宿主基因SNP对I型IFN应答的调控作用及其与结核菌感染转归的相关性研究

We found Mycobacterium tuberculosis triggered significant type I IFN response, which was considered as new diagnosis biomarkers for TB and affected Mtb-specific immune response through down-regulation of IL-1B mRNA level, but there were no reports about the association between type I IFN-related gene polymorphisms and TB. Our previous findings showed that 3 SNPs in IFNAR1/2 gene were associated with susceptibility to TB, and rs72552343 influenced SD4 domain 3-D structure of IFNAR1. In this study, since a case-control cohort and follow-up cohort have been established, we will further explore the association between IFNA/IFNB/IFNAR gene SNPs and clinical outcomes (susceptibility, clinical phenotypes, and prognosis) of Mycobacterium tuberculosis infection. Then we will make haplotype and gene-gene interaction analysis to clarify whether SNPs individually or cooperatively affect outcomes of TB. We will observe the influence of SNPs on the type I IFN response, involving type I IFN-related gene mRNA expression, STAT-1/2 phosphorylation, and IRF-1/7 expression. Through above analysis, we could identify functional SNPs associated with TB. Finally, we will investigate the mechanism how those SNPs modulate type I IFN response, such as if SNPs influence promoter activity, or the binding efficiency to transcription factor, or protein 3-D structure and function. The project would contribute to elucidation of TB pathogenesis and provide new insight to personally preventive strategy for TB.

前期发现结核菌能够诱导强烈I型IFN应答，这不仅可以作为结核诊断分子标识，并通过下调IL-1B mRNA影响结核特异性免疫应答，但该通路基因多态性与结核病的关系尚无报道。我们在IFNAR1/2基因中已经发现3个SNP与结核发生密切相关，其中rs72552343对IFNAR1蛋白SD4结构域空间构象具有重要影响。本项目拟借助前期建立的病例-对照横向队列和病例随访纵向队列，探讨I型IFN及受体基因SNP与结核菌感染转归（发生、临床表型、预后）的相关性，通过单倍体和基因-基因交互作用分析明确SNP的关联性；其次观测SNP对I型IFN相关基因mRNA表达、STAT1/2磷酸化和IRF1/7蛋白表达的影响，鉴定功能性SNP；最后探讨SNP对启动子活性、转录因子结合效率以及蛋白三维结构的影响，阐明SNP调控I型IFN应答的分子机制。项目完成将进一步阐明结核发病机制，为结核个体化免疫防治策略提供新思路。

我们首先探讨IFN通路基因SNP与结核病的关系，通过建立高通量飞行时间质谱技术（MassARRAY）和TaqMan探针技术，利用“十一五”期间建立的结核-对照人群队列进行SNP分型，在I型和II型IFN通路基因中分别发现2个SNP位点与结核病易感性密切相关，包括IFNA8基因启动子区rs1330322 G>A，IGNG基因rs2430561 A>T。进一步分层分析发现，rs1330322 G>A位点尤其与女性、年龄<25岁人群结核易感性密切相关。其次我们进行SNP功能学实验，通过体外诱导分化成熟的巨噬细胞，感染不同结核分枝杆菌菌株后，有两点有趣的发现：一是结核菌可以诱导IFNAR1、IFNAR2基因表达下调，二是不同毒力结核菌株之间对IFNAR基因表达的影响无显著差异。此外我们检测不同基因型个体PBMC上清、细胞中相应基因的表达水平，虽然我们发现rs1330322 G>A对IFNA8基因表达无显著影响，但rs2430561 TT型个体PBMC上清IFN-γ水平显著升高，提示SNP具有调控作用。最后我们进行SNP调控机制研究，由于前期发现rs2430561 A>T为功能性SNP，但由于该位点位于内含子区，从而进行机制研究具有一定困难，而最近《Nature》杂志（2014，511（7507）：99-103）报道，I型IFN与IL-1β之间存在“信号会话”（crosstalk），为此我们对IL1B基因SNP与结核病的关系进行分析，发现-31位点C>T多态性通过调控转录因子与启动子的结合活性而影响结核菌感染转归。