/c or THP1-macrophages compared to LPS stimuli. Meanwhile, LPS stimulation decreased Nlrp12 levels in BALB/c macrophage. The levels of miR-294 in murine macrophage and miR-372 and miR-373 in human macrophages increased after infection, compared to LPS-stimulated and uninfected ones. The miRNAS and NLRP12 possess a few predicted genes in literature and databases, and some of these might affect the inflammatory profile of the macrophage. As for predicted targets possibly regulated by NLRP12, we found an increase of Tnf and Nos2 levels in BALB/c stimulated with LPS macrophages, but none or very little increase in infected macrophages. Conclusions: The Leishmania infection might produce distinct effects in the modulation of NLRP12 and miRNAS expression compared to LPS. The increase in predicted targets’ levels in LPS stimulated macrophages to indicate that they can respond to an inflammatory stimulus. Still, the absence of this response in infected samples suggests that the infection might be blocking such immune reaction through repression signals mediated by NLRP12.

A comprehensive peptide library of P. vivax induces the expression of activation markers in CD4+ T cell from symptomatic and asymptomatic individuals.

Introduction: Malaria is a severe disease transmitted by the Anopheles vectors infected with Plasmodium protozoa. In Brazil, most cases are reported in the northern region and caused by Plasmodium vivax (P. vivax). Plasmodium infection does not induce long-term protection, but both humoral and cellular immune responses are essential for partial protection against malaria. The identification and measurement of antigen-specific T cell response are hampered mainly by the lack of well-defined immunogenic protein antigens of P. vivax. Objective: The aim of this study was to evaluate the specific-antigen response of CD4+ T cells and memory subpopulations from symptomatic and asymptomatic individuals infected with P. vivax, using a library with 310 peptides from P. vivax (MPv310). Methods: Peripheral blood mononuclear cells from 17 symptomatic before (SY-I) and 21 after (SY-R) treatment, 29 asymptomatic individuals infected with P. vivax, and 27 healthy donors (CTL) were stimulated in vitro with MPv310 or an equimolar dose of DMSO (negative control). The production of cytokine (IFN-𝛾 e TNF) and activation markers (CD69 e CD154) was analyzed by flow cytometry. Results: MPv310 induced the production of IFN-𝛾 and TNF in memory CD4+ T cells in SY-R and ASY. Overall frequency of responders was 29,41% (SY-I), 52,38% (SY-R), 55,17% (ASY) and 55,55% (CTL) for the co-expression of IFN-𝛾 and TNF. In addition, P. vivax-infected individuals presented increased frequencies of CD4+ T cells and effector and effector memory CD4+ T cells expressing CD69. On the other hand, only SY-I and SY-R individuals showed an increase in CD4+ T cells and their subpopulations expressing CD154 and co-expressing CD154 and CD69. Conclusion: Our results indicate that the MPv310 activate CD4+ T cells in symptomatic and asymptomatic individuals, evidencing the presence of memory CD4+ T cells specific for the P. vivax antigens.

Acrocomia acuelata (Jacq.) Lodd. ex Mart. incorporated in nanoparticles demonstrate potential antitumor activity in breast cancer cells

Introduction: The most recent data indicate that female breast cancer has become the most diagnosed type in the world. The available therapies have several serious side effects for patients, which makes treatment difficult. Vegetable oils are an alternative to attenuate these effects while providing effective therapy. Among these, the Acrocomia aculeata (Jacq.) Lodd. ex Mart., popularly known as “Macaúba” is a plant species with promising bioactive compounds for tumor treatment. The pharmaceutical application of the oil from its pulp faces some limitations caused by its physicochemical properties. The incorporation into micellar nanocarriers appears to be a viable solution to overcome this obstacle. Thus, the present work aims to develop polymeric micelles containing macaúba pulp oil to evaluate the antitumor activity against triple-negative breast cancer lines. Methods and Results: The formation of micellar nanoparticles (PM-MO) was constructed through the sonication method. Microscopic characterization was performed using the Zetasizer equipment and showed a mean hydrodiameter (MHD) of 105 nm ± 0.7, with a polydispersity index (PdI) of 0.1 ± 0.03 and a Zeta potential of -17, 20 mV ± 0.35. PM-MO cytotoxicity in the MDA-MB-231 line was evaluated by MTT at a concentration of 0.386 mg/ml and demonstrated a significant drop in viability in breast cancer cells of approximately 30% in 72h. In healthy fibroblast lines (L929), no drop in viability was observed at any of the times. For the clonogenicity assay, two concentrations of PM-MO were used, and both reduced the formation of tumor colonies, where 0.386 mg/ml had a proliferation reduction of about 95% in relation to the control group, and 0.0965 mg/ml, with 30% reduction. Antimigratory activity was quantified using the Wound Healing assay. At 48 hours, the free area of the control group was 23.1% ± 12.7, while PM-MO presented 61.9% ± 4.7, indicating a decrease in cancer cell migration. Conclusion: Our results indicate that PM-MO demonstrated promising cytotoxic, antiproliferative, and anti-migratory activity in triple-negative breast cancer cell line. In addition, our polymeric micelle formation method proved to be successful in providing particles with optimal characteristics for biomedical application.

ACTIVATION PATHWAYS OF MURINE MACROPHAGES BY LIPOPHOSPHOGLYCAN FROM STRAINS OF LEISHMANIAMAJOR (FV1 AND LV39)

INTRODUCTION: The lipophosphoglycan (LPG) corresponds to a glycoconjugate well characterized due to its association with virulence in different Leishmania species. This molecule is the main constituent of the membrane of the promastigote forms of the parasite and acts by modulating the activation of phagocytes during the infection process. The Leishmania is phagocytosed by vertebrate host macrophages triggering a wide arsenal of leishmanicidal mechanisms. It is known that LPG is related to the modulation of the immune response of macrophages during infection, however the activation mechanisms triggered by this glycolipid complex are not yet fully elucidated. This study aims to investigate the role of purified LPG from two different strains of L. major, the main etiologic agent in the Old World clinical form of Tegumentary Leishmaniasis, in the activation of macrophages. METHODS: Bone marrow derived macrophages from C57BL/6 mice were stimulated with 10ug/mL of purified LPG from LV39 and FV1 strains and we followed with the analysis of the following parameters: nitric oxide (NO) production, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), cytokine production and the activation of MAP kinases. RESULTS: As a result, we observed that the LPG of strain LV39 that presents a higher number of side branches induced a more pro-inflammatory profile when compared to the LPG of strain FV1 and a higher production of nitric oxide (NO) and prostaglandin E2 (PGE2), higher expression of COX-2 and iNOS enzymes could be observed. Induction of phosphorylation of ERK-1/2 and JNK was also increased in macrophages activated with LPG from strain LV39. Finally, TNF-α cytokine production was also increased in cells stimulated by LPG from strain LV39, however there was no difference in IL-10 production between cells stimulated by LPGs. CONCLUSION: The results found suggest that the interspecific structural differences of LPG between LV39 and FV1 strains of L. major could result in distinct cellular activation responses in the host cell. Thus, the combined results of this work may contribute to clarify the inflammatory role of the LPG of an Old World Leishmania species.

ACUTE PHYSICAL EXERCISE INDUCES ANTI-INFLAMMATORY FUNCTIONS IN MACROPHAGES ASSOCIATED WITH EFFEROCYTOSIS

Physical exercise (PE) helps to regulate immune responses, increasing the phagocytic/microbicide capacity of macrophages (MØ), and the resolution of the infection/inflammation. Phagocytosis of apoptotic cells (efferocytosis) by the TAM receptor, Mer, is a mechanism to induce the regulatory/anti-inflammatory phenotype/function of MØ during homeostasis. Thus, we hypothesized that PE may modulate the inflammatory status caused by deficiencies in Mer-mediated efferocytosis by MØ. Material and methods: C57BL/6 wild-type (WT) and Mer deficient mice were subjected to acute exercise (WT-Ex or Mer-Ex: swimming, 7 days, 4% load) or control (WT-C and Mer-C). On the eighth day, the peritoneal cavity (PC) was washed and the immune cell populations were analyzed by flow cytometry. Peritoneal macrophages (pMØ) were isolated to investigate their phenotype, functional and metabolic capacity; also cell death (CD). Results: We didn't find differences in the percentage of B and T lymphocytes, DCs, NK or pMØ.However we've found more inflammatory pMØ/monocytes in the Mer-C and Mer-Ex compared to WT. However, after PE, there was less of this inflammatory population in the Mer-Ex compared to the Mer-C. On the other hand, more of the intermediate population of pMØ was observed in WT-C than Mer-C; and PE decreased this population in both groups, WT-Ex and Mer-Ex. After 24 hours of pMØ culture, we verified that PE induced a lower nitric oxide (NO) production between groups and it was even lower in Mer-Ex compared to WT-Ex. We’ve found that more CD occurs in PC of Mer-C compared to WT-C and Mer-Ex. However, CD was higher in Mer-Ex compared to WT-Ex. On the other hand, there was less CD in pMØ in the Mer-Ex groups compared to the WT-Ex after 24 and 48 hours of culture. Finally, we verified higher lactate levels in PC of Mer-C compared to the WT-C; and that PE negatively modulated lactate release in Mer-Ex. pMØ of WT-Ex produced high levels of lactate 48 hours after stimulation with LPS, which was not observed in the pMØ of the Mer-Ex. Conclusion. Collectively, our data suggest that PE is capable of reversing the frequency of inflammatory pMØ populations found in Mer deficient mice. Specifically, PE may be increasing anti- inflammatory pMØ populations due to a decrease in NO release in trained groups. Although this characteristic was not altered in pMØ Mer, we can find a decrease in lactate release in the PC of trained mice, suggesting that less pMØ is being classically activated.

ADENOSINE DERIVED FROM EXPANDED CD39-EXPRESSING PLASMABLASTS PROMOTES DYSREGULATION OF IMMUNE RESPONSES AFTER SEPSIS

Introduction: sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Here we report a new subset of splenic B cells that express CD39 expands in sepsis-surviving mice and suppresses the immune response against secondary infections by elevating circulating adenosine. Methods: we utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent secondary infection with L. pneumophila. High-dimensional flow cytometric analysis of the expression on the cell populations of naïve and sepsis-surviving mice were performed in immune cells collected on canto, verse, or fortessa flow cytometers and analyzed using FlowJo software. Septic and naive B cell and macrophages were also sort-purified, using a FACS Aria II, from mice spleen and peritoneal cavity, respectively. B cell were co-culture with macrophages and stimulated with L. pneumophila. Results: A2aR- or CD39-deficient mice showed improved resistance to post-sepsis infections. We performed a high-dimensional flow cytometric analysis of the heterogeneous CD39+ cell populations from the spleen of naïve and sepsis-surviving mice. Representative t-SNE maps color-coded according to cluster annotation for immune cell populations and expression intensity revealed that sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine derived from CD39hiCD138hi plasmablasts promoted suppression of the immune response by impairing the microbicidal activity of macrophages and enhanced interleukin-10 production via A2aR activation, rendering sepsis-surviving mice highly susceptible to secondary infections. Myeloid-specific deletion of A2aR improved the microbial resistance of sepsis-surviving mice. Septic patients have adenosine accumulation and an increase of immunosuppressive CD39hiCD138hiCD19+ B cells in the blood. Conclusion: our finding reveals an undescribed suppressive function of CD39-expressing B cells that is critical for developing sepsis-induced immunosuppression.

A HIGH CMV-SPECIFIC T CELL RESPONSE ASSOCIATES WITH SARS-COV-2-SPECIFIC IL-17+ T CELL PRODUCTION

Introduction. Human cytomegalovirus (CMV) is a widespread persistent herpes virus requiring lifelong immune surveillance to maintain latency (Virology, 483: 83–95, 2015). Such long-term interactions with the immune system may be associated with deleterious effects including immune exhaustion and senescence (Am. J. Epidemiol., 172: 363–71, 2010). We asked whether CMV-specific cellular and humoral activity could influence immune responses towards SARS-CoV-2 and/or disease severity. Methods and Results. This study was approved by the Moinhos de Vento Hospital Institutional Review Board and UFCSPA’s Ethics Committee. PBMCs from adults with mild (n= 15) and severe (n= 14) COVID-19 were stimulated in vitro with CMV or SARS-CoV-2 peptides to evaluate the production of IFNγ, TNFα, and IL-17 in CD4+ and CD8+ T cells. Anti-CMV IgG/IgM and anti-SARS-CoV-2 IgA/IgG antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using Fisher’s exact test, the nonparametric Mann Whitney test, and the Spearman correlation, with a <0.05 p-value. All adults with mild and severe COVID-19 were seropositive for anti-CMV IgG, but negative for IgM antibodies. Antibody titers did not correlate with COVID-19 severity or anti-SARS-CoV-2 antibodies. Six COVID-19 patients exhibited frequencies of CMV-specific cells above the third quartile for at least five of the six subpopulations analyzed, designated as CMV high responders (hiT CMV). In comparison to low CMV responders (loT CMV), hiT CMV individuals exhibited elevated frequencies of SARS-CoV-2-specific CD4+IL-17+ (hiT: 32.64% vs loT: 0.08%), CD8+IFNγ+ (hiT: 7.9% vs loT: 0.44%), CD8+IL-17+ (hiT: 24.7% vs loT: 0.2%) and CD8+TNFα+ (hiT: 19.2% vs loT: 0.58%) T cells (p<0.01), with an overall IL-17-dominated proportion of response towards SARS-CoV-2 peptides in the hiT CMV group. Conclusion. These results indicate that high frequencies of CMV-specific T cells may be associated with a SARS-CoV-2-reactive profile skewed towards Th17-dominated immunity.

AhR is important to sustain IFN-y-induced macrophage inflammatory profile against Leishmania (L) amazonensis

Airway administration of postbiotic protects mice against respiratory syncytial virus infection promoting immune tolerogenic profile

Respiratory syncytial virus (RSV) is a seasonal pathogen responsible for the highest percentage of viral bronchiolitis in pediatric patients. There are currently no vaccine available and therapeutic methods to mitigate the severity of RSV bronchiolitis are limited. OM-85, an oral standardized bacterial lysate isolated from human respiratory strains and widely used to prevent recurrent infections and/or exacerbations in populations at risk, has been shown to be effective and safe in children and adults. Here, we demonstrate that airway administration of OM-85 in Balb/c mice prior to infection prevents RSV-induced disease, resulting in inhibition of viral replication associated with less perivascular and peribronchial inflammation in the lungs. These protective effects are dose and time-dependent with complete protection using 1mg dose of OM-85 only four times intranasally. Mechanistic insights using this topical route in the airways revealed increased alveolar macrophages, a selective set of tolerogenic DCs, Treg and Th1 expansion in the lung, even in the absence of infection, contributing to a better Th1/Th2 balance and preventing ILC2 recruitment in the airways and associated inflammatory sequelae. OM-85 preventive treatment also improved antiviral response by increasing IFNβ and its responsive genes in the lung. In vitro, OM-85 protects against RSV infection in a type I interferon pathway. Our animal model data suggest that intranasal use of OM-85 should be considered as a potential prophylactic product to prevent RSV bronchiolitis once human studies confirm these findings.

Akkermansia muciniphila PROMOTES THE DENDRITIC CELL CD103+ DIFFERENTIATION, REGULATORY T LYMPHOCYTE INDUCTION AND AMELIORATES THE TYPE 1 DIABETES IN MURINE MODEL

Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive destruction of insulin-producing pancreatic β-cells by an inflammatory cell infiltrate and the production of autoreactive antibodies. It is known that alterations in the gut microbiota (dysbiosis) are capable of inducing abnormal immune responses in the gut-associated lymphatic tissue (GALT) and that these alterations can compromise the systemic immune response. In addition, a variety of metagenomic studies have associated the inverse abundance of Akkermansia muciniphila in the gut microbiota of obese, pre-diabetic, and diabetic humans or mice. In this context, we investigated whether A. muciniphila administration is able to ameliorate streptozotocin (STZ)-induced T1D. Methods: The T1D was induced in vivo by inoculating 1 daily dose of STZ 40mg/kg for 5 consecutive days, and the viable or inactivated A. muciniphila was administered every other day from the one day before the first dose of STZ until day 10 in C57BL/6 male mice (CEUA 169/2020). Euthanasia was performed 15 days after the first dose of STZ. We also evaluated the effect of A. muciniphila on the differentiation of bone marrow-derived dendritic cells (BMDC) in vitro. Results: Our data showed that In vivo administration of the A. muciniphila was able to control glycemia levels, reduced the incidence of T1D and degree of insulitis in mice with STZ-induced T1D. The modulation of T1D, in mice that ingested the probiotic, was related to the differentiation of CD11b-CD103+ (DC1) and CD11b+CD103+ dendritic cells (DC2) in the cecal lymph node and peyer's patches, induction of Treg lymphocytes in the pancreatic lymph nodes (PLN) and reduction of pathogenic Th17 lymphocytes. Of interesting manner, these mice had lower IgA+ B lymphocytes in the PLNs and reduction of IgA-labeled bacteria in feces when compared to the diabetic group without A. muciniphila administration. Then, we confirmed in vitro that A. muciniphila is efficient in inducing the differentiation of BMDC with a tolerogenic profile (CD103+CD11b+PDL-1+), reported in the previous studies as important immunoregulators that act to protect against the development of T1D. Conclusion: The A. muciniphila confers immunomodulatory properties able of attenuating T1D in mice, making this probiotic a promising target as new therapeutic tools against this disease.

A method for analyzing the genetic and specificity repertoire of immunoglobulins from human and murine germinal center B cells through single cell cultures

During immune responses against vaccines or pathogens, "specific" immunoglobulins may be generated, but immunoglobulins that do not react with the immunizing antigen or pathogen-related antigens are also formed. There are few studies characterizing these "nonspecific" immunoglobulins, regarding their repertoires, both for VH:VL usage and immunoreactivities. This is, in part, due to the lack of an efficient method for in vitro expansion of antigen experienced B cells, such as germinal center B cells (B-GC), allowing the analysis of the repertoire in a cost and time effective way. We have previously developed an alternative method to study the immunoglobulin repertoire based on single cell cloning cultures, for simultaneous characterization of Igs variable genes, clonal size and antigen specificity, without the need for cloning and Igs expression in vitro - J Immunol Methods. 376:143-149, 2012. In the present study, initially we adapted the culture method to study mice GC-B cells using 3T3 fibroblasts expressing BAFF (B-lymphocyte activation factor) and CD40-ligand, a key component for GC generation in T-dependent responses, 40LB cells - Nat Commun. 2:465, 2011. The efficiency of B-cell cloning cultures and the frequency of IgM and IgG secreting B-GC cells were tested using distinct polyclonal stimulus. B-GC cells were obtained from draining lymph nodes of mice immunized with non-replicating Zika virus-like particles (VLP) associated with different adjuvants (Alum, CpG, LPS and R848) and cultured for 7 days in a monolayer of 40LB cells. After primary and secondary doses of immunization the best culture condition was established using LPS+IL-21. Then, we obtained a 40LB feeder cells transfected to secrete IL-21, NB21 cells – Immunity. 44:542–552, 2016 – by which we achieved, after LPS stimulus, the best protocol to IgG secretion suggesting a improvement IgG amount secreted per clone through reached 10-fold than obtained using NB40L cells. The COVID-19 outbreak led us to adapt our system to culture B human cells from patients infected by SARS-COV-2. Therefore, we used flow cytometry to isolate spike-binding B clones. We reached the best in vitro stimulus condition using IL-2 + R848. This approach has been applied to correlate the antibody specificity repertoire with the variable Ig gene repertoire to better understand the mechanisms of generation of "specific" and "non-specific" antibodies after vaccination protocols in mice or in human infected by viruses.

ANALYSING THE INFLUENCE OF INTERMITTENT FASTING AND CAFETERIA DIET ON BREAST CANCER PROGRESSION: THE ROLE OF ADIPOSE TISSUE

Introduction: Breast malignancies are the most common and lethal tumors among the female population. Triple-negative breast cancer (TNBC) associates with neoplasm augmented aggressiveness and metastasis. The evolution of this disease is influenced by the individual lifestyle, including the diet. The increasing consumption of cafeteria diet (CAF) associates with the alarming overweight and obesity statistics, phenotypes characterized by metabolic syndrome, inflammation, and adipose tissue (AT) dysfunction. In contrast, intermittent fasting (IF), an efficient obesity therapy, correlates with metabolic efficiency, diminished inflammatory processes, and AT adequate activity. Although the impact of dietary patterns on breast cancer is well documented, there is scarce information regarding the effects of TNBC on adipose depots and about the impact of AT modulated by diets on this tumor type. In the present study, we evaluated the influence of intermittent fasting and a cafeteria diet on triple-negative breast cancer progression and investigated the role of AT in this context. Methods and Results: We used eight weeks female BALB/c mice to compare the effects of 24 h-period IF and CAF ad libitum on the progression of 4T1-mediated TNBC and showed that the latter feeding pattern coped with tumors with increased mass, size, and vascularization, and correlated with systemic inflammatory processes, including in the BAT. Considering that 4T1- impacted animals consuming cafeteria items show BAT inflammatory processes, we sought to investigate the impact of molecules secreted by ATs on 4T1 cells in vitro. First, we informed that molecules secreted by BAT presented higher cytotoxic effects on 4T1 cells compared to white AT (WAT), an effect that was enhanced in mice submitted to IF and diminished in rodents in CAF. We also discovered that molecules released by gonadal (g) WAT and IgWAT of animals submitted to IF decreased the secretion of IL-6 by 4T1 cells and secretion products of BAT derived from CAF animals augmented the secretion of this pro-cachetic cytokine by 4T1 cells. Moreover, molecules derived from gWAT and subcutaneous WAT from mice in IF led to increased 4T1 secretion of the immunogenic IL-1β. Conclusion: Our data suggest that molecules secreted by brown adipose tissue of animals submitted to IF may present antitumoral properties by affecting 4T1 cell death profile and immunogenicity and by diminishing the secretion of pro-tumoral mediators by these cells.

ANALYSIS OF PLATELET ACTIVATION AND INTERACTION WITH EPITHELIAL CELLS AGAINST SARS-COV-2 INFECTION

Introduction: Platelets are fundamental in hemostasis and in the architecture of thrombus in lesions, but they are also capable of recognizing viral pathogens and participating in the mediation of the inflammatory process in the face of infections. It is already known that platelets are able to induce interferon, extracellular neutrophil traps (NETs), increase leukocyte recruitment and modulate tissue factor (TF) expression in monocytes, which collaborates with the clotting process and thrombosis. However, an exacerbated response of platelets can lead to a worsening of the inflammatory condition, which can contribute to thrombus inflammation and generate aggravation of the cases. In immunothrombosis , activated neutrophils and monocytes interact with platelets and the clotting cascade, leading to clot formation within small and large vessels. The worsening of COVID-19 cases can progress to severe acute respiratory syndrome (ARDS), cause pneumonia, hyperactivation of the immune response, and immunothrombosis. Based on previous studies, we know that platelets are activated and interacting with other cells during SARS-CoV-2 infection and may contribute to host´s response to infection. Therefore, our objective in this work is to evaluate role of platelets in the thromboinflammatory process, and thus corroborating the lesion in the pulmonary epithelium. Methods and Results: Healthy donors were negative for SARS-CoV-2 by the reverse transcription polymerase chain reaction (RT-PCR) technique. Blood samples were collected and platelets were purified and selected by magnetic beads . At first, we evaluated whether the platelet could be activated directly by infection with SARS-CoV-2, through labeling with CD41+ CD63+. Once we confirmed that platelets are activated by the virus, we went to assess whether the presence of the platelet in the infected microenvironment could lead to changes in a more central cell during covid 19. Lung epithelial adenocarcinoma cell line-ATCC/HTB-55 (CALU-3) were infected, and 6 hours later platelets from healthy donors were added, and we observed an increase in viral replication within 48 hours, which was corroborated by the increase in RNA double-stranded labeling. Conclusion: The data confirm that platelets can be activated directly by infection with SARS-CoV-2 and suggest that platelets may play an important role in inflammatory amplification and thromboinflammation by interfering with viral replication.

ANALYSIS OF THE ANTIHERPETIC ACTIVITY OF THE EXUDATE AND MAJOR PRODUCT OF Salvia Uliginosa

Herpes Simplex Virus-2 (HSV-2) is a dermotropic virus, recent studies indicate that the percentage of HSV-2 infections in Brazil reaches 11.3%. HSV-2 is a sexually transmitted infection that leads to severe problems in individuals with a weakened immune system or undergoing immunosuppressive treatment. Furthermore, primo-infection or recurrence infections present a high risk of transmission from mother to child during delivery. The existing antiviral therapies are not effective during the latency phase and some strains are already resistant to the primarily used drug acyclovir. Therapies with plant derivatives have proven to be effective in treating herpes. Some species of Salvia have already been studied and demonstrated efficacy against viruses and other microorganisms. This study aims to evaluate and characterize the possible anti-herpetic effects of Salvia uliginosa exudate and its major component, icetexone (ICT) against HSV-2 in vitro and in vivo. Salvia uliginosa was collected and identified by a botanist, the extracts were produced and the major compound ICT was isolated from the exudate by high-performance liquid chromatography (HPLC). Initially, we tested the cytotoxicity of S. uliginosa exudate and ICT using the MTT assay in VERO cell lineage. The CC50 was also determined. The pre-treatment, virucidal effect, and time of addition (adsorption and replication) of ICT and the exudate on HSV-2 will be evaluated by a plaque assay. The toxicity analysis showed that Salvia exsudate at a concentration of 50.81μg/mL and ICT at a concentration of 8.319μM were able to kill 50% of VERO cells (CC50). We are now performing the antiherpetic activity of S. uliginosa compounds and evaluating in which part of the viral cycle it functions, to perhaps use it as a therapeutic alternative. With these results we will be able to continue the project analyzing if they have antiviral activity when tested against HSV-2 in vivo.

ANALYSIS OF THE RELATIONSHIP BETWEEN SEROPOSITIVITY FOR CYTOMEGALOVIRUS AND T LYMPHOCYTE PROFILE IN OBESE INDIVIDUALS

Introduction: Cytomegalovirus (CMV) is a vírus that infects humans with a high frequency, is endemic throughout the world. There are studies that indicate that the inflammatory chronic condition associated with obesity may be linked with CMV reactivation in the host. Obesity is a worldwide problem, and obese individuals present a chronic and subclinical inflammatory condition, which is characterized by the production of molecules and cells with an inflammatory profile. An obese individual may present different characteristics in the inflammatory process when CMV activation starts, characterized by synergism of the antiviral response and inflammation resulting from obesity or overweight, impacting metabolic and serum changes as well as inflammatory patterns. The present study evaluated the relationship between BMI and CMV seropositivity with their biochemical profile and phenotypic profile of T cells. Methods and Results: Data were obtained from a database of 113 individuals who were classified as eutrophic, overweight, and obese according to the Body Mass Index (BMI). All participants were male with a mean age of 34.72 years. After organizing the patients into groups according to their BMI, individuals were classified according to CMV seropositivity. Data related to the biochemical profile such as Glucose, Triglycerides, Cholesterol, HDL, and LDL to the phenotypic profile of T cells were evaluated according to the markers: CD4+, CD8+, CD4+CD25+CD39+, CD4+CD25-CD39+, CD8+CD27+CD28+, and CD8+CD27-CD28-. A prevalence of 48,63% of seropositive individuals for cytomegalovirus was observed. There were no statistical differences found regarding the biochemical sample data when comparing the groups’ results. The body mass index and seropositivity for cytomegalovirus did not influence the frequency of CD4+, CD8+ T, and CD8+CD27+CD28+ cells. The frequency of CD8+CD27-CD28- cells according to the BMI groups was: eutrophic CMV negative group 16.49 ± 9.18, CMV positive group 19.82 ± 6.66, overweight CMV negative 22.23 ± 4.64, CMV positive 19.33 ± 7.28, obese CMV negative 19.33 ± 7.28 and CMV positive 30.64 ± 9.54. This group of cells presents a difference between overweight CMV positive and overweight CMV negative as well as obese CMV positive against obese CMV negative. Conclusion: The results indicate that the presence of CMV contributes to an alteration in the frequency of highly differentiated memory T cells in overweight and obese individuals.

Analysis of the secretome profile of PBMC of individuals immunized with the ChAdOx1 nCoV-19 vaccine

INTRODUCTION.The ChAdOx1 nCoV-19 vaccine is one of the most relevant that emerged during the worldwide effort to immunize against COVID 19, which was developed from sequences of the Spike protein of SARS-CoV-2 inserted in an adenoviral vector - virus attenuated recombinant vaccine. The development of an immune response is expected after vaccination, which allows the body to impair the severity of SARS-CoV-2 infection. Secreted protein profile, or secretome, can be used to analyze factors modulated in an experimental situation or to ongoing disease. The secretome strategy involves mass spectrometry technology and subsequent in silico analysis to determine the profiles of secreted proteins and factors. Quantitative and qualitative analysis of proteins profile of participants, before and in different time-points after vaccination, would assess the predominant immune response with or without natural infection by SARS-CoV-2 VOCs. METHODS. Longitudinal study with 60 volunteer participants. Inclusion criteria: (1) age ≥ 18 years, (2) Non-smokers. Non-probabilistic sampling for convenience. Blood samples were collected on days 0 (1st dose), 30 (2nd dose), 60, 120, 210, 390 after the 1st dose. PBMC were extracted from peripheral blood and subsequently stimulated with synthetic peptides from the S proteins of 4 VOCs of SARS-CoV2: Wuhan (Original), Alpha, Delta and Omicron. The following immunological parameters were evaluated: (1) qualitative and quantitative proteomics by analyzing the Nano LC-ESI-Q-TOF system and software; (2) cytokine profile in patients’ sera by CBA; and (3) cellular response by ELISPOT. The proteomics and CBA data obtained were imported into the SPSS® program and parametric or non-parametric tests were used, with a confidence level of 95%. RESULTS. In the conditioned medium, immune system molecules related to intercellular signaling processes, such as cytosine and chemokines, and immunoglobulin subunits were detected, with different expression between untreated treated cells. CONCLUSION. Despite the challenges to detect and analyze the presence of signaling molecules of the immune response by this approach, was possible to identify them through mass spectroscopy analysis. The next step will be to correlate secretome data with sera cytokine profile and ELISPOT results.

ANDROGEN HORMONES MODULATE LUNG CELLS INFECTION BY SARS-COV-2

Introduction. The higher morbidity and mortality in men with COVID-19 may be attributable to many sex-related differences, including androgens or estrogens’ regulation of immune responses. SARS-CoV-2 infection depends on host proteins such as ACE2 and TMPRRS2, whose expression are up regulated by testosterone (TT) or dihydrotestosterone (DHT), after binding to androgen receptors (AR). Then, we aimed to understand the relationship between androgens and SARS-CoV-2 in lung cells, which are the primary target of this viral infection. Methods. The male epithelial cell lineage NCI-H460, was cultivated in RPMI 1640 medium and viral stocks of SARS-CoV-2 lineage B (isolate BRA/SP02/2020, alpha variant) or lineage P.1 (GISAID EPI_ISL_2499748, gamma variant) were produced in Vero E6 cells. Subsequently, 2x105 cells/well (24-well plates) were seeded and incubated for 24h, followed by another 24h incubation with TT at 10 µM; DHT 0.1 µM or flutamide (10 µM), followed by infection with SARS-CoV-2 (alpha or gamma variant) at 0.2 or 2.0 MOI. After additional 48h-culture, at 37ºC, 5% CO2, cells were collected for mRNA evaluation and supernatants assay for IL-6. Results. DHT, the most potent testosterone metabolite, increased ACE2 expression in lung epithelial cells infected with both virus lineages. This expression was partially dependent on AR signaling, especially in the lineage B infection with a smaller MOI, while the concomitant DHT treatment with AR blockage increased ACE2 in infection with higher viral load. The AR was markedly augmented after virus B infection at MOI 0.2 and treatment with DHT, followed by reduction when AR was blocked. Otherwise, ACE2 augmented after TT treatment in infected cells independently on AR. TMPRRS2, which accounts for virus invasion, tend to augment in most conditions related to androgens treatment, similarly to the increased SARS-CoV-2 detection in cells infected with virus B, at a lower MOI and exposed to both hormones. Notably, the elevated estrogen receptor 2 expression, which may counter regulate responses after infection and exposure to androgens, seems to be dependent on AR signaling when stimulated by TT, but not DHT, similarly to the IL-6 production induced by infection with virus lineage B and androgens treatment. Conclusion. These results corroborate the clinical findings of worse COVID-19 outcomes in men and points to an important role of androgen hormones in the regulation of the lung cells responses to the infection.

A NOVEL INSIGHT ON SARS-CoV-2 S-DERIVED FRAGMENTS IN THE CONTROL OF THE HOST IMMUNITY

Introduction: Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screening new drugs and to compress the organism's response to the disease. Methods and results: Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The procedures were approved by the Ethics Committee (CEUA) of the Medicine Faculty of University of Sao Paulo and registered under protocol number 1514/2020 and by the Ethics Committee (CEUA) of the University of Parana and registered under protocol number 1410. Conclusion: The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

ANTIGEN BINDING EVALUATION OF ANTI-ACINETOBACTER BAUMANNII MONOCLONAL ANTIBODIES

Introduction: healthcare-associated infections (HAI) are a worrying issue in Brazil, along with the worldwide increase in antimicrobial resistance. Acinetobacter baumannii is a Gram-negative, commensal, and opportunistic bacteria related to nosocomial infections that result in pneumonia associated with mechanical ventilation, bloodstream infections, among others. This pathogen is considered a health threat due to its resistance and virulence mechanisms. In 2017, the World Health Organization (WHO) published a list of pathogens of global priority to guide research and development (R&D) for new antibiotics, and A. baumannii resistant to carbapenem is on the top, being considered a critical priority. The emergence of multi- and pan-resistant strains has limited the treatment of infections caused by this bacterium, and polymyxin resistance, often a last-line antibiotic, is a growing concern. Hence, there is an urgent need to develop new therapeutic options for the treatment of infections caused by this pathogen. In this scenario, non-traditional antibacterials, such as monoclonal antibodies (mAbs), emerge as a highly specific and promising approach. Therefore, the aim of this work is to characterize antigen binding to previously obtained anti-A. baumannii mAbs. Methods: mAbs targeting a specific A. baumannii protein were previously developed by hybridoma technique. ELISA and Western blot assays were performed to evaluate recombinant protein and native protein mAbs binding. Purified recombinant target protein (previously obtained in Escherichia coli) or bacterial lysates of different strains (ATCC19606, AB307/ AB307.30 – capsulated and non-capsulated, and ST162) of A. baumannii were utilized. Results: both mAbs tested were able to specifically recognize the target protein even at low concentrations (pM), including the native protein in the bacterial lysates. Binding specificity also demonstrated that the antigen target is conserved between different A. baumannii strains. Furthermore, functional and animal studies are scheduled to demonstrate mAbs antibacterial activity. Conclusion: considering the results obtained, both tested mAbs demonstrated a potential use for treatment and diagnostics of A. baumannii infections.

ANTI-HER2 PIGGYBAC TRANSPOSON-BASED CAR T CELLS PRODUCTION AND PHENOTYPE CHARACTERIZATION

The PiggyBac (PB) system consists in a non-virus transposon/transposase gene delivering tool. Chimeric antigen receptors (CARs) are molecules capable of redirecting immune cells against a specific tumor antigen (Chicaybam et al, 2020); several studies elect the HER2 receptor as a good target due to its specific overexpression in different solid tumors. The aim of this work was to evaluate the transposition efficacy of two different anti-HER2 CARs (4D5 and FRP5) on primary peripheral blood mononuclear cells (PBMCs) isolated from different healthy donors, using the PB system. The two clones of anti-HER2 CARs were synthetized and cloned into the PBCAG plasmid vector; for transposition, PMBCs up to 6 donors were isolated and electroporated with 20ug of the transposase and 10ug of the 4D5 or FRP5 carrying plasmid. After the electroporation, the cells were cultured for up to 12 days and the receptor expression, memory and exhaustion phenotype were analyzed at different times by flow cytometry. A constant expression of the two receptors was observed, reaching an average of 25% of expression 12 days after transduction. On the eighth day of expansion, the phenotypes of central memory (CD45RO+ CCR7+) and effector memory (CD45RO+ CCR7-) were also evaluated, in addition to exhaustion-related markers (PD1, TIM3 and LAG3) in CAR-T CD4+ and CD8+ populations. In both cases we observed a similar phenotype in the evaluated anti-HER2 CAR receptor populations, evidencing a higher frequency of cells with central memory phenotype. For exhaustion-associetes receptors, we observed a lower frequency of expression among CD8+ CAR-T cells expressing the FRP5 clone. For the CD4+ subset, we could observe an increase of the markers for either of the CAR constructs. In summary, the two evaluated anti-HER2 CARs demonstrated consistent expression in PBMCs from different donors, with predominant central memory phenotype and low frequency of inhibitory receptor expression. Future analysis will be performed to evaluate the functional potential of the produced cells against HER2-positive tumor cells.

ANTITUMOR POTENTIAL OF MOXIDECTIN IN BREAST CANCER CELL LINE

Introduction: The immune system is intrinsically related to cancer, as it is responsible for maintaining homeostasis through the elimination of cells with oncogenic potential. Tumor cells capable of escaping immune surveillance have clinical oncological potential. Therefore, the study of antineoplastic agents capable of expanding therapeutic approaches is necessary, especially in chemotherapy-resistant tumor cells. Moxidectin (MOX) is a broad-spectrum anthelmintic belonging to the macrocyclic lactone family. In Scabies in vivo model, MOX treatment showed mast and CD3 T cells infiltration, suggesting immune modulation. Unlike drugs from this same family, MOX lacks studies that investigate its antineoplastic activity. Therefore, our study aimed to evaluate the antitumor potential of MOX in a breast cancer cell line. Methods and Results: Cytotoxicity tests were performed with MOX at concentrations of 10, 50, 100, 250, 500 and 1000 nM for 24, 48 and 72 hours (h) in MCF-7 (tumor) and MCF-10A (normal) cell lines. The selectivity index (SI) and the half maximal inhibitory concentration (IC50) were calculated. Tumor cells morphology, cell cycle and apoptosis after treatment with MOX at 25, 50 and 100 nM for 48 hours were also evaluated. The results indicated that MOX did not cause significant decrease of viability in the tumor cell line after 24h treatment. The cytotoxic effect was observed only in 48h, followed by a slight recovery of viability after 72h. The viability of normal cells reduced 20-30% in the first 24h of treatment and the number of viable cells remained constant in subsequent times. IC50 and SI revealed that MOX had a selective effect, mainly at 48 (SI=37.89) and 72h (SI=2.78). Detected morphological variations like decreased cell volume, cytoplasm granulation and adherence and nuclear alterations, are indicative of apoptosis. At concentrations of 50 and 100nM MOX induced cycle arrest in S phase, indicating antiproliferative effect. Previous studies have already shown that MOX reduced the expression of CDK2 and cyclin E, proteins linked to DNA synthesis in the S phase in a glioma lineage. Annexin V apoptosis assays confirmed the morphological findings. Conclusion: MOX presents high selectivity in the antitumor treatment and is capable of inducing morphological changes and death by apoptosis. The high frequency of cells arrest in the S phase may be one of MOX’s mechanisms of action.

A promising multi-stage, multi-antigen chimeric recombinant protein as an anti-Plasmodium vivax vaccine candidate

Introduction: Plasmodium vivax is the main malaria cause outside Africa, including Brazil. The development of an effective vaccine formulation against this parasite is a global concern. Decades of efforts have demonstrated how challenging is to achieve this goal. Key proteins in the process of parasite entry into target cells generated promising results, highlighting chimeric proteins with antigens from different stages of the parasite’s life cycle. Therefore, our aim is to evaluate the immunogenicity of multi-antigens and multi-stage vaccine formulations against malaria vivax. Methods and Results: A new hybrid PvCSP-MSP119 protein, composed by the conserved C-terminus of P. vivax Circumsporozoite Protein (PvCSP) and its variant repeat domains, and 19kDa portion of Merozoite Surface Protein 1 (PvMSP119), were expressed in Pichia pastoris, purified and confirmed by immunoblotting. Female C57BL/6 and Balb/C mice (6-8 weeks) were vaccinated in prime-boost homologous or heterologous regimen (3 doses, 21 days interval) with 10 µg (recombinant protein/dose) using Poly (I:C) as adjuvant or 108 p.f.u. virus (dose/animal). These protocols were approved by the Laboratory Animal Research Ethics committee (CEUA/FCF/USP 054.2021 – CEUA P614). Blood samples were collected to assess antibodies titers and longevity. Antibody-secreting cells (ASC) were also evaluated on bone marrow. Balb/C mice were challenged with Pb/PvMSP119-GFPNK65 to evaluate the formulation’s protective immunity. The chimeric protein induced significant and stable antibodies titers against PvMSP119 (10^5) and PvCSP regions (10^2, 10^2, 10^4 and 10^6 against VK210, VK247, P. vivax-like and C-terminal regions, respectively), when compared to immunizations with PvCSP or PvMSP119 isolated or combined with adenoviruses. It also induced high frequency of ASCs 21 days after the third dose (10^1-10^2 cells/millon). Preliminary results demonstrated that heterologous prime-boost immunization induces the production of neutralizing antibodies, partially protecting infected mice. Conclusion: The results demonstrate that the chimeric protein is a good malaria vaccine candidate once it induces high titers of durable antibodies and ASCs against two antigens from different stages of the parasite’s life cycle, in the different immunizations regimen.

Arginase 1 is a marker of protection against illness in contacts of leprosy patients

Introduction: Leprosy household contacts are generally more prone to develop the disease compared to the general population. Previous studies have demonstrated that genes related to the alternative activation (M2) profile in macrophages are associated with the increased bacillary load in multibacillary leprosy patients (MB), and that contacts of MB patients have a higher risk of contracting the disease. In addition, positive serological responses to PGL‑1 or LID‑1 are associated with a higher risk of disease. Methods and Results: We performed a 5‑year follow‑up of contacts of leprosy patients and evaluated the pattern of gene and protein expression in cells from contacts that developed leprosy during this period. Leprosy household contacts had decreased soluble CD163 and heme oxygenase 1 (HO‑1) serum levels when compared with healthy donors and leprosy patients. In contrast, arginase 1 (ARG) activities were higher in contacts when compared with both healthy donors and leprosy patients. Of the contacts, 33 developed leprosy during the follow‑up. Gene expression analysis revealed reduced ARG1 expression in these contacts when compared with contacts that did not develop disease. Arginase activity was a good predictive marker of protection in contacts (sensitivity: 90.0%, specificity: 96.77%) and the association with serology for anti‑PGL‑1 and anti‑LID‑1 increased the sensitivity to 100%. Conclusion Altogether, the data presented here demonstrate a positive role of arginase against leprosy and suggest that the evaluation of arginase activity should be incorporated into leprosy control programs in order to aid in the decision of which contacts should receive chemoprophylaxis.

ARYL HIDROCARBON RECEPTOR IMMUNOMETABOLIC ROLE DURING A MURINE BETACORONAVIRUS EXPERIMENTAL INFECTION

Introduction The ongoing COVID-19 pandemic has already caused over 6 million deaths worldwide, making the better understanding of the mechanisms of SARS-CoV-2 infection an imperative. Aryl hydrocarbon Receptor (AhR) is a xenobiotics receptor and its activation by aromatic compounds has been associated with immune suppression. AhR activation on the lungs of COVID-19 patients is responsible for increased mucus release, leading to aggravation of respiratory distress. AhR activation has also been identified as a proviral factor in an experimental murine Betacoronavirus infection. Moreover, AhR activation has been demonstrated to regulate T cell metabolism and differentiation. However, the mechanisms underlying the role of AhR in cell and systemic metabolism are still poorly understood. In this work, we investigated the effect of AhR deletion on the disease induced by murine hepatitis virus (MHV-3) experimental infection. Methods and Results We intranasally innoculated WT and AhR-/- mice with MHV-3 and collected lung and liver samples 3 and 5 days post infection (dpi). Infected WT mice have severe weight loss and die 6 dpi. Infected WT mice also show a progressive decrease of platelet and leukocyte count in the blood, increased levels of ALT/AST and proinflammatory cytokine release in the plasma by the 5th dpi, together with systemic viral dissemination. Although AhR deletion did not affect weight loss and survival, it prevented platelet and leukocyte count drop while also preventing plasma ALT/AST increase. Mice lacking AhR also have reduced proinflammatory cytokine release in the plasma. Yet, infected AhR-deficient mice presented increased viral burden in the lungs and liver at 3 dpi, while presenting lower viral burden in the liver at 5 dpi, when compared to WT infected mice. Also, AhR deletion induced increased expression of glycolytic genes in the liver during MHV-3 infection, suggesting AhR as a regulator of glycolytic flux during viral infection. Conclusions Infected AhR-/- mice have mitigated systemic manifestations of disease, reduced inflammatory cytokines release and increased expression of glycolytic genes. Therefore, our results suggest AhR activation as an important step in the pathogenesis of MHV-3 infection.

Aryl hydrocarbon receptor is necessary to restrain tissue macrophage inflammatory tonus and systemic glucose homeostasis during obesity

Macrophages (MØ) are present in all tissues of vertebrates and play a key role in both host defense and tissue homeostasis. The aryl hydrocarbon receptor (AHR) is one of the central regulators of immune cell fitness, directly impacting MØ polarization. Once activated, AHR can modulate distinct cell functions, including toll-like receptor signaling cascades to adjust MØ activity in response to endogenous and exogenous ligands. Recent studies with AHR-null mice suggested that AHR signaling appeared to be a critical factor in obesity development, a disorder that involves the accumulation of inflammatory MØ in adipose tissue (AT). However, we still do not know if AHR signaling in MØ contributes to the development of obesity. We aim to determine how AHR function in MØ contributes to obesity development. First, we analyzed the transcriptomic profile of AT MØ in chow (CD) and high-fat (HFD)-fed mice and identified that Ahr signaling is downregulated in AT MØ from obese mice. To uncover Ahr contribution to MØ regulation during obesity, we fed Ahrfl/flLysMcre+ (KO) and Ahrfl/flLysMcre- (WT) mice with a CD or a HFD for 12 weeks. KO mice presented higher glucose intolerance when fed with a CD or HFD, with no changes in weight gain. In addition, fasting KO mice challenged with insulin presented decreased AKT phosphorylation in the liver, muscle, and perigonadal AT, indicating systemic insulin resistance. Also, CD KO had larger pancreatic islands, a sign of metabolic stress. HFD-fed KO mice presented higher lipid depots in the liver and increased TNFa production by splenic MØ and to a lower extent by AT MØ. In vitro, stimulation with palmitate (palm), the most abundant FFA in serum of obese subjects, increased the secretion of TNFa in KO MØ, which also presented elevated glycolysis. Treatment of MØ with an AHR agonist prior to palm decreased the expression of Tnfa and glycolysis compared with palm exposure alone, highlighting the role of AHR for MØ fitness upon obesogenic conditions. Therefore, our results indicate that the loss of AHR signaling in MØ exposed to an obesogenic environment affects cell metabolism and inflammatory tonus, impacting obesity-induced inflammation and the subsequent insulin resistance.

Aryl hydrocarbon receptor is necessary to restrain tissue macrophage inflammatory tonus and systemic glucose homeostasis during obesity

Macrophages (MØ) are present in all tissues of vertebrates and play a key role in host defense and tissue homeostasis. The aryl hydrocarbon receptor (AHR) is one of the central regulators of immune cell fitness, directly impacting MØ polarization. Once activated, AHR can modulate distinct cell functions, including toll-like receptor signaling cascades to adjust MØ activity in response to endogenous and exogenous ligands. Recent studies with AHR-null mice suggest that AHR signaling appeared to be a critical factor in obesity development, a disorder that involves the accumulation of inflammatory MØ in adipose tissue (AT). However, we still do not know if AHR signaling in MØ contributes to obesity-induced inflammation and metabolic syndrome. Aim: We aim to determine how AHR function in MØ contributes to obesity-induced inflammation and insulin resistance. Results: First, we analyzed the transcriptomic profile of AT MØ in chow (CD) and high-fat (HFD)-fed mice and identified that Ahr signaling is downregulated in AT MØ from obese mice. Next, we established that in vitro stimulation with palmitate (palm), the most abundant FFA in serum of obese subjects, also decreased AHRr signaling, increasing the production of proinflammatory cytokines in MØ. These data indicate that palm stimuli is a valuable proof of concept model on how the Ahr pathway contributes to MØ activity in an obesogenic environment. To uncover the contribution of Ahr to MØ regulation during obesity, we fed Ahrfl/flLysMcre+ (KO) and Ahrfl/flLysMcre- (WT) mice with a CD or HFD for 12 weeks. KO mice presented higher glucose intolerance when fed either with a CD or HFD, with no changes in weight gain. In addition, fasted KO mice challenged with insulin presented decreased AKT phosphorylation in the liver, muscle, and perigonadal AT, indicating systemic insulin resistance. Also, CD KO mice had larger pancreatic islands, a sign of systemic metabolic stress. HFD-fed KO mice presented higher lipid-depots in the liver, increased TNFa production by splenic MØ and, to a lower extent, by AT MØ. In vitro, palm treatment increased the secretion of TNFa in KO MØ, which also presented elevated glycolysis. Treatment of MØ with an AHR agonist prior to palm decreased the expression of Tnfa and glycolytic enzymes to levels compared to palm alone, highlighting the role of AHR for MØ fitness upon obesogenic conditions.

ASSESSING TEMPORAL TRANSCRIPTIONAL PROFILES OF CUTANEOUS LEISHMANIASIS TO IDENTIFY NEW TARGETS FOR THERAPEUTIC INTERVENTIONS

Introduction – Cutaneous Leishmaniasis (CL) is characterized by the appearance of ulcerative skin lesions that can cause severe and permanent scars. Previous studies have provided large-scale transcriptome datasets of human skin lesions caused by Leishmania braziliensis elucidating key issues related to immunopathology. However, due to the inherent limitations of human lesion sampling, limited progress has been achieved in understanding the early mechanisms of infection, progression and the healing process. Moreover, there are gaps in understanding the communication between the immune responses occurring in the skin lesion and the draining lymph node (LN). In view of these limitations, and the existence of an animal model that recapitulates the disease caused by L. braziliensis, we performed a time-course transcriptomic profiling study of lesions and LNs in this model. Methods and Results - BALB/C mice were infected with L. braziliensis parasites in the ears. Progression and resolution of lesions were monitored weekly for up to 77 days. Infected ears and corresponding draining LN were collected at 2, 6 and 48h, and 14, 31 and 77 days after infection. Bulk RNA-seq was performed using three biological replicates per time-point for both tissues (ear and LN). CEMItool was used to identify modules of genes that are significantly co-expressed in the different phases of lesion progression and resolution. Six modules were identified (M1-6): three related to the immune response and three related to tissue remodeling. The M1 module revealed the presence of genes related to the phagosome formation pathway, M2 to the LXR/RXR activation pathway and M5 to the IL-7 signaling pathway. Modules M3 and M4 were enriched for wound healing process and M6 for apical junction formation. Analysis of interaction networks using the Ingenuity Pathway Analysis (IPA) revealed the down-regulated miR-let7-e as a central gene of M3 module, associated with the polarization of inflammatory macrophages and possibly contributing to infection resolution. Conclusion - Altogether, the transcriptomic profile provided here highlights previous and new pathways associated to L. braziliensis infection, which could lead to new avenues for therapeutic interventions as the use of miRNA mimics and inhibitors.

ASSESS OF CELLULAR AND HUMORAL RESPONSE OF VACCINATED VOLUNTIERS AND VALIDATION OF SpiN AS A BOOSTER CANDIDATE OF COVID-19 VACCINE

The fast spread of the SARS-CoV-2 lead to a pandemic that hurried the scientists to develop vaccines in a record time. Several vaccines were developed with great efficacy in diminishing the morbidity and lethality, but none of them offer a remarkable protection against the infection. Projections indicate that SARS-CoV-2 will continue among us for a long time and this realization maintains the search for an effective vaccine that blocks the infection. We generated a chimeric protein that comprises the receptor binding domain (RBD) from Spike (S) and the nucleocapsid (N) antigens (SpiN) from SARS-CoV-2. Taking into account that vaccination with SpiN elicits a protective immune response in rodents, our goal is to evaluate the specific anti-SpiN cellular and humoral responses of vaccinated donors. Blood of healthy, convalescent and vaccinated volunteers, 30 days after the second dose and 180 days after the first dose, were collected. PBMC and serum were separated to assess IFN-γ production by Interferon-Gamma Release Assay (IGRA), antigen-specific and neutralizing IgG levels and cellular profile upon SpiN stimulus. In a general way, we observed that cellular and humoral responses to SARS-CoV-2 antigens tends to drop in vaccinated people 180 days after the first dose, mainly in CoronaVac vaccinated volunteers. We found that IFN-gamma levels decrease in CoronaVac, but not in AstraZeneca volunteers. Levels of IgG anti-S dropped in both, CoronaVac and AstraZeneca volunteers, while anti-N decreased only in CoronaVac, as expected. Interestingly, the titers of neutralizing antibodies drop significantly 180 days after the first dose. Stimulating cells of vaccinated, controls and COVID-19 convalescent individuals with SpiN, we observed that both, convalescent and vaccinated volunteers presented memory CD4+ and CD8+ T cell response. The vaccines applied in Brazil induces a specific response against SARS-CoV-2, but these responses don’t last for several months, what lead us to be frequently boosted with another dose. Stimulation of PBMC with SpiN showed a good response. Further studies need to be finished to evaluate also the humoral response, but these previous results are promising, revealing SpiN as a potential booster candidate, indeed SpiN is a proteic vaccine, what makes it safer to use as boost, including in children.

Association between adipomyokines levels with immunometabolic markers and dysfunctional adipose tissue. Highlighting sdLDLc profile and C3 levels in sedentary adults

Introduction. In adipose tissue the interaction between immune cells and adipocytes helps adipomyokines production; while the response to overnutrition leads to a low-grade inflammation process, with systemic effects on the outcomes of the metabolism. These processes equally involve irisin and adiponectin, adipomyokines secreted by adipocytes in lean and healthy state, or under-expressed in obesity. They stimulate adipocyte browning, and fat acid oxidation, respectively. The aim was to establish the association between the serum levels of irisin indicators and adiponectin-oligomers with immunometabolic markers and dysfunctional adipose tissue of sedentary subjects with an overweight-obesity state. Methods and Results. This study included 316 sedentary individuals, classified according to World Health Organization criteria by body mass index in lean, overweight and obesity. Body fat mass distribution, metabolic and inflammatory markers were measured using anthropometric, enzymatic, and immuno-turbidimetry routine methods. ELISA was used to evaluate serum insulin and adipomyokines. Statistical analysis differentiating between males and females yielded correlations between adiposity and adipomyokines measurements (P < 0.05 was considered significant). An inverse pattern of levels of irisin and adiponectin-oligomers is observed between women and men, besides negatively correlate with lipid profile, C3, and relative total body and absolute lower limb fat mass. In contrast, MMW-Adiponectin and irisin levels, and absolute total body non-fat mass positively correlated. The adiposity status categorized as lower, median, or upper (quartiles), showed differences in metabolic and inflammation markers, insulin resistance status, and elements of the lipid profile, mainly between upper and lower body fat mass. This coincides with the differences in fat mass in the trunk and model multivariate regression analyses. For irisin levels, the differences were between lower and upper groups in relative total body fat and non-fat mass, body adiposity index, lipid profile, and metabolic markers. While the differences observed between the lower and median groups were for waist to height ratio, HDLc, sdLDLc and CRP. We note that C3 levels were consistently associated with differences between all groups. Conclusion. In a dyslipidemic state with the presence of low-grade subclinical inflammation, the irisin indicators represent the leading role in dysfunctional adipose tissue.

ASSOCIATION BETWEEN VARIANTS IN THE GENES OF METALLOPROTEINASES 1, 8, 9 AND PERIODONTITIS

Introduction: Periodontitis is a chronic inflammatory disease characterized by the destruction of the supporting tissues of the teeth and triggered by the host's immune response to the presence of a dysbiotic subgingival biofilm. Metalloproteinases (MMPs) are proteolytic enzymes involved in the inflammatory process in periodontal disease, and MMPs seem to be the main enzymes related to periodontal destruction. The increased frequency of some single nucleotide MMP variants (SNVs) in individuals with periodontitis in different populations suggests that polymorphisms in MMP genes may alter protein expression, predisposing to the development of periodontitis. Methods and results: This is a cross-sectional study, carried out with blood samples from adults and individuals with periodontitis, recruited at the Health Center of the Bahia Asthma Control Program (ProAR). Genomic DNA from these individuals was extracted and genotyped using the Illumina Multi-Ethnic Global Array (MEGA, Illumina) platform. The study population consisted of 506 individuals of both sexes, with 83.2% (n=421) women and 32.8% (n=166) aged less than or equal to 39 years, 74.7% (n=166) = 378) with an income of less than one minimum wage and 83.2 (n=421) self-declared black/brown. In the genetic analysis for genotypic evaluation, three models were tested: additive, dominant and recessive, and when expanding the p-value it was noted that there was no MMP8 variant with a p-value lower than 0.05. For MMP9, in the additive and recessive models analyzed, there was no association with periodontitis. In the dominant model, SNV rs13925 (allele A) was associated with periodontitis (OR=0.56 and 95% CI 0.33-0.95), proving to be protective against periodontitis. The SNV rs2071230 (smallest G allele) of MMP1 was associated with periodontitis in the additive (OR=1.42 and 95% CI 1.002-2.03) and dominant (OR=1.53 and 95% CI 0.97-2) models. ,4). In the recessive model, there was no association. The rs13925 variant of the MMP9 gene had the highest frequency of the polymorphic allele in the population studied, MAF of 0.153. Another rs2071230 variant of the MMP8 gene showed a frequency of the lowest allele (polymorphic allele), MAF equal to 0.22. Conclusion: The data from this study suggest that there is an association of MMP9 SNV rs13925 and MMP1 SNV rs2071230 and periodontitis, however more studies are needed to clarify this association.

ASSOCIATION OF THE -308 G>A POLYMORPHISM OF TNF GENE WITH SEVERE COVID-19

Introduction: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2). Serum tumor necrosis factor-alpha (TNF-alpha) levels are elevated in patients with severe disease form. TNF-α is an inflammatory cytokine that promotes tissue damage and can affect the COVID-19 pathogenesis. Genetic polymorphisms in the TNF gene, such as -308 G>A (rs18000629), are associated with modified TNF-α production. Therefore, this study aimed to evaluate the effect of rs18000629 on the severity of COVID-19 in individuals from the northern/northeast region of Paraná. Methods: A case-control study was performed, with moderate and severe patients that were classified according to the World Health Organization (WHO), by Clinical Hospital (HC) of the State University of Londrina (UEL). Forty (40) moderate and forty (40) severe patients were selected for the study, matched according to sex, with exclusion criteria for patients with heart disease, liver disease, other respiratory diseases, HIV, and cancer. The -308 G>A polymorphism was genotyped by PCR-SSP and statistical analysis was performed using logistic regression in the SNPStats software. The groups were compared in relation to genotypes and alleles Results: The genotype distribution was according to the expected in the Hardy-Weinberg equilibrium (P > 0.05). There was an association between the G/A-A/A genotypes with the severe form of COVID-19 compared to the G/G genotype (OR=3.48, 95% CI 1.03-11.73, P=0.03). The frequency of genotype G/G was 87.5% in moderate patients compared to 67.5% in severe patients; and the G/A-A/A was 12.5% in moderate patients and 32.5% in severe patients. The allele frequencies of G were 0.92 and of A was 0.08 in moderate patients, and 0.84 and 0.16 in severe patients, respectivelly. The mean age was 58.68 ± 15.96 for moderate patients and 67.18 ± 17.63 for severe patients. Student's t-test showed a difference in mean age between moderate and severe patients (P=0.02), so adjustment for the age variable was included in the logistic regression analysis. Conclusion: We can conclude that the G/A-A/A genotypes (in a dominant genetic model) of the polymorphism -308 G>A are a risk factor for severe Covid-19 independent of age, but genotype determination should be done in a high number of patients.

ASSOCIATION OF THE INFLAMMATORY PROFILE IN PATIENTS WITH TEMPOROMANDIBULAR DISORDER ACCORDING TO DISEASE SEVERITY

Introduction: During the processes involving temporomandibular joint diseases (TMD), a complex inflammatory response is developed involving the synthesis of different cytokines by synovial and inflammatory cells that infiltrate the joint and tissues during the onset and progression of the disease. The aim of this study was to investigate the relationship between the severity of TMD and systemic inflammatory markers in patients with or without other inflammatory or immune-mediated diseases. Methods and Results: 30 patients of both genders, over 18 years old, who met the inclusion and exclusion criteria were recruited and were evaluated to define the degree of TMD clinically and by imaging exams. They were divided into three groups: 1) (N=8) with mild/moderate TMD; 2) (N=10) with severe TMD and 3) (N=12) with severe TMD presenting inflammatory or autoimmune associated comorbidity. Circulating levels of IL-1β, IL-6, IL-10, IL17, MCP1, TNF-α and TGF-β1 were assessed by ELISA. After the analysis of the cytokines, no difference in their concentration was observed between IL-1 β and IL-10. The cytokines IL-6, IL-17, and MCP-1 showed an increase in the group with advanced disease compared to the initial phase of the disease. The cytokine TNF-α showed an increase in the severe and severe groups with associated comorbidities compared to the mild group. TGF-β showed a lower level in the severe group compared to the mild group with the advance of the disease the inflammatory cytokines appear higher, reducing the expression of the cytokines that could be performing tissue repair, such as TGF-β. Conclusion: This study confirms the hypothesis that TMD worsening is proportional to systemic inflammation, placing the group with systemic or autoimmune inflammatory diseases as an important candidate for TMJ degenerative processes. Since it can’t be predicted when systemic comorbidity will be established, we understand that the group with severe signs should already be considered as a risk group for temporomandibular joint progression and degeneration, as well as for the group with systemic or autoimmune inflammatory diseases.

ASSOCIATION STUDY OF VARIANTS IN THE LEP AND RETN GENES WITH ASTHMA SEVERITY AND EXACERBATION IN OBESE INDIVIDUALS

Asthma and obesity are chronic diseases with increasing prevalence that, in addition to environmental and genetic factors, have inflammation as a common link. In this sense, cytokines secreted by adipose tissue cells (adipocytes and macrophages) with receptors expressed in the lung are pointed at the link between the immune regulation involved in the pathogenesis of both diseases. Variations in the genes responsible for the proteins Leptin (LEP) and Resistin (RETN) has been associated with their unbalanced serum levels and consequent metabolic dysfunction that promote impact on asthma severity. Thus, the aim of this study is to investigate the association between LEP and RETN variants with asthma outcomes in a Brazilian population and whether obesity modifies the observed effects. The study involved 1063 individuals, followed by ProAR (Program for Control of Asthma and Allergic Rhinitis of Bahia). Asthma and severity were defined according to GINA and obesity was defined by BMI, according to WHO. Genotyping was performed using Illumina Infinium kit Multi-Ethnic AMR / AFR-8. Logistic regression was performed to identify associations between variants in the studied gene with asthma severity, stratifying or not the population by BMI. Variants in the LEP, rs6966536 (G allele) and rs4731426 (C allele), were associated with risk of asthma severity and exacerbation in severe asthma, respectively. In RETN, rs3745367 (G allele) and rs3219178 (G allele) were associated with protection for asthma severity, only in subjects without obesity. Our results demonstrate the modifying effect of overweight on the observed association between variants in the LEP and RETN genes. Functional studies are needed to clarify the inflammatory hypothesis of the associations.

AXL AND MER RECEPTORS MODULATE TISSUE REDOX BALANCE AND PERITONEAL MACROPHAGE METABOLISM.

Macrophages (MØs) are key cells for innate immunity and efferocytosis (EF). Efferocytic TAM receptors, Axl and Mer, indirectly recognize phosphatidylserine expressed by apoptotic cells through Gas6 bridge molecules, and its activation results in EF and inhibition of inflammatory responses. EF may impact the functional differentiation of MØs and is accompanied by modulation of energy metabolism. In this context, we believe that the energetic metabolic state of MØ changes according to the dynamic functional modifications of these cells in response to EF during homeostasis. The aim of this study was to investigate how EF mediated by Axl and Mer receptors modulate the energy metabolism of peritoneal macrophages (pMØs) in homeostasis, as well as its role in tissue redox modulation. pMØs were isolated from wild mice C57BL/6 (WT) and Axl and Mer deficient to investigate their phenotype, function and metabolic capacity. Adipose and lung tissues were collected for analysis of oxidative stress biomarkers (OSB). Using pMs from WT, and deficient in Axl and Mer, we verified that these receptors induce alterations in the density of populations of immune cells in the PC, especially pMs, whose numbers are high in Axl and Mer mice. We also showed an increase in nitric oxide (NO) in the PC of Axl mice, however, pMs-Axl released less NO in vitro. Metabolically, we verified in the multivariate metabolomic analysis that fatty acids, la ctate and citrate are altered between pMs-Axl, pMs-Mer and pMs-WT. Additionally, we found a significantly higher lactate production in pMs-Axl and pMs-Mer, with or without LPS stimulation after 24 hours, compared to pMs-WT. Furthermore, we found elevated lactate levels in the culture supernatants of pMs-Axl and pMs-Mer cultured with antimycin (6, 24 and 48 hours) compared to pMs-WT. Finally, we verified that EF mediated by Axl and Mer receptors altered OSB in adipose and lung tissues, inducing an increase in lipid peroxidation, antioxidant capacity, in the activity of superoxide dismutase and catalase enzymes, inducing a decrease in carbonyl protein levels and in the formation of thiol groups. Conclusion. Collectively, our results suggest that EF mediated by Axl and Mer receptors contribute to alterations in the number, function and metabolism of pMØs, inducing an anti-inflammatory phenotype and promoting alterations in the tissue redox balance.

B-1 CELLS ACTIVATION IN SLEEP RESTRICTED MICE INTRAPERITONEALLY CHALLENGED WITH Candida albicans AND Paracoccidioides brasiliensis

Introduction: B-1 cells are subpopulation of B lymphocytes mostly distributed in peritoneal and pleural cavities. These cells are a source of natural antibodies and can express both myeloid and lymphoid commitment factors allowing their differentiation into phagocytes. B-1 cells respond to several stimulus but their response in chronic stress is still poorly known. This study evaluated the activation of peritoneal B-1 cells from mice submitted to sleep restriction, a model of chronic stress, and intraperitoneally challenged with Candida albicans and Paracoccidioides brasiliensis. Methods and Results: C57Bl/6 mice were sleep restricted for 21 days and intraperitoneally infected with C. albicans (B-1CA group) or P. brasiliensis (B-1PB group) on the 20th day. Control group was infected without sleep restriction induction. After 24 h of infection, peritoneal B-1 cells were collected by peritoneal lavage and submitted to flow cytometry for evaluating of activation molecules expression and microbicidal molecules, or purified for RT-PCR analysis to analyze expression of the lymphoid (EBF, E2A IL-7R) and myeloid (G-csfr, M-csfr, Spi1) commitment factors, cytokines (TNF, IL-6 , IL-12 and IL-10), arginase (ARG) and TLRs (2, 6 and 9) genes. Sleep restriction (SR) led to a decrease in CD86 expression in B-1CA group as compared to B-1 cells from control group (P<0.05). However, no differences were observed in activation molecules of B-1 cells from control and B-1PB group. Comparing microbicidal molecules, B-1 cells from sleep restricted mice produced higher amounts of NO after infection with P. brasiliensis (P<0.05), and higher levels of ROS after C. albicans infection. In addition, B-1CA group showed higher expression of ARG genes (P< 0.0001), TLR-2 (P<0.01), TNF-α (P<0.01), IL-6 (P<0.001), IL-12 (P<0.0001). On the other hand, B-1PB group had a significant increase in TLR2 (P<0.001), and IL-12 (P<0.0001). The analyze of myeloid and lymphoid commitment factors showed that B-1 cells from sleep restricted mice had a significant increase in expression of all myeloid factors analyzed after both infections (P< 0.05). However, B-1CA group had an increase in IL-7R in B-1 cells from sleep restricted mice (P<0.0001). Conclusion: Our findings suggests that sleep restriction alone can alter B-1 cell activation; however, the infectious stimulus can also significantly influence B-1 cell changes.

Baseline activated cellular immune profile relates to response to neoadjuvant chemotherapy in TNBC patients.

Triple negative breast cancer (TNBC) is a cancer with aggressive behavior and is defined by the absence of hormone receptors (estrogen and progesterone) and the lack of HER2 gene expression. In the neoadjuvant chemotherapy (QT) scenario, 50% of the patients achieve pathological complete response, while patients considered non-responders proceed to tumor progression and present higher mortality. The immune system plays opposite roles in patients with TNBC, acting in the antitumor defenses as well as in disease progression. Methods: Patients diagnosed with TNBC treated in AC Camargo Cancer Center were included according to institutional ethical approval CEP #2496/18D. Response assessment was performed by pathologists who classified the patients as pathologically responsive (R, n=28) and non-responsive (NR, n=16). Using peripheral blood samples collected at baseline, we characterized the systemic cellular immune profile by high dimensional flow cytometry (FACSymphony A5, BD Bioscience). PBMC were evaluated after polyclonal stimulation (αCD3+αCD28) and antigen-specific stimulation (MUC1+Surv+MamA +αCD28). We compared the immune cell populations frequencies between R and NR using appropriate tests and p-value<0.05 was considered statistically significant. Results: We evaluated the frequency of circulating immune cells populations in R and NR groups. Responder patients showed higher expression of PD1+(p=0.011) and CD69+(p=0.024) in CD4+ T cells, as well as higher PD1+ expression in CD8+ T cells (p=0.017). After polyclonal stimulation, the R group showed higher expression of TIM3+CD127+ in CD4+ T cells (p=0.041) and CD8+ T cells(p=0.017), which also showed higher expression of GzB (p=0.041) and CD161 (p=0.015). After antigen-specific stimulation, NR group showed higher frequency of CD4+ T cells (p=0.018) while R group showed higher frequency of CD8+ T cells (p=0.018), including GzB+ cells(p=0.008). Furthermore, the R group showed higher expression of PD1+CD95+ in DN T cells (p=0.030), as well as higher frequency of DN NKT cells (p=0.030). Together, these results show an activated immune profile in responder patients, even before starting treatment. Conclusion: We conclude that the peripheral cellular immune profile can predict the response to neoadjuvant treatment in TNBC patients.

BCG-TRAINED MACROPHAGES ELICIT A PROTECTIVE INFLAMMATORY RESPONSE AGAINST THE PATHOGENIC BACTERIA Brucella abortus

Bacillus Calmette-Guérin (BCG) is an attenuated vaccine derived from virulent Mycobacterium bovis and used against tuberculosis. Currently, BCG has been focus of intense research due to its ability to trigger non-specific effects based on improved activity of the innate immune system, a phenomenon known as trained immunity. When induced at bone marrow (BM) level, it results an improved immune response, by virtue of hematopoietic stem cell lifespan and proliferative capacity. Brucella abortus is a Gram-negative bacterium that causes brucellosis, a systemic debilitating infection. This disease is the most widespread bacterial zoonosis in the world. Macrophages represent its main replicative niche in the host. In this study, we investigated whether BCG-induced trained immunity is protective against B. abortus infection. C57BL/6 WT mice were administered with BCG i.v. After, they were treated with antibiotics in order to achieve BCG clearance. Subsequently, bone marrow-derived macrophages (BMDMs) were obtained and challenged with B. abortus in vitro. We demonstrate BCG-trained macrophages showed higher MHC-II expression and greater production of IL-6, IL-12 and IL-β against B. abortus compared to untrained control. Moreover, BCG-trained cells showed increased caspase-11 and caspase-1 processing. From a metabolic point of view, BCG training triggered an AKT/mTOR/S6K axis overactivation in macrophages prior to and upon B. abortus infection, which corroborated the higher level of glycolysis. Furthermore, these cells showed increased NO production and improved macrophage-killing capacity against B. abortus in vitro. In vivo, we observed a significant reduction in bacterial burden in organs of BCG-trained versus untrained mice. In addition, naïve recipient mice that received BM transfer (BMT) from BCG-trained donors showed greater control of B. abortus burden when compared to untrained-BMT counterparts. These results demonstrate BCG-induced trained immunity in mice results in better control against B. abortus infection in vivo and in vitro.

BIOCHANIN A INHIBITS CHIKUNGUNYA VIRUS INFECTION IN HUMAN FIBROBLAST-LIKE SYNOVIOCYTES (HFLS): IN VITRO AND IN SILICO STUDY

Introduction: Chikungunya fever is a viral arthropod-borne disease which causes intense joint pain on most patients, and other symptoms such as fever, headaches, and myalgia. There are no preventive or specific antiviral treatment licensed. In this way, several compounds from natural sources, as polyphenols, have been studied aiming to find antiviral properties. The aim of this study was to evaluate the antiviral activity of the biochanin A, an isoflavone, against CHIKV in human fibroblast-like synoviocytes (HFLS) in vitro. Methods and Results: To evaluate biochanin A maximum non-toxic concentration (MNTC), HFLS were seeded in 96 well plate and 24h later the cells were treated with several concentrations of the compound (from 400 to 3.125 µM) followed by incubation for 48h (37ºC, 5% CO2). Cell viability was assessed by MTT colorimetric assay which showed 200 µM as the MNTC. The antiviral activity of biochanin A was evaluated by a posttreatment assay. HFLS were incubated with CHIKV (MOI 0.5) for 2h viral adsorption followed by the treatment with biochanin A at concentrations from 200 to 50 µM and viral inhibition was accessed by MTT assay. Viral inhibitions of 61.36 % and 30.35 % were detected in HFLS treated with 200 and 100 µM, respectively. To evaluate the percentage HFLS-infected cells after biochanin A treatment, intracellular flow cytometry was performed labeling fixed and permeabilized cells with an anti-CHIKV monoclonal antibody/anti-mouse IgG-Alexa Fluor 488. As result, a significant reduction from 27.06% to 8.42% in the percentage of CHIKV-infected cells was detected in HFLS treated with biochanin at 200 µM compared to the untreated cells. In silico molecular docking was performed to evaluate biochanin A interaction with several CHIKV structural and nonstructural proteins by using GOLD v.5.8.1 software by using Chemical Piecewise Linear Potential (CHEMPLP) scoring function. It was verified that biochanin A was placed into a pocket in the nsP3 structure (FitScore=65.55) suggesting this isoflavone could be a potential viral inhibitor since this protein plays a role in virus genome replication. Conclusion: This study showed biochanin A as a suitable antiviral candidate against CHIKV in human cells in vitro and suggests nsP3 as its putative target by in silico analysis thus contributing for the discovery of new bioactive compounds against this arbovirus.

BIOLOGICAL FEATURES ACCOMPANYING LYMPHOCYTE DEATH INDUCED BY Aedes aegypti SALIVA

INTRODUCTION: Aedes aegypti is a hematophagous insect best known as the vector of dengue, yellow fever, Zika and chikungunya. Its saliva is composed of a wide range of molecules with anti-hemostatic and immunomodulatory activities, among which, a selective cytotoxicity for lymphocytes is described. However, a more detailed molecular characterization of this cell death process is still needed for a correct classification of this death. Thus, the objective of this work is to provide new biological features associated with the lymphocyte death induced by Ae. aegypti salivary gland extract (SGE). METHODS AND RESULTS: To evaluate the cytotoxic activity of the Ae. aegypti SGE, murine spleens were used as a source of lymphocytes. After incubation for different time periods, these cells were evaluated by flow cytometry and Western Blot. The culture supernatant was collected to evaluate lactate dehydrogenase (LDH) and cytokines using commercial kits. Initially, we identified a transient exposure of phosphatidylserine, characterized by positive staining with annexin V, in both T and B lymphocytes incubated with SGE. We also observed that this exposure appears to be caspase-independent, as we detect neither an increase in active caspases after SGE incubation nor changes in the presence of Q-VD-OPh, a pan-caspase inhibitor. Furthermore, B and T cells appear to react differently to SGE over time; while B lymphocytes showed a low percentage of death (Annexin V+/Live-Dead+) in relation to the negative control, T lymphocytes showed a growing increase in death, with about 50% increase of double positive cells in relation to the control after 10 hours of incubation. This increase was accompanied by a greater release of LDH over time. Interestingly, LDH release occurred mainly in T cell cultures, while IL-1β was detected in B cell cultures. CONCLUSION: Further experiments are under way in order to properly characterize the SGE-induced cell death and to evaluate its effect on the host organism in vivo. These data may improve our understanding on the microenvironment created by the saliva of Ae. aegypti in the skin of the vertebrate host at the time of the bite and contribute to future studies to generate new alternatives to fight the mosquito and the pathogens transmitted by this vector.

