Em países tropicais como o Brasil, as micoses superficiais (dermatofitoses) são comumente encontradas. O Dermatófito mais comum é o Trichophyton rubrum (Tr). Mananas e galactomananas na parede do Tr podem suprimir a resposta celular ao fungo. Quanto à resposta imune antifúngica, sabe-se a importância da via Th17. Algumas lectinas do tipo C (CLRs) como o receptor de manose e/ou receptores similares a Toll (TLRs) regulam o equilíbrio entre as vias Th1 e Th17. Nossos objetivos foram obter um extrato antigênico de Tr que induza resposta imune celular; quantificar e qualificar a resposta imune de indivíduos controles com lesão branda e de pacientes com dermatofitose extensa e/ou persistente causadas pelo Tr e por fim, avaliar a expressão de CLRs em monócitos do sangue periférico nos mesmos grupos. Para tanto, produzimos 11 extratos antigênicos de Tr. Pudemos observar na eletroforese em gel de poliacrilamida proteínas com pesos moleculares de aproximadamente 70 kDa e 38 kDa para os extratos fúngicos: Extrato TCA - Meta 1, Extrato tindalizado G1 e Extrato Coca 1. Avaliamos a resposta linfoproliferativa de células mononucleares por incorporação de timidina triciada em controles e pacientes ao peptídeo YIIDTGIDID do fungo Tr (Tri R2) e aos extratos antigênicos produzidos em nosso laboratório. Utilizamos como estímulos: PWM, CMA, Tri R2, PMA/Ionomicina. Para os ensaios funcionais avaliamos quatro pacientes e 6 indivíduos controles. Para a fenotipagem das células Th17, Th17MEM, Tc17 e Tc17MEM por citometria de fluxo, utilizamos a análise Booleana no software FlowJo X. A avaliação da expressão dos CLRs: CD206 (Receptor de Manose), Dectin 1 e Dectin 2 em monócitos do sangue periférico de controles e pacientes foi efetuada por citometria de fluxo. Dos 11 extratos produzidos de Tr, 7 se mostraram bons estimuladores para pelo menos um dos controles analisados, expressos como ponto máximo de índice de estimulação (p.m.I.E.). Dentre eles destacamos: Extrato TCA - Meta 1 (p.m. I.E. = 14,49 em 5 ug/mL), Extrato Tindalizado G1 (p.m.I.E. = 23,00 em 2,5 ug/mL) e Extrato Coca 1 (p.m.I.E. = 173,36 em 0,31 ug/mL). Na avaliação da expressão dos receptores das células Th17 e Tc17 (Th17R e Tc17R, respectivamente) após seis dias de estímulo por: Tri R2, extrato Coca 1 e o extrato TCA - Meta 1, o extrato Coca se mostrou o melhor estimulador para as populações Th17, com a frequência de 8,40% (controle) e 12,30% (paciente 1). Na avaliação da expressão de Th17R e Tc17R por 6 horas ao estímulo por PMA/Iono, todos os controles (n=3) se mostraram responsivos e no grupo de pacientes (n=3) pudemos observar maior frequência para o paciente 1 nas populações Th17 (1,64%) e Th17MEM (3,27%), e para as células Tc17 (10,70%) e Tc17MEM (3,58%). Observamos redução da expressão de CLRs nos pacientes: CD206: média 60,24% (controles) e 21,27% (pacientes), Dectin 1: 22,42% (controles) e 12,06% (pacientes) e Dectin 2: 20,26% (controles) e 4,99% (pacientes). Controles (n=6) e pacientes (n=3). A inovação na produção de extrato antigênico Extrato TCA - Meta 1 encoraja o estudo dos extratos fúngicos, para se obter melhores condições de avaliações imunológicas em pacientes com dermatofitose. Caracterizamos e qualificamos a resposta imune celular frente ao peptídeo TriR2 e aos extratos antigênicos, além de avaliarmos a expressão dos CLRs nesse grupo especial de pacientes / In tropical countries like Brazil, superficial fungal infections (dermatophytosis) are commonly found. The most common dermatophyte is Trichophyton rubrum (Tr). Mannans and galactomannans of Tr cell wall can suppress cellular responses to the fungus. Regarding the antifungal immune response, the importance of Th17 pathway is warranted. Some C-type lectins (CLRs) as the mannose receptor and / or Toll-like receptors (TLRs) regulate the balance between Th1 and Th17 pathways. Our objectives were to obtain an antigenic extract of Tr to induce cellular immune response; to quantify and classify the immune response of control subjects with mild injury and patients with extensive and / or persistent dermatophytosis caused by Tr, and finally evaluating the expression of CLRs in peripheral blood monocytes in the same groups. Therefore, we produced 11 antigenic extracts of Tr. Proteins with molecular weights of approximately 70 kDa and 38 kDa were evidenced in polyacrylamide gel electrophoresis for the following fungal extracts: extract TCA - Target 1, Tindalized extract G1 and extract Coca 1. We assessed the lymphoproliferative response of mononuclear cells by tritiated thymidine incorporation in the controls and patients, stimulated by YIIDTGIDID peptide fungus Tr (Tri R2) and the antigenic extracts produced in our laboratory. We used as stimuli: PWM, CMA, Tri R2, and PMA/Iono. For functional assays we evaluated four patients and 6 control individuals. For the phenotyping of Th17 cells, Th17MEM, Tc17 and Tc17MEM by flow cytometry, we used a Boolean analysis performed by FlowJo X software. Evaluation of the expression of CLRs: CD206 (mannose receptor), Dectin 1 and Dectin 2 in peripheral blood monocytes from patients and controls was performed by flow cytometry. Of the 11 extracts produced from Tr, seven proved to be able to stimulate proliferation of peripheral blood mononuclear cells of at least one of the analyzed controls, expressed as peak stimulation index (p.m.I.E.). Among them, were included: extract TCA - Target 1 (pmIE = 14.49 at 5 ug /mL), Tindalized G1 Extract (pmIE = 23,00 at 2.5 ug /mL) and extract Coca 1 (pmIE = 173.36 at 0.31 ug /mL). In the evaluation of the expression of receptors of Th17 cells and Tc17 (Th17R and Tc17R, respectively) after six days of stimulation by: Tri R2, Coca extract and the extract TCA 1 - Meta 1, Coca extract showed to be the best stimulator