IL1B/TGFB/IL6/IL17A基因SNP与结核病易感性的关联分析及其作用机制研究

Tuberculosis is a complex diseases caused by Mycobacterium tuberculosis infection. Host genetic plays an important role in controling susceptibity or resistance to tuberculosis.In particular, polymorphisms in cytokine genes regulate cytokines response against mycobacterium tuberculosis infection, which, in turn,contribute to different susceptibility to tuberculosis. Although it has been recognized that Th17 cell and its effector cytokine, IL-17A, play an protective role in tuberculosis,the association between genetic polymphism in IL17A gene and tuberculosis has not been established. Our previous findings showed that not only single-nucleotide polymorphism (SNP) in Il17A, but also SNPs in IL6/TGFB gene , which encode cytokines for Th17 differentiation,are associated with tuberculosis. In this study, we are going to investigate the association between IL1B/TGFB/IL6/IL17A SNP and tuberculosis in large disease-control samples. The risk of individal SNP and cumulative risk of SNPs will be calculated. In addition, the function of those SNPs associated with TB on regulation of cytokine response in tuberculosis will be defined. Finally, we will investigate the mechanism how those SNPs modulate IL-17 response and its clinical significance in pathogenesis of tuberculosis. The results of this study will provide new insight to the pathogenesis of tuberculosis and highlight potential target for the immunotherapy of tuberculosis. At the same time, SNPs associated with susceptibility to tuberculosis could be used for assessing the risk of tuberculosis in high risk population.

宿主遗传背景对细胞因子应答的调控是结核易感性的重要机制。Th17及其效应分子IL-17A在结核保护性免疫中起着重要作用，但IL17A基因多态性与结核病易感性的关系尚无报道。我们前期发现IL17A基因及调控Th17分化的IL1B/TGFB/IL6基因的多个SNP与结核病相关。本项目拟采用高通量芯片技术对IL1B/TGFB/IL6/IL17A基因SNP进行大样本病例对照研究，明确上述基因SNP及其交互作用与结核病发生的相关性；研究易感基因SNP对IL1B/TGFB/IL6/IL17A基因转录和表达的影响，明确易感基因SNP对IL-17A应答的调控作用；探讨易感基因SNP对基因的核蛋白结合能力、启动子活性、以及IL-17A信号通路蛋白STAT3活化的影响，阐明其对IL-17A应答的调控机制。项目完成，不仅为结核病免疫防治提供新的思路，同时为结核高危人群的早期筛查及其预防干预决策提供科学依据。

Th17细胞在结核免疫发病中发挥重要作用，其分化和功能受多种细胞因子调控，包括IL-1β、TGF-β、IL-6以及IL-17A等基因，同时宿主遗传背景也扮演重要角色，目前尚无关于Th17轴相关基因多态性与结核病相关性的报道。.本项目研究内容包括三方面：（1）明确Th17轴相关基因SNP与结核病易感性的相关性；（2）明确结核病易感Th17轴相关基因SNP对IL-1β/TGF-β/IL-6/IL-17A应答的调控作用；（3）阐明功能性SNP调节IL-17A应答的机制。.针对研究内容一，我们先后在IL-1β、TGF-β、IL-6以及IL-17A基因鉴定8个SNP与结核病易感性密切相关。进一步分层分析发现IL1B基因rs1143627 T>C与肺外结核易感性密切相关，提示T等位基因更增加肺外结核风险。另外有趣的是TGFB基因易感SNP中仅rs2317130和rs4803457具有连锁不平衡性，单倍体CT增加结核病风险。.针对研究内容二， SNP功能学实验证实rs1143627 T等位基因型CD14单核细胞在受到Mtb或19kD脂蛋白刺激后，分泌高水平IL-1β；rs4803457 T等位基因型分泌高水平TGF-β；与GG基因型相比，rs1800796 CC和CG基因型表达高水平IL-6；rs8193036 TT基因型PBMC在受到Mtb或anti-CD3/28刺激后表达高水平IL-17A。.针对研究目标三，我们首先体外克隆上述基因启动子并构建不同基因型表达载体，发现rs1143627 T、rs4803457 T、rs1800796 C等位基因型增加启动子活性，而IL17A基因rs8193036 T启动子仅在共转染RORγt、Runx1时增加启动子活性。EMSA实验证实rs1143627通过影响转录因子C/EBPβ、PU.1亲和活性而调控IL-1B基因表达，而rs1800796则影响C/EBPα与启动子亲和活性而调控IL-6表达。TGFB基因rs2317130 、rs4803457则影响与核蛋白的亲和力。.本项目研究结果进一步验证我们前期有关Th17细胞及宿主遗传背景与结核病发生密切相关的科学假说，系统阐明宿主基因多态性调控Th17应答的分子机制，同时为结核高危人群的早期筛查及其预防干预决策提供科学依据。