BIOMARKERS OF COVID-19 SEVERITY IN ELDERLY POPULATIONS OF ENDEMIC AND NON-ENDEMIC ZONES

SARS-CoV-2 is a β-coronavirus capable of causing COVID-19. Since the first reported cases of the disease, it was noted that the elderly was more susceptible to more severe cases and death, even when compared to other groups with inflammatory comorbidities, such as cardiovascular disease and obesity. In 2000, Claudio Franceschi described a chronic, systemic, low-grade inflammation that accompanies aging and called it inflammaging (FRANCESCHI, 2000). This inflammatory state is related to several inflammatory and degenerative diseases in frail elderly people in European countries. Brazilian populations are distintic from the ones originally recruited for these studies on immunosenescence. Some regions of Brazil are still characterized as an endemic zone for many infectious diseases. Our group has shown that, in these areas, high and continuous exposure to infectious stimuli accelerated aging of individuals residing there leading to to unexplored consequences in terms of senescence. Therefore, the hypothesis of this study is that inflammation would be one of the determining factors in the outcome of COVID-19 and this process would be more exuberant in individuals residing in endemic zones. Our objective was to evaluate the inflammatory profile of adults (18 to 59 years old) and elderly (over 60 years old) with COVID-19 in Belo Horizonte-MG and Governador Valadares-MG (GV) (endemic region). For this, 346 individuals with 1 to 7 days of symptoms were recruited, tested for SARS-CoV2 infection using the RT-PCR technique and divided into the following groups: adults/elderly with mild symptoms and adults/elderly with moderate/severe symptoms. All subjects had their blood plasma analyzed by Luminex assay (using Bio-Plex® Pro Human Cytokine Kit from BioRad) for 27 cytokines and chemokines. The results confirm our original hypothesis showing that cytokines such as IFN-γ, IL-6, IL-10 and CCL-2, which are present both in inflammaging and in the cytokine storm of COVID-19, are increased in individuals residing in an endemic area when compared to the volunteers from Belo Horizonte. The results showed statistical differences in the plasma of adults when compared to the elderly, in both regions. In addition, the elderly from GV have a very marked inflammatory profile suggesting an accentuated inflammaging. Further analysis of epidemiological data in the region would help to confirm that the individuals in the endemic are more prone to severe outcomes at earlier ages.

Biomarkers such as CCL3, CCL5, IL-15, IL-1Ra, and VEGF are important to discriminate classes of adverse events after primary 17DD-YF vaccination according to cause-specific definitions

Yellow fever (YF) is a viral hemorrhagic vector-borne disease considered one of the most lethal viral infections. The large-scale vaccination coverage is essential to controlling disease spread and the reemergence of the YF epidemic. The YF vaccine is well tolerated, and adverse events following YF immunization (YEL-AEFI) are usually mild and occasionally reported. Several hypotheses have already been proposed to explain the basis of the YEL-AEFI, but the precise mechanisms involved in the development of these events remain unknown. In the present study, a range of serum biomarkers was quantified in suspected cases of YEL-AEFI to propose a reliable laboratory algorithm to discriminate confirmed YEL-AEFI cases (“A1” class) from those with other illnesses (“C” class). The analysis of serum biomarkers, including chemokines, inflammatory and regulatory cytokines, and growth factors, was performed using the Bio-Plex Pro Human Cytokine 27-plex Assay. Our findings demonstrated that increased levels of CXCL8, CCL2, CXCL10, IL-1, IL-6, and TNF- were observed in YEL-AEFI (“A1” and “C” classes) as compared to primary vaccines without YEL-AEFI [PV(day 3–28)] and reference range (RR) controls. Notably, increased levels of CCL3, CCL4, CCL2, CCL5, IL-1, IL-15, IL-1Ra, and G-CSF were found in “A1” as compared to ‘‘C” class. Venn diagrams analysis allowed the pre-selection of biomarkers for further investigation of performance indices. Data demonstrated that CCL3, CCL5, IL-15, and IL-1Ra presented high global accuracy (AUC = 1.00) to discriminate “A1” from “C”. Decision tree was proposed with a reliable algorithm to distinguish YEL-AEFI cases according to cause-specific definitions with outstanding overall accuracy (91%). CCL3, CCL5, IL-15, and IL-1Ra appear as root attributes to identify “A1” followed by VEGF as branch nodes to discriminate Wild Type YFV infection (“C(WT-YFV)”) from cases with other illnesses (“C*”). Together, these results demonstrated the applicability of serum biomarker measurements as putative parameters toward establishing accurate laboratory tools for complementary differential diagnosis of YEL-AEFI cases.

Blocking drug-induced autophagy with chloroquine in HCT-116 colon cancer cells enhances DC maturation and T cell responses induced by tumor cell lysate.

Autophagy is an important mechanism for tumor escape, allowing tumor cells to recover from the damage induced by chemotherapy, radiation therapy, immunotherapy and contributing to the development of resistance. The pharmacological inhibition of autophagy contributes to increase the efficacy of antineoplastic agents. The exposure of the tumor cells to low concentrations of select autophagy-inducing antineoplastic agents increases their immunogenicity and enhances their ability to stimulate dendritic cell (DC) maturation. We tested whether the application of an autophagy-inhibiting agent, chloroquine (CQ), in combination with low concentrations of 5-fluorouracil (5-FU) increases the ability of tumor cells to induce DC maturation. DCs sensitized with the lysate of HCT-116 cells previously exposed to such a combination enhanced the DC maturation/activation ability. These matured DCs also increased the allogeneic responsiveness of both CD4+ and CD8+ T cells, which showed a greater proliferative response than those from DCs sensitized with control lysates. The T cells expanded in such cocultures were CD69+ and PD-1- and produced higher levels of IFN-γ and lower levels of IL-10, consistent with the preferential activation of Th1 cells. Cocultures of autologous DCs and lymphocytes improved the generation of cytotoxic T lymphocytes, as assessed by the expression of CD107a, perforin, and granzyme B. The drug combination increased the expression of genes related to the CEACAM family (BECN1, ATGs, MAPLC3B, ULK1, SQSTM1) and tumor suppressors (PCBP1). Furthermore, the decreased expression of genes related to metastasis and tumor progression (BNIP3, BNIP3L, FOSL2, HES1, LAMB3, LOXL2, NDRG1, P4HA1, PIK3R2) was noted. The combination of 5-FU and CQ increases the ability of tumor cells to drive DC maturation and enhances the ability of DCs to stimulate T cell responses.

BONE MARROW TRANSPLANTATION MODULATES LYMPHOCYTE GENE EXPRESSION IN SPONTANEOUS LEAN TYPE 2 DIABETIC GOTO­KAKIZAKI

INTRODUCTION Hyperglycemia causes changes associated with persistent activation of inflammatory pathways. In addition, lymphocyte profile is altered in type 2 diabetes mellitus (DM2) leading to an imbalance of immune system. The aim of this study was to investigate the modulation of peripheral lymphocyte profile from GK rats after bone marrow transplantation from normoglycemic Wistar rats. METHODS Bone marrow transplantation was performed from Wistar (WT) to Goto Kakizaki (GK) rats, a model of spontaneous lean T2D. Male rats (28 days old) were submitted to a myeloablative regimen (2 days of 20 mg/kg busulfan; 1day of 150 mg/kg cyclophosphamide) and the bone marrow transplantation was performed in WT (WT to WT, n=4) and GK (WT to GK, n=5) rats. Glucose and insulin tolerance test were performed in the day 90 after transplantation. In day 100, rats were euthanized and Th1, Th2, Th17 and Tregulatory lymphocyte (Treg) related gene expression were evaluated by real time PCR. This project was approved by the Animal Use Ethics Committee (CEUA) of the Cruzeiro do Sul University, registered under Nº 012-2020. RESULTS During immunosuppression we observed a decrease in total leukocytes, lymphocytes and segmented neutrophils, as well as a decrease in body weight in recipient animals. We also observed a decrease in blood glucose after 90 days of transplantation in GK animals. In the glucose tolerance test, we observed an increase of area under curve in GK rats when compared to the WT animals, showing that they continue to be resistant to insulin. In the insulin tolerance test, we observed a decrease until the time of 10 minutes, however, in the analysis of the area under the curve there was an increase for the GK animals. In the cellular evaluation, we observed an increase in the expression of the IL-35 gene, which is an important regulatory cytokine involved with Treg cell function. CONCLUSION So as preliminary conclusions we can observe that the transplantation can provide a decrease in blood glucose in diabetic animals, as well as an increase in Treg cytokine, demonstrating a beginning in the improvement of inflammation in these animals.

C5a in the presence of IFNg decreases the expression of immune checkpoint co-receptors in NSCLC cell lines

Cancer is marked by unrestrained proliferation of genetic altered cells, which can invade organs and spread to other sites. According to the International Agency for Research on Cancer, about 19.3 million cases were reported in 2020, demonstrating the need for new therapies. Immunotherapy based on immune checkpoint blockade has shown promising results, especially in patients with Non-Small Cell Lung Carcinoma (NSCLC). The inflammation is involved in antitumor response, although innate immunity role is not clear yet. The complement system (CS) is a set of proteins, characterized by sequential proteolysis of plasma proteins, and is part of innate and adaptive immunity. Its activation is related to an increase in inflammatory response. Initial studies about CS associated to tumors indicate that activation of this system contribute to antitumor response. Recently, the role of CS as a pro-tumor factor has been described in murine models. Therefore, our hypothesis is that CS activation decreases the tumor immune response, modulating immune checkpoint co-receptors. Our objective is to evaluate the role of complement system in the modulation of immune checkpoint co-receptors in NSCLC cells. We assessed the expression of CS components and receptors in cancer cell lines and observed the presence of C2, C3, C4, C5 and C5aR1/2 in the two NSCLC cell lines (H1975/A549). We also evaluated the modulation of the expression of these components against the inflammatory stimulus IFNg, which increased the expression of C2 and C4. As C5a receptors were expressed in these cell lines, we investigated whether C5a ligand would influence the expression of immune checkpoint co-receptors in NSCLC cells, but no difference was observed. Once IFNg increases the expression of PD-L1, PD-L2 and HVEM, we noticed that the combined treatment of IFNg and C5a led to a decreased expression of these co-receptors, suggesting an antitumor action of C5a. In silico analysis has shown an increased expression of CS components in normal tissue compared to tumor tissue. Also no direct correlation between PD-L1, C5, C5aR1/2 was observed. We concluded so far that in NSCLC cell lines the CS is expressed and may be affected by C5a fragment, which seems to influence the expression of immune checkpoint co-receptors in an inflammatory environment. As well, expression of C5 component has not being directly related to PD-L1 expression, suggesting that other factors of CS activation might be involved.

CAMELID NANOBODY AGAINST METALLOPROTEINASE P-I FROM Bothrops jararacussu: TOOLS FOR THE TREATMENT OF BOTHROPIC ENVENOMING

Introduction: To address the global antivenom crisis, novel antivenoms need to present high therapeutic efficacy, broad neutralization ability against systemic and local tissue damage, sufficient safety, and cost-effectiveness. Due to biological characteristics of camelid single-domain antibodies (VHH) such as small size (~15 kDa), ease of genetic manipulation, high affinity and solubility, low immunogenicity, ability to penetrate dense tissues, recognition of normally inaccessible epitopes, and stability due to changes in temperature and pH, their application in antivenoms has expanded considerably. Although serum therapy consisting of IgG fragments is the official treatment for snakebites and is effective against systemic damage, it has limitations related to the rapid evolution of tissue damage at the bite site, the inefficiency of antivenom constituents in dense tissues, and the possible development of hypersensitivity reactions to heterologous proteins. Such disadvantages, together with limited access to antivenoms, have instigated different strategies aimed to overcome these issues. Methods and Results: Therefore, this work proposes the in silico and in vitro characterization of Lama glama nanobodies previously selected against metalloproteinase P-I from B. jararacussu venom. After isolation of BjussuMP-II, a camelid was immunized with the purified toxin in order to construct the recombinant phage library. Following a round of biopanning, 52% of the selected clones were able to recognize BjussuMP-II in an ELISA assay. After sequencing, seven sequence profiles were identified. The specificity of anti-MPII nanobodies against toxin and whole venom was determined by Western blot. In the evaluation of the proteolytic activity of metalloprotease on casein, the anti-MPII 61 VHH showed inhibitory potential on the proteolytic activity of BjussuMP-II in all tested ratios. Through in vitro tests, it was possible to observe a 25% reduction in the levels of LDH released by murine endothelial cells (t-END), demonstrating the ability of anti-MPII 61 VHH to neutralize part of the toxic effect triggered by BjussuMP-II. In silico analysis, through molecular docking of anti-BjussuMP-II VHHs with metalloprotease, revealed their potential interaction with amino acids present in regions critical for the toxin’s conformation and stability. Conclusion: The findings suggest that anti-BjussuMP-II VHHs may be beneficial in the development of next-generation antivenoms.

CAN THE ACTIVATION OF THE β2-ADRENERGIC RECEPTOR (β2AR) MODULATE IN VITRO ASTROCYTE-MICROGLIA INTERACTION?

Astrocytes are important glial cells that play a key role in maintaining CNS homeostasis. In addition, astrocytes can both influence the biology of other CNS cells and be influenced by such cells and thus participate in inflammatory processes that occur in diseases such as Parkinson's, stroke and Multiple Sclerosis (MS). Our group is interested in understanding whether and how β2-adrenergic receptor (β2AR) signaling can interfere with astrocyte function during inflammatory stimuli. Primary cultures of mouse astrocytes and microglia were obtained and purified by cell sorting. After stimulating or not with LPS (1µg/mL) the microglia cultures, we obtained microglial conditioned medium (MCM) for the treatment of astrocytes. Here, we demonstrate that purified astrocytes upregulate the expression of CCL2, TNF-a, NOS2 and IL10 when treated with LPS-stimulated microglia conditioned medium (LPS-MCM, used as an inflammatory stimulus). Pretreatment with Fenoterol (β2AR agonist) is capable of inducing an up-regulation of IL10 and a down-regulation of the expression of CCL2, TNF-a and NOS2 in LPS-MCM-stimulated astrocytes. Furthermore, prior activation of β2AR in LPS-MCM-stimulated astrocytes reduced the levels of secreted CCL-2 as detected by ELISA in the culture supernatant samples. We also found that β2AR signaling reduces TNF-α production by LPS-stimulated microglia cultures and CCL-2 production in LPS-MCM-stimulated astrocyte cultures. Finally, we describe that in the absence of β2AR activation, the use of a blocking anti-TNF-α antibody partially reverses the effect of LPS-MCM on CCL-2 secretion by purified astrocytes. In conclusion, our results indicate that the microglia-astrocyte interaction plays an important role in the inflammatory microenvironment, especially in the secretion of TNF-α by the microglia, which induces the expression of important cytokines and chemokines in astrocytes. Furthermore, our results demonstrate that β2AR signaling down-regulates the pro-inflammatory effect of activated microglia on astrocytes and the microglia response to the LPS stimulation itself.

CARDIAC CHAGAS DISEASE IS ASSOCIATED WITH EXPANSION OF SPECIFIC T AND B CELL SUBSETS

Introduction: Chagas disease still represents an important public health problem that affects approximately 7 million people worldwide, with 75 million individuals at risk of infection in the Americas. Knowledge about Chagas disease has significant gaps, especially regarding the pathogenesis of the disease that causes different clinical manifestations. The improvement of knowledge about immunological mechanisms and the identification of possible biomarkers is relevant. Objectives: The study aims to identify and characterize follicular helper T cells and other T cell and B lymphocyte subpopulations in patients with Chagas disease. Methods: Peripheral blood mononuclear cells from healthy donors (CTL) and patients in the chronic phase of Chagas disease presenting the indeterminate (asymptomatic) and cardiac forms B1 (mild) or B2, C, and D (advanced) were analyzed by flow cytometry with multidimensional strategies. Results: The preliminary data showed that patients with cardiac disease had higher frequencies of effector CD4+ T cells than CTL, with p=0.03/p=0.05, mean= 3.14/3.79 and median= 1.03/1.30. Moreover, patients with advanced cardiac disease displayed higher frequencies of central memory phenotype among naive cells than CTL, p=0.01, mean= 10.20 and median= 8.18. Patients with mild cardiac disease displayed increased proportions of effector memory CD4+ T cells p=0.01, mean= 8.34 and median= 7.99 and follicular helper T cells (Tfh).The expression of CD57 was increased in all patients with cardiac Chagas disease according heatmap of multidimensional analyses. Asymptomatic patients showed decreased frequencies of CD27+ central memory CD4+ T cells with p=0.01, mean= 17.15 and median= 16.62 and increased frequencies of B lymphocytes (LB) with p=0.01, mean= 21.07 and median= 15.85. Conversely, mature LB with isotype change are more frequent during mild cardiac disease p=<0.001, mean= 34.07 and median= 30.45 while TACI and CD85j molecules are less expressed in cardiac clinical phase. Conclusion: Overall, the results showed that Chagas disease induces an inflammatory environment with highly differentiated cells in patients from the symptomatic cardiac group, reinforcing the complexity of the immune response triggered by the disease.

CASP4/11 is activated in response to SARS-CoV-2 and contributes to pulmonary inflammation and disease exacerbation in COVID-19

Introduction: Infection with SARS-CoV-2 induces COVID-19, an inflammatory disease that is usually self-limited, but depending on patient conditions may culminate with critical illness and patient death. The virus triggers activation of intracellular receptors, such as the NLRP3 inflammasome, which promotes inflammation and aggravates the disease. Thus, identification of host components associated with NLRP3 inflammasome is key for understanding the physiopathology of the disease. The aim of this study was to investigate if caspase-11 (Casp11) is important for inflammasome activation and contributes to disease severity in patients and in mice infected with SARS-CoV-2. Methods and Results: Here, we reported that SARS-CoV-2 induces upregulation and activation of human Caspase-4/CASP4 (mouse Caspase-11/CASP11) and this process is key for NLRP3 inflammasome activation in response to SARS-CoV-2. CASP4 was expressed in lung autopsy of lethal cases of COVID-19 and CASP4 expression correlates with expression of inflammasome components and inflammatory mediators such as CASP1, IL1B, IL18 and IL6. In vivo infections performed in transgenic hACE2 humanized mouse, deficient or sufficient for Casp11, indicate that CASP11 does not influence viral replication in mouse lungs. However, hACE2 Casp11–/– mice were protected from disease development, with reduced body weight loss, reduced temperature variation, increased arterial oxygen saturation, increased pulmonary parenchymal area, reduced clinical score of the disease and increased survival. hACE2 Casp11–/– mice had significantly less NLRP3 and ASC puncta formation in their lungs, compared to ACE2 Wild type mice, suggesting that Casp11 is important for NLRP3 activation upon SARS-CoV-2 infection in vivo. The reduction of NLRP3 puncta formation was also observed in bone marrow derived macrophages infected in vitro with SARS-CoV-2. Conclusion: We demonstrated that CASP4/11 participates in the inflammasome activation in response to SARS-CoV-2 and promotes exacerbation of the disease in mouse models of infection. Our results effectively account for understanding the physiopathology of the COVID-19 and establish CASP4 as a potential target for immune-mediated therapies to treat patients in moderate and critical conditions.

Caspase-1 impairs the microbicidal capacity of microglia in response to Zika virus infection by inhibiting IFN-I secretion.

Introduction: Inflammasomes are multi-protein platforms present in the cytosol capable of activating the protease caspase-1, leading to IL-1beta/IL-18 release and cell death by pyroptosis. Zika virus (ZIKV) induces inflammasome activation in myeloid cells, leading to inflammatory responses through IL-1beta secretion. However, ZIKV infects mainly Central Nervous System (CNS) cells and the impact of inflammasome activation on those cells is not well established. Thus, the aim of this work is to investigate the role of these molecular machineries on microglia upon ZIKV infection. Methods and results: ZIKV was able to replicate in microglia cells obtained from cortex of newborn mice in a MOI and time-dependent manner. Interestingly, ZIKV induced the secretion of IL-6 and TNF-alpha but not IL-1beta by microglia. By analyzing the expression of inflammasome pathway components, we observed that the expression of IL-1beta was down-regulated in infected microglia, suggesting that ZIKV inhibits the priming phase of inflammasomes in microglia. In fact, ZIKV infection attenuated IL-1beta secretion in microglia in response to nigericin and flagellin, classic inflammasome activators. Interestingly, microglia from caspase-1-/- mice presented lower viral load in comparison to the wild-type cells, an effect that was correlated with the higher levels of type I interferon secretion. Conclusion: Our data demonstrated that ZIKV infection inhibits inflammasome activation in microglia, which could result in the protection against neuroinflammation. Moreover, caspase-1 seems to inhibit type I interferon secretion by ZIKV-infected microglia, thus impacting their microbicidal capacity. Taken together, our data suggest that caspase-1 has a deleterious role on microglia in response to the ZIKV infection.

Caulerpine, from Caulerpa racemosa, ameliorate the inflammatory process and tissue damage in a murine model of Acute Lung Injury

Introduction: The appearance of adverse effects associated with the use of corticosteroids has driven the search for compounds derived from natural products that can be used as an alternative anti-inflammatory therapy. The present study sought to assess the anti-inflammatory potential of caulerpine (CLP), an alkaloid derived from seaweed Caulerpa racemosa, in murine model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Methods and Results: All experimental procedures were approved by the CEUA of UFRN (CEUA no. 007/2019). Swiss mice were treated orally with two diferent doses of caulerpine (4 and 2 mg/kg) or dexamethasone (1 mg/kg). One hour later, the mice received LPS (167µg/ml) intranasally. After 24 hours, bronchoalveolar lavage (BAL) was obtained to determine the total number of leukocytes. With the dose of caulerpine that best inhibited the cell infiltration, we evaluated the kinetics of cell migration in the BAL at 4, 24 and 48 hours after ALI induction. The levels of pro-inflammatory cytokines and total proteins were determined in the BAL supernatant and one sample of the lungs was collected for histological analysis. As the two doses of caulerpine similarly inhibited the cell migration to the lungs in model of ALI, the dose of 2 mg/kg was used for the other analyses. Caulerpine (2mg/kg) inhibited the migration of leukocytes to the lung at the three times analyzed, with a significant inhibition of polymorphonuclear cells, the main cells involved in lung damage in this model. There was a decrease in the lung damage of animals treated with caulerpine, associated a lower inflammatory infiltrate, with less edema, similar to that observed in the lungs of animals treated with dexamethasone. Was also observed a decrease in the levels of IL-1β, IL-12 and TNF-α and total proteins in the BAL, especially at 24 and 48 hours. Conclusions: The treatment with caulerpine, at a dose of 2 mg/kg, was effective in reducing the inflammatory process and the damage observed in the lung in a murine model of ALI.

CD39 as a key enzyme for restricting P2X7-mediated lung necrotic lesions in experimental tuberculosis

Severe tuberculosis (TB) is associated with a hyperinflammatory condition that results in necrotic lung lesions and bacterial spread. Massive ATP-mediated stimulation of P2X7 in hyperinflammatory disease aggravates pneumonia and lung necrosis, contributing to rapid disease progression. As a regulatory molecule that limits extracellular accumulation of ATP and generate adenosine, CD39 activity can shape the quality of the immune response. In this study, we evaluated how modulation of the ATP-adenosine axis by CD39 impacts the immune response to Mycobacterium tuberculosis (Mtb) infection. Upregulation of ENTPD1 (CD39) and P2RX7 gene expression was observed in 8 of the 9 analyses of public data from patients with active TB. In mice infected with mycobacteria of the hypervirulent Beijing 299 strain (n = 6 - 10, per group), lung immune cells co-expressed CD39 and P2X7 at higher levels in macrophages. Entpd1-/- mice infected with 200 bacilli developed a lethal TB, reaching morbidity up to 27 days post infection (p.i.), while 75% of C57BL/6 mice at day 42 p.i. were alive. When infected with 100 bacilli, Entpd1-/- mice showed greater loss of body weight, bacterial burden, neutrophil recruitment to the lungs and inflammatory cytokine (IL-1β, TNF-α, IL-12, IL-6 and IFN-γ) production compared to C57BL/6 mice. Notably, reduced numbers of macrophages were found in the lungs of infected Entpd1-/- mice. Pulmonary histopathological analysis showed extensive necrotic areas with higher numbers of extracellular bacilli in Entpd1-/- mice, while intracellular bacilli in solid granuloma-like structures predominated in C57BL/6 mice. Bacterial dissemination to the spleen and liver was also more pronounced in the absence of CD39. In addition, CD39 expression protected in vitro infected macrophages from death and reduced extracellular bacterial release. Furthermore, pharmacological inhibition of P2X7 with brilliant blue G (BBG) during early infection prevented the development of necrotic lesions in Entpd1-/- mice infected with 200 bacilli, leading to reduced lung weight and necrotic white nodes. An increase in the lung macrophage population was also observed in infected Entpd1-/- mice treated with BBG. We conclude that CD39 has an important protective role in the development of severe TB. CD39 ATPase activity attenuates P2X7-mediated macrophage killing and mycobacterial release, limiting the develop of necrotic lesions and the spread of bacilli and thus increasing host survival.

CD4+ T CELLS OF CHILDREN AND ADOLESCENTS WITH OBESITY PRESENT AN ALTERED PATTERN OF EXPRESSION OF CYTOKINES, TRANSCRIPTION FACTORS, AND COSTIMULATORY MOLECULES

Introduction: Childhood obesity is an increasing concern worldwide due to the morbidity it can subject children and adolescents. CD4+ T lymphocytes have been little explored in this population, even though the literature has shown that these cells are altered in obesity and play a significant role in the establishment of the inflammatory state characteristic of this condition. Thus, the present study aims at comparing the profile of CD4+ T cells of children and adolescents with obesity (OB group) with that of children and adolescents with normal weight (NW group). Methods and Results: Using flow cytometry, we compared the profile of expression of lymphocyte markers (CD3 and CD4), transcription factors (T-bet, GATA-3, RORγt, and FOXP3), cytokines (IFN-γ, IL-4, IL-17A, and IL-10), and costimulatory molecules (CD28 and CTLA-4) in CD4+ T cells of children and adolescents of the OB group (n=10, age range: 8-16 years) and the NW group (n=9, age range: 9-15 years). We also correlated the frequency of cells of the CD4+ subsets with anthropometric and biochemical data. This study was developed in compliance with ethical standards (Brazil Platform protocol number CAAE 32804720.5.0000.5149). Children and adolescents of the OB group present lower frequency of CD3+ T cells, as well as decreased frequency of CD4+ T cells expressing T-bet, GATA-3, IL-4, and IL-17A compared to the NW group. Moreover, the proportion of cytokine- and transcription factor-positive cells indicates a shift in the immune balance, with a higher proportion of cells expressing IFN-γ and T-bet and lower proportion of GATA-3 and IL-4 in the OB group. The frequency of CD28 is increased in all Th subsets in the OB group compared to the NW group, while CTLA-4 follows the opposite trend, except for Treg cells. Importantly, Th2 and Th17 subsets are positively correlated in the OB group and these subpopulations are negatively correlated with the anthropometric and biochemical profiles of this group. Conclusion: In general, our results show that the CD4+ T cells have an altered pattern of expression in children and adolescents with obesity, which may contribute to the inflammatory state and clinical characteristics of these patients.

CD57 EXPRESSION IN LEUKOPLAKIA AND SQUAMOUS CELL CARCINOMA OF THE MOUTH

Introduction: The lesions in the oral cavity have risk factors in common, and there is the possibility that human papillomavirus (HPV) infection may favor malignant transformation in leukoplakia progressing to oral squamous cell carcinoma (OSCC). The present study aims to correlate the presence of Natural Killer cells by CD57 expression with HPV detection in patients with leukoplakia and OSCC. Methods: Participants were selected in the period from 2018 to 2020 in the outpatient service of the Clinics of the School of Dentistry (FAODO/UFMS) and the Center for Dental Specialties (CEO) of the Municipal Health Service of Campo Grande - MS. CEP/UFMS 2.621.049. For HPV detection by Nested PCR, exfoliated cells were collected from the surface of lesions and after this collection, patients underwent surgical procedures for biopsy of the lesions and subsequent histopathological analysis. The samples were classified according to the type of lesion, being 5 samples with Leukoplakia Without Dysplasia (LWD), 8 samples with Leukoplakia With Dysplasia (LD) and 7 samples of Epidermoid Carcinoma of the Mouth (ECM). The obtained slides were evaluated by the technique of single-label immunohistochemistry for CD57 expression. The photodocumentation was performed with a Moticam 2300 3.0 Megapixel digital camera coupled to a Nikon Eclipse E200 optical microscope, using 400x magnification. The Motic Images Plus 2.0 software was used for image capture and the digital image analysis software ImageJ (NIH, USA) with the plug-ins package was used for quantification of immunolabeled cells. Twenty samples were analyzed, and 10 fields on each slide were photographed randomly. Immunolabeled cells in each of the images were counted using the "Cell Counter" plugin. Results: Considering the 20 samples analyzed, the average was 296.81 cells/mm². In the LWD group the mean number of labeled cells was 350.52 mm2, while in the LD group the mean was 267.77 mm2. In the ECM group we obtained an average of 291.63 mm2. Among the samples analyzed three were positive for HPV, being one ECM sample, 01 LD and 01 LWD. The mean of cells immunolabeled by CD57 in HPV negative samples was 316.91 mm², while the mean of cells immunolabeled in HPV positive samples was 182.92 mm². Conclusion: New samples will be analyzed and statistical analysis will be performed to verify the significance of the results.

CELL METABOLISM AND TUMORIGENESIS: ROLE OF GROWTH HORMONE AND MITOCHONDRIAL DYNAMICS IN AN EXPERIMENTAL ZEBRAFISH MODEL

Introduction: Cancer is one of the major public health problems worldwide. Globally, except for non-melanoma skin carcinomas, breast cancer is the most frequent malignant tumor among women and the fifth with the highest mortality rate. Recent studies have shown that cell metabolism acts as an important factor that contributes to tumor development and in the plasticity of the antitumor immune response. Several studies have demonstrated a positive relationship between the concentrations of GH (growth hormone) and IGF-1 (insulin-like growth factor) with the risk of cancer. Here, we formulate the hypothesis that events related to mitochondrial dynamics participate in the metabolic reprogramming that occurs in tumor cells and can influence GH-induced signaling pathways. Methods and Results: To confirm our hypothesis, we will study tumor progression and protein and gene expression of molecules related to the PI3K/Akt signaling pathway, metabolic reprogramming (PDK1, ERK1/2 and p53) and mitochondrial dynamics (Mfn1/2 and Drp1). We will evaluate the morphological, metabolic and redox profile of the mitochondria present in breast cancer cells (MDA-MB-231) monolayer and three-dimensional cultures. In in vivo assay, we will evaluate the same mechanisms induced by GH in spheroids implanted in zebrafish. Results: An increase in the number of tumor cells in 2D culture was observed when exposed to GH. We also demonstrated by PCR and WB that the protein DRP1 and MFN2 were overexpressed in the same model. We suggest through preliminary experiments that these effects may be starting with an increase in ROS, followed by an increase in hypoxia levels, an increase in TP53 and DRP1. Preliminary data acquired in 3D cultures show several effects of GH on these cells, however, these effects are not like data acquired in 2D culture. Conclusion: Growth hormone has shown an important role in the imbalance of mitochondrial metabolism in monolayer culture and in three-dimensional cultures, but in different ways. We believe that mitochondria have a central role in regulating the metabolism of cancer cells and that cells depend on this metabolic reprogramming for their proliferation, ability to metastasize or resist apoptosis. The identification of a signaling network involved in the progression of cancer has a potential application in clinical medical and can be used as a new target or synergistically associated with other drugs in the treatment of cancer.

CELLULAR AND MITOCHONDRIAL METABOLISM OF MACROPHAGES INFECTED WITH DORMANT CRYPTOCOCCUS NEOFORMANS

Dormancy is an adaptation present in a wide range of cells and organisms, in which cells reduce their metabolism, transcription and translation, to stay alive under stressful conditions, preserving the capacity to reactivate active growth once the environment reverts to favorable conditions. Dormancy occurs with Cryptococcus neoformans, a fungus responsible for causing cryptococcal meningitis in immunocompromised patients. Our previous results show that different growth stages of this fungus induces a distinct gene expression profile in infected murine macrophages. Given that that LPS stimulation and microbial infections cause alterations in macrophage metabolism, which impact macrophage functions, here we analyzed the influence of dormant cells of C. neoformans (DCn) in the metabolism of infected macrophages. We infected bone marrow-derived macrophages (BMDM) with either DCn, C. neoformans in the stationary growth stage (Stat or ura5Δ - auxotrophic uracil strain to reduce growth outside macrophages), and heat-killed (HK) + 1% Stat for 24h. Then, we analyzed mitochondrial mass, polarization of mitochondrial membrane, oxygen-reactive species (ROS) production, glucose and fatty acids uptake by flow cytometry. Only Stat induced mitochondrial depolarization, while the other parameters were unchanged in all infections. We also used oxymetry to analyze the respiratory profile of the infected cells using Oroboros and Agilent Seahorse instruments. Infection with fungus, induced a higher proton leak and extracellular acidification rate, indicating an increase in glycolysis. Infection with HK + 1% Stat induced higher basal respiration and ATP production in infected macrophages, while Stat induced higher non-mitochondrial respiration. We observed that infection with only Stat fungus prevents the increased glucose uptake induced by treatment with LPS and IFN-𝛾. Moreover, cells treated with LPS and IFN-𝛾 and infected with DCn increased their fatty acid uptake to a greater extent that other fungal forms. We conclude that DCn infection causes few changes in macrophage mitochondrial metabolism, but induces an increase in lipids uptake. Avoidance of metabolic changes is in line with the stealth strategy used by C. neoformans, preventing activating the immune system. It is also conceivable that mobilizing of lipids as an alternative fuel source to be hijacked by fungal cells will permit DCn reactivation and proliferation inside the phagolysosome.

CHADOX-1 VACCINE PRESENTS BETTER ANTIBODY RESPONSE AND IMMUNOGENICITY THAN CORONAVAC IN A GROUP OF PHYSICALLY ACTIVE OLDER ADULTS

Introduction: It is well-known that immunosenescence is a process that, due to the progressive decline in immune function associated with aging, leads to a reduction in the vaccine response in the older adult’s population, and this fact raises concern in the current pandemic situation caused by the SARS-CoV-2 virus, since, according to epidemiological data, this population is the most affected by COVID-19. Although our group has shown that older adults who regularly practice an exercise-training program have a better antibody response to vaccination, specifically against the Influenza virus, when compared to non-practicing or sedentary older adults, the restrictions imposed by the pandemic have altered the routine of this population and the maintenance of the regular practice of physical exercises by the older adult’s population have been a challenge for all involved. Thus, the objective of this study was to evaluate the antibody response to vaccination for COVID-19 in older adults who regularly practiced an exercise-training program, for at least 12 months, before the pandemic. Methods: Sixty-nine volunteers of both sexes, aged between 60 and 85 years, were enrolled and separated into two groups according to the vaccine received: CoronaVac (CO, n=46) and ChadOx-1 (CH, n=23). Blood samples were collected before and 30 days after the administration of the second dose of the vaccine against COVID-19 (CoronaVac or ChadOx-1), to determine the serum levels of immunoglobulins A (IgA) and G (IgG). Results: Higher serum IgG levels were observed in the 2 groups of volunteer groups post-vaccination (CO with p<0.05 and CH with p<0.001) than pre-vaccination values, with the immunogenicity of 39,4% in the CO group, and 56.5% in the CH group. Furthermore, higher serum IgA levels were found only in the CH group (p<0.001) post-vaccination than pre-vaccination. Conclusion: Our findings showed that older adults who regularly practiced an exercise-training program pre-pandemic period vaccinated with ChadOx-1 showed a better response of antibodies (IgG and IgA) and immunogenicity when compared to the CoronaVac vaccine.

Chagasic cardiomyopathy is marked by a unique activation pattern of CD4+ T cell populations

Chagas’ disease is a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic chagasic cardiomyopathy (CCC) is the most prominent clinical form of Chagas’ disease and can lead to heart failure and sudden death. Both the infection by Trypanosoma cruzi and host factors are proposed causes of CCC, considered to be mostly immune mediated. Although reports address the role of CD4+ T cells in the prognosis of the disease, a comprehensive analysis of activation markers during different clinical forms is lacking. Here, we assessed the expression of 27 markers combining unsupervised and supervised analyses on circulating CD4+ T cells in patients with distinct forms of Chagas’ disease. Supervised and unsupervised analyses of CD4+ T cells reveal increased frequencies of CD69+ cells during Chagas’ disease in all memory compartments and among naïve. However, CD69 expressing CD4+ T cells are decreased in patients with mild CCC compared to either asymptomatic patients or those with established CCC. Regulatory T cells expressing CD39 are reduced in mild CCC patients compared to controls. On the contrary, cytotoxic CD4+ T cells co-expressing granzyme B and perforin, are expanded in chagasic patients. Among these cells, higher levels of granzyme B and perforin are observed in patients with mild CCC compared to controls. Furthermore, patients with mild CCC produced inflammatory cytokines and displayed higher frequencies of multifunctional effector memory CD4+ T cells, here defined by the simultaneous production of IFN-gamma, TNF and the expression of CD154. Altogether, our results showed an expansion in activated CD4+ T cells and a decrease in a functional subset of regulatory T cells in the arise of Chagas cardiomyopathy, being potential markers of diseases’ aggravation and revealing the immune response during the establishment of cardiac lesions.

CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES IN MURINE BREAST CANCER CELL LINES

INTRODUCTION: Breast cancer is the most frequent type of cancer in women and the one with the biggest death occurrence. It is already known that the distant metastasis development is responsible for the majority of deaths but not the primary tumor. The mechanism, in which breast tumors metastasize specifically to different sites, is still unknown. Cell lines subcloned from a murine spontaneous carcinoma were described to have the capacity to metastasize to different sites. The cell line 67NR is the only non metastatic, whilst 66cl4 metastasized majority to lungs and 4T1 to lungs and liver. Since 4T1 is a heterogeneous line, single cell cloning showed that a subclone called 4T1.2 metastasize to the bone which is a clinically relevant site, since it is one of the most common metastatic sites. METHODS AND RESULTS: Public deposited samples from GSE160101, obtained from Gene Expression Omnibus were analyzed using a pre-established pipeline of differential gene expression analysis. The comparison was performed between each metastatic cell line and the non-metastatic one. We found 661 genes with differential expression possibly related to metastasis in the cell line 66cl4, 2299 in the 4T1 and 2274 in the 4T1.2. Furthermore, we performed GeneSet Enrichment Analysis and found that 4 genesets related to immune response were enriched (response to cytokine, defense response to other organisms, innate immune response and response to interferon gamma) in 66cl4. In the 4T1 cell line we found that the positive regulation of cytokine production and regulation of cytokine production were enriched. For last, in the 4T1.2 genesets related to embryonic skeletal system development and bone regeneration are enriched. CONCLUSION: Our results suggest that some enriched genesets could be related to site-specific metastasis.. The enrichment of bone-related genesets in the cell line 4T1.2, may be related to its capacity to metastasize to the bone, helping create the environment needed for tumor growth. Enrichment of genesets related to immune response may mean that neoplastics cells can modulate the immune response, preventing an effective response. Identifying the upregulated candidate genes from those enriched genesets will be important to design RNAi and investigate if its silencing would impact metastasis development in vivo. Understanding molecular mechanisms responsible for those metastases can shed light to the design of new therapeutic strategies.

CHARACTERIZATION OF EXTRACELLULAR VESICLES ISOLATED FROM THE PLASMA OF PEOPLE LIVING WITH HIV

Introduction: Immune cells release extracellular vesicles (EVs) composed by cytosolic contents surrounded by a lipid bilayer, enclosing a cargo of protein, sugar, and nucleic acids. These components could mediate intercellular communication under physiological or pathological conditions. Thereby, EVs may display essential functions in the elicitation of the immune system. In addition, HIV infection may trigger regulatory processes not completely elucidated so far, which could be related to the immune system modulation. Therefore, the identification of molecules into EVs that are related to the progression of the virus infection may led to a set of markers to aid in the diagnosis and treatment of people living with HIV (PLWH). Here, we sought to extract EVs from plasma and to characterize them in terms of size, concentration, and constitution. Methods and Results: We employed a differential centrifugation and ultracentrifugation protocol on plasma from healthy donors (HD) and PLWH to obtain cleaner EV samples, with higher quality and yield. First, we analyzed the EV morphological characteristics through the nanoparticle tracking analysis (NTA), then we evaluated protein constitution by coomassie blue staining, and western blotting for tetraspanin. We visualized a fraction of EVs between 100 and 400 nanometers corresponding to microvesicles and a second fraction smaller than 100 nanometers corresponding to exosomes by NTA. EVs from both donor groups showed distinct patterns of protein enrichment by coomassie blue staining and typical markers of EVs by westerns blotting. Conclusion: Thus, it was possible to isolate and characterize vesicles from plasma of people living with HIV and control subjects, therefore, once the patterns of protein seems to differ between the groups, the contents must be evaluated to understand the immunomodulatory effects.

CHARACTERIZATION OF GLOBAL REPERTORY OF SERUM IMMUNOGLOBULINS IN PATIENTS INFECTED WITH SARS-COV-2 BEFORE AND AFTER PLASMAFERESIS.

Currently, COVID-19 is the highest priority disease in terms of research, vaccine development and treatments due to its high degree of transmission and morbidity. One of the most pertinent lines of research concerns the humoral immune response during SARS-CoV-2 infection. Many studies have been carried out along these lines, such as understanding the aspects of immunization and cases of reinfection by the virus for the development of vaccines that provide long-term protection. In this project, the dynamics of the antibody response will be studied, analyzing the global profile of serum immunoreactivity in hospitalized SARS-CoV-2 infected individuals who received convalescent plasma transfusion. This study aims to characterize the immunoreactivity profiles, identifying possible changes after plasmapheresis, correlating with the clinical path of each patient. To carry out this study, the immunoblot technique was used (among others), where antigenic extracts were fractionated by electrophoresis in a polyacrylamide gel and then transferred to a nitrocellulose membrane. After transfer, the membrane was incubated with the different serum samples from the infected individuals in the Cassette Miniblot System (Immunetics Inc.). This system allows the incubation of the samples in parallel channels separated from each other, allowing the comparison of the immunoreactivity of the different samples. Immunoreactivities were revealed with appropriate secondary antibodies for detection of serum Ig of the human IgG isotypes. The preliminary results show a trend in the increase of anti-S and anti-RBD IgG in all patients (n=10) who received plasma transfusions and a maintenance of high titer of these IgG in patients who already had high concentrations before the plasmapheresis. In addition, we observed a higher profile of self-reactivity of these hospitalized patients when compared with sera from individuals who had mild and moderate symptoms of COVID19, as well as negative controls and pre-COVID19 sera. Other experiments and data analysis are still being done with a larger sample number. Therefore, this study can serve as a guide for the development of vaccines.

Characterization of initial humoral and cellular immune responde in different clincal outcomes of COVID-19

Introduction: COVID-19 is a broad-spectrum disease, ranging from asymptomatic infection to critical illness, which can be fatal. Numerous host characteristics have been associated with disease severity, such as age, comorbidities and genetic factors. Several cohorts of hospitalized patients show that elements of the immune response, such as inflammatory mediators and antibodies, play an important role in the pathogenesis of severe COVID-19. However, more detailed information about the early immune events before the need for hospitalization would help predicting a severe disease progression in these patients. In this study, samples were collected from patients with mild symptoms during the first days of onset, some of which later on progressed to more severe disease. Our aim is to identify early prognostic markers of severe COVID-19 before clinical deterioration. Methods and results: We studied the early immune response of COVID-19 patients (≤ 10 DSSO) using Luminex plasma cytokine analysis and immunophenotyping by flow cytometry. We selected ten patients who reported some degree of ventilatory support over the course of SARS-CoV-2 infection, with symptom onset between March and September 2020. These patients had a median of seven days of symptoms at the time of sample collection, with a median age of 49 years; 40% were female. Age and gender-matched uninfected controls and mild confirmed COVID-19 cases were included as control groups. Patients requiring oxygen support had lower levels of IFNγ than mildly symptomatic patients and IL-6, a known marker of disease severity, was notably elevated. They also exhibited lower levels of IL-12 and lower percentages of circulating NK cells. IP-10 and TNF-α levels were comparable to patients with mild symptoms. They also exhibited lower levels of IL-2 and IL-7, which stimulate T cell survival, and higher levels of IL-9 and IL-17, indicative of higher Th9/17 response. In addition to our cohort of local patients, we studied 28 severely ill patients from the state of Amazonas who were transferred to Rio de Janeiro in the context of a new epidemiological wave caused by a more transmissible variant of SARS-CoV-2. These patients reported symptom onset in January 2021, had a median of 14 days of symptoms at the time of sample collection, with a median age of 45 years; 22% were female. The cytokine profile was similar between these two groups of severe patients, despite the slightly later time point of sample collection.

CHARACTERIZATION OF LYMPHOCYTE METABOLIC PROFILE IN OBESE INDIVIDUALS

INTRODUCTION Obesity can cause multiple changes in the immune response, and in this way, it can interfere with the process of cell activation and inactivation. The study of the energy metabolism of lymphocytes can contribute to the understanding of these alterations. Therefore, this study aims to evaluate the metabolic alterations of lymphocytes in obese individuals METHODS AND RESULTS Initially, 32 men (18 to 45 years) were evaluated, of which 15 were characterized as eutrophic (BMI < 25 Kg/m2) and 17 as obese (BMI > 30 Kg/m2). Plasma leptin dosage was determined by the ELISA method. Lymphocytes were isolated from the venous blood for the analysis of percentage of Th1, Th2, Th17 cells and glucose uptake in the presence of PMA and ionomycin by flow cytometry; gene expression of Glut 1, Acyl Coa Oxidase, Beta-Hydroxyacyl-Coa-Dehydrogenase (HADH) and Hypoxia Inducible Factor 1 alpha (HIF-1α) by real-time PCR and kinetics of the citrate synthase enzyme. Obese subjects showed leptin levels 3.9 times higher when compared to eutrophic volunteers. In the obese group, higher percentages of Th1 and Th17 cells, higher expression of Glut 1, Acyl CoA oxidase and HIF1 α were observed when compared with the eutrophic group in the presence of stimulation. Lymphocytes from obese individuals showed a significant increase in lactate production. On the other hand, they showed lower activity of the citrate synthase enzyme. CONCLUSION So far, we have concluded that lymphocytes of obese men demand faster energy obtainment (a fact confirmed by the increase in lactate and reduction in citrate synthase). These changes are related to a pro-inflammatory condition demonstrated by Th1 and Th17 profile predomination.

CHARACTERIZATION OF PLASMODIUM VIVAX RIBOSOMAL PROTEINS AS MALARIA VACCINE CANDIDATES

The bottleneck of the malaria vaccine is the challenge to find multi-stage antigens conserved in different Plasmodium spp. Most malaria vaccines focus on antibody induction to the pre-erythrocytic stage, even though the sterile vaccine protection induced by attenuated sporozoites is directly related to cytotoxic T cells (CTLs). Our group demonstrated that P. vivax (Pv)-infected reticulocytes (iRetics) activate cytotoxic T lymphocytes (CTLs), through MHC-I, describing a protective mechanism against blood-stage parasites. In unpublished data, we performed an immunopeptidomics analysis to identify HLA-I-associated peptides of Pv-iRetics. Sixty percent of peptides were Ribosomal proteins that are conserved in Plasmodium stages and species. We selected 50 peptides and their source proteins to evaluate as malaria vaccine candidates. P. yoelii (Py) infection is an excellent experimental model for investigating malaria immune response. First, we tested the peptides in an IFN-g ELISPOT assay using Py-infected mice. To the most relevant peptides, we generate antigen-specific CTLs for a killing assay against Py iRetics. Hence, we produced five Pv Ribosomal proteins in the E. coli expression system for the immunization protocol. Mice were immunized three times with each protein associated with Alum + CpG as an immunological adjuvant. Twenty-one days after the last boost, mice were challenged with 106 PyX17NL-infected red blood cells, and the parasitemia was monitored for 30 days. To ELISPOT assay, Twenty-three Pv-peptides were immunogenic in the Py-acute infection. Moreover, 12 remain in the convalescent phase, suggesting memory response. We described that Pv antigen-specific-CTLs were able to kill Py-iRetics, which was not observed with unstimulated CD8 cells or uninfected reticulocytes. Regarding the immunization assessment, we identify that 4 of 5 tested proteins can induce antigen-specific IgG levels, additionally decreasing from 30 - 80% the parasitemia compared to the control group. Therefore, we unprecedentedly identify Pv antigens from ribosomal proteins that can induce a protective response to Py infection, indicating cross-species protection and a potential vaccine candidate.

CHARACTERIZATION OF THE BIOCHEMICAL, HEMATOLOGICAL, AND INFLAMMATORY PROFILE OF SICKLE-CELL MICE: CORRELATIONS WITH LIPID METABOLISM AND EICOSANOID PRODUCTION IN A SKIN ULCER MODEL

INTRODUCTION: Sickle Cell Anemia (SCA) is the most common form of Sickle Cell Disease, presenting high prevalence in the state of Bahia. Evidence has demonstrated that the inflammatory response, specifically sterile inflammation, plays a key role in the pathophysiology of SCA leading to systemic manifestations and multiple organ injury. In this context, the development of skin ulcers stands out as one of the most common complications. This study used a transgenic mouse model of sickle cell disease to characterize the hematological, biochemical, and inflammatory parameters of animals with different disease profiles and evaluate the local and systemic changes induced by skin ulcer in these animals. METHODS: Healthy (AA), sickle cell trait (AS) and SCA (SS) transgenic (Townes B6:129) mice had whole blood, serum, organs and skin fragments collected for the determination of baseline differences in their biochemical, hematological and inflammatory parameters. In the murine model of sickle cell ulcer, skin lesions of 4 mm were induced on the back of the animals, and samples of these lesions, as well as blood samples were collected at 0, 24, and 72h after induction. Inflammatory mediator production was analyzed by ELISA, whereas gene expression was analyzed by PCR. RESULTS: At baseline, SCA mice presented severe hemolytic anemia and extreme leukocytosis associated with increased concentrations of hepatic biochemical markers and hepatosplenomegaly in comparison with AA and AS mice. The SCA mice presented elevated concentrations of prostaglandin E2 and reduced levels of leukotriene B4 when compared to the other groups, indicating unbalanced lipid metabolism. In the skin ulcer model, the SCA mice presented a significantly higher leukocyte infiltrate after 24 and 72h. Furthermore, these animals presented increased expression of COX-2 and PLA2, providing a link between the lipid unbalance and the exacerbated inflammatory response observed in SCA animals. CONCLUSION: In summary, this pioneering work showed that mice with SCA have alterations in lipid metabolism and eicosanoid production that can both be directly related to a chronic inflammatory state an amplify the tissue inflammatory response triggered by skin injury in the sickle cell ulcer model.

CHARACTERIZATION OF THE IMMUNE RESPONSE AGAINST AN EFFLUX PUMP PROTEIN OF GRAM-NEGATIVE BACTERIA ASSOCIATED WITH ANTIMICROBIAL RESISTANCE

Introduction: Antimicrobial resistance occurs when bacteria develop the ability to bypass the pharmacological mechanisms used against them. Globally, it is estimated that approximately 700,000 deaths per year are attributed to antimicrobial resistance, and this number could increase to 10 million deaths per year by 2050 (O'Neill, 2014; Harbarth et al., 2015). The rapid spread of resistant bacteria may render the currently available arsenal of antimicrobials obsolete. Therefore, the development of alternatives to antimicrobials or adjuvants to these drugs is of utmost importance (Fernandes, P., 2006; López-Jácome et al., 2019). Among the mechanisms of antimicrobial resistance is the overexpression of efflux pumps, that may be inhibited by specific antibodies reducing bacterial resistance to antibiotics and infection. This work aims to evaluate the cellular and humoral immune response against the Gram-negative outer membrane efflux pump protein TolC from Escherichia coli for the discovery of potent antibodies. Methods and Results: Recombinant TolC protein cultured with RAW264.7 macrophages induced production of nitric oxide, IL-6 and TNF-, evaluated by Griess assay and ELISA, respectively. C57/Black6 mice were immunized with 10 g of TolC in Alum, intraperitoneally. TolC in vitro stimulation of lymph node cells obtained from TolC-immunized mice led to increased percentage of T cell proliferation, evaluated by flow cytometry. Immunization induced increased percentage of TolC-specific memory B cells in spleens and lymph nodes, assayed by flow cytometry. Through an ELISA developed in the lab, we observed that TolC immunization stimulated anti-TolC IgM and IgG production, at 7 and 10 days post immunization, respectively. Finally, we showed that anti-TolC IgM and IgG are also present in human serum, especially in patients with Gram-negative infections. Conclusion: Our results showed that TolC is immunogenic, activating macrophages, T and B cells, culminating in the production of specific antibodies in mice. Also, Gram-negative bacteria can stimulate the production of anti-TolC antibodies in humans as well. Further experiments are needed to evaluate the activity of anti-TolC antibodies against antimicrobial resistant Gram-negative bacteria.

CHARACTERIZATION OF THE LYMPHOCYTES FROM MESENTERIC LYMPH NODES OF SPONTANEOUS TYPE 2 DIABETIC GOTO-KAKIZAKI RATS

Background: Type II diabetes mellitus (DM2) is the most prevalent form of diabetes and is related to genetic and environmental factors, being a pathology caused by excess glucose, resulting from defects in the action of insulin on its receptor. DM2 is accompanied by chronic inflammation with changes in the functioning of immune system cells related to an imbalanced auxiliary T cell profile (Th). The development of obesity has been one of the main causes associated with insulin resistance (IR). However, there is a high number of non-obese patients who have DM2. Goto-Kakizaki rats (GK) are insulin-independent diabetes mellitus animals that are not obese, being an experimental model that develops a well-defined picture of IR and DM2. Thus, the aim of this study is to characterize the lymphocyte profile of the mesenteric lymph nodes of Goto-Kakizaki rats in comparison to the Wistar animals. Methods: Initially, 8 male Wistar and GK rats with 16 weeks-old were compared. In the 15th week after birth, the glucose tolerance test (GTT) and the insulin tolerance test (ITT) were performed. Lee index was calculated, and white adipose tissues (retroperitoneal, epididymal and subcutaneous) were weighed after euthanasia. Lymphocytes were isolated from mesenteric lymph nodes and determination of Th1, Th2, Th17 and regulatory T lymphocytes (Treg) was performed by flow cytometry. Cytokines secreted by lymphocytes stimulated with concanavalin A were evaluated by Cytometric Bead Array. Results: In relation to the GTT and ITT test we observed a higher area under the curve in GK rats. For the Lee index and the weight of white adipose tissues, GK rats presented a lower value compared to Wistar rats. We observed an increase in the percentage of Th1 and Th17 cells in GK animals stimulated with PMA + Ionomycin compared to Wistar. The expression of IL17 and TNF-alpha was increased in lymphocyte of GK rats. In addition, GK lymphocytes have increased secretion to the IL-18 pro-inflammatory cytokine, an important cytokine for activating Th1 response. Conclusion: These results indicate that 16-week-old GK animals, although not obese, present glucose intolerance, as well as an inflammatory lymphocyte profile with increased polarization for Th1 and Th17 cells. Approval from Ethical Committee: CEUA 010-2020

CHARACTERIZING THE SPECIFIC T CELL RESPONSE AGAINST IN SILICO PREDICTED PEPTIDE EPITOPES IN COVID-19 CONVALESCENT INDIVIDUALS

Introduction: The pandemic unleashed by SARS-CoV-2 has infected over 185 million people worldwide and took more than 4 million lives. Cellular and humoral immunity induced after infection/immunization is key for viral control and avoidance of clinical recurrence of COVID-19. Nevertheless, the durability of such responses is a concern that needs to be addressed. Here, we address this question by characterizing the specific T cell response against in silico predicted peptide epitopes in samples from SARS-CoV-2 convalescent individuals, collected 30 and 180 days after infection aiming to find epitopes for a candidate vaccine. Methods and results: We scanned the proteome of the SARS-CoV-2 (Wuhan strain) to predict in silico of specific and promiscuous peptides recognized by CD4+ and CD8+T cells in the ProPred bioinformatics tool, according to binding stability to HLA I or HLA II or in the context of the 10 most frequent HLA class I and class 2 alleles in the world population. The set of peptides was predicted to bind to HLA molecules of over 99% of the population. To analyze the promiscuity of the predicted peptides, PBMC samples from 121 convalescent individuals were collected 30 days after symptoms onset. A second PBMC sampling was performed 180 days after infection in 52 returning individuals. The number of specific IFN-g producing T cells was assessed by ELISPOT assay. The results indicated that the predicted CD4 and CD8 peptides were highly recognized by convalescent individuals, each peptide recognized by at least 70% of tested individuals. The magnitude of response was significantly higher in hospitalized patients compared with those with mild symptoms. In addition, males and patients >50 years old displayed a lower number of IFN-g producing cells than female and younger patients. The analysis of the samples collected after 180 days revealed a reduction in the numbers of specific circulating IFN-g producing T cells, indicating a decreased immunity against viral peptides. Conclusions: Our data show that in silico predicted epitopes were highly recognized by T cells of convalescent individuals independently of HLA alleles of the participants, and characteristics of the participants such as sex, age and disease outcome suggesting a possible application to vaccine design. However, the number of specific T cells decreased 180 days after infection, which could be associated with reduced protection against reinfection along time.

CHIKUNGUNYA VIRUS INFECTION IMPACTS BIOMECHANICAL PROPERTIES AND MAPK SIGNALING PATHWAYS IN HUMAN FIBROBLAST-LIKE SYNOVIOCYTES (HFLS) IN VITRO

Introduction: Chikungunya virus (CHIKV) is a transmitted-mosquito Alphavirus, that causes a disease manifested as severe persistent polyarthralgia in some cases. The synovial tissue is compromised during the infection, and human fibroblast-like synoviocytes (HFLS) may be the some of the cells affected. Thus, the aim of this study was to understand biomechanical changes and cell signaling pathways involved in the HFLS-CHIKV interaction in vitro. Methods and Results: HFLS were infected with CHIKV (MOI 0.5) for 48 hours and the percentage of CHIKV-infected cells was detected by intracellular flow cytometry by using an anti-CHIKV monoclonal antibody/anti-mouse IgG Alexa Fluor 488. Quantification of chemokines was performed in the cell supernatant by using the CBA method (Cytometric Bead Array). CHIKV-infected HFLS were submitted to Atomic Force Microscopy (AFM) and the cellular elasticity was evaluated by using a scanning tip. F-actin cytoskeleton was labeled with Alexa Fluor 488 Phalloidin and the nucleus was stained with blue-fluorescent DAPI at 0h, 12h, 24h, and 48h after infection followed by fluorescence microscopy analysis. The activation of mitogen-activated protein kinases (MAPK) was evaluated by detection of ERK1/2, p38 and JNK phosphorylated proteins by using PE Anti-ERK1/2 (pT202/pY204), Alexa Fluor 647 Anti-p38 (pT180/pY182), and PE Mouse Anti-JNK (pT183/pY185) antibodies by intracellular flow cytometry (BD Phosflow). The cytopathic effects were observed in HFLS and 46,8% of cells were positive to CHIKV 48h after viral infection. An increase in the IP-10 and a reduction in MCP-1 levels were detected. AFM analysis showed CHIKV-infected HFLS with morphological changes and rougher surface. AFM nano-indentation shows an increase of 107.46% in the average of Young’s elastic modulus in the CHIKV-infected cells. Also, significant changes in the F-actin filaments, disruption in the cytoskeleton arrangement and a significant increase in the phosphorylation of ERK1/2, p38 MAPK and JNK were detected. Conclusion: CHIKV infection in HFLS can lead to cytoskeleton disruption, changes in cellular elasticity, activation of MAPK pathways (ERK 1/2, p38 and JNK) and modulation of chemokines. The elucidation of the mechanisms of CHIKV-HFLS interaction is important to understand the viral pathogenesis and discovery of new therapeutic targets for this arbovirus disease.

CHRONIC COMBINED TRAINING MODULATES SYSTEMIC LEVELS OF CYTOKINES IN PATIENTS WITH SCHIZOPHRENIA

Introduction: Schizophrenia is a serious psychiatric condition that affects approximately 24 million individuals around the world (American Psychiatric Association, 1994). It is characterized by symptoms such as hallucinations, delusions, cognitive impairment, and mainly a limitation in social function that leads to the isolation of schizophrenic patients. An inflammatory environment with intense oxidative conditions can be considered a factor in the progression of schizophrenia. Physical exercise is capable of promoting immunoprotective and immunoregulatory effects through the balance between pro and anti-inflammatory cytokines resulting in a systemic reduction of inflammation. This study aimed to investigate the effect of Concurrent Training of Strength and Aerobic Fitness on cytokines levels in schizophrenic patients during 9 months. Methods and Results: 14 individuals (age 43.12±6.25; 75% male) were evaluated through blood analysis at the following times: pre-intervention, 30, 90, 180, and 270 days. A significant increase in IL-10 levels was found at 90 (p = 0.038), 180 (p=0.033) and 270 (p=0.027) days. There was a significant reduction in TNF-alpha in 180 (p = 0.02), 270 (p=0.001) and leptin in 90 (p=0.02) and 270 (p=0.028) days. Conclusion: It is suggested that the combined training can have a beneficial effect depending on the intervention time and modulate the inflammatory profile.

Chronic inflammation as a progression factor for Human Papillomavirus-associated cancer

Cervical cancer is caused by high oncogenic risk Human Papillomavirus (HPV). Although the virus infection is essential for the carcinogenic process, other factors play a role in cellular transformation. One of them is the microbiota. The feminine genital tract is colonized in most women by Lactobacillus species. These bacteria secrete lactic acid that maintains an acidic vaginal pH and protects the tissue against other infections. It has been shown that dysbiosis is associated with cervical cancer progression. However, a causal relationship has not been characterized yet. We have sequenced bacteria from patients with low grade and high grade cervical lesions (the last ones considered precursors for cercical cancer) and cervical cancer and have shown prevalence of Lactobacillus in low grade lesions, and higher genre diversity in high grade lesions and cancer samples. Working with the hypothesis that changes in the microbiota is a risk factor for cervical cancer development, we will test bacterial produts and bacteria on epithelial cell lines immortalized with high oncogenic risk HPV genomes. We will test cell proliferation, density, dependence on subtrate for cell growth, migration and invasion. Moreover, we will test the interaction between the immortalized cells, bacteria products and macrophages derived from peripheral blood monocytes. The rationale for this test is the role that cells in the microenvironment play on tumor progression. Macrophages play an important role in the tumor microenvironment, promoting angiogenesis and tolerance torward tumor antigens. Macrophages are also plastic cells that can respond to alterations in the environment, including bacteria and their products. We expect to be able to characterize a potential causal role for microbiota alterations and carcinogenesis initiated by HPV.

CHRONIC INGESTION OF PRIMEX-Z, COMPARED WITH OTHER COMMON FAT SOURCES, DRIVES WORSE LIVER INJURY AND ENHANCED SUSCEPTIBILITY TO BACTERIAL INFECTIONS

Introduction: Intake of food containing high levels of fat is one of the risk factors for development of hepatic disorders. However, patients from different countries remain consuming industrialized high fat foods despite the common knowledge of the harmful effects on liver. Therefore, it is of major relevance to determine safer common fat options in diets that could lead to lower risks of liver injury and inflammation. Here we investigated the impact of different industrialized high fat diets (HFD) in the hepatic immune and metabolic profile during NAFLD development in mice. Methods and Results: Male C57BL/6 mice were fed a control diet, and four different high-fat diets (HFD: 40% calories from fat) for 16 and 30 weeks. HFDs had different common fat sources, including trans-fat, non-trans-fat palm oil (Primex-Z), palm oil alone, and corn oil alone. Mice were sacrificed and samples were collected for analysis. Using an unprecedented combination of in vivo imaging with immunometabolic phenotyping, we revealed that a HFD induced a major increase in hepatic lipid droplet deposition compared with control mice, being significantly higher in Primex-Z-fed mice (Percentage of liver fatty area: CT=2.982±0.628; Primex-Z=42.14±2.102). All HFD mice had similar or less weight gain as control mice; however, Primex-Z ingestion led to a higher increase in adiposity index (~90% increase) compared with other fat sources. Gene expression of isolated liver immune cells revealed large changes in expression of several inflammatory pathways, which were also more elevated in Primex-Z-fed mice, including Tnf (~20-fold), Il1b (~60-fold), and Tgfb (2.5-fold). Immunophenotyping and in vivo analysis showed that the frequency of hepatic immune cells was also disturbed during different HFD contents, rendering not only Kupffer cell depletion (in vivo F4/80+ cells per field: CT=84.41±4.676; Primex-Z=31.07±2.987), but also reduced bacterial arresting ability (E. coli+ Kupffer cells: CT=36.92±3.463; Primex-Z=18.49±2.846). Conclusion: Different fat dietary sources imprint different immune and metabolic effects in the liver during consumption of an HFD. The present data highlighted that Primex-Z – a novel non-trans-fat regularly used in human nutrition – is not only able to damage hepatocytes, but also to impair liver ability to clear blood-borne infections.

CLINICAL AND IMMUNOLOGICAL MARKERS IN CHILDREN INFECTED WITH RESPIRATORY VIRUSES IN A PEDIATRIC HOSPITAL IN MINAS GERAIS

INTRODUCTION: The new coronavirus pandemic has affected adults and children and became an important cause of Severe Acute Respiratory Syndrome (SARS) nowadays. Among children, 12,921 cases of SARS were notified in 2021 resulting in 727 deaths. Since the beginning of the pandemic, 23,277 SARS cases due to COVID-19 and 1,449 deaths occurred in Brazil. Although children of all ages can contract COVID-19, those younger than 14 years of age are frequently less affected than adults and display mild symptoms. Compared to other pediatric respiratory syndromes COVID-19 is apparently less severe and caused less deaths. Among infants under one year age, Respiratory Syncytial Virus (RSV) is estimated to cause more deaths than any other single infectious agent with exception of malaria. The purpose of this study is to compare the clinical and immunological markers involved in the response to SARS-CoV-2 and RSV in children treated at Hospital Infantil João Paulo II, Belo Horizonte, Minas Gerais. METHODS AND RESULTS: Serum or plasma were obtained from children infected with RSV (n=29), SARS-COV-2 (n=105) and healthy controls (n=35). Levels of interleukins, chemokines and growth factors were measured by multiplexed bead assay (Human 27-Plex, Biorad). The biomarker profile of these children, with respiratory infection, was analized and compared with clinical, hematological and biochemical evaluation. There was an increase in cytokines, growth factor and chemokines in children infected with respiratory virus. We observed differences in the levels of most biomarkers analyzed between healthy and children infected either by SARS-COV-2 or RSV. Levels of CCL2 were significantly lower in SARS-COV-2 group compared to RSV. CONCLUSION : A strong pro-inflammatory profile is consistent in both respiratory diseases. As perspectives, we aim to associate data from the biomarker profiles with other clinical and hematological parameters to assess a biomarker signature useful for clinical management.

CO-CULTURE OF HUMAN TUMOR CELLS WITH ZEBRAFISH BLASTODERM CELLS: STUDY OF THE TUMOR MICROENVIRONMENT

Introduction: Breast cancer has a high mortality rate among women, and the low non-individualized treatments cause several adverse events. In precision oncology, the analysis of specific clinics, genetics, and molecular factors allows a preventive treatment that increases the effectiveness of individual treatment. Considering this, zebrafish (Danio rerio) was highlighted as a study model for tumors due to mimicking the pathophysiology of various tumors. Furthermore, the genomes of zebrafish and humans are highly conserved, especially in oncogenes and tumor suppressor genes. In this study, we aim to establish a 3D co-culture between the blastoderm of the zebrafish embryo and the MDA-MB-231 triple-negative breast cancer cells. Methods and Results: 5,000 tumor cells, belonging to the lineage-MB-MB-231 mkate2, and 2,500 blastoderm cells, derived from the zebrafish embryo explant, will be used for the production of each spheroid, produced by the nanoparticles that induce the magnetization of the cells. For the setting of the 3D hybrid co-culture, tumor cell spheroids and blastoderm cell spheroids will be pooled in a single well, forming hybrid spheroids. Glucose and lactate dosages will be performed. Confocal microscopy will also be used to assess the formation of spheroids. With the co-culture established, the hybrid spheroids will be injected into adult zebrafish exposed or not to dexamethasone. Furthermore, this method will facilitate the implantation of human tumor cells into the zebrafish model. Because we implant tumor cells together with other elements of the tumor microenvironment, part of this implanted complex will come from the animal model itself. Conclusion: We hope to eliminate the need for immunosuppression in transplanted organisms. 3D co-cultures would mimic the human organism and benefit the xenotransplantation and the tumor microenvironment, making it possible to evaluate the tumor progression, animal survival, tumor microenvironment, mitochondrial morphology, and metabolism of tumor cells.

COMPARATIVE ANALYSIS OF DISEASE-MODIFYING THERAPIES ON THE COMPOSITION OF T CELL SUBSETS IN PATIENTS WITH MULTIPLE SCLEROSIS

Recurrent remitting multiple sclerosis (RRMS) is a chronic inflammatory autoimmune disease of the central nervous system considered to be the main cause of chronic neurological disability in young adults. The rapid diagnosis and treatment with disease-modifying therapies (DMT), such as interferon-β (IFN-β), natalizumab (NTZ), fingolimod (FGL) and dimethylfumarate (DMF), can alter the clinical course by controlling immune events mediated by encephalitogenic T cells associated with relapses and neuronal injury. However, studies comparing the effects those drugs on the composition of different T cell subsets are lacking, which is the objective of the present study. Thus, lymphocytes from MS patients (treated or not with DMD) and healthy subjects (HS) were stimulated for 3h with the combination of PMA and ionomycin and subsequently labeled using different fluorochrome monoclonal antibodies directed against the CD3, CD4, CD8, CD45RA, CD62L, IFN-γ, CD28 and CD57 markers. The determination of different cell subpopulations was carried out by flow cytometry. Here, no difference in the frequency of naïve, central memory (CM), effector memory (EM) and TEMRA phenotypes was observed in CD4+ and CD8+ T-cells from non-treated patients and HS. Among DMT, FGL reduced the proportion of naïve and CM T cells, while DMF decrease the frequency of EM T cells. In contrast, the percentage of EM was higher among FGL- and NTZ-treated patients. The proportion of TEMRA cells, most of them CD28-CD57-, was significantly higher in patients under DMF and FGL therapy. With regard to cytokine production, the frequency of naïve, CM, EM and TEMRA CD4+ and CD8+ T cells able to produce IFN- was significantly higher in non-treated MS patients as compared with HS. Among DMT, FGL and DMF were more efficient in reducing the frequency of those IFN-+T-cell subsets. Interestingly, the severity of neurological disabilities was mainly associated with the frequency of naïve and EM IFN-+ T-cells. Although preliminary, our data suggest that higher therapeutic efficiency in MS depends on the ability of DMT to reduce the frequency of naïve and effector memory T IFN-+ cells. In addition, the ability of DMF and FGL to increase the frequency of non-exhausted TEMRA cells could help the patients to maintain immune surveillance against different pathogens.

Comparison of adaptive immune response induced by standard and fractional doses of 17DD-YF vaccine against yellow fever

The yellow fever vaccine is one of the most successful prophylactic vaccines, probably due to the activation of T and B lymphocytes and induction of neutralizing antibodies, along with a mixed cytokines profile. However, increase on worldwide demands and manufacturing limitations of the vaccine created the need to use its fractional doses (FD). The FD induced the production of neutralizing antibodies against the yellow fever virus and cytokine responses similar of that generated by the standard dose (SD). Our aim was to compare the viremia kinetics after vaccination with the FD or SD of the YF-17DD, their capacity to induce the production of antibodies, inflammatory and anti-inflammatory mediators, and the expansion and activation of T and B cells and their subpopulations. Study participants were selected from the population immunized with FD or SD in the campaigns against yellow fever in the state of São Paulo, Brazil. Blood and serum samples were taken before vaccination, 7 and 10-15 days after vaccination. Serum samples were also taken 30-45 days after vaccination. Cells were purified from blood clots with density gradient and phenotyped by flow cytometry (FACSFortessa). The production of soluble factors was measured using a 27-plex assay, and specific IgM, IgG and neutralizing antibodies were measured by ELISA and MicroPRN assays, respectively. Viremia was measured by reverse transcription quantitative real-time PCR. Our results show that that although FD viremia kinetics was prolonged when compared to the SD, the antibody production was indeed similarly induced by the different doses. The production of soluble markers induced by both doses was similar after 7 days, with a rapid decrease occurring after SD vaccination and slower decrease after FD administration. The cell response profile showed that the FD immunization led to the expansion of T cells expressing activation markers, along with a higher proliferation of Tfh cells, while the SD induced higher levels of activated plasma cells. Finally, a correlation-based network built 7 days after immunization with FD presents higher number of connectivity when compared to the one generated 7 days after immunization with SD. Together, these findings indicate that FD and SD induced different immune response profiles, with the same outcome of protection against the yellow fever virus.

COMPARISON OF DIFFERENT METHODOLOGIES FOR EXPANSION OF NATURAL KILLER CELLS FROM PERIPHERAL BLOOD AND UMBILICAL CORD BLOOD FOR IMMUNOTHERAPY APPLICATIONS

Several clinical studies with Natural Killer (NK) cells have shown promising results in the treatment of onco-hematological diseases due to their high cytotoxicity against tumor cells. Thus, we propose the development of a protocol for NK cell expansion and its use as cell therapy products. Our aim was to compare different methodologies and establish a standardized protocol for the isolation and expansion of NK cells from peripheral blood (PB) and umbilical cord blood (UCB). The experiment was carried out at the Laboratory of Medical Investigation (LIM31) of the HC-FMUSP. Fresh PB samples were obtained from healthy donors and the cryopreserved UCB units were obtained from a public Cord Blood bank. Cells were expanded ex vivo for 14 days in culture medium supplemented with different combinations of cytokines, such as IL-2 alone, IL-2 and IL-21, IL-15 and IL-18 and IL-12, co-cultured or not with CSTX002 feeder cells in a 1:2 ratio. The number of NK cells increased significantly when they were co-cultured, in comparison to conditions without feeder cells. Pre-activation conditions with IL-12, IL-15 and IL-18 for “cytokine-induced memory-like” (ML NK) cells in both PB-NK and UCB-NK expansion yielded a significantly greater fold expansion (12-fold and 8-fold) when compared to expansion with IL-2 alone or IL-2 and IL-21. Moreover, the addition of IL-21 did not have a significant impact in the expansion of CD56+CD3- cells when compared to conditions that used IL-2 alone. Furthermore, feeder cells mediated expansion yielded a predominantly CD56brightCD16bright mature phenotype in both PB-NK and UCB-NK. To evaluate the functionality of NK cells, co-culture in the presence of tumor target cells was performed and UCB-NK conditions with IL-2 alone or IL-2 and IL-21 showed enhanced degranulation capacity and lytic granules (Granzyme B and Perforin) and cytokine production (IFN-y and TNF-a) than PB-NK cells. ML NK cells from PB produced higher amounts of cytokines and lytic granules when compared to ML NK cells from UCB. This may be related to a lower expression of NKp46, NKG2D and CD69 activation markers in ML NK UCB, and possibly because the UCB units were cryopreserved. The results suggest that ML NK cells show good ex vivo expansion, but do not show proper functionality against tumor cells. It is possible that with the addition of IL-2, ML NK cells could improve the production of cytokines and lytic granules, as seen in the IL-2 alone and IL-2 and IL-21 conditions.

