Critical roles of endogenous TLR signaling in driving Th17 effector responses

描述（由申请人提供）：本提案将研究通过TLR 2和TLR 4信号通路直接调节CD 4 + T淋巴细胞功能。我们先前已经表明，T细胞中的TLR 2活化可以驱动Th 17细胞的产生和效应子功能。我们的初步数据表明，TLR 4信号传导还可以通过促进增殖和存活在T淋巴细胞内以类似的方式起作用。此外，CD 4 + T细胞中TLR 2或TLR 4通路的直接激活促进了实验性自身免疫性脑脊髓炎（EAE）（多发性硬化症的小鼠模型）的发展。重要的是，发现CD 4 + T细胞通过内源性产生的信号激活TLR 2来促进EAE的发病机制，所述信号作为炎症的副产物释放。内源性TLR 4信号传导是否也是如此尚未确定。因此，我们将首先使用WT和TLR 4缺陷型CD 4 + T细胞转移模型在不存在外源性TLR 4刺激的情况下进行EAE，以确定该途径在驱动自身免疫性炎症中的潜在重要性。此外，我们的目标是确定这种内源性TLR信号能够激活TLR 2和TLR 4途径在Th 17细胞的身份。已经鉴定了两种候选分子，HMGB 1和双糖链蛋白聚糖，它们都刺激TLR 2和TLR 4，并且在发炎的CNS组织中发现。我们还将研究靶向这些内源性TLR配体，而不是TLR本身，是EAE/MS治疗的可行策略。这些研究将包括通过抗体处理直接中和内源性配体。我们的体外观察清楚地表明，TLR 2信号转导促进幼稚CD 4 + T细胞增殖和直接极化为Th 17谱系。另一方面，TLR 4信号传导对谱系定型没有这种影响，而是增强了Th 17细胞的增殖和存活。这些结果共同表明，在次优条件下，通过TLR的信号传导可以增强Th 17细胞因子的产生和该炎症亚群的持续性。因此，我们将分析表观遗传，信号和转录机制TLR利用调节增殖，生存，和生产的IL-17在CD 4 + T淋巴细胞。我们的实验室已经发现，在IL-17位点富集的特定组蛋白修饰允许在Th 17分化期间允许转录机器的进入。此外，TLR 2激活Th 17细胞增强了Th 17相关转录因子的表达，如ROR？t.因此，我们将确定组蛋白H3乙酰化和甲基化事件是否在Th 17分化过程中通过TLR 2信号转导被放大。此外，我们的目标是通过微阵列分析，在全球范围内研究TLR 2和TLR 4信号转导在CD 4 + T细胞的下游目标。作为TLR信号传导的结果，对控制T细胞存活和细胞因子产生的新型信号传导和转录途径的鉴定将提供新的实验路线，这将允许候选人建立一个独立的实验室，从当前导师的研究兴趣中分离出来，并帮助申请R 01资金。

DESCRIPTION (provided by applicant): This proposal will investigate the direct regulation CD4+ T lymphocyte function through the TLR2 and TLR4 signaling pathways. We have previously shown that TLR2 activation in T cells can drive the generation and effector function of Th17 cells. Our preliminary data indicates that TLR4 signaling additionally may function in a similar manner within T lymphocytes through the promotion of proliferation and survival. Furthermore, direct activation of the TLR2 or the TLR4 pathway in CD4+ T cells promotes the development of experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. Importantly, CD4+ T cells were found to promote the pathogenesis of EAE through the activation of TLR2 by endogenously generated signals that are released as a byproduct of inflammation. Whether the same is true for endogenous TLR4 signaling has yet to be determined. Thus, we will first perform EAE using a model of WT and TLR4-deficient CD4+ T cell transfer in the absence of exogenous TLR4 stimuli to determine the potential importance of this pathway in driving autoimmune inflammation. Furthermore, we aim to ascertain the identity of such endogenous TLR signals capable of activating the TLR2 and TLR4 pathways in Th17 cells. Two candidate molecules have been identified, HMGB1 and biglycan, both of which stimulate TLR2 and TLR4 and are found in inflamed CNS tissue. We will also investigate is targeting these endogenous TLR ligands, rather than TLRs themselves, is a viable strategy for EAE/MS therapy. These studies will include directly neutralizing endogenous ligands through antibody treatment. Our in vitro observations have clearly demonstrated that TLR2 signaling promotes proliferation and the direct polarization of naive CD4+ T cells into the Th17 lineage. TLR4 signaling, on the other hand, had no such effect on lineage commitment but instead enhanced proliferation and survival of Th17 cells. These results collectively suggest that under sub optimal conditions, signaling through either TLR could enhance Th17 cytokine production and the persistence of this inflammatory subset. Thus, we will analyze the epigenetic, signaling, and transcriptional mechanisms TLRs utilize to regulate proliferation, survival, and the production of IL-17 in CD4+ T lymphocytes. Our laboratory has found that specific histone modifications enriched at the IL-17 locus allow permissive access of the transcriptional machinery during Th17 differentiation. Furthermore, TLR2 activation on Th17 cells enhanced the expression of Th17-related transcription factors such as ROR?t. Thus, we will determine if histone H3 acetylation and methylation events are amplified through TLR2 signaling during Th17 differentiation. Additionally, we aim to globally investigate the downstream targets of TLR2 and TLR4 signaling in CD4+ T cells through microarray analysis. Identification of novel signaling and transcriptional pathways governing T cell survival and cytokine production as a result of TLR signaling will provide new lines of experimentation that will allow the candidate to establish an independent laboratory, gain separation from the research interests of the current mentor, and aid in the application for R01 funding.