Complement Induces Inflammasome Assembly in Human Endothelium: Mechanisms and Consequences for Graft Rejection

实体器官移植--终末期肾、肝、肺、心的最佳治疗方法 失败，可能是由于慢性免疫排斥反应，通常采取同种异体血管病变的形式。同种异体移植物 血管病变是由于产生干扰素-γ的移植物重新聚集到移植物血管壁并在移植物血管壁内激活所致 通过移植内皮细胞反应宿主T细胞。识别非我的供体特异性抗体(DSA)的发展 移植物内皮细胞(ECs)表达MHC分子的等位基因和固定补体是 同种异体血管病变。补体通过激活内皮细胞的免疫功能来增强这一过程 调节同种异体反应性T细胞的募集和激活。高效价群体反应性抗体的结合 (PRA)来自同种异体致敏的移植候选者，用于模拟DSA，沉积补充膜攻击 人内皮细胞上的复合体(MAC)，无论是在培养中还是在免疫缺陷小鼠宿主的血管移植中，以及 以依赖MAC的方式诱导黏附分子和趋化因子的基因表达 内化和非规范的NF-κB信号转导。MAC增强T细胞的机制(S) 激活，测量为体外或增强的效应记忆T细胞增殖和细胞因子的产生 体内血管病变的变化尚不清楚。我的初步数据显示，MAC诱导形成了一个活跃的 内皮细胞中的炎性小体，这是以前没有描述过的现象。炎症小体中活化的caspase-1 将前IL-1β处理为活性IL-1β，并调节其释放。阻断caspase-1活性或抑制IL-1 用IL-1受体拮抗剂(IL-1ra)阻断内皮细胞下游炎症基因的表达 继发于典型的NF-κB激活，也阻断了同种异体T细胞反应的增强 体外培养。我假设内皮细胞中炎性小体的补体激活增强了宿主T细胞的反应 通过增加局部IL-1的产生来移植动脉内皮细胞，增强同种异体血管病变。以特定的目标 1，我将描述MAC诱导的炎症体的特征，并确定将MAC与 炎性小体组件。我将研究非规范的NF-κB信号通路是否与 炎性小体组装或IL-1mRNA和蛋白质合成。在具体目标2中，我将调查生物 同种异体抗体诱导内皮细胞炎性小体激活对同种异体反应性T细胞反应的影响 导致同种异体血管病变。我将确定这种反应是否也是MAC依赖的，以及成熟的IL-1是否 从MAC诱导的炎性小体释放出来的物质作用于内皮细胞和/或T细胞。我将描述IL-1依赖的特征 TCR DEEP检测同种异体反应性记忆T细胞克隆库和亚群的变化 测序和流式细胞仪分析表型及抑制IL-1ra或caspase-1对同种异体反应的影响 体外培养的人内皮细胞对T细胞的反应。我将使用成熟的人性化的老鼠模型 同种异体移植血管病变评估活体内EC炎症小体的作用。本项目圆满完成 可能揭示治疗的新靶点，以改善移植医学中的患者结果。

Solid organ transplantation, the best available treatment for end-stage kidney, liver, lung and heart failure, may fail due to chronic immunological rejection, often taking the form of allograft vasculopathy. Allograft vasculopathy results from recruitment to and activation within the graft vessel wall of IFN-γ-producing graft- reactive host T cells by graft ECs. The development of donor specific antibodies (DSA) that recognize non-self alleles of MHC molecules expressed by graft endothelial cells (ECs) and that fix complement is a major risk for allograft vasculopathy. Complement enhances this process by activating immune functions of the ECs that mediate both recruitment and activation of alloreactive T cells. Binding of high titer panel reactive antibody (PRA) from allo-sensitized transplant candidates, used to model DSA, deposits complement membrane attack complex (MAC) on human ECs, both in culture or in vessel grafts in immunodeficient mouse hosts, and induces gene expression of adhesion molecules and chemokines in a manner dependent upon MAC internalization and non-canonical NF-κB signaling. The mechanism(s) by which MAC potentiates T cell activation, measured as effector memory T cell proliferation and cytokine production in vitro or augmented vasculopathic changes in vivo is unknown. My preliminary data show that MAC induces formation of an active inflammasome in ECs, a previously undescribed phenomenon. Activated caspase-1 in the inflammasome processes pro-IL-1β to active IL-1β and mediates its release. Blocking caspase-1 activity or inhibiting IL-1 signaling with IL-1 receptor antagonist (IL-1Ra) blocks both downstream inflammatory gene expression in ECs secondary to canonical NF-κB activation and also blocks the augmentation of allogeneic T cell response in vitro. I hypothesize that complement activation of the inflammasome in ECs intensifies the host T cell response to graft arterial ECs by increasing local production of IL-1, potentiating allograft vasculopathy. In Specific Aim 1, I will characterize the MAC-induced inflammasome and determine the mechanisms linking MAC to inflammasome assembly. I will examine if kinases of non-canonical NF-κB signaling are linked to inflammasome assembly or IL-1 mRNA and protein synthesis. In Specific Aim 2, I will investigate the biological consequences of alloantibody-induced inflammasome activation in ECs on the alloreactive T cell response that drives allograft vasculopathy. I will determine if this response is also MAC-dependent and if mature IL-1 released from MAC-induced inflammasomes acts on ECs, T cells or both. I will characterize IL-1 dependent changes of the clonal repertoire and subsets of activated alloreactive effector memory T cells using TCR deep sequencing and FACS-analytic phenotyping and the effect of IL-1Ra or caspase-1 inhibition on the alloreactive T cell response in vitro using cultured human ECs. I will use well-developed humanized mouse models of allograft vasculopathy to assess the role of the EC inflammasome in vivo. Successful completion of this project may reveal novel targets for therapies to improve patient outcomes in transplant medicine.