A insulina regula a expressão de GLUT4, porém os mecanismos envolvidos nesta regulação não estão definidos. Alguns fatores de transcrição e proteínas cinases estão relacionados com a expressão de GLUT4. Assim, o objetivo desta pesquisa foi investigar a participação dos fatores de transcrição MEF2, HIF-1<font face=\"Symbol\">a e NF-<font face=\"Symbol\">kB, e das proteínas cinases mTOR, PI3K e AKT na regulação da expressão de Slc2a4/GLUT4 induzida pela insulina. Para isso, músculos sóleos de ratos foram incubados por 3 horas em tampão Krebs, tratados ou não com insulina, wortmanina, rapamicina, ML-9 ou TNF-<font face=\"Symbol\">a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-<font face=\"Symbol\">kB, AT-rich element e E-Box do promotor do gene Slc2a4 (EMSA). O tratamento com insulina aumentou a expressão de Slc2a4/GLUT4 no músculo sóleo, in vitro, ativando os fatores de transcrição MEF2A/D e possivelmente MyoD, através da via da PI3K/AKT e diminuindo a expressão e atividade de NF-<font face=\"Symbol\">kB. / Insulin regulates the GLUT4 expression, but the mechanisms involved in this regulation are not defined. Some transcription factors and protein kinases are related to the expression of GLUT4. Thus, the aim of this research was to investigate the role of the transcription factors MEF2, HIF-1<font face=\"symbol\">a and NF-<font face=\"Symbol\">kB, and the proteins kinases mTOR, PI3K and AKT, in regulation of Slc2a4 and GLUT4 expression by insulin. For this, rat soleus muscles were incubated for 3 hours in Krebs buffer, treated or not with insulin, wortmanina, rapamycin, ML-9 or TNF-<font face=\"Symbol\">a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-<font face=\"Symbol\">kB, AT-rich element and E-Box in the promoter of the gene Slc2a4 (EMSA). Insulin treatment increased the expression of Slc2a4/GLUT4 in the soleus muscle in vitro, activating the transcription factors MEF2A/D and possibly MyoD, via PI3K/AKT and decreasing the expression and activity of NF-<font face=\"Symbol\">kB.

Incontinentia pigmenti (IP, OMIM # 308300) est une maladie génétique liée au chromosome X qui est létale chez les garçons. Chez les filles, une dermatose débute peu après la naissance et évolue selon une séquence complexe d’événements associant de l’inflammation, de l’hyperprolifération cellulaire et de l’apoptose. En dehors de ce problème cutané, les patientes IP peuvent aussi souffrir d’anomalies oculaires, dentaires et cérébrales. Les défauts cérébraux affectent environ 30% des patientes et se caractérisent par de l’épilepsie et des troubles cognitifs et/ou moteurs. Le gène causant IP code pour la protéine NEMO, un composant essentiel de la voie de signalisation NF-kB, qui régule les processus immunitaires, inflammatoires, ainsi que la prolifération et la mort cellulaire. Chez approximativement 70% des patientes IP, le même réarrangement chromosomique élimine presque intégralement le gène NEMO, générant une perte d’activation de la voie NF-kB. L’invalidation du gène Nemo chez la souris (femelles Nemo +/-) fournit un modèle pour l’étude de la dermatose associée à IP en récapitulant les événements cutanés observés chez les patientes.Dans le but de mieux définir les anomalies cérébrales des patientes IP ainsi que leur origine qui reste obscure, nous avons analysé le cerveau des souris Nemo +/-. Nous montrons ici, en utilisant un protocole d’IRM adapté à des cerveaux de sept jours (P7), que des anomalies sont détectées dans une fraction des échantillons. Ces anomalies sont hétérogènes et localisées de manière aspécifique, comme chez l’Homme. Elles incluent des zones hémorragiques diffuses, des cavités et de l’atrophie du corps calleux. Cela démontre l’utilité du système murin Nemo +/- pour étudier également les défauts cérébraux des patientes IP. De manière intéressante, ces lésions cérébrales peuvent être détectées avant la naissance, au jour 18 du développement, et ne sont pas causées par un dysfonctionnement neuronal, astrocytaire ou des oligodendrocytes. Utilisant une approche immunitaire couplée à une analyse in toto de cerveaux P7 après mise en transparence (Technique 3DISCO), nous avons détecté des