MRN-ATM通路在慢性乙型肝炎患者CD4+ T细胞衰老过程中的作用

Hepatitis B virus (HBV) infection is a major problem for public health in China and worldwide. Despite considerable therapeutic advancements, the mechanisms of HBV persistence are unclear and overcoming chronic HBV infection remains a great challenge. T cell senescence can be induced by persistent viral antigenic stimulations, and as a result, telomere erosion and/or unrepaired DNA damage occur, probably contributing to the viral persistence and immune tolerance. DNA damage response (DDR) involves the activation of critical sensor molecules, such as the MRN complex (MRE11, RAD50, and NBS1), which subsequently recruits and mediates the activation of DNA repair kinase ataxia telangiectasia mutated (ATM) that can phosphorylate several downstream checkpoint proteins (such as p53, BRCA1, Chk1 and particularly, Chk2). CD4+ T cells play a crucial role for HBV clearance or persistence; however, the detailed profile of CD4+ T cell senescence and precise mechanisms of DNA damage repair during HBV infection remain largely unknown. Our recent studies revealed that in patients with chronic HBV infection, the process of CD4+ T cell senescence is accelerated, as demonstrated by the expansion of memory T cells , overexpression of aging markers (such as KLRG1 and SIRT1) when compared to age-matched healthy subjects (HS). Notably, ATM phosphorylation (pATM) is significantly higher in CD4+ T cells derived from patients with chronic hepatitis B (CHB) compared to HS. These results indicate that CD4+ T cell senescence may be involved in HBV persistence. Based on our innovative preliminary studies on CD4+ T cell senescence, in this proposal, we will further investigate CD4+ T cell senescence and its clinical significance in CHB patients. Also, we will explore the MRN-ATM pathway that control DNA damage repair capabilities in primary CD4+ T cells of CHB patients and HBV transgenic mice models. These experiments will provide new information regarding the role of T cell senescence in HBV persistence as well as the mechanisms of DNA damage repair mediated CD4+ T cell senescence during HBV infection and may develop novel immunotherapy to combat this global epidemic viral infection.

乙型肝炎病毒（HBV）慢性感染的机制是乙肝防治的关键问题之一，持续病毒刺激导致T细胞衰老可能是机体对HBV免疫耐受形成的重要机制。细胞衰老伴随着DNA损伤，MRE11/RAD50/NBS1（MRN）-ATM是DNA损伤修复反应的关键信号通路。CD4+T细胞在抗HBV免疫反应中发挥关键作用，有关慢性HBV感染者CD4+T细胞衰老及其与DNA损伤修复的关系尚不明了。我们前期研究发现，慢性乙型肝炎（CHB）患者有记忆性CD4+T细胞增多、衰老分子表达升高等衰老表现，以及MRN-ATM通路核心分子ATM磷酸化显著升高，提示T细胞衰老可能参与了HBV持续感染的形成。因此，本研究拟以CHB患者和小鼠模型为研究对象，分析慢性HBV感染导致CD4+T细胞衰老及其临床意义，并探讨MRN-ATM通路在其中的作用。以期揭示CD4+T细胞衰老在HBV感染慢性化的作用及机制，为慢性HBV感染的免疫治疗提供新思路。

乙型肝炎病毒（HBV）慢性感染的机制是目前慢性乙型肝炎（CHB）防治的关键问题之一。持续病毒感染可以引起T细胞衰老，可能是导致HBV持续感染和免疫耐受形成的原因之一。CD4+T细胞在抗HBV免疫反应中发挥关键作用，有关慢性乙型肝炎（CHB）患者CD4+T细胞衰老及其具体机制尚不明了，本研究旨在观察慢性HBV感染者CD4+ T细胞及其各亚群细胞衰老现象及与临床指标之间的关系，并探索DNA损伤修复通路MRN-ATM在慢性HBV感染导致的CD4+ T细胞衰老中的作用。结果发现，与健康对照相比，慢性HBV感染者总CD4+T细胞细胞衰老分子KLRG1水平显著升高，且与血清转氨酶呈正相关。此外，慢性HBV感染者CD4+T细胞端粒长度显著缩短，DNA损伤标记分子γ-H2AX表达显著增加，γH2AX/53BP1双阳性的DNA损伤聚集点明显增多。表明慢性HBV感染者CD4+T细胞存在衰老现象，并存在DNA损伤。与健康对照相比，虽然慢性HBV感染者CD4+T细胞DNA损伤感知分子MRN复合物—MRE11、RAD50和NBS1基因表达并无明显变化，但NBS1蛋白表达显著升高，DNA损伤修复分子p-ATM及其下游通路蛋白p-CHK2和p-p53显著升高，表明慢性HBV感染者CD4+T细胞DNA损伤修复通路MRN-ATM处于激活状态。之后，采用KU-60019（ATM抑制剂）处理CD4+T细胞后，慢性HBV感染者端粒长度进一步缩短，γ-H2AX及γH2AX/53BP1双阳性的DNA损伤聚集点进一步增多，ATM/CHK2/p53通路的活性被抑制，CD4+T细胞分泌IFN-γ和IL-2的水平均显著下调。说明MRN-ATM通路在CD4+T细胞衰老、DNA损伤修复及细胞功能的调节中起重要作用。有关NBS1对DNA损伤修复关键分子ATM的调节机制及在体研究尚在进行中。上述结果表明，慢性HBV感染者CD4+T细胞存在衰老现象及DNA损伤，同时DNA损伤修复系统被激活，使细胞状态和功能保持平衡。本研究揭示了CD4+T细胞衰老在HBV感染慢性化中的作用及机制，可能为慢性HBV感染的免疫治疗提供新的思路。