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;使他们容易受到 癌症、感染和非对抗性组织炎症。较老的T细胞容易分化成炎性细胞， 效应细胞，迅速侵入外周组织并促进炎性组织损伤。多 生物学过程与介导T细胞衰老有关;包括DNA修复缺陷、端粒 损伤、膜重组为侵入性突起和细胞生物能量学重编程。T细胞 在患有炎性综合征类风湿性关节炎（RA）的患者中衰老加速20-30年， 为研究老年T细胞的分子、结构和行为异常提供了极好的模型系统。 在上一个资助期内支持的工作已经确定DNA核酸酶MRE 11 A是 T细胞老化来自RA患者和来自老年健康供体的T细胞共享以下转录抑制： MRE 11A。MRE 11 A蛋白含量低的T细胞积累受损的端粒并诱导强烈的组织炎症 先天免疫和适应性免疫不受控制初步数据显示，MRE 11 Alow T细胞 也缺乏线粒体中核酸酶的表达，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NLRP 3炎性小体组装， 触发半胱天冬酶1活化并最终诱导T细胞溶解性死亡。在这里，我们检验假设， 线粒体应激是T细胞衰老的驱动因素， MRE 11 A使T细胞暴露于线粒体DNA泄漏、炎性小体活化，并最终导致细胞凋亡。 自燃死亡我们已经收集了关键的使能资源，以机械地研究线粒体如何 MRE 11 A保护T细胞免于老化;包括一个大型RA患者队列，其中T细胞是预老化的， 嵌合小鼠模型，其中在移植的人滑膜中诱导组织炎症，以证实 体内研究的体外数据。目的1研究MRE 11 A如何机械地影响线粒体功能， ATP和ROS的产生、葡萄糖利用和脂肪生成。其目标是将线粒体DNA修复 电子转移链中的氧消耗和三羧酸循环中的乙酰辅酶A氧化。 目的2研究MRE 11 A如何通过阻止mtDNA释放来指导线粒体应激防御程序 进入细胞质。在功能丧失和功能获得实验中，我们将探索MRE 11 A如何调节 炎性小体组装、半胱天冬酶1活化和T细胞焦亡。目标3将通过以下方式确定机制： 该MRE 11 A缺陷诱导组织炎症。我们将测试半胱天冬酶1依赖性切割的细胞， 促炎细胞因子IL-1 β和IL-18以及膜造孔剂gasdermin 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， alarmins释放到细胞外空间并引发非感染性炎症。

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.