anomalies vasculaires qui suggèrent que ce compartiment serait à l’origine des défauts cérébraux des patientes IP. / Incontinentia pigmenti (IP, OMIM # 308300) is an X-linked genetic disease which is lethal in boys. In girls, it causes a skin disease that begins soon after birth and evolves along a complex sequence of events involving inflammation, cell hyperproliferation and apoptosis. IP patients can also suffer from ocular, dental and brain anomalies. Brain defects affect about 30% of patients and are characterized by epilepsy and/or cognitive/motor disorders. The IP-causing gene encodes the protein NEMO, an essential component of the signaling pathway NF-kB that regulates immunity, inflammation, proliferation and cell death process. In approximately 70% of IP patients, the same chromosomal rearrangement eliminates almost completely the NEMO gene, generating a loss of activation of the NF-kB pathway. Invalidation of the gene in Nemo mice (Nemo +/- females) provides a model for the study of the cutaneous events observed in IP patients.In order to better define the IP brain anomalies of patients and their origin, which remains obscure, we analyzed the brains of Nemo +/- mice. We show here, using a MRI protocol adapted for brain of seven days (P7), that anomalies are detected in a fraction of the samples. These anomalies are heterogeneous and localized in a non-specific manner, as in humans. They include diffuse hemorrhagic areas, cavities and atrophy of the corpus callosum. This demonstrates the usefulness of the murine system Nemo +/- to also study the IP patient's brain defects. Interestingly, these brain lesions can be detected before birth, at day 18 of development, and are not caused by a neuron, astrocyte or oligodendrocyte dysfunction. Using an immune approach coupled to an in toto analysis of P7 brain, after treating them by chemicals to induce their transparency (iDISCO technique), we detected vascular anomalies, suggesting that this compartment causes the brain defects in IP patients.

Le système immunitaire inné est un mécanisme de défense commun à tous les métazoaires. Son activation peut être délétère lorsqu'elle est incontrôlée. L'étude des mécanismes qui sous-tendent cet équilibre entre l'activation ou non de la réponse immunitaire innée est à la base de mes travaux de thèse. La similarité entre les voies moléculaires - comme la voie NF-KB - relayant la réponse immunitaire innée chez les insectes et les mammifères fait de la drosophile un excellent modèle pour explorer la réponse immune. Après une stimulation immunitaire, l'arrêt des voies moléculaires de l'immunité est nécessaire pour éviter le développement de maladies auto-immunes ou du cancer. Mon premier projet s'est attaché à comprendre un mode de régulation original dépendant du temps, dans une des voies NF-KB de la drosophile. Mon deuxième projet··concerne l'activation de la réponse immunitaire. Une· protéine nucléaire contrôle l'implication de machinerie épigénétique dans le contrôle de l'expression d'une des voies NF-KB de la drosophile. Le tout permet de mieux saisir la dynamique de régulation de la réponse innée. / The innate immune system is a defense mechanism common to all metazoans. lts activation can be deleterious when it is uncontrolled. The study of the mechanisms underlying this balance between the activation or not of the innate immune response is the basis of my thesis work. The similarity of the molecular pathways - such as the NF-KB pathway - relaying the innate immune response in insects and mammals makes Drosophila an excellent model for exploring the immune response.After immune stimulation, stopping the molecular pathways of immunity is necessary to prevent the development of autoimmune diseases or cancer. My first project focused on understanding a time-dependent mode of regulation in one of Drosophila's NF-KB pathways. My second project concerns the activation of the immune response. A nuclear protein contrai the involvement of epigenetic machinery in controlling the expression of one of Drosophila's NF-KB pathways. Ali this makes it possible to better grasp the dynamics of regulation of the innate response.

