The NLRP3 inflammasome in regulating injury with lung transplant

NLRP3炎症小体调节肺移植损伤

基本信息

批准号：
10009823
负责人：
SHAMPA CHATTERJEE
金额：
$ 40.47万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-20 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10009823
关键词：
Adaptor Signaling Protein Affect Anoxia Apoptosis Automobile Driving Binding Biology Blood Circulation Blood flow CCL2 gene Calcium Calcium Channel Cell Death Chelating Agents Clinical Closure by clamp Data Databases Endothelial Cells Endothelium Epidemiologist Event Failure Functional disorder Generations Genetic Goals Hilar Human Hypoxia IL8 gene In Situ In Vitro Inflammasome Inflammation Inflammatory Injury Interleukin-1 beta Interleukin-18 Interleukin-6 Interleukins Intervention Investigation Ischemia Knockout Mice Left Link Lipid Peroxidation Lung Lung Transplantation Mibefradil Modeling Monitor Mus NADPH Oxidase Nifedipine Null Lymphocytes Organ Outcome Pathology Permeability Pharmacology Plasma Play Process Proteins Reactive Oxygen Species Regulation Reperfusion Injury Reperfusion Therapy Reporter Reporting Research Personnel Risk Factors Role Signal Transduction Source Stimulus TNF gene Techniques Tertiary Protein Structure Therapeutic immunosuppression Transcription Factor AP-1 Transcriptional Activation Transplant Recipients Transplantation Work cell type chemokine cytokine in vivo inhibitor/antagonist lung injury lung ischemia mouse model multidisciplinary neutrophil oxidation post-transplant receptor release of sequestered calcium ion into cytoplasm side effect therapeutic development tool transcription factor transplant model transplantation medicine voltage

项目摘要

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.

项目概要肺移植涉及一段储存期（缺血），然后进行移植（重新附着或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。我们之前已经表明，肺储存会导致移植失败。几个部分的表达可以使移植物（新移植的肺）“易感”炎症和其中包括 NOD 样受体蛋白 3 (NLRP3) 炎性体，这是一种很好的损伤。 NLRP3 亚基和接头分子的特征平台，其表达和组装是驱动我们的初步数据表明，炎症因素会导致许多病理学中的细胞死亡。（小鼠）肺 I/R 增加了 NLRP3 的表达和活性，并且 NLRP3 阻断减少了 I/R 损伤。此外，血浆中可检测到 NLRP3 蛋白的移植后（人类）受者进行了 PGD。表明 NLRP3 蛋白可能是 PGD 的潜在危险因素，但其机制是什么。炎症小体随肺 I/R 激活并导致损伤，但肺 I/R 与全身 I/R 的不同之处尚不清楚。肺部 I/R 不仅仅反映缺氧/缺氧-脱氧效应；代表与“感知”肺 I/R 停止和重新启动相关的信号，正如我们之前的研究一样。显示，启动 NADPH 氧化酶 2 (NOX2) 激活和活性氧 (ROS) 生成，然后通过激活转录因子 NFB 和 AP-1，我们还报道了细胞内钙的增加[via]。电压门控钙通道 (VGCC)] 我们的假设是 NOX2 激活 NLRP3。炎症小体；一旦激活，该炎症小体是 I/R 损伤（即 PGD）的主要驱动因素。利用我们过去关于肺 I/R 信号传导的工作中的模型、工具、技术和信息来确定是否 NLRP3 受 NOX2（目标 1）和/或通过多种来源（包括细胞内钙的增加）的调节 VGCC（目标 2）。最后我们将确定激活的 NLRP3 炎症小体的驱动机制。 PGD（目标 3）。对于此应用，使用的肺 I/R 模型将包括体外（肺微血管）。流动室中的内皮细胞）、原位（分离的小鼠和人肺）、体内（肺门十字夹）和小鼠肺移植模型以及肺移植结果小组储存的人血浆 (LTOG)。这些模型将用于 NOX2 null、细胞类型特异性 NOX2 null、VGCC null 和 NLRP3。报告小鼠评估 NLRP3 调节机制及其在 I/R 损伤中的作用。追求这些目标的研究小组包括 2 名人类离体供体肺专家、一名流行病学家拥有肺损伤方面的专业知识、小鼠移植小组和 PI（一位肺生物学家），他首先发现了这种联系 NOX2 与肺储存和移植引起的炎症之间的关系中性粒细胞生物学方面的专业知识， NLRP3 炎症小体将由顾问提供。