for Th17 populations, with the frequency of 8.40% (control) and 12.30% (patient 1). In the evaluation of the expression of Th17R and Tc17R after 6 hours of stimulation by PMA / Iono, all controls (n = 3) responded and in the group of patients (n = 3) we observed response more frequently for the patient #1, in Th17 populations (1.64%), Th17MEM (3.27%), Tc17 cells (10.70%) and Tc17MEM (3.58%). We observed a reduction of expression of CLRs in patients: CD206: average 60.24% (controls) and 21.27% (patients), Dectin 1: 22.42% (controls) and 12.06% (patients) and Dectin2: 26% (controls) and 4.99% (patients). Controls (n = 6) and patients (n = 3). Innovation in the production of antigenic extract extract TCA - Target 1 encourages the study of fungal extracts to obtain better conditions of evaluation of the immune response in patients with dermatophytosis. We characterized and qualified the cellular immune response to the TriR2 peptide, to antigen extracts, and evaluated the expression of CLRs in this special group of patients

Le Lupus Erythémateux Systémique est une pathologie inflammatoire chronique. L’étiologie de cette maladie auto-immune est encore méconnue bien que certains facteurs génétiques et environnementaux aggravants aient été mis en évidence. Les traitements proposés aux patients ont pour but de réduire les symptômes et aucun remède curatif n’a encore été mis au point. Nous avons observé de forts taux de sCD95L dans le sérum de patients atteints de LES comparé à celui de sujets sains. Nos données indiquent que ce facteur soluble agit comme une cytokine pro-inflammatoire et promeut la transmigration des lymphocytes T CD4+ Th17 dans les organes, au détriment des lymphocytes T régulateurs (Treg). L’accumulation de ces cellules Th17 est responsable du maintien d’une réponse inflammatoire chronique chez les patients lupiques. Nous mettons en évidence qu’il existe une interaction directe entre le récepteur CD95 et la Phospholipase Cγ1, par l’identification du Calcium Inducing Domain impliqué dans ce recrutement. L’identification du couple CD95/CD95L comme acteur aggravant le LES et la mise en évidences des mécanismes cellulaires et moléculaires sous-jacents nous ont conduit à l’élaboration de stratégies thérapeutiques innovantes. En collaboration avec les chimistes et modélisateurs de notre Unité, nous avons généré une petite librairie d’inhibiteurs, composée de peptides et peptidomimétiques sélectifs. Parmi ces composés, le TAT-CID est une protéine piège comprenant la zone d’interaction de CD95 (domaine CID des aa 175 à 210) à la PLCγ1. L’injection de ce peptide dans un modèle de souris lupiques restaure la fonction biologique rénale de ces souris et diminue la production d’auto-anticorps (anti-DNA) et de complexes immuns, marqueurs biologiques associés à la progression de la pathologie. En parallèle, le criblage d’une librairie chimique commerciale constituée de médicaments approuvés par la FDA et l’EMA a permis d’identifier un inhibiteur efficace de notre interaction. In vivo, ce composé est capable de réduire drastiquement les signes cliniques de la pathologie lupique. Ces nouvelles données enrichissent notre compréhension du processus mis en place par le système CD95/CD95L dans l’aggravation du LES, et nous permettent de proposer des outils thérapeutiques interessants. Ces molécules pourraient représenter de nouvelles options thérapeutiques originales et attrayantes pour prévenir l’inflammation dans les pathologies inflammatoires chroniques. / Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disease. The etiology of this autoimmune disease is still unknown although some aggravating genetic and environmental factors have been identified. The treatments used for patients are intended to reduce the symptoms and no curative one has been developed yet. We observed high levels of sCD95L in the serum of patients with SLE compared to healthy subjects. Our data indicate that this soluble factor acts as a pro-inflammatory cytokine and promotes the transmigration of CD4 + Th17 T lymphocytes to the detriment of regulatory T lymphocytes (Treg) in the enflammed organs of patients. The accumulation of these Th17 cells is responsible for maintaining a chronic inflammatory response in lupus patients. We show that there is a direct interaction between the CD95 receptor and the phospholipase Cγ1, by the identification of the Calcium Inducing Domain involved in this recruitment. The identification of the CD95/CD95L couple as an aggravating factor in SLE context and the underlying cellular and molecular mechanisms led us to the development of innovative therapeutic strategies. In collaboration with the chemists and modellers of our research Unit, we have generated a small library of inhibitors, composed of selective peptides and peptidomimetics. Among these compounds, TAT-CID is a decoy peptide comprising the interaction zone of CD95 (CID domain 175 to 210 aa) with PLCγ1. Repeated treatments of lupus-prone mice with this peptide restore the biological function of these mice and decrease the production of autoantibodies (anti-DNA) and immune complexes, biological markers associated with the progression of the pathology. In parallel, the screening of a commercial chemical library consisting of FDA and EMA-approved drugs allowed us to identify an effective inhibitor of our targeted interaction. In vivo, this compound is able to drastically reduce the clinical signs of lupus pathology. These new data enrich our understanding of the process implemented by the CD95/CD95L system in the aggravation of SLE, and allow us to propose interesting therapeutic tools. These molecules could represent novel and attractive therapeutic options for preventing inflammation in chronic inflammatory pathologies.

