DNA甲基化在结核菌下调CD4 T细胞表面IL-6R中的作用和作用机制

Interleukin-6 (IL-6) is critical for protective immunity against tuberculosis, as previous reported that the IL-6 deficient mice succumbed to tuberculosis from day 50 post of infection onwards (median survival time [MST], 59 days), in comparison to all wild-type control animals survived M. tuberculosis infection (MST, 120 days). IL6 is one of the major cytokines responsible for differentiating T helper lymphocytes into Th17 cells by enhancing Th17 polarization. After binding to IL-6R, IL-6/IL-6R/gp130 a complex is formed for signal transduction and, consecutively, lead to STAT3 phosphorylation. Remarkably, theexpression of the membrane-bound IL-6R is restricted to naïve T cells and hepatocytes, thus emphasizing the significance of this cytokine for these cell types. Modifications of this pathway in CD4 T cells lead directly to RORγT, the key transcription factor for IL17 production, and, therefore, to the polarization of Th17 cells. Notably, Th17 cells and related cytokines have been demonstrated play an indispensable role for protective immunity against tuberculosis. Interestingly, we found that Mycobacterium tuberculosis infection resulted in decreased expression of IL-6R on CD4 T cells, which is correlated with inhibited Th17 cell responses in patients with active TB. In vitro data indicated that Mycobacterium tuberculosis lysate reduced IL-6R expression on CD4 T cells. Importantly, we found that CD4 T cells isolated from pleural fluid caused by Mycobacterium tuberculosis infection have significantly increased IL6R DNA methylation. Therefore, we hypothesed that Mycobacterium tuberculosis reduced IL-6R expression on CD4 T cells through enhancement of DNA methylation of IL6R. In this study, we will validate this hypothesis and understand the mechanism underlying how Mycobacterium tuberculosis reduces IL-6R expression on CD4 T cells.

IL-6是宿主抗结核保护免疫的重要因子，IL6基因缺失小鼠因不能控制感染结核菌生长，生存期不到野生小鼠的一半。通过与CD4T细胞表面IL-6R结合，IL-6在促进Th17细胞极化中起关键作用，而后者在抗结核保护免疫中起着不可或缺的作用。我们前期发现，与结核患者Th17细胞免疫降低正相关，结核患者CD4T细胞表面IL-6R显著降低，体外结核菌可直接下调CD4T细胞表面的IL-6R表达。全基因组甲基化测序分析发现，结核感染局部CD4 T细胞IL6R甲基化水平显著增高，抑制甲基化可逆转结核菌下调IL-6R的作用。据此，我们提出假设：结核菌感染通过DNA甲基化修饰下调CD4T细胞表面IL-6R的表达，抑制Th17细胞应答进而促进结核病的发生。本项目拟进一步阐明DNA甲基化在结核菌下调CD4T细胞表面IL-6R表达中的作用和分子机制，为结核病新型疫苗设计和免疫干预策略提供新思路和可能靶点。

CD4 T 细胞免疫，尤其是Th1和Th7在宿主抗结核保护性免疫中起着关键作用。我们前期发现，结核患者Th17细胞应答显著降低，并与病情进展相关。进一步发现，结核菌感染下调CD4 T细胞表面IL-6R，而后者的信号是Th17细胞应答和分化的关键。采用全基因甲基化测序，我们发现结核性胸膜炎的CD4 T细胞IL6R基因DNA甲基化水平显著增高。鉴于DNA甲基化在调控基因表达的作用，我们提出结核菌感染通过增加IL6R基因DNA甲基化，抑制其在表达水平，从而抑制Th17细胞应答。本项目围绕上述假说，首先对体内结核菌感染和体外结核菌抗原刺激对外周血培养的IL-6和可溶性IL-6R表达的影响；通过纳入160例受试者（包括健康对照、结核病患者、治愈的结核病患者、非结核肺炎对照），我们发现体内结核感染和体外结核抗原刺激并不影响IL-6和可溶性IL-6R表达。相反，采用流式细胞分析技术、qPCR 和western blot我们证实结核感染显著下调CD4 T细胞表面IL-6R。采用BSP克隆测序法(Bisulfite Sequencing PCR,BSP)对结核患者CD4 T细胞IL6R基因DNA甲基化水平，发现结核菌显著增加IL6R 3‘UTR的DNA甲基化水平。结合甲基化酶抑制剂（5-AZA）的使用和双荧光素酶报告基因分析系统，明确了结核菌通过增加IL6R 3‘UTR的DNA甲基化抑制细胞表面IL-6R的表达。进一步，利用外周血CD4 T细胞和jurkat T细胞系，明确了结核菌通过上调CD4 T细胞DNMT1、DNMT3B表达维持和/或介导IL-6R 3’UTR区CpG位点从头甲基化而抑制IL-6R转录的作用机制。我们的结果表明，通过DNA甲基化是调控宿主抗结核免疫的一个重要表观遗传机制；靶向DNA甲基化调节通路可能为结核病防治提供新的可能思路。