Les lymphocytes T CD4+FoxP3+ régulateurs (Tregs) jouent un rôle majeur dans l'homéostasie du système immunitaire et la prévention des maladies auto-immunes en régulant les réponses immunitaires. Aussi bien chez la souris que chez l'homme, il est établi que des mutations de Foxp3 entraînent une déficience en Tregs qui induit un syndrome auto-immun conduisant à la mort. Bien que Foxp3 soit essentiel à la différenciation, la fonction et la stabilité des Tregs, ce n'est pas le seul facteur de transcription impliqué dans ces processus. De plus en plus d'études suggèrent notamment un rôle important de NF- B dans le développement et la fonction des Tregs mais celui-ci reste mal défini. Nous avons donc généré des souris ayant une délétion spécifique des sous-unités RelA ou RelB de NF- KB dans les Tregs. L'invalidation de RelA dans les Tregs conduit au développement d'un syndrome auto-immun sévère qui s'explique par un défaut fonctionnel et de stabilité des Tregs. L'invalidation de RelB dans les Tregs semble, quant à elle, augmenter leur fonction suppressive. Nous montrons ainsi un rôle majeur de NF- KB dans la fonction des Tregs ouvrant la voie à de nouveaux traitements qui stimuleraient ou inhiberaient les Tregs en modulant l'activation de NF- KB. / CD4+ Foxp3+ regulatory T cells (Tregs) play a critical role in immune homeostasis and in the prevention of autoimmune diseases by regulating immune responses. In humans and mice, it is well established that Foxp3 deficiency conducts to the development of an autoimmune syndrome leading to early death. Although Foxp3 plays a critical role in differentiation, suppressive function and stability of Tregs, other transcription factors are also involved in different aspects of their biology. Even though increasing evidence shows an important role of NF-KB transcription factors in Treg cells development and function, their role is still poorly defined. To address this question we have generated conditional knock-out mice for RelA or RelB subunits of NF-KB only in Tregs. We show that the deficiency of RelA in Tregs leads to the development of a spontaneous severe autoimmune syndrome that could be explained by a defect in Tregs activation and stability. However, the deficiency of RelB seems to increase their suppressive function. Altogether, our data show a major role of NF-KB in Treg biology. This work could lead to new treatments that would stimulate or inhibit Tregs through the modulation of NF-KB activation.

A obesidade é um estado inflamatório crônico. As estatinas têm efeito antiinflamatório e podem afetar a homeostase glicêmica. Estudos, nesse sentido são contraditórios e pouco se sabe sobre os mecanismos moleculares envolvidos. Este estudo verificou em animais obesos por glutamato monossódico (MSG) que além de apresentaram resistência à insulina in vivo, o tecido adiposo branco (TAB) desses animais mostrou aumento de infiltração de macrófagos, fosforilação de IKK-a/b, expressão de mRNA de TNF-a and IL-6, e redução de mRNA e proteína de GLUT4. O tratamento com atorvastatina por 4 semanas restabeleceu a sensibilidade à insulina in vivo, reduziu a inflamação e restabeleceu a expressão de GLUT4 no TAB dos animais obesos. Adicionalmente, esse trabalho encontrou sítios de ligação de NF-kB no promotor do gene GLUT4, sugerindo ligação entre resistência à insulina e inflamação. Em conclusão, a obesidade induzida por MSG em camundongos acompanha-se de resistência à insulina in vivo e atividade inflamatória crônica no tecido adiposo, com prejuízo da expressão de GLUT4. A atorvastatina melhorou esses aspectos, sugerindo que essa estatina tenha efeitos antiinflamatórios que podem melhorar a resistência à insulina na obesidade. / Obesity is a chronic inflammatory state. Statins have anti-inflammatory effects and may affect glucose homeostasis; therefore, few are known about the molecular mechanisms. Considering that inflammation contributes to insulin resistance, the aim of the present study was to investigate if atorvastatin treatment has anti-inflammatory, and consequently insulin sensitization action in white adipose tissue (WAT) of obese mice. WAT of insulin-resistant obese mice showed increased macrophage infiltration, IKK-a and IKK-b phosphorylation, TNF-a and IL-6 mRNA expression and decreased GLUT4 mRNA and protein expression. Atorvastatin restored whole-body insulin sensitivity, decreased macrophage infiltration and normalized IKK-a/b phosphorylation, TNF-a, IL-6 and GLUT4 mRNA and GLUT4 protein to control levels. Moreover, NF-kB binding sites were found in GLUT4 gene promoter, pointing out an association between insulin resistance and inflammation. Together, atorvastatin anti-inflammatory effects on WAT may be important to its local and whole-body insulin sensitization effects.