Toxoplasma gondii est un parasite intracellulaire obligatoire qui infecte plus du tiers de la population mondiale. La toxoplasmose oculaire (TO), qu’elle soit d’origine acquise ou congénitale, est une infection fréquente et redoutable où le pronostic visuel est fortement engagé. La présence du parasite dans le tissu rétinien, ainsi que la réponse immunitaire de l’hôte, qu’il génère participent au développement des lésions. Cependant, les mécanismes physiopathologiques aboutissant à la destruction de la rétine et ceux conduisant à la latence et à la réactivation parasitaires ne sont toujours pas clairement identifiés. De plus, des améliorations des traitements actuels semblent nécessaires afin de traiter au mieux l’ensemble des patients et d’agir également sur la forme latente du parasite et les récidives qui en découlent. Nos travaux sur des modèles murins de la phase aiguë de la TO démontrent que le facteur de virulence parasitaire ROP16 est responsable de l’augmentation de la charge parasitaire et des réponses inflammatoires oculaires Th1 et Th17. De plus, lorsque cette protéine se trouve dans un génotype parasitaire différent de celui d’origine, la pathologie oculaire en est exacerbée. Nous avons également démontré que la cytokine IL-23 participe à la destruction de la rétine et à la multiplication parasitaire. Notre modèle murin de réactivation de la TO met en évidence le rôle protecteur de la voie Th1 à travers l’IFN-γ et celui fortement délétère de la cytokine IL-6. La neutralisation de cette cytokine dans notre modèle diminue la charge parasitaire oculaire ainsi que l’ensemble de la réponse inflammatoire et n’altère en rien la structure rétinienne. Il serait donc très intéressant d’utiliser une thérapie ciblant le récepteur de l’IL-6 afin de contrecarrer les effets des récurrences de la TO. / The obligate intracellular parasite Toxoplasma gondii infects more than one third of the world population. Ocular toxoplasmosis (OT), whether acquired or congenital, is a common and frightening infection which can strongly impair the visual function. OT is considered as a major cause of posterior uveitis and retinochoroiditis is its most common manifestation. The presence of the parasite in the retina as well as the immune response it generates in the host are two factors involved in the development of ocular lesions. However, up to now, the pathophysiological mechanisms leading to the destruction of the retina and those implied in the parasite latency and reactivation have not been clearly identified. Morever, improvements of current treatments are necessary in order to give all patients a better care but also to fight the parasite latent form and relapses they may cause. Our mouse models of OT acute phase have demonstrated that the parasite virulence factor ROP16 is responsible for the parasite load increase and the Th1 and Th17 ocular inflammatory responses. When this protein is in a different genotype than its original one – this has been made possible by recombining strains – the ocular pathology is more severe. We also showed that the IL-23 cytokine was involved in the destruction of the retina and in parasite multiplication. Our mouse model of OT reactivation highlighted the protective role of the Th1 pathway through IFN-γ as well as the highly deleterious IL-6 cytokine. By neutralizing this cytokine in our model, we reduced the ocular parasite load and the global inflammatory response without modifying the retinal structure. It would be very interesting to elaborate a therapy targeting the IL-6 receptor in order to counter the effects of OT recurrences.

