Diabetes tipo 1 (DM1) e uma doenca autoimune que se inicia devido a defeitos na tolerancia imunologica a auto-antigenos, resultando na destruicao autoimune das celulas pancreaticas em individuos geneticamente suscetiveis. Os receptores NOD-like (NLRs) sao receptores intracelulares responsaveis pelo reconhecimento de padroes moleculares associados a patogenos (PAMPs) e padroes moleculares associados ao dano (DAMPs). Estudos recentes tem demonstrado que os receptores NOD1 e NOD2 desempenham um importante papel na ativacao da imunidade inata contra patogenos e na regulacao da imunidade adaptativa, uma vez que sua ativacao leva a producao de citocinas relacionadas a diferenciacao de linfocitos T auxiliares produtores de IL-17 (Th17). Porem, a importancia desses receptores no DM1 ainda e incerto. Nesse sentido, investigamos o papel dos receptores NOD1 e NOD2 na patogenese do DM1, com enfoque na diferenciacao de linfocitos Treg/Th17/Th1 e na plasticidade desses subtipos celulares. Nossos resultados mostram que camundongos deficientes de NOD2, mas nao NOD1 ou RIP2, sao resistentes ao DM1, como comprovado por menor incidencia, hiperglicemia, diminuicao do infiltrado inflamatorio e normalizacao dos niveis de insulina quando comparado aos controles. Foi observado tambem que animais NOD2-/- tiveram uma reducao da populacao de linfocitos Th17, Tc17, Th1 e T citotoxicos nos linfonodos pancreaticos, o que correlaciona com a inibicao da producao de IL-23p19 e IFN- no pancreas. Em paralelo, foi evidenciado o aumento do numero de celulas T reguladoras, macrofagos do perfil M2 nos linfonodos pancreaticos e elevada producao de IL-10 no pancreas de animais NOD2-/-. Alem disso, foi observado que animais NOD2-/- apresentaram uma menor populacao de linfocitos T duplo-positivos (Foxp3+RORt+ e IL-17+IFN+). Posteriormente, foi detectado menor producao de IL- 1, IL-6, IL-23p19 e IL-12p40 por celulas dendriticas de animais deficientes de NOD2. De forma interessante, foi observada a translocacao de bacterias para os linfonodos pancreaticos de animais diabeticos. Adicionalmente, animais tratados com antibioticos tornaram-se resistentes ao DM1, o que nos fornece indicios da contribuicao da microbiota intestinal na inducao da doenca. Por fim, comprovamos alta expressao genica de NOD2 nos linfonodos pancreaticos e no pancreas na fase inicial (pre-diabetica) em outro modelo de DM1, utilizando camundongos NOD (nonobese diabetic mice). Portanto, nossos dados indicam que a ativacao do receptor NOD2 por componentes bacterianos da microbiota intestinal induz a producao de citocinas pro-inflamatorias com subsequente diferenciacao/conversao de linfocitos do perfil Th17/Th1 e progressao do DM1. Dessa forma, estes dados apontam o bloqueio do receptor NOD2 como uma potencial terapia imunomoduladora para o DM1 em humanos. / Type 1 diabetes is an autoimmune disease that precipitates due to defects in the self tolerance to auto- antigens, resulting in the autoimmune destruction of the pancreatic cells in genetically susceptible individuals. NOD-like (NLRs) receptors are intracellular receptors responsible for the recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Recent studies have shown a role of NOD1 and NOD2 receptors in the innate immune response against pathogens and in the adaptive immune response, since its activation leads to the generation of cytokines related to the differentiation of IL-17-producing T helper cells (Th17). However, the role of these receptors in T1D remains elusive. Therefore, we investigated the role of NOD1 and NOD2 receptors in the pathogenesis of T1D, focusing on the differentiation of Treg/Th1/Th17 lymphocytes and in the plasticity of these subtypes. Our data demonstrate that NOD2-/- mice, but not NOD1-/- or RIP2-/-, are resistant to T1D, as shown by the lower incidence, hyperglycemia, less insulitis and normal insulin production when compared to wild type mice. It was also observed that NOD2-/- mice have a reduction in the Th17, Tc17, Th1 and cytotoxic T lymphocyte population within the pancreatic lymph nodes (PLNs), which correlates with the inhibition of IL-23p19 and IFN production in the pancreas. In parallel, there was an increase in Treg cells, M2 macrophages in the PLNs and IL-10 production in the pancreatic tissue of NOD2-/- mice. Also, NOD2-/- mice presented a downregulation of Foxp3+RORt+ and IL-17+IFN+ double-positive T cells. Later, it was shown that IL-1, IL-6, IL-23p19 and IL-12p40 production was downregulated in mice deficient to the NOD2 receptor. Interestingly, we observed a bacterial translocation to the pancreatic lymph nodes in diabetic mice, what could be triggering NOD2 activation, thus contributing to T1D development. As expected, mice pre-treated with antibiotics failed to become diabetic, suggesting a possible role of the gut microbiota in the development of the disease. Lastly, we observed a higher relative expression of NOD2 in the PLNs and pancreas of pre-diabetic mice, using another mouse model of the disease, the nonobese diabetic (NOD) mouse. Collectively, our data suggest that components from the gut microbiota are capable of translocating to the PLNs, thus triggering the activation of NOD2, which in turn induces the production of proinflammatory cytokines related to the differentiation of Th1/Th17 cells, thus contributing to T1D development in a mouse model of the disease. Therefore, the blockade of NOD2 appears as an interesting therapeutical target in the treatment of type 1 diabetes in humans.

