Regulation of encephalitogenic T cells by CRAC channels

项目摘要 多发性硬化症(MS)是一种以中枢神经系统炎症为特征的自身免疫性疾病 中枢神经系统(CNS)和局部破坏大脑和脊髓，导致神经衰弱 症状。多发性硬化症潜在的疾病过程在一种名为 实验性自身免疫性脑脊髓炎(EAE)，这极大地帮助开发了几个批准的 MS的治疗在MS和EAE中，CD4辅助T细胞错误地识别由 脑细胞被视为异物，并对其产生自身免疫反应。这种炎症反应是由 主要是通过两个辅助T细胞亚群，即所谓的Th1和Th17细胞，它们渗透到中枢神经系统并产生 促炎症细胞因子干扰素γ和IL-17。干扰幼稚的CD4T细胞的发育 分别通过基因缺失转录因子T-bet和RoRγt进入Th1和Th17细胞，保护 来自EAE的老鼠。我们的实验室表明，CD4T细胞需要钙离子的内流才能激活和 产生干扰素γ和IL-17的能力。钙离子内流是由CRAC(钙释放激活钙)介导的 位于细胞质膜并由ORAI1蛋白形成的通道。ORAI1已激活 通过两种胞内蛋白STIM1和STIM2以及这三种蛋白中的任何一种的缺失或突变 减弱CRAC通道功能和钙内流。利用ORAI1、STIM1和STIM1基因缺失的小鼠 STIM2以及特定的CRAC通道抑制剂，我们发现Th1和Th17细胞更依赖于 钙离子内流比其他T细胞的功能和能力更能引起自身免疫性炎症。T的治疗 小鼠或人的细胞经CRAC通道特异性抑制剂后，IL-17和干扰素γ的产生减少，但 不影响T调节(Treg)细胞。以剂量依赖的方式。通过基因消融ORAI1， 在T细胞中表达STIM1或STIM2，可以预防或改善EAE和中枢神经系统炎症的发展。 老鼠。重要的是，在出现EAE症状后，T细胞中ORAI1或STIM1的缺失停止 疾病的发展。同样，对EAE发展为CRAC通道的小鼠的治疗 抑制剂也显著降低了疾病的严重程度，没有明显的不良反应。这些数据表明 钙内流是EAE中枢神经系统炎症的重要介质，抑制钙内流可能是一种新的选择 为了治疗多发性硬化症，抑制CRAC通道的药物已经开发出来，但尚未进行测试 它们在治疗MS和其他T细胞介导的自身免疫性疾病方面的有效性和安全性。的目标 这一应用有两个方面：(1)了解钙通过CRAC内流的分子机制 通道控制CD4T细胞向致病T细胞的发育，特别是Th1和Th17细胞， 介导CNS炎症和EAE/MS(2)评价CRAC通道作为免疫治疗药物靶点的价值 在复发-缓解型EAE小鼠模型中，通过研究它们在人类T细胞中的作用，研究它们在EAE和最终MS中的作用 以及对感染的免疫力，作为抗炎治疗的潜在并发症。

Project Summary Multiple Sclerosis (MS) is an autoimmune disorder that is characterized by inflammation of the central nervous system (CNS) and localized destruction of the brain and spinal cord resulting in debilitating neurological symptoms. The disease processes underlying MS have been studied extensively in an animal model called experimental autoimmune encephalomyelitis (EAE), which greatly helped to develop several approved therapies for MS. In both MS and EAE, CD4+ helper T cells mistakenly recognize myelin protein expressed by brain cells as foreign and mount an autoimmune response against it. This inflammatory response is mediated mainly by two subsets of helper T cells, so-called Th1 and Th17 cells, which infiltrate the CNS and produce the proinflammatory cytokines IFNγ and IL-17, respectively. Interfering with the development of naive CD4+ T cells into Th1 and Th17 cells by genetic deletion of the transcription factors T-bet and RORγt, respectively, protects mice from EAE. Our lab showed that CD4+ T cells require the influx of calcium ions for their activation and ability to produce IFNγ and IL-17. Calcium influx is mediated by CRAC (calcium release-activated calcium) channels that are located in the plasma membrane of cells and formed by ORAI1 proteins. ORAI1 is activated by two intracellular proteins, STIM1 and STIM2, and deletion or mutagenesis of either of these three proteins attenuates CRAC channel function and calcium influx. Using mice with genetic deletion of ORAI1, STIM1 and STIM2 as well as specific CRAC channel inhibitors, we found that Th1 and Th17 cells are more dependent on calcium influx than other T cells for their function and ability to cause autoimmune inflammation. Treatment of T cells of mice or humans with a specific CRAC channel inhibitor reduced the production of IL-17 and IFNγ but did not affect T regulatory (Treg) cells. in a dose dependent manner. By genetically ablating either ORAI1, STIM1 or STIM2 in T cells, we could prevent or ameliorate the development of EAE and CNS inflammation in mice. Importantly, deletion of ORAI1 or STIM1 in T cells after EAE symptoms had already developed stopped the progression of disease. Similarly, treatment of mice in which EAE had developed with a CRAC channel inhibitor also significantly reduced disease severity without apparent adverse effects. These data suggest that calcium influx is an essential mediator of CNS inflammation in EAE and that its inhibition may be a new option for the treatment of MS. Drugs inhibiting CRAC channels have been developed, but have not been tested for their efficacy and safety in the treatment of MS and other T cell mediated autoimmune diseases. The goals of this application are twofold: (1) To understand the molecular mechanisms by which calcium influx via CRAC channels controls the development of CD4 T cells into pathogenic T cells, in particular Th1 and Th17 cells, that mediate CNS inflammation and EAE/MS. (2) To evaluate CRAC channels as drug targets for immunotherapy of EAE and eventually MS by studying their role in human T cells, in a mouse model of relapsing-remitting EAE and in immunity to infections as a potential complication of antiinflammatory therapy.