DNA Repair and Mitochondrial Dysfunction in T Cell Aging

项目摘要 T淋巴细胞的老化对老年人的健康有不利影响；使他们容易患上 癌症、感染和非对抗性组织炎症。较老的T细胞容易分化为炎症性细胞 效应细胞，迅速侵入周围组织，促进炎症组织损伤。多重 生物过程与调节T细胞衰老有关，包括DNA修复缺陷、端粒 损伤、膜重组为侵袭性突起以及细胞生物能量学的重新编程。T细胞 在炎症性综合征类风湿性关节炎(RA)患者中，衰老会加速20-30年， 为研究老年T细胞的分子、结构和行为异常提供了一个很好的模型系统。 在前一个资助期支持的工作已经确定DNA核酸酶MRE11A是 T细胞老化。来自RA患者和老年健康捐赠者的T细胞分享转录抑制 MRE11A。低表达MRE11A蛋白的T细胞积聚受损的端粒并引发强烈的组织炎症 带有不受控制的先天免疫和适应性免疫的特征。初步数据显示，MRE11Alow T细胞 线粒体中也缺乏核酸酶的表达，导致氧化损伤和细胞质渗漏 线粒体DNA(MtDNA)在细胞质中，氧化的mtDNA促进NLRP3炎症体组装， 触发caspase1激活，最终诱导T细胞裂解性死亡。在这里，我们检验一下假设 线粒体应激是T细胞衰老的驱动因素，与年龄相关的DNA修复核酸酶下降 MRE11A使T细胞暴露于线粒体DNA泄漏，炎性小体激活，最终导致 嗜热性死亡。我们已经收集了关键的使能资源来机械地研究线粒体如何 MRE11A保护T细胞免受衰老；包括一大批RA患者，在这些患者中，T细胞提前老化并 一种嵌合小鼠模型，在该模型中，植入的人滑膜中诱导了组织炎症，以证实 通过体内研究获得体外数据。目标1从机制上研究MRE11A如何影响线粒体功能， ATP和ROS的产生，葡萄糖的利用和脂肪生成。目的是将线粒体DNA修复 与电子转移链中的氧耗和三元酸循环中的乙酰辅酶A氧化有关。 目的2研究MRE11A如何通过阻止线粒体DNA的释放来指导线粒体应激防御计划 进入细胞质。在功能丧失和功能获得实验中，我们将探索MRE11A是如何调节的 炎性小体组装、caspase 1激活和T细胞下垂。目标3将通过以下方式确定机制 哪种MRE11A缺乏症会导致组织炎症。我们将测试caspase 1依赖的切割如何 促炎细胞因子IL-1、和IL-18及膜孔的前驱动剂D驱动组织损伤。 这些实验将在体外和体内探索线粒体DNA修复失败如何导致T细胞溶解， 将警报释放到细胞外间隙，并启动非感染性炎症。

Project Summary Aging of T lymphocytes has detrimental effects on the health of older individuals; rendering them susceptible to cancer, infection, and unopposed tissue inflammation. Older T cells are prone to differentiate into inflammatory effector cells, rapidly invading into peripheral tissues and promoting inflammatory tissue damage. Multiple biologic processes have been implicated in mediating T cell aging; including defective DNA repair, telomeric damage, membrane restructuring into invasive protrusions and reprogramming of cellular bioenergetics. T cell aging is accelerated by 20-30 years in patients with the inflammatory syndrome rheumatoid arthritis (RA), providing an excellent model system to study molecular, structural and behavioral abnormalities in old T cells. Work supported during the previous funding period has identified the DNA nuclease MRE11A as a key player in T cell aging. T cells from RA patients and from older healthy donors share the transcriptional repression of MRE11A. T cells low in MRE11A protein accumulate damaged telomeres and induce robust tissue inflammation with a signature of uncontrolled innate and adaptive immunity. Preliminary data show that MRE11Alow T cells also lack expression of the nuclease in mitochondria, leading to oxidative damage and cytoplasmic leakage of mitochondrial DNA (mtDNA). In the cytoplasm, oxidized mtDNA promotes NLRP3 inflammasome assembly, triggers caspase 1 activation and ultimately induces T cell lytic death. Here, we examine the hypothesis that mitochondrial stress is a driver of T cell aging and that the age-related decline of the DNA repair nuclease MRE11A exposes T cells to mitochondrial DNA leakage, inflammasome activation and ultimately to pyroptotic death. We have assembled key enabling resources to mechanistically study how mitochondrial MRE11A protects T cells from aging; including a large cohort of RA patients in whom T cells are pre-aged and a chimeric mouse model in which tissue inflammation is induced in engrafted human synovium to corroborate in vitro data by in vivo studies. Aim 1 examines mechanistically how MRE11A affects mitochondrial function, generation of ATP and ROS, glucose utilization and lipogenesis. The aim will connect mitochondrial DNA repair to oxygen consumption in the electron transfer chain and acetyl-CoA oxidation in the tricarboxylic acid cycle. Aim 2 investigates how MRE11A guides a mitochondrial stress defense program by preventing mtDNA release into the cytoplasm. In loss-of-function and gain-of-function experiments we will probe how MRE11A regulates inflammasome assembly, caspase 1 activation and T cell pyroptosis. Aim 3 will identify mechanisms through which MRE11A deficiency induces tissue inflammation. We will test how caspase 1-dependent cleavage of the pro-inflammatory cytokines IL-1 and IL-18 and of the membrane pore former gasdermin D drive tissue damage. These experiments will explore in vitro and in vivo how failed mitochondrial DNA repair causes T cell lysis, release of alarmins into the extracellular space and initiation of noninfectious inflammation.