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The activation of naïve T cells results in their proliferation and differentiation into a particular T-helper (Th) phenotype, namely Th1, Th2 or Th17 cells. This thesis focuses on the role of pro-inflammatory Th1 and Th17 cells in the induction of autoimmune disease of the central nervous system (CNS), using murine experimental autoimmune encephalomyelitis (EAE) as the model. Classically, EAE has been considered to be a Th1-mediated disease. However, since the emergence of the Th17 cells, there has been a paradigm shift towards Th17 cells being the key pathogenic subset in autoimmune disease. This thesis established robust protocols for the differentiation of naïve T cells into myelin-reactive Th1 or Th17 cells, producing ‘clean’ populations devoid of any contaminating cells. Passive T cell-transfer experiments revealed that myelin-reactive Th1 cells could induce EAE, whereas Th17 cells could not. This lack of disease correlated with the inability of the Th17 cells to accumulate in the non-inflamed CNS. Myelin-reactive Th1 cells did have this capability and only once inflammation was established, could Th17 cells be identified in the CNS, potentially exacerbating the disease. After these differences were observed, the project investigated two main aims: 1) to identify differences in homing molecule expression on Th1 and Th17 cells which could explain the difference in their ability to home to the CNS, and to investigate the functional significance of such differences, by molecular blockade; 2) to investigate the requirements for three key cytokines in EAE pathogenesis in passive T cell transfer models, investigating IFN-γ,IL-17 and TNF-α. P-selectin glycoprotein ligand-1 appeared to be important for the initial entry of inflammatory T cells into the CNS. Th1 cells deficient in IFN-γ were capable of IFNinducing EAE. A proportion of the mice developed “atypical” clinical signs, which correlated with T cell infiltration predominantly of the brain, rather than the spinal cord. This atypical EAE may be IL-17 dependent. In conclusion, this thesis indicates the importance of not focusing all resources and therapeutic approaches on Th17- induced inflammation as Th17 cells may not play such a major role as previously thought.

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Orientador: Alexandra Ivo de Medeiros / Resumo: A prostaglandina E2 (PGE2) é um mediador lipídico que participa tanto na diferenciação como na expansão de linfócitos T helper (Th) Th1 e Th17. Esse prostanoide vem sendo descrito como um importante mediador envolvido no agravamento da Encefalomielite Autoimune Experimental (EAE). A EAE é uma doença mediada por células Th1/Th17 autorreativas, responsáveis pela intensa resposta inflamatória contra antígenos do sistema nervoso central (SNC). Alguns estudos descrevem que a inibição da síntese desse prostanoide, ou o bloqueio de seus receptores EP, reduzem os níveis de IL-17A e IFN- e atenuam drasticamente o desenvolvimento da doença. A coexistência de linfócitos Th1 e Th17 na EAE, assim como a presença de células Th17 produtoras de IFN-γ (Th1-like) no SNC sugerem uma possível plasticidade destas subpopulações de linfócitos. No entanto, até o momento, não há relatos na literatura se a presença de PGE2, presente no SNC, estaria envolvida na plasticidade de linfócitos Th17 em Th1 nessa autoimunidade. Portanto, a hipótese desse estudo é que as células Th17 migrariam para o SNC e desencadeariam o recrutamento de células inflamatórias e o aparecimento dos primeiros sinais clínicos da doença. A presença de PGE2, associado esse microambiente inflamatório, favoreceria a plasticidade das células Th17 para um padrão Th1, resultando na diferenciação de células T CD4+ patogênicas (IL17+IFN+) e células Th1-like. Os resultados obtidos demonstram que as células Th17, quando cultivadas em cond... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

La Sclérose en Plaques (SEP) est une maladie d'origine multifactorielle qui se développe chez des individus génétiquement susceptibles en présence de facteurs environnementaux inducteurs. Ma thèse avait pour objectif d'analyser les effets d'un facteur génétique, le variant R63W du gène Vav1 et d'un facteur environnemental, la souche "Escherichia coli Nissle 1917", sur le développement de l'encéphalomyélite auto-immune expérimentale (EAE), un modèle animal de la SEP. Une région de 1cM comportant un polymorphisme dans le gène Vav1 a en effet été identifiée au laboratoire comme étant responsable de la résistance des rats Brown-Norway à l'EAE. Afin d'établir formellement le rôle de ce polymorphisme dans ce modèle, une souris Knock-In Vav1R63W a été générée. Nous avons montré que les souris Vav1R63W développent une EAE moins sévère. Ceci est associé à un défaut de production de cytokines inflammatoires intrinsèque aux lymphocytes T (LT) CD4 qui n'est pas lié à une augmentation de la fréquence de LT régulateurs. Sur le plan moléculaire, Vav1R63W présente une activité