<p>No</p> / Dissertation

Conformément à l'hypothèse de l'hygiène, les infections peuvent protéger des désordres immunologiques tels les maladies autoimmunes ou allergiques. Nous avons ainsi montré que la stimulation des TLR qui détectent les infections, peut protéger du diabète de type 1. En effet, nous démontrons que le traitement de souris NOD, développant un diabète spontané, par des agonistes des TLR2, 3, 4 et 7 protège les animaux du développement de cette pathologie. Bien que toutes ces structures exercent un effet protecteur, les mécanismes impliqués s'avèrent différents en fonction du récepteur considéré. Néanmoins, la stimulation de ces récepteurs semble mettre en jeu des populations de cellules régulatrices et des cytokines immunorégulatrices. Nous avons d'ailleurs montré que la protection induite par des agonistes de TLR2 et TLR3 sont dépendantes des cellules iNKT avec en plus une implication de l'IL-4 pour TLR3. Par ailleurs, nous avons mis en évidence dans un modèle d'asthme allergique que la protection induite par un agoniste de TLR7 était portée par les lymphocytes iNKT et l'IFN-γ. Afin de comprendre les mécanismes de ce ciblage iNKT par les TLR, nous avons réalisé différentes expériences in vitro et avons pu observer que l'agoniste de TLR7 ciblait directement les iNKT et que la réponse ne nécessitait pas la présence de cellules présentatrices de l'antigène. En conclusion, nos travaux ont permis de mettre en évidence que le ciblage des voies de signalisation TLR permet de moduler les réponses immunitaires, et notamment celles dues à une perte de tolérance comme dans le cas de désordres immunologiques.

No description available.

Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/ BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1b-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15 min prior to IL-1β stimulation for 24 h. AS-1 treatment significantly attenuated IL-1β-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.

We examined whether blocking the MyD88 mediated pathway could protect myocardium from ischemia/reperfusion (I/R) injury by transfecting Ad5-dnMyD88 into the myocardium of rats (n = 8) 3 days before the hearts were subjected to ischemia (45 min) and reperfusion (4 h). Ad5-GFP served as control (n = 8). One group of rats was (n = 8) subjected to I/R without transfection. Transfection of Ad5-dnMyD88 significantly reduced infarct size by 53.6% compared with the I/R group (15.1 ± 3.02 vs 32.5 ± 2.59) while transfection of Ad5-GFP did not affect I/R induced myocardial injury (35.4 ± 2.59 vs 32.5 ± 2.59). Transfection of Ad5-dnMyD88 significantly inhibited I/R-enhanced NFκB activity by 50% and increased the levels of phospho-Akt by 35.6% and BCL-2 by 81%, respectively. Cardiac myocyte apoptosis after I/R was significantly reduced by 59% in the Ad5-dnMyD88 group. The results demonstrate that both inhibition of the NFκB activation pathway and activation of the Akt signaling pathway may be responsible for the protective effect of transfection of dominant negative MyD88.

Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii, and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF -/- and MyD88 -/- mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis, partially explaining the susceptibility of MyD88 -/- mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.

A asma ocupacional é a forma mais comum de doença pulmonar relacionada ao trabalho e vários dos casos reportados estão correlacionados à exposição de proteases. A serino protease subtilisina foi bastante utilizada na década de 60 e foi a principal responsável pela alta incidência de asma na indústria de detergente. Este projeto visou a desenvolver um modelo murino de inflamação alérgica pulmonar à subtilisina e caracterizar os mecanismos principais envolvidos nessa resposta. A sensibilização e desafio com subtilisina induziu doença alérgica pulmonar, verificada pela eosinofilia às vias aéreas, produção de muco, IgE total, hiper reatividade brônquica e produção de citocinas tipo II no pulmão. Estas respostas foram dependentes da atividade enzimática da subtilisina, PAR-2, receptor de IL-33 ST2, IL-1R e da sinalização via MyD88. Em conjunto, nossos resultados estabelecem um novo modelo experimental de asma ocupacional induzida por subtilisina e fornece os principais mecanismos moleculares responsáveis pela inflamação alérgica. / Occupational asthma is the most common form of pulmonary disease related to work. Most of occupational asthma cases reported are strictly correlated with proteases exposure. Serine protease subtilisin was widely used in the detergent industry during the 60s, which resulted in increased incidence of occupational asthma. We aimed to develop and characterize a murine model of occupational asthma using subtilisin as allergen. Briefly, sensitization and challenge with subtilisin triggered lung allergic inflammation, as accessed by eosinophilic influx to the airways, mucus production, and increased levels of type II cytokines. Subtilisin induced total IgE and airway hyperactivity. Allergic responses to subtilisin were dependent on its serine protease activity, protease-activated receptor (PAR)-2, IL-33 receptor ST2, IL-1R, and Myd88 signaling. Together, these data establish a new murine model of occupational asthma induced by subtilisin and provide the main molecular mechanisms responsible for allergic inflammation.

