Metabolic control of systemic autoimmunity

描述(申请人提供)：系统性红斑狼疮(SLE)的特征是T细胞的异常激活和死亡，这一过程严重依赖于线粒体中活性氧中间产物(ROI)和ATP的受控产生。线粒体跨膜电位已明确地成为ATP合成和细胞死亡的关键检查点。在正常T细胞中，我们首先发现线粒体跨膜电位升高，即线粒体超极化(MHP)，其次是ATP耗竭，这是T细胞激活和凋亡的早期和可逆步骤。相反，在SLE患者中，我们发现T细胞表现出持续的MHP以及ATP和谷胱甘肽的耗竭，这减少了激活诱导的凋亡，相反地，T细胞易于坏死，从而刺激了SLE的炎症。因此，确定持续性MHP的分子基础和后果对于了解狼疮T细胞活化和死亡信号改变的机制是至关重要的。 我们发现，持续性MHP与T淋巴细胞线粒体质量增加、线粒体和胞浆钙含量增加以及单核细胞一氧化氮(NO)产生增加有关。NO诱导的正常T细胞线粒体生物发生加快了CD3/CD28共刺激的快速期，降低了钙内流平台期，从而模拟了狼疮T细胞的钙信号转导模式。由于线粒体是主要的钙储存库，NO依赖的线粒体生物发生可能解释了钙处理的改变。在狼疮T细胞中，我们发现了控制关键代谢途径的基因的表达变化：导致谷胱甘肽耗竭和MHP的转醛醇酶(TAL)的过度表达，调节NO分区化产生的eNOS相互作用蛋白(NOSIP)的低表达，以及雷帕霉素受体FKBP12的过度表达。在接受雷帕霉素治疗的患者中，我们观察到疾病活动性的改善，CD3/CD28诱导的钙通量的正常化，以及MHP的持续，表明改变的钙通量是下游的或独立于线粒体功能障碍的。 这些研究将检验这样一种假设，即在NO存在下，谷胱甘肽耗竭通过S亚硝化抑制电子传递链导致持久的MHP，而MHP反过来激活哺乳动物雷帕霉素靶点(MTOR)途径。首先，我们将测量分离的线粒体电子传输链的功能容量，并确定GSH耗竭和TAL激活在MHP和ATP耗竭狼疮T细胞中的作用。其次，我们将确定内源性和外源性NO产生的作用，eNOS的区区化表达，以及对NO的反应性。第三，我们将考察mTOR作为MHP的感受器和下游效应器以及钙离子通量增加的控制者的作用。第四，我们将系统地绘制MHP上游和下游的代谢检查点，并验证候选基因的参与，这些候选基因可以靶向使SLE的T细胞激活正常化。

DESCRIPTION (provided by applicant): Systemic lupus erythematosus (SLE) is characterized by abnormal T-cell activation and death, processes which are crucially dependent on the controlled production of reactive oxygen intermediates (ROI) and of ATP in mitochondria. The mitochondrial transmembrane potential has conclusively emerged as a critical checkpoint of ATP synthesis and cell death. In normal T cells, we firstly identified the elevation of the mitochondrial transmembrane potential, i.e., mitochondrial hyperpolarization (MHP), and, secondly, also ATP depletion, which are early and reversible steps of T-cell activation and apoptosis. Conversely, in SLE patients, we found that T cells exhibit persistent MHP as well as ATP and glutathione depletion which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. Therefore, determining the molecular basis and consequences of persistent MHP is essential for understanding the mechanism of altered activation and death signaling in lupus T cells. We found persistent MHP to be associated with increased mitochondrial mass and increased mitochondrial and cytoplasmic Ca2+ content in T lymphocytes and also with enhanced nitric oxide (NO) production in monocytes. NO-induced mitochondrial biogenesis in normal T cells accelerates the rapid phase and reduces the plateau of Ca2+ influx upon CD3/CD28 costimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Since mitochondria are major Ca2+ stores, NO-dependent mitochondrial biogenesis may account for altered Ca2+ handling. In lupus T cells, we identified changes in expression of genes that control key metabolic pathways: over-expression of transaldolase (TAL) which induces glutathione depletion and MHP, low expression of eNOS-interacting protein (NOSIP) that regulates compartmentalized production of NO, and over-expression of the rapamycin receptor FKBP12. We observed improvement of disease activity, normalization of CD3/CD28-induced Ca2+ fluxing, and persistence of MHP in rapamycin-treated patients, suggesting that altered Ca2+ fluxing is downstream or independent of mitochondrial dysfunction. The proposed studies will test the hypothesis that inhibition of the electron transport chain via S-nitrosylation stemming from glutathione depletion in the presence of NO causes persistent MHP which, in turn, activates the mammalian target of rapamcyin (mTOR) pathway. First, we will measure functional capacity of the electron transport chain in isolated mitochondria and determine the role of GSH depletion and TAL activation in MHP and ATP depletion of lupus T cells. Second, we will determine the role of intrinsic and extrinsic NO production, compartmentalized expression of eNOS, and responsiveness to NO. Third, we will examine the role of mTOR as a sensor and down-stream effector of MHP and controller of increased Ca2+ fluxing. Fourth, we will systematically map metabolic checkpoints upstream and downstream of MHP and validate the involvement of candidate genes that can be targeted to normalize T-cell activation in SLE.