Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17. / Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.

Nos últimos anos, um importante destaque tem sido dado aos linfócitos Th17 para o desenvolvimento e manutenção da inflamação associada à autoimunidade. A esclerose múltipla é uma doença autoimune desmielinizante do SNC, cuja patogênese está associada à resposta do padrão Th17. Evidências têm demonstrado que estas células são submetidas a uma reprogramação metabólica após serem ativadas, sendo essa adequação essencial para sua completa diferenciação e aquisição de funções efetoras. A enzima Piruvato Quinase M2 (PKM2) participa da etapa final da glicólise convertendo fosfoenolpiruvato em piruvato. Estudos recentes demonstraram que a fosforilação de PKM2 a torna capaz de translocar para o núcleo, onde adquire um papel no controle da expressão gênica. Nesse sentido, o objetivo deste estudo foi avaliar o envolvimento da PKM2 na diferenciação de linfócitos Th17, bem como sua participação no desenvolvimento da encefalomielite autoimune experimental (EAE), um modelo animal de esclerose múltipla. Observou-se que durante o processo de diferenciação, os linfócitos Th17 aumentam a expressão gênica de PKM2 bem como a sua forma fosforilada (Y105). De forma interessante, tanto a inibição farmacológica como a deleção gênica da PKM2 especificamente em linfócitos T promoveram uma redução da diferenciação e expansão da subpopulação Th17, que foi associada com diminuição na expressão de moléculas efetoras e fatores de transcrição chave para o estabelecimento do fenótipo Th17. Em um contexto de resposta autoimune, notou-se que PKM2 é superexpressa nos órgãos linfóides periféricos e sistema nervoso central de animais com EAE, sendo correlacionada com o infiltrado de células inflamatórias. Corroborando com os dados in vitro, a deficiência de PKM2 em linfócitos T promoveu redução dos sinais clínicos da EAE, acompanhada de baixa frequência de linfócitos Th17 e menor expressão de moléculas inflamatórias do perfil Th17. Adicionalmente, o tratamento farmacológico com o inibidor da PKM2 atenuou a progressão e gravidade da EAE. Portanto, esses achados implicam um importante papel para PKM2 em doenças autoimunes por regular o desenvolvimento e função de linfócitos Th17. / Over the past few years, an important highlight has been given to Th17 lymphocytes for the development and maintenance of autoimmunity-associated inflammation. Multiple sclerosis is a CNS demyelinating autoimmune disease that is associated to Th17-mediated response. Some evidences have demonstrated that those cells undergo metabolic reprogramming after being activated, which is essential for their complete differentiation and acquisition of effector functions. The enzyme Pyruvate kinase M2 (PKM2) participates at the final step of glycolysis by converting phosphoenolpyruvate into pyruvate. Recent studies have demonstrated that PKM2 phosphorylation allows its translocation into the nucleus, where it plays a role in controlling gene expression. Thus, the aim of this study was to evaluate the involvement of PKM2 in Th17 lymphocytes differentiation, as well as its role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. It was perceived that during differentiation process, Th17 lymphocytes increase PKM2 gene expression, and also its phosphorylated form (Y105). Interestingly, both pharmacological inhibition and T-lymphocyte-specific PKM2 gene deletion promoted a reduction in differentiation and expansion of Th17 subpopulation, being associated to diminished expression of effector molecules and key transcription factors for the establishment of Th17 phenotype. In the context of an autoimmune response, it was noticed that PKM2 is overexpressed in peripheral lymphoid organs and central nervous system of EAE-bearing mice, which was correlated with the inflammatory cell infiltration. Corroborating with in vitro data, the deficiency of PKM2 in T lymphocytes led to a reduction of EAE clinical score along with low Th17 frequency and diminished expression of Th17-related inflammatory molecules. Additionally, pharmacological treatment with the PKM2 inhibitor attenuated EAE progression and severity. Therefore, these findings imply an important role for PKM2 in autoimmune diseases by regulating the development and function of Th17 lymphocytes.