COMPARISON OF NEUTRALIZING ANTIBODY DEVELOPMENT BY PSEUDOVIRUS METHODOLOGY FOR INFECTION WITH SARS-COV-2 AND/OR VACCINATION

Introduction: In the current situation of the COVID-19 pandemic, obtaining information related to host immunity is essential to develop strategies for prevention. The determination of neutralizing antibodies, nAb, for SARS-CoV-2 is of great importance for evaluating possible protection against new infections, immunity status, and prognosis in case of infection. Methods and Results: In this work, samples were selected from a cohort of patients infected with SARS CoV-2 (n=50) with mild to moderate symptoms, and from vaccinated volunteers without a history of infection, followed longitudinally, to determine the presence of neutralizing antibodies using pseudovirus as detection methodology. The construction of a pseudovirus was performed, based on the expression of the Spike protein from the Wuhan sequence, in the envelope of the modified lentiviral vector, for specific inhibition of the adsorption of viral particles on 293T(ACE2). High correlations of IC50 values with PRNT were obtained. In infected individuals, the production of neutralizing antibodies is more intense at the beginning of the infection, with a reduction over time. Still, the NT50 constancy is justified by the potency, specialization, and maturation of the immune response. For those infected vaccinated with coronavac, there is a peak of neutralizing antibodies 14 days after RT-qPCR, being surpassed only after the 3rd dose with Pfizer. After the coronavac doses, the patients showed maintenance of the ELISA index for IgG against N protein, a significant increase for anti-S IgG 208 days after RT-qPCR, and for anti-S1 IgG, there was a significant increase 471 days after RT-qPCR, after a booster dose with Pfizer. Regarding potencies, anti-S1 showed a higher value during infection compared with anti-S, a lower decrease with advancing post-infection period, and a greater effect of Coronavac doses. After the 3rd dose of Pfizer, however, the increase in potency for anti-S was significantly greater. For vaccinated patients without a history of infection: Coronavac showed lower comparative efficiency in inducing a robust and lasting immune response, within the aspects analyzed in this work; AstraZeneca indicated that it is an immunizing agent with a greater comparative capacity to boost the adaptive system. Conclusion: SARS-CoV-2 infection remains the alternative that induces more nAb, and the association with the heterologous vaccine protocol is the best option to guarantee protection.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis

“Composition of immunoglobulin CDRs: geography and co-evolution with fever-inducing pathogens”

Computational modeling and heterologous expression of nanobodies as potential VLA-4 inhibitors

Integrins are heterodimeric proteins formed by non-covalently linked α and β subunits. In vertebrates, 18 α subunits and eight β subunits are known, resulting in 24 different integrins, with specific and non-redundant functions involved in distinct and critical biological mechanisms, which are evidenced, for example, by the specificity of ligands. The α4β1 (or VLA-4) integrin is a transmembrane protein widely expressed on the surface of immune cells, mainly on monocytes and T lymphocytes, being involved in the mechanism of leukocyte extravasation, an essential step for the development of inflammatory processes. As a result, VLA-4 is associated with chronic inflammatory diseases of significant public health impact, such as asthma, rheumatoid arthritis and multiple sclerosis, thus being an important target for therapies for human pathologies. A commercialized anti-VLA-4 compound is Natalizumab, a monoclonal antibody used in the treatment of multiple sclerosis, associated, however, with high production cost and increased risk for the opportunistic infection of Progressive Multifocal Leukoencephalopathy, in addition to not being specific for this integrin. Given these informations, the importance of developing VLA-4 antagonists in the VHH format is highlighted. The Natalizumab antibody presents itself as an α4β1 antagonist by targeting the α4- subunit, but it is not specific for this integrin. On the other hand, this work presents the initial results derived from computational and experimental techniques. Therefore, this work presents the initial results of developing three specific nanobodies (VHH-1, VHH-3 and VHH-4) and potential inhibitors of VLA-4, obtained by computational and experimental techniques. Three-dimensional structures, obtained by comparative modeling using the MODELLER program, of these antibodies are proposed. Furthermore, the soluble expression in E. coli SHuffle pLysS, at 15 ºC, for 16 h, and efficient purification by affinity chromatography is presented. In summary, the results are promising in developing anti-VLA-4 nanobodies and guide the subsequent work steps, which include the refinement of the modeling of the CDRs of the predicted structures, refinement of the purification methodology, quantification of the yield and functional analysis of these nanobodies.

Connection of immunometabolic and molecular markers (serum levels of CD5L and the miR-33 cluster) with sdLDLc phenotypes in sedentary subjects with cardiovascular disease risk

Introduction. Scientific evidence shows the intrinsic relationship between the activation of macrophages and microRNAs (miRs) expression in regulating metabolism and the immune response, both in adipose tissue (AT). As well as, in dysfunctional AT, the inflammatory process, lipid synthesis and metabolism may be immunometabolic processes regulated by miRs expression. In juxtaposition, the pleiotropic protein CD5L is an element of activated macrophage that maintains tissue homeostasis, and it is commonly expressed in macrophages inside swollen tissues. The aim was to establish the connection of CD5L levels and miR-33 cluster (miR-33a/ miR33b) with sdLDLc phenotypes, metabolic and inflammation markers, and dysfunctional AT in sedentary subjects with CVD risk status. Methods and Results. This cross-sectional study included 338 men and women from 20 to 59 years classified by dyslipidemia: healthy and hyperlipoproteinemia (LDLc 130 mg/dL). In a further analysis, we stratified according to the relative proportion of sdLDLc of LDLc, (30% 70% phenotype B or ≥50% phenotype A/B). Adiposity status metabolic and inflammatory markers were measured using bioimpedance, enzymatic, and immuno-turbidimetry routine methods, respectively. By ELISA, we evaluated serum insulin levels, free and total CD5L, CD36, and adipomyokines. The TaqMan qPCR 2CT method was used to quantify miR-33 cluster expression. Statistical analysis differentiating between hyperlipoproteinemia and phenotypes A/B and B, and we determined correlations of CD5L levels with adiposity and immunometabolic markers measurements (P < 0.05 was considered significant). In subjects with dyslipidemia state and both phenotypes sdLDLc, the low CD5L-CD36 levels revealed a leading role. The miR-33 cluster relative expression levels showed an inverse pattern of phenotype A/B regarding phenotype B. Adipomyokines setting imbalance and the presence of inflammation favor the increase of CDV ratios. Body obesity indices and the negative association with CD5L levels correspond to the degree of dysfunctional AT. Conclusion. We suggest that in sedentary individuals dysfunctional AT might be represented by the CD5L levels. Simultaneously, the imbalance in adipomyokines levels could be supported by the contribution of the low-grade inflammatory process. We are highlighting that the relative expression of the miR-33 cluster parallels to the presence of hyperlipoproteinemia and the B or A/B phenotype of sdLDLc.

CONSEQUENCES OF METABOLIC PROGRAMMING BY LITTER SIZE REDUCTION TO GOBLET CELLS IN THE ILEUM BALB/C MICE

Introduction: A high-fat diet has direct effects on the intestinal epithelium, affecting the metabolic biochemical pathways and triggering inflammatory processes, as long-term it has been linked to the development of Metabolic Syndrome (MetS). The gastrointestinal tract, especially the ileum, beyond essential as an immune barrier, has an important role in the systemic immunity of organisms. Despite this, there is little knowledge about changes in the intestinal epithelium and the effects of MetS on goblet cells, fundamental mucin producers in the epithelial barrier. Hence, the objective of this work was to verify the effects of MetS on intestinal goblet cells in the ileum of male BALB/c mice. Methods and results: Eight newborn male BALB/c mice were divided into a control group (n=4) and a group programmed for MetS (n=4) by Litter Size Reduction (SL) in the proportion of 3 animals per lactating. The research was approved by the Ethics Committee on the Use of Animals (CEUA), number 8137280920/002850. After 180 days, the animals were euthanized and 1 cm of the ileum was collected for histological processing. Each organ was fixed, embedded in paraffin, subjected to semi-serial cross-sections of 5 μm and stained by Alcian-Blue (AB) technique at pH 2.5 and pH 1.0. The stained sections were analyzed using light microscopy with a 100x objective. The proportion of Goblet Cells (GC) per 100 Epithelial Cells (EC) was calculated in a total of 2,560 EC per animal in each staining. After verifying the normality of the data, the T-Student Test (p<0.05) was performed using the GraphPad Prism Software v.8.0.1. According to the results, the proportion of GC (cells/100EC) AB pH 1.0 were 16.87±4.04 for SL and 14.24±0.80 for the CG group, while for AB pH 2.5 they were SL 16.88±2.84 and CG 15.53± 2.96 cells/100EC, respectively. Thus, although there was no statistically significant difference, we observed a slight increase in the number of sulfomucin goblet cells (AB pH 1.0, approximately 18%). Conclusion: The slight increase in goblet cells found in animals with MetS could reflect changes in mucus composition, an important component for the maintenance of the intestinal barrier.

CONTRIBUTION OF IL-1Ra IN OBESITY-INDUCED METABOLIC SYNDROME MODEL

The alterations in the dietary patterns of the population is characterized by the greater consumption of processed foods to the detriment of natural foods, leading to an increase in body fat, a risk factor for several chronic diseases, such as obesity and metabolic syndrome (MS). In this context, it was found increased serum lipopolysaccharide (LPS) levels named of endotoxemia, which occurs in consequence of increased intestinal permeability, due to the reduction of the tight junction proteins (TJ) in epithelial barrier. Type 3 innate lymphoid cells (ILC3) contribute to the maintenance of the intestinal epithelium, as they produce IL-22 and IL-17 cytokines, which promote the expression of TJ, mucins and antimicrobial peptides under the stimulus of IL-1β, via its IL-1R1 receptor, and IL-23. In this context, our group found a correlation between lower expression of IL-17 and IL-22 cytokines in the ileum and high intestinal permeability during the progression of MS. Additionally, our group observed that IL-1R1-deficient mice developed severe obesity and MS worsening associated with reduced IL-17 and IL-22 expression in the ileum, inferring the role of the IL-R1 receptor in the differentiation/activation of intestinal ILC3 and reduction in obesity-induced MS. Therefore, the objective of this project is to evaluate whether the IL-1Ra (interleukin-1 receptor antagonist) deficiency interferes in obesity outcome and in metabolic changes associated with obesity and MS. C57BL/6 mice were fed with standard diet (SD) or high fat diet (HFD) and IL-1Ra-/- mice fed with HFD for 12 weeks and had their weight, food and caloric intake measured once a week. At the end of the experiment, the mice were euthanized for the collection of their adipose tissue, pancreas, ileum, iliac lymph nodes and blood. Notably, it has been observed that IL-1Ra-/- mice fed with HFD exhibited a significantly reduced food and caloric intake when compared to WT animals fed with SD. In addition, these mice feeding HFD show lower weight gain and normalization of glycemic levels compared to WT fed HFD. Therefore, previous results suggest that the deficiency of IL-1Ra has a protective role in obesity-induced MS.

CONTRIBUTIONS OF EXTRACELLULAR VESICLES IN THE PATHOPHYSIOLOGY OF HUMAN CUTANEOUS LEISHMANIASIS

INTRODUCTION: Human cutaneous leishmaniasis (CL) is a relevant public health problem in Brazil, being endemic in several states and caused by Leishmania (V.) braziliensis. The clinical features affect the skin and mucous membranes that may heal spontaneously or by antimonial treatment. The disease outcome is dictated by parasite destruction performed by macrophages/dendritic cells activated by T lymphocytes. The communication between cells are important mechanisms for an effective immune response and may be conducted by direct contact between cells or biomolecules release. Extracellular vesicles (EVs) have key roles in these communication processes, transferring biomolecules and genetic information, being important regulators of cellular functions and physiopathogenesis mechanisms. OBJECTIVE: The aim of this study was to standardize pre-analytic procedures and flow cytometry protocol to identify and characterize EVs in plasma from CL patients for understanding their contributions in host-immune response against Leishmania. METHODS: Calcein, annexin V and monoclonal antibodies were used to detect and phenotype EVs and the samples was acquired by the nano-flow cytometer CytoFlex. RESULTS: Through FCM analysis, we observed low frequencies and concentrations of EVs, when analyzing plasma samples from before treatment CL patients, as well as high frequencies and concentrations from during treatment CL patients. In addition, we observed that most EVs had a platelet phenotype. CONCLUSION: Since well-standardized, flow cytometry is useful to identify, quantify and to phenotype EVs from ex-vivo plasma samples. Besides, the EVs function investigation in the CL pathophysiology is essential for the generation of knowledge and might define biomarkers for distinct phases of the disease and therapeutic targets.

CONTROL OF CD8 T CELL EXHAUSTION BY THE POLYCOMB MEMBER EZH2

Introduction: Antigen specific CD8 T cells can enter in a state of hyporesponsiveness, progressively losing their cytokine production, proliferation and cytotoxic potential, while overexpressing inhibitory receptors. These functionally impaired cells, known as exhausted CD8 T cells, are usually generated during neoplastic processes and chronic infections, also being associated with limited outcomes in immunotherapies such as CAR (Chimeric Antigen Receptor) T cells in solid tumors treatment. Our prior results in chronic strain LCMV clone 13 infected mice demonstrated that early generated exhausted CD8 T cells displayed lower levels of the epigenetic modulator Ezh2 (Enhancer of zeste homolog 2) in comparison to effector cells from LCMV Arm 5 acute infection. Ezh2 is the catalytic subunit of the Polycomb Repressor Complex 2 that trimethyles histone 3 at the lysine residue 27, and has been reported as critical to CD8 T cell differentiation and antitumor response, whereas its decreased levels can lead to loss of effector function. Thus, we pursue to evaluate the role of Ezh2 in CD8 T lymphocyte exhaustion as well as its applicability in heightening CAR T cells immunotherapy efficacy. Methods and Results: Ezh2 deficient CD8 T cells from Ezh2fl/fl Lck-Cre mice activated in vitro exhibited increased expression of inhibitory receptors and the ectoenzyme associated to exhaustion CD38, as well as decreased IFN-γ and TNF production and proliferation marker Ki67 levels, which correlated with low expansion. Consistently, preliminary data on pharmacological inhibition of Ezh2 partially recapitulates this phenotype resulting in increased TIM3 and CD38 expression and decreased IFN-γ and TNF production. Finally, our pilot results demonstrated that forced expression of Ezh2 in CD8 CAR T cells targeting a mice melanoma model could mitigate the exhausted phenotype by increasing proliferation and granzyme B production, along with the reduction in tumor size. Conclusion: Overall, these results demonstrate that deficiency in Ezh2 protein expression leads towards CD8 T lymphocyte exhaustion, while its re-establishment, through Ezh2 overexpression, is able to rescue, at least partially, the effector function of tumor-specific T lymphocytes, suggesting that modulation of Ezh2 levels might be an effective strategy for improvements in CAR T cells immunotherapies.

Correlation of activation markers (CD69, HLA-DR and CD71) on CD4+ and CD8+ T cells with IFN-g release in QuantiFERON-TB-Gold-Plus

Introduction: The World Health Organization launched the End TB (tuberculosis) strategy until 2035. To reach this goal is essential to diagnosis and treat active and also latent TB infection (LTBI). Active TB is usually diagnosed by clinical symptoms. LTBI is characterized by a persistent immune response to Mycobacterium tuberculosis (Mtb) antigens without clinical or microbiological evidence of TB. LTBI is diagnosed by in vivo tuberculin test or interferon-gamma release assays (IGRA). QuantiFERON-TB-Gold-Plus (QTF) is the world widely used IGRA. Due to the recent inconsistent results obtained in all sciences fields, the NIH-USA came up with The Human Immunology Program suggesting laboratories to use the same cell immune assays to produce more reliable and comparable results among different sites. Our laboratory established the same goal. We aimed to use universal methodologies (IGRA) and immunophenotyping panels to search for biomarkers in TB. Herein we standardize a universal protocol to evaluate activation of CD4+ and CD8+ T cell in whole blood collected in QTF from patients with LTBI diagnostic hypothesis and correlated with IFN-g quantification by IGRA. Methodology: Blood samples from LTBI suspected donors (n=38), from Institute Clemente Ferreira out-clinic was collected and incubated. IFN-g on stimulated plasma was quantified by ELISA. The remaining blood in the 4 QTF tubes was stained based in universal procedures with monoclonal antibodies against CD45, CD3, CD4, CD8, CD69, CD71 and HLA-DR. The samples were hemolyzed and evaluated in Cytoflex S. Results: From 38 LTBI suspected samples, only four were considered as possible LTBI infection using QTF. The MFI of activation markers were correlated with IFN-g released by T cells. There were no correlation of CD69 expression in CD4+ and CD8+ T cell with IFN-g release in tubes nill, TB1, TB2 or Mitogen. For CD71 marker, there were a positive correlation of CD71 expression and IFN-g in CD8 T cells stimulated with mitogen (PHA). HLA-DR expression in CD4 T cells were positively correlated with IFN-g production. Conclusion: The biomarkers discovery in TB field is dynamic, all approaches are valid. Our preliminary results showed that only CD71 and HLA-DR is correlated with IFN-g detected in QTF-Plus tubes. More patients diagnosed with LTBI and active TB must be included in this study to validate these findings.

Correlation of activation markers (CD69, HLA-DR and CD71) on CD4+ and CD8+ T cells with IFN-g release in QuantiFERON-TB-Gold-Plus assay used for latent tuberculosis infection diagnosis

Introduction: The World Health Organization launched the End TB (tuberculosis) strategy until 2035. To reach this goal is essential to diagnosis and treat active and also latent TB infection (LTBI). Active TB is usually diagnosed by clinical symptoms. LTBI is characterized by a persistent immune response to Mycobacterium tuberculosis (Mtb) antigens without clinical or microbiological evidence of TB. LTBI is diagnosed by in vivo tuberculin test or interferon-gamma release assays (IGRA). QuantiFERON-TB-Gold-Plus (QTF) is the world widely used IGRA. Due to the recent inconsistent results obtained in all sciences fields, the NIH-USA came up with The Human Immunology Program suggesting laboratories to use the same cell immune assays to produce more reliable and comparable results among different sites. Our laboratory established the same goal. We aimed to use universal methodologies (IGRA) and immunophenotyping panels to search for biomarkers in TB. Herein we standardize a universal protocol to evaluate activation of CD4+ and CD8+ T cell in whole blood collected in QTF from patients with LTBI diagnostic hypothesis and correlated with IFN-g quantification by IGRA. Methodology: Blood samples from LTBI suspected donors (n=38), from Institute Clemente Ferreira out-clinic was collected and incubated. IFN-g on stimulated plasma was quantified by ELISA. The remaining blood in the 4 QTF tubes was stained based in universal procedures with monoclonal antibodies against CD45, CD3, CD4, CD8, CD69, CD71 and HLA-DR. The samples were hemolyzed and evaluated in Cytoflex S. Results: From 38 LTBI suspected samples, only four were considered as possible LTBI infection using QTF. The MFI of activation markers were correlated with IFN-g released by T cells. There were no correlation of CD69 expression in CD4+ and CD8+ T cell with IFN-g release in tubes nill, TB1, TB2 or Mitogen. For CD71 marker, there were a positive correlation of CD71 expression and IFN-g in CD8 T cells stimulated with mitogen (PHA). HLA-DR expression in CD4 T cells were positively correlated with IFN-g production. Conclusion: The biomarkers discovery in TB field is dynamic, all approaches are valid. Our preliminary results showed that only CD71 and HLA-DR is correlated with IFN-g detected in QTF-Plus tubes. More patients diagnosed with LTBI and active TB must be included in this study to validate these findings.

Co-treatment with Amido-Coumarin protects against breathing and neuro damage during severe experimental malaria

The Plasmodium berghei ANKA(PbA) infection in mice closely recapitulates many aspects of severe malaria in humans, including cerebral malaria and acute respiratory distress syndrome. Coumarins are a class of secondary metabolites that are present in plants and exhibit several biochemical and therapeutic effects. We aimed to investigate the potential therapeutic of an amido-coumarin compound in treating severe experimental malaria. C57Bl/6 mice were inoculated with 105red cells parasitized with PbA, and 3 days after infection(dpi) were orally treated daily with amido-coumarin compound MJM220(10mg/kg) alone and/or in combination with chloroquine(CQ-30mg/kg) once per day. Was analyzed, parasitemia, body weight, survival, clinical score, memory, and immune cell profiles in the spleen, brain, and bronchoalveolar lavage (BAL) by flow cytometry; lung conditions was evaluated by spirometry. Our results showed that the treatment with MJM220 alone reduced the parasitemia at 6° and 7dpi, and improved the clinical score from 8° to 12dpi when compared with infected untreated mice. The animals treated with MJM220+CQ showed a reduction in parasitemia in all analyzed kinetics (3°to 47dpi). Treatments with MJM220 and MJM220+CQ increased the survival of infected mice (80% and 60%;28°dpi and 47°dpi, respectively). Treatment with MJM220 resulted in the protection of cognitive ability at 5dpi, and the combined treatment of MJM220+CQ preserved cognition at 5dpi and 47dpi. Treatment with MJM220+CQ resulted: in the brain, a reduction in the numbers of macrophages, dendritic cells, CD4 and CD8T cells IFNγ+, CD4T cells IL17+ and IL10+, and in the spleen, a reduction in the number of neutrophils, CD4T cells IFNγ+ or IL17+, and increased number of CD8T cells IFNγ+. Spirometry results showed that, compared with infected untreated mice, or mice treated with MJM220 or CQ alone, the animals treated with MJM220+CQ improved their lung capacities/functions. Treatment with MJM220+CQ significantly increased the number of alveolar macrophages IL10+, and CD4T cells IL17+ in the BAL. Collectively, our results suggest that the coumarin-based compound MJM220 has potentially beneficial effects on neuroprotection and respiratory capacity during severe experimental malaria.

COUMARIC ACID DERIVATIVES CONTRIBUTE TO THE CONTROL OVER MELANOMA CELL GROWTH

Cancer still represents one of the greatest challenges of modern medicine, and the available therapies involve several side effects and are not always accessible to the general population. In this sense, studying compounds of plant origin for cancer treatment meets the search for efficient and low-cost therapies. As biological effects are often achieved only at high concentrations, chemical modification of these natural compounds appears as an alternative to enhance their therapeutic use. Hence, our work aimed to determine the antitumor potential of two coumarate esters obtained from the esterification of coumaric acid (p-CA), a phenolic acid found in plants and mushrooms. To this end, murine (B16-F10) or human (SK-MEL-25) melanoma cells were treated with p-CA and its derivatives. Cell viability analysis by measuring lactate dehydrogenase in the supernatant and staining viable cells with crystal violet showed that the compounds have cytotoxic action at concentrations above 100 µM. In contrast, p-CA has this effect only at concentrations above 1000 µM. Furthermore, the analysis of proliferation and cell cycle by flow cytometry showed that both compounds have cytostatic activity on both cell lines. In addition, in ex vivo assays, compounds derived from p-CA did not exert cytotoxic activity on spleen cells from C57Bl/6 animals. Also, one of the compounds increased the expression of CD69, a molecule indicative of early cell activation, in T and B lymphocytes and NK cells. Therefore, our data show that compounds chemically derived from p-CA have cytotoxic and cytostatic activity against two different melanoma cell lines without negatively impacting the viability of immune cells. In conclusion, besides the antitumor activity, one of these compounds can contribute to the activation of the immune system, suggesting that these molecules may improve the prognosis of cancer patients, including complementing other conventional therapies.

CROSSTALK BETWEEN MICROBIOTA AND HISTONES MODIFICATION IN THE HEALTHY GUT

Introduction: The gut, especially the colon, is home of an ecosystem of microbes that are important for digestion and for training of the immune system. The microbiota influence gene expression in the intestinal epithelium and this is important for the normal metabolism of epithelial cells and innate immunity. One way by which the microbiota affect gut physiology and gene expression is by generating short-chain fatty acids that, in turn, influence histone post-translational modifications [1]. In this study, we show that global levels of many histone crotonylation marks are responsive to depletion of the microbiota and highlight the core effect associated with the microbiota via comparison of germ-free and antibiotic-treated animals. Methods and Results: Male C57BL/6 at age 8–12 weeks was treated with 200 µl of a mixture of antibiotics cocktail daily for 3 days by gavage. Colon epithelium was extracted using EDTA, followed by enzymatic dispersion of cells[2]. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed as described previously [2]. Anti-Crotonyllysine antibody was used to tag lysine crotonylation. For population mRNA sequencing (mRNA-seq), cDNA libraries for sequencing were generated using standard non-diretional kit for Illumina®. Sequencing was done by Novagen company. Bioinformatic analysis of ChIP-seq and mRNA-seq was done using SeqMonk and R packages. Raw sequenced RNA-seq data from germ-free vs conventional animals were obtained from published data [3]. In animals treated with antibiotics, 248 genes were upregulated and 137 were downregulated (Fold change 1.5). About 44% and 48% of these genes, respectively, overlapped with up- and down-regulated genes in germ-free animals compared to conventionally reared mice. Upregulated and overlapping genes return GO terms of immune response regulation and T cell activation and differentiation. There was a general decrease in the lysine crotonylation of histones from the antibiotic-treated animals, however, the relative proportion of this modification in the promoter regions of genes (<=1kb) increased. We saw a significant overlap of genes that show changes in expression and changes in crotonyation dependent on the microbiota. Conclusion: Histone crotonylation changes reflect microbiota-dependent gene expression of a subset of genes. References 1. Mol Metab. 38:100925, 2020; 2. Nat Commun. 9:105, 2018; 3. Nat Microbiol. 5:610-619, 2020.

Culture medium matters: Mayaro virus-induced macrophage death is dependent on specific nutrients present in RPMI but not in DMEM

INTRODUCTION Mayaro virus (MAYV) is an emerging arbovirus that usually causes fever, arthralgia and skin rash, but might evolve to severe and prolonged. The development of disease chronic phase is related to the extension of viral replication and the maintenance of an inflammatory response in the joints, where the cellular infiltrate consists predominantly of macrophages. Macrophages can play different roles in the immune response, being able to polarize to an anti- or pro-inflammatory profile that will be important in the maintenance of homeostasis or in the coordination of adaptive responses to different stresses, respectively. Macrophage polarization is driven by changes in cellular metabolism, and the role of different metabolites in the profile change is under investigation. In this context, the objective of this work is to understand the effect of MAYV infection in murine macrophages cultured in different culture media, focusing on cell viability and secretion of inflammatory mediators. METHODS AND RESULTS RAW 264.7 cells were cultured in RPMI or DMEM and infected with MAYV (MOI=1). After different times after infection (12, 24 and 48 hours), cell viability was evaluated using Cell Titer Blue, and the conditioned culture medium was harvested for lactate dehydrogenase (LDH) activity, determination of infectious virus production by plaque assays, and cytokines quantification by ELISA. We found that infection promoted a significant loss in viability for the cells grown in RPMI while no death was observed when the cells were grown in DMEM. Interestingly, we did not observe significant differences in the levels of TNF and IL-6, nor in the formation of infectious viral particles in each culture medium. To understand which culture medium component was related to the virus-induced cell death, we supplemented the culture media with each nutrient that was missing in DMEM but present in RPMI and repeated the viability experiments. We found that when DMEM was supplemented with a solution of non-essential amino acids, we observed a similar profile of loss of viability to that of cells cultured in RPMI, which was not seen when other supplementations were performed. CONCLUSIONS The microenvironment in which the macrophage is immersed plays an important role in cell death caused by MAYV infection.

DANGEROUS LIAISONS: CO-HOUSING WITH MIF-/- MICE TRIGGERS EARLY AND SEVERE COLITIS IN IL10-/- MICE

Introduction: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine present in immune and epithelial cells of the gastrointestinal tract. Humans with Inflammatory Bowel Disease display increased MIF plasma concentration, and mice lacking MIF are protected from intestinal inflammation. Conversely, Il10-/- mice are highly susceptible to intestinal insults, developing spontaneous colitis. We wondered which phenotype – i.e., high/low tolerance to disease - would be dominant in a double deficient Mif-/-Il10-/- mouse. During animal breeding, we unexpectedly noticed that Il10-/- mice died few days after Mif-/- mice co-housing. This observation led us to hypothesize that Mif-/- mice shelter a pathogenic microbiota able to trigger disease in Il10-/- mice. Methods and Results: Indeed, Il10-/- mice co-housed with Mif-/-, but not WT mice, develop acute and severe Th1-driven colitis - succumbing within two weeks. Disease was marked by an increase of colitogenic IFNγ+ cells, double positive IFNγ+IL-17+ cells and neutrophils within colonic lamina propria. Exposure to Mif-/- fecal pellets or fecal transplant also triggers lethal disease. Microbial analysis of fecal samples using 16S rRNA gene sequencing revealed that Mif-/- mice have the highest species diversity among the groups. A pronounced difference in microbial composition was found, highlighting that Mif-/- mice display the highest Bacteroidetes/Firmicutes ratio, lack taxa commonly associated with healthy phenotype and harbor increased Prevotellaceae and Allobaculum bacteria. Antibiotic-resistant Klebsiella oxytoca, Proteus mirabilis and Enterococcus spp. were isolated, becoming putative candidates for triggering colitis. Additionally, Mif-/- mice microbiota is enriched in IgA non-coated bacteria. Finally, double deficient Mif-/-Il10-/- mice die prematurely, and escapers develop communicable disease - suggesting that hampering MIF function is not sufficient to prevent IL-10 deficiency mediated disease. Conclusion: The ability to endure dysbiosis whilst sustaining a healthy phenotype underscore Mif-/- mice high tolerance to disease in the mucosa and strongly suggest that MIF is a critical regulator of the gut microbiota. In particular, our data suggest that animals of tolerant genotype shelter a gut microbiota enriched in pathogens/pathobionts, which can trigger disease in susceptible individuals.

Death among hematologic cancer patients presenting COVID-19 is defined by a broad T cell exhaustion and a cytokine-release syndrome profile.

Background: COVID-19 is an infectious disease caused by the coronavirus SARS-CoV-2. The severe form of COVID-19 is characterized by a cytokine-release syndrome combined with an impaired response from innate immune cells to combat the virus directly. During the early years of the pandemic, much knowledge about the immune factors involved in the development of severe COVID-19 in the general population became clearer. However, identification of the immune mechanisms behind severe disease in specific populations, such as oncologic patients, remains uncertain. Methods: Here we evaluated the systemic immune response through the analysis of blood immune factors, anti-SARS-CoV-2 antibodies, and immune cells, within the early days of a positive SARS-CoV-2 diagnostic in patients with hematological malignancies. To do so, we took advantage of high-dimensional flow cytometry and a bead-based immunoassay platform. Results: Patients that presented severe leucopenia, low antibody production against SARS-CoV-2 proteins, and elevated production of innate immune cell recruitment and activation factors went on to die. These patients also displayed intricate correlation networks of factors resembling a cytokine-release syndrome profile. On the other hand, hematologic cancer patients that showed highly coordinated anti-SARS-CoV-2 antibody production, and a central regulatory hub involving T cells, presented only mild COVID-19. Furthermore, cytometry analysis revealed that patients which presented low T cell and high NK cell frequencies went on to develop severe COVID-19 and die. These patients presented many lymphocyte populations with a strong exhaustion profile. On the other hand, patients with T cell subpopulations that presented the marker CD161, and markers associated with B cell activation and differentiation, had only mild disease and recovered well. Further analysis revealed that the exhausted immune cell populations are highly correlated. These same populations correlated negatively with cells with the marker CD161, and with activated and differentiated B cells. Conclusion: Patients that present severe leucopenia respond in an inflammation-based profile, producing many factors associated with cell recruitment and proliferation. The lack of cells also induces a high immune pressure that culminates in a broad exhaustion profile, especially in T cells. Combined, these factors may be paramount for the death outcome seen in patients with hematologic malignancies.

DECREASED MICROBICIDAL ACTIVITY OF MACROPHAGES CO-CULTURED WITH CD8 T LYMPHOCYTES IN Encephalitozoon cuniculi INFECTION

Introduction: Microsporidia are opportunistic pathogens in individuals with immunodeficiencies. The activity of CD8+ T lymphocytes is essential for the elimination of these pathogens. Less resistance to experimental infection with microsporidia Encephalitozoon cuniculi was demonstrated in B-1 (Xid)-deficient mice, despite the increased population of CD8 T lymphocytes. The aim of the present study was to evaluate the effects of B-1 cells on the cytotoxic activity of CD8 T lymphocytes co-cultured with macrophages infected with E. cuniculi. Methods and Results: CD8 T lymphocytes were purified from the spleens of Balb/c and Balb/c Xid (B-1 cells deficient) mice and then co-cultured with RAW 264.7 macrophages, previously infected for two hours with E. cuniculi. After 36h of incubation with infected macrophages plus CD8 T lymphocytes, we evaluated the proliferation, viability and presence of activating molecules (CD62L, CD69, CD107a) in CD8 T lymphocytes and identified the viability and microbicidal activity of macrophages. There was no difference in CD8 T lymphocyte survival. CD8 T lymphocytes obtained from Balb/c mice showed a higher proliferative capacity (low CFSE median fluorescence intensity, MIF) and higher percentage of expression of CD69 activating molecules. In contrast, CD8 T lymphocytes from Xid mice proliferated (high CFSE-MIF) and had lower expression of activating molecules (low expression of CD69 and high expression of CD62L). The data showed an intense proliferation capacity in RK cells of spores obtained from macrophages without the presence of lymphocytes or co-cultured with CD8 T lymphocytes from Xid mice, suggesting that E. cuniculi spores can subvert the microbicidal capacity of macrophages, surviving inside them, and preserving its proliferative competence. Macrophages co-cultured with CD8 T lymphocytes from Balb/c mice showed higher microbicidal activity demonstrated by the low amount of spores recovered. Conclusion: The absence of B-1 cells in Xid mice decreased the expression of activating molecules in CD 8 T lymphocytes and their cytotoxic activity.

DECTIN-1 AND TLR-2 ARE IMPORTANT INNATE IMMUNE RECEPTORS FOR BOTH EXPANSION AND ACTIVITY OF MYELOID-DERIVED SUPPRESSOR CELLS IN PULMONARY PARACOCCIDIOIDES BRASILIENSIS INFECTION

Introduction: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cell populations that impair immune responses in cancer, infectious diseases, and autoimmune diseases. The innate immune receptor of MDSCs, Dectin-1, is important in pathogenic fungal infections. Also, Toll-like receptor 2 (TLR-2) participates in the activity of MDSCs in different infectious contexts. However, the role of these innate immune receptors of MDSCs is still unknown in paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. Methods and Results: We evaluated the production of indoleamine 2,3-dioxygenase 1 (IDO-1), IL-10, PD-L1, and nitric oxide (NO) by MDSCs after anti-Dectin-1 and anti-TLR-2 antibodies treatments followed by Paracoccidioides brasiliensis-yeast challenge. Coculture of MDSCs with activated lymphocytes allowed us to use T cell proliferation and Th profiles as targets to verify the effect of Dectin-1 and TLR-2 expression in MDSCs activity. We also investigated the presence and immunosuppressive mechanisms of MDSCs using C57Bl/6WT, Dectin-1KO, and TLR2KO mice after infection with P. brasiliensis-yeasts. We observed that the anti-Dectin-1 and anti-TLR-2 treatments decreased the production of IDO-1, IL-10, and NO by MDSCs. Anti-TLR-2, but not anti-Dectin-1, blocked MDSCs resulted in elevated T cell proliferation in coculture assays. The impairment of immunosuppressive molecules due Dectin-1 absence was confirmed in vitro using MDSCs derived from WT and Dectin-1 KO mice. In vivo, a reduced number of M-MDSCs was observed in the lungs of Dectin-1KO animals after 72h of infection. Furthermore, MDSCs from Dectin-1KO mice presented reduced expression of IDO-1 and NO after 72 h, 2, and 8 weeks of infection. Decreased levels of IL-10 were observed 2 weeks post-infection in Dectin-1-deficient animals. In addition, coculture assays revealed that Dectin-1 absence on MDSCs resulted in higher T cell proliferation along with partial restoration of Th2 and Th17 lymphocyte profiles, which were impaired in coculture with Dectin-1-sufficient MDSCs. Preliminary data regarding TLR-2 absence revealed a higher number of MDSCs in the lungs of TLR-2 KO mice 72 h post-infection compared to the WT controls. However, after 2 and 8 weeks, a diminished number of MDSCs were recruited to the lungs of TLR-2-deficient animals. Conclusion: Our results indicate that MDSCs act through Dectin-1 and TLR-2 in the immune response during infection by P. brasiliensis in mice.

DEFICIENCY OF ANNEXIN A1 INCREASED SUSCEPTIBILITY TO EXPERIMENTAL SEVERE MALARIA

Introduction: Plasmodium falciparum infection leads to malaria in humans, which has a high morbidity and mortality. P. berghei ANKA (PbA) infection in mice is very similar to human malaria in many aspects, such as respiratory distress, severe anemia, liver dysfunction, and hypoglycemia. Annexin A1 (AnxA1) is a protein involved in the modulation of inflammatory processes regulating phagocytosis, cell signaling, apoptosis, and leukocyte migration. AnxA1 plays an important role in the modulation and resolution of sterile and infectious diseases, but its function during Plasmodium sp infection is unclear. Herein, we investigated the involvement of AnxA1 in the development of pathogenesis in an experimental model of severe malaria. Methods and Results: BALB/c (WT) and ANXA1 knockout (KO) mice were infected with 105 PbA-parasitized red cell. Parasitemia, weight loss and clinical score were evaluated daily, starting on the 3rd day post-infection (dpi). Vascular permeability was also evaluated, at 12 dpi. The absence of ANXA1 increased the susceptibility of PbA-infected mice, decreasing the survival, with deaths starting at 8 dpi (75 % of the infected mice), when compared with WT mice that starting to die only at 15 dpi. In addition, infected ANXA1 KO mice showed an early (5 dpi) and worst clinical scores at 5, 6, 7, 8, 9, 10, 11, 12 and 13 dpi, despite, surviving mice to present reduced parasitemia from 12 to 15 dpi when compared to infected WT mice. Moreover, annexin A1 seems to play an important role in modulating weight loss and vascular permeability in the liver, since in its absence, these parameters was more severe/worst. Conclusion: Collectively, the results suggest that ANXA1 plays an important role in the early phase protection of Balb/c mice during PbA infection and may be important for the development of an effective treatment against severe malaria. Financial support: FAPEMIG, CNPq, CAPES. CEUA (ethics committee on the use of animals): 97/2018.

Deficiency of formyl 2 peptide receptor: effects in the modulation of immune response and development of severe malaria caused by Plasmodium berghei anka infection

Severe malaria caused by Plasmodium falciparum in humans can results in high morbidity and mortality. The Plasmodium berghei ANKA (PbA) infection in mice closely recapitulates many aspects of human severe malaria, including respiratory distress and cerebral malaria. Formyl peptide receptor 2 (FPR2) is involved in the organism response to infections and plays na important role in the anti-inflammatory/resolution pathway presenting high affinity for lipoxin A4 (LXA4). FPR2 has been associated with the pathogenesis of sterile and infectious inflammatory diseases. However, the role of FPR2 in malaria is unclear. C57BL/6 (WT) and FPR2 knockout (KO) mice were inoculated with PbA and were treated or not with chloroquine (30mg/kg) once per day. Parasitemia, body weight, survival, brain histology/immunofluorescence and fluxo cytometry analysis was evaluated. Despite similar parasitemia found between WT and FPR2 KO mice, deficiency of FPR2: increased the numbers of macrophages producing IL-10; but decreased the numbers of CD4+ T cell producing IL-10; and decreased the numbers of CD8+ T cells producing IFN-gamma in the bronchoalveolar lavage (BAL) at 5 days after infection when compared with WT counterparts. In the brain, absence of FPR2 increased the frequency of PbA-infected erythrocytes detected/adhered in the vein when compared with WT. Moreover, at 5 dpi, a dramatically decreased of the number of CD4+ and CD8+ T cells producing IL-17 and CD8+ T cells producing IFN-gamma was observed in PbA-infected FPR2 KO mice when compared with WT counterparts. Collectively, our results suggested that during the PbA infection the FPR2 receptor is important controlling brain parasitism and orchestrating the immune response development.

DEVELOPMENT AND PROTOTYPING OF A RAPID TEST FOR THE DIAGNOSIS OF HEPATITIS B AND D

Hepatitis D is an infectious disease that affects 15 to 20 million people worldwide. The hepatitis D virus (HDV) is only able to infect people in the presence of the hepatitis B virus (HBV). HDV is responsible for the most severe form of viral hepatitis due to its rapid progression to cirrhosis and liver decompensation, what increases the need for an early diagnosis. However, there are scant diagnostic tools to detect HDV infections. The rapid test (RT) through lateral flow immunocromatography stands out as a quick and easy platform to assist in the diagnosis of both forms of hepatitis. Here we report on the development of a multiplex RT, capable of simultaneously detecting both HBV and HDV infection. Initially, 2 prototypes were developed, one for the diagnosis of HBV through the detection of HBsAg, and the other for the diagnosis of HDV by the detection of anti-HDV IgG, followed by the development of the multiplex test, capable of detecting both markers at the same time. The RT for HBsAg uses a pair of commercial monoclonal antibodies (mAb), one immobilized on the nitrocellulose membrane as the test line and the other conjugated to colloidal gold nanoparticles. The prototype developed had sensitivity and specificity of 100% (n = 29 samples). For the anti-HDV IgG RT, the recombinant delta antigen of HDV (rHDAg) was expressed in E. coli system and purified by affinity chromatography. The antigen was conjugated to colloidal gold nanoparticles and the test line was composed by anti-human IgG. The sensitivity of the test was 91.7% and the specificity was 100% (n = 29 samples). The multiplex RT has two test lines, one with a mAb immobilized (detection of HBsAg) and other with an anti-human IgG antibody (for anti-HDV IgG), and a mixture of mAb anti-HBsAg and the rHDAg conjugated to colloidal gold as conjugate. The multiplex test showed a sensitivity of 82,86% for HBsAg and 100% for anti-HDV IgG and specificity of 100% for both markers (n = 48 samples). The RT prototypes developed for HBsAg and anti-HDV IgG separately as well as the multiplex test showed high sensitivity and specificity, with potential application in the serological diagnosis of both diseases.

DEVELOPMENT OF THIRD GERENERATION ANTI-BCMA CAR-T CELLS VECTORS FOR MULTIPLE MYELOMA TREATMENT IN A LOW-MIDDLE INCOME COUNTRY

Multiple myeloma is a common hematological neoplasm whose treatment has been improved in the last years with the introduction of new drugs. However, relapsed, and refractory patients still experiment a dismal outcome. An additional challenge to low-middle income countries is closed related to the economic toxicity related to the high cost of the new treatment, mainly immunotherapy and cellular therapy. In this scenario, the B-cell maturation antigen (BCMA/TNFRSF17) based treatments, such as CAR T-cells, have been emerged as very promise treatment. In order to create local technology that might impact the access and cost of CAR T-cell therapy, this project aimed to develop a manufacture platform in Brazil to generate genetically modified T-cells to express anti-BCMA CAR. For this purpose, two new BCMA clones were selected based on a biopanel methodology. Selected antibodies of high specificity against BCMA gave rise to the scFv fragments that were cloned into a lentiviral vector with a co-stimulatory molecule (41BB) and a security and labeling gene (EGFRt), for expression in T cells. Lentiviral particles were produced in HEK293T cells and titer defined by flow cytometry by Jurkat cell line transduction and, the plasmids generated, produced satisfactory amounts of lentiviral particles. The next steps will be to transduce T-cells and evaluate the cytotoxic effect against multiple myeloma cell lines (U266 and RPMI8226). In addition, we used the CRISPR/Cas9 technique to knock out BCMA in the cell lines used. Then, it is expected that such a project will support the use of new vectors in the manufacture of anti-BCMA CAR T cells in accordance with standard of good manufacturing practices (GMP) and compatible for use in a phase I clinical trial in Brazilian patients.

Dietary fibers improve the recovery of injured intestinal epithelium

Introduction: Inflammatory Bowel Diseases (IBD), which include Crohn's disease and ulcerative colitis, are characterized by severe intestinal inflammation with intense damage in the intestinal epithelium, that is repaired through activation of intestinal stem cells (ISCs) and +4 progenitors. Diet plays a critical role in driving host inflammatory responses, mainly through changes in the composition and metabolic products of the intestinal microbiota, such as short-chain fatty acids (SCFAs), which constitute important mechanisms in the communication between the host-microbiota. In this study, we investigated the effects of SCFAs and fiber diet on intestinal regeneration after DSS-induced colitis. Methods and results: Firstly, C57BL/6/J mice were treated or not with 2.5% DSS in drinking water for five days. After this period, the animals in the DSS group were subdivided into four other groups: 1) NaCl, 2) butyrate, 3) acetate and 4) crotonate. Treatments were performed for sixteen days after DSS removal. We found no significant differences between treatments in the context of intestinal epithelial regeneration. In addition, we treated animals with a control diet and a high-fiber diet at different times (3, 7 and 16 days) after DSS removal to evaluate the effects of this diet on the recovery of the intestinal epithelium. We found that the high-fiber diet did not interfere with the clinical aspects of the disease. However, we observed that the treatment after seven and sixteen days increased the size of the colon, a beneficial effect that was associated with an improved recovery of the intestinal crypts and goblet cells numbers. Despite that, we found no reduction in the histopathological score suggesting that diet does not interfere with tissue damage. We found an increase in the number of proliferative cells in the colon of fiber-diet mice and in the expression of Hopx, an intestinal stem cell marker, on the seventh day compared with control-fed animals. Conclusion: The high-fiber diet did not clinically interfere with the disease but improved colonic recovery after injury, an effect that may be due to an improvement of intestinal stem cells numbers or function.

DIFFERENCES IN THE NEUTROPHIL RESPONSE IN DISCORDANT COUPLES FOR SARS-COV-2 INFECTION

DIFFERENTIAL GENE EXPRESSION OF RECEPTORS OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM FROM PATIENTS WITH SEVERE COVID-19 D

Introduction SARS-CoV-2 is the etiological agent of COVID-19, characterized by the Spike protein, which is critical for viral binding to the angiotensin-converting enzyme-2 (ACE2), a fundamental cellular receptor in the renin-angiotensin-aldosterone system (RAAS) that acts as a homeostatic regulator of vascular function. Studies demonstrate that SARS-CoV-2 can impact ACE/ACE2 activation of the RAAS, leading to different types of progression in COVID-19. Based on this, we evaluated the gene expression of the main receptors of the RAAS system (ACE, ACE2, MAS1, AGTR1, AGTR2, and TMPRSS2) in peripheral blood mononuclear cells (PBMC) and protein quantification (ACE2) in plasma from individuals hospitalized with COVID-19 severe at the Evandro Chagas National Institute of Infectious Diseases-INI-Fiocruz/RJ. Methods and Results Were enrolled 47 individuals not reporting the use of ACE inhibitors or angiotensin receptor blockers, and with blood collections between June 2020 and December 2021. Samples from baseline (Day 0) and from day 300 after the first collection were evaluated. RNA extractions, cDNA synthesis and qPCR were performed. The quantification of soluble ACE2 was performed using the ELISA assay. The expression differences were analyzed by Volcano plot analysis with fold change Boundary=2, T-test Statistical (P-Value=0.05). The quantification of soluble ACE2 was performed using the ELISA assay. Out of 47 patients, 42.6% were female, mean age was 55.8 years and the mean time of hospitalization was 18.1 days. 78.7% of individuals required oxygen supplementation or ventilatory support and only 7% were tobacco smokers. The frequency of comorbidities such as systemic arterial hypertension, chronic obstructive pulmonary disease, and diabetes mellitus were 42.6%, 8.5%, and 38.3%, respectively. The differential expression by fold-change for each RAAS receptor were ACE, 2.95 (P=0.036) and MAS1, 7.06 (P=0.002) up-regulated; ACE2, 0.41 (P=0.038) down-regulated; TMPRSS2, 0.38 (P=0.620), AGTR1 and AGTR2 had low expression level in D0 and D300. When analyzing soluble ACE2 protein detection mean, 1.74 pg/mL and 234 pg/mL were observed at D0 and D300 respectively. Conclusion Our data shows repression of the ACE2 gene in the acute phase of COVID-19 which can be contributing to the disease's inflammatory effect. Further investigations are necessary to understand the disease’s biology and assist in the development of effective treatments for the SARS-CoV-2 infection.

DIFFERENTIAL HISTONE MODIFICATIONS IN IMMUNE RESPONSE-RELATED GENES PROMOTERS IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF SEPTIC PATIENTS WITH DIFFERENT CLINICAL OUTCOMES

Introduction: Sepsis is one of the world's leading causes of morbidity and mortality. Its pathophysiology is complex, and its mechanisms are still not fully understood. Peripheral blood mononuclear cells (PBMCs) exhibit functional plasticity during sepsis, moving from an inflammatory to an immunosuppressive phenotype. The mechanisms that modulate gene expression that govern this inappropriate response during sepsis are still unclear; however, epigenetic mechanisms has been shown to play a crucial role in this setting. Methods: PBMC samples were obtained from five surviving septic patients, five septic patients who died, and seven healthy volunteers, matched for sex and age. Chromatin immunoprecipitation was performed for each epigenetic mark of interest (H3K4me3, H3K9ac, and H3K27me3), followed by enrichment analysis in the target gene promoters (TNF-α, IL-12, IL-10, CAMP, FPR1, IFN-β, HLA-DR). Results: Activation (H3K9ac, H3K4me3) and gene repression (H3K27me3) marks present different behaviors for some genes among to the groups. Regarding the inflammatory cytokines, IL-12 and TNF-α, the surviving septic patients show a decrease in activation marks compared to controls. Between the two septic groups, those who died presented increased epigenetic enrichment of H3K9ac in the anti-inflammatory interleukin IL-10 promotor, as well as in the antimicrobial FPR1 and CAMP. Compared to the healthy group, the H3K4me3 marker was decreased in the FPR1 promoter in surviving septic patients and increased in the IFN-β promoter in non-surviving patients. Interestingly, H3K27me3 also showed an increase in IL-10 and HLA-DR promoter enrichment in septic patients who died compared to septic patients who survived, with a tendency to decrease IL-10 in the survivor group compared to the healthy group. Conclusion: There is a change in epigenetic enrichment in genes that participate in the inflammatory response between septic patients and healthy individuals, as well as between surviving and non-surviving septic patients, with apparent differentiation according to the genes functions, inflammatory, anti-inflammatory, and antimicrobial activities.

DIFFERENTIAL IMPACT OF B CELL DEFICIENCY ON THE PERIPHERAL HOMEOSTASIS OF CD4+FOXP3+ REGULATORY T CELLS IN MESENTERIC LYMPH NODES AND EXTRA-INTESTINAL SECONDARY LYMPHOID ORGANS

Introduction: The homeostatic balance between regulatory (TREG, Foxp3+) and conventional (TCONV, Foxp3-) CD4+ T cell numbers in secondary lymphoid organs is indispensable for the establishment of immune homeostasis and peripheral tolerance. The factors influencing the extrathymic maintenance of stable TREG/TCONV ratios are, however, still poorly understood. At the steady-state, reduced levels of TREG cells in extraintestinal lymphoid sites are found in mice devoid of B lymphocytes; however, in gut-associated mesenteric lymph nodes the results are contradictory. Furthermore, the mechanisms whereby B lymphocytes control peripheral TREG cell homeostasis remain largely unknown. The goal of this work was to determine whether the population dynamics of TREG and TCONV cells in peripheral lymphoid organs associated or not with the intestinal mucosa is differentially affected by B lymphocytes at the steady-state. Methods and Results: The levels of TREG and TCONV cells in the spleen, extraintestinal lymph nodes (eLN) and mesenteric lymph nodes (mLN) were compared, by flow cytometry, between mice deficient for B lymphocytes (µMT) and lymphoreplete wild-type (WT) controls under homeostatic condition. Reduced frequencies of TREG cells were seen in all analyzed lymphoid organs of µMT mice relative to WT controls. The absolute numbers of TREG cells were significantly decreased in the spleen, although remained unchanged in the lymph nodes of µMT mice. Accordingly, an increased frequency of Annexin-V+ apoptotic TREG cells were found in the spleen, but not in the lymph nodes, of B cell-deficient animals. Notably, the percentage of mitotic (Ki67+) CD25+ TREG cells was increased in the mLN, but not in the spleen or eLN of µMT mice. The proliferation and survival of CD25+ TCONV cells were also selectively increased in the mLN of animals devoid of mature B lymphocytes. Consistent with this observation, adoptive transfer of B lymphocytes to μMT hosts precociously restrained the proliferation of the CD25+ TCONV population in the mLN, but not in the spleen. Conclusion: Our findings suggest that physiological levels of TREG cells in the mLN may be indirectly regulated by the B lymphocyte through its negative effect on the survival/proliferation of IL-2-producing CD25+ TCONV cell pool. Collectively, the data here shown reveal that B lymphocytes distinctly impact the population dynamics of TREG and TCONV cells in secondary lymphoid organs associated or not with the intestinal mucosa.

DIFFERENTIAL REGULATION OF IGE AND IGG1-POSITIVE B CELLS IN MEDIASTINAL LYMPH NODE AND SPLEEN BY CPG-OLYGONUCLEOTIDE ADSORBED TO ALUM ADJUVANT

Follicular B (FOB) cells contribute to humoral response by generating both primary antibody forming plasma cells and germinal center B cells. Different adjuvants drive activation, proliferation, and class switching of B cells. Toll-like receptors (TLRs) agonists that signal through MyD88 pathway are viewed as Th1 adjuvants and induce IgG2a/c antibody, a Th1-associated isotype while alum, that is considered a Th2 adjuvant, favor IgE and IgG1 production. Our group using the respiratory model of allergy showed that TLR9 agonist, CpG-oligodeoxynucleotides (CpG) when adsorbed to Alum adjuvant, inhibited IgE production signaling via MyD88 expressed on dendritic cells (DCs). Here we investigated directly by FACS analysis the regulation by CpG of IgE+ and IgG1+ B cells present in draining mediastinal lymph nodes (MedLD) and systemically in spleen. For this we used the OVA model of respiratory allergy. We first sensitized WT mice via subcutaneous route with 4ug OVA with or without CpG adsorbed to alum on days 0 and 7 followed by intranasal OVA challenge with 10ug OVA delivered into the nostrils on days 14 and 21. We analyzed the FOB (CD220+CD38+), positive for IgE or IgG1, on days D14, D22 and D28 in MedLN and spleen. We found that CpG inhibited FOB IgE+ cells in MedLN and spleen. In contrast, CpG increased FOB IgG1+ cells in MedLN while in spleen IgG+ B cells were decreased when compared with alum group. To further investigate the mechanism of this regulation, we used mice made MyD88-deficient in DCs by CD11c-CRE MyD88-flox (DC-MyD88-/-) or their counterpart (DCMyD88+/+) mice that express MyD88 on DCs. We found that CpG inhibited completely FOB IgE+ cell in MedLN and Spleen in DCMyD88+/+ while in DCMyD88-/- mice this inhibition was partial when compared to Alum group. These results confirm that MyD88 expression on DCs is important for the full inhibition of IgE. Regarding FOB IgG1+ cells CpG increased IgG1-producing cells in MedLN of DCMyD88+/+ but not of DCMyD88-/- mice. In spleen, CpG decreased significantly FOB IgG1+ cells DCMyD88+/+ but not of DCMyD88-/- mice. These results confirm that CpG augments IgG1+ cells only in draining MedLN, but not in spleen and that the increase of IgG1+ cells is dependent on MyD88 expression on DCs. Altogether, CpG inhibits the development of IgE+ cells and increases IgG1+ cells in draining LN but decreases in spleen suggesting that IgG1+ cells are necessarily associated with Th2 immunity.

DIFFERENT THERAPEUTIC APPROACHES TO INDUCE SUBSTANTIAL CELL DEATH IN GLIOBLASTOMA CELL LINE

Conventional dendritic cells (DCs) play a critical role in anti-tumor T cell responses. Anti-cancer therapies promote different types of cell death, such as apoptosis, which can impact the DCs activation dramatically in the tumor microenvironment (TME). The clearance of apoptotic cells promotes anti-inflammatory mediators production in the TME which favors an immunosuppressive microenvironment capable of inhibiting the antitumor response. Recent studies have shown that different metabolic pathways are activated in macrophages during efferocytosis of ACs that support the tolerogenic profile acquired by these cells. In this context, the metabolic reprogramming of immune cells becomes a new alternative for the treatment of tumors to generate an immunogenic response. Therefore, we hypothesized that efferocytosis of glioblastoma apoptotic cells activates intracellular metabolic pathways that promote a tolerogenic DC phenotype that contributes to tumor immunosuppression. To evaluate the metabolic changes that occur within the DCs during the efferocytosis of apoptotic-cancer cells, initially, we tested different approaches to induce dying tumor cells. A glioblastoma cell line ( U87MG) was treated with UV-C radiation (1-10mJ) for 18h in the presence or absence of the gold standard chemotherapy for glioblastoma, temozolomide (TMZ), at different concentrations (100–500µM) for 24h. After treatment, cells were labeled with an anti-caspase-3 antibody and the rate of apoptosis was determined by flow cytometry. So far, preliminary results have shown that 3mJ of UV-C radiation, in the absence of TMZ, was able to induce 55% of apoptosis in U87MG cells. Surprisingly, the treatment with TMZ alone in all concentrations evaluated had no effect on cell death. Moreover, the combination of UV-C and TMZ was able to induce 65% of late apoptosis, with evolution to necrosis, when the cells were submitted to the combined treatment. Since TMZ in combination with radiotherapy is still the standard gold treatment of glioblastoma, new strategies will be tested, such as adjustment of treatment time and drug concentration, to obtain a better apoptosis rate under the effect of chemotherapy.

DIGITAL MAGAZINE IMUNOQ?: PUBLISHING THE IMMUNOLOGICAL SYSTEM ON SOCIAL MEDIA

The work presents and discusses scientific disclosure material on the immune system in comic book format. It began with the COVID-19 pandemic, motivated by the wave of fake news about viruses, the immune system, vaccines and their function. The digital magazine ImunoQ? is born, a comic book aimed at teenagers with the proposal to demonstrate situations experienced by the body in everyday life, its interactions, cells and mechanisms in action. Thus, we chose to focus on complex concepts, not so understandable to a particular audience. The creation process was outlined in a scientific way, but artistic and semiotic references were used to complement and facilitate the understanding of the content created, in order to maintain didactics and scientific information. The main characters are the cells of the immune system, which were characterized differently from their original shapes, with simpler shapes and different colors. However, accessories and personifications were added to represent their biological functions. The main immunological organs were also stylized, with their functions preserved. In addition, the work aimed to demonstrate basic knowledge about cells and other systems that interconnect the immune system, such as lymphatic and cardiovascular, therefore, background details are explored, and parallel stories developed, so that it represents the dynamics of the organism. Once concluded the first chapter, introduction to the immune system, the disclosure of the material on social media was possible and, thus, there was a return about the understanding of the target audience, and, consequently, the perception of gaps in the material. In this way, new developments of the work emerged. Adaptations and further research of the public were primordial, in order to adapt the material to their knowledge, in which allowed a greater involvement with social media, such as Instagram. During the production of the comic, there were obstacles, the main one was the adaptation of the complex content in an understandable and visually interesting language. Due to this, there were several graphic and script adjustments. Therefore, the use of social networks enabled a greater polishing of the content, in addition to expanding the project. The work is still in progress, but, with the results obtained, it is possible to evaluate that the proposal of ImunoQ? has potential as a vehicle for disclosing immunological content to students, as well as to different audiences.

DISTINCT PLASMA METABOLIC PROFILE IN HIV-1 ELITE CONTROLLERS

Introduction: Elite Controllers (EC) are a group of individuals who are able to control the viremia of HIV infection, maintaining normal CD4+ T cell counts. Identification of metabolomic profile could help to comprehend important factors related to infectious diseases, such as course of infection and therapeutic interventions. Methodology and Results: In order to investigate a differential metabolic profile of ECs compared to other people living with HIV (PLHIV) and healthy controls, we performed untargeted metabolomics of plasma from 62 PLHIV, classified as Viremic (VR), Successful Treated (ST), Long Term Non-Progressor (LTNP) and EC. Twenty individuals were recruited and analyzed as controls (Healthy Donors, HD). Blood samples of all patients were collected, and plasma were used to perform the untargeted metabolomics using a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Bioinformatic tools were used to generate results and statistical analyses. A distinct abundance of metabolites, the majority were observed to be exclusive for each group. Despite all groups presenting a unique profile, in ECs plasma the number of differentially abundant metabolites were increased, most of them being downregulated, compared to VR and HD. Metabolic pathways were differentially expressed in this group, including lipid and amino-acids pathways. From all different expressed metabolites in EC, stands out Paraxanthine, Glutamate and Sphinganine, the first being positively correlated to CD4+ T cell counts. Glutamate was found in lower levels in ECs when compared to other groups. Sphinganine were observed in higher levels in HD and lower levels in VR group. Similar profiles, associated with activation, metabolism and biosynthesis of fatty acids were also detected in VR and LTNP groups, compared with HD. Conclusion: Our results indicate significant alterations in the metabolism of PLHIV and EC, when compared to other individuals, highlighting important pathways that may have a major role in HIV course of infection. These findings suggest an important correlation between metabolic profile and the mechanisms of spontaneous control of HIV infection, which should be further investigated and validated with cell culture experiments.

Distinguished role of MerTk and Axl receptors-mediated efferocytosis in alveolar macrophages on pulmonary mucosal regulation

Efferocytosis is paramount to regulating homeostasis and inflammation, particularly in an environment with high cell turnover and colonized by microbiota, such as the lungs. The TAM receptor family (Tyro3, Axl and MerTk) mediates efferocytosis and inhibits pro-inflammatory pathways through Gas6 or Protein S binding to phosphatidylserine on apoptotic cells. Here we investigated how MerTk and Axl regulate pulmonary mucosa homeostasis and inflammation, using silicosis as a study model. During homeostasis, lung cells and alveolar macrophages (AMs) from wild-type (WT) mice showed high expression of Axl, MerTk, and Gas6. Although we have found increased numbers of total airway cells, lower levels of TGF- and IL-10, and higher levels of nitric oxide in the BALFs from both Axl-/- and MerTk-/- compared to WT mice, enhanced numbers of lung total cells, higher numbers of AMs, monocytes, and neutrophils were found in BALFs and lungs of MerTk-/- compared to Axl-/- or WT mice. We also found increased expression of CXCL1, CXCL2, TNF-, and IL-6, which are associated with inflammatory cell infiltration, and enhanced numbers of MHCII+ AMs in the lungs of MerTk-/- compared to Axl-/- or WT mice. Furthermore, AMs from Axl-/- mice upregulated the expression of MerTk receptor, suggesting that this receptor plays an important role in controlling pulmonary mucosa homeostasis. In contrast, during silicosis, we found higher mortality, impaired lung function by increased airway resistance, increased levels of TGF- and protein permeability, as well as granuloma formation in silicotic (SIL)-Axl mice compared to the SIL-MerTk or SIL-WT groups. BALFs from SIL-Axl had an increased number of total airway cells, mostly represented by AMs and neutrophils, and higher levels of CXCL1 compared to the SIL-MerTk or SIL-WT groups. In addition, SIL-Axl showed a reduced number of CD206+ AMs and an enhanced number of apoptotic, late apoptotic, and necrotic AMs compared to SIL-MerTk or SIL-WT groups. Finally, we found that both SIL-WT and SIL-MerTk upregulated mRNA levels for Axl post silica exposure compared to its PBS groups, suggesting a critical role of Axl receptor in limiting pulmonary inflammation. Collectively, our data suggest that MerTk and Axl receptor-mediated efferocytosis are dedicated to regulating homeostasis/tolerance and controlling inflammation in the lungs, respectively, pointing out TAM receptors as targets to design new therapeutic strategies for inflammatory lung diseases.

DNA and histones are involved in the interaction of hRSV with NETs in a nonspecific manner

Respiratory Syncytial Virus (hRSV) has been the main etiological agent of acute lower respiratory tract viral infections. After a drastic drop in cases in the last two years, due to the pandemic by Sars-Cov2, the incidence of hRSV rose again this year, in the absence of a specific treatment or an authorized vaccine. Patients infected with hRSV have great difficulty breathing because the accumulation of mucus into lung. Research has shown that much of this mucus consists of NETs released from stimulated-hRSV neutrophils which migrated from blood to lung tissue. Serine proteases from NETs are partially responsible for the virucidal effect of the NETs because they damage the hRSV viral particles. In parallel, viral particles are found co-located with the NETs in the lung tissue, but the molecules involved in this interaction is not clear yet. The beneficial or deleterious role of NETs, in a context of hRSV-induced respiratory syndrome, reinforces the need to broaden our knowledge about the possible interactions between NETs and hRSV. Considering the hypothesis that complex NETs/hRSV occur by electrostatic forces between the negative charges from DNA chains and the positive charges from viral surface proteins (F and G), our aim was to evaluate whether this hypothesis would be confirmed or not. Thus, hRSV or mock (supernatant recovered from uninfected Hep-2 cells) were incubated with neutrophil-isolated DNA or NETs and submitted to electrophoresis assays were performed on agarose gel (0.8%). Staining gels using GelRed presented the following results: (i) both hRSV and Mock form complexes with neutrophils-isolated DNA, followed by degradation; (ii) incubation of NETs with hRSV generates complexes in the absence of degradation, (iii) while the presence of Mock, the DNA chains from NETs are degraded. Taken together, these data suggest that hRSV interacts with DNA strands, or their histones, in a unspecific manner because the same results were obtained in the presence of mock. In addition, the fact that the electrophoretic profile presented by NETs/hRSV and NETs/mock complexes are different, suggests that the interaction NETs/hRSV is specific and non-dependent of nitrogenous bases from DNA or the histones found in it. In conclusion, the initial hypothesis has not been confirmed but opens new perspectives around the identity of hRSV binding molecules in the structure of NETs.

DP2 receptor activation by endogenous PGD2 attenuates Schistosoma mansoni infection-induced fibrogenic response of hepatic granuloma

Introduction: During the protective immune response to S. mansoni infection, long-lasting egg-encasing hepatic granulomas display severe fibrosis which contributes to schistosomiasis-related morbidity/mortality. Thus, identification of molecular candidate capable of controlling this fibrotic process is of potential therapeutic relevance. One such molecule is the immunomodulatory lipid prostaglandin D2 (PGD2) which has been proposed as a potential inducer of pro-fibrotic TGFβ production in vitro by eosinophils and S. mansoni infection-driven hepatic stellate cells. Our aim was to evaluate the potential role of PGD2 and its cognate receptor, DP2, in fibrosis of S. mansoni-induced eosinophilic hepatic granulomas. Methods and Results: PGD2 role in S. mansoni infection was analyzed 55 days after infection by both (i) impairment of PGD2 synthesis by HQL-79 (2.65 µg/day), an inhibitor of the H-PGD synthase; and (ii) blockage of DP2 receptor activation by CAY10471 (1.75 µg/day), a selective antagonist. Both treatments were continuously administered through subcutaneous-implanted osmotic pumps, starting24 days after infection. Of note, HQL-79 did impair S. mansoni infection-induced PGD2 synthesis detected in both peritoneal wash and liver tissue, therefore validating the systemic reach of the treatment maneuver. Surprisingly, HQL-79 and CAY10471 amplified the fibrotic response within schistosome hepatic granulomas, increasing hepatic deposition of collagen fibers. The already enhanced hepatic levels of the pro-fibrotic cytokines TGFβ and IL-13 in infected animals were further increased by HQL-79 and CAY10471. Meanwhile, LTC4 synthesis, which was shown to have an anti-fibrotic role in schistosomiasis, inversely correlated with collagen, TGFβ and IL-13 levels. As PGD2-activated eosinophils represent a key cell source of LTC4, it is of note that: (i) HQL-79 and CAY10471 impaired systemic eosinophilia, drastically decreasing eosinophils within peritoneum and hepatic granulomas; (ii) S. mansoni-induced peritoneal eosinophils are a cell population responsible for LTC4 synthesis in vivo; and (iii) eosinophils isolated from S. mansoni-induced hepatic granuloma synthesize LTC4 in vitro. Conclusion: While PGD2 acting on DP2 receptors does contribute to S. mansoni-driven type 2 eosinophilic response, it simultaneously attenuates fibrosis of hepatic granulomas. Hence, therapeutic strategies targeting PGD2/DP2 axis during schistosomal infection should be addressed with caution

DRP1 IS NECESSARY FOR MACROPHAGE METABOLIC ADAPTATION AND PHENOTYPE DURING COLD

During acute (6h) or chronic (3 days) cold exposure (CE), macrophages (macs) are critical cells regulating adaptative thermogenesis (AdT), which are mediated by specialized fat cells (brown/beige adipocytes) via Uncoupling Protein 1 (UCP1) to produce heat. Depending on the milieu, adipose tissue (AT) macs can assume different phenotypes known as inflammatory (M1) or resolution (M2). Mitochondria dynamics is regulated by fragmentation/fission and elongation/fusion events, which are regulated by Drp1 or Mitofusin 1 and 2, respectively, and can affect macs phenotype. We hypothesized that modulation of mitochondrial dynamics will impact the metabolic adaptation of macrophages, affecting thermogenesis. Macs from mice with specific deletion of Drp1 in myeloid cells (Drp1Δmacs) have reduced mitochondria fission. After chronic cold exposure, we observed that Drp1Δmacs mice have no alteration neither in body weight nor in subcutaneous AT (scAT) and brown AT (BAT) mass compared to flox control mice (WT), but display reduced expression of UCP1 and frequency of M2 macs in BAT and scAT of WT. Next, 9B cells (a brown-like adipocyte lineage) were differentiated and treated with CL-316243, a beta-3 adrenergic agonist that mimics CE. Macs derived from WT and Drp1Δmacs mice were culture with CL-316243-treated 9B cell conditioned media (CM). WT macs displayed decreased protein levels of mitochondrial complexes I, III, IV and V, but increased complex II levels. Macs from Drp1Δmacs mice showed an even greater reduction all complexes. Next, macs from WT and Drp1Δmacs mice were cultured with CL-316243-treated 9B cell CM. WT macs displayed increased F4/80+CD11b+CD301+ (resolution/M2) and decreased F4/80+CD11b+CD11c+ (inflammatory/M1) percentages, while Drp1Δmacs macs had higher frequency of F4/80+CD11b+CD11c+ (inflammatory/M1) macs. These indicate that stimulus from adipocytes mimicking CE milieu enhances the resolution/M2 phenotype, which is abrogated upon Drp1 deletion in macs. Next, to evaluate the metabolic consequences in macs, we measured oxygen consumption and observed that WT macs had greater O2 consumption after culture with CL-316243-treated 9B cell CM than Drp1Δmacs macs. Together, our data shows that Drp1 is required for the resolution profile and metabolic adaptation of macrophages when brown adipocytes are exposed to cold.

DUAL DEFICIENCY IN MIF AND IL-10 CAUSES EARLY LETHAL DISEASE IN MICE

Introduction: The macrophage migration inhibiting factor (MIF) is a proinflammatory cytokine that plays an important role in inflammatory responses and is also relevant in the context of chronic inflammatory conditions, such as Inflammatory Bowel Diseases (IBDs). IBDs include two pathologies: Crohn's disease and Ulcerative Colitis and are considered multifactorial disorders with high morbidity, in which the host's genetics and the environment determine its onset, progression and severity. Previous data show that without MIF, mice exhibit reduced intestinal damage, which in infectious models does not correlate with a reduction in infectious burden. However, the anti-inflammatory cytokine IL-10 has a critical role in the immune system, mainly in the maintenance of intestinal homeostasis. Defects in the coding regions of the IL-10 genes and its receptor lead to inflammatory disorders, which can cause severe enterocolitis in humans and mice. There are two mechanisms by which the host can handle a pathogen load: resistance and tolerance. Resistance represents the host's ability to limit the infectious load, while tolerance means the host's ability to limit damage to the pathogen. As the absence of MIF is able to confer tolerance in colitis models and the absence of IL-10 is known to confer susceptibility to colitis, we questioned which phenotype would be dominant in an animal with double deficiency (Mif -/- and Il10-/-). Methods and Results: For this study, Il10-/- animals were placed in co-housing with Mif -/- to mate. During this process, we noticed that the Il10-/- animals died prematurely, making the breedings difficult. Mif -/-Il10-/- mice were born in a lower proportion than expected according to Mendelian law, and died prematurely. The few survivors show a serious illness: their weight loss compared to their littermates, the presence of rectal prolapse, soft stools, stooped posture - signs that initially indicate an intestinal disease. We co-housed them with Il10-/- and they were able to transfer disease like their Mif -/- parents. Conclusion: Dual deficiency compromises fetal development or its first days of life and appears to be lethal. Taken together, they suggest that while MIF deficiency is responsible for allowing colonization by different pathobionts and pathogens, it is the presence of IL-10 that gives the animal its tolerant status and has a dominant effect on the phenotype in terms of susceptibility to intestinal inflammation.

DUAL DYNAMICS OF B-1 AND B-2 CELL SUBSETS IN LYMPHOID AND NON-LYMPHOID ORGANS DURING CALORIC RESTRICTION

Introduction: Caloric restriction (CR) is a dietary intervention known for promoting longevity. Among several factors that contribute to its benefits, one is the decrease in fat deposits that slow down various physiological and metabolic changes. Recently, it was described that CR is capable of reducing the number of immune cells in different peripheral tissues. However, little is known about how it affects the B cell subsets. B-1 cells, or innate-like B cells, are present majorly in the visceral fat and the peritoneal/pleural cavities. In addition, B-1 cells are known to have an increased metabolic demand compared to other subtypes of lymphocytes. Still, it is not known how CR affects the metabolism and, consequently, the functionality of these cells. We hypothesize that CR differently interferes with the B cells' subsets metabolism. Methods and results: To access how CR interferes directly with genes related to cellular metabolism, we performed in silico analysis from CR and control (ad libitum) mice liver samples by RNA microarray. CR mice showed upregulation in pathways related to mitochondria-associated genes and calcium regulation. In addition, to understand the metabolic differences between the B cells subsets, we evaluated the transcriptome and enrichment analysis of B-1a, B-1b, and B-2 lymphocytes from C57BL/6 mice. The signature genes among B-1a, B-1b, and B-2 subsets showed enrichment of pathways related to metabolic function in all B-1 cells, especially regarding responses to cytokine signaling, lipids metabolism, and nutrient sensors. Furthermore, we submitted C57BL/6 mice to a 40% CR for 4 weeks to assess the effects of the dietary changes in the frequencies of B cell subsets by flow cytometry and confocal microscopy. Surprisingly, the frequencies of B-1 and B-2 cell subsets are different in lymphoid and non-lymphoid organs during CR, and the B-1 subsets were less affected than B-2 cells in the periphery. By confocal microscopy, we observed that B-1a cells were conserved in the visceral adipose tissues even after nutritional deprivation. Conclusion: CR exerts changes in the cell dynamics of B cell subsets in the periphery. We believe that it is a consequence of different metabolic reprogramming in these subsets, interfering with their activation and maintenance. Understanding how CR exerts its beneficial effects on the immune system will enable the emergence of new therapeutic targets for the control of inflammatory processes.

EARLY TRANSCRIBED MEMBRANE PROTEINS PURIFICATION AND SEROLOGICAL CHARACTERIZATION FOR MALARIA ENZYME LINKED IMMUNOASSAY

EBI3 SUBUNIT CONTROLS THE RECRUITMENT OF ACTIVATED PHAGOCYTES IN LESIONS OF Leishmania braziliensis-INFECTED MICE

Introduction: American cutaneous leishmaniasis is endemic in almost the entire Brazilian territory characterized by located ulcers on the skin, at the site of the sandfly bite, which may spontaneously heal or progress to the mucocutaneous form. An understanding of the mechanisms of control of Leishmania-induced lesions is necessary to prevent tissue damage. The heterodimeric cytokines IL-27 (formed by EBi3 and IL-27p28 subunits) and IL-35 (EBi3 and IL-12p35 subunits) belong to the IL-12 family. These cytokines play an immunomodulatory role, directly modulating the inflammatory response through inducing IL-10 secretion and inhibiting Th17 cell differentiation. In this sense, we aim to understand the role of these cytokines in Leishmania braziliensis (LB) infection. Methods and Results: Interestingly, biopsies of lesions from LB-infected patients showed increased expression of EBI3, IL-27p28, IL-12p35, and gp130 subunits evaluated by immunohistochemistry and RT-PCR. Furthermore, augmented expressions of pro-and anti-inflammatory cytokines were also observed in these lesions. In addition, mice deficient of IL-27 receptor (IL-27R-/-) or EBI3 subunit (EBI3-/-) and control mice (WT) were infected in the ear with LB and the size of the lesion, parasite burden, and the immune cells recruited to the site of infection at 5 weeks post-infection (wpi) determined. The lesion size significantly increased in the ear of EBI3-/- mice compared to WT and IL-27R-/- mice from the 3rd to 5th wpi. Meanwhile, the parasite burden in the ear of EBI3-/- and IL-27R-/- mice decreased compared to WT at 5th wpi. Regarding the cellular profile, a reduction in the number of myeloid cells was found, with a decrease in the number of neutrophils, dendritic cells, and inflammatory monocytes in the ear of EBI3-/- mice compared to WT mice. On the other hand, we found an increased percentage of activated TNF-producing phagocytes and a reduction in those producing NOS-2 or IL-12 compared to WT mice. About lymphoid cells, it was also observed a reduction in the number of IFN-g or IL-10-producing T CD4+ in the ear of EBI3-/- mice compared to WT mice. Conclusion: Together, our data suggest that the EBI3 subunit is important in the control of a localized immune response induced by L. braziliensis infection.

