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

实体器官移植，终末期肾、肝、肺和心脏的最佳治疗方法 失败，可能由于慢性免疫排斥而失败，通常采取同种异体移植物血管病的形式。移植 血管病变是由产生IFN-γ的移植物在移植物血管壁内的募集和激活引起的， 反应性宿主T细胞的移植EC。供体特异性抗体（DSA）识别非自身抗体 移植物内皮细胞（EC）表达的MHC分子的等位基因，固定补体是移植物内皮细胞（EC）的主要风险。 同种异体移植血管病补体通过激活内皮细胞的免疫功能来增强这一过程， 介导同种异体反应性T细胞的募集和活化。高滴度组反应性抗体的结合 (PRA)来自同种异体致敏的移植候选者，用于模拟DSA，沉积补体膜攻击 复合物（MAC）对人EC的作用，无论是在培养物中还是在免疫缺陷小鼠宿主的血管移植物中， 以依赖于MAC的方式诱导粘附分子和趋化因子的基因表达 内化和非经典NF-κB信号传导。MAC增强T细胞增殖的机制 激活，测量为效应记忆T细胞增殖和细胞因子产生，在体外或增强 体内血管病变的变化是未知的。我的初步数据显示，MAC诱导形成一个活跃的 炎性小体在内皮细胞，以前未描述的现象。炎症小体中激活的caspase-1 将pro-IL-1β加工成活性IL-1β并介导其释放。阻断caspase-1活性或抑制IL-1 IL-1受体拮抗剂（IL-1 Ra）信号传导阻断EC中下游炎症基因表达 继发于典型的NF-κB活化，也阻断了同种异体T细胞反应的增强， 体外我推测补体激活内皮细胞中的炎性小体增强了宿主T细胞的反应 通过增加局部IL-1的产生，增强同种异体移植血管病变。具体目标 1，我将描述MAC诱导的炎性小体，并确定MAC与 炎性小体组装。我将检查非经典NF-κB信号传导激酶是否与 炎性小体组装或IL-1 mRNA和蛋白质合成。在具体目标2中，我将研究生物学 同种异体抗体诱导的内皮细胞炎性小体活化对同种异体反应性T细胞应答的影响， 导致同种异体移植血管病变。我将确定这种反应是否也是MAC依赖性的， 从MAC诱导的炎性体释放的细胞因子作用于EC、T细胞或两者。我将描述IL-1依赖 利用TCR深穿透技术观察同种异体反应性效应记忆T细胞克隆库和亚群的变化 测序和FACS分析表型以及IL-1 Ra或半胱天冬酶-1抑制对同种异体反应性 使用培养的人EC的体外T细胞应答。我将使用发达的人源化小鼠模型， 同种移植物血管病以评估EC炎性体在体内的作用。成功完成该项目 可能揭示新的治疗目标，以改善移植医学中的患者结局。

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.