Les lymphocytes Th17 forment une population de cellules T CD4+ pro-inflammatoires, impliqués non seulement dans l'élimination d'agents pathogènes, mais ayant aussi un rôle délétère dans l'induction de maladies inflammatoires chroniques. Ils expriment spécifiquement le récepteur de chimiokines CCR6, qui a pour ligand le CCL20 mais aussi les β-defensine-1, 2 et 3, peptides ayant une activité antimicrobienne. Les cellules souches mésenchymateuses (CSMs) représentent une population cellulaire hétérogène exerçant diverses propriétés immunomodulatrices.Les résultats obtenus dans ce travail de thèse montrent que l'environnement inflammatoire contribue à augmenter l'adhésion des lymphocytes Th17 aux CSMs, et qu'elle est régulée par l'interaction du CCR6 avec ses ligands ; que les CSMs exercent, en partie via la sécrétion de PGE2, des effets anti-inflammatoires en faisant acquérir un phénotype régulateur aux lymphocytes Th17 différenciés, soulignant ainsi la plasticité de ces derniers.De plus, nous avons montré que les lymphocytes Th17 activés par l'antigène produisent du CCL20 et induisent, via la production de l'IL-17 et de l'IL-22, la sécrétion d'hBD-2, mais pas celle des hBD-1 et 3, par des kératinocytes épidermiques humains et de la peau reconstituée; que le CCL20, ainsi que la hBD-2, induisent l'arrêt de ces cellules sur l'endothélium enflammé in vitro en conditions de cisaillement. Finalement, l'activation spécifique d'antigène des lymphocytes Th17 entraîne une perte de l'expression de CCR6, ce qui provoque ainsi un état transitoire de non réponse à une nouvelle stimulation de ces cellules avec les ligands de CCR6, permettant leur migration ultérieure hors du tissu enflammé. / Th17 cells form a population CD4+ T cells with strong pro-inflammatory properties that are not only involved in the clearance of pathogens, but also play a deleterious role of in the pathogenesis of inflammatory disease. Th17 cells specifically express CCR6, a chemokine receptor that binds to its unique chemokine ligand, CCL20, as well as to human β-defensin (hBD)-1, 2 and 3, peptides with anti-microbial activity. Mesenchymal stem cells (MSC) represent a heterogenous population that exert broad immunomodulatory effects.The results from the studies carried out during this thesis show that the inflammatory environment contributes to increased adhesion of Th17 cells to MSCs, which is mediated via the interaction of CCR6 with its ligands, and that MSCs exert, in part via the secretion of PGE2, anti-inflammatory effects through the induction of a T regulatory cell phenotype in fully differentiated tissue-infiltrating Th17 cells, thereby underscoring the plasticity of the latter cells.Furthermore, the results show that antigen-activated Th17 cells produce CCL20 and induce, via the production of both IL-17 and IL-22, the secretion of hBD-2, but not 1 and 3, by normal human epidermal keratinocytes and reconstituted skin, and that CCL20, as well as hBD-2, induce arrest of these cells onto inflamed endothelium in vitro under conditions of shear stress. Finally, antigen-specific activation of Th17 cells also causes a loss of CCR6 expression from their cell surface and thus results in a transitory state of non-responsiveness to further stimulation of these cells with CCR6 ligands, which is likely to permit their subsequent migration out of inflamed tissue.

Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome cansignificantly vary among patients within the same stage. Data collected from largecohorts of human cancers has demonstrated the impact of immune-classification, which has a prognostic value that may add largely to the significance of the AJCC/UICC TNM-classification. In our study we examined the immune cells that infiltrated the tumor tissues of colorectal and ovarian cancer patients. In a cohort of newly diagnosed colorectal cancer patients we examined the correlations between the KRAS mutational status, patterns of tumor-infiltrating immune cells and the presence of tumor recurrence. Our data suggest that colorectal cancer patients with low levels of tumor-infiltrating lymphocytes, a high CD1a/DC-LAMP tumor-infiltrating dendritic cells ratio, and a KRAS mutation in codon 13 are at a high risk of disease recurrence. In ovarian cancer patients we focused on the dynamics of the tumor-infiltrating...