Introdução: A sepse é causada por uma resposta desregulada a uma infecção cujo tratamento é de suporte, inexistindo alternativas imunomoduladoras. Linfócitos T reguladores são responsáveis por limitar a inflamação, mas podem causar imunossupressão e os Th17 são pró-inflamatórios e responsáveis pela imunidade de mucosas; ambos apresentam-se elevados nos pacientes com sepse. A PARP é uma enzima sensor de dano ao DNA que é continuamente ativada na sepse, sendo importante também na diferenciação¸ dos linfócitos T reguladores e como coativador de NF-kB. Neste estudo, avaliamos se o tratamento com inibidor de PARP é capaz de manter os linfócitos Th17 e T reguladores próximos aos valores basais, impedindo que ocorra a resposta exacerbada causada por estas células e servindo, portanto, como opção de tratamento imunomodulador. Métodos: Camundongos machos da linhagem C57Bl/6 com 7 semanas de idade e pesando entre 20-25 gramas foram submetidos à ligadura e punção cecal e receberam tratamento com olaparibe (10mg/Kg) após 30 minutos e após 8 horas da cirurgia. Baço, timo e sangue foram coletados e utilizados para análise das populações de linfócitos T reguladores e Th17, citocinas e miRNAs. Resultados: O modelo de ligadura e punção cecal foi capaz de mimetizar a linfopenia encontrada em pacientes e o aumento de linfócitos T reguladores e Th17. O tratamento com olaparibe reduziu os linfócitos T reguladores no baço tanto em porcentagem quanto em quantidade de células. Tanto o nível de IL-10 quanto a expressão do miRNA 146a-5p caíram em ambos os grupos CLP, sugerindo menor atividade supressora destes linfócitos. No sangue houve aumento dos linfócitos T reguladores, mas apenas o grupo não tratado apresentou alta de IL-10, sugerindo que o tratamento conteve o perfil supressor. No timo o tratamento parece agir por uma forma diferente; embora ocorra aumento dos linfócitos T reguladores, o grupo tratado teve aumento da expressão do miRNA 17a-5p, que reduz a atividade supressora desses linfócitos, mostrando que as células produzidas tem sua atividade supressora alterada, o que é corroborado pelo não aumento de IL-10 nesse grupo. Os linfócitos Th17, que são pró-inflamatórios, foram controlados com o tratamento no baço e no sangue. Isso possivelmente ocorreu pela ação da PARP que impediu o aumento de citocinas como IL-1beta, IL-6, TNF-alfa, IL-17A, INF-y, que estavam elevadas apenas no grupo não tratado. Além disso, a relação entre linfócitos Th17 e T reguladores foi controlada, sugerindo melhora no desfecho clínico. Conclusões: O tratamento com olaparibe se mostrou eficiente em reduzir as respostas inflamatória (causada pelo Th17) e supressora (causada pelo Treg) neste modelo, talvez pela alteração de citocinas e da expressão dos miRNA 17a-5p e 146a-5p / Introduction: Sepsis is caused by a dysregulated response to an infection whose treatment is supportive, and there are no immunomodulatory alternatives. Regulatory T lymphocytes are responsible for limiting inflammation but may cause immunosuppression and Th17 are proinflammatory and responsible for mucosal immunity; both are elevated in patients with sepsis. PARP is a DNAdamaging enzyme that is continuously activated in sepsis, also important in the di_erentiation of regulatory T lymphocytes and as a cofactor of NF-kB. In this study, we evaluated whether treatment with PARP inhibitor is able to keep the T regulatory and Th17 lymphocytes close to the baseline values, preventing the exacerbated response caused by these cells and therefore serving as an option for immunomodulatory treatment. Methods: C57Bl male mice at 7 weeks of age weighing between 20-25 grams were submited at cecal binding and puncture and received treatment with olaparib (10mg/kg) after 30 minutes and after 8 hours of the surgery. Spleen, thymus and blood cells have been used for analysis of T regulatory and Th17 lymphocytes populations, cytokines and miRNA. Results: The cecal ligation and puncture model was able to mimic the lymphopenia found in patients and the increase of T regulatory and Th17 lymphocytes. Treatment with olaparib reduced the T regulatory lymphocytes in the spleen in both percentage and number of cells. Both the IL-10 level and the 146a-5p miRNA expression fell in both CLP groups, suggesting lower suppressor activity of these lymphocytes. In the blood there was an increase in the T regulatory lymphocytes, but only the untreated group showed high IL-10, suggesting that the treatment contained the suppressor profile. In the thymus the treatment seems to act in a di_erent way; although there is an increase in the T regulatory lymphocytes, the treated group had increased expression of 17a-5p miRNA, which reduces the suppressive activity of these lymphocytes, showing that the cells produced have their supressor activity altered, which is corroborated by the non-increase of IL- 10 in this group. Th17 lymphocytes, which are proinflammatory, were controlled with treatment in the spleen and blood. This was possibly due to the action of PARP which prevented the increase of cytokines such as IL-1beta, IL-6, TNF-alpha, IL-17A, INF-y which were raised only in the group not treated. In addition, the ratio between Th17 and T regulatory lymphocytes was controlled, suggesting improvement in clinical outcome. Conclusions: The treatment with olaparib was e_cient in reducing inflammatory responses (caused by Th17) and suppressor (caused by Treg) in this model, perhaps due to the alteration of cytokines and the expression of miRNAs 17a-5p and 146a-5p

Neste trabalho foi estudado o potencial imunorregulador da crotoxina (CTX) obtida do veneno de C. d. terrificus, em modelo experimental de colite induzida pelo TNBS em camundongos. A CTX foi capaz de diminuir a perda de peso, o score clínico e histológico, síntese de MPO e citocinas pró-inflamatórias. Menor número de neutrófilos e macrófagos com fenótipos M1 e M2 na lâmina própria foi observado nos grupos TNBS/CTX em relação ao TNBS. A CTX induziu TGF-b e IL-10, PGE2 e LXA4. A neutralização in vivo dessas citocinas ou o bloqueio da síntese desses eicosanoides indica que estas moléculas exercem papel relevante na ação moduladora da CTX no quadro inflamatório. As análises das diferentes populações celulares da lâmina própria, linfonodos e Placas de Peyer mostraram que não houve diferença nos linfócitos CD4+Tbet+ entre os grupos TNBS e TNBS/CTX. No entanto, a CTX promoveu aumento de CD4+FoxP3+ e diminuição de CD4+RORg+. Estes resultados indicam que a CTX é capaz de modular a resposta inflamatória aguda intestinal melhorando o quadro clinico observado nos animais. / In this work it was analyzed the immunomodulatory effect of crotoxin (CTX) isolated from C.d. terrificus snake venom, on the experimental model of colitis induced by TNBS in mice. The CTX was able to inhibit the weight loss, clinical and histological score, MPO synthesis and pro-inflammatory cytokines. Lower number of neutrophils and macrophage (M1 and M2) in lamina propria was observed in TNBS/CTX mice compared with the TNBS group. In contrast, the CTX induced increased TGF-b, IL-10, PGE2 and LXA4. The in vivo neutralization of these cytokines or eicosanoids synthesis indicates that these molecules exert significant role in the modulatory effect of CTX. The analyzes of distinct cell populations from lamina propria, lymph nodes and Peyers pathes showed no difference in CD4+Tbet+ between TNBS or TNBS/CTX mice. However, CTX induced an increase of CD4+FoxP3+ and decreased CD4+RORgt+. Together, these results indicate that CTX is able to modulate intestinal acute inflammatory response induced by TNBS improving the clinical status of the mice.