INTRODUÇÃO: O receptor tipo Toll 4 (TLR4) pertencente a uma família de receptores do sistema imune inato, reconhece o padrão molecular de lipopolissacarídeos (LPS), expressos por bactérias Gram negativas. Sua cascata de sinalização, nas células apresentadoras de antígeno, ocorre por duas vias principais: MyD88-dependente, que resulta na ativação de NF-B e na expressão de genes de resposta inflamatória e MyD88-independente, responsável pela ativação dos fatores IRF3 e IRF7, culminando na síntese de interferons e , envolvidos na resposta anti-viral e anti-bacteriana. Células não-imunes, de diversos tecidos, também expressam TLR4, incluindo células pancreáticas murinas e humanas. Devido ao seu papel nos processos inflamatórios, os TLR estão implicados em doenças crônicas como obesidade e diabetes. Estudo anterior do grupo identificou TLR4 como uma molécula que ativa sinais inflamatórios e provoca alterações na homeostase das células . Neste trabalho, investigamos qual via é ativada por LPS e quais os efeitos da expressão do TLR4 na viabilidade celular e na produção de insulina em células murinas. MÉTODOS: Células MIN6 (linhagem celular de insulinoma de camundongo) foram cultivadas em condições de hipo (2,8mM glicose), normo (5,6mM glicose) e hiperglicemia (11,2mM glicose), por 4 dias. Após esse período, foi adicionado LPS (50 ng/mL) por 48h e foram feitas análises por PCR em tempo real, Western Blot, ELISA e citometria de fluxo. RESULTADOS: Os resultados confirmam o aumento de TLR4 em células em condições de hiperglicemia e a via de sinalização ativada por LPS é a via MyD88-dependente, envolvida na produção de citocinas pró-inflamatórias. A via de indução de intérferons tipo 1 está ausente nestas células. Além disso, TLR4 ativado por LPS aumentou secreção de insulina em resposta a glicose, mas não induziu a morte celular. CONCLUSÃO: A expressão de TLR4 em células pancreáticas murinas é induzida em resposta ao aumento da glicemia, constituindo um novo elo entre a agressão à célula causada por altos níveis de glicose e a alteração da função celular induzida por LPS / INTRODUCTION: Toll-like receptor 4 (TLR4) belongs to a family of innate immunity receptors and recognizes the molecular pattern present in lipopolysaccharides (LPS), typical of Gram-negative bacteria. There are two TLR4 signaling pathways, typically in antigen-presenting cells: one is MyD88-dependent, activating NF-kB transcription factor and triggering inflammatory cytokine production and the other is MyD88-independent, leading to activation of IRF3 and IRF-7 and production of interferons e , involved in antiviral and antibacterial immune responses. Non-immune cells in several tissues also express TLR4, including human and murine pancreatic cells. Due to their role in inflammatory processes, TLRs have been implicated in chronic diseases like obesity and diabetes. Our previous study identified TLR4 as a molecule which activates inflammatory signals and induces changes in cell homeostasis. In this study, we investigated which of the TLR4 pathways is activated by LPS and the effects of glucose levels on cell viability and insulin production in a mouse insulinoma cell line. METHODS: MIN6 cells were maintained in low (2,8mM), normal (5,6mM) and high (11,2mM) glucose levels for 4 days, and then incubated with LPS (50 ng/mL) for 48 hours. Analyses were done by real-time PCR, Western Blot, ELISA and flow cytometry. RESULTS: Analysis confirmed increase in TLR4 gene expression in hyperglycemic conditions and showed that the signaling pathway activated by LPS is MyD88-dependent. The interferon induction pathway is absent in these cells. Furthermore, upon activation by LPS, TLR4 impacts on insulin secretion in response to glucose, but without triggering cell death. CONCLUSION: We conclude that TLR4 expression in mouse pancreatic cells is induced in response to increased glucose levels, constituting a new link in the chain of events leading to cell stress caused by high glucose levels with concomitant changes in cell function induced by LPS