L'épilepsie est un trouble chronique du système nerveux central qui touche 70 millions de personnes dans le monde. Un tiers des patients souffrent d'épilepsie réfractaire (ER), caractérisée par des crises récurrentes malgré l’utilisation de médicaments antiépileptiques (MAE) appropriés. Plusieurs données soutiennent un lien biologique entre la neuroinflammation et l'épilepsie chronique. De plus, des études suggèrent que certains MAE de nouvelle génération comme le brivaracétam présentent des propriétés immunomodulatrices qui pourraient contribuer à leur bénéfice dans l’ER et d’autres pathologies neurologiques. Les objectifs de cette thèse étaient tout d’abord de caractériser ex vivo le profil des cellules immunitaires périphériques, les niveaux de cytokines pro- et anti-inflammatoires et les biomarqueurs neurodégénératifs dans le sang périphérique de sujets souffrant d’épilepsie (ER vs. bien contrôlée) en comparaison à celui de donneurs sains. Par la suite, nous avons évalué l’impact du brivaracétam sur l’activation des cellules immunitaires humaines in vitro et son potentiel immunomodulateur et neuroprotecteur in vivo dans le modèle de l'encéphalomyélite auto-immune expérimentale (EAE) active, un modèle animal de la sclérose en plaques, la pathologie inflammatoire du SNC la plus fréquente. Méthodologie : Après isolation des cellules mononucléées et du sérum à partir du sang périphérique d'adultes souffrant d'épilepsie focale et de donneurs sains, nous avons effectué des analyses par cytométrie en flux ex vivo, ELISA multiplex et technique ultrasensible de détection de biomarqueurs par technologie single molecule array (SIMOA). Nous avons comparé l'influence du brivaracétam et du lacosamide sur l'activation des cellules immunitaires périphériques humaines in vitro et in vivo dans l'EAE active induite par immunisation avec le MOG, le modèle animal le plus commun de la sclérose en plaques. Résultats : Nous avons rapporté une augmentation de la proportion des lymphocytes T CD4 dans le sang périphérique des adultes souffrant d’ER, avec une fréquence plus élevée de lymphocytes pro-inflammatoires Th17/Th1 en comparaison avec les contrôles. Nous avons également rapporté des niveaux significativement plus élevés du marqueur de lésion neuronale sNfL chez les sujets plus âgés avec l’ER par rapport aux témoins appariés pour l’âge. De plus, nous avons montré que l'administration prophylactique du brivaracétam ou du lacosamide ne retardait pas l'apparition de l'EAE mais était associée à une évolution clinique significativement moins sévère dans la phase chronique de l'EAE active chez les souris femelles C57BL/6 par rapport au contrôle (véhicule). Conclusion : Dans l'ensemble, nos données soutiennent d'une part que l’ER est associée à un profil immunitaire pro-inflammatoire des lymphocytes Th17/Th1 dans le sang périphérique et à des niveaux pathologiques de sNfL, soutenant la présence de composantes inflammatoire et neurodégénérative potentielles dans l’ER. D'autre part, les MAE de nouvelle génération (brivaracétam et lacosamide) n'altèrent pas de façon importante la réponse à l'immunisation avec le peptide MOG, mais améliorent l'évolution de l'EAE par le biais principalement de mécanismes neuroprotecteurs. / Epilepsy is a chronic central nervous system condition affecting 70 million people around the world. One third of patients suffer from drug-resistant epilepsy (DRE), characterized by recurrent seizures despite appropriate trials of anti-epileptic drugs (AEDs). Accumulating data support a biological link between neuroinflammation and chronic epilepsy. Recent studies suggest that some of the new generation AEDs, like brivaracetam, can display immunomodulatory properties that could contribute to their beneficial impact in DRE and other neurological disease. We aimed to investigate differences in immune cell populations, cytokines, and neurodegenerative biomarkers in the peripheral blood of subjects with epilepsy (DRE vs. well-controlled) compared to healthy controls and to assess the impact of brivaracétam on activation of human immune cells in vitro and its immunomodulatory and neuroprotective potential in vivo in the active experimental autoimmune encephalomyelitis (EAE), a common model of the prototypical CNS neuroinflammatory disease multiple sclerosis. Methodology: Using peripheral blood mononuclear cells and serum isolated from the peripheral blood of adults suffering from focal onset epilepsy and healthy donors, we performed flow cytometry analysis ex vivo, multiplex immunoassays, and ultrasensitive single molecule array. We compared the influence of brivaracetam and lacosamide on activation of human peripheral immune cells in vitro and in vivo in MOG-induced active EAE, the most common animal model of multiple sclerosis. Results: We observed an increased proportion of CD4 T cells in the peripheral blood compartment of adults suffering from DRE, with a higher frequency of proinflammatory Th17/Th1 cells compared to controls. We also reported significantly higher levels of the marker of neuronal injury sNfL in aging subjects with DRE compared to age-matched controls. Furthermore, we showed that prophylactic administration of brivaracetam or lacosamide did not delay EAE onset but was associated with a significantly less severe clinical course in the chronic phase of active EAE in C57BL/6 female mice compared to control (vehicle).Conclusions: Taken together, our data support that DRE is associated with a proinflammatory Th17/Th1 CD4 T cell immune profile in peripheral blood and pathological levels of sNfL, supporting both potential inflammatory and neurodegenerative components in DRE. On the other hand, the novel generation AEDs (brivaracetam and lacosamide) do not impair the response to immunization with MOG peptide but improve the course of EAE through mostly neuroprotective mechanisms.