O trato gastrointestinal é um sítio de alta exposição antigênica, por isso requer a presença de mecanismos de regulação imunológica mediada por linfócitos T reguladores e T auxiliares produtores de IL-17 (Th17) na mucosa intestinal. Se houvera falha na indução desses mecanismos, pode ocorrer o desequilíbrio das populações de bactérias comensais da microbiota intestinal, denominado de disbiose, geralmente associado à ruptura da barreira intestinal e translocação de bactérias ou LPS para o sangue. Neste sentido, alguns estudos têm evidenciado a importância dos linfócitos Th17 no intestino, já que estas células tem a capacidade de manter a integridade da barreira intestinal e, como conseqüência controlar a colonização e translocação bacteriana. Em adição, em pacientes e animais diabéticos têm sido observada a correlação de altos níveis de LPS circulantes e resistência à insulina. Baseado nessas evidências, nosso objetivo foi avaliar o papel da citocina IL-17 no controle das alterações inflamatórias e metabólicas no modelo de diabetes do tipo 2 (DM2). Para isso, foram utilizados camundongos C57BL/6 selvagens (WT) ou deficientes do receptor da citocina IL-17 (IL-17R-/-) submetidos à dieta controle (DN), composta por 10% de gorduras, 70% de carboidratos e 20% de proteínas ou à dieta hiperlipídica (DH), composta por 60% de gorduras, 20% de carboidratos e 20% de proteínas. Nossos dados demonstraram que a deficiência do receptor de IL-17 protegeu os animais contra a obesidade, mas os mesmos desenvolveram maior hiperglicemia e hiperinsulinemia decorrente da resistência à insulina. Além disso, foi verificada a hiperplasia das ilhotas pancreáticas, anormalidades na arquitetura e intenso infiltrado inflamatório no intestino (íleo) dos animais IL-17R-/- comparados aos WT após DH. Esse fato parece estar correlacionado a um defeito da migração de neutrófilos para a mucosa intestinal, uma vez que foi detectada reduzida expressão gênica da quimiocina CXCL-1 e do receptor CXCR-2 no íleo desses animais. De maneira interessante, as populações de neutrófilos (CD11b+Ly6G+) e de macrófagos anti-inflamatórios (CD11b+CX3CR1+) mostraram-se aumentadas nos linfonodos mesentéricos dos animais IL-17R-/- após DH. Em seguida, foi constatada maior translocação bacteriana no sangue tanto de animais IL-17R-/- submetidos à DN como DH. Entretanto, a análise metagenômica do gene 16S revelou a prevalência de bactérias Bacteroidetes e Proteobacterias, principais representantes de bactérias gram-negativas, somente nas fezes dos animais IL-17R-/- submetidos à DH. Em conjunto, estes dados indicam que o eixo IL-17/IL-17R é importante na manutenção da homeostase intestinal e na regulação das alterações inflamatórias e metabólicas associadas ao DM2 / The gastrointestinal tract is a high antigenic exposure site, so it requires the presence of immune regulation mechanisms mediated by regulatory T lymphocytes and IL-17- producing T helper lymphocytes (Th17) in the intestinal mucosa. If there is a failure in the induction of these mechanisms, may occur the imbalance in the populations of commensal bacteria of the intestinal microbiota, called dysbiosis, generally associated with the break of the intestinal barrier and translocation of bacteria or their products like LPS into the blood. In this regard, some studies have evidenced the importance of Th17 lymphocytes in the intestine, since these cells have the ability to maintain the integrity of the intestinal barrier, and consequently controlling the colonization and bacterial translocation. In addition, in patients and diabetic animals have been observed correlation between high circulating levels of LPS and insulin resistance. Based on this evidence, our objective was to evaluate the role of IL-17 cytokine in the control of inflammatory and metabolic changes in the type 2 diabetes (T2DM). For this reason, were used C57BL/6 wild-type mice (WT) or lacking of IL-17 cytokine receptor (IL-17R-/-) mice undergoing to the control diet (ND) comprising 10% fat, 70% carbohydrate and 20% protein or high fat diet (DH), comprising 60% fat, 20% carbohydrates and 20% protein. These data demonstrate that IL-17 receptor deficiency protected the animals against obesity, but these mice developed hyperglycemia and hyperinsulinemia due to insulin resistance. Furthermore, we verified a hyperplasia of the pancreatic islets, abnormalities in architecture and intense inflammation in the intestine (ileum) of IL-17R-/- animals undergoing DH compared to WT. This appears to be correlated to a defect in the neutrophil migration to the intestinal mucosa, since was detected reduced gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum of these animals. Interestingly, the populations of neutrophils (CD11b+Ly6G+) and antiinflammatory macrophages (CD11b+CX3CR1+) were shown to be increased in the mesenteric lymph nodes of IL-17R-/- animals after DH. Later, it was found more bacterial translocation in blood, both in IL-17R-/- mice with ND or DH. However, the metagenomic analyzes of the 16S gene revealed increased of Proteobacteria and Bacteroidetes phyla, the main representatives of gram-negative bacteria, only in the faeces of IL-17R-/- mice underwent DH. Together, these data indicate that IL-17/IL-17R axis is important in maintaining intestinal homeostasis and the regulation of inflammatory and metabolic alterations associated to T2DM