EFFECT OF 5'-DEOXY-5'-METHYLTHIOADENOSINE TREATMENT ON NK CELL SURFACE RECEPTOR EXPRESSION OF PEOPLE LIVING WITH HIV

Introduction: With the emergence of Anti Retroviral Therapy (ART), there was an improvement in the quality of life and a reduction in the mortality of people living with HIV (PLWH). This fact changed the status of disease to a chronic infection, since the treatment greatly reduces the viral load, but keep the viral reservoirs. New approaches have been pursued the effective cure, such as shock and kill, which uses a latency reversal agent (LRA) combined with ART, aiming at the elimination of viral reservoirs. LRAs can affect immune system cells, influencing their function and activation profile. Here, the aim of this work was to evaluate the effect of an ARL (5'-deoxy-5'-methylthioadenosine - MTA) on the phenotype and effector function of NK cells from PLWH. Methods and Results: NK cells isolated from human PBMCs from control and PLWH subjects were incubated at 37°C for 1 h with MTA (500 µM). Afterwards, they were rested overnight with IL-12/15/18 (10 ng/mL, 10 ng/mL, 50 ng/mL) or only IL-15 (10 ng/mL). The effector function of NK cells was evaluated by IFN-γ secretion. Subpopulations of NK cells were evaluated by flow cytometry through the expression of CD56 and CD16 and characterized the expression of NKp46, NKB1 and CD69 receptors. Both groups showed an increase in IFN-γ levels under cytokine stimulation (CTRL:93.4±60.2; PLWH:77.4±25.8), although MTA suppressed IFN-γ production in both groups (CTRL:29.1±14.8; PLWH:22.8±14.9). PLWH had lower frequency of CD56lowCD16high when not stimulated (p<0.001), but stimulation with cytokines led to a decrease in this population in both groups, with no difference being observed between groups regardless of the presence of MTA. Furthermore, MTA inhibited CD69 expression in CD56lowCD16high cells in PLWH (p<0.0001), which was not observed in the CTRL group (p=0.34). Nonetheless, MTA inhibited NKp46 activating receptor expression more strongly in CD56lowCD16high and CD56highCD16low/neg cells in the CTRL group (p=0.006, p=0.05) than in PLWH (p=0.96, p=0.99). Finally, PLWH showed a lower frequency of NKB1+ cells in the CD56lowCD16high population, regardless of stimulus and MTA (p<0.0001). Conclusion: Although MTA inhibits IFN-γ production equally in both groups, differences in receptor expression were observed between CTRL and PLWH cells after stimulation with cytokines and under treatment with MTA, suggesting that this epidrug could change the dynamic of innate immune response beyond the expected latency reversal effects.

EFFECT OF AN EPISODE OF ACUTE GASTROINTESTINAL INFECTION ON PATHOGENESIS AND PROGRESSION OF TYPE 1 DIABETES

The gastrointestinal tract houses several commensal microorganisms that actively participate in the modulation of the host's metabolism. In addition to the microbiota, this tissue receives several daily antigens from the diet, so a highly specialized immune system is needed, which allows the host to tolerate the microbiota and diet antigens and to develop responses against potential pathogens. This balance between the immune system and microbiota is essential for maintaining homeostasis in the intestinal tissue and disturbances in this balance are commonly observed in patients with autoimmune diseases, such as type 1 diabetes mellitus (T1D). Previous work by our group have showed that infection by the Yersinia pseudotuberculosis (YP) leaves an immunological scar in the intestine, even after its complete elimination, which is associated with the breakdown of the intestinal mucosa barrier and the lymphatic system, leading to a deviation of the migration route of intestinal dendritic cells for draining lymph nodes and, ultimately, blocking the induction of canonical adaptive responses. In addition, unpublished results from our group indicate the presence of an inflammatory infiltrate in the pancreas of C57BL/6 animals after infection by YP. However, this event per se does not lead to the development of spontaneous T1D. Preliminary results from our laboratory showed that NOD mice, which develop diabetes spontaneously, when infected with YP before the onset of symptoms, had a lower incidence of T1D compared to the control group. Thus, in this project, the impact of YP infection on the development of autoimmune diabetes, the mechanisms and cells involved in this process will be investigated, in addition to assessing the mechanisms that are leading to the appearance of the inflammatory infiltrate in the pancreas using a animal model of autoimmune diabetes induced by multiple doses of streptozotocin and also NOD mice.

Effect of BRD4 and mTOR pathway inhibitors in immune checkpoint expression in AML

Introduction: Acute myeloid leukemia (AML) is a hematological cancer characterized by poor prognosis, fail to standard treatment (chemotherapy and bone marrow transplant) and high relapse rates. Hence, new therapies are needed for this disease. In this context, BRD4 protein has been studied as a target for AML treatment, but some trials demonstrated innate and acquired resistance to BRD4 inhibitors. Our research group has observed that the combined treatment with BDR4 inhibitor and mTOR pathway inhibitor is able to break the innate resistance, showing a synergic anti-leukemic effect. Also, immune checkpoints have grown in significance in cancer research during the past few years. Thus, we aim to investigate if our combined treatment can modulate the expression of immune checkpoints in AML cells. Methods and Results: We used HL60, HEL and MOLM cell lines, which were treated with JQ1 (BRD4 inhibitor), Rapamycin (mTOR pathway inhibitor), Combo (both treatments) and DMSO (vehicle). Gene expression of four checkpoints (PD-L1, PD-L2, HVEM, GAL-9) were evaluated by qPCR. In HL60 we observed a tendency of HVEM and PD-L1 decreased expression by JQ1 and Combo treatments and a low expression of both receptors in this cell line. Also, an increased expression of GAL-9 by Rapamycin treatment and a higher expression of this checkpoint. In HEL we noticed an effective modulation of checkpoints. Combo treatment has decreased PD-L1 expression, and we have also seen increased expression of HVEM and GAL-9, the first one in Combo and Rapamycin and the second one in Rapamycin treatment. In MOLM we have seen no modulation of PD-L1, which had low expression, a tendency of decreased expression of HVEM by JQ1 and Combo, and GAL-9 expression were decreased also by JQ1. No PD-L2 expression were detected in any of the cell lines. Conclusion: These results indicate that the anti-leukemic effect seen in the combined treatment apparently occurs independently of the modulation of checkpoints. In addition, it seems that immune checkpoint blockade (ICB) therapies would not be effective in AML. This is justified for the lack of PD-L2 expression and because two of the three cell lines had low PD-L1 expression. Once these two checkpoints are ligands of PD-1 and the major checkpoint inhibitor in use today is anti-PD-1 antibody, ICB would not have a considerable action, at least not in these cell lines. Financial support: FAPESP - 2020/16491-7

EFFECT OF CANNABINOID RECEPTOR TYPE 2 AGONIST GP1a ON MACROPHAGE ACTIVATION DURING M. BOVIS-BCG INFECTION

Introduction: Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis which is disseminated by aerosols mostly affecting the lungs. It is an important public health problem presenting high mortality rates in Brazil and worldwide. The actual treatment causes many side effects and lasts a long period, which contributes to the treatment abandonment and emergence of resistant bacteria. Cannabinoid receptor type 2 (CB2) has been pointed out as a potential new target to modulate the immune response, including infectious diseases. Data from our group demonstrated that in vivo treatment with CB2 receptor agonist modulates the inflammatory response to mycobacteria. Thus, our study aims to evaluate the effects of the CB2 receptor-selective agonist GP1a on the activation of macrophages during M. bovis BCG infection. Methods and Results: Bone marrow-derived macrophages were infected with M. bovis BCG expressing green fluorescent protein (GFP) at MOI 5, and post-treated with CB2 receptor agonist GP1a (10 and 20 uM) for 24 hours. Lipid droplets (LD) area was determined by Oil Red O staining and bacteria burden by the quantification of GFP area, analyzed by fluorescence microscopy. Cytokines were quantified in culture supernatant by ELISA. Compound cytotoxicity was measured by violet crystal quantification and lactate dehydrogenase activity on the supernatants. The statistical test used to analyze these data was ANOVA test and Turkey or Student’s t-test (p<0.005). GP1a at 0,16-10 uM did not present any cytotoxic effects in macrophages infected or not with BCG. Cells treated with GP1a at 20 uM presented approximately 70% of viability. And 40 and 80 uM concentrations were highly cytotoxic (30% cell viability). We observed that GP1a (10 and 20 uM) treatment inhibited the number of lipid droplets increased in BCG-infected macrophages but did not affect the area of bacteria. In addition, GP1a (20 uM) treatment also inhibited the production of IL-1beta, IL-6, IL-10, and TNF-alpha by macrophages during BCG infection. Conclusion: These results show that GP1a downmodulates macrophage response to mycobacterial infection; however, further investigation is necessary to further elucidate if CB2 activation would improve to host immunity or favors the pathogen and how it modulates macrophage activation during M. bovis BCG infection.

EFFECT OF GUT DYSBIOSIS ON EPIGENETIC MODULATION OF NEUTROPHILS

Neutrophils constitute the most abundant population of circulating leukocyte cells in humans and are the first cells to migrate and accumulate in tissues during inflammation and microbial infections. In addition to their ability to phagocyte and eliminate infectious agents through their antimicrobial mechanisms, they also constitute a relevant source of cytokines and eicosanoids that regulate the activation and recruitment of other cells. Microbiota signals, including the short-chain fatty acids (SCFAs), regulate the recruitment, phagocytic capacity and production of cytokines by these cells. However, there is scarce information on how modifications of gut microbiota composition and functions impact on neutrophils in humans. In this study, we evaluated the effect of antibiotics on SCFA production and their impact and relevance on epigenetic modulation and effector responses of neutrophils. Healthy male volunteers underwent 3 days of treatment with the antibiotics neomycin, vancomycin and metronidazole. Blood and stool samples were collected before and after antibiotic treatment. Circulating neutrophils were isolated and analyzed for their cytokine production, gene expression profile and chromatin accessibility. Antibiotic-induced gut dysbiosis model was validated by 16S rRNA sequencing and SCFAs quantification of subject’s feces. After antibiotic treatment, microbial diversity and relative abundance was reduced (Pielou’s Evenness 0.687 ± 0.067 before treatment (T1), and 0.351 ± 0.076, after treatment (T2); Weighted-UniFrac 0.486 ± 0.145 (T1), and 0.847 ± 0.080 (T2)). These effects were accompanied by marked changes in SCFA production. Acetate concentrations significantly decreased after treatment with antibiotics (0.833 ± 0.1080 mg/g of feces (T1) and 0.173 ± 0.036 mg/g of feces (T2)). Furthermore, propionate and butyrate were not detected after treatment. We believe that this study will bring new perspectives on the mechanisms and contribution of the gut microbiota on neutrophil modulation and effector response.

EFFECT OF HEMODIALYSIS ON THE INFLAMMATORY AND NUTRITIONAL PROFILE OF PATIENTS IN A TWO-YEAR FOLLOW-UP

Hemodialysis (HD) is an effective method for treating stage 5 Chronic Kidney Disease. Malnutrition inflammation, and atherosclerosis (Mia Syndrome) which occur in chronic kidney patients on HD due to several factors such as anorexia, obesity, metabolic disorders, and the therapy itself. MIA induces a reduction in serum proteins and an increase in the production of pro-inflammatory cytokines, raising the risk of cardiovascular mortality, which is the leading cause of death in these patients’ profiles. This work aimed to verify the existence of the association of nutritional parameters with inflammatory biomarkers. Methods and Results: Prospective observational study was carried out at the Dialysis Unit of Complexo Hospitalar Santa Casa de Misericordia de Porto Alegre, linked to the Federal University of Health Sciences in Porto Alegre. The sample comprised 81 patients, 42 men, and 39 women, who were followed for 24 months. The age in years averaged 52.51±17.42 and all patients with chronic kidney disease were on HD 3 times a week. The C-reactive protein and serum albumin were evaluated through blood sampling. Nutritional status was assessed through bioimpedance and phase angle. The mean time of dialysis was 54.18 ± 51.64 months. The minimum dialysis time was 3 months, and the maximum was 202 months. Body mass index and phase angle after dialysis were 25.86±5.93km/m² and 6.01±1.58 respectively, with no significant changes in the period. CRP levels was 12,14 ± 15,26ng/ml and serum albumin 3,89 ± 0,38 without associations observed with nutritional status. It was observed that the lower weight and BMI presented, the greater the probability of death in the first 18 months, while a phase angle lower than 4° showed an unfavorable outcome at 6 months of follow-up. Conclusion: Monitoring nutritional markers are an important predictor of more severe clinical outcomes, highlighting the importance of nutritional care in these patients.

EFFECT OF HEMODIALYSIS ON THE MORTALITY OF PATIENTS IN A TWO-YEAR FOLLOW-UP

Chronic kidney disease (CKD) is a growing public health problem worldwide. In Brazil, it is estimated that more than 10 million Brazilians have some degree of renal impairment. The prevalence of cardiovascular disease is markedly higher among individuals with CKD. This work aimed to verify the mortality risk among individuals with CKD in Hemodialysis (HD). Methods and Results: Prospective observational study was carried out at the Dialysis Unit of Complexo Hospitalar Santa Casa de Misericordia de Porto Alegre, linked to the Federal University of Health Sciences in Porto Alegre. The sample consisted of 81 patients, 42 men and 39 women, followed for 24 months. The age averaged was 52.51±17.42 years, and all patients with chronic kidney disease were on HD 3 times a week. The C-reactive protein (PCR), serum albumin, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were evaluated through blood sampling. The mean time of dialysis was 54.18 ± 51.64 months. The minimum dialysis time was 3 months, and the maximum was 202 months. The main components analysis was performed to verify the relationship of variables in the impact of the death outcome: Ratio_CRP_albumin (0.965), CRP (0.959), and inflammation_CRP (0.802) are highly correlated with deaths outcome in hemodialysis patients. Death during follow-up was related to the CRP/Albumin Ratio (0.965) and CRP (0.959). Conclusion: Monitoring clinical markers such as albumin, PCR, HDL, and LDL is an important predictor of the risk of cardiovascular mortality in these patients.

Effect of obesity on the protective immune response developed during experimental Paracoccidioides brasiliensis infection

Obesity affects the functionality of most organs and stimulates deficient immune response. Paracoccidioidomycosis is a severe systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The aim of this study was to verify whether the immune response of obese mice is altered in terms of susceptibility to infection by this fungus. Obese and normal mice were inoculated with 5x106/mL virulent Pb intraperitoneally (followed during 120 days) or in the subcutaneous air pouch (followed for 13 days) and evaluated for weight, survival rate, abdominal fat, lipid and glucose profiles, subcutaneous exsudate cell number, viability, activity, production of IL-4, IL-6, IL10, IL-12, IL-17, IFN-y and KC cytokines and of reactive oxygen and nitrogen species. After 120 days of intraperitoneal infection, delayed type hypersensitivity reactions and antibody concentrations were determined and organs were collected to quantify viable fungi. Obese mice showed significant increase in abdominal fat and total cholesterol, compared to controls and reduction of viability but not of the numbers of cells attracted to the inoculation site. Cell activity and nitric oxide production was significantly lower in the obese group than in controls, but production of reactive oxygen species increased significantly. There was significant reduction in IL-4, IL-6 and IL-12 in obese animals but significant increase in IL-10 and KC production at different times analysed. There was a decrease in IFN-y production by obese animals at all times examined, while IL-17 production was not significantly different. We found no significant differences in delayed type hypersensitivity reactions and in IgG, IgG1a and IgG2b antibody titres. However obese mice produced higher concentrations of IgM at 120 days post-infection. The highest number of viable fungi were found in the lungs and epiploon. Our results suggest that Pb infection tends to be more severe in obese animals than in controls, due to the development of a less effective immune response.

EFFECT OF ÔMEGA-3 (DHA) ON CARCINOGENIC PARAMETERS OF MELANOMA CELLS IN VITRO

INTRODUCTION: Ômega-3 is an essential fatty acid acquired from fish and plant oils. Among other ômega-3 fatty acids, docosahexaenoic acid (DHA) has been studied to prevent and treat several diseases, including cancer. Melanoma is a skin cancer derived from melanocytes, the cells that produce the pigment melanin. This cancer is a highly aggressive primary cutaneous malignancy and is responsible for a majority of skin cancer–related deaths. Considering this, the present project aimed to analyze the possible effects of ômega-3 stimulus on B16F10 cells, derived from murine melanoma. METHODS: B16F10 cells were stimulated or not with DHA at different times and concentrations. Mitochondrial viability was assessed by MTT assay and analyzed by spectrophotometry. Cell proliferation was evaluated by CFSE staining and analyzed by flow cytometry. Membrane pore formation was assessed by propidium iodide uptake and analyzed by fluorescence spectrophotometry. Lipid droplet biogenesis was analyzed by BODIPY staining and analyzed by flow cytometry. RESULTS: Melanoma cells treatment with DHA triggered cell viability loss in a time and dose-dependent manner. DHA at 50µM induced membrane pore formation, indicating an occurrence of DHA-induced lithic cell death in those melanoma cells in vitro. In addition, DHA was able to reduce melanoma cell proliferation at 50 µM concentration. None of the concentrations had a significant effect on lipid droplet biogenesis. CONCLUSION: Our data support the antineoplastic potential of DHA. Our results suggest that DHA can reduce the mitochondrial viability of melanoma cells, by the induction of a type of lithic cell death that still need to be clarified. Thus, this project shows novel effects of docosahexaenoic acid, DHA, on murine melanoma cells, supporting evidence on the potential adjuvant antitumor therapy of the ômega-3 DHA

EFFECT OF PHARMACOLOGICAL INHIBITION OF SREBP TRANSCRIPTIONAL ACTIVITY ON SARS-COV-2 REPLICATION AND INFLAMMATORY MEDIATOR PRODUCTION IN CALU-3 CELLS

EFFECTS OF AQUEOUS EXTRACT LEAVES OF PASSIFLORA ALATA CURTIS ON INFLAMMATORY CYTOKINES, NITRIC OXIDE PRODUCTION AND CELL VIABILITY IN LPS-TREATED MACROPHAGES.

INTRODUCTION: P. alata is a plant/fruit consumed in Brazil as food and its leaves stands out due to their pharmacologial properties. Polyphenols and flavonoids are bioactive compounds present in this plant. The study of these compounds leads to a better comprehension about their mechanism of action, toxicity and immunomodulatory capacity. Previously, we demonstrated that the aqueous leaves extract of P. alata presented antiinflammatory, anti-apoptotic and antioxidant activity in non-obese diabetic (NOD) mice, decreasing the diabetes incidence and inflammation in pancreatic islets (Int. Immun., 18: 106-115, 2014). Since macrophages are a key player in pancreatic islet inflammation, we addressed the effects of P.alata aqueous leaves extract in viability and cellular immune response of murine macrophages cell lines RAW264.7 and J774. METHODS AND RESULTS: The cells were cultured for 48 hours with 0.5μg/mL LPS and 50 UI/mL IFN-γ (inflammatory stimulation), and treated with the doses of 50, 100, 200, 300, 400 e 500μg/mL of P. alata extract. To evaluate nitric oxide (NO) production, the cell lineages were seeded at 2x105/well and the NO measured by Griess reaction. The inflammatory stimulation induces a significant production of NO in both lineages when compared with control. Stimulated RAW264.7 cell lineage presented a decrease in NO production when treated with 400 and 500µg/mL of P.alata extract in comparison with control. J744 cell lineage without stimulation presented an increase of NO production in 100, 200, 300 and 400µg/mL, of P.alata extract, and a decrease in stimulated cells in the dose of 500µg/mL. The cell viability was carried out by flow cytometry assays using annexin V-FITC and 7-AAD probe staining to analyze viable, early apoptotic and late apoptotic/necrotic cells frequencies. The dose of 500µg/mL of P.alata extract increased the apoptotic cell population, decreasing viable cells in both cell lineages. Cell culture supernatants were collected to quantify the cytokines: IL-1α, IL-6, MCP-1, GM-CSF and IL-10, by multianalyte bead based flow cytometry assays. The P.alata treatment (300µg/mL) increases IL-6 and MCP-1 production in non-stimulated RAW264.7, whereas in stimulated J774, the IL-1α, IL-6, GM-CSF and IL-10 production were reduced in comparison with the controls. CONCLUSIONS: The aqueous leves P. alata extract was able to modulate the inflammatory profile of stimulated macrophages, suggesting its potential use in inflammatory diseases.

EFFECTS OF CHILD UNDERNUTRITION AND ITS TREATMENT WITH COTRIMOXAZOLE IN THE INTESTINAL MICROBIOTA AND IMMUNE SYSTEM IN MICE MODEL

Introduction: Despite little progress in some areas, childhood undernutrition remains a major global health concern because it affects child development and increases the death risks of infectious diseases. Therefore, the World Health Organization recommends prophylactic treatment with Cotrimoxazole (SXT) and nutritional recovery to overcome this issue. Paradoxically, wide-spectrum antibiotic therapy can lead to gut dysbiosis, which in turn induces undernutrition besides antibiotic-resistant bacteria development. Moreover, the effects of cotrimoxazole on the gut microbiota and the immune system of undernourished infants are unknown. Methods And Results: To understand how SXT prophylaxis could affect gut microbiota in undernutrition, we induced protein-energy undernutrition in weaning C57BL/6 mice for three weeks and treated animals with SXT for two weeks. Using 16S rRNA gene sequencing, we compared the taxonomic composition and metabolic pathways between controls, animals submitted to undernutrition (UND), treated with SXT, or undernourished/SXT treated (UND+SXT). Undernutrition protocol was responsible for increasing Bacteroidetes and decreasing Firmicutes abundance. We identified that UND mice had a significant increase in predicted pathways related to metabolic syndromes later in life. The prophylactic SXT treatment alone resulted in a significant loss in community richness and beta diversity. In addition, we identified the reduction of 6 families in SXT-treated mice, including the butyrate producers Lachnospiraceae and Ruminococcaceae. The double challenge (UND+SXT) resulted in a reduction in the Clostridiaceae family and in the urea cycle pathway, both related to the fermentation of amino acids, the intestinal epithelial permeability, and the healthy gut environment. We also found that undernourished mice have differential development in the B cell compartment compared to controls, with an elevated number of total, follicular, and memory B cells in the spleen. Conclusion: Our results show that SXT prophylaxis during an undernourishment period in infant mice did not re-establish the undernourished microbiota community composition similar to controls but instead induce a distinct dysbiotic profile, with functional metabolic consequences and the undernutrition can disrupt the normal B cell development.

EFFECTS OF CYANIDIN-3-GLUCOSIDE IN THE SECRETOME OF MESENCHYMAL STEM CELLS

Background: The Cyanidin-3-glucoside (CY3G) is a type of anthocyanins that presents a capacity for anti-inflammatory, antioxidant and has the ability to modulate the inflammatory response for an immunosuppressed profile. Cyanidin has attracted attention for its potential biomedical applications in immune system. Mesenchymal Stem Cells (MSCs) are a group of multipotent adult stem cells that has a high of immunoregulatory capacity. In order to verify the role of cyanidin in potentiating the immunoregulatory response of MSCs in an inflammatory environment. Aims: In this study, we investigated the capacity of cyanidin to modulate the profile of mesenchymal stem cell in cytokine production under Lipopolysaccharide (LPS) stimulation. Methods: This work analyses the production of cytokines using Mesenchymal stem Cell lineage (C3H10T1/2) was maintained in medium DMEM at 37°C and 5% of CO2. This cell line was divided into four groups: control – treated with DMEM, CY3G- treated with 50μM of Cyanidin and DMEM, CTR/LPS- treated with DMEM and 1,25μg/mL of LPS and CY3G/LPS- treated with 50μM of Cyanidin, DMEM and 1,25μg/mL of LPS. Next, the cells were incubated for 24 hours. So, we performed ELISA assay for cytokines like IL-6, IL-1β, IL-10 and TGF-β. Results: The cytokines like IL-6 (n=6) and IL-1β (n=6) that induces a pro-inflammatory response in MSCs were reduced in the group of MSCs stimulated with LPS. Nonetheless, cytokines with immunosuppressive characteristics like IL-10 (n=6) present no significant result, and TGF-β (n=6) that presents an increased in the production of this cytokine. Conclusion: Preliminary results showed that cyanidin negatively regulated the production of inflammatory cytokines. Therefore, this anthocyanin can modulate MSCs for an immunosuppressive profile in vitro model with an inflammatory stimulus. Disclosures: The authors declare no conflict of interest.

EFFECTS OF METABOLIC SYNDROME INDUCED BY LITTER SIZE REDUCTION ON IMMUNE AND GOBLET CELLS IN THE JEJUNUM OF BALB/C MICE

Introduction: Metabolic Syndrome (MS) is a complex disorder, represented by the presence of a set of risk factors that can contribute to the development of cardiovascular disease and/or type 2 diabetes. The MS was not understood completely, and yet, affects about a quarter of the population globally, causing changes in several organs, such as the intestine, an important immune and endocrine organ, which is related in different ways to MS. Thus, the aim of this work was to evaluate the effects of a programmed MS during lactation on the number of cells that participate in the immune response of the jejunum of mice. Methods and Results: Eight male BALB/c mice were used and divided into a control group (CG) and a programmed MS by litter size reduction (SL) group, which consisted of the standardization of 3 animals per lactating, which increased milk supply, leading to overweight and metabolic dysfunction. The Ethic Committee in Animal Experimentation of the State University of Maringá approved this study (CEUA 8137280920/002850). At 180 days of age, the animals were euthanized and 1 cm of the jejunum was collected for histological processing and staining using Toluidine Blue and Fuchsin G, Hematoxylin and Eosin and Alcian Blue (AB) pH 1.0 and 2.5 for counts of mast cells, intraepithelial lymphocytes (IEL) and goblet cells (GC), respectively. The number of mast cells was quantified using an optical microscope in 100 microscopic fields of each animal (9.96 mm²) and calculated the proportion of these cells/mm². For the quantification of IEL and GC, the number of cells in 2,560 epithelial cells (EC) (160 epithelial cells/quadrant/section/animal) was determined and the proportion calculated for 100 EC. All data presented normal distribution, and the T-test was applied to compare the groups with p < 0.05 using GraphPad Prism version 8. Results were expressed as mean ± standard deviation. There was not a significant difference in the number of mast cells (294.20 ± 1.64 cells/mm²), neither was the IEL count (9.85 ± 0.67 IEL/100EC) altered in the SL group when compared to the CG group (303.0 ± 7.13 cells/mm²; 14.29 ± 4.93, respectively). The proportion of GC stained by AB pH 1.0 (SL6.07 ± 0.71 vs CG4.44 ± 1.37 cells/100EC) or by AB pH 2.5 (SL7.25 ± 1.59 vs CG6.90 ± 2.25cells/100EC) also remained unchanged. Conclusion: Our results suggest that, in this experimental model of MS, the intestinal immune response is not affected by quantitative changes in these cells.

EFFECTS OF ORAL ADMINISTRATION OF OLEIC ACID IN IMQ-INDUCED PSORIASIS MOUSE MODEL

INTRODUCTION: Thousands of people worldwide suffer with psoriasis, a disease caused by dysregulated immune reactions in skin. Fatty acids have been a successful strategy for the treatment of inflammatory diseases. However, there are few studies that have investigated the effects of oleic acid (OL) on psoriasis. The aim of this study was to evaluate the effects of OL on imiquimode (IMQ)-induced psoriasis mouse model. METHODS AND RESULTS: To psoriasis induction was applied 60 mg of Ixium® (5% of IMQ) on shaved back skin of C57BL/6 mice from day 1 to day 5. Oral administration with 12.5µL of OL occurred from day 1 to day 10. We observed that supplementation with OL reduced the percentage of skin thickness on 3th day (134%±9 (IMQ); 117±4 (IMQ+OL12.5µl)) until 7th day (168%±10 (IMQ); 145±7 (IMQ+OL12.5µl) n=5/group). OL supplementation did not change body weight, food or water intake. By gas chromatograph we showed that 12.5 µL of oleic fatty acid increased oleic serum incorporation (129 µg/mL±55 (IMQ); 313±97 (IMQ+OL12.5µl); n=4-5/group). After that, we collected skin and axillary lymph nodes on 3th day. By rt-PCR we observed that IMQ increased IL-23 and IL-22 in comparison to control mice and administration of oleic fatty acid reduced IL-23 in comparison to IMQ group (1.3fold change±0.94 (Control); 4.6 ±0.94 (IMQ); 3.2 ±0.47 (IMQ+OL12.5µl); n=6/group). By flow cytometry, IMQ group increased the percentage of neutrophils (36±14 (Control); 60±11 (IMQ); 40±12 (IMQ+OL12.5µl), monocytes (6±3 (Control); 18±11 (IMQ); 7±3 (IMQ+OL12.5µl) and Langerhans cells (20%±6 (Control); 61±13 (IMQ); 32±8 (IMQ+OL12.5µl); n=6/group) and OL reduced these cells percentages in skin. In axillary lymph nodes, IMQ group increased neutrophils, monocytes and dendritic cells percentages in comparison to control mice. No alterations were observed in oleic group. Moreover, cells isolated from bone marrow were stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF, 10 ng/mL) for 7 days and treated for 24 hours with 10 µM of OL and LPS (1µg/mL). By ELISA, we observed that OL decreased IL-23 (266pg/mL±51 (Control); 170±44 (10µM OL) and IL-12 (294±108 (Control); 198±41 (10µM OL) production but not altered IL-1β, TNFα, IL-22. CONCLUSION: Oral supplementation with oleic acid reduces inflammatory response, attenuating the local effects of IMQ in psoriasis mouse model.

EFFECTS OF THERAPEUTIC TREATMENT WITH PROPIONATE BRONCHIOLITIS CAUSED BY RESPIRATORY SYNCYTIAL VIRUS

Introduction: Bronchiolitis is a disease that affects children under two years and is characterized by inflammation and edema of the airways, causing an increase in the production of secretion and necrosis of the bronchioles. Bronchiolitis is mainly caused by respiratory syncytial virus (RSV), representing 50-80% of cases. Preclinical studies have shown that short-chain fatty acids (SCFA) protect against RSV infection, with acetate being the most evident. This study aimed to evaluate the role of therapeutic treatment with propionate during RSV infection. Methods: Mice were given 200mM sodium propionate in water during RSV infection. Mice were weighted daily and at 5 days post infection mice were euthanized, and lungs were collected for real-time PCR analysis. All animal procedures were performed according to the protocols approved by CEUA/UNICAMP (protocol number 4022-1). For the clinical study, patients of both sexes, younger than 1 year, with signs and symptoms of acute pulmonary viral infection admitted to Hospital São Lucas/PUCRS were recruited. The person responsible signed the informed consent and nasal lavage was later collected. Nasal lavage was processed to collect supernatant and obtain cells. The cells obtained were plated and treated or not with propionate (260 µM) for 24 hours, followed by RNA extraction and subsequent synthesis of complementary DNA and real-time PCR for RSV, IFNB1, DDX58, MAVS, ISG15 and OAS1. The study was approved by the Institutional Review Board (number 2.471.678). Results: Treatment with propionate protected the animals from weight loss caused by the infection. This was confirmed by the significant reduction in viral load in the lungs of animals treated with propionate. In addition, clinical data indicate that ex vivo propionate treatment of nasal cells obtained from patients with RSV bronchiolitis significantly reduced viral load compared to untreated cells (p=0.0156). Conclusion: therapeutic treatment with propionate can be considered an alternative against RSV infection. Further studies should be carried out to confirm such an antiviral effect.

Effects of zileuton in vitro and in vivo against Leishmania major

Cutaneous leishmaniasis is a public health problem causing a range of diseases from self-healing infections to chronic disfiguring disease. Currently, there is no vaccine for leishmaniasis, and drug therapy is often ineffective. 5-lipoxygenase (5-LO) is an enzyme required for production of leukotrienes and lipoxins and interferes with parasitic infections. Parasite as Toxoplasma gondii carries an enzyme like lipoxygenase activity that can interact with host biosynthetic circuits for endogenous negative signals that divert the host immune response and limite acute inflammation. Here, our aim was to evaluate the effect of zileuton (5-LO inhibitor) treatment during Leishmania major infection in vivo, and mainly to investigate if L. major itself wielding its own lipoxygenase. For this, promastigotes form of L. major was incubated with 5-LO inhibitor at 100, 33, 10, 3 and 1 micromolar or dimethyl sulfoxide (DMSO; used as a vehicle/solvent). Treatment of L. major-infected C57BL/6 with zileuton reduced the parasite titer in the ear at 3 and 4 week after infection when compared with infected untreated mice. In vitro, zileuton induced a dose-dependent reduction of parasite numbers in an axenic culture. Collectively, these results suggest that zileuton treatment reduce the number of parasite in vivo and in vitro (axenic culture), mainly indicating that L. major promastigotes may carry a lipoxygenase-like activity that itself could respond to zileuton treatment.

Efferocytosis of Renca-apoptotic cells promotes suppressive effects on dendritic cells supported by glycolysis

Tumor cells are resistant to cell death, however, many conventional therapies used in cancer treatments lead to intense cell death, such as apoptosis in the tumor microenvironment (TME). Dendritic cells (DCs) and macrophages are the major phagocytes involved in the clearance of these apoptotic cells (ACs) through a process termed efferocytosis. The internalization and digestion of apoptotic tumor cells favor macrophages polarization toward M2 phenotype and generation of an immunosuppressive microenvironment. There is a growing appreciation that cellular activation is closely linked to changes in cellular metabolism. It has been shown that glycolytic and mitochondrial metabolism sustain the M2 macrophage-like polarization after efferocytosis. DCs play a fundamental role in the presentation of tumor cell antigens to TCD4/TCD8 lymphocytes, meantime, the immunometabolism of DCs during efferocytosis of apoptotic-tumor cells and its implications on DCs phenotype remain unknown. Thus, we hypothesized that the efferocytosis of apoptotic-tumor cells promotes metabolic reprogramming on DCs that favors a tolerogenic phenotype. Renca cells were treated with 2, 5, 10, and 20uM of doxorubicin (DOXO), for 24 hours. DOXO treatment was able to induce about 80% of cleaved caspase-3+ cells, compared with 2% on the untreated condition. Renca-apoptotic cells (Renca-ACs) were stained with PKH and co-cultured with DCs for 18h, and we observed about 85% of efferocytosis. Efferocytosis of Renca-ACs results in a slight increase in CD86 expression and an expressive increase in PD-L1 expression, without any modulation of MHC II molecules. Interestingly, there was an increased IL-10 and IL-6 production, compared with resting BMDCs, which appears to be dependent on pyruvate transport into mitochondria, lactate production, and fatty acid synthesis. Moreover, we observed the absence of TIM4 and MertK receptors reduces the efferocytic capacity of BMDCs and IL-10 production seems mostly dependent on recognition by TIM4. Furthermore, there was an increased expression of glycolytic enzymes Slc2a1, Ldha, and Pkm2. Taken together our results suggest that efferocytosis of Renca-ACs is mediated by TIM4 and MertK, and drives tolerogenic BMDCs characterized by increased PD-L1 expression, IL-10, and IL-6 production, which seem to be supported by glycolytic metabolism.

Efferocytosis of ZIKV infected apoptotic neuroblastoma drives dendritic cells activation

Zika virus (ZIKV) infection can cause an intense neuronal cell death that triggers a large number of neurological disorders, such as Guillain-Barré syndrome (GBS) and acute disseminated encephalomyelitis (ADEM). Studies have shown that efferocytosis of infected apoptotic cells (iAC) by dendritic cells (DC) can play an essential role in the development of autoreactive clones. Therefore, we hypothesized that during ZIKV infection, efferocytosis of infected apoptotic cells could induce the activation of DC and autoreactive CD4(+) T cells. To address our hypothesis, we started by standardizing the infection of the human neuroblastoma cell line SH-SY5Y with the PE243 strain of ZIKV. For this, 1.5x106 SH-SY5Y cells were cultured with ZIKV inoculum (MOI 1). After 1h, cells were washed and cultured in DMEM-F12 complete medium. The MOCK supernatant was used as a control. After 48 hours of incubation, the cells were fixed and permeabilized and then labeled with anti-4G2 antibody to determine the rate of infection by flow cytometry. We observed that after 48h, ~ 32% of cells are infected with ZIKV. Next, to induce cell death, MOCK or ZIKV-cultivated cells were treated with the chemotherapeutic Doxorubicin (DOXO) (10µM), for 24h. The percentage of apoptotic cells was determined using anti-cleaved Caspase-3 antibodies. DOXO treatment resulted in ~ 60% of Caspase-3+ cells compared to 7.45% in untreated cells. Next, to evaluate the efferocytosis, ZIKV-iAC was labeled with PKH67, and then co-cultured with Bone Marrow-Derived Dendritic Cells (BMDCs) (ratio -1BMDC:3ZIKV-iAC) for 18h, and we observed about 97% of efferocytosis (CD11c+PKH67+ cells). The internalization and digestion of iAC lead to a significant increase in the mean fluorescence intensity (MFI) of MHC-II, CD86, and PD-L1 molecules compared with MOCK. Furthermore, we also observed an increase in the production of IL-10 and IL-6 in the supernatant from ZIKV-iAC co-culture. These results demonstrate that neuroblastomas of the SH-SY5Y lineage are susceptible to ZIKV infection and DOXO-induced apoptosis. Moreover, our partial results indicate that efferocytosis of ZIKV-iACs leads to activation of BMDCs capable to produce IL-10 and IL-6. Next, we will evaluate the differentiation of naive CD4 T cells after culture with conditioned supernatant of BMDCs co-cultured with ZIKV-iAN.

ELIMINATION OF RASSF9 EXPRESSION IN B16F0 MELANOMA CELL LINE BY CRISPR/CAS9 TECHNOLOGY

Title: ELIMINATION OF RASSF9 EXPRESSION IN B16F0 MELANOMA CELL LINE BY CRISPR/CAS9 TECHNOLOGY Author(s): JULIA SOUZA E COSTA; JOÃO GUSTAVO PESSINI AMARANTE MENDES Institution: Biological Sciences Institute (ICB) - USP Text: Melanoma is the form of cancer caused by the transformation of melanocytes. Importantly, melanoma is the most lethal among all skin cancers. Melanoma can be divided into four groups depending on the type of mutation present in the melanoma cells. The more frequent alterations were mapped in B-raf and ras genes. The Ras protein may initiate different signaling cascades, thereby regulating processes such as proliferation, differentiation, morphology, and apoptosis. The main effectors of Ras related to cell death are members of the RASSF family (Ras-association Family). RASSF9 has been implicated in keratinocyte differentiation and dermal homeostasis and, interestingly, its expression can be induced by sun exposure. To study the role of RASSF9 in melanomas, we will use the CRISPR/Cas9 system to eliminate the expression of this gene in the murine lineage B16F0. After selecting stable transfectants, we will evaluate the new melanoma strain (B16F0.R9KO) regarding in vitro and in vivo tumor growth, as well as resistance to chemotherapy. Finally, the B16F0.R9KO strain will serve as a tool for further studies on the role of RASSF9 on intracellular biochemical pathways and other aspects of tumor biology. Footnote: FAPESP

Encapsulation and characterization of the immunodominant epitope SSIEFARL from HSV-2: a nanostructured immunoprophylactic system against herpetic recurrences.

Herpes simplex virus type 2 (HSV-2) is one of the most common sexually transmitted infections (STIs). HSV-2 commonly causes genital herpes affecting approximately 22% of adults over 12 years old. There are no vaccines or curative treatments for HSVs, and antiviral therapy has little impact on viral relapses. Viral replication occurs in epithelial tissue establishing dormancy in sensory neurons, periodically reactivating as localized recurrent lesions, and viral recurrence at specific sites is controlled by Tissue Resident Memory lymphocytes (TRMs). TRMs are epitope-specific and therefore SSIEFARL – an immunodominant epitope from the viral envelope’s glycoprotein B - could boost cell population and function. The increasing of SSIEFARL-specific TRMs could reduce viral relapses in HSV hosts. However, applying SSIEFARL directly to the mucosa is impracticable because of its microenvironment degradation. Therefore, the aim of this study is to encapsulate SSIEFARL as a delivery nanosystem to an HSV infected mucosa. In order to standardize this production system, size and uniformity analyses were performed by Dynamic Light Scattering (DLS) which showed an average size of 196 nanometers; the size was also measured by Mastersizer 2000 with an average of 222 nanometers and a span of 1.496. Moreover, we used Zetasizer to assess stability with a zeta potential of -7,85mV. The aforementioned stability was also verified storing nano/SSIEFARL at 4-8ºC for 30 days, and no microorganisms or precipitation were observed. Our method was standardized and validated for its linearity, precision, and accuracy. The release coefficient was measured for newly produced nano/SSIEFARL in High Performance Liquid Chromatography (HPLC) on day 1 and on day 30 after storing the formulations at 4-8ºC, presenting an average of 45% release after 4h. The encapsulation efficiency was performed by the ultrafiltration/centrifugation method using 100 kDa filters and quantified by HPLC showing a 43.99% efficiency. Toxicity analyses were performed by the MTT technique, demonstrating that nano/SSIEFARL do not present toxicity to splenocytes. In conclusion, we have standardized a peptide delivery system that is stable, biocompatible and noncytotoxic. Together, these promising results show a nanostructured immunoprophylactic system suitable for mucosal application that would not only resolve acute infection but would also prevent HSV recurrences.

ENDOCANNABINOIDS AND PAF PATHWAY CORRELATION TO THE SYSTEMIC INFLAMMATORY RESPONSE IN COVID-19 PATIENTS

INTRODUCTION: COVID-19, caused by SARS-CoV-2 virus, lead mild to severe complications, mainly through increased inflammation, cytokine storm, and production of bioactive lipids. The endocannabinoid system (eCB) is involved in several physiological processes and in the regulation of the immune response. PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is another lipid mediator that can be produced by different cell types, being a product of the cleavage of phospholipids from cell membranes during inflammatory processes. The study of these lipids pathway represents an advance in the understanding of COVID-19 disease pathology, since few treatments have significantly improved the clinical outcome, and it could be important therapeutic targets. Our objective was to profile the production of eCB and PAF in individuals infected with SARS-CoV-2 in the different clinical presentations of COVID-19 and to correlate with their effects on the immune response. METHODS: This is a cross-sectional study with 200 patients infected with SARS-CoV-2 and 35 control subjects. Data from quantification of lipid mediators in plasma were obtained by Mass Spectrometry and the patients clinical data compilation by medical/nursing questionnaire. RESULTS: Besides, established an analytical method for sample extraction and analysis, we demonstrated that the severity of COVID-19 influenced the production of 2- Arachidonylglycerol (2-AG) and decrease the production of PAF. This amplified production of eCB negatively correlated with systemic inflammatory parameters such as IL-6 and neutrophil counts in blood. However, PAF levels positively correlated with lymphocytes counts. The use of glucocorticoids influenced these lipids pathway by decreasing PAF and increasing 2-AG production. Furthemore, in the transcriptome analysis, we showed genes up-regultaion for the enzyme monoacylglycerol lipase (MGLL) and down-regultaion for phospholipase A2 (cPLA2). CONCLUSION: The increased formation of eCB and the reduction of PAF in severe cases of COVID-19 treated with glucocorticoids could be another point of inflammation and thromboembolism regulation, also representing an indirect pharmacological benefic side effect.

ENDOCYTOSIS OF COMMENSAL ANTIGENS BY INTESTINAL EPITHELIAL CELLS REGULATES MUCOSAL T CELL HOMEOSTASIS

EVALUATION OF ANTITUMORAL ACTIVITY OF RESVERATROL ANALOGS AGAINST BREAST CANCER

Introduction: Resveratrol (RVT) is a natural phytoalexin synthetized by plants as an answer to stress environment. This compound can be found in food sources including grape, red wine, nuts and soybean. Among its biological effects, the antitumoral activity can be highlighted due to its effect over several mediators in the initiation, promotion, and progression of cancer, beyond interfering in apoptosis and cell cycle. Despite its importance, RVT present low bioavailability, limiting its use in mammals. In order to solve this matter, a practicable alternative is to develop analogs that present improved chemical structure and preserve the biological functions of RVT. Thus, this study aimed to evaluate the antitumoral potential of six analogs of RVT (AR07, AR08, AR22, AR25, AR26, and AR33) in a murine model of breast cancer. Methods and results: First, the influence of compounds on cell viability was evaluated using J774A.1 (murine macrophage) and 4T1 (murine mammary adenocarcinoma), with MTT method. The next step was the evaluation of the analog's impact on 4T1 proliferation and migration with the MTT method and wound healing assay, in vitro. Then, in vivo, tumor growth and metastasis were evaluated in a murine model of breast cancer (protocol 020/2018). Results showed that analogs AR26 and AR33 presented the most promising activities with acceptable impact on J774A.1 cell viability (viability higher than 70%), being cytotoxic to 4T1 (AR26: 25% and AR33:70%). Furthermore, in 4T1 cells, compounds lowered proliferation (around 50%) and inhibited wound healing (AR26: 50% and AR33: 80%). In in vivo assays, both analogs inhibited tumor growth in 20% (AR26) and 43% (AR33), as well as lungs metastasis (AR26: 45% and AR33: 60%) and bone metastasis (AR26: 12% and AR33: 40%). Conclusion: The results showed that RVT analogs – AR26 and AR33 – have an important antitumoral activity, demonstrating a potential to compose new therapeutic strategies against breast cancer.

EVALUATION OF CELLULAR REPAIR OF DECELLULARIZED ZEBRAFISH HEART USING STEM CELLS: INVOLVEMENT OF PRO-REGENERATIVE MACROPHAGES

Introduction: Tissues obtained by bioengineering are a promising option for the shortage of transplants. There are several techniques for the synthesis of these materials, including decellularization, which uses as its main component the extracellular matrix (ECM) as a structure for repopulation of new cells. The advantage of using decellularized ECM is the low immunogenicity, one of the main factors responsible for organ rejection. Therefore, we aim to standardize the decellularization technique in the zebrafish (Danio rerio) model and to analyze the immune cells that are activated, in the process of re-cellularization, in particular the pro-regenerative M2 macrophages. Methods and results: Hearts were extracted from zebrafish for the application of the decellularization technique. The efficacy of this procedure was analyzed by DNA quantification (each group presented n=15 hearts, and on average the decellularized group presented a 90% reduction when compared to the control group). Histology of the sample groups was performed, and it was observed that the decellularized group did not present the presence of cells in the framework, demonstrating great effectiveness of the protocol. For the next stage of re-cellularization, the generation of transgenic zebrafish is in progress. In this regard, two synergistic genes related to high stem cell production (gata2a and runx1) have been combined with fluorescent proteins (GFP and mCherry); these constructs will be injected into zebrafish eggs and, in adults, will facilitate cell extraction from the zebrafish kidney. Future steps include differentiating these stem cells into M2 macrophages and injecting them along with stem cell-derived myocytes into the ECM scaffold. Conclusion: The zebrafish has been shown to be an auspicious model for organ bioengineering. In addition, the decellularization technique was effectively applied in removing cells from the ECM scaffold. And finally, elucidating the role of pro-regenerative M2 macrophages in regenerative processes could be helpful in understanding the role of this cell in transplantation dynamics.

EVALUATION OF HEMATIN ANHYDRIDE AS A NANOCARRIER IN CROSS-PRESENTATION.

Introduction: Hematin anhydride (HA) has been suggested as a potential adjuvant in vaccines. During the synthesis of HA gaps are formed between the nanocrystal domains, where it is possible, hypothetically, to couple substances. In addition, the synthesized crystals can also be coated due to their amphiphilic properties. High immunogenic adjuvants are required to promote T cells immune responses. In some diseases, such as leishmaniasis, CD8+ T cells activation is usually important, but it may be done by CD4+, hence the cross-presentation importance. Some patents used HA as an adjuvant in the context of bacteria and virus immunization, however, none of them explore the cross-presentation aspect in the vaccine context. The present study evaluates HA coupled or coated with ovalbumin (OVA) in CD8+ T cells activation through cross-presentation, aiming at its potential as an antigen nanocarrier. Methods and Results: HA was generated using the fast method, in some experiments to generate the coupled HA, OVA (25 mg) was added. To generate the coated HA the crystals were incubated for 30 min with OVA (0.5 – 1.5mg), washed, and used for the experiments. To determine the presence of proteins in the HA, SDS-PAGE was performed followed by silver staining. To validate cross-presentation, dendritic cells (DC2.4) were co-cultured were with MF2.2D9 (CD4+) or RF33.7 (CD8+) T cells line labeled with CFSE (10µM). The co-culture was maintained for 3-5 days and subsequently analyzed by flow cytometry. The results showed that treatment of DC cells with 50 and 200µg/mL of OVA-coupled HA induced 8.3% and 13.7% in CD4+ T cells proliferation, and 6.7% and 26.4% for CD8+ T cells. DC loaded with HA-OVA coated with (0.5, 1.0 and 1.5 mg of OVA) stimulated only CD8+ T cells proliferation (5.0%, 4.2% and 15.5%). Conclusion: The results indicated that HA coupled or coated with OVA induced T cells proliferation in the context of MHC class I and II, suggesting its potential nanocarrier capacity of activating antigen cross-presentation. Future studies will be carried to evaluate the role of HA in immunization against leishmaniasis.

EVALUATION OF IMMUNOMODULATION OF HUMAN ADIPOSE TISSUE INFECTED IN VITRO BY TRYPANOSOMA CRUZI

Adipose tissue (AT) has been gaining prominence in the last years due to its immunological properties and its potential as a reservoir of microorganisms. One of the parasites that can use AT as a reservoir tissue is Trypanosoma cruzi (T), which causes Chagas disease. The drug recommended for the treatment in Brazil is Benzonidazole (BZ), but its effectiveness in the chronic phase is controversial. However, the low effectiveness of the treatment may be related to the AT acting as a reservoir. Thus, this study investigated the effectiveness of treatment with BZ in human AT infected with T and examined the immunomodulation caused by the drug. Initially, we cultured human adipose tissue-derived stem cells (PT-5006, LONZA) and subsequently performed adipogenic differentiation, according to the manufacturer's recommendations. After differentiation, we performed four co-cultures where we infected the differentiated AT with the trypomastigotes forms of the Y strain of T at a ratio of 5:1 parasites/cell, and 48 hours post-infection, Bz (1µg/mL) was added. After 72 hours of treatment, the infected AT was submitted to qPCR for quantification of the parasite load in addition to transmission electron microscopy (MET). The supernatant was collected for cytokine dosage (IL-6, IL-10, IFN-γ and TNF) by cytometric bead array. Statistical analysis was performed by Wilcoxon test and taken at 5% significance level. The ethical aspects of the study are approved by the Research Ethics Committee - Fiocruz (CAE: 97930918.3.0000.5190). We observed that adipogenic differentiation was successful, since lipid vesicles were visualized. The same was observed in the quantification of the parasite load, where AT+T showed higher parasitaemia than AT+T+BZ, although there was no statistically significant difference between them. Regarding the production of cytokines, we observed that all the cytokines evaluated showed similar values between the conditions of culture, except the IL-6 where we found that AT+T+BZ showed lower levels compared to AT+T, being statistically significant (p=0.05). Therefore, we verified that although treatment with BZ decreased the parasite load, there was no statistically significant difference indicating that AT could explain the therapeutic failure. The decrease in IL-6 caused by BZ suggests that the drug may act by reducing the exacerbated inflammation in the tissue, which is beneficial for the host.

EVALUATION OF LIPOGENIC AND LIPOLYTIC PATHWAYS IN ADIPOCYTES DERIVED FROM HUMAN ADIPOSE TISSUE STEM CELLS (ADSC) UPON INFECTION WITH TRYPANOSOMA CRUZI AND TREATMENT WITH BENZNIDAZOLE

The adipose tissue (AT) is the regulatory organ of energy homeostasis, responsible for lipid metabolism, secretion of molecules such as hormones, cytokines, and adipokines. The dysregulation of AT can lead to the development of metabolic syndromes such as Diabetes Mellitus and insulin resistance. Several studies using murine models point the AT as a possible reservoir of Trypanosoma cruzi. However, it is not clear which are the alterations caused by T. cruzi in the lipid metabolism of AT in humans and the implications of these changes in the etiologic treatment. In the present study we evaluated the metabolic effects induced by Trypanosoma cruzi in human adipose tissue-derived stem cells (ADSC) differentiated into adipocytes, during Benznidazole (Bnz) therapy. Given this, after differentiation of ADSCs into adipocytes, we evaluated the genes involved in lipolysis and lipogenesis using relative expression (ΔCt) by RT-qPCR technique, we evaluated the genes involved in lipolysis: Adipose triglyceride lipase (PNPLA2), (Hs00982040_g1) and lipogenesis: fatty acid synthase (FASN) (Hs01005622_ m1) and Acetyl-CoA Acetyltransferase (ACAT1) (Hs00608002_m1), upon T. cruzi infection and treatment with Bnz for 72h. The parasite load in adipocytes was evaluated by q-PCR and high amounts of T. cruzi DNA were observed, demonstrating that this cellular model is permissive to parasite multiplication. However, treatment with Bnz had no significant impact on decreasing the parasite load. In addition, T. cruzi infected cells increased the expression of PNPLA2 (RQ: 3.934) when compared to non-infected cells (RQ: 1.000). A similar result occurred when we evaluated the expression of FASN (2.138). However, we found that Bnz induced more expression of PNPLA2 (RQ: 14.326) but not ACAT1 (RQ: 2.275) in infected and treated cells and in treated only cells. Given this, since PNPLA2 was the most expressed gene, we conclude that the lipolytic pathway is more utilized by adipocytes in both T. cruzi infection and Bnz treatment.

EVALUATION OF MICRORNAS IN ASTROCYTES DURING IMMUNOPATHOGENESIS OF MICROCEPHALY CAUSED BY ZIKV.

The Zika Virus (ZIKV) is a flavivirus that leads to neurological impairment characterizing the Congenital ZIKV Syndrome (CZS). Although neuronal precursor cells (NPCs) and neurons are the most affected cells. The astrocytes are more susceptible to ZIKV infection, since they tolerate higher viral loads, suffer less apoptosis and are used for viral replication. Curiously, it is known that several regulators of biological processes are involved in the susceptibility to CZS, including microRNAs (miRNAs) that have a fundamental function for embryonic development4 and in antiviral immune response5. MiRNAs work downstream of transcription factors by interacting with complementary mRNA target sequences for further post-transcriptional repression6. In this context, the aim of this study is to understand the role of miRNAs in the development of microcephaly caused by ZIKV in astrocytes. We showed that ZIKV activates transcription factors C/EBP and NF-B to increase Nlrp12 and Larp7 transcription and, consequently, miR-295 and miR-302d in SJL astrocytes. These miRNAs lead to decreased Ahrr, Neurod4 and Neurod6 gene expression, contributing to CZS development in susceptible SJL mice, but not in C57BL/6 WT resistant mice. Taken together, we suggest a mechanism of susceptibility to CZS development. Therefore, our data point miRNAs as potent regulators against ZIKV infection, which provide important clues not only about the pathogenesis of microcephaly caused by ZIKV, but also reveal mechanisms related to susceptibility and provide possible targets for therapeutic intervention.

EVALUATION OF MURINE MELANOMA CELLS (B16F10) TREATED BY PHOTOTHERMAL THERAPY THAT INTERNALIZED PLASMA FUNCTIONALIZED CARBON NANOTUBES.

New therapies against cancer, often based on nanotechnology, have been studied. The literature shows that multi-walled carbon nanotubes (MWCNTs) can be used for different medical applications. However, the most significant restriction for MWCNTs is the formation of aggregates in contact with water due to their hydrophobicity. The chemical plasma ablation process can alter the surface of MWCNTs, making them more hydrophilic. Exposure to plasma breaks the bonds in the surface layer of MWCNTs, allowing the binding of functional groups that make them more hydrophilic. This study aimed to evaluate a photothermal therapy (PTT) using 660 nm LED after the uptake of plasma-treated MWCNTs by murine melanoma cells (B16F10) in vitro. The uptake of MWCNT treated with plasma for 30 (MWCNT-F-30s) and 60 seconds (MWCNT-F-60s) by B16F10 and L929 murine fibroblast (non-tumor control cells) was evaluated. Then, we analyzed the ability of plasma functionalized MWCNTs to cross the membrane of those cells compared to non-functionalized nanotubes (MWCNT-0). After the internalization of the nanotubes, the cells were exposed to LED for 5 minutes for three consecutive days, and their viability was assessed by MTT. The results showed that the plasma ablation process increased the dispersion of MWCNT-F 30 and 60s in water without affecting their crystalline structure. Internalization and LED treatment were more effective in cells that internalized MWCNT-F-30s and MWCNT-F-60s after 72h. In L929, the same treatment stimulated the growth of these cells in vitro. In summary, our findings suggest that the plasma treatment reduced the hydrophobic character of MWCNT-F-30s and MWCNT-F-60s. Therefore, they were more internalized by B16F10 and L929 in vitro. This greater internalization of the nanotubes boosted the effects of the PTT. Our results are of great relevance and may help to understand and apply the plasma-functionalized MWCNTs as a therapy against cancer.

EVALUATION OF NETs IN POST-COVID PATIENTS WITH THROMBOEMBOLISM

Evaluation of plasmacytoid dendritic cell differentiation and migration during COVID-19 infection in an experimental model

Infection with the SARS-CoV-2 virus causes severe and mild clinical manifestations. In severe cases, there is a decrease and loss of function of plasmacytoid dendritic cells (pDC), which are important for viral replication control. Studies have also revealed that the gut microbiota of critically ill patients has a significant reduction in beneficial bacteria producing short-chain fatty acids (SCFA), such as butyrate. Bacterial metabolites may play a role in the hematopoiesis of immune cells, suggesting that butyrate may play a role in COVID-19 outcome. Therefore, the objective of this work is to compare the differentiation and migration of plasmacytoid dendritic cells during SARS-CoV-2 virus infection in an two experimental models (resistance and susceptibility). Therefore, experimental models of susceptibility (C57Bl/6 mice transgenic for human ACE2) and resistance (wild-type C57Bl/6 male mice) of COVID-19 were established, and the clinical parameters were evaluated during 6 days of infection. Infected hACE2 mice exhibited a severe clinical score when compared to infected WT, characterized by loss of body weight, decline in body temperature, score and mortality. Also, histopathology and viral load analyzes were performed in the lungs of the mice on the sixth day of infection. Notably, more diffuse inflammatory infiltrates, increased septa, decreased alveolar space and extravasation of red blood cells into the lung tissue were observed in the infected hACE2 lungs. In addition, a high viral load of SARS-CoV-2 was also detected in the lungs of infected transgenic mice. On the other hand, in the lung of the infected WT group, we found a scarse inflammatory infiltrate and an increase in viral load. In parallel, flow cytometry analysis to identify pDC population showed that, in the bone marrow, there was a decrease in pDC in the WT infected group, compared to the WT non-infected control group. However, the infected ACE2 group displays an increase in pDC cells in the bone marrow, compared to its non-infected control ACE2 group. The lung analyzes revealed that infected WT mice showed a trend to increase pDC, compared to the control WT, but the infected ACE2 micehad a lower pDC population in the lungs, compared to the ACE2 control group. In conclusion, the data indicate that in the susceptibility model there may be a failure of pDC migration from the bone marrow to the lungs. Future analyzes will be carried out to evaluate the role of butyrate in hematopoiesis.

EVALUATION OF THE ANTI-INFLAMMATORY POTENTIAL OF EXTRACTS FROM THE LEAVES OF MICONIA ALBICANS (CANELA-DE-VELHO)

Introduction: Miconia albicans (Melastomataceae), popularly known as canela-de-velho, is a plant species used in folk medicine. It can be found in pharmacies or herbalists in different pharmaceutical forms, such as tea, capsules, ointment, and gel. This species is used for arthritis and arthrosis treatment, pain relief, and inflammatory processes. Despite its popular use, scientific evidence of its activities is still scarce, suggesting that further studies need to be carried out to support its rational and safe use. Thus, given the demand for new alternatives related to the treatment of inflammatory diseases, as well as the importance of studies on popularly used plant species, the objective of this study is to evaluate the anti-inflammatory potential of ethanolic (MaEt), aqueous (MaAq) extracts. and hydroethanolic acid (MaAqEt) from M. albicans leaves. Methods and Results: First, the cytotoxic potential of the extracts was evaluated in J774A.1 cells (MTT test). Then, in order to assess whether the extracts are capable of acting on inflammatory processes, the production of nitric oxide (NO - Griess reaction) and cytokines (IL-6 - ELISA) in the supernatant of J774A.1 cells previously stimulated was evaluated. The results revealed that at the concentrations tested (200, 100, 50 and 25 µg/mL) all extracts have shown acceptable cytotoxicity (viability above 80%) for the tested cell.. Regarding anti-inflammatory activity, the extracts MaAqEt and MaAq proved to be very effective, significantly reducing the production of nitric oxide by 100 and 83.93±2.11%, respectively, at the highest concentration tested. Similarly, these extracts also impacted the production of IL-6, reaching a reduction of 100% (MaAqEt) and 95.97±0.40% (MaAq) at the highest concentration. Conclusion: So far, the results obtained propose a relevant anti-inflammatory activity of Miconia albicans extracts. Despite this, more comprehensive studies are being carried out to confirm this activity and assess other impacts on the immune system.

EVALUATION OF THE HUMORAL RESPONSE OF HTLV-1 CARRIERS AGAINST INFECTIONS CAUSED BY GRAM-NEGATIVE BACTERIA

Introduction: The Human T-cell Lymphotropic Virus type 1 (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic inflammatory disease that affects the central nervous system. Little is known of the humoral response of infected individuals, which currently suffer with recurrent opportunistic infections, such as urinary infections caused by E. coli (Nat Rev Microbiol. 13:269-284, 2015). Gram-negative bacteria express an efflux pump, involved in antimicrobial resistance, called AcrAB-TolC. TolC is conserved phylogenetically in bacteria and represents a new target to investigate the specific humoral response. Thus, this work evaluated the humoral response of people living with HTLV-1 against Gram-negative bacteria antigens such as LPS and TolC. Methods and results: For this purpose, sera were obtained from people living with HTLV-1 (HAM/TSP patients and asymptomatic carriers-AC) or uninfected individuals. IgM and IgG serum levels were evaluated by ELISA assays. Plates were sensitized with TolC-protein from E. coli (strain K12) or LPS (O111:B4). The median levels of IgG anti-TolC in the serum were similar between HAM/TSP patients and AC. However, there was a discrete increase in IgG anti-TolC in HTLV-1 carriers when compared to uninfected individuals. Moreover, approximately, 34% of people living with HTLV-1 presented high levels of IgG anti-TolC while only 7% of uninfected individuals exhibited the same levels. There was no significant difference between groups on IgG anti-LPS, while for IgM anti-LPS a significant higher level was observed in uninfected individuals, when compared to HTLV-1 carriers. Though, 10% of HAM/TSP patients presented high levels of IgM anti-LPS, suggesting an active infection. A positive correlation was found between IgG anti-LPS and IgG anti-TolC, but only 5 patients presented higher levels of IgG anti-LPS and anti-TolC simultaneously, and 15 out of 58 HTLV carriers exhibited elevated levels of anti-TolC. Conclusion: Our results suggested that IgG anti-TolC levels can be used to follow up the humoral response against Gram-negative bacteria in people living HTLV-1. Moreover, our results indicated that HTLV-1 carriers seem to have a more intense humoral response due to constant exposure to microorganisms, corroborating the susceptible state of these patients. Further studies are needed to better understand the differences in the humoral response of HTLV-1 virus carriers.

Evaluation of the immune response in different vaccine formulations containing the rSm14 of Schistosoma mansoni in mice

Introduction: Vaccines have revolutionized public health. However, the immunological mechanisms that lead to protection are still not fully understood. Adjuvants have gained importance due to their role in enhancing and directing the immune response in vaccination. Also, advances in understanding the activation of the innate immune system through pattern recognition receptors is improving the mechanistic understanding of adjuvants, which has enabled the development of new adjuvants that maintain their property of activating the immune response with low toxicity. Therefore, the selection of adjuvants to be used in vaccine formulations is an important matter to improve vaccine development. Objective: To evaluate the immune response induced by the rSm14 antigen of S. mansoni formulated with different adjuvants in mice. Methods: Mice were immunized (three doses in a fifteen-day interval) with rSm14 plus Freund’s; or rSm14 plus MPLA; or rSm14 plus MPLA plus Alum. The control groups were inoculated with saline and either Freund or MPLA or MPLA plus Alum, respectively. Serum and blood samples were obtained 15 days after each immunization dose to evaluate the production of specific anti-rSm14 IgG by ELISA and the cellular immune response, respectively. Results: The rSm14/MPLA induced a higher proportion of circulating total and effector CD4+ T cells compared to control group. Mice immunized with rSm14/MPLA plus Alum showed a higher proportion of effector memory CD4+ T cells than mice treated with saline/MPLA plus Alum. Decrease in the proportions of CD8+ T cells was observed in mice treated with rSm14/MPLA. The rSm14/Freund’s formulation decreased the proportion of effector and effector memory CD8+T cells, and increased proportion of circulating central memory CD8+ T cells. In addition, this formulation increased the proportion of circulating and memory B cells compared to saline/ Freund’s group. Higher levels of anti-rSm14 IgG antibodies were observed 15 days after the second immunization dose when rSm14/Freund’s or rSm14/MPLA were used as the vaccine formulations. After the third dose, the levels of IgG antibodies increased in all immunized groups. After the second and third doses, antibody levels were higher in mice immunized with rSm14/Freund compared to the other immunized groups. Conclusion: Our data show that the Sm14/Freund formulation induced a stronger effector and memory B cell response while Sm14/MPLA plus Alum induced triggers CD4+ memory response.

EVALUATION OF THE IMMUNE SIGNATURE IN RESPONSE TO CHEMOTHERAPY IN MMTV-PYMT MOUSE MODEL

Introduction: The knowledge of the leukocyte infiltrate allows us to understand and evaluate the efficiency of different antitumor therapies. Currently, anthracyclines followed by taxane are used for the chemotherapeutic treatment of breast cancer, but there is no biological basis to support this order. The NeoSamba Clinical Trial (INCA) demonstrated that there was better overall and disease-free survival in patients treated with the reverse order of chemotherapy arms: taxane followed by anthracyclines (FAC). Objectives: Based on the rationale of the NeoSamba protocol, our goal is to evaluate the impact of neoadjuvant treatment with combinations of FAC followed by taxane and the reverse order (taxane followed by FAC) on the leukocyte infiltrates in the model of breast cancer development in PyMT animals. Methodology: PyMT mice were treated for three weeks with each chemotherapy arm (FAC - 5-fluorouracil, doxorubicin, cyclophosphamide and/or taxane - docetaxel) with an interval of one week between arms. Tumors (untreated; treated only with FAC or with taxane; FAC-T and T-FAC) were collected when they reached a volume between 800mm³ and 1400mm³ or after the end of treatment and were referred for flow cytometry, immunohistochemistry and RNA sequencing (RNA-seq). Results: We performed initial studies to characterize the leukocyte tumor infiltrate in untreated and FAC-only (group FAC) or Taxane-only (group T) treated mice. In the FAC group, there was an overall increase in the CD4+ T cell population expressing IFNγ (Th1) compared to the untreated control and T groups. In the FAC and T groups, there was an overall decrease in the cancer stem cell population compared to the untreated control. There was also a decrease in endothelial cell population (CD34+) in the T group compared to the FAC and control groups. We also observed decreased percentage of TIM-3+ cells amongst CD3+ cells in the FAC and T groups when compared with untreated animals. Furthermore, we observed that TIM-3 is mostly expressed in CD4+ cells while PD-1 is expressed in CD8+ cells, although we observed no differences between treated and untreated groups. Next steps: As next steps, we will continue the treatments of the animals and qualitatively and quantitatively evaluate the leukocyte infiltrates after each of the chemotherapy blocks. Gene expression analysis will be performed in the samples already collected. By doing so, we hope to characterize the impact of different chemotherapeutic regimen.

EVALUATION OF THE MECHANISMS INVOLVED IN BCG IMMUNOTHERAPY IN A SYNGENIC MELANOMA MURINE MODEL

Introduction: Although the BCG vaccine has been clinically applied in the treatment of bladder cancer for more than 40 years and proposed in clinical trials for the treatment of melanoma since 1970, its mechanism of action still not fully understood. In the context of melanoma, BCG could act as an agonist to induce different innate immune pathways involving Toll-like receptors (TLRs), cytosolic DNA sensors or inflammasomes. Furthermore, BCG is also important to activate the adaptive immune response against cancers. Melanoma represents 4% of dermatological neoplasms diagnosed worldwide and is the most aggressive skin cancer due to the high incidence of metastasis. The purpose of this study is to analyze the role of innate immune pathways to better understand the mechanisms involved in the successful BCG immunotherapy against melanoma. Methods and Results: We first investigated the ability of BCG to infect the murine melanoma cell line B16-F10 by measuring the colony unit formation (CFU) and found that BCG can be internalized by the tumor cells, but less than bone marrow derived macrophages (BMDMs). BCG reduced the B16-F10 viability in a dose response manner after 24h and 48h of infection. We infected B16-F10 and BMDMs (positive control) with BCG (MOI 5, 10, 20 and 40) or stimulated with agonists for different innate immune pathways, such as Pam3CSK4, ultrapure LPS, CpG-ODN, Poly I:C and dsDNA90. After 24h, we measured the IL-6, TNF-α and CXCL10 production by ELISA and IFN-β expression by RT-PCR. B16-F10 were only capable to respond to poly I:C and dsDNA90 stimuli inducing CXCL10 and IFN-β. Moreover, we evaluated BCG intratumoral treatment in a B16-F10 subcutaneous mouse tumor model in C57BL/6 wild type (WT) and several knockout (KO) mice such as MyD88, TLR3, TLR4, TLR7, TLR3/7/9, cGAS, STING, IFNAR, Caspase 1, Caspase 11 and Rag. So far, none of the above studied molecules investigated seems to be related to the drastic BCG impact for tumor reduction. Conclusion: BCG induces IL-6 and TNF-α in BMDMs, but not in B16-F10. Tumor cells were only activated by poly I:C and dsDNA90, that may be considered as potential adjuvants for cancer treatment. BCG intratumoral injection dramatically reduces melanoma. BCG intratumoral injection dramatically reduces melanoma in WT and KO mice. Our future perspective is to better understand the immune response induced by BCG in melanoma using other tumor models.

EVALUATION OF THE PARTICIPATION OF MACROPHAGES IN THE DEVELOPMENT OF FIBROSIS AND REGENERATION IN CARDIAC CRYOINJURY MODEL IN ZEBRAFISH

Introduction Infarction is a pathological process characterized by the death of a region of cardiac tissue caused by a lack of blood supply at that site, generating a fibrotic scar. This condition affects a large part of the world population, around 126 million people. In zebrafish, cardiac tissue regeneration occurs, without loss of function, when there is a simulation of infarction, with macrophages being one of the cells that orchestrate the regenerative process. Studying the processes leading to this complete regeneration amplifies understanding and allows physiology extrapolation to humans and new therapies development. Methods and Results All analyses were made 3, 7, 13, and 21 days after injury. Ablation of macrophages will be performed using zoledronic acid (ZA) encapsulated in liposomes, and lipidomic and single-cell RNA sequencing analyses will be conducted. We established a quantification methodology of the ZA inside the liposomes using high-performance liquid chromatography (HPLC), and their size was measured using the dynamic light scattering (DLS) technique. We injected liposomes in adult wild-type zebrafish every three days before the analysis. The liposome sizes were larger than expected, and the amount of ZA chosen for the tests was toxic to the fish. The cryoinjury model was standardized through histology and electrocardiogram (ECG). The histological analysis showed an environment characteristic of the recovery injury, but this still needs further characterization. The ECG data analysis is currently in process. Moreover, tests for lipidomics were carried out, and a preliminary assay was able to establish that an N of 7 animals per group is sufficient for the tests. We have samples of macrophages on all days of analysis, frozen, and waiting for the lipidomic tests. Conclusion We have established various methods, and preliminary analysis suggests that our model agrees with other similar models from the literature. The analysis shown here along with a more detailed description of the macrophages’ profiles will help construct a scientific basis for innovative technology generation.

EVALUATION OF THE PROLIFERATIVE EFFECT OF IN VITRO BREAST CANCER CELLS EXPOSED TO LATEX FROM AVELOS (Euphorbia tirucalli) AND JANAUBA (Synadenium grantii Hook f.)

In popular culture, Avelos (Euphorbia tirucalli) and Janauba (Synadenium grantii) are used to treat different types of diseases. These plants are known as “milk that cures cancer” in several regions of Brazil. Breast cancer is the most common malignancy among women. Cancer progression to metastasis is the leading cause of death in these patients. Different people report that they have already used this latex to treat breast cancer, regardless of the stage of the disease or whether treatment. In a study carried out by our research group, we observed that specific concentrations of bottled Avelos and Janauba stimulated the growth of the lung carcinoma strain in vitro, which could be disastrous for patients who use this type of resource. The objective of this work was to evaluate the activity of Avelos and Janauba latex diluted in water at different concentrations (100, 50, and 25%) natural (AN and JN) and filtered (AF and JF) on breast cancer cells (MCF- 7) in vitro. MCF-7 cells were distributed in a 96-well plate and treated with different concentrations of latex for 24, 48, and 72h. MTT method determined the cell viability. Cell dead was measured by the incorporation of annexin V and propidium iodide (PI). The results showed that both avelos and janauba at different concentrations stimulated the growth of MCF-7 in vitro compared to the untreated control. The assessment of concomitant use with a chemotherapeutic agent was tested when cells were exposed to AN, AF, JN, and JF solutions (100%) together with 5μg/mL of Doxorubicin (Dox). The results showed that the latexes did not cancel the antiproliferative effect of Dox in vitro. However, these solutions induced cell death by apoptosis (Annexin V+/PI-) in MCF-7 cells. However, the addition of Dox changed this pattern of death to necrosis (Annexin V+/PI+). The results are preliminary but suggest that latex from avelos and janauba diluted in water can stimulate the growth of breast adenocarcinoma cells in vitro. The use of latex with Doxorubicin induces necrosis in MCF-7 in vitro, activating the inflammatory response.

EVALUATION OF THE ROLE OF P2X7 RECEPTOR ON GC-TFH CELL DEATH IN EXPERIMENTAL MALARIA

Introduction Amongst cells that participate in the immunological response against Plasmodium, germinal center-T follicular helper (GC-TFH) cells play a key role in antibody production, which is essential for symptom resolution and parasite control. GC-TFH cells express high levels of the ATP-gated P2X7 receptor (P2X7R). Persistent stimulation of P2X7R has been associated with distinct cell death pathways in different cell types, including caspase-1-mediated pyroptosis and caspases-3 and 7-mediated apoptosis; however, the consequences of P2X7R activation in TFH cells remain unclear. In this study, we evaluated the role of P2X7R in TFH cell death in an experimental malaria model. Methods and Results C57BL/6 mice expressing P2X7 (B6) and knockout for P2X7 (P2rx7-/-) were evaluated after 14 days of infection with Plasmodium chabaudi; spleen cells were kept in culture for 0, 1, 3 or 4h. GC-TFH cells from B6 mice, but not from P2rx7-/- mice, showed increased mortality over time when compared to non-TFH cells, indicating that P2X7R-dependent cell death is a phenomenon specific to TFH cells. Moreover, TFH cells from B6 mice, but not from P2rx7-/- mice, showed increased caspases-1 and 3/7 activation over time, suggesting that downstream events of P2X7R stimulation may be the activation of these caspases. Next, we analyzed spleen cells from B6 mice on day 14 p.i. in presence of caspases-1 and 3/7 inhibitors (YVAD-FMK and Z-DEVD-FMK, respectively). There was a decrease in GC-TFH cell death in the presence of both inhibitors, indicating that GC-TFH cells may be susceptible to both P2X7R-dependent pyroptosis and apoptosis. Conclusions In our experimental malaria model, GC-TFH cells are more susceptible to P2X7R-mediated cell death than non-TFH cells. The mechanism triggered after P2X7R stimulation may be the activation of caspases-1 or 3/7, which would characterize the cell death as pyroptosis or apoptosis. Further experiments are needed to completely understand the cell death pathways present in these cells.

EVALUATION OF THE ROLE OF RUBICON IN THE REGULATION OF MACROPHAGE METABOLISM

Introduction: Autophagy plays an important role in the regulation of cell homeostasis upon stress, but its components play non-canonical roles in diverse cell processes. For instance, autophagy components can be recruited to phagosomes (LC3-associated phagocytosis, or LAP) and endosomes (LC3-associated endocytosis, or LANDO), regulating cell responses from these compartments. Besides the recruitment of the autophagic components, LAP and LANDO require the recruitment of specific proteins, such as the molecular adaptor Rubicon (RUBCN). RUBCN is essential in the elimination of dying cells by macrophages, leading to the secretion of anti-inflammatory cytokines and functional polarization of these cells into an anti-inflammatory phenotype. Conversely, defects in Rubicon impair dead cells processing in macrophages, with an associated production of inflammatory cytokines, such as IL-6, IL-1B and IFNB .Herein, we investigated the role of Rubicon in the regulation of macrophage function in response to stimuli that can trigger non-canonical autophagy. Methods: Wild-type (Rubcn+/+) or RUBCN-deficient (Rubcn-/-) bone marrow-derived macrophages (BMDM) were stimulated with apoptotic thymocytes (AC) or LPS to evaluate the role of glycolytic metabolism in RUBCN-dependent regulation of macrophage function. We first evaluated the expression of glycolytic enzymes in Rubcn-/- versus Rubcn+/+ BMDM by qPCR. We also evaluated the role of RUBCN in the regulation of cytokine secretion by ELISA. Last, we investigated if RUBCN deficiency affects HIF1a transcription factor that regulates macrophage responses. Results: RUBCN ablation increases the expression of enzymes of the glycolytic cascade in response to AC. Rubcn-/- BMDM produce low levels of IL-6 in response to AC, which was inhibit in the absence of glucose. Additionally, HIF-1α expression is increased in Rubcn-/- macrophages treated with AC. Conversely, the expression of glycolytic genes and HIF-1a were reduced in Rubcn-/- BMDM in response to high dose LPS, known to be endocytosed. HIF-1α stabilization was reduced in response to LPS in Rubcn-/- macrophages. Conclusion: Our data suggest that RUBCN modulate glycolytic pathway and cytokine secretion in response to the engulfment of apoptotic cells and endocytosed LPS. Our preliminary findings showing the opposite role of RUBCN in responses driven by these stimuli suggest that RUBCN is a rheostat for the regulation of macrophage metabolism and HIF1a function in macrophages.