La fibrose kystique (FK) est la maladie autosomique récessive la plus fréquente chez les individus de race caucasienne. Elle est secondaire à la mutation du gène Cystic Fibrosis Transmembrane Regulator (CFTR). Grâce à des traitements plus agressifs, la médiane de l’espérance de vie des individus atteints de la FK a augmenté et cette augmentation est associée à l’émergence du diabète secondaire ou associé à la FK (DAFK), une complication associée à une augmentation du taux de mortalité. La pathophysiologie du DAFK n’est pas parfaitement comprise. Par exemple, la cause de l’accélération de la perte de la fonction pulmonaire, qui débute des années avant l’apparition du DAFK, n'est pas élucidée. Tous les patients atteints de la FK, même ceux sans le DAFK, présentent de l’hyperglycémie et des fluctuations glycémiques. D’ailleurs, une étude a démontré que la réactivité immunitaire est affectée par l’hyperglycémie dans un modèle animal de la FK et il y a des évidences que les lymphocytes sans CFTR fonctionnel ou en présence d’un excès de glucose ont des réactions inflammatoires anormales. Donc, nous avons émis l’hypothèse que les patients atteints de la FK, surtout ceux non-diabétiques et pré-diabétiques, auront une plus grande proportion de lymphocytes Th17 et Treg produisant la cytokine pro-inflammatoire IL-17A comparativement aux sujets sains et que l’augmentation de cette cytokine pourrait influencer la chute accélérée des fonctions pulmonaires avant l’apparition du DAFK. Des niveaux élevés d’IL-17A sont retrouvés dans les poumons des patients atteints de la FK et dans le sang périphérique des patients avec le diabète de type 1 (DT1) et de type 2 (DT2). L’IL-17A peut aussi être produite par les lymphocytes Treg dysfonctionnels. Habituellement, ces lymphocytes atténuent les réponses inflammatoires excessives, mais lorsqu’ils sont dysfonctionnels, ils peuvent produire de l’IL-17A, contribuant ainsi à l’état inflammatoire. De plus, nous avons supposé que les proportions de Th17 et Treg produisant de l’IL-17A seront associées aux fonctions pulmonaires des patients atteints de la FK et que l’alimentation, l’activité physique et la composition corporelle influenceraient ces relations. Les résultats de cette thèse ont montré que, malgré une association entre la proportion de lymphocytes dans le sang périphérique et les indices de fluctuations glycémiques, celles-ci n’influençaient pas les proportions de lymphocytes Th17 et Treg produisant de l’IL-17A lorsqu’ils étaient mis en culture pour 24 ou 48 heures dans des milieux contenant soit 5 mM ou 25 mM de glucose et stimulés par le phorbol 12-myristate 13-acetate (PMA) et le phytohemagglutinine (PHA) ou, encore, non stimulés. De plus, ces proportions étaient semblables entre les patients atteints de la FK et les individus en santé. Toutefois, les proportions de lymphocytes Treg stimulés produisant de l’IL-17A des sujets sains étaient plus élevées que les proportions de lymphocytes Treg non stimulés de tous les participants (patients atteints de la FK et individus en santé). Tout ceci suggérant donc que les Treg des sujets sains et atteints de la FK ne réagissaient pas de la même façon à la stimulation. D’ailleurs, la durée d’incubation affectait les proportions de Th17 produisant de l’IL-17A, mais elle n’avait aucun effet sur les proportions de Treg produisant cette cytokine. Donc, ces types cellulaires réagissaient différemment dans les mêmes milieux de culture. De plus, nous avons observé que seulement l’énergie provenant des glucides affectait modestement les indices de fluctuations glycémiques et que les proportions de Th17 et Treg produisant de l’IL-17A n’étaient pas associées aux fonctions pulmonaires des patients atteints de la FK. En conclusion, les patients atteints de la FK avaient plus d’hyperglycémie et de fluctuations glycémiques, mais elles n’influençaient pas les proportions de lymphocytes Th17 et Treg produisant de l’IL-17A ex vivo. Dans des études futures, il faudrait étudier le rôle de l’IL-17A dans les poumons des patients avec et sans le DAFK et réaliser une