adaptatrice défectueuse conduisant à la diminution de la phosphorylation de ERK, AKT et p38 mais à une activité enzymatique normale. Nos résultats montrent un rôle de la fonction adaptatrice de Vav1 dans les fonctions des LT CD4 et son implication dans la susceptibilité à l'inflammation du système nerveux central (SNC). L'analyse de l'effet d'un traitement oral par le probiotique E. coli Nissle 1917 (ECN) montre un effet bénéfique sur le développement de l'EAE. Ceci est associé à un défaut de la sécrétion de cytokines par les LT CD4, ainsi qu'à une diminution de l'infiltration de LT CD4 auto-réactifs dans le SNC. De plus, la barrière intestinale est moins altérée chez les souris traitées par ECN au cours du développement de l'EAE. L'effet bénéfique de ECN semble être dû à la production d'une génotoxine, la colibactine. Par contre, la colonisation néonatale des souris C57BL/6 par ECN ne reproduit pas le même effet observé à l'âge adulte. Dans l'ensemble, nos résultats montrent un effet bénéfique du changement de la fonction de Vav1 ainsi que du traitement par ECN sur le développement de l'EAE. L'analyse approfondie des mécanismes mis en jeu, permettra une meilleure compréhension de la pathogenèse de la SEP et pourrait contribuer à l'identification de nouvelles options thérapeutiques. / Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. It develops in genetically susceptible individuals when they encounter specific environmental factors. The aim of my thesis was to analyze the role of a genetic factor (Vav1R63W variant) and an environmental factor (Escherichia coli Nissle 1917) in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Previous genetic studies of my team suggest the implication of a polymorphism in the Vav1 gene in the resistance of Brown-Norway rats to EAE. In order to analyze the role of the identified polymorphism in the susceptibility to EAE, we generated a Knock-In mouse bearing the same polymorphism (Vav1R63W). Using this model, we showed that Vav1R63W mice develop less severe EAE due to a defect in cytokine production by CD4 T cells. This defect is intrinsic to CD4 T cells and is not linked to the increased proportion of regulatory T cells observed in Vav1R63W mice. We also showed that Vav1R63W present an altered adaptor function as shown by reduced ERK and AKT phosphorylation and decreased calcium flux after TCR stimulation, with no effect on Vav1 enzymatic activity. Thus, our results highlight the role of Vav1 adaptor function in CD4 T cell functions and susceptibility to central nervous system inflammation. Next, I analyzed the impact of the treatment with the probiotic E. coli Nissle 1917 (ECN) on EAE development. Our results showed that the daily oral treatment of adult C57BL/6 mice with ECN ameliorates the course of the disease. In addition to their defect in cytokines production, MOG specific CD4 T cells from ECN treated mice were increased in the periphery. Consequently, ECN treated mice exhibited reduced CD4 T cell infiltration in their central nervous system. Furthermore, analysis of intestinal permeability revealed that its alteration after MOG immunization was partially reversed after ECN treatment. The reduced EAE seems to be due to the secretion of a genotoxin by ECN, the colibactin. In contrast, neonatal colonization of C57BL/6 mice did not protect against EAE. Together, our data showed a beneficial role of the imbalance of Vav1 function and ECN treatment in EAE development. Further analysis of the involved mechanisms will help us to better understand the pathogenesis of MS and to develop new therapeutic strategies for MS.

Inhibitors of apoptosis (IAPs) modulate cell death and play critical role in signal transduction that promotes inflammation. Recently, Smac mimetics, which are IAP antagonists, have attracted attention as novel cancer therapeutics. Cellular Inhibitor of Apoptosis 2 (cIAP2), a member of IAP family, positively affects both NF-κB and MAPK activation in response to many inflammatory stimuli. We observed that the lack of cIAP2 ablates LPS-induced neuroinflammation. Also, cIAP2-/- macrophages demonstrated diminished antigen presentation potential that could contribute to ablated immunity. In addition to these functions, we have previously reported that cIAP2 also regulates the activation of Interferon Regulatory Factor 1 (IRF1). Since IRF1-/- mice are resistant to experimental autoimmune encephalomyelitis (EAE), we hypothesized that cIAP2-/- mice will be protected from the disease. Surprisingly, induction of EAE in cIAP2-/- mice resulted in exaggerated infiltration of immune cells increased expression of proinflammatory cytokines and demyelination within CNS. We found that the lack of cIAP2 induces caspase-8 expression in microglia derived macrophages, contributing to their activation and polarization towards M1 phenotype, and exacerbates the symptoms of EAE. These findings suggest that cIAP2 limits neuroinflammation in the CNS and thus the use of Smac mimetics as chemotherapeutics needs to be reevaluated.