O conhecimento dos eventos biológicos que ocorrem no periápice dos dentes com necrose pulpar se torna importante para compreender o desenvolvimento das lesões periapicais. Muitas são as moléculas e mediadores que participam na instalação da lesão periapical, a partir da infecção bacteriana que ocorre no interior dos canais radiculares. Assim, o objetivo do presente trabalho foi avaliar moléculas do sistema imune inato, da osteoclastogênese e metaloproteinases em lesões periapicais (LP) induzidas experimentalmente em camundongos knockout e wild type. Para esse objetivo, o presente estudo foi dividido em dois trabalhos distintos. O primeiro teve como objetivo avaliar a expressão de metaloproteinase 2 (MMP2) e metaloproteinase 9 (MMP9) durante a progressão da LP em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO), em comparação com camundongos wild type (WT). O segundo estudo avaliou a correlação da expressão gênica e imunomarcação de RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da LP em camundongos WT. No primeiro estudo lesões periapicais foram induzidas em molares inferiores de 54 camundongos TLR2 KO, MyD88 KO e WT (n=18/grupo). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. Os cortes histológicos foram submetidos a imunohistoquímica e posteriormente foi avaliada presença ou ausência de MMP2 e MMP9 nos diferentes grupos. No segundo estudo, 35 camundongos WT foram utilizados. As lesões periapicais foram induzidas nos primeiros molares inferiores de ambos os lados. Após 0 (G0), 7 (G7), 21 (G21) e 42 (G42) dias, os animais foram anestesiados e eutanasiados para que as mandíbulas fossem dissecadas e divididas ao meio.O lado direito das mandíbulas foi para o processamento histotécnico, para posterior marcação de RANK, RANKL, OPG, TLR2 e MyD88, por meio da imuno-histoquímica do lado esquerdo da mandíbula foi utilizado para a extração de RNA, para a determinação da expressão gênica de RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) e MyD88 (Myd88) utilizando quantificação em Tempo Real da Reação da Polimerase em Cadeia (qRT-PCR). Para ambos os estudos, testes paramétricos e não paramétricos foram realizados com nível de significância de 5%. Foi possível observar, no primeiro estudo, que nos períodos iniciais da progressão da lesão periapical, houve um aumento na imunomarcação de MMP9 nos camundongos TLR2 KO e MyD88 KO, quando comparados aos WT, diferente da MMP2 que não se observou nenhum aumento na imunomarcação. No entanto, aos 42 dias observou-se uma redução da imunomarcação de MMP2 e um aumento da MMP9 nos camundongos TLR2 KO. Adicionalmente, no segundo estudo, foi possível observar um aumento da imunomarcação para RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da lesão periapical (p<0,05). O aumento da expressão de Tnfrsf11 foi diferente entre os grupos G0 e G42, e G21 e G42 (p=0,006). No entanto, a expressão de Tnfrsf11b foi diferente entre os grupos G0 e G7, G7, G21 e G42, sendo possível observar uma diminuição dessa expressão ao longo do tempo (p<0,001). Tlr2 foi mais expresso entre os grupos G0 e G42 (p=0,03). E a expressão da molécula Myd88 foi estatisticamente significante entre os grupos G0 e G7, G21 e G42 (p=0,01). A razão Tnfrsf11/Tnfrsf11b aumentou durante a progressão da lesão periapical (p=0,002). Também foi possível observar uma correlação moderada entre Myd88 e Rankl (r=0,42; p=0,03) e entre Myd88 e Tlr2 (r=0,48; p<0,0001). Após as metodologias empregadas e os dados analisados, concluímos que a produção de MMP2 e MMP9 foi modulada por TLR2 e Myd88 durante a progressão da lesão periapical. Alem disso, podemos sugerir que existe uma correlação positiva entre o sistema RANK/RANKL/OPG e as proteínas do sistema imune inato, TLR2 e MyD88, durante a perda óssea decorrente da infecção bacteriana dos canais radiculares e posterior progressão da lesão periapical. / Knowledge of the biological events occurring inteeth apex with pulp necrosis becomes important to understand the development of periapical lesions. There are manymolecules and mediators that participate in the installation of the periapical lesion, from the bacterial infection that occurs inside the root canals. Thus, the aim of the present study was to evaluate molecules of the innate immune system, osteoclastogenesis and metalloproteinases in experimentally apical periodontitis (AP) induced in knockout and wild type mice. For this purpose, the present study was divided into two distinct studies. The first one aimed to evaluate the expression of metalloproteinases 2 (MMP2) and metalloproteinases 9 (MMP9) during the progression of AP in TLR2 knockout mice (TLR2 KO) and MyD88 knockout mice (MyD88 KO), compared to wild type mice (WT). The second study evaluated the correlation of gene expression and immunostaining of RANK, RANKL, OPG, TLR2 and MyD88 during LP progression in WT mice. In the first study AP were induced in lower molars of 54 TLR2 KO, MyD88 KO and WT mice (n = 18 / group). After 7, 21 and 42 days, the animals were euthanized and the jaws were dissected and submitted to histotechnical processing. The histological sections were submitted to immunohistochemistry and subsequently the presence or absence of MMP2 and MMP9 in the different groups was evaluated. In the second study, 35 WT mice were used. Periapical lesions were induced in the lower first molars on both sides. After 0 (G0) to 7 (G7), 21 (G21) and 42 (G42) days, the animals were anesthetized and euthanized so that the jaws were dissected and divided in half. The right side of the jaws was for the histotechnic processing, for subsequent imunostaining of RANK, RANKL, OPG, TLR2 and MyD88, through immunohistochemistry and the left side of the jaws was used for the extraction of RNA, for the determination of expression of RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) and MyD88 (Myd88) using Quantification Real Time of Polymerase Chain Reaction (qRT-PCR). For both studies, parametric and non-parametric tests were performed with significance level of 5%. It was possible to observe in the first study that in the initial periods of AP progression there was an increase in MMP9 immunostaining in TLR2 KO and MyD88 KO mice when compared to WT, different from MMP2 that no increase in immunostaining was observed. However, at 42 days there was a reduction in MMP2 immunostaining and an increase of MMP9 in TLR2 KO mice was observed. Additionally, in the second study, it was possible to observe an increase in the immunostaining for RANK, RANKL, OPG, TLR2 and MyD88 during periapical lesion progression (p <0.05). The increase in Tnfrsf11 expression was different between groups G0 and G42, and G21 and G42 (p = 0.006). However, the expression of Tnfrsf11b was different between the G0 and G7, G7, G21 and G42 groups, and a decrease in expression over time (p <0.001) was observed. Tlr2 was more expressed between the G0 and G42 groups (p = 0.03). And the expression of the Myd88 molecule was statistically significant between the G0 and G7, G21 and G42 groups (p = 0.01). The Tnfrsf11 / Tnfrsf11b ratio increased during the AP progression (p = 0.002). It was also possible to observe a moderate correlation between Myd88 and Rankl (r = 0.42, p = 0.03) and between Myd88 and Tlr2 (r = 0.48, p <0.0001). After the methodologies used and the data analyzed, we conclude that the production of MMP2 and MMP9 was modulated by TLR2 and Myd88 during the AP progression. In addition, we can suggest that there is a positive correlation between the RANK / RANKL / OPG system and the proteins of the innate immune system, TLR2 and MyD88, during bone loss due to bacterial infection of the root canals and subsequent progression of the apical periodontitis.