étude prospective pour déterminer si une augmentation des niveaux d’IL-17A chez les patients sans le DAFK se traduit par une chute accélérée des fonctions pulmonaires avant l’apparition de cette complication. / Cystic fibrosis (CF) is the most common autosomal recessive genetic disease among Caucasians and it is cause by a mutation in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. With the emergence of more aggressive therapies to treat CF, the median life expectancy of patients with CF has increased and new complications, such as CF-related diabetes (CFRD), have emerged. CFRD is associated with increased mortality. The physiopathology of this complication is not fully understood. For instance, the reason why people with CF have an accelerated decline in lung functions years before the diagnosis of CFRD is not known. Patients with CF, even those without CFRD, have increased hyperglycemia and glucose fluctuations. In addition, a study has reported that hyperglycemia affected immune reactivity in a mouse model of the disease. Furthermore, studies have shown that lymphocytes without a functional CFTR or that have increased uptake of glucose have abnormal immune responses. Therefore, we hypothesized that patients with CF, specifically those with normal and impaired glucose tolerance, would have increased proportions of Th17 and Treg lymphocytes producing the pro-inflammatory cytokine IL-17A and that the increase in IL-17A levels would contribute to the accelerated decline of lung functions before the onset of CFRD. Increased levels of IL-17A have been found in the lungs of patients with CF and the peripheral blood of patients with type 1 (T1D) and 2 diabetes (T2D). Dysfunctional Treg lymphocytes can also produce IL-17A. These lymphocytes usually attenuate excessive immune responses but, in certain cases, can be dysfunctional and produce the pro-inflammatory cytokine IL-17A. Moreover, we hypothesized that the proportions of these cells producing IL-17A would be associated to lung functions in patients with CF and that nutrition, physical activity and body composition could influence the relationship between inflammation and glucose fluctuations. Although an association between the proportion of lymphocytes in the peripheral blood of participants and glucose fluctuations was observed, glucose fluctuations did not affect the proportions of Th17 and Treg lymphocytes producing IL-17A stimulated with PHA and PMA or not stimulated in media containing either 5 or 25 mM of glucose for 24 or 48 hours. Furthermore, these proportions were similar between healthy individuals and patients with CF with the exception of the proportion of stimulated Treg lymphocytes producing IL-17A of healthy individuals that was greater than the proportions of non-stimulated Treg lymphocytes producing IL-17A of all participants (patients with CF and healthy individuals). This suggests that Treg lymphocytes of healthy individuals and CF patients do not respond in the same manner to stimulation by phorbol 12-myristate 13-acetate (PMA) and phytohemagglutinin (PHA). Also, the duration of incubation affected the proportions of Th17 cells producing IL-17A but did not affect the proportion of Treg producing this cytokine. Therefore, these two types of lymphocytes are differently affected in the same culture media. Also, only the proportion of calories from carbohydrates affected modestly glucose fluctuations and the proportions of Th17 and Treg lymphocytes producing IL-17A were not associated to lung functions in CF patients. To conclude, patients with CF had increased hyperglycemia and glucose flucutations when compared to healthy individuals but this glucose variability did not affect the proportions of Th17 and Treg lymphocytes producing IL-17A ex vivo. Future studies are needed to explore the role of IL-17A in the lungs of patients with CFRD and a prospective study would be important in order to determine if an increase in IL-17A in patients without CFRD is associated to an accelerated decrease in lung functions before the onset of this complication.