In neurodegenerative diseases, the CNS becomes inflamed through activation of pathways, including the NF-B pathway. Some of the therapies for those diseases target neuroinflammatory pathways. Here, we explore the mechanisms for the upregulation of a subset of genes following a restimulation of the NF-B pathway. We discover that this upregulation occurs independent of IRF1 expression and type 1 interferon signaling. A knockdown of IRF1 using siRNA and an inhibition of JAK proteins using inhibitor AG490 both had no effect on priming. A secreted factor was found to upregulate the expression of both this subset of genes and genes encoding pro-inflammatory cytokines induced by NF-B activation. We also explored the role of IRF1 in a mouse model of multiple sclerosis. We found that the deletion of IRF1 from oligodendrocytes diminished EAE severity. A deletion of IRF1 from myeloid cells within mice did not diminish EAE severity, however showed a promising decrease in the expression of certain inflammatory genes. Thus, IRF1 plays a critical role in fine-tuning inflammatory responses in the brain.

Numerosos estudios han demostrado a lo largo de los años, el papel fundamental que juegan las células de microglía en el funcionamiento del SNC. No sólo en condiciones normales, donde controlan la correcta homeóstasis del tejido, sino también en todas aquellas situaciones que, como consecuencia de alteraciones y procesos patológicos diversos, conllevan a una pérdida de esta homeóstasis. En respuesta a todas estas situaciones, las células de microglía son capaces de detectar rápidamente el daño y actuar de una manera específica en función del tipo de perturbación que se produzca en su entorno. Esta respuesta microglial ha sido ampliamente estudiada en daños agudos, en los que la microglía actúa como parte del sistema inmune innato, sin embargo los procesos que subyacen a la reactividad microglial ante una situación de inmunidad adquirida, así como la comunicación que se establece entre estas células microgliales y las células inmunes periféricas infiltradas permanecen en muchos aspectos sin esclarecer. En el presente trabajo hemos caracterizado el patrón de reactividad microglial y su relación con las diferentes poblaciones de linfocitos infiltrados a lo largo de las diferentes fases de la evolución que acontecen en un modelo agudo de encefalopatía autoinmune experimental (EAE). Nuestros estudios demuestran que las células de microglía se activan en respuesta a la inducción de la EAE y presentan un patrón de activación específico en cada una de las fases. Durante la fase de inducción y pico del proceso patológico, en estrecha relación con el aumento de la sintomatología clínica que se manifiesta por un progresivo deterioro de las funciones motoras, estas células microgliales experimentan cambios morfológicos y en su distribución acumulándose alrededor de los vasos sanguíneos. Además de microglía reactiva y posiblemente de macrófagos de origen sanguíneo, se observa también un gran número de linfocitos infiltrados, mayoritariamente del tipo T-cooperador y subtipo Th1 (pro-inflamatorio), si bien también se observan linfocitos T-citotóxicos y T-γδ. En estas fases, la activación microglial se caracteriza a nivel fenotípico por el aumento en la expresión de moléculas del complejo mayor de histocompatibilidad clase I y clase II (MHC-clase I y MHC-clase II) sin expresión concomitante de moléculas co-estimuladoras B7.1 o B7.2, es decir las células de microglía activadas presentan en estas circunstancias un fenotipo característico de células dendríticas inmaduras; hecho que hemos corroborado con la demostración de la expresión del marcador CD1. En este contexto, la señal que inducen estas células de microglía a los linfocitos infiltrados, podría estar implicada en la modulación del proceso inflamatorio induciendo la apoptosis o anergia linfocitaria. Durante la fase de recuperación, a pesar de que los animales experimentan una progresiva mejora sintomatológica, las células de microglía siguen mostrando una morfología, distribución y patrón de expresión de moléculas característicos de células reactivas. Si bien, en general, las células de microglía siguen mostrando el mismo patrón fenotípico (CD1+, MHC-I+, MHC-II+, B7.1- y B7.2-), aquellas localizadas en el entorno de algunos vasos sanguíneos expresan diferencialmente la molécula B7.2. Durante esta fase, además, el número total de linfocitos T-cooperadores, T-citotóxicos y T-γδ se mantiene muy elevado con valores similares a los observados en las fases anteriores. Es interesante señalar que ya no encontramos linfocitos Th1 y la población de T-cooperadores está constituida por los subtipos Th17 y T-regs. Los linfocitos que se acumulan en las inmediaciones de los vasos sanguíneos expresan CTLA-4, uno de los co-receptores de B7.2. En este nuevo contexto, la interacción de las células de