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）和脑和脊髓的局部破坏导致神经衰弱 症状MS潜在的疾病过程已经在一种称为MS的动物模型中进行了广泛研究。 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 在MS和EAE中，CD4+辅助性T细胞错误地识别MS表达的髓鞘蛋白， 脑细胞作为外来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性T细胞亚群，即所谓的Th1和Th17细胞，它们浸润CNS并产生免疫应答。 促炎细胞因子IFN γ和IL-17。干扰初始CD4 + T细胞的发育 分别通过基因缺失转录因子T-bet和ROR γ t进入Th1和Th17细胞， EAE小鼠。我们的实验室表明，CD4 + T细胞需要钙离子的流入来激活， 产生IFN γ和IL-17的能力。钙内流由CRAC（钙释放激活钙）介导 位于细胞质膜中并由ORAI1蛋白形成的通道。ORAI1激活 通过两种细胞内蛋白质，STIM 1和STIM 2，以及这三种蛋白质中的任一种的缺失或诱变， 减弱CRAC通道功能和钙内流。使用基因缺失ORAI1、STIM1和 STIM2以及特异性CRAC通道抑制剂，我们发现Th1和Th17细胞更依赖于 钙内流比其他T细胞的功能和能力，导致自身免疫性炎症。治疗t 具有特异性CRAC通道抑制剂的小鼠或人的细胞减少了IL-17和IFN γ的产生， 不影响调节性T细胞（Treg）。以剂量依赖的方式。通过基因切除ORAI1， 通过在T细胞中表达STIM 1或STIM 2，我们可以预防或改善EAE和CNS炎症的发展。 小鼠重要的是，EAE症状发生后T细胞中的ORAI1或STIM1缺失已经停止 疾病的发展类似地，用CRAC通道治疗已经发展EAE的小鼠， 抑制剂还显著降低疾病严重程度而没有明显的副作用。这些数据表明 钙离子内流是EAE中枢神经系统炎症的重要介质，抑制钙离子内流可能是EAE治疗的新选择 已经开发了抑制CRAC通道的药物，但尚未测试用于MS的治疗。 它们在治疗MS和其它T细胞介导的自身免疫性疾病中的功效和安全性。的目标 这一应用是双重的：（1）了解钙通过CRAC流入的分子机制 通道控制CD4 T细胞发育成致病性T细胞，特别是Th1和Th17细胞， （2）评价CRAC通道作为免疫治疗药物靶点的可能性 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， 以及对作为化疗潜在并发症的感染的免疫力。

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.