DNMT3a通过调控M1/M2型巨噬细胞极化平衡参与抗结核菌免疫的机制研究

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major health issue worldwide. Macrophage polarization balance plays a pivotal role in anti-MTB immunity and the pathogenesis of TB. Much interest has recently arisen as to the role of epigenetic regulation in macrophage polarization during inflammation. Our preliminary studies show that DNMT3a is up-regulated in the PBMC of pulmonary TB patients; DNMT3a expression level in macrophage increases after infection with MTB; DNMT3a knockout promoted M1-type polarization, while inhibited M2-type polarization of macrophage in MTB infected RAW264.7 cells. DNMT inhibitor AZA treatment significantly reduces bacterial load in lung and alleviates lung tissue damage in MTB infected mice. These results indicate that MTB may interfere with M1/M2 polarization through up-regulating DNMT3a in macrophages, hence promotes its intracellular survival. In this proposal, the role of DNMT3a in DNA methylation profile of MTB infected macrophages, macrophage polarization and the function of eliminating MTB will be investigated using MTB infected macrophage, MTB infected mice model and patient samples, which might be helpful to understand the significance of Dnmt3a to pathogenesis and host-pathogen relationship of MTB , and will reveal novel biomarkers for TB diagnosis and targets for intervention of MTB infection as well.

巨噬细胞极化平衡在抗结核菌免疫及结核病转归中有重要作用。近年来利用表观遗传调控巨噬细胞极化成为治疗感染的热点。DNA甲基转移酶（DNMT）通过甲基化修饰参与巨噬细胞极化平衡。目前DNMT3a在抗结核免疫中的作用不详。我们前期研究发现：结核患者外周血单个核细胞中DNMT3a的表达高于健康对照；结核菌感染巨噬细胞后DNMT3a表达上调；而敲除DNMT3a后巨噬细胞M1型极化比例升高；DNMT抑制剂显著减少结核感染小鼠肺部荷菌量，从而减轻肺部病理损伤；这些提示DNMT3a可能通过促进巨噬细胞M2极化参抗结核免疫。本项目拟从细胞、动物模型和临床样本中探索DNMT3a在巨噬细胞抗结核免疫中的作用，并寻找DNMT3a在巨噬细胞极化中的靶基因。这些靶基因甲基化水平有望成为诊断结核的生物标记，且改变靶基因的水平有望提升机体抗结核的免疫效率，为结核病防治提供新思路。

本研究利用结核菌感染巨噬细胞模型，检测野生型巨噬细胞与DNMT3a缺失巨噬细胞在结核菌感染过程中的M1/M2型巨噬细胞表面标记物、细胞 M1/M2 基因表达、分泌细胞因子的水平及清除结核菌的能力，分析DNMT3a基因对巨噬细胞极化的影响。检测结核菌感染前后野生型及DNMT3a缺失的巨噬细胞差异甲基化区域，结合RNA-seq技术及Real-time PCR实验，筛选出影响巨噬细胞极化过程的DNMT3a靶基因。染色体免疫共沉淀（chromatin-immunoprecipitation， ChIP）实验验证DNMT3a确实与靶基因的启动子区结合。利用siRNA敲降靶基因，观察能否回复DNMT3a缺失在结核菌感染过程中巨噬细胞极化改变的表型，从功能上确定DNMT3a对其的调控作用。 本研究收集确诊肺结核病人和健康体检者的外周血标本，分离PBMC，real-timePCR检测DNMT3a的mRNA表达量，流式细胞术检测外周血巨噬细胞 M1/M2 比例，ELISA检测血清中炎症因子，分析DNMT3a表达量与巨噬细胞极化状态的相关性。同时检测肺结核患者及健康对照PBMC中DNMT3a靶基因启动子区甲基化水平及mRNA表达水平，分析DNMT3a靶基因与肺结核发病的相关性。研究发现，DNMT3a敲除降低DNA甲基化水平。抑制/敲降DNMT3a影响结核分枝杆菌刺激下巨噬细胞极化。以及DNMT3a促进Arg-1甲基化水平、抑制Arg-1转录水平。与研究计划基本符合。研究共纳入5例支气管内膜结核患者，选取患者结核感染组织及正常组织进行对照，通过组织基因检测，血PBMC检测，通过转录组学联合甲基化分析，得出4个关键基因的甲基化与支气管内膜结核的发病相关。该基因的发现对于进一步分析甲基化对于支气管内膜结核发病的影响有至关重要的作用。