Metabolic Regulation of Caspases and Survival in T Cells

描述（由申请人提供）：效应T细胞存活至记忆状态对于经历线粒体氧化磷酸化（OxPhos）而不是糖酵解的那些T细胞更有效。IL-15驱动T细胞中的OxPhos，并与几种自身免疫性疾病有关，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。巴德博士和林孔的研究小组已经提出了两个关键的初步观察结果，提供了一个解释。首先，Rincon博士已经表明，称为MCJ（甲基化控制的J蛋白）的新定义的蛋白质负调节线粒体复合物I活性和OxPhos（6）。其次，Budd博士已经表明，与IL-2培养的T细胞相比，IL-15下调MCJ表达，允许增加的复合物I活性、升高的OxPhos和反应性自由基。这导致位于酶口袋中的关键Cys 163处的S-亚硝基化和S-谷胱甘肽化以及半胱天冬酶-3的失活。因此，该R21申请提出了一种新的假设，即IL-15通过MCJ的下调促进效应T细胞存活，导致线粒体OxPhos、反应性自由基的增强以及通过S-亚硝基化的半胱天冬酶-3的失活。然后，我们将我们的小鼠研究结果应用于富含IL-15的实际人类炎症条件，类风湿性滑膜。具体目标1将确定IL-15培养的T细胞中MCJ的逆转录病毒再表达是否抑制线粒体复合物I活性和OxPhos，并增加半胱天冬酶-3活性。然后，我们将确定IL-15是否也促进超复合物的形成，如在MCJ-/- T细胞中观察到的。具体目标2将定义人类风湿性滑膜中存在的IL-15是否通过MCJ下调促进OxPhos、ROS/RNS、降低的半胱天冬酶-3活性和对细胞死亡的抗性。通过Seahorse通量分析、ROS/RNS产生、MCJ水平、半胱天冬酶-3活性和对细胞死亡的抗性来评估风湿性关节炎滑膜组织或滑液T细胞的代谢状态。这项新的研究结合了PI（一位研究流变学家）在T细胞中通过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及其在复合物I中调节线粒体呼吸的作用。这些发现将为未来扩大IL-15和MCJ在免疫反应和自身免疫性疾病（如类风湿性关节炎）中的作用提供框架。所有工具都已到位，可用于进行这些研究，包括MCJ-/-小鼠和类风湿性滑膜组织培养。这些实验还代表了一项集中的探索性研究，具有高产量的人类炎症状况的潜力，并建议通过调节MCJ，IL-15和代谢进行治疗干预。

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.