Metabolic Regulation of Caspases and Survival in T Cells

Caspases 的代谢调节和 T 细胞的存活

• 批准号: 9110491
• 负责人: Ralph C Budd
• 金额: $ 19.3万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9110491
• 关键词: Autoimmune Diseases; Autoimmune Process; CASP3 gene; Caspase; Cell Death; Cell Survival; Complex; Down-Regulation; Electron Transport; Environment; Future; Generations; Genus Hippocampus; Glycolysis; Health; Human; Immune response; Infection; Inflammatory; Interleukin-1 beta; Interleukin-15; Interleukin-2; Link; Memory; Metabolic; Metabolism; Methylation; Mitochondria; Modeling; Mouse Protein; Multiple Sclerosis; Mus; Oligomycins; Oxidative Phosphorylation; Pathogenesis; Predisposition; Protein Methylation; Proteins; Psoriasis; Reactive Nitrogen Species; Reactive Oxygen Species; Regulation; Research; Resistance; Respiration; Rheumatoid Arthritis; Role; Rotenone; Series; Site; Suggestion; Syndrome; Synovial Fluid; Synovial Membrane; T memory cell; T-Cell Proliferation; T-Lymphocyte; TNF gene; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Ulcerative Colitis; autoreactive T cell; cytokine; driving force; extracellular; inhibitor/antagonist; mitochondrial metabolism; novel; protein expression; research study; rheumatologist; tissue culture; tool

DESCRIPTION (provided by applicant): The survival of effector T cells to the memory state is more effective for those T cells undergoing mitochondrial oxidative phosphorylation (OxPhos) rather than glycolysis. IL-15 drives OxPhos in T cells and has been associated with several autoimmune disorders, such as rheumatoid arthritis, where the inflamed synovium is rich in IL-15. It is currently unknown how IL-15 promotes OxPhos in T cells or how this promotes T cell survival. Dr. Budd's and Rincon's groups have made two key preliminary observations that offer an explanation. First, Dr. Rincon has shown that a newly defined protein known as MCJ (Methylation- Controlled J protein) negatively regulates mitochondrial Complex I activity and OxPhos (6), Second, Dr. Budd has shown that IL-15 downregulates MCJ expression, allowing increased Complex I activity, elevated OxPhos and reactive radicals compared to IL-2-cultured T cells. This results in S-nitrosylation and S- glutathionylation and inactivation of caspase-3 at critical Cys163, which resides in the enzymatic pocket. This R21 application thus posits a novel hypothesis that IL-15 promotes effector T cell survival through downregulation of MCJ, resulting in enhanced mitochondrial OxPhos, reactive radicals, and inactivation of caspase-3 through S-nitrosylation. We then apply our murine findings to an actual human inflammatory condition rich in IL-15, rheumatoid synovium. Specific Aim 1 will determine whether retroviral re-expression of MCJ in IL-15-cultured T cells inhibits mitochondrial Complex I activity and OxPhos, and increases caspase-3 activity. We will then determine whether IL-15 also promotes supercomplex formation, as observed in MCJ-/- T cells. Specific Aim 2 will define whether IL-15 present in human rheumatoid synovium promotes OxPhos, ROS/RNS, reduced caspase-3 activity and resistance to cell death through MCJ downregulation. Rheumatoid synovial tissue or synovial fluid T cells will be assessed for their metabolic state by Seahorse flux analysis, ROS/RNS generation, MCJ levels, caspase-3 activity, and resistance to cell death. This novel study combines the expertise of the PI, a research rheumatologist, in regulation of caspases by cytokines in T cells, with those of Dr. Rincon who first described MCJ in T cells and its role in regulating mitochondrial respiration at Complex I. The findings would provide the framework for future expanded studies on the role of IL-15 and MCJ in immune responses and autoimmune disorders, such as rheumatoid arthritis. All the tools are in place to conduct these studies, including MCJ-/- mice and rheumatoid synovial tissue cultures. The experiments also represent a focused exploratory study with the potential for high yield for human inflammatory conditions and suggestions for therapeutic intervention through modulation of MCJ, IL-15, and metabolism.

描述（由申请人提供）：效应T细胞存活至记忆状态对于那些经历线粒体氧化磷酸化（OxPhos）而不是糖酵解的T细胞更有效。 IL-15 驱动 T 细胞中的 OxPhos，并与多种自身免疫性疾病相关，例如类风湿性关节炎，其中发炎的滑膜富含 IL-15。目前尚不清楚 IL-15 如何促进 T 细胞中的 OxPhos 或如何促进 T 细胞存活。巴德博士和林康的研究小组做出了两项重要的初步观察结果，并给出了解释。首先，Rincon 博士表明，一种新定义的蛋白 MCJ（甲基化控制 J 蛋白）可负向调节线粒体复合物 I 活性和 OxPhos (6)，其次，Budd 博士表明，IL-15 下调 MCJ 表达，与 IL-2 培养的 T 细胞相比，可以增加复合物 I 活性、升高 OxPhos 和反应性自由基。这导致 Caspase-3 在位于酶袋中的关键 Cys163 处发生 S-亚硝基化和 S-谷胱甘肽化以及失活。因此，R21 的应用提出了一个新的假设，即 IL-15 通过下调 MCJ 促进效应 T 细胞存活，从而增强线粒体 OxPhos、反应性自由基，并通过 S-亚硝基化使 caspase-3 失活。然后，我们将小鼠研究结果应用到富含 IL-15 的实际人类炎症状况（类风湿滑膜）中。具体目标 1 将确定在 IL-15 培养的 T 细胞中逆转录病毒重新表达 MCJ 是否会抑制线粒体复合物 I 活性和 OxPhos，并增加 caspase-3 活性。然后我们将确定 IL-15 是否也促进超复合物形成，如在 MCJ-/- T 细胞中观察到的那样。具体目标 2 将确定人类类风湿滑膜中存在的 IL-15 是否会通过 MCJ 下调促进 OxPhos、ROS/RNS、降低 caspase-3 活性和抵抗细胞死亡。将通过 Seahorse 通量分析、ROS/RNS 生成、MCJ 水平、caspase-3 活性和细胞死亡抵抗力来评估类风湿滑液组织或滑液 T 细胞的代谢状态。这项新颖的研究结合了 PI（一位研究风湿病学家）在 T 细胞中细胞因子调节半胱天冬酶方面的专业知识，以及 Rincon 博士的专业知识，Rincon 博士首次描述了 T 细胞中的 MCJ 及其在调节复合物 I 线粒体呼吸中的作用。这些发现将为未来关于 IL-15 和 MCJ 在免疫反应和自身免疫性疾病（如类风湿）中的作用的扩展研究提供框架 关节炎。进行这些研究的所有工具均已到位，包括 MCJ-/- 小鼠和类风湿滑膜组织培养物。这些实验还代表了一项重点探索性研究，具有对人类炎症状况产生高产潜力的潜力，并为通过调节 MCJ、IL-15 和代谢进行治疗干预提供建议。