Th17细胞中RORγt转录调控机制的研究

Th17 cells produce IL-17A, IL-17F, and IL-22, cytokines involved in neutrophil, tissue remodeling, repair production of antimicrobial proteins. Th17 helper cells are abundant at mucosal interface, where they inhibit infection with pathogenic bacteria and fungi. Th17 cells differentiation in response to the STAT3-activating cytokines IL-6, IL-21, and IL-23 along with TGF-β and IL-1β. The transcription factor RORγt is necessary and sufficient for Th17 cell differentiation. In addition to RORγt, multiple other transcription factors have shown to be required for the expression of IL-17 in polarized T helper cells, this include IRF4, STAT3, Runx1, C-Maf, BATF and Ahr. RORα also contribute to some IL-17 expression in the absence of RORγt. STAT3, IRF4, BATF and Runx1 are required for expression of RORγt in Th17 cells, yet each contributes additionally, in cooperated with RORγt, to induce IL-17 expression. Most of the current studies are focused on the mechanism by which IL-17 was regulated. However, there is barely any study on the mechanism by which the Th17 lineage specific transcription factor RORγt was regulated. Besides, the relationship between TGF-β and IL-6 signaling with the transcription factors are still unclear. Identifying the mechanism by which RORγt was regulated is essential to find important components that could be used to manipulate or improve strategies to control infectious disease, vaccine design strategy or autoimmune disease. In this project, our goal is to elucidate the molecular basis for Th17 cell differentiation. The objective of this project is to identify the mechanism by which transcription factors such as Runx1 or some unknown transcription factors regulate RORγt expression. We formulated this hypothesis based on the literature and our previous research on transcription regulation of IL-17 expression as well as our current preliminary data showing that mutation of the Runx1 or E proteins binding site, RORγt promoter activity decreased significantly. We are prepared to undertake the proposed research because we have extensive experience in studying transcription regulation of T cell differentiation, and we were the first to report that Runx1 play a very important role in regulating Th17 cell differentiation. In addition, our preliminary identification of mutation Runx1 binding site and E protein binding site resulted in reduction of RORγt expression has positioned us well to perform the experiments described in this project. By using molecular biology technique such as reporter assay, ChIP assay, molecular immunology technique such as retroviral transduction, siRNA,, construct KO mice, and disease model such as EAE. Our expertise will insure that our discoveries are linked basic concepts of Th17 differentiation and potential application of manipulate immune response.

本课题主要针对目前Th17细胞研究领域的热点和难点问题，利用分子免疫学手段和转基因动物模型，对Th17细胞的转录调控机制进行全面系统的研究和探索。通过构建RORγt 启动子区的reporter，定点突变，染色体免疫沉淀，Knock down及利用Knock out小鼠和疾病模型等手段，研究参与调节RORγt的特异性转录因子及这些转录因子在体内和体外调节RORγt的机制，同时研究这些转录因子与TGF-β和IL-6信号途径的关系。据此进一步阐明TGF－β和IL-6调节Th17和Treg细胞间相互抑制的分子基础。课题在选题，立意及所用的技术方法上都处于国际领先水平。研究成果将全面、准确地阐明Th17细胞的转录调控机制及与Treg细胞的关系，在Th17 和Treg细胞的研究领域将会是一个重大的突破，也将对疾病的诊断，治疗和控制提供依据。

E蛋白是一种对TCR信号敏感，且对胸腺T细胞发育必不可少的转录因子。我们的研究发现：E蛋白的缺失会促进TGFb诱导产生的iTreg(适应性调节性T细胞)及胸腺来源的nTreg(天然调节性T细胞)细胞的分化。与之对应的，Id蛋白作为E蛋白的抑制分子，它的缺失会抑制nTreg细胞的分化。通过对这一现象进行深入研究，发现：E蛋白的缺失使得对Foxp3表达有抑制作用的信号通路受到抑制，即E蛋白的缺失使得其结合IL-2受体a链（即CD25）的能力下降，进而促进了TCR信号诱导CD25的表达。此外，E蛋白活性的降低还能促进TCR诱导的NF-kB的活化，引起c-Rel的大量产生。另外，我们还做了基因芯片分析，与我们的实验结果一致，即E蛋白的缺失会导致还没有表达Foxp3的细胞中IL-2及NF-kB信号通路的活化，还有Foxp3相关基因的表达增加。最后，为了进一步验证TCR信号与E蛋白活性及Treg细胞分化之间的关系，我们利用了Nur77-GFP的转基因小鼠，Nur77的表达强弱可以在一定程度上反应TCR信号强弱，再用GFP对Nur77进行标记，这样，GFP的强弱即反映了TCR信号的强弱。利用这种转基因小鼠进行实验，发现：如果一个细胞的TCR信号强度能够达到诱导Treg细胞分化的水平，那么这个细胞的E蛋白水平是被下调的。综上，TCR信号能够通过下调E蛋白活性从而诱导Treg细胞分化。

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