EVALUATION OF THE TRYPTOPHAN METABOLISM IN THE MASSIVE GENERATION OF ANTIBODY-SECRETING CELLS IN DENGUE PATIENTS THROUGH THE TRANSCRIPTOME OF SINGLE BLOOD CELLS

Introduction Dengue is the most prevalent arbovirus infection worldwide, which manifestations range from asymptomatic to severe. Of note, an exacerbated and transient antibody-secreting cell (ASC) response is detected in the blood of patients nearly 7 days of symptomatology. During that period, the ASC frequency may represent more than 50% of all circulating B cells. This quantification is higher than those obtained upon viral infections, immunizations, and even ASC-related neoplasias (e.g. J Virol 86:2911-8, 2012). Moreover, the magnitude of this response directly correlates with the disease severity. Hence, it has been demonstrated that DENV particles can stimulate the B cell differentiation into ASCs upon incubation with human PBMCs derived from healthy donors. This process was associated with an enhanced expression of IDO1 and IDO2 in the PBMCs and increased tryptophan (TRP) consumption and kynurenine (KYN) accumulation in the culture supernatants (e.g. Front. immunol. 11:20, 2022). If the activation of the TRP metabolism is the cause or consequence of the ASC generation in this model, it remains elusive. Methods and Results To investigate the TRP role in the ASC generation derived from Dengue infection in vivo, we have analyzed the public transcriptome (GSE116672 study) of blood single cells. Using the RStudio program and the Seurat package, we initially performed a pre-processing analysis to reinforce the data quality control, normalization and dimensional reduction. Then, distinct cell subsets derived from blood were clustered (T and B cells, natural killer cells, monocytes and dendritic cells). This procedure enabled the identification of distinct B cell subsets from different groups (healthy controls, moderate or severe Dengue infected patients). Finally, we evaluated the expression of the main genes related to the TRP metabolism pathways using the MetaCyc platform in each patient group. Conclusion The knowledge about what cell types are involved in the TRP metabolism and the ASC response may facilitate the understanding of the Dengue pathogenesis.

EVALUATION OF TICK ANTIMICROBIAL PEPTIDES BY THE TRANSCRIPTOMIC ANALYSIS OF DEFENSINAS IN RHIPICEPHALUS MICROPLUS SALIVA

Pathogen resistance to antimicrobial drugs is one of the main threats to public health and has mobilized investigators in the quest for alternative antimicrobials that can replace conventional therapies. Antimicrobial peptides (AMPs) are a promising category of such molecules, including those secreted by hematophagous arthropods, such as Rhipicephalus microplus ticks. This parasite secrets diverse and abundant repertoire of AMPs, which participate in its innate immunity to control entomopathogens within it, and possibly affect its host’s skin microbiota. Herein, we aim to evaluate with in silico tools the potential as antimicrobials of select AMPs from R. microplus. Initially, we screened an annotated sialotranscriptome R. microplus to find a specific AMP family, the Defensins. Thirteen coding sequences were selected using the following criteria: they were secreted (i.e., contained a signal peptide) and complete (initiating with methionine). The protein domains were found with the Pfam algorithm (EMBL-EBI). The Random Forest algorithm of CAMP platform evaluated the amino acid sequences in order to find the antimicrobial-determining regions, i.e., peptides of approximately 20 amino acids within the protein that are responsible for the microbicidal activity. Sequences found with a score greater than 0.5 were selected within the AMPs Rm-nr-107206, Rm-tb2-114592, Rm-nr-114593, Rm-nr-32621, Rm-nr-113771, Rm-nr-89218, Rm-nr-77882, Rm-nr-77883, Rm-nr-103279, Rm-tb2-103420, Rm-nr-78288, Rm-nr-77881, Rm-tb2-7185. Among these, Rmnr-78288 was the one with the highest probability in one of the sequences, with a 0.933 score. The physical and chemical parameters were evaluated using ProtParam (Expasy). The Robetta algorithm was used to predict the peptide’s 3D structure, and aggrescan3D 2.0 demonstrated the high solubility, a positive feature for clinical use. Thus, our results elected 13 peptides (~20 amino acids each) most likely to be tested against pathogens with resistance to conventional therapies.

Evolutionary loss of Neu5Gc influences intestinal microbiota and natural antibody repertoire

Every cell in the body is decorated with a layer of glycans known as the glycocalyx. These glycans are attached to proteins and most of them terminate in sialic acid, a nine-carbon sugar that mediates many protein-ligand interactions. Sialylated glycoproteins from human cells terminate only with the N-acetylneuraminic acid (Neu5Ac) isoform of sialic acid, a pattern that differs from the majority of other mammals, whose glycans can also terminate with N-glycolylneuraminic acid (Neu5Gc), since humans lost the Cmah gene which encodes the enzyme that hydroxylates Neu5Ac in Neu5Gc. Because sialic acids can act as self-associated molecular patterns (SAMPs) and previous studies revealed that Neu5Gc loss increased activation and effector functions of immune cells in humans, we sought to investigate if the absence of Neu5Gc in humans could be related to the autoreactivity profile of natural antibodies (NAbs). To test this hypothesis, we blotted serum or B cell culture supernatants (SNs) from WT and human-like Cmah-null C57BL/6 mice against WT and Cmah-null mice proteins from muscle lysates. Our data show increased binding of IgM from Cmah-null mice serum and SN to both WT and Cmah-null mice muscle lysates when compared to Igs from WT mice, suggesting that the absence of Neu5Gc might influence IgM reactivity against self-antigens. Moreover, SNs’ binding is directly influenced by glycoproteins present in the muscle extract, since peptide N-glycosidase F (PNGase-F) and neuraminidase treatment directly modulated both WT and Cmah-null SNs IgM binding to the extract. Since gut microbiota directly interferes with the NAb repertoire, we evaluated IgA bound to fecal bacteria using flow cytometry. Results show a higher number of IgA+ bacteria in WT mice compared to Cmah-null mice in a Neu5Gc-dependent manner. To further evaluate the dynamics of the microbiota, we performed a WT versus Cmah-null mice co-housing experiment, evaluating IgM binding at 0, 15 and 30 days post co-housing. Our data show increased binding of serum IgM after 15 or 30 days in co-housing in Cmah-null, but not in WT mice serum IgM, associated with the modulation of the alpha- and beta-diversity of the gut microbiota, as seen in RNAseq analysis of 0 and 30 days post co-housing feces. Taken together, our data suggest that the evolutionary loss of Neu5Gc directly interferes in the natural antibody repertoire and that gut microbiota plays an important role in this modulation.

Exhaustive exercise-induced metabolic and inflammatory response is disturbed via TLR-4 pathway

Acute exhaustive exercise leads to metabolic and inflammatory changes in serum, skeletal muscle, liver and adipose tissue, and was previously demonstrated that these alterations might be dependent of the inflammatory pathway such as Toll-like receptor 4 (TLR-4). This study aimed to investigated the role of TLR-4 on metabolic and inflammatory in adipose tissue after acute exhaustive exercise. Mice wild type C57BL/6J (WT) and TLR-4 knockout (TLR-4(-/-)) submitted to euthanasia immediately (E0 group), 2 (E2 group), and 6 h (E6 group) after the exhaustive exercise, which consisted of running on a treadmill (approximately 70% VO2max) for 50 min and immediately followed by 1m/min increased increment running until exhaustion. The sedentary control group (C group) was not subjected to exercise. The metabolic profile (glucose, lactate, triacylglycerol, and free fatty acid concentrations), gastrocnemius and liver lipid extraction, liver and soleus muscle glycogen content, as well as, inflammatory profile Interleukin 6 (IL-6), Interleukin 10 (IL-10) and Tumor necrosis factor α (TNF-α) of the mesenteric and retroperitoneal adipose tissues were evaluated to determine the role of TLR-4. Results: A higher concentration of TAG and lower gastrocnemius and lipid content were found in TLR-4(-/-) group compared with WT group (p<0.05). In mesenteric adipose tissue a significantly lower IL-10 concentration in TLR-4(-/-) immediately after (0h) exhaustive exercise compared with WT group (p<0.05) and a significantly lower TNF-α concentration in TLR-4(-/-) after 2h of exhaustive exercise compared with WT group (p<0.05). Regarding to cytokines content in RPTA adipose depot, there was a significantly lower IL-6 and IL-10 concentrations in TLR-4(-/-) immediately after (0h) acute exhaustive exercise compared with WT. Furthermore, the lower IL-6 concentration in TLR-4(-/-) group was also observed after 2h acute exhaustive exercise compared with WT group. Conclusion: TLR-4 are involved not only in host defense but also act as an immunomodulatory effector of exercise-induced metabolic and inflammatory response and its absence may alter inflammatory cytokines release.

EXPRESSION AND CHARACTERIZATION OF ANTIBODY FRAGMENTS AGAINST SARS-CoV-2 FUSION PEPTIDE

Introduction In December of 2019 a new coronavirus was isolated in Wuhan province, China. SARS-CoV-2 is the etiological agent of covid-19 and since the second half of 2020, multiple variants of SARS-CoV-2 have emerged. Glycoprotein Spike (S) is the main target for neutralizing antibodies among the virus proteins. It binds to the ACE2 cell receptor via the Receptor Binding Domain (RBD), mediating fusion to the cell membrane, which culminates in viral entry. Spike glycoprotein (S) is a homotrimer, and each of its 180kDa monomers may be divided into two subunits, S1 and S2. The RBD is located at the S1 subunit. The S2 subunit contains the Fusion Peptide (FP), which is a small conserved region among coronaviruses and consequently among SARS-CoV-2 Variants of Concern (VOC), suggesting it contains broadly neutralizing epitopes. Here we isolated human antibody fragments reactive with a designed mimetic FL as putative SARS-CoV-2 neutralizing antibodies. Methods and Results Antibodies against the SARS-CoV-2 synthetic FP were selected by Phage Display technology using a human V gene combinatorial library. The selected VH and VL domains were determined by combining NGS and bioinformatic approaches. After that, two antibodies in scFv (single chain fragment variable) format were proposed combining the best selected VHs and VLs. Synthetic genes encoding the scFvs were designed and synthesized chemically. The genes were cloned into the pET-SUMO vector, which allows high expression of proteins in the soluble fraction. The resulting vector was confirmed and used to transform Shuffle pLys Y strain bacterial cells, which facilitate the expression of antibodies due to their oxidant cytoplasm. ELISA assays were performed to analyze the binding of scFvs to SARS-CoV-2 synthetic FP. Conclusion We obtained good results in the expression and purification of the scFvs, which also showed the ability to bind to SARS-CoV-2 synthetic FP. Next, the scFvs will be analyzed for the binding to different SARS-CoV-2 variants and neutralization tests by PRNT (plaque reduction neutralization test) will be performed.

EXPRESSION OF AN ANTIGENIC EFFLUX PUMP PROTEIN FROM E. COLI ASSOCIATED WITH ANTIMICROBIAL RESISTANCE, FOR THE DISCOVERY OF POTENT ANTIBODIES

Introduction: The increase of infections caused by multidrug-resistant bacteria has been a public health problem in the last decades. Bacteria develop resistance mechanisms dodging the antimicrobials that already exist. The frequent emergence of new resistant strains threatens to turn the existing antibiotics useless and, the development of new antimicrobials does not keep up with demand, since few antibiotics were approved in the last years (COPHAR. 48:48–56, 2019). The objective of this study was to express the TolC protein, which composes the MacA-MacB-TolC efflux pump of E. coli, that contributes to resistance of antibiotics (Nat Microb. 2, 2017), and evaluates its immunogenicity in order to discover potent antibodies against E. coli. Methods and results: The TolC protein was expressed in E. coli BL21(ƛDE3). Competent BL21(ƛDE3) was transformed with a pET-30a plasmid carrying the TolC gene by heat shock and cultured in a Luria-Bertani (LB) broth with 50 μL/mL of kanamycin. The positive clone was subjected to an expression test in LB medium with antibiotic at 15 ºC, overnight with agitation and 1mM of IPTG (isopropyl β-D-thiogalactopyranoside) as an inducer. The induced and non-induced clones were analyzed by SDS-PAGE. Expression was then performed in 4 L of medium. The buffer used to recover the protein included 50 mM Tris-HCl pH 7.5, 250 mM NaCl, and 0,01 % Chaps. The protein was expressed as inclusion bodies, which were solubilized in the buffer containing 8 M urea. The purification was performed in a His-Trap affinity column in the presence of 8 M urea. Fractions were combined after elution, concentrated, and re-folded the protein using a buffer composed of 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 200 mM L-arginine, 1 mM glutathione reduced, 0,1 mM glutathione oxidized e 10 % glycerol. Our preliminary results show the TolC protein can be expressed by this protocol and re-folded efficiently (0,589 mg/mL of purified protein), keeping its structure. The protein was recognized by IgG and IgM antibodies from the serum of TolC-immunized BALB/c mice (CEUA 109-19), tested through ELISA assays. Conclusion: TolC recombinant protein was successfully expressed in E. coli and re-folded. The recombinant protein was able to bind serum IgM and IgG from TolC-immunized mice. Further studies are necessary to test the antibodies against antimicrobial resistant E. coli.

EXPRESSION OF RECOMBINANT CHIKUNGUNYA VIRUS E2 PROTEIN IN EUKARYOTE CELLS FOR USE IN DIAGNOSTIC PLATFORMS

Chikungunya fever, caused by the Chikungunya virus (CHIKV), includes symptoms such as fever, arthralgia, myalgia, and headache. About 40% of patients develop a chronic pathological condition for months or years. The symptomatology and epidemiological profile of CHIKV is similar to other arboviruses, which makes clinical diagnosis difficult in Brazil. POC (point of care) serological tests are cheap and easy to apply, however, highly antigenic recombinant proteins are required to compose a sensitive and specific test. Recombinant proteins expressed by prokaryotes are absent in post-translational modifications, which may affect antigenicity. This project aims at the expression of CHIKV E2 protein in eukaryotic platforms with prospection for the development of serological tests in the Lateral Flow Immunochromatography and ELISA format. The E2 envelope protein was chosen in this project because it is the main target of the humoral response. E2 covers most of the CHIKV spike and has 3 putative glycosylation sites and 4 disulfide bonds. The E2 sequence was obtained by creating a consensus sequence based on Brazilian samples of Asian lineage and ECSA deposited in GenBank. The TPA (tissue plasminogen activator) signal peptide sequence was added for protein secretion and a histidine tag for purification. The transmembrane and hydrophobic regions were predicted by TMHMM and Protscale-Expasy softwares and removed. The constructed E2 sequence will be stably expressed in mammalian cells, HEK293T (Human embryonic kidney cells) and CHO2353 (Chinese hamster ovary cells), by integration into the genome by a lentiviral vector. The RFP (Red Fluorescent Protein) gene was integrated into the genome of HEK293T cells by lentiviruses for standardization of transduction. After selection with puromycin, the fluorescence of the RFP was evaluated by flow cytometry, and about 99% of the cells showed emission of RFP fluorescence. RFP fluorescence was also visualized in 100% of the cells for one month using fluorescence microscopy. A preliminary essay of transfection of HEK293T cells with the pcDNA 3.1 plasmid containing the E2 gene was performed to assess transient expression. Anti-CHIKV E2 antibodies recognized a protein of about 60 kDa. HEK and CHO cells were recently transduced with the E2 gene, the expressed recombinant protein will be evaluated for its antigenicity by ELISA, and the structure evaluated for glycosylations.

Expression profile of mac-1 and vla-4 cell adhesion molecules in pulmonary eosinophils and macrophages in mouse experimental model of bystander suppression.

The bystander suppression effect can be defined as immunomodulatory mechanisms generated in tolerogenic microenvironments to co-localized antigens, suppressing harmful immune responses to uncorrelated antigens. This phenomenon can be a promising strategy to improve allergen specific immunotherapy (AIT) protocols to Hymenoptera venom allergy, since the induction of allergen specific tolerance, by classical AIT protocols, in some cases, can induce side effects such as intense inflammatory reactions and anaphylaxis. Here we evaluate the effect of oral tolerance induction protocol to ovalbumin (OVA) in an experimental mouse model of allergen sensitization to Polybia paulista (P. paulista) wasp venom, analyzing, after the antigen challenge, the frequency of macrophages and eosinophils in bronchoalveolar lavages (BAL) as well as the expression of MAC-1 (CD11b) and VLA-4 (CD49d) in both cells’ populations. METHODS AND RESULTS: BALB/c mice (n=10) were previously sensitized with 6 weakly intradermal doses of 20μg of P.paulista venom. After 7 days, half of the mice (Treated, n=5) were submitted to oral treatment with OVA (4mg/mL in drink water), and the control group (n=5) received just water for 7 days. After this period, mice received 2 intraperitoneal (i.p.) doses containing 10μg of OVA, and after seven days were challenged with an i.p. dose containing 10μg of OVA and 20μg of P.paulista venom. After 30 minutes from the challenge, the mice were sacrificed, and BAL obtained through intratracheal injection of 1mL of Hank's balanced saline solution (HBSS) and aspiration by 3 times. All the procedures were approved by Institutional Ethical Committee (Protocol#5654-1/2020). The BAL cells suspensions were used to cytospin slides preparation using Panoptic staining and to flow cytometry assays to quantify the frequencies (%) of F4/80 + CCR3 + (eosinophils) F4/80 + CCR3 neg (macrophages) and the CD11b and CD49d expression (MFI). After the final challenge, oral OVA-treated mice showed no differences in the macrophage or eosinophils frequencies. However, presented na increased expression of VLA-4 (CD49d), but not in MAC-1 (CD11b) in both cell populations, when compared to the control group. CONCLUSION: These results suggest that oral tolerance, induced to uncorrelated protein antigens, could interfere in the expression of cell adhesion molecules in the effector cells (macrophages and eosinophils) involved in the immune response to P.paulista wasp venom allergens.

EXTRACELLULAR VESICLES CARRYING MIRNA-181B CARGO DERIVED FROM PLASMA OF BREAST CANCER PATIENTS MODULATE GSKIP AND INDUCE DENDRITIC CELL PHENOTYPE TOWARDS TOLEROGENIC PROFILE

INTRODUCTION: The mechanisms by which tumor cells can drastically alter the systemic immune response are still elusive, but suggest a systemic mode of cell communication. The secretion of EVs by the tumors may be a possible mechanism for this modulation. METHODS: We evaluated the ability of EVs, purified from plasma of patients with invasive ductal breast cancer (BC-EVs) and healthy donor (HD-EVs) to promote the phenotypical and functional modulation of monocyte-derived dendritic cells (Mo-DC) in vitro. Mo-DCs were differentiated in the presence of BC-EVs (Mo-DC/BC-EV) or HD-EVs (Mo-DC/BC-EVs) and then activated with LPS and analyzed by flow cytometry. The ability of these DCs to activate T cells was tested in a mixed lymphocyte reaction (MLR). In vivo murine aggressive breast tumor 4T1 or mild breast tumor E0771 were evaluated for expression miR-181b. RESULTS: BC-EV/Mo-DC showed signs of maturation arrest with decreased expression of HLA-DR, CD80, CD86, CD11c, and increased expression of PD-L1 when compared to HD-EV/Mo-DCs. These changes were associated with impaired ability of BC-EV/Mo-DCs to induce CD4+ and CD8+ T cell proliferation and expression of pro-inflammatory cytokines such as IFNγ, but increased induction of Tregs, IL-10 and PD1 expression by T cells. These changes were associated with impaired mTOR activation and maintenance of GSK3β pathway in Mo-DC/BC-EVs after LPS activation of DCs. BC-EVs were enriched for miR-181b-5p a miRNA that regulates GSK3β interaction protein (GSKIP), which promotes the inactivation of GSK3β. Inhibition of miR-181b with specific siRNA restored BC-EV/Mo-DCs pro-inflammatory phenotype upon LPS activation. Finally, the presence of miR181b was also evaluated in two different murine models of breast cancer and the presence of miR181b was related to the aggressive metastatic model. Indeed, high levels of miR-181b in breast cancer are associated with a higher mortality rate. CONCLUSION: miR-181b carried by BC-EVs can impair the DC maturation through modulation of the GSK3β/mTOR axis inducing a suppressive immune state. This mechanism may be associated with cancer immune evasion and the formation of metastases.

Extracellular vesicles directly inhibit T cell activation – a cancer immune escape mechanism

Cancer cells display several immune escape mechanisms, many of them are constricted to the tumor microenvironment, while others like soluble mediators and extracellular vesicles (EVs) can have local and systemic effects. EVs are an important communication tool among cells. In general, cancer cells not only secrete more EVs than their normal counterparts, but they also change their cargo. EVs have a role in the establishment of pre-metastatic niches, resistance to therapy and immune suppression. We have shown that cervical cancer displays several local and systemic immunomodulatory effects, promoting tolerance towards tumor antigens. We are working on the hypothesis that EVs secreted by cervical cancer cells are part of the immunomodulatory effects displayed by these cells. We have isolated EVs from different cervical cancer derived cells and have shown that they are up taken by monocytes and B cells, but not by T cells after 24 hours of incubation. However, EVs added to peripheral blood mononuclear cells treated with phytohemagglutinin were able to significantly inhibit T cell proliferation in 30%, IL-2 secretion (1-fold less), and induce a 3-fold increase in IL-17 secretion. Now, we are working on replenishing IL-2 to the EVs treated cell cultures to verify if it can rescue T cells. Taken together our data strongly suggests that EVs are part of cervical cancer immunomodulatory mechanisms acting as immunosuppressants on the immune system.

Extracts from the roots of Lonchocarpus cultratus decreases the secretion of pro-inflammatory mediators from RAW 264.7 macrophages.

In folk medicine, the use of extracts from plants happens from decades and it has been the focus of many scientific researches for the search of new components to treat many conditions. Lonchocarpus cultratus is a type of tree, different parts of this plant may possess medicinal uses, such as anti-parasitic effect from the extracts of the seeds and the roots of this plant, seeing in previous research in our lab. Together with previous anti-trypanosoma and leishmanicide effect, and with the presence of terpens in the extract, our aim was to evaluate the possible immunomodulation effect of the methanolic extract from the roots (LMR) L. cultratus. Previously LMR were dissolved in dimetilsulfoxide (DMSO), and then RAW 264.7 macrophages were treated under different conditions: only medium DMEM (control); lipopolysaccharide (LPS) 0,1µg/Ml; LMR 175 µg/mL; LPS 0,1µg/mL + LMR 175 µg/mL; and DMSO control. After 30 minutes of treatment the expression of phospho-ERK 1/2 protein was measured using Western blot technique. After 24 hours of treatment the following markers was measured: the nitrite secretion using Griess reaction; anion superoxide activity; tumoral necrosis factor alpha (TNF-α), interleukin-10 (IL-10) using enzyme-linked immunosorbent assay; and arginase activity. LMR 175 µg/mL inhibited the secretion of nitrite by RAW 264.7 stimulated by LPS, together with that the anion superoxide activity was also decreased under LMR 175 µg/mL treatment. The extract also did interrupt the secretion of TNF-α by RAW 264.7 stimulated by LPS, whereas it did not influence IL-10 secretion and arginase activity. The phosphorylation of ERK 1/2 protein was also not influenced by LMR 175 µg/mL extracts. Altogether LMR 175 µg/mL extracts abrogates the pro-inflammatory profile of RAW 264.7, being promising to be used as an immunomodulatory component.

FEEDING WITH Lactococcus lactis EXPRESSING HSP65 INDUCES REGULATORY T CELLS IN PANCREATIC LYMPH NODE AND CONTROLS HYPERGLYCEMIA IN MICE WITH TYPE 1 DIABETES

INTRODUTION: Heat-shock proteins (HSPs) are chaperones highly expressed after cellular stress, which acts by degrading misfolded proteins. As they are highly conserved across species, heterologous HSPs are being associated with probiotics and recently used as therapeutic strategies to induce tolerance in autoimmune disease models. Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by an inflammatory process in the pancreas that culminates in the destruction of insulin-producing β cells, leading to hyperglycemia. New therapeutic strategies capable of preventing the development of the disease or controlling blood glucose are important to avoid the complications associated with T1D. AIM: In this work, we evaluated the effects of recombinant HSP65 (rHSP65) on the activation of bone marrow-derived dendritic cells (BMDC) from C57Bl/6 mice and evaluated the effects of L. lactis expressing HSP65 in the STZ-induced type 1 diabetes (T1D) experimental model (CEUA 007/2020). RESULTS: Our results demonstrate that BMDC stimulated with rHSP65 have increase in percentage and expression by mean fluorescence intensity (MFI) of MHC-II and CD80 in CD11c+ cells after 24h of stimulation when compared to control group. Furthermore, rHSP65 increased CD11c+CD11b+CD103+ cell percentage and higher expression of MHC-II and CD80 by MIF after 24h of stimulation. In addition, rHSP65 also increased CD11c+CD11b-CD103+ cell percentage after 24h, with intermediate levels of MHC-II and CD80 expression, indicating a semi-mature state of these cells. Interestingly, rHSP65 induced increased production of IL-10 by BMDC when compared to LPS-treated cells. In vivo, we observed that diabetic mice feeding with L. lactis expressing HSP65 had lower hyperglycemia and higher insulin expression in the pancreas when compared to the diabetic group of diabetic mice (STZ only). Furthermore, administration of L. lactis-HSP65 increased the number of cDC1 (CD103+ CD11b-, tolerogenic profile) TLR2+ in the cecal lymph node (CLN) and the number and percentage of regulatory T cells (CD3+ CD4+ Foxp3+) in the pancreatic lymph nodes compared to mice that received only STZ. Our data show that HSP65 has an immunoregulatory role, and that the association between L. lactis and HSP65 may be a potential therapeutic alternative to glycemic control in patients with T1D.

FOOD ALLERGY DIAGNOSTIC TEST TO PEANUTS AND CASHEW NUTS: INFLUENCE OF THE WAY OF OBTAIN PROTEIN EXTRACTS

Introduction: Food allergy is a pathological process, and its diagnosis is not always easy. Contact with a certain food triggers a cell-mediated inflammatory immune response, which can cause respiratory, cardiovascular, gastrointestinal and/or neurological symptoms. The specificity of the diagnosis of food allergies still falls short of what is desired, as false-negative and/or false-positive reactions are frequently verified. Previous works have shown that the type of protein extraction from the food matrix and its processing can generate different results in an ELISA test. Objective: To identify the homologous and heterologous antigen/antibody reactivity pattern of peanut (Arachis hypogaea) and cashew nut (Anacardium occidentale L.) extracts obtained with four extraction buffers. Methods and Results: For the extraction of peanut and cashew nut seed proteins, four buffers with different pH were used: pH 10 - Borate Buffer without mercaptoethanol (TB), pH 8 ,8 - Borate Buffer with β- mercaptoethanol (TB-2βME), pH 7.2 - Saline Buffer (TS) and pH 6.8 - Tris / HCl buffer (TT /HCl). The band profile was made by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Using serum from C57BL/6 mice, the specific immunogenicity of each peanut and cashew nut allergen was established using the Western Blot technique. From the use of different protein extracts, different protein profiles were generated. The buffer that extracted the highest amount of protein was borate with the addition of β-mercaptoethanol for both seeds, however, it was the also one that obtained lower reactivity in the western blot compared to other extracts. The buffers used in this work successfully extracted the main allergens from both seeds. The reactivity of western blots showed a very heterogeneous pattern for peanuts, while for cashew nuts the reactivity profile of the bands was more homogeneous for bands with Ana o 1 presence, but the same does not occur for a band that appears in the presence of Ana o 2. Conclusion: The type of protein extraction influences the diagnostic test due to the difference in reactivity when crossing homologous and heterologous extracts and sera in peanuts and cashew nuts.

FUNCTIONAL STUDY OF THE ROLE OF CD99 PROTEIN IN THE TUMORIGENESIS PROCESS OF CERVICAL CANCER: PARTIAL RESULTS

Gasdermin-D activation in response to Leishmania infection induce a transient cell permeabilization to promote NLRP3 activation and host resistance to infection

Leishmania is an obligate intracellular parasite that causes Leishmaniasis, a disease that affects millions of people worldwide. Leishmania evades immune response by inhibiting specific processes on parasite-containing immune cells, yet the NLRP3 inflammasome activation is key for disease outcome. The molecular mechanisms upstream of the inflammasome activation are still unclear and there is no evident host cell death in Leishmania-infected cells. Here, we investigated the participation of Gasdermin-D (GSDMD, a pore-forming effector protein associated with pyroptosis) during Leishmania infection in macrophages and in vivo. We demonstrated that despite the absence of pyroptosis, GSDMD is active at the early stages of L. amazonensis infection in macrophages, allowing a transient cell permeabilization and potassium efflux, promoting NLRP3 inflammasome activation. Gsdmd–/– macrophages exhibit less ASC puncta formation and IL-1β production in response to infection, suggesting that the transient GSDMD-mediated permeabilization contributes for NLRP3 inflammasome activation. Mouse and macrophages deficient in GSDMD were highly susceptible to infection by several Leishmania species, including L. amazonensis, L. major, L. braziliensis and L. mexicana, confirming a key role of Gasdermin-D for inflammasome-mediated host resistance to infection. Finally, ASC/NLRP3 puncta and cleaved Gasdermin-D were present in skin biopsies of leishmaniasis patients, supporting the role of these molecules during active disease in humans. Altogether, our findings reveal that Leishmania infection triggers a transient activation of GSDMD and this molecule is critical for inflammasome activation and immunity in Leishmaniasis.

GENERATION AND CHARACTERIZATION OF ANTI-CD19 CAR-T CELLS OVEREXPRESSING THE PROTEIN PHF19

Introduction: CAR T cells are lymphocytes genetically modified to express a synthetic receptor that recognizes surface antigens. CAR-T therapy cost is largely impacted by using clinical-grade viral vectors but can become more economically feasible with the use of transposon-based systems. Another limitation is short-term persistence of functional T-cells in vivo, restricted by acquisition of an exhausted phenotype. Recent work showed that Phf19, a member of the PRC2 silencing complex, can induce formation of memory cells and avoid terminal differentiation of CD8+ lymphocytes. Thus, we aimed to overexpress Phf19 on CAR-T cells generated with transposon systems Sleeping Beauty (SB) and piggyBac (PB). Methods and Results: Phf19 sequence followed by an LNGFR reporter was cloned in the pT3-19BBZ and PBCAG-19BBZ CARs, transposon plasmids for the SB and PB systems, respectively. To generate human CAR-T cells, we electroporated peripheral blood mononuclear cells from healthy donors with the SB or PB transposase along with the respective transposon plasmid. Cells were activated with anti-CD3/CD28 beads and expanded for 8 to 12 days in media with IL-2. T cell phenotype was assessed by flow cytometry, and we evaluated lytic capacity through Calcein-AM lysis assay in a co-culture with CD19+ leukemia cell line Nalm-6. We successfully established a co-expression system of the CAR and the Phf19 protein along with a LNGFR reporter in a single tri-cistronic construct (Phf19+ CAR-T). We obtained around 2 million CAR+ cells per well with transfection rates of 20-30% on day one that decreased to 10-15% on day 12. We did not observe significant differences in CAR-T cell lytic capacity in Phf19+ cells. However, Phf19 overexpression led to increased formation of central memory-like cells (21.5% +- 3.2 19BBz vs. 37.2% +- 5.4 Phf19+, p=0.018), and reduction of effector memory-like phenotype. Surprisingly, we also observed increased levels of the inhibitory receptor PD-1 in Phf19+ cells, particularly within the CD4+ compartment (MFI 772 +- 130 control vs. 1355 +- 340 Phf19+, p=0.009), but not of TIM-3. Conclusion: These results suggest an ambiguous role for Phf19 in CAR-T cells that seems to differ between CD8+ and CD4+ subtypes and paves the way for assessment of the in vivo function of these cells as well as transcriptional and epigenetic characterization. We hope this work can support new strategies to generate cost-effective CAR-T cells while ameliorating cell phenotype.

GENERATION AND OPTIMIZATION OF anti-CD19 CAR-T CELL FOR LEUKEMIA IMMUNOTHERAPY

CAR-T cell immunotherapy has achieved high response rates in treatment-refractory patients with B-cell malignancies. This therapy involves the genetic modification of T cells isolated from patients to express an anti-CD19 CAR (Chimeric Antigen Receptor). Those cells are expanded in vitro and then re-infused into the patient. CAR-T cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe and recently in Brazil. To adapt this protocol to our local reality, we are developing simple and less costly manufacturing protocols to meet the increasing demand for this therapy. The main goal of this work was to generate anti-CD19 CAR-T cells with a short in vitro expansion protocol based on the non-viral Sleeping Beauty (SB) transposon-based vector system. The 19BBz CAR sequence was cloned in the transposon vector pT4/HB. PBMCs were collected from healthy donors after signed board–approved informed consent. Mononuclear cells were isolated by density gradient centrifugation and electroporated with plasmids enconding 19BBz CAR and the SB100x transposase. The expansion of CAR-T cells was performed using G-REX culture wells for 8 to 12 days. For the xenograft mouse model, NOD-SCID IL2R gamma null (NSG) mice were injected on the tail vein with Nalm-6 Luc-GFP cells and treated 2 days later with 2.5 x105, 5 x105 CAR-T or 3 x106 cells or control cells expanded but not expressing the CARs. For in vivo imaging, mice were injected i.p. with d-luciferin and tumor burden was evaluated by bioluminescence. For cell analysis, cells or organs were processed and analyzed by flow cytometry. Using the protocol described herein we generate from 9 x107 total PBMCs a mean of 1.34 x107 CAR-T cells after 8 days of expansion. CAR-T cells generated showed cytotoxic effect against CD19+ leukemia cells in vitro. Furthermore, CAR-T cells treatment improved the survival rates (40% at 2,5 x105 dose after 37 days, 77% at 5 x105 dose after 86 days and over 60% at 3 x106 after 68 days) of NSG mice inoculated with 1x 105 Nalm-6 B-cell leukemia. The improved survival was accompanied by a significative reduction in the tumor burden. Finally, infused CAR-T cells persisted for up to 50 days, showing that they are capable of long-term persistence and antitumor response. Our current protocol can generate a cellular product compatible with regulatory requirements and sets the groud to test these CAR-cells in a phase I clinical assay.

GENETICALLY MODIFIED LACTOCCUS LACTIS INDUCES ORAL TOLERANCE AND PREVENTS FOOD ALLERGY TO β-LACTOGLOBULIN IN MICE

Introduction: oral tolerance is an active, antigen-specific response that starts in the gut following contact of local immune cells with diet and microbiota antigens. This tolerogenic response prevents unnecessary inflammation and promotes gut homeostasis. As such, disturbances in this physiological process are associated with intestinal disorders like inflammatory bowel disease, celiac disease, and food allergies, including milk allergy, which is a common food allergy disorder affecting children and adolescents worldwide. Thus, a therapeutic tool aiming for oral tolerance reinforcement capable of treating milk allergy would be of great interest. In mice, milk allergy is associated with increased serum levels of anti-β-lactoglobulin IgE and IgG1 antibodies, as well as weight loss and aversion to antigen consumption. Objective: in this present study we tested a protocol for oral tolerance induction to reverse experimentally-induced milk allergy in mice. Methods: a genetically-modified Lactococcus (Lactococcus lactis) containing a β-Lactoglobulin (BLG)-producing plasmid was used for feeding. Mice were treated for four days with either BLG-producing L. lactis or L. lactis containing an empty plasmid. This was done before and after sensitization with 20µg of BLG absorbed into AlOH3 and a later boost containing BLG and saline. To assess efficacy, one week later animals were orally challenged with a solution containing 20% of whey protein for seven days, mimicking allergen consumption. Results: pre-treatment of mice with BGL-producing L. lactis prevented weight loss, decreased serum levels of anti-β-lactoglobulin IgE and IgG1 antibodies, and reduced aversion to allergen consumption. However, the protocol failed to reverse allergy signals after sensitization. Conclusion: oral treatment of mice with BGL-expressing L lactis avoids milk allergy development but fails to treat previously sensitized animals.

GENETIC PANEL GENERATED BY MACHINE LEARNING PREDICTS RISK OF SEVERE EXACERBATION IN REGULARLY TREATED SEVERE ASTHMA PATIENTS

Introduction: Severe exacerbations of asthma are the main contributors to the increase in health costs related to asthma, morbidity and mortality. However, there is a need for additional predictors for this outcome. The aim of this study was to evaluate the performances of ten machine learning algorithms in the prediction of severe asthma exacerbations using top Single Nucleotide Variants (SNVs) from a previous Genome Wide Association Study (GWAS) for severe asthma exacerbations in individuals with regurlaly treated severe asthma. Methods and results: This study was carried out with 364 Brazilians with severe asthma of the ProAR cohort. Severe exacerbation was defined as use of systemic corticosteroids ≥ 3 days and/or emergency room visit and/or hospital admission in the last year. Genotyping was performed using the Illumina Multi-Ethnic Global Array (MEGA, Illumina) in 223 exacerbators cases and 141 non-exacerbators controls with severe asthma of the ProAR study. After performing a GWAS for severe asthma exacerbations, the variants with p value < 0.05 were filtered and the feature selection method was done by Boruta. The prediction models were built using the following algorithms: K-nearest neighbors (KNN), Naive Bayes, Artificial neural networks, Support Vector Machine, Bagging, AdaBoost, Classification and Regression Trees (CART), C5.0, Random Forest and XGBoost. After the integration of all algorithms, the best predictive algorithm was the C5.0. The final panel had 14 variants, that were considered the most important markers for the C5.0 algorithm to effectively predict the chances of being an exacerbator among individuals with severe asthma, with an overall accuracy of 81%, an Area Under the Curve (AUC) of 98%, a specificity of 83% and a sensibility of 76%. Conclusion: Our results suggests a genetic panel with 14 variants for identification of the exacerbators, among patients with severe asthma. More studies are needed to assess the functional effect of these variants in the risk of exacerbation of severe asthma.

GENETIC POLYMORPHISM OF Plasmodium falciparum PROTEINS MSPDBL-1 AND MSPDBL-2 AND ITS IMPACT ON B AND T IMMUNODOMINANT EPITOPES IN BRAZILIAN MALARIA ENDEMIC AREAS

Introduction: Merozoite Surface Protein Duffy Binding-like -1 and -2 (MSPDBL-1 and MSPDBL-2) are proteins that bind directly to MSP-1 on the surface of the parasite, forming a complex on the erythrocyte surface through its DBL domain facilitating the invasion of P. falciparum into the erythrocyte. Considering that the extensive genetic diversity exhibited by P. falciparum is an important factor for the parasite's evasion of the host immune system, we believe that the presence of polymorphisms in the genetic regions encoding the MSPDBL-1 and -2 proteins may modulate specific immune response. Here, we propose to identify genetic polymorphisms and to evaluate the impact of these polymorphisms on the potentially antigenic regions in the Duffy Binding-like (DBL) and Secreted Polymorphic Antigen Associated with Merozoite (SPAM) domains of the MSPDBL-1 and MSPDBL-2 proteins in P. falciparum isolates circulating in Brazilian-malaria endemic areas. Methods: Sixty-five isolates from Cruzeiro do Sul and Mâncio Lima (Acre State) and Guajará (Amazonas State), Brazilian Amazon, were collected. Genomic DNA was extracted, the gene region encoding DBL and SPAM domains was sequenced and the impact of polymorphisms on the potentially antigenic regions was evaluated. Intrapopulation genetic diversity and Tajima’s D test values were calculated using specific software’s. B-cell and T-cell epitopes were identify using BCPreds and IEBD, respectively. Results: Twelve and forty polymorphisms were identified in the SPAM and DBL domains of MSPDBL-1, respectively. The MSPDBL-2 protein was more conserved, with three and one polymorphism in the respective SPAM and DBL domain. In the genetic analysis of the population, the DBL domains presented positive values for the TDT, while the SPAM domains were negative, however we did not observe statistically significant values. The nucleotide diversity was higher in MSPDBL-1 (0,00430 ± 0,00054) than in MSPDBL-2 (0,00046 ± 0,00015). In silico prediction analysis of immunodominant B and T epitopes, considering the 3D7 reference sequence, showed fifteen B-cell epitopes and fifteen T-cell epitopes that are recognized with a frequency of 50% to 82% of MHC antigens. Conclusion: Our data suggest that P. falciparum isolates that circulate in Brazilian Amazon present an extensive genetic diversity and the gene region that encodes MSPDBL-1 presents a greater genetic diversity when compared to MSPDBL-2, mainly in the gene region encoding DBL domain.

GLOBOTRIAOSYLCERAMIDE ACCUMULATION INDUCES INFLAMMASOMES ACTIVATION IN A CATHEPSIN-DEPENDENT MANNER

Fabry disease (FD, OMIM 301500) is a X-linked lysosomal storage disease (LSD), characterized by deficiency or total absence of α-galactosidase A (α-Gal) enzyme due to mutations in GLA gene. This leads to progressive accumulation of globotriaosylceramide (Gb3) in lysosomes of a variety of cells, affecting several organs as kidney and heart. However, Gb3 accumulation itself does not fully explain the different phenotypes observed among FD patients and it is believed that secondary physiopathological processes are elicited upon Gb3 storage. In this context, an increasing number of studies indicate a pro-inflammatory profile in FD patients, suggesting the activation of innate immune pathways, including inflammasomes. Inflammasomes are intracellular multiproteic complexes responsible for caspase-1 activation, and the consequent maturation and secretion of IL-1b and IL-18 cytokines. The role of inflammasome in LSDs remains to be elucidated and could represent an important therapeutic target since these complexes significantly affect the progression of diverse inflammatory diseases. Thus, the aim of this study was to evaluate the activation of inflammasomes in response to the accumulation of Gb3 in the presence or absence of deoxygalactonojirimicin (DGJ), an inhibitor of α-Gal, thus mimicking the profile observed in FD patients. IL-1β levels and ASC punctas formation in PMA- differentiated THP-1 cells were significantly higher in cells treated with Gb3 and DGJ, compared to these treatments alone (IL-1β: Gb3 vs Gb3+DGJ: p = 0.0064; DGJ vs Gb3+DGJ: p = 0.0017) (ASC punctas: Gb3 vs Gb3+DGJ: p = 0.0042; DGJ vs Gb3+DGJ: p = 0.0031), indicating the inflammasomes assembly and activation in response to the Gb3 accumulation. To understand the mechanism involved in inflammasome activation in response to the Gb3 accumulation, THP-1 cells were treated with Ca-074Me, a cathepsin B inhibitor. Upon treatment with Ca-074Me, IL-1β levels significantly decreased in both Gb3 and Gb3+DGJ treatments (IL-1β: Gb3 vs Gb3+Ca: p = 0.04; Gb3+DGJ vs GB3+DGJ+Ca: p = 0.025). Together, these data suggest that Gb3 accumulation induces inflammasome activation in a cathepsin B- dependent manner. Further studies remain to be performed to clarify the impact of inflammasome on progression and physiopathology of Fabry disease.

GLUCOSE METABOLISM IS UPREGULATED IN THE MONONUCLEAR CELL PROTEOME DURING SEPSIS AND SUPPORTS ENDOTOXIN-TOLERANT CELL FUNCTION

Introduction: Metabolic adaptations shape the function of immune cells. During sepsis, leukocytes from patients show inhibition of cytokine production while other functions such as phagocytosis and production of reactive oxygen species (ROS) are preserved, which characterizes immunomodulation similar to that observed in endotoxin tolerance in vitro. Here, we sought to investigate how cellular metabolism is related to the modulation of the immune response in sepsis and endotoxin tolerance. Methods: Peripheral blood mononuclear cells were obtained from 24 patients with sepsis and from 9 healthy volunteers, lysed and used for label-free proteomic analysis. Altered metabolic pathways were identified in the patients’ proteome with Ingenuity Pathway Analysis, and metabolism-related pathways were filtered out of the canonical pathways with a p-value corrected for BH < 0.05 and a z-score as a predictor of inhibition or activation. PBMCs from healthy volunteers were pre-stimulated for 48h with lipopolysaccharide (LPS), and incubated with a challenge dose of LPS (100 ng/mL) for 24h. Tolerance induction was assessed by the production of TNF-α by flow cytometry. Metabolic activity was analyzed by ATP quantification, glucose uptake and lactate quantification. The influence of metabolic pathways in cellular function was evaluated in phagocytosis, ATP and cytokine production using specific metabolic inhibitors. Results: Proteomic analysis in PBMC from septic patients obtained at ICU admission showed an upregulation of proteins related to glycolysis, pentose phosphate pathway (PPP), production of reactive oxygen species and nitric oxide, and downregulation of proteins in the TCA cycle and oxidative phosphorylation compared to healthy volunteers. Using the endotoxin-tolerance model in healthy PMBCs, we observed increased lactate production and glucose uptake in control cells upon LPS stimulation, while endotoxin-tolerant cells presented inhibited TNF-α and lactate production along with preserved glucose uptake and phagocytic capacity. Conclusion: Our study describes that PBMCs from patients with sepsis presented a phenotype compatible with a metabolic modulation towards glycolysis. In addition, analyses of PBMC from healthy volunteers in an endotoxin-tolerance model indicate that glucose metabolism supports leukocytes’ functions in a condition of immunomodulation, similar to sepsis, with both preserved (phagocytosis) and suppressed (TNF-α production) cellular response.

GLUTAMINE METABOLISM AND ITS EFFECTS ON THE TUMOR MICROENVIRONMENT.

Tumor cells display metabolic reprogramming, of which preferential use of glycolysis is the best known alteration. However, many tumor cells concomitantly use glutaminolysis, and some of them are known as glutamine addicted. The use of glutamine, by glutaminolysis, provides metabolic intermediates for the Krebs cycle, for the synthesis of macromolecules and for the redox balance. Tumor cells and immune cells share metabolic similarities and tumors are capable of modulating the immune response through the competition for nutrients and the generation of metabolites. In our laboratory, we have previously demonstrated the influence of lactate on the tumor associated macrophages (TAM) phenotype. Studies in the literature show that the inhibition of glutaminase, the enzyme that converts glutamine in glutamate, in TAM, negatively impacts the M2 phenotype, which potentially has pro-tumor activity. Cervical cancer has a complex tumor microenvironment, infiltrated by different leukocyte populations, including macrophages, which our group and others have shown to display a M2 biased phenotype and to be important for tumor growth. Our hypothesis is that the inhibition of glutaminolysis will decrease tumor growth, both due to the direct negative effects on tumor cell metabolism, but also by facilitating a change in macrophages towards a cytotoxic phenotype. We worked with the cervical cancer derived cell line, SiHa, in co-culture with peripheral blood mononuclear cells, or the THP-1 monocyte lineage. We used tumor spheroids, seeking to better mimic what would be a microenvironment in vivo. Preliminary results demonstrated cytotoxicity of glutaminase inhibitors BPTES and compound 968 in SiHa monocultures and cocultures of tumor cells and monocytes. Both inhibitors led to about a 50% reduction in the number of live cells, both in monocultures and cocultures. In addition, cell cycle analysis of these treated cultures, from DAPI labeling, revealed an accumulation of tumor cells in the SUB-G1 phase (dead cells). It is worth mentioning that we had very interesting preliminary data, the increase in the percentage of dead tumor cells when co-cultured with THP-1 cells previously incubated with LPS and treated with glutaminolysis inhibitors. Now, we are evaluating surface markers of monocytes after culture in cocultures with tumor cells and with the blocking of glutamine metabolism in order to understand the influence of this environment on the phenotype of the immune cell.

GOLD NANOPARTICLES CONJUGATED WITH ANTI-RBD ANTIBODIES FOR HIGHLY SENSITIVE DETECTION OF SARS-COV-2

GUT EPITHELIAL CELL DYNAMICS DURING ANTIBIOTIC-INDUCED MICROBIOME DEPLETION

The crosstalk between the gut microbiota and host metabolism is a complex and important process for normal physiology. The intestinal epithelial cells (IEC) play a fundamental role in this relationship, building mucosal barriers, secreting immunological mediators and providing bacterial antigens. Furthermore, the metabolites produced by the microbiome also plays direct effects on the metabolism of these cells. However, the consequences of microbiota-host crosstalk remain incompletely understood, as well as the mechanisms in which it occurs. We dissected this using single-cell transcriptomics in 9,019 colonic IEC from 4 individuals between controls and antibiotic-induced microbiome depleted (AIMD) mice. Here we show the effects of AIMD that generate an inter-and intracellular remodeling of the colonic epithelium. We identified the main IEC present in the colon, including progenitor, secretory and absorptive cell types. By comparing the control and treated groups, we mapped the impact of AIMD on maintaining IEC proportions. We saw that treatment leads to a significant reduction in the number of enteroendocrine cells and a decrease in progenitor cells. In addition, we reveal the cellular specificity of the host response to the microbiome, as AIMD leads to a different transcriptional profile modulation for each IEC. We show that reduced cell types also exhibit reduced activity. Progenitor cells show lower expression of genes involved with the cell cycle, and hormone genes for enteroendocrine cells are also reduced. Furthermore, AIMD results in a functional change in principal mature IEC. We describe two populations of enterocytes that show different gene expression profiles relevant to the AIMD. One of these populations is more observed in the control group and has a transcriptional profile focused on the interaction with the microbiota, presenting genes involved in the metabolism of microbiota-derived metabolites and response to bacteria. In the AIMD group, another population with an altered transcriptional profile related to energy-metabolism emerges. We hypothesized that the remodeling generated by AIMD is directly related to chromatin modifications. Especially such modifications that are known to be microbiota- and short-chain fatty acids dependent. Altogether, this study highlights a new overview of how AIMD generate a remodeling of the colonic epithelium and suggests a link between these changes and epigenetics modifications.

GUT MICROBIOTA FROM PATIENTS WITH MILD COVID-19 CAUSE ALTERATIONS IN MICE THAT RESEMBLE POST-COVID SYNDROME

There is mounting evidence that SARS-CoV-2 targets tissues beyond the respiratory tract. Long-term sequelae after COVID-19 are frequent and of major concern. Prolonged virus detection in the gut has been particularly intriguing. Of note, SARS-CoV-2 infection also disturbs the gut microbiota composition, a finding linked with disease severity in patients with COVID-19. Here, we aimed to characterize the functional role of the gut microbiota in the long-term consequences of COVID-19. To this end, we characterized the gut microbiota from COVID-19 human subjects and followed the effects of human fecal transfer to germ-free mice. The gut microbiota of post-COVID subjects (up to 4 months from the initial positive test) revealed a remarkable predominance of Enterobacteriaceae strains with multidrug-resistance phenotype compared to healthy controls. After fecal transfer to germ-free mice, animals receiving samples from post-COVID subjects displayed higher lung inflammation and increased susceptibility to pulmonary infection caused by an antimicrobial resistant Klebsiella pneumoniae strain. These mice also showed poorer cognitive performance associated with increased expression of TNF-, reduced levels of brain-derived neurotrophic factor-BDNF and postsynaptic density protein-PSD-95 in the brain, as well as alterations of several biochemical pathways. These alterations were observed in the absence of SARS-CoV-2, suggesting that alterations in the gut microbiota caused them. Our results show prolonged impact of SARS-CoV-2 infection in the gut microbiota that persists even after the individuals have cleared the virus. Increased Enterobacteriaceae with antimicrobial resistance phenotype were of particular concern. Moreover, microbiota transfer from post-COVID subjects induced loss of brain cognitive functions and impaired lung defense in mice. Altogether, our work emphasizes the importance of microbiota as a target for therapies to help treat post-COVID sequelae.

HEMATOLOGICAL PARAMETERS CAN BE USED AS PREDICTIVE FACTORS FOR COVID-19 OUTCOME

Introduction: COVID-19, caused by the SARS-CoV-2 virus, is a novel infectious disease that has been characterized by the World Health Organization as a pandemic. The disease can manifest itself in different forms classified as asymptomatic, mild, moderate, severe, or critical, with the need for hospitalization. Thus, biomarkers of disease outcome are essential to stratify patients' risk, consequently improving clinical management and efficient use of scarce medical resources. A complete blood count (CBC) is a viable option to find biomarkers that predict the severity of COVID-19, since the infection can alter various blood parameters. Therefore, the goal of the present study is to identify a possible association between hematological parameters and different prognosis of COVID-19, using them as predictors of disease outcome. Methods: We performed a CBC with 300μL of whole blood samples from 101 individuals from Belo Horizonte, Brazil, using the automated hematology analyzers Sysmex XN-550 and Bayer ADVIA 60 Hematology Analyzer. These individuals were divided into three groups: Healthy Controls (n=51), Mild COVID-19 (n=38) and Hospitalized COVID-19 (n=12). All subjects were tested using RT-qPCR for SARS-CoV-2 and had their blood collected between 1-4 days of symptoms. Kruskall-Wallis and Dunn’s multiple comparisons tests were used to identify correlations between CBC parameters and disease severity. Receiving Operating Characteristic (ROC) curve was performed to validate the parameters’ accuracy. The research protocol was approved by COEP-UFMG and the National Ethics Committee. Results: Among all hematological alterations, the only ones capable of differentiating the Mild COVID-19 from Hospitalized COVID-19 groups were the percentage of monocytes (MON%) (p=0.0046), hemoglobin concentration (p=0.0370) and granulocyte to lymphocyte ratio (GLR) (p=0.0365), while the platelet to lymphocyte ratio (PLR) was the best at differentiating control groups from COVID-19 patients (p<0.0001). ROC curve validated MON% as the best parameter to differentiate mild COVID-19 from hospitalized COVID-19 patients (p=0.0014) and from healthy controls (p<0.0001), while GLR best differentiates healthy controls from hospitalized COVID-19 patients (p<0.0001). Conclusion: Our data suggests that MON% and GLR could be used as early predictors of severe disease, discriminating patients at hospital admission in the beginning of infection.

HEME IMPAIRS CELLULAR RESPIRATION AND INDUCES MITOCHONDRIAL FISSION IN MACROPHAGES

Introduction. Heme is a cofactor of hemoglobin and can be released during hemolysis. Free heme is a potent inducer of reactive oxygen species (ROS), including mitochondrial ROS (mtROS), and has pro-inflammatory effects on macrophages. Early activation of spleen tyrosine kinase (Syk) and mitogen-activated protein kinases (MAPK) ERK, JNK and p38 in cells exposed to heme is critical for TNF and Cxcl1 release and depends on ROS and mtROS generation, suggesting a role for mitochondrial function in this signaling axis. Also, scavenging mtROS protects mice against death by sterile hemolysis. Thus, this work aims to investigate the effects of heme on mitochondria and its potential role as a signaling platform for the activation of Syk and MAPK in macrophages. Methods and Results. Firstly, we show that heme abrogates mitochondrial oxygen consumption in bone marrow-derived macrophages (BMDM) as early as 5 minutes upon stimulation, as indicated by a 50% reduction in routine respiration and by an 86% decrease in reserve respiration capacity when compared to control cells. Also, 5 minutes of heme treatment brings down oxygen consumption related to ATP synthesis in 50%. This condition is sustained for 30 and 60 minutes after heme stimulation and is accompanied by morphological changes in mitochondria, which rapidly change from a fused network to individual swollen organelles. Mitochondrial fission is an expected response of the cell upon acute stress, such as damage to the electron transport chain (ETC), mtROS generation and mitochondrial membrane depolarization, which are all observed in heme-stimulated BMDM. Interestingly, coincubation of BMDM with heme and inhibitors of complexes I, II and III of the ETC undermines all MAPK activation, particularly ERK phosphorylation, impairing TNF and Cxcl1 production. Considering mtROS generation is Syk-dependent and essential only to ERK signaling, our results suggest other signaling pathways are at play in mitochondria refunctioning by heme. Conclusion. Collectively, our results show that impairment of mitochondrial function happens fast and is likely one of the first effects of heme in macrophages. It is followed by early mitochondrial fission, which likely triggers a signaling cascade involving Syk and MAPK activation, culminating in pro-inflammatory cytokine production.

HEME INDUCTION OF PARADOXICAL SIGNALING PATHWAYS LEADING TO PROGRAMMED CELL DEATH IN MACROPHAGES

Introduction. The prosthetic group heme can be released from hemoproteins in situations like hemolysis, acting as a DAMP recognized by receptors present in immune cells. This leads to pro-inflammatory cytokine expression by macrophages, such as TNF, which induce programmed necrosis (necroptosis) in bone marrow-derived macrophages (BMDM) in an autocrine and paracrine manner. Studies from our lab show that heme strongly induces immediate early genes, like the PI3K/Akt pathway. As the major role of the PI3K/Akt axis is in cell survival signaling, it is paradoxical that the activation of this pathway precedes programmed cell death. Since Akt activation can induce apoptosis, we hypothesize that this pathway could also contribute to necroptosis in BMDM exposed to heme. This work intends to investigate the mechanism that connects the PI3K/Akt axis to necroptosis in macrophages. Methods and Results. Firstly, we confirmed the early activation of Akt and its inhibition by MK2206 (iAKT). PI3K, which acts upstream of Akt, is constitutively expressed in BMDM. Upon 30 minutes of heme exposure, both Akt and PI3K seem to be degraded. Using a method based on quantifying lactate dehydrogenase (LDH), we observed increased cell death in iAkt-treated BMDM exposed to heme. Additionally, cells pretreated with Necrostatin (Nec-1/ allosteric RIP1 inhibitor) are protected from heme-induced cell death. This suggests that the PI3K/Akt axis works oppositely to the necroptosis pathway, indicating a paradoxical signaling pathways of cell survival and death triggered by heme in macrophages. Thus, our next step is to study these pathways in different tissue-resident murine macrophages, analyzing cell death rate and the effect of iAkt and Nec-1 on the activation of heme metabolism-related transcription factors, such as NRF2, BACH1 and SpiC, in search for the mechanism promoting the shift from cell survival to necroptosis. We will start with splenic red pulp macrophages, which were successfully isolated using Magnetic-Activated Cell Sorting (MACS), since these cells may be more resistant to heme due to their role in the erythrophagocytosis. Conclusion. Strong early activation of Akt protects against cell death in BMDM but is not sufficient to prevent necroptosis. The current stage of this study is focused on understanding how these two signaling pathways interplay in BMDM and splenic red pulp macrophages.

Hexosamine biosynthetic pathway/OGT as a nutrient sensor for control of Tregs stability during obesity and insulin resistance development

Adipose tissue located FoxP3+ cells (Fat Tregs) are critical for the maintenance of glucose homeostasis in healthy mice, and are reduced in murine models of insulin-resistance and obesity. Finding new check points for control of Tregs differentiation and stability could be a promising target against obesity-related insulin-resistance. We aimed to study the role of OGT (O-linked N-acetylglucosamine transferase), a critical nutrient sensor, as a crucial controller of Tregs metabolism program. Firstly, we found an increase of HBP (hexosamine biosynthetic pathway) enzymes and OGT expression dependent of TGF- concentration during in vitro Tregs (iTregs) differentiation mainly in stable FoxP3+ cells. Indeed, iTregs culture supplemented with GlcNac (N-acetylglucosamine) showed an increase in FoxP3 levels while pharmacological and genetic inhibition of HBP or OGT (iOGT) promoted a reduction of FoxP3+ cells. We also observed the iOGT impaired TGF- signaling, as observed by the reduction of Smad2/3 expression and phosphorylation in T cells. Aerobic glycolysis has been linked to loss of Tregs stability and function. In this context, iTregs treated with iOGT have shown an increase of glycolysis related genes expression and ECAR (extracellular acidification rate). We also observed iTregs differentiated with iOGT presented a reduction of FoxP3 stability after second round of culture upon TCR stimulation. In order to investigate the impact of OGT in the fat Tregs stability we have performed the HFD (high-fat diet) model in Tregs induced OGT depletion by using KO mice (Foxp3EGFP-cre-ERT2/OGTfl/fl). Both groups, controls (OGTfl/fl) and KO did not show any differences in glucose homeostasis and weight gain before HFD. OGT in Tregs was depleted by tamoxifen injection at week 2 and 8 after HFD protocol. Although we did not find differences in body weight gain in the early point of obesity development (4 weeks after HFD), KO mice have shown a compromised glucose homeostasis, evaluated by GTT (glucose tolerance test) and ITT (insulin tolerance test). Interestingly, 12 weeks after HFD, KO mice have shown resistance to obesity development with lower body weight, reduced glucose tolerance and increased insulin sensitivity compared to control mice. Taken together, these results suggest HBP/OGT have a crucial role in Tregs differentiation and stability, having a possible biphasic role during obesity and insulin resistance development.

HHV-8 VIRAL LOAD AND NFKB EXPRESSION IN B LYMPHOCYTES IN HIV/HHV-8 COINFECTED INDIVIDUALS

Introduction: In people living with HIV/AIDS, co-infection with Human Herpesvirus type 8 (HHV-8) is associated with one of the most common cancers in this population, Kaposi's sarcoma (KS). The nuclear factor Kappa B (NFκB) is one of the immunogenetic factors related to the host, which is capable of increasing cell survival and directing viral replication. The aim of the study was to verify in HIV/HHV-8 coinfected patients whether there is a correlation between the expression of the HHV-8 viral load and the expression of NFκB. Methods and Results: The study population consists of HIV/HHV-8 coinfected individuals, followed up at the Outpatient Clinic for Infectious and Parasitic Diseases (DIP) of the Hospital das Clínicas of the Federal University of Pernambuco (HC-UFPE), Recife - PE. We selected 28 patients with a confirmed diagnosis of HIV and who had IgG antibodies to HHV-8 infection and with a detectable viral load for HHV-8, the study was approved by the Research Ethics Committee of te Health Sciences Center of the Federal University of Pernambuco (protocol number: 45156215.5.0000.5208). For the analysis of NFκB expression in B lymphocyte populations (CD19+) and (CD3+), it was labeled and analyzed by immunophenotyping. When we evaluated the expression of HHV-8 viral load with the expression of NFκB, we found a strong and positive correlation (p=0.0001; r=0.068) in the lymphocyte population, thus demonstrating a directly proportional relationship between the two variables. It is known that the activation of NFkB in the face of a viral infection, initiates processes of the immune response against the pathogen, however, some studies, with HHV-8, demonstrate that it is able to develop strategies to take advantage of this pathway to direct the expression and viral gene replication, in addition preventing cellular apoptosis, and our current results corroborate this evidence. Conclusion: Therefore, we believe that the high expression of NFκB in HIV/HHV-8 coinfected patients does not present an advantage for the viral response in the fight against the virus in the body and we still suggest in future studies the investigation of NFKB, as a possible biomarker, which may assist in the clinical evaluation of these patients.

HIF1Α-MEDIATED GLYCOLYSIS PROMOTES THE MIGRATORY CAPACITY OF DENDRITIC CELLS IN TUBERCULOSIS

Introduction Mycobacterium tuberculosis (Mtb) is a highly successful pathogen that interferes with dendritic cells (DCs) functions impairing the onset and development of adaptive immunity, favoring Tuberculosis (TB). Since several studies are now revealing the importance of metabolic pathways involved in DCs functions, we wondered how the metabolic pathways influence DCs activation in response to Mtb. Methods Buffy coats from healthy donors were prepared at the Garrahan Hospital (Buenos Aires), according to institutional guidelines (CEIANM-664/07). Peripheral blood samples from patients with TB were provided by the Muñiz Hospital. Written informed consent was obtained in accordance with the guidelines of the hospital's ethics committee (protocol number: 2110/21). Monocyte-derived DCs were stimulated with equivalent doses of either irradiated Mtb or viable Mtb. After 24h, the metabolic profile of DCs was determined by Seahorse and SCENITH technologies, lactate release and glucose consumption measured by colorimetric assays and HIF-1α and LDHA expression, which was assessed by qPCR and flow cytometry. Mitochondrial morphology was determined by transmission electron microscopy. When indicated, blocking antibodies against TLR2 or TLR4 were added. Besides, HIF-1α activity was modulated by using either PX-478, a HIF-1α inhibitor, or DMOG, a HIF-1α activator. To assess the participation of glycolysis, the drug GSK28378 or sodium oxamate were used. Finally, the capacity of DCs to migrate was evaluated by chemotactic assays towards CCL21 and 3D migration through fibrillar collagen. Results Mtb-stimulated DCs displayed a predominant glycolytic profile in a TLR2-dependent manner. Additionally, Mtb-triggered HIF-1α-mediated glycolysis was required for DCs to adopt a migratory phenotype, since either HIF-1α activity or glycolysis inhibition abolished the trafficking properties.. Besides, the stabilization of HIF-1α promoted the migration towards CCL21 and the 3D-migration of cells throughout the collagen matrix in tolerogenic DCs induced by dexamethasone. Finally the stabilization of HIF-1α promotes the chemotactic activity in DCs derived from TB patients. Conclusions Our data provide novel insights into the metabolic pathways involved in the trafficking of Mtb-activated DCs to the lymph nodes that could be targeted to generate sterilizing vaccine-induced immunity against TB.

HIF2α SIGNALING PATHWAY IN MYELOID LEUKOCYTES PROMOTES SUSCEPTIBILITY TO INFECTION WITH MYCOBACTERIUM TUBERCULOSIS

Introduction: Tuberculosis (TB) is caused by the infection with Mycobacterium tuberculosis (Mtb) bacillus and represents one of the deadliest infectious diseases in the world. Approaches targeted at host biologic processes involved in disease pathogenesis have been proposed as potential new strategies to help controlling TB. Signaling via hypoxia inducible factor (HIF) transcription factors play important roles in the modulation of leukocyte activation. HIF1α in particular has been shown to induce the expression of inducible nitric oxide synthase (iNOS) and promote resistance to Mtb infection. Differently from HIF1α, HIF2α is known to induce the expression of arginase 1 (Arg1) in macrophages. The role played by this transcription factor in TB, however, is unknown. Methods and Results: To start assessing the role of HIF2α in TB, we infected C57BL/6 mice with 100 colony forming units (CFU) of Mtb strains H37Rv or H37Rv mCherry and quantified the expression of HIF2α in the lungs. Although Mtb-infected mice presented lower HIF2α mRNA expression compared to naïve animals, at 4 weeks post infection (wpi), the expression of HIF2α, as quantified by flow cytometry, was higher in infected pulmonary myeloid cells compared to non-infected counterparts from the same mice. Moreover, in vitro, the infection of C57BL/6 mice bone marrow-derived macrophages (BMDM) with Mtb resulted in increased expression of HIF2α, as quantified by western blot. The inhibition of HIF2α (TCS7009 treatment) in Mtb-infected C57BL/6 BMDM resulted in improved control of bacterial replication following IFNγ activation. In vivo, the treatment of Mtb-infected C57BL/6 with another HIF2α inhibitor (PT2385 – daily for three weeks, starting at four wpi), also resulted in reduction of pulmonary bacterial loads. Mice with conditional deletion of HIF2α in myeloid leukocytes (Epas1fl/flCsfr1Cre+) displayed lower pulmonary bacterial loads compared to wild type (WT) counterparts (Epas1fl/flCsfr1Cre-) at 4 wpi with 1000 CFU or 7 wpi with 100 CFU of Mtb. In vitro, BMDM from Epas1fl/flCsfr1Cre+ mice were more resistant to Mtb infection compared to WT cells, which was associated with lower Arg1 expression. Conclusion: Our preliminary results indicate that the induction of HIF2α signaling pathway in TB promotes susceptibility to infection. In addition, they suggest that repression of HIF2α in myeloid leukocytes might represent a potential target for therapeutic intervention against TB.

HIGHER SELPLG EXPRESSION AND TOTAL LEUKOCYTE COUNT ARE ASSOCIATED WITH SEVERITY IN COVID-19 PATIENTS

INTRODUCTION: COVID-19, a disease caused by the SARS-COV-2 virus, has its pathophysiology related to hyperinflammation and immunothrombosis. Recent research indicates that arginase-1 (ARG-1), nitric oxide synthase-2 (NOS2), α4β1 integrin (ITGA4), and P-selectin-1 (SELPLG) are involved in dysregulated immune responses. The immunopathological mechanisms involving these genes in the severity of COVID-19 are still under investigation. OBJECTIVE: To evaluate the expression of ARG-1, NOS2, ITGA4, and SELPLG genes in total leukocytes from patients with COVID-19. METHODS, PRELIMINARY RESULTS: In this case-control study, patients with positive PCR for SARS-CoV-2 were recruited and grouped accordingly to disease severity. Whole blood was collected, and mRNA was isolated from total leukocytes. The RT-qPCR assay was performed to analyze relative gene expression using the 2-∆CT method. The human ethical committee approved this study (4,014,165). Our results show males (n=38; OR=2.77) and the elderly (n=32; OR=7.04) were more likely to have a severe outcome (p<0.0001). Comorbidities associated with the severe outcome were hypertension (n=23; OR= 2.57, p<0.026), diabetes mellitus (n=27; OR=16.26, p<0.0001), and cardiovascular diseases (n=27; OR= 24.39, p<0.0001). Red blood cells count, hemoglobin, and hematocrit (%) were reduced in patients with severe COVID-19, indicating anemia (p<0.0001). Platelet count and leukocytosis were significantly higher in critically ill patients (p<0.001). A positive correlation between neutrophilia and lymphopenia was observed in the severe group (r=0.78), p<0.0001. Analysis of gene expression indicated that the relative expression of the SELPLG gene was increased in the mild and severe groups (p<0.005 - p<0.01, respectively) compared to the endogenous gene HPRT-1. There was a trend toward higher expression of the SELPLG gene in critically ill patients compared to mild patients. There was no significant differential expression of ARG-1, NOS2, and ITGA-4 (p>0.05) between the groups. ROC curve analysis for SELPLG showed significant diagnostic value (AUC=0.79; p>0.006; 58.82% – 83.33%). CONCLUSION: Age, males, hypertension, diabetes, cardiovascular diseases, dysregulated hematological parameters, and the overexpression of SELPLG were associated with the severity of COVID-19. Our results suggest that surveillance of the peripheral biomarker SELPLG and blood parameters may be useful in the clinical management and prognosis of severe COVID‐19.

High intensity interval training effects in postmenopausal overweight women: Inflammatory and epigenetic modulation

Breast cancer (BC) is the most common woman neoplasm. Evidences have pointed out the involvement of inflammatory profile and epigenetic imbalance in breast cancer hallmarks and pathogenesis, specifically linked to the overexpression of oncogenes or silencing of tumor suppressor genes in overweight/obese patient. This pilot study aimed to investigate the effects of the High Intensity Interval Training (HIIT) protocol on inflammatory markers, tumor cell viability (MCF-7), and overall levels of histone H3 acetylation in overweight and obese postmenopausal women at risk for developing BC. Six participants were submitted to do the HIIT protocol (performed on a cycle ergometer for 4 weeks at a frequency of twice a week) and blood samples were collected at baseline, immediately after 1st session and 24h after the last exercise bout. The results show that there were no changes in IL-6 levels, while a significant decrease immediately after 1st session on IL-8 (P=0,023) and TNF-α (P=0,004). TNF-α levels also decreased when compared directly after 1st session and after the training period (P=0,003). Regarding LDH, a significant increase was observed immediately after 1st session (P=0,001) while a decrease was found after training (P=0,006). The oxidative stress marker (ROS) had a significant increase immediately after 1st session (P=0,024). Finally, a significant reduction in AcH3 acetylation levels of MCF-7 cells after the training period compared to the baseline was found (P=0,046). There was no significant difference in tumor cell viability. These findings suggest that HIIT may improve physical performance, which could be reducing inflammation and increasing histone H3 hypoacetylation status, supporting the idea that it has great potential as a non-pharmaceutical intervention for BC management and prevention.

High levels of serum glycans monovalent IgG immune complexes detected by dissociative ELISA in experimental visceral leishmaniasis

Visceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme-linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000-3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6-13 sugar residues using MALDI-TOF analysis. Glycan-BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia

HISTONE ACETYLATION AND CROTONYLATION PATTERN CHANGES IN EPITHELIAL CELLS DURING INTESTINAL INFLAMMATION

INTRODUCTION: The intestinal epithelial cells (IECs) form the first physical barrier of the gastrointestinal tract, recognizing and responding to components of the intestinal microbiota for the maintenance of homeostasis. This interaction may modulate gene expression in these cells via histone post-translational modifications (HPTMs), including acetylation and crotonylation, which promote an active transcriptional profile. Aberrant regulations of HPTMs have been associated with different diseases in this environment, such as inflammatory bowel disease, but little is known about their role in the development of intestinal inflammation and the factors that influence them. METHODS AND RESULTS: In our study, we analyzed six HPTMs (H3K18ac, H3K18cr, H4K8ac, H4K8cr, Kac, and Kcr) in epithelial cells obtained from animals treated with 2.5% dextran sulfate sodium (DSS, colitis model) and observed an increase in the levels of all on the third day after DSS administration. This treatment time is considered the beginning of inflammation, with no significant difference in the structure of the crypts, the amount of inflammatory infiltrate, and the concentration of cytokines compared to the control condition. On the seventh day, corresponding to the acute phase with exacerbated inflammation and a drastic reduction in the colonic concentration of short-chain fatty acids (SCFAs), there is a decrease in H3K18cr and H4K8ac, while the other HPTMs have similar levels to the control. CONCLUSION: These findings indicate that HPTMs may be important in the changes that occur before and after the establishment of inflammation in the colon.

HISTONES DEACETYLASES’ ROLE IN REGENERATION AND MACROPHAGE POLARIZATION POST-ACUTE KIDNEY INJURY INDUCTION IN ZEBRAFISH

Introduction: Acute kidney injury (AKI) is defined as an abrupt decrease in renal function. After the injury, immune cells are recruited and participate in processes such as promoting and resolving inflammation. The epigenetic control, which is characterized by chromatin rearrangement to modulate gene expression, is one of the regulatory mechanisms responsible for the transcription of genes related to tissue regeneration. Thus, we hypothesize that histone deacetylases (HDACs) act in the transition of macrophage phenotypes, hence modulating the regeneration process. To this end, the present study aims to investigate the role of HDACs in renal regeneration in zebrafish and identify metabolic changes in macrophages at different times after injury. Methods and Results: The use of zebrafish in this study was approved by the University Committee (CEUA/ICB-USP) under the n° 2026070720. To develop a new AKI model in zebrafish larvae, a highly nephrotoxic drug – cisplatin - was administrated in the water. We assessed the expression of genes related to inflammation and kidney damage through qRT-PCR and macrophage infiltration through fluorescence imaging of transgenic animals for macrophage protein expressed 1 (mpeg1.1). Cisplatin had a dose-dependent effect in 5 days post-fertilization larvae (dpf). Also, larvae exposed to an intermediate dose (0.075 mg. mL-1) demonstrated an increase in the macrophage infiltration in the gut and a decrease in the kidney. Despite presenting inflammation, the model was not efficient for further regeneration studies, since the mortality of the animals is high after a few days of exposure to the drug, and the AKI has not been confirmed by the analyzes performed. Now, experiments are being carried out to develop a model of AKI by genetic ablation, using the nitroreductase-metronidazole (NTR-Mtz) system, affecting proximal convoluted tubule cells of these transgenic animals. Sequentially, we look forward to studying specifically the acetylation of histones in genes involved in the polarization of macrophages during kidney regeneration. Conclusion: Cisplatin was efficient in promoting AKI in adult zebrafish in recent work from our laboratory and promoting inflammation; however, this treatment was too aggressive for the establishment of a kidney regeneration model in larvae. In this sense, a new genetic model using the NTR-Mtz system is being generated to investigate the mechanisms for macrophage polarization in the regenerative process.