A exposição à radiação ultravioleta (RUV) pode provocar desequilíbrio no balanço oxidante/antioxidante da pele, causando prejuízos à sua integridade e levando a diversas alterações, entre as quais o envelhecimento precoce e o câncer de pele. Considerando a estreita relação entre o aumento do estresse oxidativo e os efeitos danosos causados pela RUV na pele, aliado ao fato de que estudos epidemiológicos demonstram que o uso de protetores ou bloqueadores solares não é completamente efetivo na prevenção dos diversos malefícios causados pela exposição à RUV, o uso de antioxidantes aparece como importante alternativa nas terapias de fotoproteção. A administração tópica de antioxidantes, como a quercetina, poderia afetar as alterações moleculares desencadeadas pela RUV e conseqüentemente as seqüelas biológicas e clínicas resultantes das mesmas. Desta forma, na presente pesquisa, sistema microemulsionado para a liberação cutânea de quercetina foi obtido, caracterizado e avaliado quanto a sua capacidade em promover maior penetração cutânea deste ativo, estabilidade, segurança e eficácia in vivo contra os danos na pele causados pela exposição à RUV. Além disso, o efeito da quercetina contra diferentes alterações moleculares induzidas pela RUV foi também avaliado, com o objetivo de investigar os possíveis mecanismos de ação fotoprotetora deste flavonóide. Os resultados demonstram que a incorporação da quercetina em sistema microemulsionado aumentou a penetração cutânea in vitro e in vivo deste flavonóide sem causar irritação, sendo, portanto, uma importante estratégia para melhorar a liberação tópica da quercetina. O estudo de estabilidade demonstra a necessidade de armazenamento deste sistema a 4°C para manutenção de sua funcionalidade. A microemulsão contendo quercetina inibiu a depleção do antioxidante endógeno GSH, assim como o aumento da atividade/secreção de proteinases e da atividade da MPO, induzidos pela exposição à RUVB. O pré-tratamento de queratinócitos com quercetina não alterou a indução pela RUV das MAP quinases, conseqüentemente não houve inibição na elevação dos níveis de c-Jun e c-Fos, assim como no aumento da produção das MMPs 1 e 3, mas por outro lado foi efetivo contra o aumento na produção das citocinas IL-1, IL-6, IL-8 e TNF-. Finalmente, demonstrou-se que a ação fotoprotetora da quercetina contra os danos na pele causados pela RUV é mediada principalmente pela inibição da via de sinalização do NF-kB, uma vez que, enquanto o pré-tratamento de queratinócitos com quercetina diminuiu a ativação deste fator de transcrição, nenhum efeito contra a indução da via de sinalização da AP-1 foi observado. Concluindo, este trabalho sugere a incorporação da quercetina em sistema microemulsionado como estratégia relevante no combate ao aparecimento de desordens cutâneas causadas pela exposição à RUV, além de contribuir para a elucidação, pelo menos em parte, do mecanismo de ação fotoprotetora da quercetina contra alterações moleculares induzidas pela RUV. / The ultraviolet radiation (UVR) exposition may lead to the skin oxidant/antioxidant imbalance injuring its integrity and leading to several disorders, such as ageing and skin cancer. Considering the close relationship between the increase in oxidative stress and UV-induced skin damages, together with the fact that epidemiological studies indicate that the use of sunscreen and sun block are not completely effective in preventing UV-induced damages, the use of antioxidants arises as an important approach to photoprotection therapies. The topical use of antioxidants, such as quercetin, would affect the molecular changes induced by UV and subsequent biological and clinical sequela. Therefore, in the present study, microemulsion system for topical delivery of quercetin was obtained, characterized and evaluated with regards to its capability to increase skin penetration of quercetin, stability, toxicity and in vivo effectiveness against UV-induced skin damages. Moreover, quercetin effect against different UV-induced molecular changes was also assessed, in order to investigate the possible photoprotective mechanisms of action of this flavonoid. The results demonstrate that the incorporation of quercetin into microemulsion increased the in vitro and in vivo skin penetration of this flavonoid without causing skin irritation, being an important strategy to improve the topical delivery of quercetin. The stability study demonstrate the necessity to storage this system at 4°C to maintain its functionality. The microemulsion containing quercetin inhibited the depletion of the endogenous antioxidant GSH, as well as the increase in proteinases activity/secretion and MPO activity induced by UVB irradiation exposure. The pretreatment of keratinocytes with quercetin had no blocking effect on UV activation of MAP kinases, consequently, there was no inhibition in the c-Jun and c-Fos levels, as well as in the induction of MMPs 1 and 3, on the other hand, it was effective against the increase in the production of cytokines IL-1, IL-6, IL-8 e TNF-. Finally, it was demonstrated that the photoprotective action of quercetin against UV-induced skin damages is mediated mainly by suppression of NF-kB signaling pathway, once, while the pretreatment of keratinocytes with quercetin suppressed the activation of this transcription factor, no effect was observed against UV-induced AP-1 activation. In conclusion, the present study suggests the incorporation of quercetin into microemulsion system as a relevant strategy to prevent UV-induced skin disorders, and contribute, at least in part, to the elucidation of quercetin photoprotective mechanism of action against UV-induced molecular changes.