A cromoblastomicose é uma infecção fúngica crônica que acomete pele e tecido subcutâneo. As lesões podem ser classificadas em tumoral, verrucosa, cicatricial e do tipo placa. A resposta imune é principalmente celular e a forma grave da doença correlaciona-se com citocinas de perfil Th2. Nós exploramos populações celulares do tipo T reguladoras e Th17. Foram utilizadas vinte e três biópsias da forma verrucosa obtidas de pacientes com diagnóstico clínico e histopatológico de cromoblastomicose, sem tratamento. Foi realizado o método de imunohistoquímica para detectar Foxp3, CD25, TGF-beta, IL-6, IL-17 e IL-23. A IL-17 predominou sobre os outros marcadores, embora haja número regular de Foxp3. TGF-beta, IL-6 e IL-23 raramente foram visualizados. A constituição de uma resposta imune local com alta expressão de IL-17 e baixa expressão de outras citocinas pode ser, ao menos em parte, uma tentativa de ajudar o sistema imunológico contra infecções fúngicas. Células Foxp3 poderiam ser capazes de interferir na resposta imune eficiente contra fungos, mas também beneficiar o hospedeiro, através da capacidade de reduzir os danos do tecido que seguem uma resposta imune local. Esses elementos celulares podem contribuir para a cronicidade que caracteriza esta doença / Chromoblastomycosis is a chronic fungal infection that affects skin and subcutaneous tissue. Lesions can be classified in tumorous, verrucous, cicatricial and plaque type. The immune response is primarily cellular and the severe form of the disease correlates with a Th2 pattern of cytokines. We intended to explore the populations of regulatory T cells and the Th17 pattern. Twenty-three biopsies of verrucous form were obtained from patients with clinical and histopathological diagnostic of chromoblastomycosis, without treatment. It was performed an immunohistochemistry method to detect Foxp3, CD25, TGF-beta, IL-6, IL-17 and IL-23. IL-17 predominated over the other markers in chromoblastomycosis, although there was a regular number of Foxp3. TGF- beta, IL-6 and IL-23 were rarely visualized. The constitution of a local immune response with high expression of IL-17 and low expression of other cytokines could be at least in part, an attempt to help the immune system against fungal infection. Foxp3 cells could be able to interfere with the efficient immune response against fungi, but also benefit the host, through the ability to reduce the tissue damage that follows a local immune response. They could play a role in chronicity that characterizes this disease

INTRODUÇÃO: Atualmente as células da microglia têm recebido grande atenção dentro da resposta imune, isto devido ao fato de que sua ativação por citocinas inflamatórias é capaz de promover a infiltração e destruição do sistema nervoso central (SNC) durante algumas doenças, principalmente no caso da esclerose múltipla (EM). Além de seu papel pró-inflamatório, já demonstrou-se que estas também são capazes de expressar moléculas supressoras como a indoleamina-2,3-dioxigenase (IDO), capaz de suprimir a proliferação de células T. Contudo, ainda pouco se sabe sobre seu verdadeiro papel na patogenia da EM. Recentemente tem sido descrita uma população de células T chamadas Th17, capaz de secretar grandes quantidades de IL-17, IL-21 e GM-CSF possuindo uma importância fundamental na patogenia da EM e de seu modelo murino, a EAE. Nesse contexto, a relação entre as Th17 e as células da microglia pode nos fornecer dados importantes acerca dos mecanismos envolvidos nas lesões observadas no SNC. OBJETIVO: Este trabalho teve como objetivo melhor elucidar a relação existente entre a expressão das moléculas imunes por células da microglia e a ação que estas promovem sobre as células T. MÉTODOS: Utilizamos culturas de células da microglia de linhagem, chamadas C8-B4, assim como cultura primária de células da microglia obtidas a partir sistema nervoso de camundongos C57BL/6 adultos. Caracterizamos o perfil imune da microglia, avaliando a transcrição de genes para citocinas através de PCR em tempo real assim como a expressão de suas moléculas ativadoras por citometria de fluxo. A avaliação da IDO se deu através da expressão da mesma por células da microglia ativadas ou não por LPS ou IFN-?. Ja sua capacidade funcional foi medida através da atividade proliferativa de linfócitos T CD4 específicos para MOG 35-55. RESULTADOS: Nossos resultados demonstraram que as células de ambas as culturas possuem a capacidade de expressar diversas moléculas imunes, tanto pró quanto anti-inflamatórios. Dentre estas observamos TLR-4, TLR-2, IL-6, IL-10 e TGF-?. Além disso, confirmamos a expressão da enzima IDO por estas células. O bloqueio de tal enzima impede o controle que a microglia tem sobre a proliferação dos linfócitos T CD4, tanto in vitro quanto in vivo. No modelo in vivo tal efeito repercute em uma encefalomilite mais severa, onde o quadro clínico do animal não regride. CONCLUSÃO: Os resultados aqui obtidos nos dão a certeza da influência das microglias dentro do contexto inflamatório, afirmando sua capacidade de modular a resposta imune. Além disto, fica clara a importância da enzima IDO, cuja ação dentro do controle de uma autoimunidade demonstra ser altamente necessária / INTRODUCTION: Microglia cells has gained great attention recently because its activation by inflammatory cytokines can promote infiltration and destruction of Central Nervous System (CNS) during some disease, mainly in the case of Multiple Sclerosis (MS). On the other hand, these cells may also express suppressor molecules such as the