Metabolic Regulation of Caspases and Survival in T Cells

描述(申请人提供)：对于那些正在进行线粒体氧化磷酸化(OxPhos)而不是糖酵解的T细胞来说，效应T细胞存活到记忆状态更有效。IL-15在T细胞中驱动OxPhos，并与几种自身免疫性疾病有关，如类风湿性关节炎，炎症的滑膜中含有丰富的IL-15。目前尚不清楚IL-15如何促进T细胞中的OxPhos，或者这如何促进T细胞的存活。巴德博士和林肯博士的团队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，林肯博士证明了一种名为MCJ(甲基化控制的J蛋白)的新定义蛋白质对线粒体复合体I的活性和OxPhos(6)进行了负面调节；其次，Budd博士证明了与IL-2培养的T细胞相比，IL-15下调了MCJ的表达，使Complex I的活性增加，OxPhos和反应性自由基增加。这导致了S-亚硝化和S-谷胱甘肽基化，并在关键的Cys163处使位于酶口袋中的caspase-3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，导致线粒体OxPhos增强，反应性自由基增加，并通过S亚硝化使Caspase-3失活，从而促进效应者T细胞的存活。然后，我们将我们的小鼠发现应用于一种富含IL-15的实际人类炎症情况，即类风湿滑膜。具体目的1将确定逆转录病毒在IL-15培养的T细胞中重新表达MCJ是否抑制线粒体复合体I的活性和OxPhos，并增加caspase-3的活性。然后我们将确定IL-15是否也促进了超复合体的形成，就像在MCJ-/-T细胞中观察到的那样。具体目标2将确定存在于人类类风湿滑膜中的IL-15是否通过MCJ下调促进OxPhos、ROS/RNS、降低caspase-3活性和抵抗细胞死亡。类风湿滑膜组织或滑液T细胞将通过海马流量分析、ROS/RNS生成、MCJ水平、caspase-3活性和抗细胞死亡能力来评估其代谢状态。这项新颖的研究结合了风湿病学家PI在T细胞中细胞因子调节caspase方面的专业知识，以及林肯博士的专业知识，他首先描述了T细胞中的MCJ及其在调节复合体I的线粒体呼吸中的作用。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。进行这些研究的所有工具都已就位，包括MCJ-/-小鼠和类风湿滑膜组织培养。这些实验也代表了一项有针对性的探索性研究，具有提高人类炎症条件的潜力，并建议通过调节MCJ、IL-15和新陈代谢进行治疗干预。

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.