Atherosclerosis is a major underlying cause of cardiovascular disease; however, the molecular mechanisms by which cardiovascular risk factors promote the development of atherosclerosis are poorly understood. Recent evidence from our laboratory suggests that endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)-3α/β is involved in the activation of pro-atherosclerotic processes. Previous studies from our lab show that myeloid-specific deletion of GSK3α attenuates the progression of atherosclerosis. However, the precise role(s) of GSK3α/β in atherosclerotic regression is not known. The primary goal of this thesis is to investigate the role(s) of GSK3α/β in lesional macrophages and atherosclerotic regression. Initially, we have targeted the ER stress- GSK3α/β pathway by supplementing the drinking water of low-density lipoprotein receptor (Ldlr)-/- mice with the small molecules 4-phenylbutyric acid or valproate. The results suggest that ER stress or GSK3α/β inhibition can attenuate the growth of existing atherosclerotic lesions and appear to increase lesion stability. From this study it remains unclear whether these interventions can promote atherosclerotic regression. Next, to investigate the role(s) of GSK3α/β in pro-atherogenic processes, bone marrow derived macrophages were isolated from myeloid-specific GSK3α- and/or GSK3β-deficient mice. The effects of GSK3α/β-deficiency on signaling pathways regulating atherogenic functions in macrophage were analyzed. This study revealed that GSK3α and GSK3β play distinct, and often opposing roles in macrophage polarization, inflammatory response, lipid accumulation and migration. Furthermore, both GSK3α and GSK3β appear to play redundant roles macrophage viability, proliferation, and metabolism. Lastly, we investigated the effect of macrophage-specific deletion of GSK3α and/or GSK3β on atherosclerotic regression in Ldlr−/− mice. A novel inducible knock out mouse model has been created in which GSK3α and/or GSK3β expression can be ablated by treating the mice with tamoxifen. These mice were fed a high fat diet to promote the development of atherosclerosis, and then mice were treated with tamoxifen to induce GSK3α/β deletion and switched to a chow diet for 12 weeks. All mice were sacrificed at 33 weeks of age and atherosclerotic plaques were analysed. Female mice with induced macrophage-specific GSK3α deficiency, but not GSK3β deficiency, showed regression of existing atherosclerotic lesions. Together, these studies begin to delineate the specific roles of GSK3α and GSK3β in atherosclerotic regression. Furthermore, these data suggest that GSK3α inhibition could be an effective strategy for the treatment of atherosclerotic cardiovascular disease. / Thesis / Doctor of Philosophy (PhD) / Atherosclerosis is a disease involving the build-up of fatty plaques in the arteries, making them hard and narrow, which leads to damage in the heart, coronary or peripheral blood vessels. This can cause acute cardiovascular complications (heart attacks or stroke) and potentially death. We suspect that protein named glycogen synthase kinase (GSK)-3α/β is involved in the development of atherosclerosis. The purpose of this research is to see if we can treat atherosclerosis by blocking GSK3α/β’s functions. The findings of this study demonstrate that blocking GSK3α reduces inflammation, which is a primary cause of atherosclerosis. Furthermore, blocking GSK3α promotes the regression of atherosclerotic plaques and may lower the risk of cardiovascular disease. This knowledge could aid in the development of medications to treat atherosclerosis and reduce the number of individuals who die from heart attacks or strokes.