HLA-G ALLELES IN ASSOCIATION WITH HSV-1 PLACENTAL INFECTION IN SOUTHERN BRAZIL

HOST–PATHOGEN TRANSCRIPTOMICS BY DUAL RNA-SEQ AND PROTEOMIC APPROACH REVEAL AN EFFECTIVE AND REGULATED IMMUNE RESPONSES AS WELL AS NEW FUNGAL VIRULENCE FACTORS IN LUNG GRANULOMA OF MURINE PARACOCCIDIOIDOMYCOSIS

Introduction: Granulomas are important immunological structures in host defense against the fungus Paracoccidioides brasiliensis, the main etiological agent of Paracoccidioidomycosis (PCM), the rifest systemic mycoses in Latin America. Transcriptomics and proteomics of both yeast and murine hosts may reveal important genes and proteins that act under stress conditions induced by the constant activity of the immune system within the granuloma. Thus, they may reveal potential virulence factors as well as provide high throughput data about immunity in granulomatous lesions. Methods and results: C57BL/6 mice were intratracheally infected with 1x106 yeasts. After 8 and 12 weeks of infection, the lung granulomatous lesions were surgically removed. Dual RNAseq assay was performed on total RNA extracted from the lung lesions. Additionally, a proteomic approach was performed on yeasts recovered from the lung granuloma. We detected overexpressed antibodies and complement system-related genes in the lung lesions when compared with their expression in uninfected lungs. Also, cytokine (Il6, Il10, Tnfα, and Ifnγ) and chemokine (Cxcl2, Cxcl3, Ccl8, and Ccl9) genes were upregulated in the lung lesions, suggesting the presence of innate and adaptive cytokine-producing cells. Concomitantly, transcripts related to tissue repair, such as arginase1 (Arg1) and macrophage metalloelastase (Mmp12) were also overexpressed in the lung lesions. Important regulatory genes, including those related to immune checkpoint such as Ctla4 and Pdl1 were also upregulated in the mouse granulomatous lesions. In addition, our data reveal a host lung malfunction within the granuloma, evidenced by the subexpression of actin and myosin. Moreover, when compared with transcripts in control yeasts, granuloma fungal transcripts levels indicated increased siderophore production related to iron uptake. Also, we found suppressed fungal metabolic activity within the granuloma, affirmed by proteomic data and evidenced by the subexpression of genes and proteins such as hexokinase and acyl-CoA dehydrogenase. Conclusion: Our data indicate an increase of several effective and regulatory immunological players in the host lung granulomas. At the same time, the fungus represses its energy metabolism and increases siderophore synthesis for iron uptake, revealing virulence mechanisms that have never been described in mycotic granulomas.

Human monoclonal antibodies anti-Flavivirus with ability to neutralize Zika Virus infection

Introduction: Flavivirus infections have shown recent outbreaks that lead to concerns mainly due to the complications associated such as Hemorrhagic Dengue Fever and Congenital Zika Syndrome. Effective therapeutic approaches are still challenge. To generate effective neutralizing antibodies, the choice of the target antigen is crucial stage. New human anti-Flavivirus antibodies were selected by Phage Display technology against the fusion loop region in the protein E, which is highly conserved among different Flavivirus. Methods and Results: Four rounds of selection were performed using two strategies: the first was using acidic elution of bound phages, and the second was applying a competing procedure. After panning, the selected VH and VL domains were determined by combining NGS and bioinformatics. Three different monoclonal antibodies were expressed as scFvs (single chain fragment variable) and they were further characterized. All showed a binding capacity to Zika (ZIKV) and showed cross-recognition to Yellow Fever (YFV), and Dengue (DENV) viruses. The structure of antibodies was also analyzed for the binding to the epitope and for their structural stability. Two of these antibodies, AZ1p and AZ6m, could neutralize the ZIKV infection (PRNT). Conclusion: These new antibodies have the potential to be used in therapeutic intervention against different Flavivirus illnesses and, due to the conservation of the fusion loop region, they may be resistant to scape mutations. Now, these antibodies will be evaluated by cytotoxicity assay and by in vivo experiments, measuring the reduction of viral load, the activation of the immune response and protection of some tissues, like neural and intestinal tissues.

HUMORAL IMMUNE RESPONSE IN PEOPLE LIVING WITH HIV-1 HOSPITALIZED WITH SEVERE COVID-19

Introduction: Advanced age and comorbidities such as hypertension, diabetes, cardiovascular disease, and immunodeficiencies are risk factors for developing severe COVID-19 and are associated with a high mortality rate in unvaccinated people. This population profile has increased among people living with HIV (PLHIV). However, the available information on the antibody production capacity and clinical outcome of SARS-CoV-2 infection among PLHIV is limited. Therefore, we evaluated the neutralizing antibody response of PLHIV admitted to the COVID-19/INI-FIOCRUZ hospital center and thus better understand the immune response of these individuals in SARS-CoV-2 infection. Methods: We evaluated plasma samples from 30 patients admitted to the hospital center between June 2020 and March 2021; 15 were PLHIV on combination antiretroviral therapy. The HIV-1 uninfected patients were selected based on the same length of hospital stay as the PLHIV group. Neutralization assays against HIV pseudovirus (psV) were performed using TZMBl cells and SARS-CoV-2 psV with 293T-ACE2 cells. For each antibody neutralization assay, we performed 11 serial dilutions (dilution factor=3) of the inactivated plasmas in duplicate and determined the neutralization titer ID50 of the samples for both psVs. Results: Among the 30 participants, 14 were women, and the median age was 48.5. Of these, 12 patients died, and only three were PLHIV. The most common presenting symptoms in PLHIV with COVID-19 were dyspnea (n = 10, 66.6%), cough (n = 7, 46.6%) and fever (n = 7,46.6%). The geometric mean of titers (GMT) of the SARS-CoV-2 neutralizing antibody ID50 of the PLHIV/COVID-19 group was 146 (95% CI: 57 - 962), and the COVID-19 group participants were 260 (95%, CI: 163 - 424). In addition, the group of PLHIV/COVID-19 had an anti-HIV-1 antibody GMT of 401 (95%CI: 328 - 1109). Conclusions: PLHIV can be infected with COVID-19 and are affected by similar features of disease risk and progression as HIV-uninfected. The PLHIV/COVID-19 showed no differences in anti-SARS-CoV-2 antibody production compared to individuals without HIV-1 infection. The PLHIV demonstrates maintenance of anti-HIV-1 antibody production even though hospitalized for severe COVID-19.

Hyperglycemia impairs cDC2 CCR7 expression and dampens host defense against methicillin-resistant Staphylococcus aureus (MRSA) infection in diabetic mice.

Type I diabetes patients are more susceptible to epithelial infections induced by methicillin-resistant Staphylococcus aureus (MRSA). This infection leads to intense recruitment of neutrophils to the site of infection which plays an important role during the bacteria clearance that undergoes apoptosis after hours or days. Dendritic cells (DCs) are crucial for the efficient removal of these infected apoptotic cells as well as the recruitment of Th17 cells to promote infection resolution. It has been shown in a model of MRSA-skin infection that resident DCs in the skin of diabetic mice are less capable to mature and migrate towards lymph nodes to recruit the adaptive response. Since the activation of glycolysis is crucial to sustaining DC phenotype and function, we hypothesized that continuous exposure to hyperglycemia dampens the activation of glycolysis thus impairing DCs ability to mature upon efferocytosis of MRSA-infected apoptotic neutrophils. We treated male C57BL/6 mice with streptozotocin (STZ – 40 mg/kg) injections (i.p.) for 5 consecutive days and mice were considered diabetic with blood glucose levels > 200 mg/dL after 6h of fasting. After 30 days of the onset of diabetes, we injected 1x107 CFU of MRSA USA300 strain (s.c.). The skin biopsies were collected and digested after 18h of infection. The resident DC population was isolated by magnetic separation with CD11c microbeads, stained with anti-Sirp1α, anti-Langerin, anti-CD86, anti-MHC-II, anti-CCR7, and anti-PD-L1 and assessed by flow cytometry. The negative fraction was labeled with Ly6G and Fixable Viability Stain (FVS) to evaluate neutrophil recruitment and cell death. Our results indicated that MRSA infection during 18h promoted an intense accumulation of viable neutrophils or FVS-(~80%). We also observed an equivalent percentage of cDC2 in the vehicle and STZ group (~80% FVS-). Analysis of cell maturation revealed no difference in the percentage of MHC-II+/CD86+ DCs (~6%) and no difference in the MFI of these molecules. However, these MHC-II/CD86 DC had a lower CCR7 and higher PD-L1 expression in the STZ group compared to Veh group. We also observed increased MRSA CFU recovery in the STZ group (107) compared to Veh group (103) after 7d of infection. So far, these results suggest that diabetes inhibits CCR7 and increases PD-L1 expression on cDC2. Our next step is to evaluate the expression of enzymes and transporters associated with glycolysis on cDC2 to determine the hyperglycemia impact.

IDENTIFICATION AND VALIDATION OF GMZ2.6C IMMUNODOMINANT B-CELL EPITOPES IN INDIVUALS EXPOSED TO MALARIA LIVING IN BRAZILIAN ENDEMIC AREAS

Introduction: The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the Glutamate-Rich Protein (GLURP) and the C-terminal region of Merozoite Surace Protein-3 (MSP-3), presently in phase II clinical trial. Previous study showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. Also, anti-GMZ2.6c antibodies increase with exposure to malaria infection and may contribute to parasite immunity. Therefore, identify epitopes of proteins recognized by antibodies may be an important tool to understand the protective immunity. Herein, we identify and validate the immunodominant B-cell epitopes of GMZ2.6c chimeric protein in individuals exposed to malaria living in Brazilian Amazon. Methods and Results: The study was performed using serum samples from 303 individuals from Cruzeiro do Sul and Mâncio Lima, Acre State, and Guajará, Amazonas State. Specific IgG antibodies and subclasses against MSP-3, GLURP and Pfs48/45 epitopes were detected by Enzyme-Linked Immunosorbent Assay using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 (MSP-3a, MSP-3b, MSP-3c and DG210) and GLURP (P1, P2. P3, P4, P5, P6, P7, P8, P9, P10, P11, S2 and S3) or identified using BepiPred for Pfs48/45 (Pfs48/45a and Pfs48/45b). The results showed that P11 (45%), from GLURP, and MSP-3c (48,2%) and DG210 (50%) from MSP-3 were preferentially recognized by antibodies from the studied population. Although at low frequency, responders to P1, P4, P3, S3, from GLURP, and MSP-3a and MSP-3b from MSP-3 presented higher IgG antibody levels. The IgG1 and IgG3 subclasses were more frequent for all immunodominant epitopes, supporting previous studies that these proteins are targets of cytophilic antibodies, important for the acquisition of protective immunity. Most individuals (68,5%) presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. Conclusion: The higher frequency and antibody levels against different epitopes from GLURP, MSP-3 and Pfs48/45 highlights the relevance of GMZ2.6c as malaria vaccine candidate.

IDENTIFICATION OF A BONE MARROW MACROPHAGES SUPBOPULATION WITH HIGHER PHAGOCYTOSIS OF Mycobacterium tuberculosis

INTRODUCTION: Macrophages are essential cells for immune response and homeostasis, they present diverse functions including phagocytosis and pathogen elimination. However, some microorganisms have developed strategies to survive within these cells, for example Mycobacterium tuberculosis (Mtb). This bacterium gain access to the intracellular milieu using surface molecules which can be differently express in host cell depending on the context they are found. Due to its great heterogeneity macrophages can response uniquely to infections. METHODS: Macrophage heterogeneity was explored here using two different subpopulation of bone marrow macrophages (BMM). These cells were separated by flow cytometry based on size, complexity and molecules expressed on their surface and also by RNA sequencing. RESULTS: BMMs have two subpopulations, we denominated them as small and large macrophages. Small macrophages were phenotype as F4/80intCD11bintCD206intCD195high and large macrophages F4/80highCD11bhighCD206highCD195low. Phagocytosis experiments using flow cytometry and fluorescence microscopy reveal that small macrophages phagocyte more Mtb than large macrophages. Furthermore, we confirmed this phenotype by sorting these two population and infecting them separately, in addition Mtb were able to replicate inside both cells. Although we did not see difference in Mtb killing, mediated by high doses of IFNgamma, between small and large macrophages a distinctive cytokines profile was identified. Small macrophages secrete increased levels of TNF and decreased levels of IL-10 during Mtb infection. These cytokine profiles are established before Mtb interaction as RNA sequencing showed that uninfected small macrophages have an enrichment for TNF pathway and large macrophages display IL-10 pathway enrichment. CONCLUSION: In conclusion, bone marrow macrophages have two distinct functionally and phenotypically populations. Moreover, small macrophages have a higher phagocytosis of Mtb that could act as a niche for Mtb replication and dissemination.

Identification of a CXCR3+ CD8+ T lymphocyte subset associated with diminished risk of paradoxical tuberculosis-related immune reconstitution syndrome in patients with advanced HIV infection

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a clinical aggravation of tuberculosis symptoms observed among a fraction of HIV coinfected patients shortly after the start of antiretroviral therapy (ART). Such syndrome is characterized by exacerbated inflammation and tissue damage that occurs in response to the elevated production of CD4+ T cell-derived IFN-γ. Several studies underscored the paramount role of CD8+ T cells in the settings of both TB and HIV infection, but to what degree these lymphocytes contribute to TB-IRIS immunopathogenesis remains largely unclear. Methods: In this prospective study, we followed 48 TB-HIV coinfected patients initiating ART and dissected the phenotypic landscape of CD8+ T cell activation and immunologic memory. Subsequently, we assessed the association between these T cells and the levels of circulating inflammatory molecules as well as with the risk of TB-IRIS development. Results: We observed that TB-IRIS individuals display pronounced CD4+ lymphopenia prior to ART initiation, and higher CD8+ T cell counts. Additionally, we found an ART-induced increase in frequency of cytotoxicity CD8+ T lymphocytes among TB-IRIS patients. Next, our Spearman correlation-based network analysis pointed a negative correlation between the frequencies of total CD4+ and cytotoxic CD8+ T cells, further suggesting a compensatory mechanism in the face of severe lymphopenia. We also observed an enrichment of antigen-experienced CD8+ T cells that were positively correlated with bacillar loads in TB-IRIS patients prior to ART. The dissection of CD8+ T memory subsets revealed a bias towards effector memory phenotype during TB-IRIS occurrence and a lesser degree of naïve population reconstitution after treatment. We also detected an enrichment of a subpopulation of circulating CXCR3+ lymphocytes in non-IRIS patients, which reflects a distinct cell-stage associated with a lower degree of inflammation and tissue damage. Finally, we found that naïve CXCR3+ CD8+ T cells were inversely associated with the risk of TB-IRIS development. Conclusion: Our data suggest an important regulatory role of naive CXCR3+ CD8+ T cells which is also associated with a diminished risk of TB-IRIS development. Collectively, our findings lend insights into the potential therapeutic role of targeting memory CD8+ T cell subsets to enhance protective mechanisms in TB-IRIS pathophysiology.

IDENTIFICATION OF IMMUNE SIGNATURES ASSOCIATED WITH ASYMPTOMATIC SARS-COV-2 INFECTION: WHY DO SOME PEOPLE NOT GET SICK?

Most studies that address the physiopathology of COVID-19 have focused on moderately or severely symptomatic patients. However, little is known about the immune response in asymptomatic individuals. Herein, we aimed to identify molecular signatures in transcriptomic profiles that determines protective mechanisms in SARS-CoV-2-infected asymptomatic individuals. Initially, we selected three RNA-seq datasets containing asymptomatic vs symptomatic samples in the Gene Expression Omnibus (GEO). The raw read counts were treated and normalized by edgeR and DESeq2 R packages. Differentially expressed genes (DEGs) were obtained by limma R package (Fig. 1A). Functional enrichment analysis were performed using Gene Set Enrichment Analysis (GSEA), g:Profiler, Pfam, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Regulation of transcription factors was evaluated by the Enrichr TF-PPIs. We imputed cell populations with the digital cytometry CIBERSORTx and PanglaoDB. Network interactions were generated and visualized using String and Cytoscape. The main enriched pathways in asymptomatic SARS-CoV-2-infected individuals by GO and KEGG were related to upregulation of protein and rRNA processing components and downregulation of ubiquitination and autophagy (Fig. 1B-C). Besides ubiquitin, g:Profiler and Pfam algorithm also indicated the involvement of cadherin in the asymptomatic phenotype (Fig. 1D-E). Expression profiles were enriched for genes encoding six transcription factors: ESR1, ILF3, ILF2, ATF, ATF2 and ESR2, mostly related to T cell proliferation, activity of Natural Killer (NK) cells, estrogen signaling, and homeostasis (Fig. 2A). GSEA indicated the upregulation of KLRG1 expression and downregulation of genes encoding proinflammatory cytokines (Fig. 2B). Cell type profiling showed the reduction of monocytes (Fig. 2C). The data suggests that asymptomatic SARS-CoV-2-infected individuals deploy mechanisms associated with proteasomal degradation, cadherin signaling, and reduced autophagy, which can explain the positive effect of autophagy inhibitors (such as azithromycin) on patient outcomes with limited progression of COVID-19. It also indicates the involvement of NK cells in virus clearance, since ILF and KLRG1 are related to their functions; diminished counts of monocytes are associated with milder COVID-19. Further studies may focus on these markers in order to develop novel strategies detrimental clinical outcomes in SARS-CoV2 infections.

IDENTIFICATION OF IMMUNE SIGNATURES ASSOCIATED WITH ASYMPTOMATIC SARS-COV-2 INFECTION: WHY DO SOME PEOPLE NOT GET SICK?

Most studies that address the physiopathology of COVID-19 have focused on moderately or severely symptomatic patients. However, little is known about the immune response in asymptomatic individuals. Herein, we aimed to identify molecular signatures in transcriptomic profiles that determines protective mechanisms in SARS-CoV-2-infected asymptomatic individuals. Initially, we selected three RNA-seq datasets containing asymptomatic vs symptomatic samples in the Gene Expression Omnibus (GEO). The raw read counts were treated and normalized by edgeR and DESeq2 R packages. Differentially expressed genes (DEGs) were obtained by limma R package. Functional enrichment analysis were performed using Gene Set Enrichment Analysis (GSEA), g:Profiler, Pfam, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Regulation of transcription factors was evaluated by the Enrichr TF-PPIs tool. We imputed cell populations with the digital cytometry CIBERSORTx and the PanglaoDB databases. Network interactions were generated and visualized using String and Cytoscape algorithms. The main enriched pathways in asymptomatic SARS-CoV-2-infected individuals by GO and KEGG were related to upregulation of protein and rRNA processing components and downregulation of ubiquitination and autophagy. Besides ubiquitin, g:Profiler and Pfam algorithm also indicated the involvement of cadherin in the asymptomatic phenotype. Expression profiles were enriched for genes encoding six transcription factors: ESR1, ILF3, ILF2, ATF, ATF2 and ESR2, mostly related to T cell proliferation, activity of Natural Killer (NK) cells, estrogen signaling, and homeostasis. GSEA indicated the upregulation of KLRG1 expression and downregulation of genes encoding proinflammatory cytokines. Cell type profiling showed the reduction of monocytes. The data suggests that asymptomatic SARS-CoV-2-infected individuals deploy mechanisms associated with proteasomal degradation, cadherin signaling, and reduced autophagy, which can explain the positive effect of autophagy inhibitors (such as azithromycin) on patient outcomes with limited progression of COVID-19. It also indicates the involvement of NK cells in virus clearance, since ILF and KLRG1 are related to their functions; diminished counts of monocytes are associated with milder COVID-19. Further studies may focus on these markers in order to develop novel strategies detrimental clinical outcomes in SARS-CoV2 infections.

IDENTIFICATION OF MICROBIOTA-DEPENDENT GENE EXPRESSION IN INTESTINAL CELLS

NATHALIA ARAUJO1, MARIANE FONT FERNANDES2, VINICIUS NIRELLO1, MARCO AURELIO RAMIREZ VINOLO2, PATRICK VARGA-WEISZ1,3,4 1 International Laboratory for Microbiome Host Epigenetics, Department of Genetics, Evolution, Microbiology, and Immunology, Institute of Biology, University of Campinas, Campinas, SP, Brazil 2Laboratory of Immunoinflammation, Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas, Campinas, Brazil 3 São Paulo Chair of Excellence, International Laboratory for Microbiome Host Epigenetics, Department of Genetics, Evolution, Microbiology, and Immunology, Institute of Biology, University of Campinas, Campinas, SP, Brazil 4 School of Life Sciences, University of Essex, Colchester, UK INTRODUCTION: Histone post-translational modifications at gene regulatory elements are known to affect gene expression profiles. In addition to widely studied histone acetylation, phosphorylation, and methylation, the development of high-sensitivity mass spectrometry has uncovered a plethora of novel modifications such as crotonylation, propionylation, malonylation, etc. Histone crotonylation, which is evolutionary conserved from yeast to humans, was found to be primarily associated with active chromatin. Crotonylation is particularly abundant in the intestinal epithelium and is microbiota-dependent, as depletion of gut bacteria leads to decrease of overall crotonylation in the intestine. However, little is known how microbiota-dependent chromatin modifications in intestinal epithelial cells affect gene expression at the single cell level, maybe reflecting proximity of cells to the microbiota. METHODS AND RESULTS: We performed single-cell RNA-Sequencing (scRNA-Seq) in in Intestinal Epithelial Cells (IECs) in C57BL/6 mice treated with a mix of antibiotics to deplete the microbiota. This scRNA-Seq analysis suggest that there is a reduction in the antimicrobial peptide and in the immune response post-antibiotic treatment. CONCLUSIONS: Our data suggest that the gene expression changes on antibiotics treatment were more pronounced in differentiated epithelial cells compared to progenitor cells, possibly reflecting exposure to microbiota-derived metabolites.

IDENTIFICATION OF THE IMMUNE PROFILE RELATED TO THE DIFFERENTIAL RESPONSES AND OUTCOMES OF IDENTICAL MICE TO SEPSIS

Introduction: Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. New therapies, such as immunotherapies, have been tested in sepsis; but with no success. Thus, studies using innovative methods may contribute to the identification of new therapeutic targets in sepsis. Using mice genetically equal and having the same grown-up environment we observed different responses to the cecal ligation and puncture sepsis model (CLP), in which half of the mice survive and the other half succumb. That way, our study aims to identify new therapeutic targets by comparing the activation profile of immune cells of surviving and non-surviving septic animals. Methods: We induced sepsis in C57/Bl6 male mice using cecal ligation and puncture (CLP) with an intensity that promotes mortality of 50%. Animal Research Ethical Committee: 151/2019. Results: After 6 h of CLP induction, survived and non-survived mice displayed the same serum levels of cytokines. However, at 12h, 24h, or 48h, survived animals presented reduced cytokines levels, while in non-survived mice these levels remained higher. Furthermore, non-survived mice also showed increased organs lesions and bacteremia. Likewise, non-survived mice showed increased concentrations of cytokines in the lungs, kidneys, heart, liver, and also in the primary infection focus in comparison with surved mice. Next, performing a single-cell RNA sequencing of leukocytes from survived and non-survived mice 12 h after CLP, we observed decreased expression of genes related to migration, adhesion, and cellular response to type I interferon in neutrophils of non-survived mice. In monocytes from non-survived mice, we observed decreased expression of genes associated with chemotaxis, adhesion, and regulation of cytokine production. Validating the scRNA-seq finds, we observed that after 24 h and 48 h of CLP induction, once neutrophils of surviving mice reestablish CXCR2 and CD11b expression levels similar to the control, the neutrophils of non-surviving mice remained with internalized CXCR2 and high CD11b expression. As consequence, neutrophils in non- survived mice migrate less to the peritoneal cavity and become more migrative to the organs. Conclusion: Our results demonstrate that non-survived mice had persistent hyperinflammation and extensive alterations at transcriptional and protein levels in leukocytes. Now, we are exploring the identified molecules as new therapeutic targets.

IgA quantification as a good predictor of the levels of neutralizing antibodies after vaccination against SARS-CoV-2

INTRODUCTION The pandemic caused by the SARS-CoV-2 became one of the biggest problems faced by health agencies in the recent years. Although 9 vaccines of different types are currently being used worldwide, the emergence of variants that can evade the acquired immune response highlights the importance of understanding the mechanisms involved in the immune response generated by vaccination or after SARS-CoV-2 infection. Vaccines against SARS-CoV-2 induce the production of antibodies (Abs) driven to Spike protein (S), which is present on the surface of the virion and is essential for viral entry into the host cell. The quantification of Abs is an important tool for monitoring vaccine coverage. However, not all Abs are neutralizing antibodies (NAbs), which protect against new infections. Furthermore, quantification of NAbs is very expensive, making it not applicable for surveillance. Thus, our objective was to correlate the amount of serum IgG and IgA with the serum NAbs induced in response to the complete vaccination regimen with CoronaVac (N=18), ChAdOx-1 (N=68) and BNT162b2 (N=32) vaccines in individuals without previous infection. METHODS AND RESULT A total of 250 serum samples were collected from participants before vaccination (T0), 15 to 20 days after receiving the first dose of vaccine (T1), and 15 to 60 days after receiving the second dose of vaccine (T2). Sera IgG and IgA against S were quantified using an in-house ELISA assay previously established by our group. NAbs were quantified using cPass™ kit (GenScript). We found that the 3 vaccines were able to induce high titers of IgG after the complete vaccination scheme (T2). The CoronaVac vaccine did not induce IgA production, while the ChAdOx-1 vaccine induces low IgA titers even in T2 and BNT162b2 vaccine significantly induced IgA in T1 and even more significantly in T2. On average, NAbs were found in high amounts in participants who received BNT162b2 (1086.4 UI/ml), while lower titers were found in those who received ChAdOx-1 (439.5 UI/ml) or CoronaVac (162.2 UI/ml). Although the IgG titers induced by the 3 vaccines were high, no correlation with NAbs was seen. In contrast, NAbs profile was very similar to that of IgA, revealing a strong positive correlation between these quantifications. CONCLUSION Our data suggest that IgA rather than IgG quantification may be more suitable for vaccination coverage surveillance.

IGBP-C AS A NEGATIVE REGULATOR OF THE ANGIOGENESIS PROCESS.

Hard ticks feed on blood from the bleeding pool they form on the skin of their hosts. Consequently, they ingest large amounts of immunoglobulins. To aid the female tick's blood meal, male ticks secrete saliva containing IgG-binding proteins (IGBPs). Previous studies have observed that IGBPs interfere with the effector functions of immunoglobulins. In addition, some proteins present in the saliva of different tick species have been described as modulators of the host's angiogenesis process. Gangliosides affect the growth and differentiation of tissues and, indeed, IGBP-C presents a sequence similar to a ganglioside–binding domain. Rabbit aortic endothelial cells and endothelial cells of the vena cava of rats cultured on Matrigel and treated with recIGBP (7μM) presented strong inhibition of blood vessel formation and in a qPCR array presented significant differential expression of some of 84 genes that participate in the angiogenesis process, respectively. We now verified the action of IGBP-C in the inhibition of blood vessel formation in vivo. We used the chick embryonated egg chorioallantoic membrane (CAM) assay to demonstrate an in vivo reduction in the formation of new capillary tubes. We next hope to confirm that IGBP has anti-angiogenic properties in a model where IGBP-C is used to treated mice that have been injected with oncogene-transformed tumorigenic fibroblasts and a similar line deficient in genes encoding ganglioside synthases.

IL-1α REDUCES THE PULMONARY INFLAMMATION AND THE SUSCEPTIBILITY TO M. TUBERCULOSIS INFECTION IN THE CONTEXT OF OBESITY

Introduction: Mycobacterium tuberculosis is the bacillus responsible for 1.2 million of deaths worldwide caused tuberculosis (TB) annually. Obesity is one of the comorbidities associated to the development of severe TB. Protection against TB requires IFN-γ, IL-17, IL-1β, and IL-1α. We hypothesized that in the obesity, IL-1α, an alarmin produced by M1 macrophages, would exacerbate the pulmonary inflammation during experimental TB and aggravate the progression of infection. Methods: Wild type (WT) or IL-1α deficient mice (IL-1α-/-) were fed with High Fat Diet (HFD) or Low Fat Diet (LFD) for 8 weeks, infected with M. tuberculosis and their lungs were evaluated 3 weeks post infection (11 weeks - HFD feeding). Results: WT Obese (HFD) mice produced significant levels of IL-1α in the lungs compared to lean (LFD) mice. As we reported previously, obesity induced by a high-fat diet (HFD) exacerbated pulmonary inflammation and accentuated the susceptibility to M. tuberculosis infection. However, IL-1α-/- obese mice exhibited a lower frequency of CD4+IFN-γ+ and CD4+IL-17+ cells in the lungs than WT obese mice. Their lungs showed also an increased magnitude of inflammation and a higher recovery of bacilli than WT obese mice. Treatment with recombinant IL-1α decreased the bacterial load in the lungs of IL-1α-/- obese mice compared to untreated IL-1α-/- obese. Conclusion: These findings show a protective role for IL-1α related to lung damage tolerance and to pathogen tolerance. The mechanism by which IL-1α promotes this protective effect is under investigation.

IL-27 AND IL-35 CYTOKINES REGULATE THE SEVERITY OF PLASMODIUM CHABAUDI-TRIGGERED PATHOGENESIS

Malaria is an infectious disease of great importance to global public health, caused by infection by parasites of the genus Plasmodium. Failure to establish an adequate balance between pro-and anti-inflammatory immune responses contributes to the pathogenesis of severe malaria. The cytokines IL-27 and IL-35 are formed by the p28 and EBI3 subunits and by the p35 and EBI3 subunits, respectively. Both cytokines belong to the IL-12 family, are produced by macrophages, dendritic cells, T and B cells, and are responsible for regulating the immune response. During Plasmodium infection, there is the development of a strong inflammatory response to control the parasite. However, if it is not controlled, it ends up being deleterious to the host. Therefore, understanding the mechanisms of regulation of the immune response triggered by this parasite is necessary. In this sense, main to elucidate the importance of the cytokines IL-27 and IL-35 in the pathogenesis of experimental malaria. WT, Il27rα-/- and Ebi3-/- mice inoculated with 105 red blood cells infected by P. chabaudi and different parameters such as parasitaemia, histopathological alterations, gene expression in the liver tissue, and the profile of immune cells in the spleen were evaluated by H&E, qPCR and flow cytometry. P. chabaudi infection induces high expression of p28, Ebi3 and p35 subunits in liver tissue at 8 dpi, suggesting the involvement of IL-27 and IL-35 cytokines in the course of infection. Il27rα-/- and Ebi3-/- infected mice showed a decrease in parasitemia when compared to WT animals, and in Ebi3-/- animals the reduction in blood forms was more pronounced, reaching 20%. Furthermore, Il27rα-/- and Ebi3-/- animals showed an accumulation of CD4 and CD8 cells in the spleen and an increase in the inflammatory cytokines IFN-γ and TNF-α compared to WT animals. Interestingly, Il27rα-/- and Ebi3-/- animals maintained a high production of pro-inflammatory cytokines up to 10dpi, while in WT animals there was already control of the inflammatory response. As a consequence of the lack of regulation of the immune response, Il27rα-/- and Ebi3-/- animals showed an increase in serum alanine aminotransferase enzyme when compared to WT animals, suggesting greater liver damage. Our results show that the cytokines IL-27 and IL-35 are induced during infection and participate in the regulation of inflammation triggered by P. chabaudi.

IMMUNE CHECKPOINT BLOCKADE VIA PD-L1 POTENTIATES MORE CD28-BASED THAN 4-1BB-BASED ANTI-CARBONIC ANHYDRASE IX CHIMERIC ANTIGEN RECEPTOR T CELLS

Introduction. Chimeric antigen receptor (CAR) T cells are capable to be activated directly when in contact with a specific tumor antigen, becoming a new potent strategy against cancer. The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 CAR T cells in doses equivalent to ≅108 CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses for ccRCC in adjuvant settings and metastatic scenarios, becoming an important pillar treatment. This project tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR T cells releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Methods. Lentiviruses containing the different CAR constructions to be tested were produced, concentrated, and the transduction efficiency was determined by flow cytometry and IgG secretion. The cytotoxic effects of anti-CAIX CAR T were analyzed by flow cytometry and lactate dehydrogenase activity. The secretion of IL-2 and IFNγ was determined by ELISA. The exhaustion status was determined by flow cytometry. Alanine (ALT) and aspartate (AST) transaminases activity was determined by spectrophotometry. Results. Anti-CAIX CAR T cells were able to induce around 80% decrease in the viability of ccRCC cells in vitro. Using a ≅107 CAR PBMCs cells/kg dose in the in vivo orthotopic ccRCC model showed that anti-CAIX CAR T cells that release anti-PD-L1 promoted a significant reduction in tumor volume and weight, with the construction with CD28 showing more potent results compared to 4-1BB, preventing the induction of tumor metastases. Considering T cell exhaustion, the constructions with anti-CAIX CD28 CAR and anti-PD-L1 secretion and with 4-1BB CAR with or without anti-PD-L1 secretion showed reduced co-expression of PD-1, TIM-3, CTLA-4, and CD39 in viable tumor-infiltrating T cells. We evaluated the renal and hepatic function of the mice by measuring transaminases and creatinine and did not observe any type of toxicity. Conclusion. Anti-CAIX CAR T cells secreting anti-PD-L1 can diminish T cell exhaustion and improve CAR T cell treatment of ccRCC in vivo, offering exciting new prospects for the treatment of refractory ccRCC and hypoxic tumors.

Immune traits of resistance and disease tolerance in COVID-19

Background: The vast spectrum of clinical manifestations of SARS-CoV-2 infection, from asymptomatic to lethal, keeps challenging scientists and clinicians. COVID-19 outcome depends on the control of viral load (resistance) and on the capacity to limit tissue damage (disease tolerance). Severe COVID-19 patients exhibit exacerbated inflammation and tissue dysfunction, being the paradigm of low disease tolerance. Conversely, patients with low resistance may stay oligosymptomatic despite persistent infection, being unrecognized transmission vectors. The immune profile of these patients remains unclear. Here, we aim to elucidate immune dynamics involved in resistance and disease tolerance in SARS-CoV-2 infection, and thereby identify early predictors of COVID-19 progression. Methods and results: early immune responses (<14DSSO) of non-hospitalized COVID-19 patients with distinct resistance and disease tolerance were analyzed by immunophenotyping and plasma multiplex analysis. Patients evolving either to persistent infection (qRT-PCR positive >21DSSO, n=33) or to severe disease (oxygen support, n=11) had fewer circulating plasmacytoid dendritic cells, NK cells and classical monocytes compared to mild, control patients (n=32). Plasma levels of IL-7, IFNγ and IL-12 were reduced, in contrast to higher IL-17A and MIP-1α, indicating a shift from type 1 to type 3 immunity. Long-term carriers displayed lower IFN-I responses, whereas severe patients showed elevated IFN-induced IP-10. Enhanced inflammation and tissue repair, reflected by IL-6, VEGF and PDGF-BB, were characteristic of severe COVID-19, while no increase in those markers occurred in long-term carriers. Conclusions: here, we identify distinct early immune traits in mild COVID-19 patients that define their progression to either persistent infection or severe disease. Impaired resistance via type 1 immunity and increased Th17 responses characterized both groups. However, only patients who later on required oxygen support presented elevated inflammatory and tissue repair markers, even before the appearance of severe symptoms. Further analysis of stress-response and immunoregulatory pathways will shed light on differences in disease tolerance between these two groups of patients, bringing up tolerance mechanisms of therapeutic value. Overall, our study reveals alternative immune strategies to deal with SARS-CoV-2 infection and propose early immune markers with potential predictive value of COVID-19 progression.

IMMUNIZATION MUCOPENETRATING NANOPARTICLE CONTAING RBD SARS-COV-2 RBD PROTEIN INDUCES HUMORAL AND CELLULAR IMMUNITY IN MICE

Introduction: The pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected 357 million people and caused more than six million deaths worldwide. The SARS-CoV-2 anchors the receptor-binding domain (RBD) of the spike protein on the angiotensin-converting enzyme 2 receptor (ACE2) expressed by host cells. There is some information on the correlates of protection during infection, and RBD-specific neutralizing antibodies appear to be promising candidates. Despite the success of the worldwide vaccination programs, the development of new technologies is essential for disease control. In this context, nanoparticle based vaccines represent a promissing new platform, solving problems such as temperature and molecular stability. The chemical structure which envolves the immunogen works both as carriers and as adjuvants, promoting otimization of the formulation and higher immunoestimulation. For this reason, a subunit RBD protein-based vaccine is considered a potential prophylactic or therapeutic target. In this work, we evaluated humoral and cellular immune responses using a mucopenetrating nanoparticle containg RBD admixed with adjuvant in vivo. Methods and Results: An artificial gene corresponding to the consensus sequence of RBD was cloned and used to produce a recombinant RBD protein in Expi293F cells. C57BL/6 mice received two or three doses of mucopenetrating nanoparticle containg RBD admixed with adjuvant or RBD protein in the presence of adjuvant by subcutaneous e/or intranasal route. To evaluate the specific humoral immune response, sera from immunized mice were collected 15 days after the last dose and analyzed by ELISA. Subsequently, mice were euthanized 15 days after the last immunization to analyze RBD-specific cellular response. Immunization with mucopenetrating nanoparticle containg RBD admixed with adjuvant induced higher anti-RBD IgG titer, and IFNγ secreting cells when compared to the group that received RBD plus adjuvant. Conclusion: Our findings demonstrate that immunization mucopenetrating nanoparticle containg RBD admixed with adjuvant was able to induce high specific antibody titers and robust cellular immune responses in mice.

Immunization with a RBD/Nucleocapsid fusion protein promotes neutralizing antibody-independent resistance to infection with wild type and SARS-CoV-2 variants of concern

Both T cells and B cells have been shown to be generated after infection with SARS-CoV-2 yet protocols or experimental models to study one or the other are less common. Here, we generate a chimeric protein (SpiN) that comprises the receptor binding domain (RBD) from Spike (S) and the nucleocapsid (N) antigens from SARS-CoV-2. Memory CD4+ and CD8+ T cells specific for SpiN could be detected in the blood of both individuals vaccinated with Coronavac SARS-CoV-2 vaccines and COVID-19 convalescent donors. In rodents, SpiN elicited a strong IFN-γ response by T cells and high levels of antibodies to the inactivated virus, but not detectable neutralizing antibodies (nAbs). Importantly, immunization of Syrian hamsters and the human Angiotensin Convertase Enzyme-2-transgenic (K18-ACE-2) mice with Poly ICLC-adjuvanted SpiN promotes robust resistance to the wild type SARS-CoV-2, as indicated by viral load, lung inflammation, clinical outcome and lethality. The protection induced by SpiN was ablated by depletion of CD4+ and CD8+ T cells and not transferred by antibodies from vaccinated mice. Finally, vaccination with SpiN also protected the K18-ACE-2 mice against Delta and Omicron isolates. Hence, vaccine formulations that elicit effector T cells specific for the N and RBD proteins may be used to improve COVID-19 vaccines and circumvent the immune escape by variants of concern.

“Immunogenetics profiles’ in a young adult monozygotic twin pair following simultaneous critical Covid-19”

Reports of Covid-19 in monozygotic (MZ) twins where both were infected simultaneously with similar disease outcomes, including siblings who died from SARS-CoV-2 infection within days apart provide support for the host genotype influence on the disease susceptibility and outcomes, However, successive exposures to pathogens throughout life and other environmental factors make the immune response unique for each individual. Thus, it is expected that even among MZ twins, there may be subtle differences in the immunity profile and metabolic processes, during and after viral infection. Here, we had the opportunity to report a case of long-term effects of Covid-19 in a young adult monozygotic twin pair after severe SARS-CoV-2 simultaneous infection and compare their humoral and cellular immunity profile. Whole-exome sequencing (WES) on peripheral blood DNA to identify genetic variants related to severe Covid-19 and Complementary clinical laboratory analyses including systemic biomarkers were also performed. Serological assays for SARS-CoV-2 IgA, IgG, and IgM for the receptor-binding domain (RBD) and nucleocapsid protein (NP) were performed through ELISA. Type I/III IFN response and the production of IFN-γ and IL-2 by CD4+ T lymphocytes were evaluated by RT-PCR. Similar humoral and cellular immunological responses were observed in both twins. Pos-covid hematologic and blood systemic parameters alterations observed in only one of the brothers who reported fatigue symptoms. We detected two rare missense variants in the BTK gene, which may be related to pro-inflammatory signaling pathways which require further functional assays to verify their possible role as a candidate gene.

Immunolocalization of antibodies and cytokines in mice treated with itraconazole and celecoxib in an experimental model of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a granulomatous mycosis, caused by the fungus Paracoccidioides brasiliensis. The infection occurs by the inhalation of conidia and/or mycelial fragments that are present in the soil and affects mainly male individuals. The initial infection may be exudative or granulomatous. Therefore, the presence of granulomas characterizes this disease. OBJETIVE: To evaluate the effectiveness of Celecoxib and Itraconazole combined treatment in the granulomatosa response of paracoccidioidomycosis. METHODS: In this work, the animals were infected intraperitoneally with the virulent strain of P. brasiliensis (Pb18) and after three days of infection gavage treatment was initiated in the groups with itraconazole (3mg/mL), celecoxib (6mg/mL) and combination (6 mg/mL) of itraconazole (3 mg/mL) for 15 and 120 days daily. On the last day of treatment the animals were sacrificed and collected epiploon/pancreas. The organs were processed and and immunostaining was performed with IgG and IgM antibodies and cytokines IL-17 and GM-CSF. RESULTS: The results of immunohistochemistry showed that there was a strong positivity for the presence of IgM, IgG, IL-17 and GM-CSF at different stages of infection. From these results, it can be concluded that the combined treatment presented satisfactory results both in the initial and late of this disease. This therapeutic combination reduces the intense inflammatory response in patients and may be used as a possible new treatment strategy.

IMMUNOLOCALIZATION OF THE MEROZOITE SURFACE PROTEIN-1 (MSP1) ON BLOOD STAGES OF Plasmodium vivax PARASITE USING IgG ANTIBODIES FROM INDIVIDUALS LIVING IN A LOW MALARIA TRANSMISSION AREA, BRAZIL.

Introduction: The merozoite surface protein-1 (MSP-1) is a member of the family of proteins that have functions in the life cycle of malaria parasite, but also can be immunogenic activating immune response with acquisition of antibodies that recognize different antigenic components, including the C-terminal portion of the Plasmodium vivax MSP-1 (PvMSP1-19). This protein is highly immunogenic in humans and animal models with high levels of IgG antibodies. In this study, we described the pattern of recognition of the native protein in blood stages by immune serum from individuals living in a malaria endemic area in Pará state, Amazon region, Brazil. Methods: Pools of human serum with high titers of IgG anti-PvMSP1-19 were determined by ELISA (Enzyme-Linked Immunosorbent Assay) using the recombinant protein His6-MSP1-19, and the immunolocalization was performed by Immunofluorescence Indirect assay using blood stages of the parasite. Results: Our results revealed forms indicating the presence of immune complexes formed by binding between human IgG antibody and the MSP1 protein on the blood stages of the parasite obtained from a patient diagnosed with malaria caused by P. vivax. These descriptions are relevant as contribution regarding the location and distribution of MSP1, as well as to investigate if it can be any influence related to possible structural alterations of the parasite due the formation of these immune complexes with IgG antibody and the native protein present on membrane of P. vivax malaria parasite. Conclusion: In this study, we showed that IgG antibodies against PvMSP1-19 acquired after natural exposure to whole parasite were able to recognize the native MSP1 protein expressed on surface of blood stages obtained from patient infected with P. vivax malaria parasite.

IMMUNOLOGICAL DIFFERENCES IN COVID-19 CHILDREN INFECTED BY OMICRON

Introduction: Despite the expectation that COVID-19 is not a serious illness in children, epidemiological data contradict this statement. According to UNICEF, in USA COVID-19 is the 8th cause of death among 5-11 years old children,1 in 2021 ,was the second cause of death in children 5-11 years old in Brazil.2-3 The immunological mechanisms of COVID19 in children are complex and largely uncharacterized. It is still too early to assess the effects of long-term COVID-19 on children. In this study, we aim to evaluate the immunological profile of COVID 19 infection in this population. Methods and results: Sixty-four patients up to 16 years suspected COVID19 were referred to this study. Clinical records, EDTA blood samples and swab samples were included. Study participants were divided into SARS-CoV-2 negative (n=38) and infected (n=26), according to qRT-PCR. The SARS-CoV-2 infected group was subdivided into children who acquired COVID 19 during or before the omicron epidemiological period in Rio de Janeiro. Blood samples were assessed by leucocyte counts and flow cytometry. Luminex Multiplex assay was used to evaluate 27 cytokines in plasma and swab samples. Among monocytes population, MoMDSCs, was less frequent in SARS-CoV-2 infected children and intermediate monocytes were more frequent in omicron infected children’s. A higher activation state of T cell subpopulations was observed based on the quantification of expressed HLADR and CD69. The activation of EMRA+ and EM3+ T cells was higher in children infected with omicron as define by surface HLA-DR expression. In swab samples, patients infected with omicron showed a reduction of IL1-RA, IL-10 and IL-12. Reduction of IL-15(p= 0,0681), together with IL-12 reduction (p= 0,0438) may suggest a less active state of NK cells in the nasopharyngeal mucosa. In contrast, cytokine profile assessed in plasma samples suggest a variation in Th2-type response due to an observed reduction in IL-13, IL-5 e IL-4 production in children infected with omicron. In addition, this group showed elevated levels of IL-9, CXCL-10 and MCP-1. Conclusion: Our results indicate that the immunological profile of children with COVID-19 infected by omicron is different from that induced by other strains. That might contribute to a better understanding of the clinical outcome of COVID-19 in pediatric population.

IMMUNOLOGICAL PROFILE IN SEVERE COVID-19 CANCER PATIENTS

Patients with cancer are more likely to have severe complications and even death when affected by COVID-19. Particularly in low- and middle-income countries, COVID-19 has brought a heavy burdening to the public health systems and induced new planning and adjustments in the clinical approach to cancer patients. Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity, but the determinants of severe outcome remain largely unknown. The aim of this study is to comprehend the cytokine and lymphocyte profile of COVID-19 cancer patients, to correlate with disease severity and to compare with non-cancer participants. Blood samples were analyzed from mild and severe COVID-19 hospitalized patients with cancer (n=40) and without cancer (n=38), and healthy donors (n=21). The criteria for the infection diagnosis included positive result qRT-PCR for SARS-CoV-2 from nasopharyngeal swab samples. Severe patients were considered in cases of mechanical ventilator use or death. We analyzed the presence of 46 different cytokines, chemokines and growth factors in each of the participants by Luminex. Using flow cytometry, we analyzed cell markers for T, Treg and NK cells. Our results indicate that CXCL10/IP-10 were increased in COVID-19 patients with cancer and discriminated disease severity. Levels of CXCL10/IP-10 were also increased in severe cancer patients compared to non-cancer patients. COVID-19 cancer patients show reduced T lymphocytes analyzed by CD3+ T cells. Furthermore, COVID-19 cancer patients exhibited an increased levels of exhausted lymphocyte profile evaluated by CD4+PD1+ and CD8+PD1+ T cells. In fact, exhausted T lymphocyte profile were increased in COVID-19 cancer and non-cancer patients and discriminate disease severity. In conclusion, our data suggested that COVID-19 cancer patients present an amplification of exhausted immune profile that could have a potential implication to cancer clinical outcome.

Immunology disclosure to Brazilian society by social media: Imunolog – Divulgando ciência

Introduction: The dissemination of scientific content aims to bring the academy closer to society. Access to reliable information is a social need; and nowadays the Internet, with its global power, is an important tool to facilitate and expand the dissemination and access to information on diverse areas of knowledge. The dissemination of content with an accessible language and with an attractive format may favor the democratization of knowledge, allowing the understanding of scientific work by society. Social media networks have a wide broad of users and a comprehensive portion of the Brazilian population, making them one of the main channels of communication. These networks are free and allow interactive communication in different content formats. They allow quick interaction with society through the direct response to questions regarding the content. Imunolog – Divulgando Ciência is a social media channel aiming to disseminate scientific news in an accessible and attractive language/format through social media, to favor the democratization of the knowledge about immunology. Methods and Results: In March 2020, Imunolog – Divulgando Ciência science disclosure channels were launched on the following platforms: Facebook, Youtube, and Instagram. We are currently focusing on Instagram posts, as this platform reaches out to our target audience (young 16–30-year-old Brazilians). We create content like videos and cards concerning different topics of immunology in Portuguese. Our team comprises 6 undergraduate students from Universidade Federal Fluminense and 2 researchers. In June 2022, were have 165 pots reaching 1050 followers on the Instagram platform. From March to June, we reached 2017 accounts, in which 30.7% were from Rio de Janeiro, 18.1% from Niteroi, and more than 10% from other Fluminense cities. Our main followers are from Brazil; however, we also have an audience from Portugal, Canada, and United States, presenting ages from 18-34 (69%) and 35-54 (28%) from the female gender (74%). Our account engagement is of 703 followers accounts and 1314 non-followers. Conclusion: The science channel Imunolog - Divulgando ciência has successfully disseminated reliable information about immunology. Thus, this project contributes to the democratization of information and access to scientific information in Brazilian society.

IMMUNOMETABOLIC REPROGRAMING OF NEUTROPHILS BY TROPHOBLAST CELLS IN AN IN VITRO MODEL OF EARLY MATERNAL-PLACENTAL INTERFACE

Introduction Trophoblast cells (Tb) interact with maternal immune cells at placentation favoring an anti inflammatory microenvironment required for fetal growth. Circulating neutrophils (PMN) appear activated during pregnancy and even more in pregnancies complicated by preeclampsia. Metabolic regulation underlies the functional profiling of immune cells in a number of settings but immunometabolic reprogramming in the context of pregnancy was not yet studied. The aim of this work was to evaluate the effect of trophoblast cell-derived factors in neutrophils’ metabolic, phenotypic, and functional profile. Methods Neutrophils from healthy donors were cultured with the human first trimester trophoblast cell line Swan-71 conditioned media (CM). PMN profiles were assessed by RT‐qPCR and flow cytometry. Glucose uptake, intracellular lipid accumulation, long chain polyunsaturated fatty acids uptake and reactive oxygen species (ROS) production were determined by flow cytometry using the glucose fluorescent analog 2-NBDG, BODIPY 493/503, BODIPY FL C12 or DCFH-DA respectively. Treg modulation was explored co-culturing PMN with autologous mononuclear cells and CD4, FOXP3 staining. Results PMN cultured with CM enhanced glucose uptake (MFI X±SE Basal 897±195; CM 1212±244, P<0.05) although to a lower extent than PMA (1992±194). Also, an increase in the glucose specific transporter GLUT1 was observed. Furthermore, CM increased lipid droplet accumulation (MFI X±SE Basal 998±93; CM 1318±115 P<0.05) and fatty acids uptake. Treatment with lactate -a metabolite released by Tb- or changes in the media pH modulated neutrophils’ reactive oxygen species (ROS) production. This metabolic profile of PMN was accompanied by the acquisition of a proangiogenic profile characterized by an increase in VEGF, an increased capacity to promote vascular transformation and an inhibition of PMA-induced neutrophil extracellular trap formation and ROS production. Moreover, PMN pre-incubated with CM increased the frequency of CD4+ FOXP3+ cells in co cultures (% X±SE Basal 3.8±0.6 CM 10.0±3.4, P<0.05). Conclusion Our results support an immunometabolic programing of neutrophils towards a proangiogenic profile upon trophoblast cell interaction in an in vitro model of early maternal-placental interface.

Immunometabolism in the symbiotic relationship between the Aedes fluviatilis mosquito and bacterium Wolbachia pipientis

Wolbachia pipientis is a maternally transmitted bacterium that mostly colonizes arthropods, including insects, arachnids, crustaceans, and nematodes. Was seen that Wolbachia strain (wFLU) promotes a modulation of glycogen metabolism in your natural endosymbiont, the mosquito Aedes fluviatilis. When silencing the enzyme glycogen synthase kinase 3 (GSK3), the enzyme regulating glycogen synthesis, the amount of this bacterium and glycogen in embryos Aedes fluviatilis increases. The aim of this study was described metabolic e immune changes mediated by GSK3. Furthermore, we comparing naturally Wolbachia-infected (Wolb+) and uninfected (Wolb-) embrionic cells of Ae. fluviatilis, thus increasing the knowledge about Wolbachia parasitic/symbiotic mechanisms. Comparing Wolb+ and Wolb- cells, we observed that Wolb+ have double of glycogen content followed by decreased GSK3 transcription and increased PEPCK (Phosphoenolpyruvate carboxykinase) transcription. In addition, Wolb+ cells have down-regulated gambicin. By inhibiting GSK3 activity, we observed an increase in Glycogen content and Wolbachia load, followed by a reduction in Relish2 (REL2) and Gambicin transcripts. To investigate whether the IMD pathway acts to control the amount of Wolbachia, we reduced the levels of REL2 in Wolb+ cells by RNAi. The knock-down of Rel2 leads to amount of Wolbachia increases followed by increase in Glycogen content and up-regulated PEPCK transcription. Therefore, these data set suggest a possible correlation between GSK3 and REL2.

IMMUNOMODULATION ROLE OF C57BL/6 MURINE ENDOMETRIAL MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR VESICLES IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS MODEL

IMMUNOMODULATION ROLE OF C57BL/6 MURINE ENDOMETRIAL MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR VESICLES IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS MODEL Laura Caroline de Faria, Tiago Francisco da Silva, Carolina Manganeli Polonio, Jonathan Miguel Zanatta, Lilian Gomes de Oliveira, Yan de Souza Angelo, Patrick da Silva, Sandra Marcia Muxel, Jean Pierre Schatzmann Peron Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo- SP, Brasil Introduction: Multiple Sclerosis (MS) is a multifactorial disease, primarily autoimmune, that’s affect the Central Nervous System (CNS). Although it’s not clear what the etiology of disease, it’s known to be mediated by local and systemic immune mechanisms and believed to begin with T lymphocytes activation by peripheral antigen-presenting cells (APCs), which migrate and infiltrate CNS, where they find myelin sheath antigens, leading to increased production of proinflammatory cytokines and chemokines that will orchestrate inflammation and neurological damage. Th1 and Th17 lymphocytes are responsible for pathology, and the FoxP3+ TReg cell are essential to control the response. Mesenchymal Stem Cell (MSC) has been used in different clinical trial for immune disease treatment, and the capacity of them to secret extracellular vesicles (EVs) acting in TReg and glial cells still have limited study. Objective: To evaluate the immunomodulatory capacity of EV-MSC in immune and glial cells in vitro or in vivo, during Experimental Autoimmune Encephalomyelitis (EAE) disease. Method and Results Uterus EV-MSC was cultivated, isolated and them submitted to characterization by flow cytometry. In vitro analyses were performed after EV-MSC incubation for 1 or 4 hours with primary Astrocytes from C57Bl/6 pups (1-3 days), stimulated or not with LPS (100 ng/ml). EV-MSC seems to modulate pro-inflammatory cytokines in astrocytes, such as Il-6, Tnf-α, Il-1β and Nos-2, while the opposite is observed in Il-27 gene expression, even in presence of LPS, that may be an indicative of EV content modulating astrocytes in vitro. Conclusion: EV has been standardizing for characterization and in a near future will be delivery to EAE mice, that we hope that future results demonstrate immunomodulation.

Immunomodulatory and neuroprotector effects of Tityus serrulatus toxin mimetic peptides during experimental severe malaria

Plasmodium berghei ANKA (PbA) infection in mice mimics many aspects of severe malaria in humans, including cerebral malaria. Studies demonstrated many functions of the components of the Tityus serrulatus, yellow scorpion, venom (TsV) including their immunomodulatory action as well as their antimicrobial, antitumor and antiparasitic capacity.Therefore, herein, our aim was to test the therapeutic and neuroprotective potential of TsV-derived peptides (characterized and synthesized by our group) in the treatment of experimental severe malaria.C57Bl/6 mice were inoculated with 105 red cells parasitized with PbA pre-treated or not with peptides (Pep 1a, 2a or 2b; 100µg/mL). The treated group received intraperitoneally the peptides (1mg/kg) 8 hours after infection, or orally with chloroquine (CQ-30mg/kg). All treatment was given once a day, until the seventh day after infection. Parasitemia, body weight, survival, clinical score, memory and immune cell profiles (by flow cytometry) were analyzed. Our results showed that the treatment with Pep 1a, 2a or 2b prolongated survival, decrease body mass loss, and alleviate the pathological base of the severe manifestations of malaria. In the novel object recognition test, animals treated with the peptides had a subtly better result compared to chloroquine. Of note, there was no difference in parasitemia, indicating that the protective effects of the peptides could be, at least in part, related to regulation of the immune system in different tissues/organs. So far, the treatment, mainly with Pep2a, regulates the production of IL-17 by CD4 and CD8 T cells in the brain and lung when compared with untreated infected group. Collectively, our results suggest that the peptides Pep1a, Pep2a and Pep2b have potential immunomodulatory activity, protecting the lung and brain, and improving the disease symptoms, being a promising compound for treatment of severe malaria.

IMMUNOMODULATORY EFFECTS OF OMEGA 3 SUPPLEMENTATION ON THE INTESTINAL MUCOSA OF MICE

Introduction: The mucosal surfaces of our organism constitute the region of greatest contact with the external environment and the intestinal mucosa is constantly exposed to a wide range of antigens. Feeding is the main event in which the body comes into contact with foreign proteins and other molecules with immunological relevance. Nutrients have great metabolic importance at all stages of life, being essential elements for the survival of organisms. In addition to the energy function, the components of the diet have direct effects on the cells of the immune system. Lipids, for example, can exert numerous effects on the immune system through their action on receptors expressed in immune cells. In this study, we analyzed the immunomodulatory effects of omega 3 supplementation on the intestinal mucosa of mice. Methods: Young female BALB/c mice were fed for 21 days with the AIN93G diet supplemented with w3 before the analyzes of IgA-coated to bacteria and the profiles of immune cells from the spleen, mesenteric lymph nodes and the lamina propria of the small intestine by flow cytometry. In addition, we monitored the weight and diet consumption of all mice. Results: Dietary supplementation with w3 did not alter weight gain or diet consumption, but it increased the frequency of bacteria coated to IgA suggesting that diet supplementation causes changes in the microbiota composition. Furthermore, we found alterations in the profile of T lymphocytes in the mesenteric lymph nodes, indicating a suppression of the Th1 and Th17 inflammatory response and a decrease in the frequency of regulatory T cells. In the spleen, we observed a decrease in the frequency of Th2 lymphocytes and in the lamina propria there was a change in the frequency of Th2 and Th17 lymphocytes. When analyzing the dendritic cells, we observed an increase in the frequency of CD103+ CD11b- cells and we did not observe differences in the frequency of ILCs from gut lamina própria. Considering the results obtained and the anti-inflammatory potential of w3 described in the literature in many disease models, we evaluated the potential adjuvant effect of 3 in inducing oral tolerance. However, our data indicated that dietary supplementation with w3 had no tolerogenic effects. Conclusion: Our work shows important immunological effects triggered by dietary supplementation with omega3, suggesting that this lipid may have application in preventive and therapeutical strategies for inflammatory diseases.

IMMUNOMODULATORY POTENTIAL OF NATURAL PRODUCT IN THE HEALING OF WOUNDS INFECTED BY METHICILLIN-RESISTANT Staphylococcus aureus (MRSA)

Introduction: Wound healing is a complex process that comprises cellular and molecular mechanisms. Healing therapies seek to modulate cells at the wound site and eliminate or control secondary infections, in addition to minimizing pain, discomfort and scarring. To date, there are no studies that identify the action of natural product in the repair of wounds infected by methicillin-resistant Staphylococcus aureus (MRSA). In this context, this study aimed to evaluate the immunomodulatory potential of natural product in a wound healing model infected by MRSA. Methods: Female Swiss mice were anesthetized. Subsequently, wounds were made on the skin of the animals' backs and minutes after wounding a bacterial inoculum with approximately 1.5 · 108 CFU of MRSA was added over the wound. Topical treatment was performed daily for 10 days. For the treatment of wounds, gels containing natural product were used as investigational treatment and the base gel, without the presence of natural product as a placebo (CTRL); and as a positive control, based on deoxyribonuclease (6.66%), fibrolysin (0.1%) and chloramphenicol (1%) (Fibrinase®) (DFC). On days 3, 7 and 10, animals were euthanized, and tissue samples around the wound were collected for histological analysis. Results: Regarding the inflammatory infiltrate, histological differences were observed between the PG, CTRL and DFC groups on the seventh day. The inflammatory infiltrate was classified as abundant in all groups, especially in the CTRL group, on the seventh day. On day 7, there was a greater presence of macrophages, mainly in the natural product group, while on day 10 this number decreased significantly. There was a high rate of angiogenesis in the dermis of all groups. Collagen deposition was higher in the natural group than in the CTRL and DFC groups, mainly on day 7. There was also an increase in fibroblast proliferation in the PG group compared to the DFC group, mainly on day 7. In general, mice treated with P. granatum showed increased levels of fibroblast proliferation, collagen deposition and reduction of inflammatory cells. Furthermore, natural product was able to reduce the proliferation of MRSA in infected lesions. Conclusion: The results obtained so far suggest a reduction of inflammatory response in animals treated with natural product evidenced by the decrease in macrophage levels, in addition to the increase in fibroblast proliferation and abundant collagen deposition.

IMMUNOMODULATORY POTENTIAL OF THE RESVERATROL ANALOG AR23, IN DENDRITIC CELLS.

Introduction: Resveratrol is a natural phytoalexin found in grapes and red wine, exhibiting important anti-inflammatory and immunomodulatory effects. However, this compound shows low bioavailability in mammals, and the synthesis of analog compounds could overcome this issue. Recent studies have targeted dendritic cells, in order to achieve regulation in several inflammatory diseases. Therefore, the present study aimed to evaluate the immunomodulatory activity of the Resveratrol analog AR23, in dendritic cells. Methods and Results: Possible citotoxic effects of AR23 (25uM) over bone marrow-derived dendritic cells (CEUA: 04/2018) was checked with Propidium Iodide (PI) staining on flow cytometry. Then, the activation profile of those cells was assessed by labeling CD40, CD80, MHCII and CD73 on LPS-stimulated, followed by flow cytometry. Finally, IL-6 and IL-12 production was determined in stimulated dendritic cells supernatant (ELISA). The data are presented as mean ± standard deviation of treated-stimulated group (AR+LPS) and control-stimulated group (LPS), respectively. The results shown that AR23 did not affect DCs viability, as PI staining was similar to the control (28.33±2.74% and 29.3±1.36%). Furthermore, AR23 decreased CD40 (5077±300 and 6213±266 MFI*) and MHCII (26530±614 and 33243±1540 MIF*) expression, while increasing CD73 expression (643.3±5.43 and 508.5±49.9 MFI). In addition, AR23 analog reduced IL-6 (1759±65.4 and 3479±325 pg/mL) and IL-12 (2477±77.6 and 31076±17.76 pg/mL) in the supernant of treated DCs. Conclusion: The results suggest that AR23 has modulatory activity on dendritic cells, and could induce a tolerogenic profile. However, other approaches are required in order to verify its effects in dendritic cell tolerogenesis protocols. *MFI - mean fluorescence intensity.

IMMUNOSENESCENCE AND CELLULAR EXHAUSTION TRANSCRIPTIONAL SIGNATURES ARE CO-EXPRESSED IN HUMAN TEGUMENTARY LEISHMANIASIS

IMMUNOSENESCENCE RESPONSE IN COVID-19 INFECTION

Introduction: In COVID-19, lethality is correlated with patients’ age and comorbidities. Accumulation of terminally differentiated lymphocytes and senescent cells is observed in aged adult humans. Chronic diseases, often associated with age, can also lead to immunesenescence and increased levels of inflammatory mediators (inflammaging). These observations suggest a possible link between age and comorbidities in explaining higher risk of severe COVID-19 in patients with more senescent cells. We investigated the evolution of the immunosenescence response of hospitalized COVID-19 patients from Belo Horizonte, Brazil, and Lisbon, Portugal. Methods: Non-vaccinated patients were recruited within, after presenting a positive nasopharyngeal swab test for Sars-Cov-2. The blood samples were collected at the time of enrolment (T0) and seven days later (T7). Peripheral blood mononuclear cells (PBMCs) were obtained by blood collection from COVID-19 patients. PBMCs were first stained for immunephenotype of peripheral blood mononuclear cells from patients with different disease severity. Results: The study included 20 individuals with severe cases of COVID-19 from Lisbon, and 8 individuals with mild to severe cases from Belo Horizonte. The median age of individuals from Lisbon were significantly higher when compared to individuals from Belo Horizonte. In Lisbon, our results showed an increased frequency of T cells expressing both markers of senescence/exhaustion (CD57+KLRG1+), and those expressing only senescence phenotype (CD57+) in both T cell compartments. In addition, shows a significant increase in memory effector T cells (EM) displaying CD57+KLRG1+ and CD57+KLRG1- phenotype during hospitalization, while those displaying CD57-KLRG1+ showed decreased frequency. Meanwhile, the EM subset only presented a significant result for the KLGR1+ exhaustion marker. In Brazil, we observed an increase in CD4+ T cells expressing markers of senescence/exhaustion (CD57+KLRG1+) in a seven-day period. The frequency of naive T cells (CD4+ and CD8+) showed a reduction of frequency in a seven-day interval. Differently, the severe patients from Portugal, the patients in question did not show alterations in the frequency of CD57 and KLRG1 (senescence/exhaustion) in the T cells of effector memory. Conclusion: We showed that independent of disease severity and age, the data suggests that the infection of COVID-19 is associated with senescence phenotype in T cells.

IMMUNOSUPPRESSIVE POTENTIAL OF CELL THERAPY WITH ALLOGENIC REGULATORY T CELLS AND SYGENEICS IN MURINE SKIN TRANSPLANTATION

Introduction: Regulatory T cells (Tregs) have been the subject of studies aimed at inducing operational tolerance in allogeneic transplants. Treg cells are considered central to operational tolerance and cellular therapies are being tested with autologous Tregs to improve transplantation tolerance. Our objective is to investigate if allogeneic Treg therapy using donor’s Tregs can also be used as cell therapy with better results. Methods: The immunoregulatory potential of Treg cells was evaluated in a mixed lymphocyte reaction (MLR), where CD4+ T lymphocytes (Teff) from one individual were cultured with stimulatory antigen presenting cells (APCs) from another individual in the presence or absence of autologous or allogeneic Tregs to the APCs. Tregs were added in different proportions to a fixed Teff/APC mix and cultured for 7 days. Proliferation was analyzed by CFSE dilution and the production of IFN-γ, IL-10 and TNF was evaluated in the cell culture supernatant by ELISA. The in vivo efficacy of cell therapy with allogeneic or syngeneic Tregs was evaluated using a murine skin transplant model. Tregs from the skin donor or from syngeneic mice was sorted on FACs Aria or using a Treg isolation kit and infused into skin recipients. Graft rejection assessment was performed using a score from 0 (no rejection) to 5 (total rejection). Immune responses to the transplantation was analyzed by cytokine production on draining lymph nodes and identification of allogeneic-reactive antibodies by cross-matching test. to assess the production of antibodies reactive to the donor serum were analyzed by flow cytometry. Result: Treg cells, allogeneic or autologous, were able to decrease the proliferation of Teff cells and inhibit the production of IFN-γ and TNF, but did not modulate IL-10 in MLR. However, allogeneic Tregs displayed greater immunosuppressive capacity when compared to autologous Treg cells. Mice that received skin transplants and allogeneic Tregs in therapy also showed lower frequencies of skin rejection when compared to animals that received syngeneic Tregs (xx% versus YY%) in 10 days of observation with a mean survival of graft respectively of XX days against YY days. Allogeneic Treg cells were also better regulators of humoral rejection as measured by the detection of alloreactive antibodies. Conclusion: Allogeneic Treg seems superior to syngeneic Tregs in inducing regulation and tolerance of alloreactive T cells in MLR and skin transplants.

IMPACT OF ACUTE GASTROINTESTINAL INFECTIONS IN THE HOST CAPACITY TO INDUCE MUCOSAL TYPE 2 IMMUNITY

Introduction: The intestinal mucosa is in close contact with a diverse population of commensals, which provide efficient food digestion and contribute to intestinal homeostasis. Specialized leukocytes reside in the gut mucosa, and complex regulatory networks ensure the prevention of inflammation triggered by innocuous antigens and protection against pathogenic agents. The breakdown of these canonical barrier responses can result in the development of inflammatory disorders, such as food allergy and inflammatory bowel diseases. In this context, our group has shown that a single episode of acute gastrointestinal infection by Yersinia pseudotuberculosis (YP) in mice was capable of inducing a phenomenon called “immunological scar”, which can lead to the long-term failure of mucosal immunity. This process starts with irreversible damage to gut lymphatics, which compromises the migration of dendritic cells (DCs) to the draining lymph nodes and impacts the induction of effector and regulatory intestinal T cells. Because of this, there is a permanent failure in sustaining microbiota compartmentalization and the development of mesentery remodeling, marked by the presence of a type 1 inflammatory infiltrate and the loss of type 2 homeostatic resident cells. In this study, we hypothesized that rescuing the type 2 component of the mesentery would allow for the recovery of the lymphatic structure/function post-infection and restore DC migration and induction of mucosal homeostatic responses. Methods and Results: To restore type 2 immunity in the gut/mesentery, female naive or YP-infected C57BL/6 mice were subcutaneously sensitized with ovalbumin (OVA) + alum followed by an oral challenge with OVA in the drinking water. Serum was collected for antibody detection, and the small intestine and mesentery were analyzed by flow cytometry. The OVA sensitization/challenge protocol effectively promoted a systemic and local type 2 immune response, marked respectively by OVA-specific IgG1 serum antibodies and the recruitment of eosinophils, M2 macrophages, ILC2 and Th2 cells in the mesentery and gut. However, although YP-infected mice could develop the systemic type 2 responses, the infection impaired the induction of type 2 immunity in the gut and mesentery. YP-infected mice sustained lymphatic damage even after the type 2 response induction. Conclusion: These results suggest that YP-infected mice failed to establish a type 2 mucosal response and reverse the immunological scar.

Impact of Mygalin on the inflammatory response induced by TLR2 agonists

Several natural products are being studied in order to identify new bioactive molecules with therapeutic potential for infections and immune modulation. The characterization of the biological activities and mechanisms of action of these molecules are fundamental for its application and identification of possible molecular targets. Macrophages express a wide range of receptors, including Toll-Like receptors. Mygalin is a synthetic acylpolyamine analog of spermidine, originally isolated from hemocytes of the Acanthoscurria gomesiana spider. This molecule reduces in vitro the production of pro-inflammatory mediators in LPS-activated macrophages by interacting with the TLR4/MD2 complex. Our objective was to evaluate the action of Mygalin on Toll-like 2 receptors that do not have the MD2 adapter protein, analyzing the immune response of macrophages pre-activated with Mygalin in the presence or absence of TLR 2/1 agonists (Pam3CSK4) and TLR 2/6 (Zymosan), followed by the evaluation of immune mediators: nitric oxide (NO) and pro-inflammatory cytokines TNF-α and IL-6. J774A.1 strain macrophages were pre-treated with Mygalin and further stimulated with TLR 2/1 agonists, Pam3CSK4 (300 ng/mL) and TLR 2/6, Zymosan (10 µg/mL). Cells were incubated at 37°C, in presence 5% CO2 for 20 hours. LPS (100 ng/mL) was used as a positive control for cell activation. Culture supernatants were collected for NO dosage by the Griess method and TNF-α and IL-6 by ELISA. This study was approved by the Ethics Committee for the Use of Animals (CEUA) of Instituto Butantan, CEUA nº7902090421. It was observed that pretreatment of macrophages with Mygalin and subsequent activation with Pam3CSK4 reduced NO production, regardless of the dose used. The level of IL-6 was reduced only in the groups treated with the highest dose of Mygalin (360 µM), suggesting a dose dependent effect. There was no statistically significant difference in the production of TNF-α between the groups treated or not with Mygalin. Similar results were obtained when cells were pretreated with Mygalin and stimulated with Zymosan. The pre-treatment of cells only with Mygalin (90 and 360 uM) did not induce significant production of any of the analyzed immune mediators, being the level close to that obtained with untreated cells. The results indicate that the action of Mygalin in reducing the inflammatory response does not depend exclusively on its interaction with the MD2 adapter, involving other mechanisms.

IMPACT OF STING SIGNALING PATHWAY ON THE DIFFERENTIATION AND FUNCTION OF REGULATORY T CELLS

Nucleic acids have long been described to activate the immune system. The protein STING comprises the intracellular DNA-sensing machinery, which is activated upon recognition of microbial or self- DNA, thereby promoting the activation of transcription factors, including those involved in the production of type I IFN. Although STING functions are widely described in innate immune cells, little is known about its role on lymphocytes. Regulatory T cells (Tregs) are Foxp3-expressing T cell subpopulation that have a central role on the immune system’s homeostasis and prevention of exacerbated inflammatory responses. The present study aims to investigate the role of STING on the differentiation and function of Tregs. For that purpose, naive T cells were isolated from WT and STING KO mice by cell sorting and cultured under iTreg polarizing conditions (TGF-β) in the presence or not of STING agonists (DMXAA and c-di-AMP). Additionally, peripheral regulatory T cells were isolated from Foxp3-CreYFP e Foxp3-CreYFPTmem173fl/fl mice and cultured in vitro with IL-2 or IL-2 + IL-6, in the presence or absence of STING agonists. Afterwards, the impact of STING activation on the differentiation, stability and function of Tregs was assessed by Flow Cytometry and/or RT-qPCR and ELISA. Our preliminary results, obtained during the period of my Undergraduate Research, demonstrate that STING activation not only increases the differentiation of Tregs, but also the expression of genes related with the function of these cells (such as Foxp3, Pdcd1, Entpd1, Nt5e, and Tnfrsf18), as well as the expression of the anti-inflammatory cytokine IL-10. Also, the presence of STING contributes to the stability of Tregs, as observed by the maintenance of Foxp3 expression. In conclusion, our study suggests a role of STING on Treg cells generation and function, revealing STING as a potential candidate for Treg-targeted therapies.

IMPACT OF TYROSINE KINASE INHIBITORS ON T CELLS

Introduction: Cancer is one of the most frequent causes of death. As an alternative treatment tyrosine kinase inhibitors (TKIs) have been used in several types of cancer, consisting in a way of stopping a specific signaling pathway in tumor cells. Lately, immunotherapy has been introduced in cancer treatments, known as immune checkpoint blockade (ICB), consisting in a block of co-receptors associated with negative responses of immune cells, especially T cells. The literature indicates that downstream pathways of EGFR and VEGFR are also present in immune cells, but the action of TKIs in these cells is poorly understood, and this knowledge is relevant, when we consider the increase in TKI plus immunotherapy use in cancer patients. Thus, our aim is to elucidate the role of TKIs on T cells. Methods and Results: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were separated with Ficoll-Paque, T cells were activated with anti-CD3/CD28 beads and treated with osimertinib (EGFR inhibitor), axitinib e sunitinib (VEGFR inhibitors) in different concentrations. Gene expression was evaluated by qPCR, proliferation analysis was measured using CFSE and flow cytometry (FACS) and pathways activation were evaluated by Western Blotting or FACS using Phosflow protocols. Isolation of T-Cell Subsets were performed by Cell Sorter System. PBMCs, specially T cells activated, expressed EGFR and VEGFR, and also some immune checkpoints. When treated with TKIs, lymphocytes proliferation was downregulated in higher concentrations. After isolation of CD4+ and CD8+ populations, it was observed that CD8+ had even more affected proliferation than CD4+ cells with these treatments. When we analyzed signaling pathways activation, VEGF appeared to decrease activation of the ERK pathway in PBMCs, but with AXI and SUNI treatments this pathway was more activated. The mTOR pathway appears to have been unaffected under these conditions. Conclusion: PBMCs express receptor tyrosine kinases. TKIs treatments can affect immune cells, especially T cells, decreasing their proliferation and interfering with their signaling pathways, mainly in the presence of VEGF. Although VEGF may decrease activation of the MAPK pathway in these cells, VEGFR inhibitors appear to help re-establish this activation. However, more studies need to be done to better understand the impact of TKIs on immune cells and the expression of immune checkpoints on T cells in these treatments.

IMPORTANCE OF ERK5 IN THE DIFFERENTIATION AND FUNCTION OF T REGULATORY CELLS

Introduction: FoxP3+ regulatory T cells (tregs) play important suppressive roles in maintaining the homeostasis of the immune system. TGF-β is known to be a crucial cytokine for the expression of FoxP3, a transcription factor required for the differentiation, maintenance and function of Tregs. ERK5 (extracellular signal-regulated kinase 5) is an atypical member of the mitogen-activated protein kinase (MAPK) family that has canonical kinase function and also the role of regulating the activity of transcription factors independent of this canonical function. ERK5 is known to be activated in different cell types by TGF-β, but there is nothing in the literature about its involvement in the activation or differentiation of Tregs lymphocytes. This project aims to understand the role of ERK5 in Tregs differentiation and function. Methods: naïve CD4+ T cells purified from C57BL / 6, ERK5flox/flox, CD4creERK5flox/flox, FoxP3cre or ERK5flox/flox mice were cultured under polarizing conditions for Treg. First, we checked the expression of pERK5 in Treg against a control group without TGF-β. In order to verify the role of ERK5 in Treg differentiation, the ERK5 pathway was inhibited with different drugs, such as XMD 8-92 or ERK5-IN-1. The contribution of ERK5 in the pathogenesis of autoimmune diseases was investigated by inducing a model of multiple sclerosis, termed experimental autoimmune encephalomyelitis (EAE), which is induced by subcutaneous injection of the peptide MOG35-55. Results: we found that phosphorylation of ERK5 is increased in Tregs cells when compared with the control group (Th0, no cytokines), suggesting that TGF-β can increase pERK5 expression. Then, we show that pharmacological inhibition or genetic deficiency of ERK5 in CD4 T cells decreased the differentiation of Tregs cells. Furthermore, CD4creERK5flox/flox and FoxP3creERK5flox/flox mice developed more severe EAE than ERK5flox/flox and control FoxP3cre mice, characterized by a reduction of Treg cells in draining lymph nodes. Furthermore, FoxP3+ cells extracted from the FoxP3creERK5flox/flox animal was shown to possess less stability and lower suppressive potential when compared to FoxP3+ cells from the FoxP3Cre. Our study reveals a novel role for ERK5 in modulating Treg differentiation and attenuating EAE severity. Therefore, ERK5 modulation may be a potential therapeutic target for autoimmune diseases such as multiple sclerosis.

