The NLRP3 inflammasome in regulating injury with lung transplant

项目摘要 肺移植涉及一段时间的储存（缺血），然后是移植（再附着或再移植）。 再灌注）事件。由此产生的缺血再灌注（I/R）损伤，临床上称为原发性移植物功能障碍， (PGD)是移植后失败的主要原因。我们以前已经证明，肺储存诱导 表达几个可以使移植物（新移植的肺）“易患”炎症的部分， 后续伤害。其中包括NOD样受体蛋白3（NLRP 3）炎性体， NLRP 3亚基和衔接分子的表征平台，其表达和组装是驱动 炎症因子在许多病理学中诱导细胞死亡。我们的初步数据显示， （小鼠）肺I/R增加NLRP 3表达和活性，并且NLRP 3阻断减少I/R损伤。 此外，血浆中具有可检测的NLRP 3蛋白的移植后（人）受体发展为PGD。这 提示NLRP 3蛋白可能是PGD的一个潜在危险因素。然而，这一机制 炎性小体是由肺I/R激活的，并驱动损伤尚不清楚。肺I/R不同于全身I/R。 肺中的I/R并不单独反映缺氧/缺氧-脱氧效应;相反， 表示与“感测”流的停止和重新开始相关联的信令。肺部I/R，正如我们早期的调查 如图所示，启动NADPH氧化酶2（NOX 2）活化和活性氧（ROS）产生，随后 通过激活转录因子NF κ B B和AP-1。我们还报道了细胞内钙的增加[通过 电压门控钙通道（VGCC）]。我们的假设是NOX 2激活NLRP 3 炎性小体;一旦被激活，该炎性小体是I/R损伤（即PGD）的主要驱动因素。我们的目标是 使用我们过去在肺I/R信号传导方面的工作中的模型、工具、技术和信息来确定是否 NLRP 3受NOX 2（Aim 1）和/或通过各种来源的细胞内钙升高调节，包括 VGCC（目标2）。最后，我们将确定激活的NLRP 3炎性小体驱动 PGD（目标3）。对于该应用，所使用的肺I/R模型将包括体外（肺微血管）模型。 流动室中的内皮细胞）、原位（分离的鼠和人肺）、体内（肺门交叉钳）和 小鼠肺移植模型以及肺移植结果组中储存的人血浆 （LTOG）。这些模型将用于NOX 2无效、细胞类型特异性NOX 2无效、VGCC无效和NLRP 3 报告小鼠，以评估NLRP 3的调节机制及其在I/R损伤中的作用。多学科 追求这些目标的研究小组包括2名人类离体供体肺专家，一名流行病学家， 一个专门研究肺损伤的小鼠移植小组和一位肺生物学家PI， NOX 2与肺储存和移植的炎症发作之间的关系。嗜中性粒细胞生物学方面的专业知识， 及NLRP 3炎性小体的资料将由顾问提供。

Project Summary Lung transplant involves a period of storage (ischemia) followed by the transplant (reattachment or reperfusion) event. The resultant ischemia-reperfusion (I/R) injury, clinically known as primary graft dysfunction (PGD), is a major cause of post-transplant failure. We have previously shown that lung storage induces the expression of several moieties that can “predispose” the graft (newly transplanted lung) to inflammation and subsequent injury. Among these is the NOD like receptor protein 3 (NLRP3) inflammasome, a well characterized platform of a NLRP3 subunit and adaptor molecules whose expression and assembly are driving factors in inflammation induced cells death in a number of pathologies. Our preliminary data showed that (mouse) lung I/R increased NLRP3 expression and activity and that NLRP3 blockade reduced I/R injury. Additionally, post-transplant (human) recipients with detectable NLRP3 protein in plasma developed PGD. This implied that NLRP3 protein could be a potential risk factor for PGD. Yet the mechanism(s) by which this inflammasome is activated with lung I/R and drives injury is not known. Lung I/R differs from I/R in systemic organs in that I/R in the lung does not reflect anoxia/hypoxia-deoxygenation effects alone; rather it also represents signaling associated with “sensing' stop and restart of flow. Lung I/R, as our earlier investigations show, initiates NADPH oxidase 2 (NOX2) activation and reactive oxygen species (ROS) generation, followed by activation of transcription factors NFB and AP-1. We also reported an increase in intracellular calcium [via voltage gated calcium channels (VGCC)]. Our hypothesis is that NOX2 activates the NLRP3 inflammasome; once activated this inflammasome is a major driver of I/R injury (i.e. PGD). Our goal is to employ models, tools, techniques and information from our past work on lung I/R signaling to ascertain if NLRP3 is regulated by NOX2 (Aim 1), and/or by rise in intracellular calcium via various sources including VGCC (Aim 2). Finally we will determine the mechanism by which activated NLRP3 inflammasome drives PGD (Aim 3). For this application, the lung I/R models used will comprise of in vitro (pulmonary microvascular endothelial cells in flow chambers), in situ (isolated murine and human lungs), in vivo (hilar cross clamp) and mouse lung transplant models as well as human plasma banked at the Lung Transplant Outcomes group (LTOG). These models will be used on NOX2 null, cell type specific NOX2 null, VGCC null and NLRP3 reporter mice to evaluate the mechanism of NLRP3 regulation and its role in I/R injury. The multidisciplinary team of investigators to pursue these aims includes 2 human ex vivo donor lung experts, an epidemiologist with expertise in lung injury, a mouse transplant group, and the PI, a lung biologist who first identified a link between NOX2 and onset of inflammation with lung storage and transplant. Expertise on neutrophil biology, and on the NLRP3 inflammasome will be provided by consultants.