The NOTCH Signaling Pathway in Large Vessel Vasculitis

项目摘要 巨细胞动脉炎（GCA）是一种自身免疫性和自身炎症性疾病，其靶向主动脉及其主要血管， 分支血管。GCA导致血管闭塞性疾病，导致失明和中风。约有半数患者 发生GCA动脉炎，一种由于主动脉夹层和动脉瘤形成而可能危及生命的并发症。 潜在的疾病过程是肉芽肿性动脉炎，具有CD 4 T细胞、巨噬细胞和多核 巨细胞浸润到血管壁中，引起适应不良的血管壁重塑，伴有新血管生成和管腔- 闭塞性内膜增生 我们已经确定了癌基因NOTCH 1在CD 4 T细胞中的异常表达是CD 4 T细胞中的一个关键异常。 GCA患者的免疫系统。在这里，我们将检验NOTCH信号转换为 通过抑制线粒体琥珀酸酶将保护性免疫转化为病原性免疫 脱氢酶（SDH）和截短三羧酸（TCA）循环。三氯乙酸循环的碎片化 然后导致代谢中间产物琥珀酸的积累，琥珀酸被释放到组织中， 网站和功能作为第二信使。我们认为NOTCH 1hi SDHlo CD 4分泌的琥珀酸 T细胞靶向周围细胞以重定向T效应细胞分化，以诱导多核细胞分化。 巨噬细胞和促进微血管新生。我们已经汇集了关键的有利资源， 机械地研究NOTCH指示的琥珀酸释放如何增强血管炎症;包括 临床上良好表型的GCA患者的大队列和诱导血管炎的嵌合小鼠模型 通过体内研究证实体外数据。目标1将定义分子 导致NOTCH依赖性SDH功能丧失的机制，建立在初步研究的基础上， 通过N6-甲基腺苷修饰调节SDH mRNA稳定性的RNA结合蛋白。目标2A 研究琥珀酸如何机械地重编程T效应细胞分化。实验旨在 研究琥珀酸如何麻痹NF-κ B抑制剂A20/TNFAIP 3以释放NF-κ B信号传导， 诱导多功能效应T细胞（Thpoly），包括共产生IFN-γ、IL-17、TNF-α、IL-21和 IL-22。目的2B将确定NOTCH指导的琥珀酸如何改变巨噬细胞功能，特别是通过 驱动组织破坏性多核巨细胞的形成。我们将描述琥珀酸如何使一个强大的 DNA损伤反应及其如何通过干扰纺锤体促进核分裂和停止胞质分裂 装配检查点。目的2C关注琥珀酸在诱导促血管生成内皮细胞（EC）中的作用 表型，并将探讨如何琥珀酸训练EC迁移，增殖，并失去其屏障功能。目标3 将从工作台连接到床边，并将测试是否抑制琥珀酸生产， 阻断上游酶α-酮戊二酸脱氢酶可以成功地治疗体内血管炎。

Project Summary Giant Cell Arteritis (GCA) is an autoimmune and autoinflammatory disease which targets the aorta and its major branch vessels. GCA causes vaso-occlusive disease, leading to blindness and stroke. About half of the patients develop GCA aortitis, a potentially life-threatening complication due to aortic dissection and aneurysm formation. The underlying disease process is a granulomatous arteritis, with CD4 T cells, macrophages and multinucleated giant cells infiltrating into the vessel wall, eliciting maladaptive wall remodeling with neoangiogenesis and lumen- occlusive intimal hyperplasia. We have identified aberrant expression of the oncogene NOTCH1 in CD4 T cells as a key abnormality in the immune system of GCA patients. Here, we will examine the hypothesis that NOTCH signaling transforms protective immunity into pathogenic immunity by suppressing the mitochondrial enzyme succinate dehydrogenase (SDH) and truncating the tricarboxylic acid (TCA) cycle. Fragmentation of the TCA cycle then leads to the accumulation of the metabolic intermediate succinate, which is released into the tissue site and functions as a second messenger. We propose that succinate secreted by NOTCH1hi SDHlo CD4 T cells targets surrounding cells to redirect T effector cell differentiation, to induce multinucleated macrophages and to promote microvascular neoangiogenesis. We have assembled key enabling resources to mechanistically study how NOTCH-instructed succinate release enhances vascular inflammation; including a large cohort of clinically well phenotyped GCA patients and a chimeric mouse model in which vasculitis is induced in engrafted human arteries to corroborate in vitro data by in vivo studies. Aim 1 will define the molecular mechanisms leading to NOTCH-dependent SDH loss-of-function, building on preliminary studies that implicate RNA-binding proteins in regulating SDH mRNA stability through N6-methyladenosine modifications. Aim 2A examines mechanistically how succinate reprograms T effector cell differentiation. Experiments are designed to investigate how succinate paralyzes the NF-kappaB inhibitor A20/TNFAIP3 to unleash NF-kappaB signaling and induce polyfunctional effector T cells (Thpoly), including T cells that co-produce IFN-, IL-17, TNF-α, IL-21 and IL-22. Aim 2B will determine how NOTCH-instructed succinate alters macrophage function, specifically by driving formation of tissue-destructive multinucleated giant cells. We will delineate how succinate elicits a robust DNA damage response and how it promotes nuclear division and halts cytokinesis by interfering with the spindle assembly checkpoint. Aim 2C is focused on succinate’s role in inducing a pro-angiogenic endothelial cell (EC) phenotype and will explore how succinate-trained EC migrate, proliferate, and lose their barrier function. Aim 3 will bridge from the bench to the bedside and will test whether the suppression of succinate production by blocking the upstream enzyme a-ketoglutarate dehydrogenase can successfully treat vasculitis in vivo.