IMPORTANCE OF IL-33 IN THE DEVELOPMENT OF WALLERIAN DEGENERATION AND ITS ACTIONS ON IMMUNE CELLS

Introduction: Wallerian degeneration (WD) is a process of degradation of myelin remnants and axons after injury, being important for tissue functionality. The cytokine IL-33, expressed by hematopoietic cells, belongs to the IL-1 superfamily group and has been described as important in different contexts. However, there isn’t description of its participation in the peripheral nervous system. Our aim was to investigate the role of IL-33 in the development of DW, as well as any of the underlying mechanisms. Methods and Results: We analyzed scRNA-seq data from the sciatic nerve of mice and identified the presence of IL-33 in mesenchymal cells. To understand the role of IL-33 during WD, we induced sciatic nerve crush injury (PNI) in WT and IL-33-deficient (Il33Gfp/Gfp) mice, we evaluated locomotor function (Rota Rod tests) and mechanical allodynia (von Frey filaments). The Il33Gfp/Gfp animals had a higher locomotor function from day 3 (174.750 ± 24.893) in relation to the WT (75.750 ± 33.109) and less mechanical allodynia (36 ± 8.94) from the day 5 compared to WT (60 ± 7.071). To understand the importance of IL-33 in the immune cells that are present during WD, we performed flow cytometry during different days of PNI and observed that in the Il33Gfp/Gfp animals there wasn't difference in Ly6C+ monocytes, however, the decrease in maturating monocytes Ly6Cint (3.515 ± 1.349) compared to WT (6.740 ± 2.511) on day 3 and Ly6C-(0.925 ± 0.457) compared to WT (2.577 ± 1.120) on day 5. All experiments followed the ethical standards established by the Ethics Committee for the use of animals (CEUA/FMRP-USP: 150/2020). Conclusion: IL-33 from mesenchymal cells is important for monocyte maturation in WD, this being important for the inflammatory and degenerative state of the sciatic nerve, increasing the locomotive deficit and allodynia caused by the sciatic nerve injury.

Importance of NFR2 on osteoclastogenis and its impact on bone metabolism

Bone is a rigid tissue, but it is in continuous remodeling activity, which is essential for the renewal and maintenance of its integrity throughout the individual's life. Bone remodeling is a metabolic process characterized by a precise balance between bone matrix degradation (resorption) and synthesis, which involves the activity of several specialized cells, among which osteoclasts and osteoblasts play a fundamental role. The imbalance between osteoclastic and osteoblastic activities results in changes in bone tissue and, consequently, loss of bone mass. In this context, numerous evidence has shown that reactive oxygen species (ROS) and oxidative stress are associated with the progression of bone diseases. It has been shown that ROS act as an important regulator of the fate and function of osteoblasts and osteoclasts and, consequently, alter bone homeostasis. In this context, understanding the mechanisms that regulate these processes may allow designing therapeutic strategies to control the damage caused by ROS in bone. Physiologically, cells have control mechanisms against the adverse effects of oxidative stress, such as the activation of transcription factors that lead to the induction of genes with antioxidant properties. Among them stands out a gene known as "Nuclear Factor Erythroid 2-Related Factor 2" or NFR2. The importance of this gene in controlling oxidative stress was demonstrated in NFR2 deficient animals that were highly susceptible to the negative impacts of oxidative stress. NFR2 has been described as an important protein in controlling osteoclast differentiation. In the present work, we used the compound Resveratrol as a direct activator of NFR2 antioxidant activity during osteoclasts differentiation. The preliminary results shown that Resveratrol in the 10 uM concentration inhibit the osteoclasts differentiation and reduce the genic expression of CTSK, NFATC1 and TRAP, important genes for osteoclasts function and activities. Animal models with specific deletion of NFR2 in osteoclasts are being generated to study the importance of NFR2 in the osteoclasts reabsorption functions. These results will understand the effects of antioxidant therapy in the treatment of osteoporosis and bone diseases.

IMPROVEMENT OF CAR-T CELL THERAPY WITH ULTRA-FAST PROTOCOL AND IL-15 MEMBRANE BOUND ADDITION

Despite the advancement of new technologies for immunotherapy, gene therapy is far from being widely available. CAR-T cell therapy, which has a great response rate in B-cell tumor patients, has a high cost. This therapy starts with leukapheresis to collect T cells, transport to specialized laboratories, activation to promote proliferation, genetically modification with viral vectors to insert the CAR gene and expansion for about 15 days. Cells are then frozen and returned to the hospital. In this project, we propose an ultra-fast protocol to decrease the time, cost, and complexity of CAR-T cell generation. We use transposon-based non-viral vectors such as Sleeping Beauty (SB) or PiggyBac (PB), allowing us to skip the cell activation step before gene insertion. As a result, no in vitro cell expansion is required. This protocol can be performed in less than 24h and CAR-T cells and used to treat NSG mice leukemia cells. Since this approach could theoretically be taken at the bedside, we named it a Point-of-care (POC) CAR-T production protocol. PBMC were isolated by Ficoll and electroporated using the Nucleofector IIb device with SB plasmids encoding 19BBz CAR and SB100x transposase. CAR expression on day 1 following electroporation ranged between 5-15% in all experiment with SB. NSG mice were injected iv. RS4;11 or Nalm-6 cells and treated 3 days later with electroporated CAR-T cells (doses of 1x105 and 7x105 per mice, respectively), showing improved survival when compared to mice treated with mock electroporated cells. Extensions in survival curves were accompanied by decreased tumor burden in blood and spleen. Head-to-head comparison of 19BBz cells used in POC approach or expanded for 8-12 days in vitro showed similar antitumor activity against RS4;11 cells, leading to equivalent improvements in mice survival. After that, we added an IL-15 membrane bound molecule (mbIL-15) to the CAR cassette to improve cell persistence and animal survival. To this end and we used PB vector to insert the transgene. We noticed that the tumor burden evaluated by bioluminescence of animals that had 19BBz-mbIL15 was lower when compared to 19BBz alone (dose of 3x105). Improvements in survival of the mice were also observed. We conclude that our proposed POC approach for CAR-T cell therapy can be explored as an alternative with less cost, time, and complexity. Furthermore, mIL15 added to CAR appears to bring benefits in controlling tumor growth in preclinical mice models.

INCREASED EXPRESSION OF TYPE 1 INTERFERON RECEPTOR AND INTERLEUKIN 17-A ASSOCIATED WITH SEVERITY IN COVID-19.

Introduction: COVID-19 is characterized by systemic hyper-inflammation and acute respiratory distress syndrome. Recent studies have shown that the impaired response of type-1 IFNs (IFN-α and its receptors) in COVID-19 infection and deregulated expression of the cytokine IL-17A are essential for developing cytokine storms. Moreover, the activation of human endogenous retroviruses (HERVs) may be related to the severity of COVID- 19, but their contribution to COVID-19 severity is still under investigation. Objective: To evaluate the expression of IFN-α genes and their receptors (INFAR1/INFAR2), IL-17A, and HERVs (HERV-K /HERV-W) in patients with mild and severe forms of COVID-19. Methods and Preliminary Results: The human ethical committee approved this study (4.014.165). In a case-control study with 117 patients with a diagnosis confirmed by qRT-PCR, 59 participants were in the case group (severe symptoms) and 58 in the control group (mild symptoms). We collected whole blood and performed the isolation of mRNA from total leukocytes. The RT-qPCR assay was performed to analyze the relative expression of genes (2-∆CT). Baseline characteristics showed that 65.5% were male, and 30% of elderly patients were in the severe group (p<0.0001). The patients exhibited at least one pre-existing comorbidity, with hypertension being the most common in 23 (39,7%) patients. Compared with the control group, patients with severe COVID-19 had significantly decreased levels of red blood cells, hemoglobin, percentage of hematocrit (p< 0,001), and lymphopenia positively correlated with neutrophilia (r=0.78; p<0.0001). RT-qPCR analysis showed that INFAR1 and IL-17A gene expression increased in the severe group (p<0.05). ROC curve analysis showed that INFAR1 (area under the curve [AUC] = 0.81) and IL-17A (AUC = 0.77) expression were equivalent to predicted poor outcomes in COVID-19 patients (95%CI; p<0.05). Final Considerations: Our analysis indicates that males, the elderly, hypertension, and exacerbated immune response, indicated by high expression of INFAR1 or IL-17A, were associated with the severity of COVID-19. These findings suggest the potential use of the INFAR1 and IL-17A genes as biomarkers for the severity of COVID-19.

INCREASED NADase ACTIVITY IN THE BRAIN DURING ZIKA VIRUS INFECTION IN NEONATE MICE: POSSIBLE IMPACT OF NAD METABOLISM IN THE IMMUNE RESPONSE AND THE PATHOGENESIS OF NEUROINFECTION

Introduction: Zika virus (ZIKV) infection is an important emerging health issue. During pregnancy, ZIKV infection may lead to neurodevelopmental delays and/or congenital brain abnormalities in the fetus, including microcephaly. Therefore, it is fundamental to understand ZIKV pathogenesis to better design therapies that prevent or mitigate the sequelae caused by gestational exposure. A recent study has identified NAD+ metabolism disruption during ZIKV infection in the fetal brain. Members of the Poly (ADP-ribose) polymerase (PARP) family are known to present varied roles in host-pathogen interactions, acting as an antiviral or pro-viral factor, in addition to its involvement in inflammatory processes. Likewise, CD38 and SARM1 are known to participate in the immune response against viral infections. Therefore, hyperexpression of these NAD+-consuming enzymes may contribute to NAD levels decline as well as the innate immune response against ZIKV infection and its pathogenesis. Here we investigated the modulation of NADases expression and activity in the brain of ZIKV-infected neonates. Methods: We used a mouse model of ZIKV infection in neonates, corresponding to an infection in humans in the third trimester of pregnancy. Swiss mice at P3, subcutaneously infected with 10e6 PFU ZIKV or mock, were euthanized 13 days post-infection. Brain tissue samples were collected and processed for analysis. NADase activity and changes in NAD+ transcriptome were analyzed through etheno-NAD+ assay and qPCR, respectively. Results: Our results showed an increase in NAD+ hydrolase activity by 40% in the brain of ZIKV-infected mice. We also have found a significant increase in mRNA expression of several NADases (from 3 to 30 fold increase). Conclusion: These results showed increased expression and/or activity of NAD+ hydrolases in the brain of neonates during ZIKV infection. Further experiments are needed to confirm these enzymes' contribution to the pathogenesis or the immune response against ZIKV infection in the brain.

INCREASED PLATELET ACTIVATION DURING CHIKUNGUNYA INFECTION

Introduction: Chikungunya fever is a viral disease transmitted by mosquitoes of the genus Aedes. The infection is usually symptomatic and most common symptoms are fever accompanied by joint pain and swelling. In most cases symptoms subside within a week. However, severe prolonged and disabling joint pain, that may persist for several months, even years, are reported. Although the pathogenesis of Chikungunya infection is not fully understood, the evolution to severe disease seems to be associated with the activation of immune mechanisms and the action of inflammatory mediators. Platelets are recognized as inflammatory cells with fundamental activities in the immune response, maintenance of vascular stability and pathogenicity of several inflammatory and infectious diseases. Although the involvement of platelets in the pathogenesis of viral diseases has gained attention in recent years, their activation in Chikungunya has not been explored. The aim of this study was to analyze platelet activation and the possible role of platelets in the amplification of the inflammatory response during Chikungunya infection. Methods and Results: We prospectively included 132 patients attended at the Quinta D'Or hospital and 25 healthy volunteers during the 2016 epidemic in Rio de Janeiro, Brazil. We observed increased expression of CD62P on the surface of platelets, as well as increased plasma levels of CD62P and platelet-derived inflammatory mediators indicating that the Chikungunya infection leads to platelet activation. In addition, platelets from chikungunya patients exhibit increased expression of NLRP-3, cleaved IL-1β and caspase 4, suggestive of platelet-inflammasome engagement during chikungunya infection. In vitro experiments confirmed that the Chikungunya virus directly activates platelets. Moreover, we observed that platelet activation and soluble p-selectin at the onset of symptoms were associated with development of chronic forms of the disease. Conclusion: Collectively, our data suggest platelet involvement in the immune processes and inflammatory amplification triggered by the infection.

INDIVIDUALS LIVING IN ENDEMIC AREA FOR INFECTIOUS DISEASES PRESENT ACCELERATED EPIGENETIC AGING

Introduction: Inflammaging is a low-grade chronic inflammatory state generated by the aging process that can contribute to frailty and age-related diseases in the elderly. However, it may have distinct effects in older adults living in areas endemic for chronic infectious diseases. An increased inflammatory response may confer protection against infectious agents in these areas, although this advantage may come at the cost of accelerating the epigenetic aging of the body's tissues. In this study, we evaluated the inflammatory profile and epigenetic age of infected and uninfected individuals from a leprosy-endemic area in Brazil. Methods: We compared individuals from the endemic area with individuals from non-endemic areas using a multiplex assay (BioRad Bio-Plex® Pro Human Cytokine Standard) to measure 27 inflammatory mediators in the individuals’ sera and the Illumina Infinium MethylationEPIC BeadChip to assess their methylation status and epigenetic age according to the Horwath methodology (DNAmAge). Results: The profile of cytokines, chemokines and growth factors produced by these two groups of individuals showed similarities, although infected individuals have a higher production of these mediators. A significant increase in the production of IL-1ra, CXCL8, CCL2, CCL3 and CCL4 was associated with leprosy infection. Notably, the elderly showed distinct immune responses associated with their infection status when compared to adults, suggesting an adaptive remodeling of their immune responses. Epigenetic analysis showed that 31 CpGs were differentially methylated when we compared infected and control individuals. According to the global inflammatory profile, there was no difference in epigenetic age between the two groups of subjects. However, individuals from the endemic area had a significant accelerated aging when compared to individuals from São Paulo, a non-endemic area in Brazil. Furthermore, this last cohort was also epigenetically aged relative to an Italian cohort. Conclusion: Our data show that living in areas endemic for chronic infectious diseases can result in remodeling of the inflammatory process and acceleration of epigenetic aging in individuals regardless of their infectious state. In conclusion, geographic, genetic and environmental factors influence aging and immunosenescence in its rhythm and profile.

Indoleamine 2,3 dioxygenase is important for the maintenance of cell metabolic activity in Schwann cells stimulated with Mycobacterium leprae.

Introduction: Leprosy is an infectious disease caused by Mycobacterium leprae that affect peripheral nervous system and skin. Schwann cells and macrophages are the major target of M. leprae. The injury of peripheral nervous system is the most severe symptom affecting patients with leprosy. The neural damage may be directly caused by the bacteria or by inflammatory infiltrate. It has been found that pro-inflammatory cytokines may be associated with neural damage by inducing the expression of the enzyme indoleamine 2,3-dioxygenase (IDO), that mediates the initial and rate-limiting step in tryptophan (TRP) catabolism along with the kynurenine pathway (KP). The study of the KP has demonstrated the existence of metabolites with neurotoxic or neuroprotective activity in central nervous system, but little is known of the effect of these metabolites in infections of the peripheral nervous system. Thus, we hypothesized the existence of an association between increased IDO activity and its metabolites (kynurenines) with neural damage in peripheral neuropathy observed in patients with leprosy. Methodology: Human Schwann cells from the ST88-14 lineage were treated with various concentrations of kynurenines and either dead or live M. leprae for 24 hours. After stimulation, the metabolic cell activity of Schwann cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and levels of TNF and IL-1β were assessed by ELISA. Results: In current study, we found that treatment with the KP metabolites: kynurenic acid (KynA), quinolinic acid (QUIN) and picolinic acid (PA) did not affect Schwann cell metabolic cell activity; however, a dose dependent decrease in metabolic cell activity was observed in Schwann cells treated with 3-hydroxyanthranilic acid (3-HAA). Live M. leprae was able to rescue the metabolic cell activity in 3-HAA-treated Schwann cells, but not dead M. leprae. Furthermore, we observed that the treatment of cells with IDO inhibitor 1-MT (1-methy-D- tryptophan), along with dead M. leprae, inhibited metabolic cell activity in Schwann cells. TNF and IL-1β levels increased after M. leprae stimuli and in the presence of QUIN and 3-HAA. Conclusion: Our results suggest that metabolites of kynurenine pathway may differentially modulate the Schwann cell metabolic activity and that IDO is important for the maintenance of cell metabolic activity in Schwann cells stimulated with M. leprae.

INDUCTION OF IMMUNOGENIC CELL DEATH BY VACCINATION IN AN EXPERIMENTAL MURINE MODEL OF MELANOMA AFTER THERAPEUTIC VACCINATION PROTOCOLS.

Introduction Melanoma treatments are usually systemic and present innumerable side effects. The immune system has tools to eliminate tumor cells before tissue is formed. Still, many tumors manage to repress the immune system, allowing cancer to grow and preventing the immune system from exerting its role. Mushrooms are sources of beta-glucans that present active properties, mainly immunoregulatory. In addition to activating the immune, the early presentation of tumor antigens can cause the maturation of the response against the tumor. It can improve the body's response against a future neoplasm. The use of therapeutic vaccines has been gaining ground in the treatment of tumors resistant to drugs or therapies currently available. Prophylactic vaccines have been known for a long time and act by preparing the immune system for the encounter of the antigen or organism used in the vaccination, aiming for a more effective and faster treatment of pathogens. Methods and Results In vitro assays were performed with B16 (melanoma-derived cells) culture for 24 hours with chemotherapeutic agents or mushroom fractions at different concentrations. After 24 hours, the supernatant was collected and used to determine the concentration of ATP and HMGB1 to prove immunogenic death. Mammal cells were added with treatment for more than 24 hours to access cytokine production. To prove the same theory of cell death, fluorescence microscopy was done to show the translocation of calreticulin to the membrane, which along with the increase in ATP and HMGB1 concentration in the supernatant, are known markers of immunogenic cell death. For the In Vivo experiments, the B16 cells were cultured for 24 hours with different treatments. They are administered as subcutaneous vaccines three weeks before or after tumor induction to access anti-tumoral response. The therapy that causes immunogenic death plays a more interesting role in tumor prevention through vaccination. In vitro results show that B16 cells experienced an immunogenic death when treated with doxorubicin. The treated B16 cells co-cultured with M1/M2 macrophages and dendritic cells induced a cytokine production modulation along with gene modulations. In vivo vaccination of C57bl6 mice before or after tumor, induction shows different results depending on the treatment b16 cells received. Conclusion Based on the results, immunogenic death is an essential step in activating the immune response and can be used in cases of melanoma.

INFLAMMASOME ACTIVATION IN THE LEPROSY NEUROPATHY

Introduction: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an intracellular pathogen that has tropism for Schwann cells. Neural damage during leprosy may occur by M. leprae or by inflammatory cytokines, produced mainly during the occurrence of reactions. Leprosy reactions are responsible for incapacities in leprosy and represent the major cause of permanent neuropathy. Our previous study demonstrated that the type 1 reaction outcome is associated with an impairment in autophagy process in skin lesion cells associated with the increased expression of NLRP3, caspase-1 (p10) and IL-1β production, suggestive of an involvement of inflammasome machinery in leprosy neuropathy. Methods and Results: The transcriptomic analysis of human primary Schwann cells demonstrated that M. leprae infection triggers a positive regulation of genes of the inflammasome pathway like NLRP1, CASP5, PYCARD e CARD8 after 24h. Analysis of cytokine production by ELISA revealed that both live and dead M. leprae increased IL-18 levels in supernatants from ST88-14 cells after 24h. To evaluate the role of different M. leprae antigens, ST88-14 cells were stimulated with different mycobacterial components like DNA+Hlp, soluble fraction of M. leprae (MLSA), membrane fraction of M. leprae (MLMA) and the core of M. leprae cell-wall composed by Mico arabinogalactan-peptidoglycan (mAGP) alone or in the presence of LPS. Both irradiated and sonicated M. leprae, as well as DNA+Hlp, were able to significantly increase levels of IL-1β in relation to non-stimulated cultures. Gene expression analysis by qRT-PCR revealed an upregulation of IL18 in nerve fragments from patients with pure neural leprosy when compared with other neuropathies and immunofluorescence microscopy demonstrated the expression of ASC in the perineurium. In addition, serum levels of IL-1β in serum from patients with pure neural leprosy were higher when compared with patients with other peripheral neuropathies. Conclusion: Therefore, our results suggest that the inflamassomes activation could be involved in the leprosy neuropathy and mediates the production of inflammatory factors, such as IL-1β and IL-18.

INFLAMMASOME ACTIVATION IN THE PULMONARY PARENCHYMA DEFINES TWO DISTINCT PROFILES ASSOCIATED WITH CYTOKINE STORM AND WORSENING OF LUNG FUNCTION IN COVID-19 PATIENTS

Inflammasome activation is associated with disease severity in patients infected with SARS-CoV-2 and influenza viruses. However, but the specific cell types involved in inflammasome activation as well as the balance of inflammasome activation versus viral replication in COVID-19 exacerbation and induction of patient death is still unknown. Here, we assessed human lung autopsy of 47 fatal COVID-19 patients and 12 influenza fatal casespatients , and examined inflammatory profile, inflammasome activation and the correlationed with clinical and histopathological patient’s conditions. We found an overall stronger inflammasome activation in lethal cases of SARS-CoV-2 compared to iInfluenza infected patients as well as a and found a different profile of inflammasome-activating cells during these diseases. In COVID-19 patients, inflammasome activation is mostly mediated by macrophages and endothelial cells whereas in iInfluenza, type I and type II pneumocytes contribute more significantly. Analysis of gene expression allows classification of COVID-19 patients in two different clusters, cluster 1 (n=16 patients) died with higher viral loads and reduced inflammatory profile than oppose to cluster 2 (n=31). Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, and inflammasome activation significantly differed in the two clusters. Our data reveal two distinct profiles in lethal cases of COVID-19, indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation may lead to different clinical conditions, yet both lead to patient death. Understanding this process is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.

Inflammation mediated by smoking and in chronic obstructive pulmonary disease is shown to be controlled by a new enzyme inhibitor

Introduction: Several enzymes are responsible for the regulatory pathways of factors that modulate inflammatory responses in the lungs, these processes are closely related to tissue damage and functional loss of these organs. The evaluation of enzymatic interactions related to the mechanisms of lung damage is a possible generator of intervention to improve the patient's prognosis. The inhibition of the epoxide hydrolase enzyme using “GSK2256294” has been considered promising in clinical and preclinical models for pulmonary inflammatory processes associated with tobacco and COPD, but the discrepancies between the effect sizes in the types of models have not yet been raised. Objective: Thus, the present study aimed to evaluate the regulatory activity of a new enzyme inhibitor in lung inflammation Methods: A secondary study was carried out in the scientific databases and gray literature. The searches were performed using the descriptors “COPD”, “GSK2256294” and their corresponding synonyms, delimiting the records between the period from 2012 to 2021. The records of clinical and pre-clinical in vivo or in vitro trials were included. Duplicate articles that did not fit the scope of the research were excluded. The methodological quality and consistency of evidence were analyzed. Meta-analysis was applied to compare the different variables. Result: A total of 86 studies were found in the different databases, 4 of which were selected from the gray literature. After applying the eligibility criteria, 3 studies were classified and evaluated. However, GSK2256294 was able to reduce the soluble epoxide hydrolase enzyme in both clinical and preclinical study models, but with greater effectiveness in clinical studies. In addition, it collaborates in the anti-inflammatory activity mediated by the eicosatrienoic pathway, both by reducing DHETS and Leukotoxin-diol. It also helps in controlling blood flow. Conclusion: The present study demonstrated that GSK2256294 is a promising drug in the control of the deleterious manifestations observed in lung inflammation, however, due to the small number of articles retrieved from the databases, further studies are needed to ensure consistency of evidence and allow for unraveling. other routes of biological activities mediated by the inhibition of the soluble epoxide hydrolase enzyme in clinical and preclinical studies.

INFLAMMATORY RESPONSE TO COVID-19 IN OVERWEIGHT PATIENTS FROM ENDEMIC AND NON-ENDEMIC AREA FOR INFECTIOUS DISEASE

Introduction: The Covid-19 pandemic caused by the Sars-Cov-2 virus was responsible for the death of more than six million people since 2019. Risk factors for severe forms of the disease include obesity, and associated comorbidities, such as diabetes and high blood pressure. Overweight and obese people living in endemic area for infectious diseases may present a more pronounced inflammatory response to SARS-CoV2 due to underlying infections. In this study, we compared the inflammatory profile of patients with excess weight living in endemic and non-endemic area for infectious disease in response to Covid-19. Methodology: Overweight individuals from Belo Horizonte (non-endemic area) and Governador Valadares (endemic area) presenting mild flu symptoms from up to seven days after beginning of symptoms were recruited between 2020 and 2021 (before vaccination). Sars-Cov-2 infection was confirmed by a nasopharyngeal swab test by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and blood samples were collected. The inflammatory profile was determined through the analysis of 27 biomarkers, using a Luminex assay (Bio-Plex® Pro Human Cytokine Assay). Results: A total of 87 excess weight patients from Belo Horizonte and 47 from Governador Valadares were recruited and divided into two groups: Covid-19 and Influenza-like illness (ILI). No significant differences were found between age, the proportion of overweight and obesity, or prevalence of comorbidities. Results showed that individuals living in the endemic area showed a more expressive inflammatory profile when compared to non-endemic area regardless of Covid-19 diagnosis. They presented a significantly increased production of IL-5, IL-13, IL-15, and PDGF when compared to people living in Belo Horizonte. No differences in biomarkers were observed between Covid-19 and ILI patients from Governador Valadares. Levels of CCL-2 increased in Covid-19 for both populations and were significantly higher in the cohort from Governador Valadares. Conclusion: Our investigation shows that people living in an endemic area for infectious diseases present a prominent production of inflammatory mediators in response to infections when compared to a cohort from a non-endemic area. Excess weight does not seem to enhance inflammatory response to mild cases of Covid-19 in people living in an endemic area. Underlying infections may play an important role in the inflammatory response for both Covid-19 and ILI.

Inflammatory stimuli lead to translational stress in activated macrophages

During macrophage classical activation, there is increased production of reactive oxygen species (ROS). Although the production of ROS is necessary for the inflammatory response, it is not without drawbacks: ROS are known to cause oxidative damage to nucleic acids, both DNA and RNA. This damage is primarily in the form of oxidation of guanine bases, creating 8-oxoguanosine (8-oxo-G) in RNAs. The occurrence of 8-oxo-G in mRNAs can cause ribosome stalling and collisions, hampering the translational machinery. In order to keep the protein synthesis fitness, cells have translation control mechanisms which can identify collided ribosomes and promote their rescue; these include the proteins ZNF598 and LTN1, the latter being a component of the RQC complex. Since inflammatory macrophages present high oxidative stress due to increased levels of intracellular ROS, it is possible that they are also under translational stress, highlighting the importance of their translation control mechanisms. To investigate if inflammatory activation causes translational stress, we polarized macrophages with inflammatory (LPS and IFN-γ) or regulatory (IL4) stimuli and non-lethal doses of the translation inhibitor cycloheximide (CHX). Inflammatory macrophages treated with CHX for 24h became apoptotic, in contrast to regulatory macrophages, which were still viable. This suggests that inflammatory macrophages are under translational stress, since the additional stress from CHX became lethal, which differs from regulatory macrophages. Agreeing with this, treatment with CHX led to reduced levels of the cytokines TNFα and IL6 in inflammatory macrophages. Also, CHX treatment alone was able to promote a weak expression of IL6, indicating that translational stress itself might act as an inflammatory signal. In contrast, early phagocytic and microbicidal activity of E. coli treated macrophages was not influenced by CHX, indicating that the damaging effects arise after mechanisms involved with pathogen phagocytosis/killing. Together, our results suggest that inflammatory and not regulatory macrophages are under severe translational stress, which direct impact their activity and function.

INFLUENCE OF HLA REPERTOIR IN THE OUTCOME OF HCMV INFECTION IN TRANSPLANTED INDIVIDUALS

Introduction: Human Cytomegalovirus (HCMV) is a common human herpesvirus mostly asymptomatic in immunocompetent population. In individuals under immunosupression because of transplantation, HCMV infection can reactive and affect the transplant outcome. The type I HLA is an important molecule that participates in antigen presentation to CD8+ T cells promoting their activation and elimination of infected cells. Our hypothesis is that prediction of the interactions between the HLAs and HCMV proteins might enable to understand the dynamics of virus infection post transplantation. Methods and Results: We developed a score for each HLA-I, based on the numbers of in silico predicted significant interactions that each HLA molecule performs with the 10 most immunogenic proteins of the virus. With the defined HLA-I score values, we calculated a final score for the HLA-I repertoire for each patient. To assess whether the HLA-I repertoire of transplanted patients was associated with the clinical outcome, we investigated the medical records of 65 patients who had HCMV infection. Patients with severe HCMV infection had lower HLAs repertoire scores than patients with mild HCMV infection (1017 ± 247 versus 1508 ± 188, P= <0,0001). A 1300 score cut off could predict mild HCMV evolution with 100% specificity and 85% sensitivity after transplantation. To investigate the immunological basis of this finding, blood samples from patients from both groups were submitted to stimulation with a HCMV peptide library and flow cytometry analysis. Preliminary results indicate that IFNg production by CD8 T cells correlate positively with the HLA-I repertoire score, while IL-10 production correlated negatively. Conclusion: The score of HLA-I repertoire of transplanted patients seems to be able to determine the outcome of the infection based on the capacity of CD8 T cells responses.

INFLUENCE OF OBESITY ON THE MECHANISMS OF PERIPHERAL TOLERANCE AND ON THE MODULATION OF IMMUNE RESPONSE IN THE SMALL INTESTINE OF MICE

Introduction: Studies suggest that obesity increases the onset of autoimmune diseases. However, immune mechanisms are not well elucidated. The small intestine responds to diet, microbiota, and pathogens antigens while maintaining immune homeostasis beyond participation in the peripheral tolerance. It is appreciated that obesity impairs intestinal immunity, but whether obesity exerts influence on the mechanisms of peripheral tolerance is unknown. Here, we aim to investigate whether obesity leads to the loss of peripheral tolerance focusing on the immune processes in the small intestine. Methods: We performed in silico analyses to explore whether obesity (induced by HFD or using ob/ob mice) promoted differential expression of genes of interest on small intestine epithelial cells or adipose tissue Tregs. Public datasets containing data from transcriptomic assays (microarray or RNAseq) were analyzed by GEO2R (limma) or NetworkAnalyst (EdgeR). Adjusted p-value (padj) and logarithm fold change (LogFC) values were utilized for enrichment analyses by using the platform Metascape. Genes were considered upregulated when padj<0,05 and LogFC>1, and downregulated when padj<0,05 and LogFC<-1. Results: Mice submitted to HFD showed downregulation of genes involved with the modulation of microbiota and the response to pathogens, such as Mbl2, Reg3g and Zbp1, and reduced expression of genes involved with the regulation of inflammatory response, such as Casp4 and Socs3 in the small intestine (GSE26300). In another dataset (GSE182348), the jejunum of ob/ob mice showed upregulation of Areg and genes involved with the response to interferons, and downregulation of Cfd and genes involved with the regulation of the adaptive immunity. Focusing on Tregs, the analysis of the dataset GSE174706 showed reduced expression of Il2ra and Il10 and increased expression of Cd86 and Dpp4 in Tregs isolated from the adipose tissue of HFD-submitted mice. Conclusion: In silico data showed that obesity alters the expression of genes involved with the response to intestinal microbiota and regulation of immune response and that obesity alters the gene expression profile of adipose tissue Tregs. These data suggest that obesity may influence intestinal immunity and interfere with the mechanisms of peripheral tolerance. Ongoing in vivo experiments will decipher the underlying mechanisms associated with obesity and peripheral tolerance.

INFLUENCE OF T LYMPHOCYTES ON THE CENTRAL NERVOUS SYSTEM OF MICE DURING DEVELOPMENT

ERIANE CERQUEIRA DOS SANTOS1; FELIPE HENRIQUE DA CUNHA XAVIER2; PEDRO HENRIQUE OLIVEIRA VIANNA2; YGOR PARLADORE SILVA2; ÁQUILA RODRIGUES COSTA SANTOS2; ADRIANA CESAR BONOMO2; RUDIMAR LUIZ FROZZA2; POLIANA CAPUCHO SANDRE2. 1 IFRJ, Biological Sciences, Rio de Janeiro - RJ. 2 FIOCRUZ, Oswaldo Cruz Institute, Thymus Research Laboratory, Rio de Janeiro - RJ. The role of the immune system involves not only the recognition of self versus non-self, but also the maintenance of homeostasis of the organism throughout development. For decades it was believed that the central nervous system (CNS) was not responsive to the peripheral immune machinery due to the apparent absence of lymphatic vascularization and anatomical organization of the brain. Recent research has changed the view of the CNS as immunoprivileged site and demonstrates that innate and adaptive immune cells can provide homeostatic support to the CNS through neuroimmune communication. Thus, this work aims to evaluate the presence and analyze T lymphocyte profiles different brain areas during development and their possible contributions in stages before and after the closing of the critical period in mice. The experiments were approved by the Ethics Committee on the Use of Animals (CEUA/IOC-023/2020). Through RT-PCR method, it was possible to detect the presence of T lymphocytes in different brain regions (cortex, hippocampus, cerebellum, and other brain areas) at all ages analyzed (PND7, PND21, PND49, 24 weeks and 20 months). The immunofluorescence and flow cytometry techniques were standardized for the different brain regions for further analysis of the location and profile of CD4+ T lymphocytes during CNS development. In addition, behavioral tests were standardized for further study of the possible contribution of CD4+ T lymphocytes to neurodevelopment in neonates and adults immunodeficient animals. Through these preliminary data, we can conclude that T lymphocytes are present during the development and maturation of the CNS, which may be relevant for the modulation of behavior, memory and learning. This project is essential to research improvement and the understanding of how the immune system acts in the different stages of CNS development, opening paths for elaboration of more effective therapies and manipulations of immune system in neurodevelopmental disorders.

INGESTION OF SUGARY SWEET BEVERAGES ALTERS METABOLISM, INTESTINAL MUCOSA IMMUNITY AND BEHAVIOR OF MICE

INTRODUCTION: The increased consumption of meals rich in fat and refined carbohydrates favors the development of metabolic changes, and higher inflammation in adipose and in lymphoid organs. In addition, to being associated with intestinal dysbiosis, which in turn has been correlated with the development of psychiatric disorders. Among the highly consumed foods, sugar-sweet beverages, have high content of glucose and fructose. So, we sought to clarify whether the ingestion of sugar-sweet beverages change the innate behavior and how the metabolic changes induced by sugar-sweet beverages interfere with the gut immune system. METHODS: We used an experimental model with C57BL/6 mice that were fed with AIN93G diet and received sugary drink (25% of a mixture of glucose and fructose, in the proportion 45:55, respectively). While control animals consumed the AIN93G diet and drank filtered water. The anxiety-like behavior, the metabolism and changes in the immune cell profile of intestinal mucosa cells were evaluated in the 8 th week. RESULTS: After 8 weeks of treatment, animals treated with sugar-sweet beverages showed increased total caloric intake, adiposity and had the glucose and lipid metabolism altered. In addition, animals treated with sugar-sweet beverages showed anxiolytic behavior after 8 weeks of treatment, a fact that would be harmful to the animal. Furthermore, adipose tissue inflammation in the sugar-sweetened beverage group was identified by histological and flow cytometry analysis, which showed an increased frequency of Th 17 cells (CD4+RORγT+) and a reduced frequency of FoxP3+ regulatory T cells. Also, the mesenteric lymph nodes showed an increased frequency of Th 17 cells (CD4+RORγT+) and a reduced frequency of FoxP3+ regulatory T cells. CONCLUSION: Thus, it was possible to observe that the chronic consumption of a solution containing glucose and fructose resulted in metabolic syndrome, changes in innate behavior and increased inflammation in the mesenteric lymph nodes after.

INGUINAL LYMPH NODE IMMUNOSENESCENCE MARKERS AND CD8/CD4 T CELL RATIO ARE INDUCED IN EXERCISE-TRAINED LIPODYSTROPHIC MICE

INHIBITION OF PROTEIN DISULFIDE ISOMERASE AS A POTENTIAL THERAPEUTIC MECHANISM AGAINST TUMOR ESCAPE BY PROMOTING OXIDATIVE STRESS AND REDUCING CELL ADHESION IN BREAST CANCER CELLS

INTRODUCTION: Cancer is a pleiotropic disease caused by the uncontrolled proliferation of cells and, when in metastasis, it uses several mechanisms of tumor escape, including the formation of the platelet shield in the bloodstream. Recent research points to it as the second leading cause of death in the world and an acceleration of the death toll to one in eight men and eleven women worldwide, of which lung and breast cancer are predominant. Cancer is a multifactorial disorder, with several conventional therapies in its treatment, enormous side effects due to its nonspecificity, and it can attack healthy cells. In the last decades, phytochemicals have been studied as an alternative form of treatment and an adjuvant in conventional anticancer therapy. Of the various phytochemicals that have exhibited potent anticancer properties, flavonoids have been one of the most prominent in recent years, as they have diverse pharmacological properties that include antioxidant, anti-inflammatory, anticancer and epigenetic modulating activities. METHODS: To investigate the antitumor effect of the flavonoid #2506 inhibiting PDI inducing oxidative stress in a tumor microenvironment model, with in vitro cytotoxicity assessment techniques and 3D cell culture that mimic tumor tissues closer to in vivo, quantification of H2O2 and NO and co-culture with platelets. RESULTS: #2506 generated a cytotoxic effect in normal breast tissue cells, in a discrete way, and in breast cancer cells in a more pronounced and dose-dependent way, mainly in the more aggressive strain. It was also evident in our results that #2506 reduces spheroid formation and stabilization, this effect being more manifest in MDA-MB-231 triple negative breast cancer cells. There was an increase in the production of H2O2 and NO, in a dose-dependent manner and more evident in cells with greater metastatic power. There was a dose-dependent reduction in the interaction of tumor cells with platelets and more evident in the MDA-MB-231 lineage. CONCLUSION: The studies are unanimous in stating that there is still a lot to be researched on the antineoplastic effect of #2506, as its effect is proven to be broad in several molecular targets that involve the progression of different types of cancer, confirmed by different studies.

In silico ANALYSIS OF MOLECULAR INTERACTIONS AND BINDING AFFINITIES BETWEEN SESQUITERPENES WITH MPK1 AND ATF-7

Introduction The MPK-1/ERK pathway is activated when there is an accumulation of apoptotic corpses caused by deficient clearance mechanisms that stimulate cytokines release in Caenorhabditis elegans - raising resistance to pathogenic bacteria (Virulence 12:75-83, 2020). ATF-7, associated with PMK-1, regulates most genes related to the nematode's immune response induced by pathogenic infection (FPLoS Gen 15(2):e1007830, 2019). Sesquiterpenes (ST) are terpenoid molecules extracted from different plant species linked to anti-inflammatory properties. Objective To evaluate and map the interactions between STs molecules with MPK-1 and ATF-7, besides obtaining their binding energy and affinity via in silico analysis. Methods and results The AlphaFold was used to obtain the protein structures of MPK-1 and ATF-7 (Nature 596:583-589, 2021; Nuc Ac Research 50:D439-D444, 2021). 20 STs molecules were obtained from PubChem. GRaSP was used to predict the binding sites of the proteins (Bioinf 36:i726-i734, 2020). Protein preparation and molecular docking were performed on Autodock Vina software (J Comput Chem 31:455-461, 2010). The molecules showed binding affinities between -8.3 to -6.9 Kcal/mol and -7.4 to -5.6 Kcal/mol for MPK-1 and ATF-7, respectively. Among these, the lowest binding energy with MPK-1 was represented by α-Eudesmol (-8.3 Kcal/mol), α-copaene (-8.1 Kcal/mol), β-Copaene (-8.1 Kcal/mol), and Aromadendrene (-8.1 Kcal/mol). α-Copaene (-7.4 Kcal/mol), y-amorphene (-7.1 Kcal/mol), and β-Bisabolene (-6.9 Kcal/mol) were molecules with the lowest binding energy with ATF-7. The amino acids Valine (A:49), Leucine (A:178), and Alanine (A:62) showed essential interactions at the active site of MPK-1 with STs. In ATF-7, common bonds occurred in the amino acids Phenylalanine (A:112 and A:108), Alanine (A:405), and Tyrosine (A:408). Conclusion The obtained in silico results reveal STs molecules can interact with C. elegans immune response regulators, MPK-1 and ATF-1; thus, they may be capable of interfering in the innate system of the nematode. Hence, the worm can be used as a model to elucidate the ST's anti-inflammatory properties.

In silico ANALYSIS OF MOLECULAR INTERACTIONS AND BINDING AFFINITIES OF SESQUITERPENES ON THE INFLAMMATORY TARGETS IL-6 AND TNF-α IN THE IMMUNOMODULATION OF NEUROINFLAMMATION

Introduction Neuroinflammation is a biological response to noxious stimuli protecting nervous tissue and maintaining homeostasis and repair. However, elevated levels of IL-6 and TNF-a mark neuroinflammation and their increase disrupts the blood-brain barrier (BBB) and other damages (Mol Neurobiol 59:1724-1743, 2022). Sesquiterpenes are a class of terpenes synthesized by plants used as semiochemicals with many biological activities. Objective Analyze the favoring of the sesquiterpenes' binding energy in IL-6 and TNF-α, as well as map the types of binding, interactions with different amino acids, and pharmacokinetics of sesquiterpenes. Methods and results The crystallographic molecular structures of IL-6 (PDB: 1ALU) and TNF-α (PDB: 6OP0) proteins were obtained from the protein data bank. Twenty sesquiterpenes molecules were obtained from PubChem. GRaSP was used to identify binding sites on proteins through machine learning. Molecular docking was performed in Autodock Vina software, removing water molecules and adding polar hydrogen (J Comput Chem 31:455-461, 2010). The BIOVIA software was used to visualize molecular interactions with proteins. The prediction of activity spectra for substances (PASS) was performed on the Way2Drug server (Bioinformatics. 16:747-748, 2000). Pharmacokinetics were measured on pkCSM and Swisstarget servers. The molecules showed binding affinities from -5 to -6.2 and -5 to -6.9 Kcal/mol for IL-16 and TNF-α, respectively. Epi-cedrane (-6.2 Kcal/mol), Allo-aromadendrene (-6.1), and α-Copaen-11-ol (-6.1) presented the best values for IL-6. Valencene (-6.9) and α-copaene (-6.7) obtained the best TNF-α affinity scores. The interactions between ligands and amino acids were Alkyl, Pi-alkyl, and Pi-sigma bonds. 8-epi-Dictamnol, α-Eudesmol, and Himachalol showed hydrogen bonds with IL-6. 8-epi-Dictamnol and Himachalol also showed hydrogen bonds with TNF-α. Sesquiterpenes presented intestinal absorption above 93.075%, and permeability to BBB, however any hepatotoxicity. PASS showed the estimation of immunosuppressive, immunomodulatory, and anti-inflammatory activity, as well as inhibition of TNF expression and IL-6 antagonism. Conclusion In silico analyzes favored the staging of sesquiterpenes regarding interactions and binding energy with IL-6 and TNF-a, in addition to being linked to direct actions on the immune system by probabilistic analysis. The results guarantee theoretical support for subsequent in vitro and in vivo analyses.

IN SILICO MOLECULAR INTERACTION OF Enterolobium contortiliquum TRYPSIN INHIBITOR (EcTI) AND PROSTATE CANCER METASTASIS MEDIATOR CYTOKINES

Introduction Most of the deaths caused by prostate cancer result from metastasis development induced by epithelial-mesenchymal transition, stimulated by cytokines such as IL-6, VEGF-β, CCL2, CX3CL1, CXCL1, and CXCL12 (Int J Mol Sci 21(12):4449, 2020). The Enterolobium contortisiliquum trypsin inhibitor (EcTI) is a proteinase inhibitor capable of inhibiting the action of molecules when they interact with active sites, forming inactive stoichiometric complexes (FASEB J. 31:695.1-695.1, 2017). Objective To obtain an in silico analysis of molecular interactions between EcTI and IL-6, VEGF-β, CCL2, CX3CL1, CXCL1, and CXCL12. Methods and Results The crystallographic structures of IL-6 (1ALU), VEGF-β (2C7W), CCL2 (IDOK), CX3CL1 (1F2L), CXCL1 (1MGS), CXCL12 (3HP3), and EcTI (4J2K) were gathered from Protein Data Bank. ClusPro 2.0 was used to make the protein-protein molecular dockings (Structure 28:1071-1081, 2020; Proteins 85:435-444, 2017; Nature Protocols 12: 255-278, 2017; Proteins 81:2159-2166, 2013). The PRODIGY webserver annalized the archives obtained (Front. Mol. Biosci. 8:729513, 2021). EcTI showed more affinity with VEGF-β (ΔG=-13,98±1,34 Kcal/mol; Kd=2,81±6,6E-10 M) and CCL2 (ΔG=-13,29±1,79 Kcal/mol; Kd=1,47±2,38E-9 M) than with CXCL12 (ΔG=-9,82±1,11 Kcal/mol; Kd=1,35±1,08E-7 M), CX3CL1 (ΔG=-9,58±1,1 Kcal/mol; Kd=2,97±3,75E-7 M), CXCL1 (ΔG =-9,51±0,21 Kcal/mol; Kd=1,06±0,35E-8 M), and IL-6 (ΔG= -8,11±1,97 Kcal/mol; Kd=1,89±3,3E-5 M). Conclusion The results show that EcTI can interact with cytokines related to prostate cancer metastasis development, mainly to CCL2 and VEGF-β. It indicates EcTI possibly can inhibit these cytokines' activity, and reduce prostate cancer metastasis development.

IN SILICO PREDICTION AND IN VITRO STUDY OF SYDNONES AND CHALCONE-THIOSEMICARBAZONES EFFECTS ON HTLV-1-INFECTED CELLS

INTRODUCTION: Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of people worldwide and is responsible for highly aggressive adult T-cell leukemia (ATL). ATL carriers have a poor prognosis, a low life expectancy and the infected cells are resistant to apoptosis-inducing agents (Viruses 8:31, 2016). There is still a need to develop more effective therapies, and it is important to investigate new compounds with cytotoxicity or antiviral activity. Due to their large capacity and diversity of biological activities, sydnones and chalcone-thiosemicarbazones (CTs) derivatives show promising aspects for this purpose (Basic Clin Pharmacol Toxicol. 119:41-50, 2016; Eur J Med Chem. 46:4702-08, 2011). Our objective is to study the effects of new compounds on HTLV-1-infected cells. METHODS: 13 sydnones and 3 CTs were evaluated using AdmetSAR and PASSonline softwares to verify the theoretical toxicity through predictions of the biological activity. Compounds effects were evaluated through A. salina toxicity method and in vitro assays were carried out to examine the cytotoxic activity on Jurkat and HTLV-1-infected MT-2 cell lines. RESULTS: The in silico study resulted in prediction of 12 compound effects in different cancer cells, as leukemia cells, with a low probability to exhibit toxicity to non-tumor cells. The analysis also resulted in the probability of the existence of antiviral activity against different viruses, such as Dengue and Vaccinia virus, and 2 sydnones and 2 CTs were likely to have antiviral activity against HIV. Hepato and genomic toxicity, endocrine dysregulation and absorption capacity analysis suggested that most compounds have low toxicity. A. salina toxicity assay demonstrated treatment with the compounds did not affect the survival of brine shrimp, with the exception of sydnones S3 and S4. Cytotoxicity assays demonstrated that sydnones 2 and 6 presented survival rates below 70-75% on Jurkat (S2 IC50=36.1µM; S6 IC50=66.3µM) and MT-2 cells (S2 IC50>100µM; S6 IC50=74.2µM), when treated with the higher concentration. Furthermore, preliminary results indicate that all 3 CTs have promising effects on survival of both cell lines with IC50<25µM. CONCLUSION: Chalcones are substances belonging to the family of flavonoids and their structural link with thiosemicarbazones, suggested an improvement to biological activity. Altogether our results suggest that these compounds are able to show activities in the HTLV-1-infected cell model.

INTEGRATION OF SINGLE-CELL TRANSCRIPTOME REVEALS MACROPHAGES AS AN IMPORTANT CLINICAL FACTOR IN PROGNOSIS IN TRIPLE-NEGATIVE BREAST CANCER

Introduction: Breast cancer (BC) is the most prevalent malignancy and the leading cause of cancer death among women worldwide. The choice of treatment is based mainly on the molecular subtype. Understanding the tumor immune microenvironment (TIME) is extremely important for determining the impact of different cell compositions on the patient's clinical outcome and also to understand how cell-cell communication is involved in tumor progression. Single-cell RNA sequencing (scRNA-seq) analysis enables a high-resolution understanding of the transcriptomic profile of subpopulations/states in complex tissues, such as tumor mass. Methods and Results: scRNA-seq and clinical data from 76 treatment-naive patients diagnosed with BC of all molecular subtypes — ER+, HER2+, and Triple Negative (TNBC) — as well as from 18 healthy donors were collected from five public datasets. Data were subjected to a rigorous quality control pipeline and integrated, totaling 465,948 high-quality cells. After clusterization, cell types were identified using a transfer learning method, and by analyzing the expression of markers among differentially expressed genes. Epithelial components were subdivided into basal and luminal cells. In addition, we identified vascular and lymphatic endothelial cells, pericytes, and fibroblasts from the stroma. The main diversity was found in the immune cell component, where we distinguished 168,769 cells into the following 16 cells subpopulations: Plasma and Follicular B cells, TCD8+, TCD4+, γδ T, T Reg, neutrophils, mast cells, and plasmacytoid dendritic cells (pDC); regarding myeloid mononuclear phagocytes, we identified conventional dendritic cells (cDC) type 1, type 2 and mature cDC, monocytes, and macrophages (Mø). BC cohorts from TCGA were downloaded and deconvoluted to estimate the TIME composition of each sample, using the signature of the immune cell identified by scRNASeq. Our analysis demonstrates that TNBC is the molecular subtype most impacted by the TIME landscape, and patients with Mø enrichment exhibited an increased risk of death (HR = 1.6 (1.1-2.2) p-value = 0.011). Conclusion: Integration of scRNA-seq data allowed us to transcriptionally characterize immune and non-immune cells across BC molecular subtypes. TIME heterogeneity is associated with tumor progression and patient survival. Mø negatively impacted the TNBC subtype prognosis. Future works should dissect which states or phenotypes are associated with this worse prognosis.

Integrative inflammatory and metabolic phenotypes of severity, fatality and recovery of COVID-19

Coronavirus disease 2019 (COVID-19) has been associated with physiological alterations that provide insights into the pathogenesis of the disease. Moreover, factors driving recovery from COVID-19 can be explored to identify correlates of protection. The cellular metabolism represents a potential target to improve survival upon severe disease or develop therapies that prevent the progression fo disease, but the associations between the metabolism and the inflammatory response during COVID-19 are not well understood. In this study, we analyzed blood cell counts, acute phase proteins, coagulation function, cytokines, and metabolomes of 150 individuals with mild to severe disease, including individuals that had fatal COVID-19. Twenty individuals were followed-up after hospital discharge and recovery of acute disease. We used the integration method of hierarchical community network to identify the association between markers of inflammation, coagulation, tissue damage, cytokines and metabolites. We identified significant alterations in the plasma metabolome of individuals with positive RT-qPCR status for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Carnitine and derived metabolites increased in the plasma of individuals with COVID-19, however, differential abundance of metabolites affected by infection greatly overlap with metabolites influenced by other factors, including age, sex, diabetes, hyperthension, dyslipidemia, chronic obstructive pulmonary disease, chronic renal disease, heart disease and obesity. Interestingly, metabolites and pathways whose activity varies according to the disease severity, also correlate with levels of oxygen saturation. Differential metabolism underlying death was marked by amino acids and related metabolites, such as glutamate, tryptophan and oxoproline; and lipids, including progesterone, phosphocholine and lysophosphatidylcholines (lysoPCs). Individuals that recovered from severe disease displayed persistent alterations enriched for metabolism of purines, phosphatidylinositol phosphate and glycolysis. Recovery of mild disease was associated with vitamin E metabolism. Data integration demonstrated that the metabolic response is a hub connecting other biological features during disease and recovery. In conclusion, infection by SARS-CoV-2 induces metabolic and inflammatory responses that correlate with earch other, affect disease severity and collectively predict differential ouctomes of COVID-19.

Interaction between NETs and hRSV: western blot and immunoprecipitation analysis

Human Respiratory Syncytial Virus (hRSV) is considered one of the main viral agents responsible for respiratory infections, with no approved vaccines so far. The approved treatments consider Ribavirin, a nonspecific drug with great cytotoxic potential, or the Palivizumab antibody, with a restricted indication and high cost. When infected, the patient has an intense accumulation of neutrophils in the pulmonary region, which upon stimulations, secrete an extracellular content containing a large amount of cytoplasmic and granular content, called “Neutrophil Extracellular Traps” or NETs. NETs, ​​in turn, have the ability to capture and inactivate bacteria, viruses, parasites and fungi. Furthermore, it was demonstrated that NETs have the ability to bind to hRSV particles, in addition to promoting virucidal action against it. Thus, the present work aimed to evaluate possible interactions between hRSV and molecular components of NETs. To this end, western blotting and immunoprecipitation assays were performed, using NETs obtained from human neutrophils stimulated with PMA and hRSV from the culture of Hep-2 cells. Western blotting assays demonstrated that NETs immobilized on nitrocellulose membranes interact with hRSV viral particles, because the specific antibody Palivizumab evidented the virus localized in a region corresponding to an apparent molecular weight between 50-75 kDa.In an alternative and complementary way, the same band was evidenced in immunoprecipitation assays between NETs and hRSV. Future protein sequencing assays may clarify the identity of this molecule. The data set indicates that one of the molecular components of NETs, ​​with an apparent molecular weight of 50-75 kDa, interacts with hRSV particles, suggesting that this component is, at least in part, responsible for the virucidal activity of NETs against hRSV. Continuing studies will help to understand the role of NETs during respiratory tract infections induced by the presence of hRSV.

INTERACTIONS MEDIATED BY SPHINGOSINE-1-PHOSPHATE, CXCL12 AND INTEGRINS IN THE MIGRATION OF NEOPLASTIC T LYMPHOCYTES

T-cell lymphoblastic leukemia and lymphoma (T-LLL) results from malignant proliferations of T precursors that exhibit phenotypic characteristics similar to distinct stages of normal T cell maturation. Data from our group, obtained with human T-ALL lineages suggest that interactions mediated by the sphingosine-1-phosphate receptor 1 (S1P1) are directly related to the migratory behavior of the blasts. Moreover, sphingosine-1-phosphate (S1P) and CXCL12 can modulate interactions mediated by integrins, important to migration of T cell precursors in physiological and pathological conditions. Therefore, the objective of this work is to study the role and mechanisms of interactions between S1P1, CXCR4 (CXCL12 receptor) and integrins, as well as their ligands, in the control of the migration of LLL-T blasts. We first observed, by flow cytometry, that Jurkat cells (a human T cell acute lymphoblastic leukemia cell line) express high levels of the CXCR4 and theVLA-4 and VLA-5 (both fibronectin receptors). EL-4 cells (a mouse T-lymphoblastic lymphoma cell line) express low levels of the CXCR4 and VLA-4 and high levels of VLA-5 and S1P1. In migration assays, we observed that EL-4 and Jurkat WT cells migrate in response to S1P, and such response was modulated in the presence of FN, this effect is even more pronounced in the migratory response of VLA-4 knockout Jurkat cells (Jurkat KO). Blocking S1P1 resulted in decreased migration of EL-4 and Jurkat WT cells towards S1P, while blocking VLA-5 in the presence of S1P and FN resulted in a lower migratory response of the three cell lines. Furthermore, EL-4 cells migrate in response to CXCL12, and such response was not modulated in the presence of FN. Blocking CXCR4 resulted in decreased migration of EL-4 cells towards CXCL12. In addition, we are developing an in vivo model of T-LLL consisting in intrathymic injections of EL-4 cells in C57BL/6 mice. Our preliminary data show the presence of EL-4 cells in the thymus of the animals 7 days after inoculation, suggesting that these cells are able to survive into the thymus in vivo and could therefore form a lymphoma in the organ. Using the animal model, we seek to better understand the participation of S1P1, CXCR4, VLA-4 and VLA-5, as well as their respective ligands in the migration of T-LLL blasts. Taken together, our data point to an interaction between S1P1, CXCR4 and integrins, evidencing the participation of these receptors and their ligands in the migration of leukemic cells

Intranasal administration of Lactobacillus delbrueckii UFV H2b20 improves lung defenses to regulate asthma-induced immunopathology

The interaction of the resident microbiota with the host immune system has the potential of modulating the immune response, promoting peripheral tolerance, thus mitigating hyperreactivity and the persistence of inflammation. Some probiotic species also have these immunomodulatory characteristics, and their protective effect has been demonstrated in inflammatory and allergic conditions of respiratory tract, such as asthma. Asthma is mainly characterized by an exacerbated and prolonged Th2 inflammatory reaction, mediated by IgE, with extensive eosinophilic infiltrate, secretion of IL-4, IL5, IL-13 and IL-33, in addition to mucus hyperproduction and airway hyperresponsiveness (AHR). Previous studies from our group have shown that oral administration of Lactobacillus delbrueckii UFV H2B20 (LAC) diminishes the allergic response seen in experimental asthma. Such results, combined with knowledge of the importance of the lung microbiota, are the basis of this work, which aims to investigate the impact that intranasal administration of LAC has on the lungs, and to evaluate its safety and protective efficacy. In addition, we investigated if some LAC metabolite could be responsible for this effect. Our results show that LAC and its culture supernatant (SUP) control the allergic inflammatory response to ovalbumine challenge. Treatments reduced the inflammatory infiltrate in lungs, mainly by reducing the presence of eosinophils. This lessened inflammatory state is confirmed by the reduction of production of IL-5 and IL-13, essential cytokines in asthma. LAC and SUP also reduced the typical asthma respiratory deficit, normalizing respiratory parameters, especially AHR and FEV50. Induction of a regulatory response is probably responsible for these effects, since the treatment with LAC induced multiple regulatory cell types, such as regulatory alveolar macrophages(F4/80MEDIUM), tolerogenic dendritic cells(CD103+) and regulatory lymphocytes(FoxP3+). Treatments elevated production of IL-10 and TGF-β1 responsible to maintain tissue homeostasis in lungs. Histological analysis also revealed different tissue inflammation in the group that received LAC, indicating the formation of iBALT, that could be local focuses of induction and activation of Tregs, and a key player in controlling the inflammation. Histology also showed that LAC repressed mucus hyperproduction. These results show that both treatments attenuate the general pathology seen in experimental asthma.

INTRANASAL IMMUNIZATION WITH A NON-REPLICATE INFLUENZA VIRUS PREVENTS THE INFLUX OF INFLAMMATORY MONOCYTES AND PROMOTES THE ANTIGEN-SPECIFIC CD8+ T CELL RESPONSES IN INFECTED ASTHMATIC MICE

Influenza viruses are responsible for flu epidemics each year. Vaccination plays an important role in reducing the risk of flu severity and can even prevent death in children. These infections are known as an important risk factor for asthma exacerbation, mainly in early childhood. The mechanisms of increased influenza susceptibility in asthmatic individuals are elevated serum IgE concentrations and increased FcεRI expression in plasmacytoid dendritic cells (pDC). Besides, higher FcεRI expression can induce the inhibition of virus-induced IFN-α released by those cells. Based on that, we aimed to elucidate the effect of flu vaccination in the experimental airway inflammation model. To do so, C57BL/6 mice were immunized with VNaDeL (a genetically modified Influenza lacking neuraminidase) and, then, submitted to sensitization with Alum and Ovalbumin on days 30, 37 post-vaccination (p.v.). On days 44 and 51 p.v., mice were challenged with OVA antigen and 24 hours post-challenge, the animals were infected with 105 PFU of the wild-type influenza PR8. We observed a temporary weight loss in immunized/ asthma-induced animals, that recovered on day 4. On the other hand, non-immunized/asthma-induced mice presented a dramatic weight loss and succumbed to PR8 infection. Interestingly, immunization with VNaDeL induced the production of IL-5, IL-4, and CCL17 in asthmatic mice, in comparison with the non-vaccinated asthmatic group. Besides, we could also observe increased numbers of neutrophils and eosinophils in the lungs from immunized/asthma-induced animals. In contrast, non-vaccinated asthmatic mice presented higher expression of chemokines receptors CCR2, CCR5 and CCL2 and also increased amounts of inflammatory monocytes and decreased amounts of eosinophils in lung tissue. Moreover, we found in the lung, T CD4+ and CD8+ cells, including Resident Memory CD8+ T cells and antigen-specific CD8+ T cells against influenza, in immunized/asthma-induced mice, suggesting better control of viral infection. Taken together, our results suggest that our vaccinal strategy against influenza in the murine asthmatic model is effective in protecting mice without leading to inflammatory response exacerbation.

INVESTIGATING THE ROLE OF m6A mRNA MODIFICATION DURING LEISHMANIA MACROPHAGE INFECTION

Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania, which during its life cycle shifts from an invertebrate to a vertebrate host. In the vertebrate host, Leishmania is an obligatory intracellular parasite, infecting mainly macrophages. During evolution, the parasite developed several mechanisms to subvert the normal function of these cells for its survival and proliferation. In this sense, we believe that regulatory mechanisms of gene expression of the host cell, such as posttranscriptional modifications, could be affected by Leishmania. Chemical modifications of specific nucleotides have been described as important regulatory posttranscriptional mechanisms of mRNAs life. The N6-methyladenosine (m6A) is one of the most abundant mRNA modifications and has been implicated in the regulation of mRNA stability and translation efficiency. Moreover, changes of m6A levels in the host cell have been implicated in the immune response against intracellular pathogens such as, HIV, Zika and SARS-COV-2 virus. The m6A levels are regulated by different enzymes: writers (METTL3 and METTL14), which will add the modification; erasers (FTO and ALKBH5) that removes the chemical group, and the readers (YTHDC1-2 and YTHDF1-3), which recognize the modified nucleotides affecting downstream functions of the mRNA. Considering the studies that demonstrate the role of m6A in the immune response and the ability of Leishmania to manipulate its host cell, in this work we seek to investigate the role of m6A during the Leishmania infection in macrophages. Initial analyses of public RNAseq data showed that liver samples from mice infected with L. infantum, presented higher levels of METTL3 compared to non-infected mice. To better understand the relationship of m6A and Leishmania-host interaction, we will generate RAW 264.7 macrophages knockouts for m6A regulatory proteins (METTL3, ALKBH5, YTHDF1-3) using CRISPR/Cas9 method, to investigate the parasite infection rate compared to wild-type cells. These experiments are in progress. In parallel, we are evaluating the METTL3 and m6A levels of macrophages infected or not with Leishmania by qPCR, western blot and dot blot with specific antibodies. In the end, we intend to investigate the possible involvement of m6A during Leishmania-macrophage infection, contributing to better understanding of the mechanisms used by this parasite to subvert the immune system during infection.

INVESTIGATION OF AXL RECEPTOR IMPLICATION IN MALARIA IMMUNOPATHOGENESIS

Introduction: Axl belongs to the TAM receptors family of receptor tyrosine kinases - as well as Mertk and Tyro3 - also known as efferocytosis receptors. It binds indirectly to exposed phosphatidylserine of apoptotic cells membrane through a connector protein called Gas6 and activates a signaling pathway to phagocytosis of those cells. This process has an important role in modulating immune responses since the Axl pathway is related to production of anti-inflammatory cytokines. However, the engagement of cell death and efferocytosis mechanisms can have different implications in parasitic infection outcomes such as beneficial or detrimental to the host. It is well known that Plasmodium sp. infection induces phosphatidylserine exposure in red blood cells although the implications of those mechanisms are uncertain. In this study, we focus on investigating the Axl signaling pathway in P. chabaudi infection and its consequences on immunopathogenesis of Malaria. Methods: We conducted in vivo analysis of C57BL/6 wild type and Axl-/- mice infection kinetics with P. chabaudi and performed measurements of parasitemia, body temperature and weight. Ex vivo samples (splenocytes, hepatocytes and plasma) were analyzed by flow cytometry and ELISA assays to identify expression of Axl receptor and its ligand Gas6. Results: Levels of parasitemia in Axl-/- mice were significantly lower compared to C57BL/6, especially in 8 and 10 days post infection interval. Splenocytes from infected C57BL/6 mice also showed higher expression of Axl receptor than the control groups. Conclusion: Our data suggest that Axl signaling pathway has an important role in P. chabaudi infection immunopathogenesis mechanisms. Axl-/- mice showed a more resistant phenotype and the expression of this receptor is increased during infection in wild-type mice. The principal hypothesis is based on a change of the immunoprofile - from anti-inflammatory pathway from Axl signaling to a pro-inflammatory in its absence - and it can be beneficial to the host. More investigations are being conducted to evaluate this proposition such as analysis of cytokine profile and immunophenotyping.

INVESTIGATION OF METABOLIC REPROGRAMMING IN HUMANS MONOCYTES INFECTED WITH Leishmania amazonensis AND Leishmania brasiliensis

INTRODUCTION: Leishmaniasis is a disease caused by protozoa of the genus Leishmania spp. that parasitize mammals. During infection by Leishmania spp. different cell types are recruited to the site of infection, including monocytes. The immune response against Leishmania is characterized by inflammation, where cells need energy to mature and produce biomolecules, being intrinsically related to metabolic activity and mitochondrial bioenergetic understanding in the pathogenesis of leishmaniasis, so the present study uses inhibitors of metabolic pathways in an attempt to elucidate the important pathways for the destruction or proliferation of the parasite. METHODS: Healthy individuals were recruited to obtain human peripheral blood monocytes. After processing PBMC'S, monocytes were previously treated with inhibitors of the metabolic pathways of glycolysis, oxidative phosphorylation, β-oxidation and fatty acid synthesis and subsequently infected with Leishmania amazonensis and Leishmania braziliensis. Initially, a dose-response curve was made to verify the best concentration for use, evaluating cell viability. After identification, the effect of inhibition of metabolic pathways was investigated by analyzing the infection rate and parasite load on the susceptibility of human monocytes to infection by Leishmania spp. RESULTS: The amount of metabolic inhibitor needed to inhibit metabolism while maintaining cell viability was established, and from this experiment the concentrations of use were defined, based on the percentage of viability that was obtained. In addition, further post-infection analyzes were performed to verify whether the infection could interfere with post-treatment viability. Therefore, it allows us to infer that the observations about the interference seen in the parasite load and in the infection rate is caused by the action of metabolic inhibitors in the host cells. Monocytes treated with inhibitors and infected with Leishmania spp. showed statistical difference, decreasing the infection rate and parasite load. These data indicate that metabolism is important for the persistence and proliferation of the parasite. CONCLUSION: Taking into account the aspects discussed, the metabolism of cells infected with Leishmania spp. interferes with the immune response of hosts and the elucidation of these pathways may contribute to changes in therapeutic strategies.

INVESTIGATION OF NEW PATHWAYS RELATED TO THE EARLY GENE EXPRESSION DURING T CD4 CELL ACTIVATION

T CD4 cell activation plays a central role in the immune response. Activation of these cells occurs in cooperation of three signals. The first activation signal occurs through the interaction of the TCR and the MHCII-peptide complex. The second activation signal occurs through the interaction of co-stimulatory molecules CD28, present on T cells, and CD80/86, present on antigen-presenting cells (APCs). Finally, the third signal occurs through the production of cytokines by the cell itself or by cells that are present in the microenvironment, after the first and second signals. Although the TCR-MHC-peptide binding plays a central role in the activation of T lymphocytes, other accessory molecules are also extremely important in this process, being therefore, the target of studies by our group. Therefore, we performed a next-generation sequencing (NGS) RNA-seq in purified naïve CD4 T cell activated in vitro for 1 hour to analyze early genes expression. The analysis of this experiment demonstrated differentially expressed gene groups in these initial moments of CD4 cell activation, compared to the control, with emphasis on the expression of GADD45 (Growth Arrest and DNA Damage inducible) family members. These proteins that are often induced by DNA damage and other stress signals associated with growth arrest and apoptosis. In addition, to further evaluate the GADD45 gene expression at the initial moments of activation, CD4 cells purified from lymph nodes of C57Bl/6 animals were in vitro activated with anti-CD3 plus anti-CD28 and evaluated at times 0, 24 and 48h. These cells were also labeled for the activation markers CD25, CD69, CD44 and CD62L. Then, we performed a qRT-PCR that demonstrated a decrease of GADD45B expression in both times. In order to better detail the expression of these molecules, CD4 cells purified from lymph nodes from C57Bl/6 animals were in vitro activated for 0h, 4h, 8h, 16h and 24h and had their RNAs extracted. The levels of GADD45A, GADD45B and GAD45G were evaluated by qRT-PCR. Thus, the characterization of the GADD45 family genes in these processes could shed light on the pathways related to the early gene expression during T CD4 cell activation.

“IN VITRO INFECTION RATE AND INTRACYTOPLASMIC CYTOKINE PROFILE INDUCED AFTER INFECTION OF HUMAN HEMATOPOIETIC STEM CELLS BY DISTINCT GENETIC GROUPS (TCI, TCII, AND TCVI) OF TRYPANOSOMA CRUZI”

Chagas disease, caused by the protozoan Trypanosoma cruzi, was discovered more than a century ago and remains a serious public health problem worldwide. Several aspects influence the clinical outcomes of the disease such as the host's immune system and the biological/genetic features of the parasite. Considering the genetic variability of T. cruzi and that Chagas disease can be reactivated in immunosuppressed patients, this study aims to evaluate the in vitro infection rate and the intracytoplasmic cytokine profile induced after infection of human hematopoietic stem cells (HSCs) by different genetic groups of T. cruzi. Three genetic groups of T. cruzi were used: TcI (Colombian Strain), TcII (Y Strain), and TcVI (CL Strain). Human hematopoietic stem cells (CD34highCD45low) provided from ten donors were incubated in the presence of fluorescein-labeled parasites. After monoclonal infections, the rate of the internalization in vitro of the genetic groups by HSCs and the synthesis of pro-inflammatory (IL-6/TNF-α/IL-17A) and regulatory (IL-10/TGF-β) cytokines were carried out by flow cytometry. The data showed that undifferentiated HSCs, regardless of the genetic groups evaluated, can internalize trypomastigote forms of T. cruzi [Colombian= 28.7% (IR:19.8-38.4%); Y= 21.5% (IR:17.3-30.2%); CL=34.4% (IR:26.5-40.6%)], and respond to infection with the production of pro-inflammatory [IL-6 - Colombian= 24.0% (IR:21.5-35.1%); Y= 5.5% (IR:2.5-10.5%); CL=7.6% (IR:3.0-31.2%)], and regulatory cytokines [IL-10 - Colombian= 16.6% (IR:11.2-23.1%); Y= 7.2% (IR:2.6-9.9%); CL=12.7% (IR:2.9-29.4%)]. The immune profile found is differentiated according to the genetic groups employed. Furthermore, the CL strain induced a higher rate of T. cruzi internalization than the Y strain [CL= 156.0 (IR:110.8-199.8%); Y= 81.5 (IR:21.5-111.8)]. Predominantly for the Colombian and CL strains, the CD34+ stem cells that internalized the most parasites were also those that synthesized more both pro-inflammatory and regulatory cytokines. When correlations between the evaluated attributes were performed, it was found that the internalization of CL strain by HSCs promotes a greater number of correlations (12), followed by the Y (7) and Colombian strains (5), respectively. Overall, the data from this study provides new insights for a better understanding of some biological aspects related to the host and distinct parasite strain involved in the reactivation of T. cruzi infection.

IN VITRO VITAMIN D-INDUCED CYTOKINE PRODUCTION IN CHAGAS DISEASE: EVALUATION OF CULTURE TIME KINETICS

Chagas disease is a neglected tropical disease caused by the hemoflagellate protozoan Trypanosoma cruzi (T. cruzi). Vitamin D is a fat-soluble hormone that acts as a ligand for the nuclear transcription factor, the VDR (Vitamin D Receptor) and plays important roles in the immune system. One of the manifestations of Chagas disease is severe heart disease, explained immunologically by the absence of immune homeostasis, with an exacerbated inflammatory response. The objective of this study was to evaluate vitamin D in immune response cells (lymphocytes and monocytes) of patients with chronic Chagas' disease through the production of Th1 and Th2 cytokines. Venous blood was collected from patients selected from the Chagas' disease and heart failure outpatient clinic of the Cardiology Emergency Room of Pernambuco (PROCAPE) of the University of Pernambuco, according to the inclusion criteria of the study. First, the material was used for serological confirmation for T. cruzi infection and in the experiments from the separation of peripheral blood mononuclear cells (PBMCs) stimulated with Vitamin D. Blood from patients with Chagas disease clinical form C (n=03) was collected in a vacuum system in tubes containing sodium heparin. PBMCs were separated by Ficoll Hypaque PLUSTM density gradient centrifugation and then deposited in 48-well plates (1 x 10^6 cells/mL) stimulated with Vitamin D (cholecalciferol) for 24, 48 and 72 hours. After the stimulation time, culture supernatants were stored at -20°C for subsequent cytokine (IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF) assay by Cytometric Beads Array (CBA). We found that vitamin D in vitro induces a decrease in TNF, IL-2 and IL-6 while IL-4 production is not altered throughout the studied period. IL-10 decreased at 24 and 48 hours and increased after 72 hours. As for IFN-γ, an increase in this cytokine was observed throughout the studied period. We can conclude that the pattern of cytokines known to be Th1 of patients with chronic Chagas' disease is regulated in the presence of vitamin D, suggesting that vitamin D could negatively modulate the inflammatory response in these patients.

Involvement of IL17A and IL17RA variants in interleukin-17A levels and disease activity in Ulcerative Colitis

Background: Ulcerative colitis (UC) is a condition characterised by chronic inflammation of the large intestine. Disease activity and mucosal injury are significantly affected by Th17 cells. Some researchers have studied the link between genetic variations in IL17A genes and autoimmunity susceptibility, however, the data are contradictory and limited in UC. Objective: The purpose of this study was to assess the involvement of genetic variants of IL17A and IL17A receptor (IL17RA) on susceptibility, interleukin-17A plasma levels, and endoscopic activity in patients with UC. Subjects: The case-control study included 104 UC patients and 213 healthy controls. Patients were divided into two groups of endoscopic activity: remission/mild (MAYO ≤1) and moderate/severe (MAYO >1). The IL17A (rs3819024, rs3819025) and IL17RA (rs2241043, rs2241049, rs6518661) variants were genotyped by TaqMan allelic discrimination assay on qPCR. IL-17A plasma levels were determined using microspheres immunofluorimetric assay. Results: Our data indicated that neither IL17A nor IL17RA variants were associated with UC susceptibility. However, the IL17A GA genotype (rs3819024) was associated with elevated levels of IL-17 only in UC patients. Patients with the G allele (dominant model) of IL17RA (rs2241049) showed a 2.944 more chance of developing moderate to severe disease. Conclusion: In conclusion, the IL17A GA genotype (rs3819024) was associated with elevated IL-17A plasma levels only in UC patients but not controls. In addition, AG+GG genotypes of IL17RA (rs2241049) were associated with moderate to severe UC. Our findings suggested a potential interaction between the IL17A variant (rs3819024) and other genetic, epigenetic, or environmental variables linked with UC in IL-17A level modulation.

Is a negative correlation between sTNFR1 and TNF in patients with chronic Chagas disease the key to clinical progression?

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects more than 8 million people around the world. It has a great impact on morbidity and mortality when compared to any other parasitic disease due to the different clinical manifestations presented, leading to 10,000 annual deaths in Latin American countries. In Brazil, oral infections have caused acute outbreaks, alerting to the implementation of urgent sanitary measures constituting a serious public health problem. The chronic phase of the disease is characterized by undetermined form (IND), cardiac (CARD), digestive and mixed form. Chronic chagasic cardiopathy (CCC) is the most severe form of the disease, as it affects the heart extensively leading to fibrosis with consequent loss of contractile function. It is believed that the immune response plays a central role in the evolution of clinical forms in chronic carriers of Chagas' disease. TNF-α, the most pleiotropic cytokine in the immune system, is a proinflammatory cytokine that plays a role during infection. Soluble TNF-α receptors (sTNFR1 and sTNFR2) are considered potent natural endogenous inhibitors of TNF-α and are elevated in many inflammatory, autoimmune and chronic conditions. The objective of the present study was to evaluate the levels of TNF-α and its receptors in the serum of chronic carriers of Chagas' disease. A total of 133 patients were included, of which 51 were undetermined (IND), 39 were mild (CARD 1) and 43 were severe cardiac (CARD 2), and 20 uninfected individuals (NI). The results point to an important role in the regulation of TNF-α activity performed by soluble receptors. The sTNFR1 and sTNFR2 receptors are elevated in patients with Chagas disease compared to uninfected. Although there is no statistical difference between the chronic carriers, sTNFR1 levels increase as the disease worsens. In addition, we found a negative correlation between sTNFR1 and TNF-α in IND individuals, suggesting that this relationship may prevent progression to more severe forms, such as cardiac form. We found no statistically significant difference in TNF-α levels, but lower levels of this cytokine in the IND group reveal regulatory characteristics of this clinical form. However, other cytokines may be acting synergistically or inhibiting TNF-α activity. These findings suggest the importance in endogenous balance of soluble TNF-α receptors and indicate protection and balance in patients with chronic Chagas' disease.