O reconhecimento da flagelina é compartilhado pelo receptor transmembrânico TLR5 e citosólico NAIP5/NLRC4. Entretanto, pouco se sabe sobre os mecanismos efetores individuais induzidos a partir do reconhecimento extra e intracelular da flagelina. Aqui, nós demonstramos que macrófagos estimulados com a flagelina citosólica (FLA-BSDot) induziu a expressão de iNOS, enzima responsável pela produção do óxido nítrico (NO). A expressão de iNOS foi dependente do eixo NAIP5/NLRC4/caspase-1 e independente de IL-1&beta;, IL-18 e MyD88, descartando a via de ativação dos TLRs. Ainda, esta via não requer a ativação do fator de transcrição IRF-1, mas envolve a ativação do NF-kB, assim como a clivagem da enzima PARP-1 (poly(ADP-ribose)polymerase-1). Por fim, avaliamos a relevância biológica desta via no controle das infecções por L. pneumophila e S. Typhimurium, dados que definem um mecanismo efetor adicional no controle de patógenos. / Recognition of flagellin is shared by transmembranic TLR5 and cytosolic NAIP5/NLRC4. However, little is known about the individual effector mechanisms induced by extra and intracellular flagellin. Here, we have demonstrated that cytosolic flagellin-stimulated macrophages (FLA-BSDot) induced iNOS expression, an enzyme responsible for the production of nitric oxide (NO). iNOS expression was dependent of the NAIP5/NLRC4/caspase-1 axis and independent of IL-1&beta;, IL-18 and MyD88, discarding TLRs signaling pathway. Still, this pathway do not require the activation of IRF-1 transcriptional factor, but involves NF-kB activation as well as the cleavage of the enzyme, PARP-1 (poly(ADP-ribose)polymerase-1). Finally, we have evaluated the biological relevance of this pathway in the control of the infections by L. pneumophila e S. Typhimurium, which define an additional effector mechanism to the control of pathogens.

A insulina regula a expressão de GLUT4, porém os mecanismos envolvidos nesta regulação não estão definidos. Alguns fatores de transcrição e proteínas cinases estão relacionados com a expressão de GLUT4. Assim, o objetivo desta pesquisa foi investigar a participação dos fatores de transcrição MEF2, HIF-1<font face=\"Symbol\">a e NF-<font face=\"Symbol\">kB, e das proteínas cinases mTOR, PI3K e AKT na regulação da expressão de Slc2a4/GLUT4 induzida pela insulina. Para isso, músculos sóleos de ratos foram incubados por 3 horas em tampão Krebs, tratados ou não com insulina, wortmanina, rapamicina, ML-9 ou TNF-<font face=\"Symbol\">a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-<font face=\"Symbol\">kB, AT-rich element e E-Box do promotor do gene Slc2a4 (EMSA). O tratamento com insulina aumentou a expressão de Slc2a4/GLUT4 no músculo sóleo, in vitro, ativando os fatores de transcrição MEF2A/D e possivelmente MyoD, através da via da PI3K/AKT e diminuindo a expressão e atividade de NF-<font face=\"Symbol\">kB. / Insulin regulates the GLUT4 expression, but the mechanisms involved in this regulation are not defined. Some transcription factors and protein kinases are related to the expression of GLUT4. Thus, the aim of this research was to investigate the role of the transcription factors MEF2, HIF-1<font face=\"symbol\">a and NF-<font face=\"Symbol\">kB, and the proteins kinases mTOR, PI3K and AKT, in regulation of Slc2a4 and GLUT4 expression by insulin. For this, rat soleus muscles were incubated for 3 hours in Krebs buffer, treated or not with insulin, wortmanina, rapamycin, ML-9 or TNF-<font face=\"Symbol\">a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-<font face=\"Symbol\">kB, AT-rich element and E-Box in the promoter of the gene Slc2a4 (EMSA). Insulin treatment increased the expression of Slc2a4/GLUT4 in the soleus muscle in vitro, activating the transcription factors MEF2A/D and possibly MyoD, via PI3K/AKT and decreasing the expression and activity of NF-<font face=\"Symbol\">kB.