Introduction: L’activation des cellules stellaires hépatiques (CSHs) est un point clé du processus de fibrose hépatique. Les lymphocytes T CD4+ intra-hépatiques sont une source majeure de cytokines anti-inflammatoires comme l’IL-10 et pro-inflammatoire (IL-17A), hépatoprotectrice (IL-22) produites par les Th17. Les Th17 sont impliqués dans de nombreuses pathologies inflammatoires mais l’effet de ces cellules sur les CSHs n’est pas encore élucidé. Objectif: Comprendre le rôle des cytokines de type Th17 dans le processus d’activation des CSHs. Méthodes: La lignée de CSHs humaine LX2 a été stimulée par l’IL-17A ou l’IL-22 puis comparée à des cellules traitées par le TGF-b et le tampon phosphate salin (PBS). L’activation des CSHs a été évaluée en examinant les molécules profibrotique alpha-smooth muscle actin (a-SMA), collagène de type I (COL1A1) et inhibiteur produits par les tissus des métalloprotéases matricielles I (TIMP-I) par q-PCR. L’expression protéique a été validée par immunobuvardage ou coloration au rouge de picro Sirius. L’expression membranaire de l’IL-10Rb, du TGF-b-RII et de l’IL-17RA a été mesurée par cytométrie en flux. Résultats: L’IL-17A et l’IL-22 n’activent pas les cellules LX2, car aucune induction d’a-SMA, de COL1A1 et de TIMP-I n’a été observée. Cependant, l’IL-17A et l’IL-22 sensibilisent les CSHs à l’action du TGF-b, tel que démontré par une forte expression et production d’a-SMA, collagène type I et TIMP-I. L’IL-17A, mais pas l’IL-22, induit la surexpression à la surface cellulaire du TGF-b-RII et inhibe partiellement la baisse d’expression du TGF--RII après stimulation au TGF-b. Conclusion: Nos résultats démontrent une fonction pro-fibrotique de l’IL-17A et de l’IL-22, car les deux cytokines sensibilisent les CSHs à l’action du TGF-b. L’IL-17A agit via la surexpression et la stabilisation du TGF-b-RII tandis que l’IL-22 agit probablement par des mécanismes intracellulaires. / Background: Activated hepatic stellate cells (HSCs) are key initiators of the fibrogenic process. Intrahepatic CD4+ T cells are major producers of hepatoprotective cytokines such as IL-10 produced by regulatory T cells (Tregs) or inflammatory and regulatory cytokines like IL-17 and IL-22 produced by Th17 cells. Th17 cells have been implicated in various conditions or liver damage but the mechanism of action of Th17 cytokines on HSC is still poorly understood. Aims: To understand the role of the different Th17 cytokines (IL17-A and IL-22) in modulating HSC activation. Methods: The HSC line LX2 was stimulated with increasing doses of IL-17A or IL-22, and compared to TGF-b and PBS-treated cells. Activation of HSCs was evaluated by examining the expression of the pro-fibrotic molecules alpha-smooth muscle actin (a-SMA), collagen type I (COL1A1) and tissue inhibitor of matrix metalloproteinase I (TIMP-I) by q-PCR. Protein expression was validated by either western blot or picro Sirius red stain. Cell surface expression of the cytokine receptors IL-10Rb, TGF-b-RII and IL-17RA was evaluated by flow cytometry. Results: IL-17A and IL-22 alone did not induce LX2 activation, as no induction of a-SMA, COL1A1 and TIMP-I was observed. However, both IL-17A and IL-22 sensitized HSCs to the action of suboptimal doses of TGF-b, confirmed by strong a-SMA, collagen type I and TIMP-I gene expression and protein production. IL-17A but not IL-22 upregulated TGF-b-RII cell surface expression and partially inhibited TGF-b-RII downmodulation upon TGF-b stimulation. Conclusion: Our results demonstrated a pro-fibrotic function for IL-17A and IL-22, as both cytokines sensitize HSC to the action of TGF-b. IL-17A acts through upregulation and stabilization of the TGF-b-RII while IL-22 probably acts through an intracellular mechanism.

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