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，以主动脉及其主要器官为靶点 分支血管。GCA会导致血管闭塞疾病，导致失明和中风。大约一半的患者 发生GCA主动脉炎，这是由于主动脉夹层和动脉瘤形成而导致的一种潜在的危及生命的并发症。 潜在的疾病过程是肉芽肿性动脉炎，有CD4T细胞、巨噬细胞和多核细胞 巨细胞渗入血管壁，引起适应不良的管壁重塑和新生血管生成和管腔- 闭合性内膜增生症。 我们已经发现癌基因NOTCH1在CD4T细胞中的异常表达是 GCA患者的免疫系统。在这里，我们将检验缺口信号转换的假设 通过抑制线粒体琥珀酸酶将保护性免疫转化为致病免疫 脱氢酶(SDH)和截断三羧酸(TCA)循环。三氯乙烷循环的碎片化 然后导致代谢中间产物琥珀酸的积累，并释放到组织中 站点，充当第二信使。我们认为由NOTCH1hi SDHlo CD4分泌的琥珀酸 T细胞以周围细胞为靶点，重定向T效应细胞分化，诱导多核 巨噬细胞和促进微血管新生血管生成。我们已经整合了关键的支持资源 从机制上研究切迹指示的琥珀酸释放如何增强血管炎症；包括 临床表型良好的GCA患者和诱发血管炎的嵌合小鼠模型的大量队列 通过体内研究来证实体外数据。目标1将定义分子 导致缺口依赖型SDH功能丧失的机制，建立在初步研究的基础上 RNA结合蛋白通过N6-甲基腺苷修饰调节SDH mRNA稳定性。目标2 A 从机制上研究琥珀酸如何重新调节T效应细胞分化。实验的目的是为了 研究琥珀酸酯如何麻痹核因子-kappaB抑制剂A20/TNFAIP3以释放核因子-kappaB信号和 诱导多功能效应T细胞，包括共同产生干扰素-、白介素17、肿瘤坏死因子-α、白介素21和 IL-22。目标2B将确定缺口指导的琥珀酸如何改变巨噬细胞功能，特别是通过 推动组织破坏性多核巨细胞的形成。我们将描述琥珀酸酯如何引发强健的 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 装配检查站。目标2C集中在琥珀酸在诱导促血管生成内皮细胞(EC)中的作用 表型，并将探索琥珀酸训练的EC如何迁移、增殖和失去屏障功能。目标3 将从板凳架到床边，并将测试是否通过抑制琥珀酸的产生 阻断上游酶α-酮戊二酸脱氢酶可成功治疗体内血管炎。

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.