The NLRP3 inflammasome in regulating injury with lung transplant

NLRP3炎症小体调节肺移植损伤

• 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
• 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

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.

项目摘要 肺移植涉及一段时间的储存(缺血)，然后是移植(重新附着或 再灌流)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。我们之前已经证明，肺储存诱导了 表达几种可使移植物(新移植的肺)发生炎症和 随后的伤害。其中包括结节样受体蛋白3(NLRP3)炎症体，这是一种 NLRP3亚基和表达组装驱动的接头分子的特征化平台 炎症因子在许多病理过程中导致细胞死亡。我们的初步数据显示 (小鼠)肺I/R增加NLRP3的表达和活性，阻断NLRP3可减轻I/R损伤。 此外，移植后(人类)受者在血浆中检测到NLRP3蛋白后会发生PGD。这 提示NLRP3蛋白可能是PGD的潜在危险因素。然而，这一切的机制(S) 炎症小体在肺I/R时被激活，并导致损伤尚不清楚。肺I/R与I/R在全身性 肺I/R中的器官不仅反映了缺氧/缺氧-脱氧效应；相反，它还反映了 表示与“检测”流的停止和重新启动相关的信号。肺I/R，正如我们早先的调查 显示，启动NADPH氧化酶2(NOX2)的激活和活性氧物种(ROS)的产生，随后 通过激活转录因子NFB和AP-1。我们还报告了细胞内钙离子的增加[通过 电压门控钙通道(VGCC)]。我们的假设是NOX2激活了NLRP3 炎性小体；一旦激活，这种炎性小体是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信号方面的工作中的模型、工具、技术和信息来确定 NLRP3受NOX2(Aim 1)和/或通过多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NLRP3炎症小体驱动的机制 PGD(目标3)。对于这一应用，所使用的肺I/R模型将包括体外(肺微血管 流室内皮细胞)、原位(离体鼠和人肺)、活体(肺门交叉夹)和 小鼠肺移植模型和储存在肺移植结果组的人血浆 (LTOG)。这些型号将用于NOX2空、特定于单元类型的NOX2空、VGCC空和NLRP3 以小鼠为报告动物，评价NLRP3的调控机制及其在I/R损伤中的作用。多学科的 追求这些目标的调查团队包括两名人体体外供体肺专家，一名流行病学家 在肺损伤方面拥有专业知识的一个小鼠移植小组，以及最先发现了 NOX2与肺部储存和移植的炎症发作之间的关系。中性粒细胞生物学方面的专业知识， 而在NLRP3上，炎症器将由顾问提供。