KATP channel is necessary for activation of inflammatory macrophages in a glutamine dependent manner

The role of ion channels in cell metabolism has just recently drawn scientists’ attention. Ions can be enzyme cofactors and metabolic signaling agents, which make them indispensable for initiating dynamic changes in cells function. Macrophages are immune cells with great phenotypic plasticity, changing their profile from pro-inflammatory to cellular repair and vice versa. Adaptations in the flow of ions are the key modulators of these changes. We focused on the study of a potassium channel controlled by the ATP/ADP ratio, called KATP. We observed that the modulation of KATP alters the profile of cytokines secreted by macrophage. Because blockage of KATP in pancreatic β-cells induces an influx of calcium to potentiate of glutamine metabolism and the subsequent secretion of insulin, we investigated the effects of KATP blockade with glibenclamide (GLI) in LPS-activated macrophages. KATP blockade increased the LPS-induced stabilization HIF-1α and expression of HIF-1α-target gene Pfkfb3 and activation of NF-κB. KATP blockade also increased the levels of TNF-α, IL-1β and nitric oxide in LPS-activated macrophages. In addition KATP blockade reduced the levels of IL-6 and H2O2. Next, due to the potential role of glutaminolysis in these effects, we determined if inhibion of glutaminolysis with CB839 is sufficient to reverse the changes caused by glibenclamide. We observed that CB839 treatment reversed the observed effects of KATP blockade in LPS-activated macrophages. Glibenclamide also change the phagocytic capacity of macrophages. Taken together, the results reveal that the KATP channel plays a central role in inflammatory macrophages function and metabolism.

KINETICS OF IgG1 AND IgM REACTIVITY IN MICE EXPERIMENTALLY INFECTED WITH SCHISTOSOMA MANSONI AND TREATED WITH PRAZIQUANTEL

Introduction: Schistosomiasis is a chronic and debilitating disease that affects over 200 million people worldwide. Proper diagnosis and treatment are essential to control the disease morbidity and to prevent transmission. Currently recommended diagnostic techniques, such as the Kato-Katz test, have low sensitivity for detecting individuals with low parasite load, cases of neuroschistosomiasis, or for evaluating post-treatment cure. This study aims to identify antigens and antibody isotypes useful in the development of more sensible immunological diagnostic approaches, evaluating the kinetics of different immunoglobulin isotypes during experimental infection with S. mansoni and/or treatment with a curative dose of Praziquantel (PZQ). Methods: CD1 male mice were subcutaneously inoculated with 50 cercariae of S. mansoni and evaluated at 2, 4, 6, 8, 10, 12, and 20 weeks post-infection (wpi). Uninfected mice and infected mice treated with PZQ (300mg/kg) at 8 wpi were concurrently evaluated. The number of worms recovered from blood stream, and parasite eggs in feces and trapped in intestine and liver were quantified in each infected and infected/treated mouse. IgM and IgG1 reactivity against S. mansoni egg antigens (SEA) and adult worm antigens (SWAP) were estimated in serum samples through enzyme-linked immunosorbent assay (ELISA) and Western Blot. Results: Schistosome eggs were found in the stool as early as the 6th week of infection and continued to be shed after 20 weeks in untreated mice. PZQ treatment eliminated all worms from the blood stream and significantly reduced the number of eggs retained in the tissues (infected 15539±6500 vs infected and PZQ-treated 4569±2600 eggs/left liver lobe; p=0,0006) and released in the stool; parasite eggs were still found in the tissues of treated mice at 20 wpi. Antibody response (IgG1 and IgM) against SEA progressively increased after egg production and PZQ treatment. IgG1 reactivity against SWAP increased at 4 wpi and remained elevated until 20 wpi in both treated and untreated mice. In contrast, IgM reactivity to SWAP started earlier, during pre-patent infection (2 wpi) and showed significant reduction in PZQ-treated infected mice (at 20 wpi, Infected ABS=0.922±0.206 vs Treated ABS=0.627±0.139; p=0,0013). Conclusion: The data suggest that IgM reactivity against a SWAP antigen could be valuable in the development of immunodiagnostic tests that seek to identify pre-patent infection and monitor post-treatment cure.

Leishmania braziliensis EXOSOMES INDUCE THE PRODUCTION OF INFLAMMATORY MEDIATORS IN HUMAN MONONUCLEAR PHAGOCYTES

Background: Exosomes are 30-200nm vesicles secreted by various cell types. Leishmania exosomes are composed by several proteins such as heat shock proteins, annexins, GP63, proteins with signaling activity, and also contain mRNAs and miRNA. Over the past 10 years it has been reported that these vesicles actively manipulate host signaling and immune cell functions, due to virulence factors such as GP63. Studies demonstrate that L. donovani exosomes activate tyrosine-phosphatases, downregulating IFN-γ and inhibiting the expression of microbicidal molecules (TNF and NO), which creates a microenvironment that favors parasite proliferation. L. braziliensis infection induces exaggerated inflammatory response by macrophages, with high production of IL-1β, IL-6, TNF. Despite of not having immunological memory, studies suggest that after a first stimuli mononuclear phagocytes can get “trained”, passing through epigenetic changes, and responding more effectively against a second stimuli. Thus, the sensitization of macrophages with L. braziliensis exosomes, prior to its infection by this pathogen, may be associated with higher inflammatory cytokines production, as well as inflammasome activation once the pathways involved in this process would already be activated. Methods and Results: Healthy subjects’ monocyte-derived macrophages were sensitized for 24 hours with L. braziliensis exosomes. Afterwards, these cells were infected with L. braziliensis and cultured for 24 hours. We observed higher levels of IL-1β and IL-6 in cultures that were sensitized before infection than in cells only infected. Furthermore, stimulation with L. braziliensis exosomes increased NF-κB1 gene expression and enhanced the production of IL-1β, IL-6, TNF and reactive oxygen species by macrophages. We also inhibited the secretion of exosomes by L. braziliensis prior to macrophage infection and observed that cells infected with parasites lacking exosomes induced less production of TNF and IL-1β than cells that were infected with exosomes–secreting Leishmania. Exosomes stimulation also induced the consumption of NLRP3 inflammasome in macrophages. Conclusion: Our results suggest that L. braziliensis exosomes stimulate macrophages leading to inflammation through the activation of NF-κB and NLRP3 inflammasome.

LEISHMANIA BRAZILIENSIS INFECTION ACTIVATES ATP- BINDING CASSETTE TRANSPORTERS IN HUMAN MACROPHAGES: IMPLICATIONS FOR DRUG RESISTANCE

Introduction: Protozoa of the Leishmania sp. are the causative agents of leishmaniasis and the state of Bahia is endemic for cutaneous leishmaniasis (CL). The effectiveness of antiparasitic drugs can vary substantially and the most important limitation of leishmanicidal drugs is treatment failure and emergence of drug-resistant parasites. Several cellular mechanisms can lead to multidrug resistance (MDR). Transmembrane proteins of the superfamily of ABC transporters such as MDR1, MRP and BCRP may decrease the cellular accumulation of pentavalent antimonials (SbV). The high activity of these proteins may be associated with therapeutic failure and our hypothesis is that L. braziliensis isolated from patients that failed therapy induce ABC transporters activity in human macrophages. Methods and Results: L. braziliensis isolates obtained from lesions of patients with CL that healed or failed treatment with SbV, were treated in vitro with SbV. Also, peripheral blood-derived macrophages from healthy individuals were stained with a dye that has affinity for ABC transporters, and then infected with L. braziliensis. All Leishmania isolates showed some degree of in vitro resistance to SbV. All L. braziliensis isolates induced activity of ABC transporters, and inhibition of MDR1 and MRP activity decreased dye efflux in L. braziliensis-infected macrophages. Conclusion: The infection by L. braziliensis derived from patients with CL activated MDR1 and MRP, suggesting the participation of these ABC transporter in SbV resistance.

Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils

INTRODUCTION: Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite–vector–host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. METHODS: Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. CONCLUSION: Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

LEPTIN FAVORS IMBALANCE OF ANTIGEN-SPECIFIC CD4+ T CELLS ASSOCIATED WITH SEVERITY OF CAT ALLERGY

Introduction: Cat allergies are a common allergic disease, affecting up to 20% of adult population worldwide, with usual symptoms being rhinitis, asthma and/or conjunctivitis. Fel d1 has been described as the main cat allergen, accounting to up to 96% of human sensibilization to cats. Since the hallmark of car allergy is the production of high affinity Fel d1-specific IgE, several T cell phenotypes have been implicated in the disease severity, such as Th2, TFH2 and TFH13. Other comorbidities that affect the immune system, such as obesity, have also been described to increase allergic diseases severity. This adverse relationship may be associated with high leptin production in obesity, an adipokine with T cell function modulatory properties. As such, we aimed to To investigate the ability of obesity-related leptin dose to modulate Fel d1-specific CD4+ T-cells in cat allergic patients. Methods: PBMC cultures from cat-allergic patients were stimulated with Fel d1, without or with leptin (Fel d1/Lep). After 6 days, the supernatants were harvested and both the cytokine and IgE levels were determined by Luminex and ELISA, respectively. The CD4+ T-cell phenotypes were analyzed by flow cytometry. Results: Fel d1 induced IgE and the production of cytokines related to Th2, Th9 and Th17 cell phenotypes. Interestingly, Fel d1 was more efficient at increasing the frequency of TFHIL-21- cells positive for IL-4, IL-5 and IL-13 than TFHIL-21+ cells. Regarding regulatory CD4+ T cells, Fel d1 induced Treg (CXCR5-FoxP3+IL-10+), Tr-1 (CXCR5-FoxP3-IL-10+) and TFR (CXCR5+FoxP3+IL-10+ and CXCR5+FoxP3 -IL-10+) cells, expressing or not CD39. Leptin favored the expansion Th2-like and Th9-like cells, as well as TFHIL-21+ (IL-5+, IL-9+ and IL-17+) and TFHIL-21- (IL-4+, IL-5+ and IL-13+) cell subsets. In contrast, leptin reduced the proportion of conventional and follicular regulatory CD4+ T-cell subsets. Finally, leptin not only elevated IgE production, but the levels of this antibody directly correlated with the frequency of TFHIL-21+IL-5+ and TFHIL-21- (IL-4+, IL-5+ and IL-13+), and negatively correlated with the percentage of Treg and TFR cells. Conclusions: Obesity-related leptin dose should negatively impact the severity of cat allergies by favoring the expansion of pathogenic Fel d1-specific CD4 T-cell phenotypes and damaging the functional status of regulatory CD4+ T-cell subsets.

LEUKOTRIENES IN TYPE 1 DIABETES: ACTIVATION OF A DISTINCT IMMUNOMETABOLIC PHENOTYPE IN MACROPHAGES

Type 1 diabetes (T1D) is a metabolic disease in which the imbalance of nutrients leads to systemic inflammation and consequent comorbidities. Leukotrienes (LTs) are lipid mediators that contribute to systemic inflammation and comorbidities in T1D. In this context, LTs promote the activation of macrophages and an increased mitochondrial oxidative metabolism in these cells. However, which metabolic pathways, substrates and their role for the activation of macrophages in T1D had never been studied. Herein, our bulk-RNAseq data indicated that fatty acid oxidation (FAO) associated with the uncoupling protein UCP1 is important for the activation of a distinct phenotype in T1D peritoneal macrophages in which inflammatory and reparative markers are up regulated in a LTs-dependent way. We also observed that elevated levels of mitochondrial ROS in T1D macrophages are not induced in mice that do not produce LTs. The same pattern was observed for proton leak when we measured oxygen consumption. In addition, cytokines (IL-12, IL-1β, and IL-10), higher produced in T1D macrophages in a LTs-dependent manner, came down dramatically after blocking FAO. This result suggest that FAO can be a source of ROS and consequent cytokine secretion in T1D macrophages. Also, in vitro assays showed that UCP1 protein expression in macrophages increases in a LTs-B4-dependent manner when these cells are placed in a conditioned medium derived from lipolysis-stimulated adipocytes. Likewise, in vivo, leukotrienes contribute to the production of cytokines in epididymal adipose tissue, intensifying adiposity loss in diabetic mice. Together, our results indicate that in the context of inflammation-related lipolysis in T1D, leukotrienes promote a distinct state of activation in macrophages. In such phenotype, UCP1 may exert a regulatory effect in order to reduce any damages provoked by a chronic state of cellular activation promoted by FAO and consequent mitochondrial ROS. These results open paths to the study of therapeutic strategies to reduce chronic inflammation in metabolic diseases, which is often associated with comorbidities.

LINOLEIC ACID ACCELERATES WOUND HEALING AND IMPROVES COLLAGEN DEPOSITION IN DIABETIC MICE THROUGH IL-6 DEPENDENT SIGNALING

INTRODUCTION: Wound healing is impaired due to inflammatory and angiogenic disorders, in diabetic individuals. Linoleic acid has been shown to modulate cell membrane composition and wound healing in rats. However, the effects of linoleic acid supplementation on the remodeling phase of wound healing in diabetics are not well described. This study aimed to evaluate the effects of oral administration of linoleic acid on the remodeling phase of wound healing in mice submitted to type I diabetes. METHODS: C57BL/6 male mice were divided into three groups: (C) control; (D) diabetic animals and (DLA) diabetic animals that received oral supplementation with linoleic acid. The animals received intraperitoneal injection of streptozotocin at low doses for induction of diabetes. DLA animals were supplemented with 50 µL of linoleic acid for 5 consecutive days. The wound was induced on the 5th day of supplementation. Quantification of inflammatory mediators, histological analyzes and traction assay were performed. IL-6-/- animals were used to investigate the essentiality of IL-6 in this process and to culture dermal fibroblasts. Data were analyzed by one-way ANOVA or two-way ANOVA followed by post-hoc Bonferroni with p < 0.05. RESULTS: There was a delay in wound closure in diabetic animals when compared to controls. On the other hand, the DLA group had a smaller wound area compared to diabetic animals. There was no difference in wound closure between the C and DLA groups, suggesting that linoleic acid supplementation reversed the deleterious effects of diabetes by accelerating wound closure. DLA animals showed a reduction in the inflammatory infiltrate and an increase in collagen deposition compared to diabetic animals, however, the tensile strength limit and the specific elongation of the skin were lower in the DLA group compared to C. The improvement in wound healing may be due to the modulation of IL-6, since linoleic acid restored IL-6 concentrations in diabetic animals. Deletion of IL-6 caused delayed wound healing in diabetic mice supplemented with linoleic acid; contributed to exacerbated inflammation and impaired collagen deposition in the wound area. The absence of IL-6 in fibroblasts reduced the migratory response of these cells, maintained in high glucose + linoleic medium. CONCLUSION: Linoleic acid supplementation accelerates wound closure, controlling inflammation and stimulating collagen deposition and ECM organization through IL-6-dependent signaling.

LIPID ACCUMULATION MODULATES DENDRITIC CELL ACTIVATION, INFLAMMATORY RESPONSE AND BACTERIAL BURDEN DURING M. bovis BCG INFECTION

Introduction: Tuberculosis represents a major challenge of public health, killing more than 1 million people each year. The actual available treatment is old and faces antibiotic resistance. Mycobacterium tuberculosis is transmitted by aerosol and penetrates pulmonary alveolus, where it is phagocyted by alveolar macrophages and dendritic cells, the main bacterial reservoirs and immune response initiators. This pathogen can change host metabolic pathways as a survival mechanism. Lipid accumulation has been shown to be a key-component in host-pathogen interaction, enabling bacteria replication and survival. Thus, in the present work we evaluated the role of enzymes involved in neutral lipid biosynthesis in dendritic cell (DC) activation during M. bovis BCG infection. Methods and Results: DCs were obtained from C57BL/6 bone marrows, differentiated with 10 ng/ml of GM-CSF for 10 days, and infected by M. bovis BCG at MOI 5. We identified an increase in triacylglycerol (TAG), cholesterol and cholesterol ester (CE) accumulation in DCs during BCG infection by thin-layer chromatography. To evaluate the roles of TAG and CE biosynthesis in infection, BCG-infected DCs were treated with diacylglycerol acyltransferase 1 (DGAT-1) (A922550; 20uM) and the acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) (Ci976; 10uM) inhibitors. It was observed that the inhibition of DGAT-1 reduced lipid droplet (LD) area and bacterial burden in DCs. The ACAT inhibitor treatment did not affect LD formation nor bacteria area in DCs. However, we observed by ELISA and EIA that the treatment with ACAT inhibitor inhibited the increase in IL-1beta, IL-6, IL-10, IL-12p40, TNF-alpha and PGE2 production by BCG-infected DCs while DGAT-1 inhibition only reduced PGE2 release by these cells. Furthermore, it was observed by flow cytometry that BCG infection induced an increased expression of MHC I and II and the costimulatory molecule CD80. DGAT-1 inhibition reduced CD80 expression, and both treatments reduced MHC I and II expression. Conclusion: Together, these data demonstrate that TAG biosynthesis by DGAT-1 is important to LD formation and BCG replication in DCs, whereas CE biosynthesis by ACAT is involved in host inflammatory response by these cells. Both pathways seem to represent potential therapeutic target to modulate DC response against tuberculosis.

LIPID DROPLETS ARE CENTRAL PLATFORMS IN THE CONTROL OF BACTERIAL LOADING AND INFLAMMATION INDUCED BY SEPSIS

Introduction: Recent studies indicate that lipid metabolism reprogramming is crucial for survival from bacterial sepsis, playing a central role in maintaining the support of vital functions. At the cellular level, changes in lipid metabolism are often observed in the form of lipid droplet (LD) accumulation in different tissues and leukocytes. LDs are dynamic organelles composed of a core of neutral lipids surrounded by a monolayer of phospholipids associated with diversified protein content. In the last three decades, LDs have been characterized as a key cellular platform involved in several cellular processes, including the inflammatory response. Based on it, our hypothesis in this work was that LDs are a central platform in the resistance process to bacterial infection. Methods and Results: We conducted in vitro and in vivo experiments to confirm this hypothesis. Using primary macrophages, we observed that increased oleic acid-induced LD accumulation decreased intracellular bacterial load compared to macrophage controls at 24 hours post-infection (hpi). On the other hand, when the enzyme DGAT-1 has inhibited with A922500 (iDGAT1), we observed that a significantly increased intracellular bacterial load followed the reduction of LD accumulation at 24 hpi. We also evaluated the participation of LDs in macrophages' activation and inflammatory response. We observed that inhibition of LD accumulation was accompanied by a significant decrease in levels of nitric oxide (NO), lactate, PGE2, TNF, CCL2, and IL-6, indicating that modulation of lipid metabolism impaired the macrophage's pro-inflammatory activation. To validate these in vitro data, we pretreated C57BL6 mice with iDGAT1 1h before cecal ligation and puncture (CLP)-induced polymicrobial sepsis. We observed that treatment with iDGAT1 also impaired the LD accumulation in peritoneal leukocytes in 6h and 24h after sepsis. Moreover, our results show that the inhibition of DGAT1-enzyme increased the bacterial load both in the peritoneum and blood of septic mice in both times analyzed. Moreover, the inhibition of LDs also impaired the level of NO, LTB4, and MCP1/CCL2 induced by sepsis. Conclusion: Together, this set of results supports our hypothesis that LD accumulation is a critical orchestrator of the protective inflammatory response to bacterial infection during sepsis

Lipid droplets contribute to SARS-CoV-2 infection and inflammatory process in human cells

Introduction: The coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has rapidly spread in a pandemic, representing an unprecedented health, social and economic threat worldwide. In spite of the enormous scientific efforts to understand mechanisms of SARS-CoV-2-induced disease and to develop strategies to control COVID-19 pandemic, many aspects of SARS-CoV-2 biology and pathogenesis remain elusive. Viruses are obligate intracellular parasites and interact with several intracellular structures to benefit viral replication. In this context, several RNA viruses are able to modulate the host lipid metabolism and to recruit Lipid droplets (LDs) to enhance their replication/particle assembling capacity through mechanisms that vary according to the virus and the host cell infected. LDs are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Methods and Results: We described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection increased the expression of key proteins in the regulation of lipid metabolism as CD36, SREBP-1, PPARγ, DGAT-1, and trigged LD accumulation in different human cell lines. In addition, we have found SARS-CoV-2 and/or its components associated with LDs in infected cells, suggestive that LDs are recruited as part of replication compartment in infected cells. Moreover, modulation of LD formation by pharmacological inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of pro-inflammatory response and cell death. Also, inhibition of SARS-CoV-2 replication and cell death were observed with the genetic knockdown of DGAT-1 in calu-3 cells. Conclusions: Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new perspectives for therapeutic strategies to COVID-19.

LIPID DROPLETS CONTRIBUTE TO SEPSIS-ASSOCIATED HEPATIC DYSFUNCTION THROUGH THE AMPLIFICATION OF LIPID PEROXIDATION AND INFLAMMATION.

Introduction:Sepsis is defined as an acute life-threatening organ dysfunction caused by a dysregulated host response to infection. The development of organ dysfunction is the most serious outcome of sepsis and is directly related to morbidity and mortality. The mechanisms involved in sepsis-induced multiple organ dysfunction (MODS) are multifactorial and still incompletely understood, but maladaptive inflammation in response to infection and disrupted mechanisms of tissue tolerance are believed to be the main causes of tissue damage, and mortality. Evidence indicates that sepsis-induced organ failure occurs due to immunometabolic reprogramming. Lipid droplets (LDs) accumulation in other tissues than adipose is a recurrent metabolic alteration in septic patients alongside the increase in oxidative stress. Even though liver dysfunction is commonly observed in sepsis, the role played by LD accumulation in sepsis-associated hepatic tissue damage is still not clear. Methods and Results:Here, we investigate the mechanisms and consequences of lipid droplet accumulation in the liver during sepsis. For it, C57BL/6 mice were submitted to cecal ligation and puncture (CLP). Our results demonstrate that sepsis triggers an increased accumulation of LDs in liver cells. This phenomenon occurs in parallel with hepatic dysfunction measured by serum levels of AST, ALT, and albumin. Moreover, we observed a significant increase in liver triglycerides (TAG) content and lipid peroxidation biomarkers in the CLP group compared to sham group, with a further increment in 48h after surgery. We also evaluated the impact of pharmacological inhibition of triglycerides synthesis on hepatic dysfunction induced by experimental sepsis. We observed that treatment with DGAT-1 inhibitor (A922500, iDGAT-1) reduced LDs numbers and triglycerides levels in the liver of septic mice. Moreover, iDGAT-1 reduced liver injury markers AST and ALT and improved hepatic function accessed by albumin levels. We also evaluated the impact of iDGAT treatment on hepatic oxidative stress and inflammation. We observed that iDGAT1 treatment reduced the sepsis-induced malondialdehyde, 4-HNE and 3-NT levels—accordingly in the liver. iDGAT-1 treatment also reduced inflammatory mediators in the liver, reducing MPO, CXCL1, CCL2, IFN-γ, and IL-1β levels. Conclusion: These data presented here suggest that LDs could be related to the amplification of liver damage, peroxidation, and inflammation in the late times of sepsis.

Liver X receptors are critical for T cell polarization and development

LXRs (liver x receptors) are nuclear receptors that act as sensors of cholesterol, playing a crucial role in the regulation of metabolism, transport, and efflux of lipids. LXRs are found in two isoforms, LXRα and LXRβ. Once bound to a ligand, LXRs become active and translocate to the nucleus, where in association with a retinoid receptor (RXR), act as a transcription factor. LXRs also participate in the regulation of immune cell function, displaying an anti-inflammatory role through the inhibition of NF-Kb. Although many works have described the benefic effects of LXR activation for the regulation of the inflammatory response, little is known of the effects of each LXR isoform in the polarization of T CD4 cells. First, we determined if LXR is important for T cell development. We isolated thymus from WT, LXRα-/- and LXRβ-/- knockout mice and observed that the deletion of LXRα and LXRβ decreased the percentage of CD4+CD8+, CD8+ and CD4+ cells, indicating that both isoforms play a role in the development of CD4 and CD8 cells. In the periphery, we observed a reduction in CD8+CD62L-CD44+ and CD4+CD62L-CD44+ cells and an increase in LXRβ knockout mice of CD8+CD62L+CD44-. Moreover, LXRβ isoform deletion had a bigger effect on thymic and periphery T cells, indicating its prominent role. Next, we assessed the role of LXR in CD4 T cell polarization. Naive CD4 cells were isolated from wild type (WT), LXRα-/- and LXRβ-/- knockout mice and differentiated into T helper 1 (Th1), Regulatory T cell (Treg), and T helper 17 (Th17) profiles. We observed a lower percentage of CD4+ IFN+ (Th1), CD4+ FOXP3+ (Treg) and CD4+ IL-17+ (Th17) cells in both knockout models, but LXRβ-/- knockout mice displayed an even lower percentage. These data indicate that both isoforms are important for the polarization of T lymphocytes, with a critical role for LXRβ. Finally, we determined if LXR absence would lead to functional alterations in vivo. We immunized WT and LXRα-/- and LXRβ-/- knockout mice with ovalbumin (OVA). In a recall experiment, we isolated lymph node cells and cutured these cells for 3 days with OVA. We observed increased secretion of IL-17 in both knockout models, but again, the secretion was higher when LXRβ was deleted. Together, our data indicate that LXRα/β is important for the polarization and the effector function of CD4 lymphocytes in Th1, Th17, and Treg profiles, with opposing effects in vitro and in vivo.

LONG-TERM RESISTANCE TO METABOLIC SYNDROME INDUCED BY ACUTE GASTROINTESTINAL INFECTION

Introduction: The 21st century has seen the emergence of various chronic non-communicable diseases. Social behavior like sedentarism and overfeeding are fueling the appearance of these diseases, which is seen in the increased prevalence now and predicted for the future. Some of them, like type 2 diabetes (T2D), obesity and dyslipidemias, have intrinsic metabolic characteristics and an underlying immunological factor. In this study, we are looking to shed light on the long-term bi-directional regulatory mechanisms between immunology and metabolism, particularly under infectious conditions. Previous results from our group showed that acute episodes of gastrointestinal infection with Yersinia pseudotuberculosis (YP) induce a remodeling of the mesenteric lymphatic system, causing lymph leakage and loss of compartmentalization of the microbiota in a process called Immunological scar. In parallel, YP-infected mice presented increased weight gain and chronic inflammation in the liver and white adipose tissue. Therefore, we hypothesized that YP infection could predispose the host to the development of metabolic disorders and type 2 diabetes (T2D). Methods: In this study, C57BL/6 mice previously infected with YP (after bacterial clearance) were fed with a regular or a fructose-supplemented High-fat Diet (40% fat) for 15 weeks. This diet is also known by the induction of non-alcoholic steatotic hepatitis (NASH). Insulin resistance and glucose tolerance were measured to evaluate the systemic metabolic phenotype, and mice were also weighed weekly. Further, we evaluated liver and pancreas histologies and flow cytometry data from liver and mesentery. Results: Contrary to our previous hypothesis, YP infection protected the animals against metabolic syndrome. YP-infected/NASH mice gained significantly less weight and had lower glycemic levels than the Naive/NASH mice. Infected mice receiving NASH diet had better glucose disposal and lower insulin resistance and glucose tolerance than naive/NASH mice. In addition, the liver from infected NASH mice had a higher immune infiltrate in the parenchyma compared to the naïve control. Conclusion: Unexpectedly, infected mice that received NASH diet were protected from metabolic syndrome despite chronic inflammation in the liver and white adipose tissue. With these experiments, we will better understand the metabolic response induced by previous immunological responses in the intestinal tract.

LOW DOSES OF 5-FU ASSOCIATED WITH Ganoderma lucidum POLYSACCHARIDES INCREASE THE EXPRESSION OF IMMUNO-CHECKPOINTS OF TONGUE SPINOCELLULAR CARCINOMA CELLS

LPS induce monocytic TLR activation and tolerance in COVID-19 patients

Introduction: SARS-CoV-2 infection is characterized by a hyperinflammatory state and strong innate immune cell response, which causes damage to multiple organs. Elevated levels of lipopolysaccharides (LPS) in the circulation was found in patients with severe COVID-19. Our hypothesis is that microbial translocation and immune activation during severe COVID-19 may induce endotoxin tolerance and impaired immune response against secondary infections. In this research, the inflammatory response and monocyte phenotype of COVID-19 patients after lipopolysaccharide (LPS) in vitro challenge were analyzed. Methods: Patients were recruited from the Inpatient Unit of Hospital Moinhos de Vento/Porto Alegre-RS. Whole blood samples of healthy controls (n=5), patients with mild COVID-19 (n=8) and severe COVID-19 (n=7) where incubated with lipopolysaccharide (LPS, 1 ng/mL and 10 ng/mL) for 2 hours. The levels of LPS in plasma samples were quantified by liquid chromatography-tandem mass spectrometry. The production of interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1 MCP-1/CCL2 and tumor necrosis factor-alpha (TNF)-α were analyzed by Multiplex. The soluble form of CD14 (sCD14), PEG-2 and IL-1β were measured by Elisa. The expression of toll-like receptor-4 and the activation of nuclear factor-kappaB (NF-κB) in CD14+HLA-DRlow/high monocytes were evaluated by flow cytometry. Results: Severe COVID-19 patients presented higher LPS and sCD14 levels in the plasma than healthy controls and mild COVID-19 patients. In non-stimulated in vitro condition, severe COVID-19 patients presented higher inflammatory cytokines and PGE-2 levels and lower CD14+HLA-DRlow monocytes frequency than controls. Moreover, severe COVID-19 patients presented higher NF-κB activation in CD14+HLA-DRlow, as well as higher expression of TLR-4 and NF-κB activation in CD14+HLA-DRhigh compared to controls. Endotoxin challenge with both concentrations of LPS reduced the frequency of CD14+HLA-DRlow in severe COVID-19 patients. Moreover, the increase in TLR-4 expression and NF-κB activation were more pronounced in CD14+ monocytes of healthy controls and mild COVID-19 patients compared to severe COVID-19 group. Conclusion: We conclude that acute SARS-CoV-2 infection is associated with decreased endotoxin response in monocytes.

M1 AND M2 MACROPHAGES POLARIZATION IN MEN WITH HIGH OR LOW CARDIORESPIRATORY FITNESS: ROLE OF AMPK

Metabolic disease-associated inflammation is accompanied by an imbalance in macrophage phenotypes and functionality, as well as a stronger proportion of inflammatory subpopulations. The size of adipose tissue depots and physical fitness levels are important factors relationship with inflammatory profile. However, is unclear the impact of the physical fitness levels on M1 and M2 macrophages polarization in individuals with similar body composition. Additional, role of adenosine monophosphate-activated protein kinase (AMPK) pathway need be investigate. Aim of the study was examine M1 and M2 macrophage polarization in men with low or high cardiorespiratory fitness (both with normal weight). This study has a cross-sectional design, with young subjects who exhibit different levels of maximum oxygen consumption (V̇O2max) included. V̇O2max was measured by the maximal incremental test on a cycle ergometer monitored with a gas analyzer (Quark PFT). Additionally, assessments of body composition, physical activity level, food records and blood samples were obtained. CD14+ monocytes were purified by negative immunomagnetic selection and polarized to M1 or M2 phenotypes in the absence or presence of AMPK activator (AICAR) or inhibitor (compound C). Data normality was checked by the Shapiro-Wilk test. The comparison of dependent variables between groups was performed using Student's t test for parametric variables and Mann-Whitney test for non-parametric variables. Group-treatment comparisons in macrophage culture were verified by two-way analysis of variance with Tukey post-test adjustment. The significance level adopted was p < 0.05. There were no differences in the release of tumour necrosis factor alpha (TNF-α), interleukin-6 and -10 (IL-6 and IL-10) when comparing the M1 vs M2 phenotypes intra and inter groups (p > 0.05 for all). Additionally, treatment with AMPK inhibitor or activator did not promote significant differences in the release of TNF-α, IL-6 and IL-10 between the phenotypes of each group, as well as in group and treatment comparisons (p > 0.05 for all). From the preliminary results, we concluded that the polarization of the M1 and M2 phenotypes, with or without the participation of AMPK, does not present differences between individuals with different levels of cardiorespiratory fitness and similar body composition.

M2A MURINE MACROPHAGES TRAINED FOR FIVE DAYS WITH IL-4 PRODUCE A LOW AMOUNT OF NITRIC OXIDE AFTER LPS STIMULUS.

Introduction: Macrophages are heterogenic and plastic phagocytic cells able of adapting to microenvironment signals under physiological and pathological conditions. IFNg and lipopolysaccharide (LPS) generate classical activated macrophages or M1, with great microbicidal activity and able to produce a high amount of nitric oxide (NO). IL-4 generates alternatively activated macrophages or M2a, involved in scar formation and wound healing with high arginase activity. It is unclear whether M2 macrophages can be converted to M1 macrophages after long term in culture with IL-4. Objective: Evaluate the ability of IFNg+LPS to convert IL-4-treated M2a macrophages to M1 after 10 days in culture. Methods: Murine peritoneal macrophages were cultured with IL-4 for one to ten days followed by a stimulus with IFNg+LPS. Production of NO was evaluated by the Griess method and arginase activity was evaluated based on urea production (Biochem Biophys Res Commun 206(2): 667-673, 1995) during the time. Results: Urea production of cells cultured with IL-4 without IFNg+LPS increased significantly from 1 to 5 days (p<0,05), maintaining high until 10 days (T0= 0,14±0,04; T1= 0,36±0,12; T3= 0,51±0,23; T5=0,97±0,35; T10=0,87±0,22 mg/mL). The ability of IL-4 trained M2a macrophages to produce NO after stimulus with IFNg+LPS decreased significantly from 1 to 5 days (p<0,05), maintaining low, but above of unstimulated cells, until 10 days l (T0=72,6 ± 4,6; T1=46,6 ± 8,6; T3=30,5±6,8; T5=11,4 ±2,1; T10= 7,6±0,77 uM/ cells without IFNg+LPS = 1,7±2,8 uM). Conclusion: Macrophages cultured for 5 days with IL-4 in vitro reach the maximum commitment for high arginase activity and high resistance to being converted to M1 macrophages, based on arginase activity and NO production as markers of M2/M1 macrophages respectively.

Macrophages derived from peripheral blood monocytes (MDM) of taurine and indicine breeds present different pattern of RNA expression: implications on bovine immunity to diseases and thermal stress

INTRODUCTION: Taurine and indicine are two bovine subspecies that share a common ancestor and display different levels of resistance to thermal stress, infections and parasitic diseases due its evolutionary in European and Asian regions, respectively. OBJECTIVE: Since macrophage play a key role on animal immunometabolism displaying phenotypes that acts directly on inflammatory immune response development, this study aimed to verify the transcriptome of unstimulated macrophages derived from peripheral blood monocytes (MDM) of taurine and indicine bovine breeds. MATERIAL AND METHODS: Bovine macrophages were obtained by in vitro differentiation of peripheral blood monocytes isolated by ficoll gradient from taurine (Holstein; n=4) and indicine (Gir; n=4) bovine breeds. The mRNA of these cells on homeostasis was sequenced trough NGS to identify differentially expressed genes (DEGs; Fold Change≥1; Counts Per Million>1; FDR<0.05) contrasting taurine and indicine sequences using the EdgeR package version 3.8 and R version 3.5.0 (http://www.R-project.org). The co-expression analysis using transcript abundance counting table were performed on CeTF package, in R, to identify Transcription Factors (TF) using Regulatory Impact Factors (RIF) and Partial Correlation and Information Theory PCIT) analyses. Enrichment analysis were performed on Metacore software (P <0.001; FDR<0.05) using Gene Onthology (GO) database. RESULTS: Breed-specific transcriptional signatures pointed to significant differences on response to stress, to regulation of fatty acid transport and to mitotic cell cycle GO processes (P<0.00001; FDR <0.0001). The PCIT/RIF analysis displayed a list of 37 TF of which 20 present frequency differences higher than 10 times highlighting STAT 2 and 3, JUNB, RXR, XPB1 and IRF7. CONCLUSION: Indicine breed resistance to thermal stress, infection and parasitic diseases seems to be associated to macrophage phenotype and activation of key TF that modulate inflammation amplification and metabolism of fatty acids. Modulation of this process might help to prevent economic losses on bovine production based on taurine genetics in tropics. More specific studies with pathogen and thermal challenge must be done in order to validate the modulation of TF to get resistance to thermal stress, infections and parasitic diseases on taurine cattle.

MAJOR DEPRESSION FAVORS THE EXPANSION OF TH17-LIKE CELLS AND DECREASE THE PROPORTION OF CD39+ TREG CELL SUBSETS IN RESPONSE TO MYELIN ANTIGEN IN MULTIPLE SCLEROSIS PATIENTS

Introduction: Multiple Sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disorder of the central nervous system caused by myelin-specific T cells that attack the myelin sheath, causing sensory, autonomic, cognitive and motor function deficits. As with other autoimmune diseases, the outcome of MS may be influenced by mental disorders, like Major Depressive Disorder (MDD). The prevalence of depression is three times higher in patients with MS than in the general population. Increased levels of circulating pro-inflammatory cytokines are related to MDD, and as such, may negatively affect the clinical course of MS. Serotonin, a neurotransmitter reduced in MDD, possesses immunomodulatory properties and may explain the inflammatory profile observed in theses patients. However, little is known about the impact of MDD and the effect of serotonin on the in vitro MS-derived T cell behavior in response to anti-CD3/anti-CD28 beads and myelin antigen, which was the objective of the present study. Methods: Plasma and PBMC were obtained from 60 MS patients (30 with lifetime MDD) in remission phase. The PBMC cultures were stimulated with anti-CD3/anti-CD28 beads or myelin basic protein (MBP), and effector and regulatory T cell phenotypes were determined by flow cytometry. The cytokine levels, both in the plasma or in the supernatants collected from PBMC cultures, were quantified by Luminex. In some experiments, the effect of serotonin (5-HT) was investigated. Results: Higher Th17-related cytokine levels in response to anti-CD3/anti-CD28 and MBP were quantified in the plasma and PBMC cultures of the MS/MDD group in comparison with MS patients. Further, elevated frequency of CD4+ and CD8+ T cells capable of producing IL-17, IL-22 and GM-CSF was observed in depressed patients. Interestingly, the percentage of myelin-specific IFN-γ+IL-17+ and IFN-γ+GM-CSF+ CD4+ T cells directly correlated with neurological disabilities. In contrast, the occurrence of MDD reduced the proportion of MBP-specific CD39+ Tregs subsets. Notably, the severity of both neurological disorder and depressive symptoms inversely correlated with these Tregs. Finally, the addition of 5-HT downregulated the release of Th17-related cytokines in response to anti-CD3/anti-CD28 and myelin antigen. Conclusion: Our findings suggested that recurrent major depression, by favoring imbalances of effector Th17 and Treg cell subsets, contributes to MS severity.

Malaria-induced Kupffer cells death leads to uncontrolled viral and bacterial superinfections

Kupffer cells are self-maintained tissue-resident macrophages that line the liver sinusoids and play an important role on host defense against infections. Here we investigated the consequences of Kupffer cell death in a rodent malaria model. Similar to malaria patients, infection with Plasmodium chabaudi induced liver toxicity as determined by serum ALT and AST elevation. This liver damage was associated with accumulation of hemozoin (a Plasmodium-derived insoluble iron crystal), increased Type 1 inflammatory response, Kupffer cell death and impaired clearance. Our results indicate that hemozoin act as damage-associated molecular patterns inducing necrosis cell death of Kupffer cells associated to the production of mitochondrial superoxide, lipid peroxidation and increased free iron, hallmarks of ferroptosis. In addition, we found that the malariainduced Kupffer cell death resulted in rapid spread of Escherichia coli to different organs. Likewise, we found that mice infected with P. chabaudi are highly susceptible to virus infection. We conclude that Kupffer cells play a central role in intravascular immune response in the liver, generating a highly efficient surveillance and filtering system.

MAST CELLS DECREASE THE PARTICIPATION OF WOUND REPAIR IN THE SKIN OF SENESCENT MICE

Introduction: The skin is an important organ that constitutes a protective barrier, preventing excessive water loss and the entry of harmful substances and pathogens from the environment. With aging skin occour intrinsic and extrinsic changes resulting from the impairments, throughout people life, which it is exposed. Skin aging is accompanied by genetic modifications, reduced cell proliferation and tissue renewal, accumulation of senescent cells, changes in the extracellular matrix and a pro-inflammatory environment that favors undesirable conditions, including the difficulty of repairing lesions. Mast cells are innate immune cells that participate in inflammatory responses in various tissues. They are abundant in the skin, reside near blood vessels and nerves, and degranulate in response to injury. However, the participation of these cells in the inflammation of aged skin wounds is not known for sure. Since mast cell degranulation releases important chemical mediators, influencing inflammation and consequent repair of injured tissue. Methods: This study evaluated the difference in mast cell density and maturation at the edges of skin wounds as a result of aging. Therefore, histological analyzes of skin wound biopsies from mice were performed on slides stained with Toluidine Blue and Alcian-Blue and Safranin. Results: Our results show that there is a decrease in the number and difference in the content of mast cell granules at the edges of skin wounds along the aging. Conclusion: Whence, we believe it may be a reduction of the participation of mast cells in the repair of aged skin wounds.

MATERNAL HIGH-FAT DIET PROMOTES LUNG EOSINOPHILIA AND REDUCES IgE SECRETION IN ALLERGIC OFFSPRING

INTRODUCTION: Maternal obesity raises the risk for various offspring complications including asthma at any age, by mechanisms still unknown. The concomitance of both diseases promotes distinct phenotypes characterized by either increased Th17 and neutrophilic inflammation, or aggravated Th2 phenotype and eosinophilia. In both cases, the symptoms are exacerbated and resistant to corticosteroid treatment. Our aim in this study was to explore the immunological modifications by which maternal diet can impact the risk of asthma and its aggravated symptoms. METHODS: BALB/c dams (CEUA-UFJF 33/2020) fed either a high-fat (HFD) or a standard diet (SD) were mated and their pups were used in the experiment. Asthma was induced by Ovalbumin sensitization on the 7th and the 14th days of life, followed by three intranasal challenges on the 21st -23rd days, mice were euthanized on the 24th day. Animals were weighed, lungs and bone marrow were collected for histological analysis and immunophenotyping, and blood was drawn for antibody titration. RESULTS: The results were expressed as the difference between means and standard error of the difference of means. When evaluating the effects of maternal diet on the offspring regardless of asthma status, pups from HFD dams had higher body mass (2,36±0,59g), and higher expression of PDL1 in lung cells (3,64±1,41 MFI) when compared to pups from SD dams. The maternal high fat diet also decreased the expression of CD80 in bone marrow-derived dendritic cells stimulated with LPS (-88,44±30,35 MFI), and increased the expression of PDL1 on the same unstimulated cells (6,564±55,17 MFI). When asthma was induced, asthmatic pups from HFD dams presented a greater eosinophils count in lung tissue (2,60±1,02 cells per field), higher IgG2a titers (23,95±8,99 OD), and lower IgG1 (-30,90±12,66 OD) and IgE (-41,23±17,15 OD) titers, compared with asthmatic pups from SD dams. CONCLUSION: The results demonstrate that the maternal HFD affected lung immune response and dendritic cell activation even before asthma induction, as well as antibody production and eosinophilic inflammation in asthmatic animals.

MATRIX METALLOPROTEINASES ON SEVERE COVID-19 LUNG DISEASE PATHOGENESIS

Introduction. Patients with COVID-19 predominantly have a respiratory tract infection and acute lung failure is the most severe complication. While the molecular basis of SARS-CoV-2 immunopathology is still unknown, it is well established that lung infection is associated with hyper-inflammation and tissue damage. Matrix metalloproteinases (MMPs) contribute to tissue destruction in many pathological situations, and the activity of MMPs in the lung leads to the release of bioactive mediators with inflammatory properties. We sought to characterize a scenario in which MMPs could influence the lung pathogenesis of COVID-19. Methods and Results. This observational, analytical, and prospective study was conducted using stringent and reasonable inclusion and exclusion criteria. In total, non-COVID-19 subjects (n = 13), who were hospitalized and intubated due to different clinical primary conditions, along with patients in severe/critical illness (n = 39), intubated and hospitalized in intensive care unit (ICU) who tested positive for SARS-CoV-2 infection. Although we observed high diversity of MMPs in lung tissue from COVID-19 patients by proteomics, we specified the expression and enzyme activity of MMP-2 in tracheal-aspirate fluid (TAF) samples from intubated COVID-19 and non-COVID-19 patients. Moreover, the expression of MMP-8 was positively correlated with MMP-2 levels and possible shedding of the immunosuppression mediator sHLA-G and sTREM-1. Together, overexpression of the MMP-2/MMP-8 axis, in addition to neutrophil infiltration and products, such as reactive oxygen species (ROS), increased lipid peroxidation that could promote intensive destruction of lung tissue in severe COVID-19. Conclusion. Uncontrolled protease activity and improper expression of several MMPs were correlated to lung disease in severe COVID-19. Although considered plasma prognostic biomarkers, the MMP-2 and MMP-8 pathways in the lung could become the target of specific therapies, including those proposed to diminish inflammation, oxidative stress and tissue damage during COVID-19.

MATURATION, MOBILIZATION AND INFLAMMATORY PROFILE OF NEUTROPHILS IN EXPERIMENTAL MALARIA

Cerebral malaria (CM) is responsible for thousands of deaths associated with an unbalanced inflammatory response. Neutrophils are key components of inflammation, producing inflammatory mediators that amplify the response. Chemokines and their receptors play several roles during neutrophils life cycle. In the bone marrow (BM), the CXCR4 receptor retains immature neutrophils, while the CXCR2 receptor induces the mobilization of neutrophils from BM to the bloodstream. This study aimed to investigate the role of neutrophils in the development of experimental cerebral malaria (ECM). The production and maturation of neutrophils in the BM, the kinetic of recruitment to the spleen and the activation profile of these cells were evaluated following infection of C57BL/6 and BALB/c mice with Plasmodium berghei ANKA, experimental models susceptible and resistant to ECM, respectively. Immature and mature neutrophils followed different kinetics of production and maturation in the BM of infected BALB/c and C57BL/6 mice. Spleen analysis revealed a constant increase in the total number of neutrophils in the BALB/c animals throughout the infection. On the other hand, in C57BL/6, we observed a transient increase, which was not maintained at the 6th dpi. Among the neutrophils present in the spleen of naïve animals, approximately 70% are mature cells, but their values decreased after infection. The rise in the count of neutrophils in the spleen of both mice strains is associated with an increase in cells with an immature or aging profile. Interestingly, the analysis of the neutrophil fate and activation profile showed a greater amount of late apoptotic and TNF-α+ neutrophils in C57BL/6 parasitized animals. Our data suggest that BALB/c and C57BL/6 mice respond to infection by increasing the medullary production of neutrophils, but there is a constant mobilization of immature neutrophils from the BM to the periphery in BALB/c animals. On the other hand, C57BL/6 mice are able to retain these cells in the BM for a longer time, releasing a smaller number of immature neutrophils and, thus maintaining a higher percentage of cells with a pro-inflammatory profile in the splenic neutrophil pool.

MECHANISMS OF RESISTANCE TO CAR-T CELL IMMUNOTHERAPY: INSIGHTS FROM MATHEMATICAL MODELLING

Introduction Chimeric Antigen Receptor (CAR) T-cell therapy are genetically reprogrammed T-lymphocytes that recognize an antigen in malignant cells and activate an anti-tumor response. This immunotherapy has revolutionized anticancer therapy on hematological malignancies. However, long-term studies revealed non-durable remissions in a significant number of patients: 30-60% of patients will experience either CD19+ or CD19- relapse in B cell acute lymphoblastic leukemia (B-ALL). Poor CAR T cell persistence and/or cancer cell resistance due to antigen loss or lineage switching are some of the mechanisms underlying these relapses, affecting the immune response intensity and duration. To investigate how antigen-mediated resistance mechanisms affect therapy outcomes, we develop a mathematical model that simulates interactions between CAR-T cells and a heterogeneous population of leukemic cells with different levels of antigen expression. Methods and Results The model is based on a set of integral-partial differential equations that enables in silico analysis of several therapy scenarios, providing a deeper understanding of key biological factors underlying CD19+/CD19- relapses. After calibration with clinical data, simulations revealed that the presence of CD19- clones at the start of therapy, even in small numbers, significantly impairs the therapy outcome. The mutation rate - that produces clones with low antigen expression strongly correlates with the relapse period and promotes microenvironment adaptations. These resistant CD19- cells quickly progress due to CAR-T cells’ lower cytotoxicity, leading to tumor escape. Furthermore, the developed model can capture possible changes in the therapy's effectiveness due to any intrinsic patient-specific characteristics represented by the model parameters. Conclusion Mathematical models have been demonstrated to be an indispensable tool in biomedicine. Herein, we develop a model of CAR T-cell therapy that replicates clinical trial dynamics, evaluates the impact of different resistance mechanisms, and identifies key determinants of treatment efficacy to predict patient outcomes. Through collaboration between lab scientists and clinicians, computational models like the one presented could be integrated into clinical practice as an auxiliary tool for evaluating different CAR T cell protocols or designs and their associated efficacy, guiding future experiments and saving resources.

MELANOMA-DERIVED SMALL EXTRACELLULAR VESICLES INFLUENCE THE FUNCTIONAL PROFILE OF MACROPHAGES VIA REGULATION OF GLUCOSE METABOLISM

Introduction: Extracellular vesicles (EVs) are produced by most cells in physiological and pathological conditions, such as cancer. Tumor-derived extracellular vesicles have been shown to influence the phenotype and function of various immune system cells, including macrophages. However, studies on the role of EVs released by tumor cells in the metabolism of macrophages are under investigation. In the present work, we evaluate the influence of melanoma-derived EVs on glucose metabolism and the functional profile of murine macrophages. Methods: EVs isolated from B16F10 culture supernatant were characterized by nanoparticle tracking analysis and then used in vitro assays with bone marrow-derived macrophages (BMDMs) from C57BL/6 mice. Production of Th-1/Th-2/Th17 cytokines profile in the BMDMs culture supernatant, and cellular glucose uptake were evaluated after 48 hours of culture. Results: BMDMs treated with tumor-derived EVs showed an increase in both the glucose uptake, and production of TNF-α, IL-6, and IL-10 cytokines. Analysis of the ratio between inflammatory and anti-inflammatory cytokine profiles indicated a more expressive increase in inflammatory cytokines. Conclusion: The present work showed that melanoma-derived EVs influence the phenotype of macrophages towards the pro-inflammatory profile mediated by the increase of glucose uptake. These findings are relevant and provide new information on the effect of EVs derived from melanoma cells on macrophages` metabolic and functional profiles.

Memory B cell dynamics in clinically discordant cases after SARS-CoV-2 infection

SARS-CoV-2, the etiologic agent of the COVID 19 pandemic, has been the target of many studies due to its high rate of transmission, which generally precedes symptoms. Furthermore, there is a distinct spectrum of immune response among the infected individuals, causing different clinical conditions, and the reason for these phenomena is still unclear. Viral infections trigger the clonal expansion of specific B cells that produces antibodies against structurally distinct epitopes from the viral particles. Those antibodies can prevent the binding of viruses to their cell receptors or to form immune complexes, leading to the elimination of the pathogen. Furthermore, these specific B lymphocytes undergo class switching and somatic hypermutation favoring the selection of antibodies with increased affinity to viral antigens as the time of infection progresses. In this work, we made a longitudinal study of the case of two patients with differing immunological response profiles. One follows the pattern described by the OMS, of rapid convalescence (around 14 days after symptoms onset), and the other, in contrast, showed a condition of long persistence (more than 50 days after symptoms onset). To perform this analysis, functional studies were made by limiting dilution assay of total memory B cell (MBC) IgD- culture on a NB21 feeder cell monolayer in the presence of polyclonal stimuli. Through the technique of Flow Cytometry, we were able to describe the different populations of total and specific B cells against the viral Spike protein (S), and its RBD subunit, which are the main targets for neutralization. We describe during the kinetics that, despite having a higher percentage of total MBC (MBC -CD19+CD38-CD27-IgD-) in relation to the convalescent patient, the persistent patient has a lower percentage of MBC specific for S protein. Also, the convalescent patient maintained the same Anti-S-specific B cell percentage levels during all times studied and a higher percentage of total MBC IgD+ compared to the persistent patient. Furthermore, the data provide information regarding the frequency of MBC IgD+ and IgD- and the frequency of specific responder cells to the viral antigen, which can be complemented by the analysis of the neutralizing activity of secreted antibodies. This work emphasizes the importance of the immunological history of individuals with different responsive profiles to the same viral infection, given the complexity of different B cell populations.

Metabolic characterization of Foxp3+ RORgt+ T cell population in the experimental model of colitis associated colorectal cancer

Colorectal cancer (CRC) is responsible for 700,000 deaths worldwide each year, the fourth most deadly cancer. Chronic ulcerative colitis (UC) has also been associated with an increased risk of CRC, classified explicitly as colitis-associated colorectal cancer (CAC). Recently, a population of Foxp3+ RORgt+ T cells was identified in the region of the lamina propria of the colon, which is dependent on the interaction with microbiota and recognized as an immunosuppressive component in CAC. However, its metabolic profile, as well as its participation during the progression of CAC, has not yet been clarified. Thus, we hypothesize that the inflammatory microenvironment observed in the experimental model of CAC is essential for the definition of the metabolic program of Foxp3+ RORgt+ T cells, sustained by HIF-1a function confers the immunosuppressor phenotype of double-positive cells, promoting the development and progression of the tumor. We performed in vitro differentiation assays of Foxp3+RORgt+ T cells, obtaining a double positive population corresponding to 60-70% of the total CD4+ cells at the end of the experiment. We observed that this population of double-positive cells has a higher mitochondrial capacity and the Tregs (CD4+Foxp3+). We demonstrated that the specific inhibition of HIF-1a during the in vitro differentiation of Foxp3+RORgt+ cells leads to a drop in this population and favors the stabilization of CD4+Foxp3+ T cells. Also, this leads to increased expression of mitochondrial fusion genes and decreased genes related to mitochondrial fission. Furthermore, we demonstrated that In vitro differentiated Foxp3+RORgt+ T cells are more suppressive than in vitro Tregs. We also performed the in vivo experimental model of CAC in Cre-lox mice with specific disruption of HIF-1a in RORgt cells or Foxp3 cells. By flow cytometry, we phenotyped the activation and cytokine production of the T cell population after the cancer induction. HIF1a depletion in RORgt+ cells impairs CAC progression and leads to CD4+ Foxp3+ a higher IL-17 production; Foxp3+RORgt+ T cells had higher expression of exhaustion markers and impaired IL-17 production, together with an increase in IFNg production by RORgt+ cells. Our results suggest that the expression of HIF1a is necessary for in vitro and in vivo stabilization and suppressive function of Foxp3+RORgt+ T cells.

METABOLIC DISORDER IN LONG COVID: IMMUNE PROFILE OF DIABETES ASSOCIATED WITH COVID-19

INTRODUCTION: SARS-CoV-2 infection is associated with the development of sequelae, named long-COVID (Infect Dis (Lond), 53:737-754, 2021), including the diabetes mellitus associated with COVID-19 (DAC) (Lancet Diabetes Endocrinol,10:311-321, 2021). However, little is known about immune response associated with DAC. The aim of this study was to investigate clinical and immune response features in patients with DAC. METHODS: This is a cross-sectional study with patients that presented COVID-19: previous diagnosis of diabetes mellitus (DM), DM associated with COVID-19 (DAC) and controls without history of DM or elevated HbA1c. DAC was defined as non-previous diagnosis of DM and HBA1C >6.4%. Clinical and sociodemographic characteristics were compared between DAC and DM groups. To assess the profile of inflammatory mediators, TNF-α, IL-6, LTB4 and PGE2 were measured by ELISA in the serum or the plasma of patients XX days after disease onset. Furthermore, auto-antibody, anti-GAD and anti-pancreatic islets were evaluated for DAC group. RESULTS: In total, 670 patients were recruited: 149 with DM, 83 with DAC and 230 controls. DAC and DM presented similar characteristics except for age (53 vs 60.1, years, respectively). DAC group included 49 % of non-obese patients. In total, 55 individuals were tested for antibody anti-GAD and anti-pancreatic islets, with negative results. Levels of TNF-α was higher for DAC compared to DM for individuals that presented severe COVID-19 at acute disease (169.6 vs 55.9, p=0.0193, respectively) . Besides, levels of TNF-α correlation positively with PGE2 (r=0.458; p=0.037) and LTB4 (r=0.518; p=0.011) in the DAC group. CONCLUSION: DAC presented clinical characteristics compatible with Type II diabetes and an inflammatory immune profile late after disease onset. Further studies are needed to elucidate the involvement of these inflammatory mediators in disease pathogenesis. In addition, there may be unconfirmed autoimmune mechanisms in this study.

Metabolic profiling of chronic myeloid leukemia patients during disease progression and response to tyrosine kinase inhibitors.

Introduction: Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm that expresses Bcr-abl, a constitutively activated tyrosine kinase. It is a product of a chromosomal translocation resulting in the Philadelphia chromosome in hematopoietic progenitor cells. Bcr-abl tyrosine-kinase inhibitors (TKI) do not definitively cure all the CML patients. The efficacy of TKI is reduced in CML patients in blastic phase – the most severe phase of the disease –, and resistance to this drug has emerged. There is limited knowledge on the underlying mechanisms of disease progression and resistance to TKI beyond BCR-ABL1, as well as on the impact of TKI treatment and disease progression on the metabolome of CML patients. The present study reports the metabolomic profiles of CML patients at different phases of disease treated with TKI. Methods and Results: The plasma metabolites from CML patients were analyzed using liquid chromatography, mass spectrometry, and the results were analyzed using bioinformatics tools. Distinct metabolic patterns were identified for CML patients at different phases of the disease and for those who were resistant to TKI. The lipid metabolism in CML patients at advanced phases and TKI-resistant patients is reprogrammed, as detected by analysis of metabolomic data. CML patients who were responsive and resistant to TKI therapy exhibited distinct enriched pathways. In addition, ceramides levels were higher and sphingomyelin were lower in resistant patients compared to control and CML groups. Conclusion: Taken together, the results here reported established the metabolic profiles of CML patients who progressed to advanced phases of the disease and failed to respond to TKI therapy as well as patients in remission. In the future, an expanded study on CML metabolomics may provide new potential prognostic markers for disease progression and response to therapy.

METABOLIC REPROGRAMMING OF MACROPHAGES DURING BRUCELLA INFECTION IS ORCHESTRATED BY STING VIA HIF-1Α ACTIVATION

The metabolic reprogramming of macrophages in response to microbial insults is a major determinant of pathogen growth or containment. Brucella abortus is an intracellular bacterium that causes brucellosis, an infectious disease that promotes abortion in domestic animals leading to severe economic losses and an inflammatory condition in humans. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory macrophage profile upon Brucella abortus infection. This metabolic reprogramming in macrophages is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1β release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1β release. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.

Metabolic-trained immunity potentiates SARS-CoV-2 inflammation to worsen Covid-19 disease

Covid-19 is caused by the severe-acute-respiratory-syndrome coronavirus-2 (Sars-Cov-2). Dysregulated immune-response, with an exaggerated inflammation, and enrichment of myeloid-cells in lungs are linked to severe symptoms and higher risk of death. We described that diabetic people has improved risk of severe Covid-19 because Cov-2 depends on glucose to replicate and potentiates inflammation. Because non-diabetic obese individuals have a higher risk for severe Covid-19, we aimed to determine the mechanisms involved. Monocytes isolated from healthy-donors (HD) were infected with Cov-2 and cultured with heated-inactivated-serum from obese/not-diabetic patients (OIS), which displayed higher viral-load and IL-6 and IL-1b expression compared to CoV-2-infected monocytes cultured with heated-inactivated-serum from lean/not-diabetic patients (LIS). Since levels of circulating free-fatty-acid (FFA) are increased in obesity, we used palmitate to treated HD monocytes, which displayed enhanced proinflammatory profile when infected with CoV-2 (palmitate+Cov2). Our RNAseq data indicated that palmitate+Cov2 have enhanced fatty-acid-oxidation (FAO) machinery. However, palmitate was not used as a substrate for oxygen consumption, suggesting that palmitate-derived carbons used to form acetyl-CoA during FAO have another fate. Etomoxir (FAO-inhibitor) and BTA (citrate-transporter-inhibitor) blocked the proinflammatory profile of palmitate+Cov-2 monocytes. We observed that FAO-derived citrate must exit mitochondria to exert its effects. Next, we determined whether cytoplasmic citrate-derived acetyl-CoA was used to acetylate histones. H3k18 acetylation was increased in OIS and palmitate+CoV2 monocytes. Deacetylase inhibition enhanced the pro-inflammatory profile of palmitate+Cov-2 monocytes. Mass-spectrometry of purified histones (C13-label carbons), revealed that palmitate-C13-derived carbons are found in histones. Chip analysis with H3k18-acetylated antibody showed that palmitate+Cov-2 monocytes have increased H3k18 acetylation in IL-1b and IL-6 promoter-regions. Finally, we fed palmitate to fasted-HD, collected and infected their monocytes with CoV-2, which displayed increased viral load. Together, we propose that obesity induces metabolic-trained-immunity, which leads to enhanced and dysregulates inflammation upon infection with CoV-2, aggravating Covid-19 disease.

METABOLIC UPTAKE OF DIETARY N-GLYCOLYLNEURAMINIC ACID PROMOTE COLON CANCER GROWTH BY IMMUNE AND NON-IMMUNE MECHANISMS

Every living cell is covered with an extense layer of glycoconjugates, including glycoproteins and glycolipids. The majority of these glycans present a terminal sialic acid. While most mammals synthesize both N-acetylneuraminic sialic acid (Neu5Ac) and N-glycolylneuraminic sialic acid (Neu5Gc), humans do not synthesize Neu5Gc due to a mutation in the Cmah gene, which encodes the only enzyme capable of hydroxylating Neu5Ac into Neu5Gc. Despite this, Neu5Gc can be found in healthy human tissues and, at higher levels, in carcinomas such as colorectal cancer (CRC). Ingestion of red meat is the only known source of Neu5Gc incorporation in humans and data shows an association of its intake with CRC risk. Here, we investigate if the metabolic incorporation of Neu5Gc by CRC cells could affect signaling pathways relevant to its progression. HCT-116, human CRC cells, when fed with different concentrations of Neu5Gc presented an increased Wnt3a binding to the cell surface, suggesting that Neu5Gc could influence Wnt/Fzd interaction. The increased response of Neu5Gc-fed cells to Wnt3a resulted in increased activation of the Wnt signaling pathway, as seen in luciferase reporter assay, augmented expression of Axin2 and SP5, and higher cell proliferation rates. Using a human-like Cmah-/- mice model, presenting a mutation in the Wnt pathway, that spontaneously develops CRC (APC-CPC Cmah-/- mice) we found a higher number of polyps, that were also bigger in size, in mice fed a Neu5Gc-rich diet. These results demonstrate that metabolic uptake of Neu5Gc can promote CRC growth both in vitro and in vivo. The incorporation of Neu5Gc is associated with the induction of xenosialitis, an inflammatory process provoked by circulating anti-Neu5Gc. Here, our animal model was submitted to induction of xenosialitis by the immunization with Chimp red cell membrane ghosts, we found that these animals presented an increased number of polyps and tumor load when fed a Neu5Gc-rich diet. In these mice, we also observed higher mRNA expression of regulatory genes, such as IL-10. On another hand, we observed lower mRNA expression of TGF-β and pro-inflammatory genes such as TNFα, IL-12, and IL-6 did not increase neither decrease. Our data suggest that dietary ingestion of Neu5Gc can promote CRC growth by immune and non-immune mechanisms likely contributing to the human-specific risk of CRC associated with red meat consumption.

MICA-129 FREQUENCY IN BRAZILIAN POPULATION

MICA encodes a cell surface glycoprotein expressed on endothelial, epithelial cells, monocytes, and fibroblasts. MICA is upregulated upon cellular stress (e.g., tumors, infections, and oxidative stress). MICA interacts with the activating receptor NKG2D from the surface of Natural Killer (NK) cells, TCD8+ cells, and γδT cells. There is a polymorphism at MICA codon 129, related to variant rs1051792 G>A, that modifies the alpha-2 domain by exchanging valine (Val) for methionine (Met) at residue 129. This dimorphism has a functional impact because MICA-129-Met binds to the NKG2D receptor with high affinity, with higher activation of NK cells and higher production of cytokines by NK and TCD8 cells. Conversely, MICA-129-Val has reduced affinity to NKG2D. Homozygosity for MICA-129-Val (rs1051792 G/G) appears to result in high levels of soluble MICA, an inhibitory molecule to NK cell activity. We evaluated the frequency of this amino acid exchange in Brazilians admixed individuals from the SABE cohort, in 1,170 elderly individuals from the city of São Paulo, by using next-generation sequencing (NGS) and bioinformatics pipeline to reduce alignment errors in genes from the MHC region. We detected 474 (50.4%) homozygous individuals for rs1051792/G (129-Val), 527 heterozygous (45%), and 169 (14.5%) homozygous for rs1051792/A (129-Met). Moreover, allele 129-Met is more frequent in Brazilians with major African ancestry, which is in line with the higher frequency of rs1051792/A in Africa than in Europe. Therefore, in general, half of the Brazilian population presents at least one copy of rs1051792/A (129-Met), which may be directly related to the efficient escape mechanisms of tumor or infected cells by releasing the soluble MICA protein. However, soluble proteins may also be more efficient in down-regulating NK cells in autoimmune diseases. Therefore, the MICA-NKG2D pathway may become a potential target for developing immunotherapies.

MICA GENE POLYMORPHISMS AND PROTEIN FREQUENCY DISTRIBUTION IN BIOGEOGRAPHIC REGIONS

The MICA gene encodes immunomodulatory molecules expressed upon cellular stress, such as infections and tumors (oxidative stress). When in its membrane-bound isoform (mMICA), MICA interacts with the NKG2D receptors leading to the activation of NK and γδT cytotoxicity. However, MICA can also be expressed as soluble molecules by two mechanisms, protein shedding and polymorphisms hindering the translation of the transmembrane domain. When in its soluble isoform, sMICA leads to a downregulation of NK and γδT cytotoxicity. Understanding the MICA genetic diversity might shed some light on the mechanisms regulating its expression levels and function. We evaluated the MICA polymorphisms in exons and the protein sequences using next-generation sequencing (NGS) in more than 5,000 individuals from all continents. Among these samples, there are 1,323 Brazilians from the SABE cohort. We identified 94 variable sites in exons, 52 (55%) configuring missense mutations. Most of these variants coincide with exon 4, which encodes the alpha-3 domain. This domain may suffer a proteolytic breakdown generating soluble isoforms, but it is unclear whether these polymorphisms influence MICA shedding. In worldwide samples, the MICA allele known as MICA*008 is the most frequent one (23%), followed by MICA*002 (19%), MICA*004 (11%), and MICA*009 (8%). Some MICA alleles encoded smaller MICA protein sequences due to premature stop codons, and these isoforms are secreted as soluble molecules. Our data indicate that alleles MICA*008, *015, *017, *064N, *068, *070, and *185 encode this soluble isoform, and they present a summed frequency of 27.96%. MICA*008 is frequent in all biogeographic regions, from 9.92% in South Asia to 30.6% in the Middle East. Therefore, the presence of alleles that can only generate soluble isoforms is a common feature, even in a homozygosis state. Moreover, considering that some polymorphisms in exon 4 may influence MICA shedding, the frequency of individuals prone to produce mostly sMICA might be even higher. Functional studies must be placed to understand the functional impact of a high (and sometimes restricted) production of sMICA and the modulation of the immune system in the face of different stress situations.

MICROGLIA DERIVED EXTRACELLULAR MITOCHONDRIA MODULATE NEURONAL SURVIVAL AND FUNCTION

Introduction Neuroinflammatory diseases are often associated with mitochondrial dysfunction on brain parenchyma. Extracellular vesicles, some containing mitochondria, have emerged as important in disseminating information from activated cells to quiescent or injured neural cells. In this context, we explored microglia-derived mitochondria and their effects on neurons. Methods We used timelapse image of microglia from PhamLyz mice brain, which express the fluorescent protein Dendra2 in mitochondria, co-cultured with hypothalamic neurons that express AgRP (Clu189 lineage). Results We do observe microglia release mitochondria into the extracellular medium, either isolated (< 600nm) or inside vesicles (1-6μm). Fascinating, Clu189 take up those mitochondria released by microglia. Conversely, injured Clu189 subjected to rotenone treatment (electron transport chain complex I inhibitor) uptake threefold more mitochondria, suggesting a key role of microglia-derived mitochondria for their survival. Also, neuronal death increases when, in co-culture, microglia are cultivated on inserts with 0.2μm pore size, which do not allow mitochondria transference (>200nm). It is still unknown how neurons uptake those mitochondria. To identify whether a specific activation profile induces the release of mitochondria by the microglia with neuroprotective effects, microglia were stimulated prior to co-culture with LPS (100ng-24h), Mdivi-1 (Drp1 inhibitor, 25uM-2h), or palmitate (0.4uM-6h). Mitosox labelling confirms that rotenone treated neurons had increased superoxide production, which was higher when neurons were cultured with LPS-activated microglia. However, Mdivi-1-treated microglia improved neurons function due to reduced superoxide levels. These results suggest that mitochondria transference protects neurons from damage. No effects on superoxide production were seen after palmitate stimulus. In addition, microglia-derived vesicles (>0.2μm) promote neuronal survival, regardless of the activation state of microglia. Also, neurons take up mitochondria in vivo, when mice are fed a high-fat diet, especially in the hypothalamus. Conclusion Together, these data indicate microglia may favour neuronal survival through extracellular mitochondria transfer, a future therapeutic insight for neurodegenerative and metabolic diseases.

Mitochondria fission controls obesity-induced hypothalamic inflammation

Obesity is commonly related to a high lipidic intake during feeding. Lipids that reach the bloodstream are capable to surpass the blood-brain barrier and enter the hypothalamus, the brain region responsible for controlling the systemic metabolic homeostasis. High levels of fatty acids in the hypothalamus activate the resident innate immune cells - microglia, leading to the secretion of proinflammatory cytokines and local inflammation. Hypothalamic inflammation compromise central metabolic control, affecting whole body metabolism, which contributes to the development of metabolic disorders. Obesity-induced hypothalamic inflammation precedes weight gain and contributes for such event. The metabolism of fatty acids occurs mostly at mitochondria, a highly dynamic organelle that alters its structure accordingly to the cell metabolic needs. Drp1 protein possesses a fundamental role in mitochondrial dynamics and acts inducing mitochondrial fission during immune cells activation. Our aim is to determine the role of mitochondria fission in hypothalamic microglial cells upon high fat diet (HFD)-induced obesity. Through in vitro and in vivo assays using pharmacological modulators followed by an obesogenic stimulus we determined the consequences of Drp1 inhibition for weight gain, microglial activation in hypothalamic inflammation. We hypothesize that the inhibition of Drp1 will prevent HFD-induced microglial activation in hypothalamus. Our results showed that in vitro, Mdivi-1 treatment, the mitochondrial fission blocker, followed by a 6h palmitate stimulus, was able to prevent expression and secretion of the pro-inflammatory cytokine IL-6 as well as the classic HFD-activated marker ABCa1 when compared to the untreated group. Also, Mdivi-1 treatment maintained the metabolic profile related to inactivated cells through a Mito-Stress assay. In vivo, a similar profile was observed. A single injection of Mdivi-1 in the third ventricle of chow- and HFD-fed mice prevented HFD-induced hypothalamic inflammation (reduced expression of IL-1β and TNF-α) and weight loss. Thus, inhibition of mitochondria fission in the hypothalamus reduces HFD-induced hypothalamic inflammation and prevents weight loss. References: Trends Immunol. 2018 Jan;39(1):6-18. Mol Cells. 2020 May 31;43(5):431-437 Cell Rep. 2020 Feb 18;30(7):2180-2194.e8.

Mitochondrial Metabolism as a Keratinocyte Immune Modulator During the Progression of Experimental Psoriasis

Introduction: Psoriasis is a chronic inflammatory skin disease that affects up to 200 million individuals at worldwide level. Keratinocyte (KC) hyperproliferation and lymphocytic infiltrates are the histological hallmarks of this disease. Although some treatments are available for the disease, poorly is still known regarding the initial triggers that lead to psoriasis progression. Psoriatic KCs are under constant proliferation which demands high levels of ATP to sustain cell division. Furthermore, cell metabolism can regulate immune responses, where pro-inflammatory cells usually prioritize glycolytic metabolism while anti-inflammatory cells typically rely on oxidative phosphorylation. Mitochondria is a master regulator of cell metabolism and undergoes through processes of fission and fusion to meet energetic demands, a phenomenon named mitochondrial dynamics. The knowledge regarding how mitochondrial metabolism participates in psoriasis is scarce. Objective: In this context we sought to investigate how mitochondrial dynamics is connected to psoriasis where we hypothesized that mitochondria dynamics is altered in psoriasis progression. Results: Our results show that mitochondria is affected in the epidermis of psoriatic patients as observed in our in silico analysis. Mitochondrial gene structures were upregulated in psoriatic epidermis suggesting mitochondrial biogenesis. We could confirm this by providing transcription factor enrichment analysis where the enrichment of PGC-1α was observed in both epidermis derived from human samples and imiquimod-induced model in mice. In experimental models we also observed mitochondria biogenesis in vitro using mitochondrial probes as we observed increase in mitochondrial mass and membrane potential after imiquimod treatment. Also in vivo, after retrieving epidermis from imiquimod-induced psoriasis in mice model, we suggest that Drp-1, a protein that mediates mitochondria fission, is downregulated while Mfn-2, a protein that mediates fusion, remains unchanged through the induction of psoriasis. Conclusion: So far our results show that mitochondrial metabolism is highly affected in psoriasis where KCs seem to be increasing mitochondrial content during the progression of the disease.

Modulation of antiviral molecules USP18 and ISG15 as a cellular reponse to infection and antiviral resource in DENV-2 infected A549 cells

INTRODUCTION It is well established that dengue virus (DENV) infection can alter host cell metabolism and cellular immune response. One of the most important responses to viral infection is triggered by type I interferons (IFN-I), which induce the expression of hundreds of interferon-stimulated genes (ISG). Among the products of ISGs, USP18 is an enzyme belonging to the class of deubiquitinases. USP18 catalyses protein deISGylation, i.e., it removes post-translational modifications caused by the addition of ISG15 (also a product of genes stimulated by IFN) to specific target proteins. ISGylation can cause cellular metabolic changes as well as exert antiviral effects during infections (Viruses vol. 10,11-629.2018). In this context, our objective is to investigate the impact of ISGylation on the antiviral response (P. N. Acad Sci USA.104(4).2007) and energy metabolism in DENV-2 infected cells. METHODS AND RESULTS We generated three stable sub-strains of the cell line A549 through CRISPR/CAS9 NHEJ and CRISPR ALT-R HDR systems: A549 wild type, A549 knockout control containing the empty vector (A549EV), A549 knocked out for USP18 (USP18KO). We are currently validating a fourth lineage: A549 expressing the inactive USP18 (USP18C64A/C64A), containing a mutation in its catalytic site, preventing this enzyme from performing its deISGylating function, thus increasing ISGylation levels in the cell. A549 WT was infected with DENV-2 at MOI 0.1, MOI 1, and MOI 5, with USP18 and ISG15 expression evaluated by western blotting. USP18 and ISG15 protein expression increased in cells primed with IFNalpha-2b in a non-dose-dependent manner. We found a higher increase in USP18 and ISG15 expression in cells infected with DENV-2 at MOI 1 and primed with 500UI of IFN. Intriguingly, we noted that USP18 and ISG15 expression increased in MOI 0.1 and MOI 1 with a paradoxical decrease of its expression in MOI 5. CONCLUSION The paradoxical expression of proteins observed in western blotting may be justified by a greater number of cells being infected at MOI 5 so that the paracrine signaling induced by the infected cell and seen in the situations of MOI 0.1 and MOI 1 is being blocked by the inhibition of interferon signaling by the DENV-2. Next, we intend to perform high-resolution respirometry and plaque assays to analyze cellular respiration and viral replication in A549 C64A cells infected with DENV-2.