结核菌感染上调PGAM1表达的表观调控机制及其对CD4 T细胞应答的影响

Protective immunity to TB primarily requires CD4 T cells. Their quantity and function directly affect the outcome of mycobacterium tuberculosis infection. Aerobic glycolysis plays a key role in regulating the immune function of CD4 T cells, but its role in tuberculosis, as well as key genes and underlying mechanisms, remain unclear. Interestingly, we found that the metabolic pathways, especially carbon metabolism, were highly enriched among the differentially methylated genes between CD4 T cells isolated from pleural fluid and peripheral blood through whole-genome methylation sequencing. The methylation level of PGAM1, an important metabolic enzyme that catalyzes the key steps of glycolysis, was significantly reduced in pleural fluid CD4 T cells due to mycobacterium tuberculosis infection. What’s more, elevated expression of PGAM1 was observed in CD4 T cells during mycobacterium tuberculosis infection, which promoted glycolysis, enhanced cell proliferation and IFN-γ secretion. Inhibiting PGAM1 significantly reversed the phenotypes mentioned above. Therefore, we hypothesize that host increases PGAM1 expression in CD4 T cells through interference of DNA methylation of PGAM1, thereby upregulating glycolysis and enhancing the host protective immunity against tuberculosis. In this study, we will validate this hypothesis and elucidate the mechanism underlying how mycobacterium tuberculosis infection elevates PGAM1 expression in CD4 T cells, as well as its role in fine-tuning the immune response of CD4 T cells to TB.

CD4 T细胞在宿主抗结核保护性免疫中起着不可或缺的作用，其数量和功能直接影响结核菌感染转归。有氧糖酵解是调控CD4 T细胞免疫功能的重要机制，但其是否参与调控CD4 T细胞抗结核免疫及具体机制尚不清楚。采用全基因组甲基化测序，我们发现结核患者胸腔积液CD4 T细胞碳代谢通路基因显著富集，其中糖酵解关键代谢酶PGAM1启动子区甲基化水平显著降低。进一步实验证实，结核感染时，CD4 T细胞PGAM1表达显著升高，从而增强有氧糖酵解、细胞增殖及γ干扰素分泌；抑制PGAM1显著逆转上述表型。据此，我们提出假设：宿主通过下调CD4 T细胞PGAM1甲基化，增加PGAM1表达，将能量代谢从氧化磷酸化重塑为有氧糖酵解，从而增强抗结核免疫。本课题拟进一步阐明DNA去甲基化上调结核感染者CD4 T细胞PGAM1表达的作用和机制，明确PGAM1对CD4 T细胞抗结核免疫的影响，为结核病免疫防治提供新思路。

DNA甲基化是调控细胞发育和功能的重要表观遗传机制，但是在结核病中，DNA甲基化是否参与调控CD4 T功能尚不清楚。我们利用氧化亚硫酸氢盐测序（oxidative bisulfite sequencing）和微量全基因组甲基化测序（MBS），对结核性胸膜炎（TP）、结核潜伏感染（LTBI）和健康对照（HC）中分离的CD4 T细胞全基因组DNA甲基化水平进行了检测。与外周血单个核细胞（PBMC）相比，胸水单个核细胞（PFMC）中CD4 T细胞显示出显著不同的DNA甲基化图谱，PFMC中5-甲基胞嘧啶水平要低得多。通路富集分析显示，TP PFMC中大量代谢通路（尤其是“碳代谢”）显著富集，其中，糖酵解关键代谢酶PGAM1参与了35.29% (6/17)的能量代谢通路，其启动子区甲基化水平显著降低。与胸腔积液CD4 T细胞中PGAM1启动子区域甲基化水平显著低于配对外周血相反，结核菌感染局部CD4 T细胞PGAM1 mRNA和蛋白表达水平显著升高，并从单细胞水平进一步证实胸腔积液CD4 T细胞中，PGAM1表达水平显著上升。结核菌裂解物刺激显著诱导CD4 T细胞PGAM1基因启动子DMR区域低甲基化，同时提高其表达水平；萤光素酶实验提示，结核菌感染时，CD4T细胞PGAM1 启动子DMR甲基化下降可以促进PGAM1转录。进一步实验发现，CD4 T细胞PGAM1启动子区DNA甲基化水平显著降低，从而促进PGAM1表达，结核菌感染可以通过影响DNA去甲基化酶TET2表达进而影响PGAM1启动子DNA甲基化修饰。在T细胞中敲除PGAM1后，肺部和脾脏结核菌载量显著增加，肺部病理损伤增强，提示PGAM1在宿主抗结核免疫中起保护作用。结核菌感染时，PGAM1表达上调增强宿主CD4 T细胞糖酵解、细胞增殖以及γ干扰素分泌，提示PGAM1介导的有氧糖酵解可能是调控CD4 T细胞功能的关键代谢途径。DNA甲基化水平降低，尤其是参与代谢的基因中的甲基化水平降低，是结核病感染部位CD4 T细胞的重要标志。通过PGAM1低甲基化增强有氧糖酵解是调控CD4 T细胞免疫应答的关键机制之一。这些发现为CD4 T细胞免疫应答的表观遗传调控提供了新的思路，并为结核病免疫治疗提供了潜在靶标。