Fibroproliferative disorders are the leading cause of morbidity and mortality worldwide, with one specific group of fibroproliferative disorders being interstitial lung diseases (ILD). Idiopathic pulmonary fibrosis is the most common ILD; however its pathogenesis is not entirely understood. What is known is that there is repetitive cellular injury preceding the fibrotic remodeling in the lungs that contributes to the irreversible deposition of extracellular matrix (ECM) proteins. Myofibroblasts that accumulate at the site of injury are thought to be the key drivers of ECM deposition and are often associated in the disease. Although it is poorly understood how these immune cells differentiate in the lung, one hypothesis suggests the role of alternatively activated profibrotic macrophages in this process. The data presented in this thesis suggest that there are a presence of UPR and macrophage proteins in the lungs of IPF patients and the UPR may be necessary in the polarization of alternatively activated macrophages. / Thesis / Master of Science (MSc)

A histiocitose de células de Langerhans (HCL) é uma neoplasia mieloide inflamatória comumente afetando pacientes pediátricos e apresenta frequentemente mutações ativadoras somáticas em genes da via MAPK, incluindo BRAF e MAP2K1. Vários estudos sugerem que as células lesionais da HCL podem recrutar e modular células inflamatórias e cujas citocinas parecem fornecer sinais recíprocos de sobrevivência celular. Para o presente estudo foram selecionados 15 casos de HCL (10 crianças, 5 adultos), sendo as amostras de tecido avaliadas através de imunoistoquímica utilizando marcadores para macrófagos (CD68 e CD163), células dendríticas maduras (CDm) (CD83 e CD208), linfócitos T regulatórios (LTregs) (CD4, CD25 e FOXP3) e linfócitos citotóxicos (LCs) (CD56, CD57, perforina e granzima B). Além disso, marcadores de células B (CD20), células T (CD3, CD8) e confirmatórios de HCL foram analisados. Todos os casos de HCL foram positivos para S100, CD1a, CD207 e CD4; enquanto que Bcl-2 e Ciclina D1 foram positivos em 13/15 (86,7%) casos. No microambiente imune intralesional, macrófagos M2 (CD68+/CD163+), seguidos por LTregs, foram as populações celulares mais predominantes. Em quantidade significativamente menor, foram observadas CDm, seguidas por escassos LCs. Considerando a população linfoide, linfócitos T CD3+ foram mais numerosos do que linfócitos B CD20+. Dentro dos linfócitos T, linfócitos T CD4+ foram mais numerosos do que linfócitos T CD8+ (p<0,05). Nossos resultados sugerem que a infiltração de células imunes na HCL, provavelmente através de mecanismos pró-tumorais, inflamatórios e/ou imunossupressores mediados por citocinas, pode promover o desenvolvimento e sobrevivência das células lesionais da HCL, fornecendo uma justificativa para a combinação de imunoterapia e terapia gênica (BRAF) na HCL / Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia often affecting children with constitutively somatic activating mutations in MAPK pathway genes including BRAF and MAP2K1. Several studies suggest that LCH cells can recruit and modulate inflammatory cells and whose cytokines appear provide reciprocal survival signals. For the present study, 15 cases of LCH (10 children, 5 adults) were selected, and the tissue samples were evaluated through immunohistochemistry using markers for macrophages (CD68 and CD163), mature dendritic cells (mDC) (CD83 and CD208), regulatory T-cells (Tregs) (CD4, CD25 and FOXP3) and cytotoxic lymphocytes (CLs) (CD56, CD57, perforin and granzime B). Moreover, B-cell (CD20), T-cell (CD3, CD8) and LCH markers were analyzed. All LCH cases were positive for S100, CD1a, CD207 and CD4, while Bcl-2 and Cyclin D1 were positive in 13/15 cases (86.7%). In the immune microenvironment, M2-polarized macrophages (CD68+/CD163+), followed by LTregs, were the predominant cell populations. In a significantly lower amount, mDC were observed, followed by scarce CLs. Moreover, CD3+ Tcells than CD20+ B-cells were more numerous (p>0.05), the former presenting a higher number of CD4+ than CD8+ T-cells (p<0.05). Our results suggest that immune cell infiltration in LCH, probably through cytokine-mediated pro-tumoral, inflammatory and/or immunosupressive mechanisms, can promote LCH cell development and survival, providing a rationale for combining immunotherapy and BRAF-targeted therapy in LCH