Clostridium difficile (CD) est l’entéropathogène le plus fréquemment responsable d'infections nosocomiales intestinales post-antibiotiques. L'apparition de cas graves liés à l'émergence de souches hypervirulentes ces dernières années a contribué à accroître la morbidité et la mortalité. Les toxines TcdA et TcdB contribuent directement aux lésions intestinales associées aux infections à CD (ICD), mais d'autres facteurs bactériens sont nécessaires à l’adhésion et la colonisation intestinale. Les flagelles de CD, qui confèrent la mobilité et la chimiotaxie, pourraient jouer un rôle dans la pathogenèse en contribuant à la réponse inflammatoire de l’hôte et aux lésions de la muqueuse. En effet, en activant le récepteur TLR5, les flagelles peuvent provoquer l'activation des cascades de signalisation cellulaire des MAPK et de NF-κB conduisant à la sécrétion de cytokines pro-inflammatoires. Notre objectif était d’étudier ce rôle potentiel des flagelles de CD in vitro et in vivo. Nous avons montré que l'interaction de la flagelline de CD avec le TLR5 active principalement la voie de NF-κB, et, dans une moindre mesure, la voie des MAPK, conduisant ainsi à la régulation de l'expression de gènes pro-inflammatoires et à la synthèse de médiateurs pro-inflammatoires. De plus, en utilisant un modèle murin d’ICD, nous avons démontré un effet synergique des flagelles et des toxines dans l’induction d’une réponse inflammatoire de la muqueuse caecale. Dans ce modèle, l'absence de flagelles diminue considérablement le degré d'inflammation de la muqueuse caecale et la seule présence de toxines, sans flagelles, ne suffit pas à provoquer d’importantes lésions épithéliales. Ces résultats mettent en évidence le rôle des flagelles de CD dans l’induction d’une réponse inflammatoire intestinale en synergie avec l’action des toxines bactériennes sur l'épithélium. / Clostridium difficile (CD) is the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. The appearance of severe cases related to the emergence of hypervirulent strains these last years has contributed to increased mortality and morbidity. The CD TcdA and TcdB toxins contribute directly to CD infection (CDI)-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the gut. The CD flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. Our objective was to study the potential role of CD flagella in vitro and in vivo. We reported that the interaction of CD flagellin-TLR5 predominantly activates the NF-κB, and, in a lesser degree, the MAPKs pathways, thus leading to up-regulation of pro-inflammatory gene expression and subsequent synthesis of pro-inflammatory mediators. Moreover, by using a mouse model of CDI, we demonstrated a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreased the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of CD flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

Clostridium difficile (CD) représente la première cause d'infections digestives nosocomiales dans les pays développés. Les infections à CD (ICD) induisent une inflammation intestinale importante qui se manifeste principalement par des colites pseudomembraneuses ainsi que par un taux de mortalité élevé. Les facteurs majeurs de virulence sont les toxines TcdA et TcdB. Dans la première partie des travaux de cette thèse, nous avons validé le rôle in vivo d'un autre facteur bactérien, les flagelles, dans un modèle murin d'ICD, en montrant que les flagelles induisent une réponse inflammatoire au niveau de la muqueuse caecale en synergie avec les toxines. Nous avons ensuite montré une régulation de de cette réponse par un microARN (miARN) exerçant un rôle anti-inflammatoire en modulant l'activation de la voie de signalisation de NF-KB. Le traitement des souris infectées par ce miARN réduit l'inflammation intestinale apportant la preuve du concept pour une nouvelle approche thérapeutique. / Clostridium difficile (CD) is the leading cause of nosocomial digestive infections in developed countries. CD infections (CDI) induce significant intestinal inflammation that is manifested primarily by pseudomembranous colitis and a high mortality rate. The major virulence factors are TcdA and TcdB toxins. In the first part of the work of this thesis, we validated the in vivo role of another bacterial factor, the flagella, in a mouse model of CDI, showing that the flagella induce an inflammatory response in ceacal mucosa in synergy with toxins. We then showed a regulation of this response by a microRNA (miRNA) exerting an anti-inflammatory role by modulating the activation of the NF-KB signaling pathway. The treatment of mice infected with this miRNA reduces intestinal inflammation providing proof of concept for a new therapeutic approach.