microglía B7.2+ con estos linfocitos CTLA-4+ podría ser la responsable de la resolución de la respuesta immunitaria y la inducción de la tolerancia. En su conjunto los resultados obtenidos evidencian que la microglía juega un papel clave en la evolución de la respuesta inmune adquirida modulando la activación e inactivación de las diferentes poblaciones linfocitarias implicadas tanto en la inducción del proceso inmune/inflamatorio como en su posterior resolución. La realización de este trabajo ha sido financiada por las siguientes ayudas: Beca pre-doctoral de formación de investigadores de la UAB (UAB2004-11), Fundación La Maratón de TV3, Fundación Alicia Koplowitz, Fundación Uriach y Ministerio Español de Ciencia e Innovación (BFU2005-02783, BFU2008-04407). / Over the years, numerous studies have demonstrated the fundamental role played by microglial cells in the CNS, not only in normal conditions where they control the correct tissue homeostasis, but also in all those situations in which, as a result of alterations and pathologies, homeostasis may be disturbed. Thus, when the integrity of the nervous tissue is disrupted, microglial cells are activated, showing specific activation patterns which fully depend on changes in the particular micro-environment. The microglial response has been widely studied in acute injuries in which microglia act as an intrinsic element of the innate immune system. However, the processes underlying microglial reactivity in situations of acquired immunity, as well as the relationship established between these microglial cells and infiltrating peripheral immune cells, remain poorly understood. In this study we have characterized the pattern of microglial reactivity and their relationship with the different populations of infiltrated lymphocytes, along the evolution of an acute model of experimental autoimmune encephalopathy (EAE) induced in Lewis rat. Our studies have demonstrated that microglial cells became activated in response to EAE induction, showing a specific activation pattern in each phase along disease evolution. During the inductive and peak phases, microglial cells showed changes in morphology and distribution, in close association with the increase of clinical symptoms, manifested by a progressive deterioration of motor function. These microglial cells progressively shorten their ramifications leading to amoeboid morphologies. Microglia and blood-borne macrophages increased in number and accumulated around blood vessels. In addition, a large number of infiltrated lymphocytes were also detected during the inductive and peak phases. These lymphocytes belong mostly to the T-helper phenotype (pro-inflammatory Th1 cells), although T-cytotoxic and γδ T-cells were also observed. Activated microglial cells displayed an immature dendritic cell phenotype characterized by expression of CD1 and major histocompatibility complexes class I and class II (MHC-class I and MHC-class II) without concomitant expression of B7.1 or B7.2 co-stimulatory molecules. In addition, these activated microglia expressed CD1, a marker of immature dendritic cells. In this context, the signal that these immature dendritic cell-like microglial cells induced to infiltrated lymphocytes may provoke lymphocyte apoptosis or anergy. During the recovery phase, animals experienced a gradual improvement in symptomatology, although microglial cells in this phase still showed a morphology, distribution and phenotype characteristics of reactive cells. In general, these microglial cells displayed the same immature dendritic cell phenotype (CD1+, MHC-I+, MHC-II+, B7.1- and B7.2-) observed during earlier phases. Noticeably, microglial cells located around blood vessels express B7.2. The total number of T-helper, T-cytotoxic and γδ T-cells remained very high with values close to those observed during the inductive and peak phases. Interestingly, we do not find Th1 lymphocytes during the recovery phase, and the population of T-helper cells mainly consists of Th17 and T-regs subtypes. Lymphocytes accumulated in the vicinity of blood vessels expressed CTLA-4, one of the B7.2 co¬receptors. In this new context, the interaction between B7.2+ microglial cells and CTLA-4+ lymphocytes could be responsible for the immune response resolution and the induction of subsequent tolerance. Altogether, these results show that microglia play a key role in the evolution of the acquired immune response, modulating the activation and inactivation of the different lymphocyte populations involved in both the induction of the immune/inflammatory process and its subsequent resolution. This work was supported by: UAB Pre-doctoral fellow (UAB2004-11), Fundación La Maratón de TV3, Fundación Alicia Koplowitz, Fundación Uriach y Ministerio Español de Ciencia e Innovación (BFU2005-02783, BFU2008-04407).

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