O carcinoma espinocelular oral (CECO) corresponde a aproximadamente 95% das neoplasias malignas que acometem a cavidade oral. Os fatores de risco clássicos incluem o tabagismo e etilismo; no entanto, as disfunções do sistema imune em decorrência do envelhecimento (imunossenescência) na patogênese do CECO são muito pouco estudados. Análises comparativas vêm sendo feitas para melhor caracterização do perfil de pacientes jovens e idosos acometidos pelo CECO, o qual permanece ainda controverso. Vários estudos têm mostrado que os macrófagos associados ao tumor (MATs) no CECO de pacientes idosos exibem um fenótipo M2 (pró-tumoral), com propriedades moduladoras do estroma vascular, promovendo a angiogênese. No entanto, considerando a imunossenescência, não se sabe o perfil de MATs e seus efeitos na angiogenese no CECO afetando pacientes jovens. Assim, este estudo analisou por meio da técnica imunoistoquímica, a frequência e localização de MATs em correlação com a angiogênese, no CECO, afetando pacientes jovens e idosos. Cinquenta e sete biópsias de CECO divididos em 3 grupos (I: <40 anos [n=17]; II: 40-65 anos [n=20]; III: >65 anos [n=20]) foram selecionados para compor o estudo, sendo classificados morfologicamente seguindo às recomendações da OMS (2017). Os grupos I, II e III foram comparados quanto à imunoexpressão de CD68 e CD163 para análise de MATs e de CD34 (vasos sanguíneos) e D2-40 (vasos linfáticos) para avaliação da densidade microvascular (DMV), área microvascular (AMV) e área vascular total (AVT). A análise imunoistoquímica evidenciou similar expressão de CD68 e CD163 nos três grupos (p>0,05). A avaliação da DMV, AMV e AVT sanguínea e linfática também exibiu padrões similares, com predominância significativa (p<0.05) de vasos sanguíneos, nos três grupos analisados. Não houve correlação significativa quando avaliados marcadores macrofágicos e angiogênicos. Nossos resultados mostram um similar perfil de MATs e angiogênese quando comparados os três grupos estudados, sugerindo participação de mecanismos moleculares do microambiente tumoral na manutenção da predominância de macrófagos M2 e vasos sanguíneos no CECO afetando pacientes jovens e idosos / Oral squamous cell carcinoma (OSCC) corresponds to approximately 95% of all cases of oral cavity malignancies. The classic risk factors include smoking and alcoholism; however, immune system dysfunctions due to aging (immunoscencence) in OSCC pathogenesis are poorly studied. Comparative analyzes have been made to better characterize the profile of young and old patients affected by the OSCC, which remains controversial. Several studies have shown that tumor-associated macrophages (TAMs) in OSCC of elderly patients exhibit a pro-tumoral M2 phenotype, with vascular stroma modulating properties, promoting angiogenesis. However, considering immunoscencence, the profile of TAMs and their effects on angiogenesis in OSCC affecting young patients is unknown. Thus, this study analyzed by immunohistochemical technique, the frequency and location of TAMs in correlation with angiogenesis in OSCC affecting young and elderly patients. Fiftyseven biopsies were divided into three groups (I: <40 years [n=17]; II: 40-65 years [n=20]; III: > 65 years [n=20]) and selected to compose this study, being classified morphologically following WHO (2017) recommendations. Groups I, II and III were compared for immunoexpression of CD68 and CD163 for analysis of TAMs, and CD34 (blood vessels) and D2-40 (lymphatic vessels) for evaluation of microvessel density (MVD), microvascular area (MVA) and total vascular area (TVA). Immunohistochemical analysis showed similar expression of CD68 and CD163 in the three groups (p>0.05). The evaluation of blood and lymphatic MVD, MVA and TVA also showed similar patterns, with a significant predominance (p<0.05) of blood vessels in the three groups analyzed. There was no significant correlation when evaluating macrophage and angiogenic markers. Our results show a similar profile of TAMs and angiogenesis when compared to the three groups studied, suggesting participation of molecular mechanisms of the tumor microenvironment in the maintenance of M2-polarized macrophages and blood vessels in OSCC affecting young and elderly patients