Transcriptional Regulation of T Helper Cell Differentiation in Autoimmunity




Gocke, Anne Elizabeth

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As a means to understand the significance of transcriptional regulation of Th cells in MS pathogenesis, we investigated the role of the transcription factors T-bet and PPARalpha in the differentiation and effector function of T helper cells in the EAE model. Based on its ability to induce a pro-inflammatory immune response, T-bet has been found to regulate genes that have been directly linked to pathogenicity in EAE/MS. We examined the use of T-bet specific AS oligonucleotides and siRNA to silence T-bet expression in autoreactive encephalitogenic T cells and demonstrated that this transcription factor plays a critical role in the differentiation of these cells and in the induction of EAE. siRNA and AS T-bet suppressed T-bet expression, IFN-gamma production, and STAT1 expression during antigen-specific T cell differentiation. In vitro suppression of T-bet in myelin specific cells used for adoptive transfer, and in vivo administration of AS and siRNA T-bet inhibited the induction of EAE. T-bet bound the IFN-gamma and STAT1 promoter regions, suggesting the ability of T-bet and STAT1 to regulate one another in a positive feedback loop, but was not found to regulate the IL- 12/STAT4 pathway. Moreover, we explored the role of T-bet in the IL-23/IL-17 pathway. IL-17 producing T cells have recently been implicated in the pathogenesis of EAE, but the potential role of T-bet in the differentiation of Th17 cells is not completely understood. We demonstrated that therapeutic administration of siRNA T-bet significantly improved the clinical course of established EAE. The improved clinical course was associated with a decrease in T-bet protein expression, reduced IFN-gamma production, and a reciprocal increase in GATA3 expression. We also observed a decrease in IL-23R expression both ex-vivo in splenocytes from siRNA T-bet treated mice and when cells were transfected with siRNA T-bet and subsequently activated in vitro. When T-bet was silenced we observed an ensuing decrease in both IL-23R and IL-17 expression in the CNS. This observation was correlated by the finding that T-bet directly regulates transcription of the IL-23R and, in doing so, influences the fate of pathogenic Th17 cells, which depend on optimal IL-23 responsiveness for survival. Taken together, these data demonstrate that suppression of T-bet ameliorates EAE by limiting differentiation of autoreactive Th1 cells, as well as inhibiting pathogenic Th17 cells via regulation of the IL-23R. In contrast to T-bet, PPARalpha has been demonstrated to regulate anti-inflammatory genes and the PPARalpha agonists gemfibrozil and fenofibrate have been associated with protection from EAE. However, the manner in which these agents ameliorate disease is not well understood. We investigated the mechanism by which gemfibrozil induces immune deviation and protects mice from EAE. We demonstrated that treatment with gemfibrozil increases GATA3 and decreases T-bet expression in vitro and directly ex-vivo. The protective effects of gemfibrozil in EAE were shown to be partially dependent on IL-4 and to occur in a receptor dependent manner. Moreover, PPARalpha was demonstrated, for the first time, to regulate the IL-4 and IL-5 genes and bound the IL-4 promoter in the presence the coactivator SRC-1, suggesting transactivation of the IL-4 gene. Finally, therapeutic administration of gemfibrozil and fenofibrate ameliorated clinically established EAE. The data presented herein illustrate that modulation of specific transcription factors can influence Th cell differentiation and the clinical outcome of EAE, and suggest that targeting T-bet and/or PPARalpha by gene silencing and administration of specific agonists could provide a novel therapy for inflammatory immune-mediated diseases such as MS.

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