indoleamine-2,3-dioxygenase (IDO), able to suppress T cell proliferation. However, still little is known about its role in MS pathogenesis. Recently it has been described a new population of T cells called Th17, able to secrete high amounts of IL-17, IL-21 and GM-CSF, with a fundamental importance on MS and its murine model, EAE. In this context, the relationship between Th17 and microglia cells can provide us important data about the mechanisms involved in the establishment of CNS lesions. OBJECTIVES: This work had the objective to better elucidate the relationship between the expression of some molecules by microglia and its role T cell activation. METHODS: Through a cellular lineage knowing as C8-B4 and primary cultures of microglia obtained from CNS of adult mice C57BL6 we investigated the transcription of several genes for cytokines and membrane expression of several pattern recognition receptors. The IDO evaluation was performed after activation with LPS or rIFN-?. Its functional capacity was measured trough its action over T cell proliferation. RESULTS: Our results demonstrated that both cells have the capacity of express several immune molecules, both pro and anti-inflammatory. Among this, we observed TLR-4, TLR-2, IL-6, IL-10 and TGF-?. We also confirmed IDO expression by these cells. The blockade of such enzyme prevents the control of microglia above T CD4 lymphocytes proliferation, both in vitro and in vivo. Using the in vivo model, IDO blocker rendered a encephalomyelitis more severe. Conclusion: The results here obtained give us the certainty of microglia influence in inflammatory context, stating its capacity of modulating the immune response

Im ersten Teil dieser Dissertation wurde der Einfluss des mitochondrialen Proteins TCAIM (T cell activation inhibitor, mitochondrial) auf die T-Zellaktivierung untersucht. Hierzu wurde eine transgene Mauslinie mit einem T-zellspezifischen knock-in (KI) von Tcaim in den Rosa26 Lokus generiert. Die Tcaim-Überexpression beeinflusste die Fission und Umverteilung von Mitochondrien und reduzierte die T-Zellrezeptor (TZR)-induzierte Bildung mitochondrialer, radikaler Sauerstoffspezies. In vitro stimulierte CD4+ Tcaim KI T-Zellen zeigten eine geringere Aktivierung, Proliferation und IL-2 Sekretion als Kontrollzellen. T-Zellen aus Tcaim KI Mäusen, die in Rag-1 knock-out Mäuse transferiert wurden, waren nicht fähig ein allogenes Haut-Transplantat abzustoßen und behielten einen naiven Phänotyp. Diese Ergebnisse zeigen, dass TCAIM als mitochondriales Protein wichtige Schritte in der Zellaktivierung und der Bildung von Gedächtnis-T-Zellen beeinflusst. Der zweite Teil der Dissertation beschäftigte sich mit dem Einfluss der CD44-Oberflächenexpression auf die Differenzierung von T-Helfer (TH)-Zellen. Eine hohe CD44-Expression unterscheidet Effektor- von naiven T-Zellen. Durch die allogene Stimulation von CD4+ T-Zellen bildeten sich drei verschiedene Populationen: CD44+, CD44++ und CD44+++. Sowohl in vitro als auch in vivo generierte alloreaktive TH17-Zellen wurden in der CD44+++ Population, TH1-Zellen hingegen in der CD44++ Population, detektiert. Es wurde beschrieben, dass sowohl eine geringe TZR- als auch eine geringe CD28-Stimulation eher die Bildung von TH17- als TH1-Zellen unterstützen. Unter genau diesen Bedingungen kann CD44 als kostimulatorisches Molekül die Signaltransduktion verstärken. Tatsächlich zeigten allogenreaktive CD44+++ TH-Zellen eine höhere ZAP-70-Phosphorylierung als CD44++ TH-Zellen. Diese Ergebnisse unterstützen die Annahme, dass CD44 durch die Verstärkung der Signaltransduktion die TH17-Differenzierung fördern kann. / Within the first part of this thesis, the influence of the mitochondrial Protein TCAIM (T cell activation inhibitor, mitochondrial) on T cell activation was investigated. Tcaim expression correlated negatively with the rejection of allografts and it is down-regulated during T cell activation. To study effects of TCAIM during T cell activation, we generated a T cell-specific mouse strain with a Tcaim knock-in (KI) targeted to the Rosa26 locus. Tcaim overexpression changed the mitochondrial morphology and reduced the T cell receptor (TCR)-induced mitochondrial reactive oxygen species production. In vitro activation of Tcaim KI CD4+ T cells resulted in a decreased activation, proliferation and cytokine release. Importantly, Rag-1 knock-out mice, reconstituted with Tcaim KI T cells, tolerated allogeneic skin grafts. Thus, by regulating TCR-induced mitochondrial distribution and ROS production, TCAIM controls important steps during T cell activation and memory formation. The second part dealt with the influence of CD44 surface expression level for T helper cell (Th cell) differentiation. By association with lymphocyte-specific protein kinase (LCK) it can enhance T cell signaling. Allogeneic stimulation of CD4+ T cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo-reactive TH17 cells were mainly CD44+++. This is in contrast to TH1 cells which were dominantly CD44++. Titration experiments revealed that low TCR- and co-stimulation supports TH17 rather than TH1 development. Under exactly these conditions it was reported that CD44 can act as co-stimulatory molecule and replace CD28. Indeed, CD44+++CD4+ T cells contained already more phosphorylated ZAP-70 as compared to CD44++ cells. Our results support the notion that CD44 enhances TCR signaling strength by delivering LCK, which is required to support TH17 development.