Chez la souris, les cellules dendritiques plasmacytoïdes (pDC) et natural killer (NK) contribuent à la résistance contre les infections systémiques par les virus herpétiques tels que le cytomégalovirus murin (MCMV). Les pDC représentent la source majeure d’interférons de type I (IFN-I) lors d’une infection par le MCMV. Cette réponse est dépendante de MyD88 et des récepteurs de type Toll 7 et 9. D’autre part, les cellules NK, qui expriment le récepteur d’activation Ly49H, peuvent détecter et lyser les cellules infectées par le MCMV. La perte de l’une de ces réponses augmente la sensibilité à l’infection. Cependant, la façon dont ces réponses antivirales interagissent est mal connue. Chez l’homme, bien que les réponses dépendantes des IFN-I soient essentielles, MyD88 semble superflu pour l’immunité antivirale. Cependant, les mécanismes susceptibles de compenser l’absence de MyD88 chez l’homme sont inconnus. Il a été supposé que les souris déficientes pour MyD88 ne parvenaient pas à monter de réponse protectrice dépendante des IFN-I lors d’infections par le MCMV. Afin d’évaluer cela, nous avons comparé la résistance de souris déficientes pour MyD88, les récepteurs aux IFN-I (IFNAR) et/ou Ly49H lors de cette infection. La déplétion sélective des pDC ou l’absence de MyD88 diminue drastiquement la production d’IFN-I, mais n’empêche pas l’établissement d’une forte réponse aux IFN-I dans la rate. De plus, l’absence de MyD88, mais pas celle d’IFNAR, peut être compensée par l’activité antivirale des cellules NK dépendant de Ly49H. Par conséquent, chez la souris, MyD88 est redondant pour l’établissement d’une réponse splénique aux IFN-I lors d’une infection systémique par le MCMV. / In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses has been reported to increase susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 appears to be dispensable for antiviral immunity. However, the mechanisms that could compensate MyD88 deficiency in humans have not been elucidated. Moreover, it has been assumed, but not proven, that MyD88-deficient mice fail to mount protective IFN-I responses to systemic herpes virus infections. To address these issues, we compared resistance to MCMV infection between mouse strains deficient for MyD88, the IFN-I receptor (IFNAR) and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 drastically decreased production of IFN-I, but not the protective antiviral responses mediated by these cytokines. Moreover, MyD88, but not IFNAR, deficiency could be compensated by Ly49H mediated antiviral NK cell responses. Thus, contrary to the current dogma, but consistent with the situation in humans, we conclude that, in mice, MyD88 is redundant for splenic IFN-I responses against a systemic herpes virus infection.