等位基因介导转录因子C/EBPβ和PU.1协同调控IL-1B表达的分子机制及其对结核菌感染转归的影响

IL-1β plays a dual role of protective and pathogenic immunity during Mycobacterium tuberculosis infection, and its regulatory mechanism remains unclear. Our previous findings showed that M.tb infection triggered pro-IL-1B expression in macrophage, which positively correlated with transcription factor C/EBPB and PU.1 expression. As a functional SNP, the rs1143627 allele in the IL-1B gene promoter region affected IL-1β and IFN-γ secretion, meanwhile, the rs1143627 allele was associated with TB susceptibility and clinical phenotypes. Furthermore, the rs1143627 allele influenced the binding ability of C/EBPβ and PU.1 to the IL-1B promoter, which synergistically regulated pro-IL-1B expression. In this study, we will first investigate how to regulate pro-IL-1B expression by C/EBPβ and PU.1, involving the interaction between them, and interaction with other proteins. In addition, we will clarify the functional domains of C/EBPβ and PU.1 through designing series of truncated proteins. Secondly, we will identify the key bacterial factors, recognized receptors, and signal pathways to induce C/EBPB and PU.1 expression. Finally, since a LTBI cohort has been established during Twelve-fifth mega project, we will further explore the diagnosed value of IL-1B expression and polymorphisms to predict the clinical outcomes of M.tb infection. The results of this study will contribute to clarifying the transcription regulatory mechanism of IL-1B after M.tb infection, and provide a novel personally preventive strategy for TB.

IL-1β在结核菌感染过程中发挥免疫保护性和病理性双重作用，其表达调控机制异常复杂。我们前期发现：结核菌诱导巨噬细胞表达pro-IL-1B，并与C/EBPB、PU.1表达呈正相关；等位基因rs1143627影响IL-1β、IFN-γ分泌，并与结核病易感性、临床表型（肺内、肺外）密切相关；C/EBPβ、PU.1协同调控pro-IL-1B表达，rs1143627影响两者与IL-1B启动子结合能力。在此基础上，本项目拟探讨：1）C/EBPβ、PU.1协同调控IL-1B表达的作用形式，包括两者的直接作用、功能效应区以及与其它蛋白的交互作用；2）鉴定结核菌诱导C/EBPB、PU.1表达的关键细菌因子、受体及信号通路；3）借助“十二五”队列，评估IL-1B表达水平、多态性对预测结核菌感染转归（未进展、发病）的价值。项目将进一步阐明结核菌诱导IL-1B表达的转录调控机制，为结核病个体化免疫防治提供依据。

IL-1β的产生受多重因素调控，即包括转录因子介导的转录水平调控，也包括miRNA介导的转录后调控，同时pro-IL-1β还需要通过以NLRP3为主的炎症小体切割加工才能具有活性，我们前期证实IL-1β产生还受宿主遗传背景调控。本项目正是从转录水平和宿主遗传背景两个层面探讨结核菌诱导IL-1β表达的调控机制。本项目研究内容包括：（1）C/EBPβ、PU.1协同调控IL-1β转录的作用形式以及功能效应区的鉴定；（2）结核菌诱导核转录因子C/EBPβ和PU.1表达的机制研究；（3）IL-1β等位基因多态性与结核菌感染转归的相关性研究。针对研究内容一，研究发现等位基因rs1143627介导转录因子与启动子结合活性差异，核转录因子C/EBPβ和PU.1可以结合IL-1β基因启动子，C/EBPβ和PU.1可以协同调控IL-1β基因启动子活性，siRNA干扰技术反向验证C/EBPβ和PU.1协同调控IL-1β，构建C/EBPβ和PU.1系列截短载体，通过双荧光素酶系统鉴定C/EBPβ和PU.1调控IL-1β基因启动子的功能效应区。针对研究内容二，研究发现结核菌刺激上调巨噬细胞IL-1β、PU.1、C/EBPβ表达，PU.1、C/EBPβ表达与pro-IL-1β表达水平呈正相关，并且PU.1、C/EBPβ表达与pro-IL-1β表达具有时间先后性，结核菌感染巨噬细胞激活ERK1/2、JNK、NF-kβ等多条信号通路。针对研究内容三，发现IL-1β基因rs1143627 SNP与结核易感性的相关性，T等位基因为结核易感基因，进一步分层分析发现IL-1β基因-31位点T>C SNP与活动性肺结核临床类型相关，携带rs1143627 T等位基因的个体更易患肺外结核，等位基因rs1143627不仅影响肺脏的病理严重程度，而且与肺结核治疗转归息息相关。项目不仅进一步阐明结核菌诱导IL-1β表达的转录调控机制，同时所鉴定的等位基因可以作为新型分子遗传标签，为结核病个体化免疫防治提供依据。