Die Myasthenia Gravis (MG) ist ein Prototyp einer Antikörper-vermittelte Autoimmunerkrankung. Die Autoantikörper richten sich hauptsächlich gegen den Acetylcholinrezeptor (AChR). Welche Bedeutung TH17-Zellen für die Pathogenese der MG haben, konnte bisher noch nie direkt gezeigt werden. Mithilfe des Tiermodells Experimentelle Autoimmune Myasthenia Gravis (EAMG) sollte die Rolle der TH17-Zellen im Rahmen dieser Arbeit analysiert werden. Eine signifikante Anzahl tAChR-spezifischer CD4+ T-Zellen, die IL17 exprimieren, konnte nach der Immunisierung mit torpedo AChR in CFA in Wildtyp-Mäusen (WT) beobachtet werden. Die IL17ko Mäuse entwickelten weniger oder keine EAMG Symptome, obwohl weder die Frequenz tAChR-spezifischer CD4+ T-Zellen, die IL2, IFNgamma oder IL21 sezernierten noch der prozentuale Anteil der FoxP3+ Treg-Zellen einen Unterschied aufwiesen. Im Gegensatz dazu waren die Level pathogener anti-muriner AChR Antikörper statistisch geringer in IL17ko Mäusen, während bei anti-tAChR Antikörpertitern kein Unterschied festzustellen war. Ähnliche Resultate erbrachten TCRbeta/delta ko Mäuse rekonstituiert mit entweder WT oder IL17ko CD4+ T-Zellen. Die Depletion von Treg-Zellen mithilfe von DEREG Mäusen in der frühen Erkrankungsphase zeigte keine signifikanten Unterschiede bezüglich der analysierten Parameter. Zusammenfassend lässt sich hier festhalten, dass die Frequenz und Differenzierung Antigen-spezifischer CD4+ T-Zellen sowie der Antikörpertiter gegen den tAChR nicht durch die IL17-Defizienz im EAMG Modell beeinflusst wird. Auch hat eine frühe Treg-Zell-Depletion keinen Einfluß auf die Erkrankungsstärke. Allerdings scheint das Durchbrechen der B-Zell Toleranz, das zur Produktion von pathogenen Anti-mAChR-spezifischen Antikörpern und somit zu einer Induktion der Erkrankung führt, abhängig von IL17-produzierenden CD4+ T-Zellen zu sein. Der Einsatz von Anti-IL17-Antikörpern könnte insofern auch für die MG eine Therapieoption darstellen. / Myasthenia gravis (MG) is an antibody-mediated autoimmune disease. The autoantibodies are directed against the acetylcholine receptor (AChR). The importance TH17 cells have for MG pathogenesis has never been directly demonstrated. Therefore, the analysis of TH17 cells in the Experimental Autoimmune Myasthenia Gravis (EAMG) animal model was the aim of this work. Here, it is shown that in wildtype mice (WT) significant numbers of IL17-producing tAChR-specific CD4+ T cells could be observed after immunization with torpedo AChR in CFA. IL17ko mice developed less or no EAMG symptoms, although frequencies of tAChR-specific CD4+T cells secreting IL2, IFNgamma or IL21 as well as percentage of FoxP3+ Treg cells were similar. In contrast, pathogenic anti-murine AChR antibody levels were significantly lower in IL17ko mice, while anti-tAChR antibody levels were equal. Similar results were obtained by the reconstitution of TCR beta/delta ko mice with CD4+ T cells of either WT or IL17ko origin. For the depletion of Treg cells using DEREG mice in the initial phase of the disease no significant differences could be detected in terms of the analyzed parameters. In summary, this thesis demonstrates, that frequencies and differentiation of antigen specific CD4+ T cells as well as the level of anti-tAChR specific antibody titers are not affected by IL17-deficiency in the EAMG model. Likewise, an early Treg cell depletion seems to have no impact on disease severity. However, breaking of B cell tolerance resulting in pathogenic anti-murine AChR specific antibodies and subsequent disease induction, seems to be dependent on IL17 producing CD4+ T cells. In this respect, the application of anti-IL17 antibodies could also become a MG therapy option.