TRPM2 mediates neutrophil transendothelial migration and inflammation

TRPM2介导中性粒细胞跨内皮迁移和炎症

• 批准号: 9260918
• 负责人: CHINNASWAMY TIRUPPATHI
• 金额: $ 39.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9260918
• 关键词: Address; Adherens Junction; Adhesions; Bacteria; Bacterial Infections; Blood Vessels; CRISPR/Cas technology; Calcium; Cell Communication; Cell surface; Cell-Cell Adhesion; Cells; Complex; Data; Dissociation; Endothelial Cells; Endothelium; Event; Extravasation; Generations; Guide RNA; Host Defense; Immunity; Infection; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Invaded; Knock-out; Lead; Leukocyte Rolling; Leukocytes; Lipopolysaccharides; Lung; Lung Inflammation; Mediating; Modeling; Molecular Genetics; Mus; NADP; Nuclear; Oxidants; Oxidases; P-Selectin; PRKCA gene; Phase; Phosphorylation; Physiological; Play; Polymorph; Production; Pseudomonas aeruginosa; Reactive Oxygen Species; Recruitment Activity; Role; Route; SRC gene; Secondary to; Seminal; Signal Transduction; Site; System; Testing; Therapeutic; Time; Tissues; Toxin; Transgenic Mice; cadherin 5; defense response; improved; killings; leucyl-phenylalanine; leukocyte activation; microorganism; migration; mouse model; neutrophil; novel; pathogen; public health relevance; receptor; response; sulfated glycoprotein 2; trafficking

 DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) produced by polymorph nuclear leukocytes (PMNs) and endothelial cells (ECs) during the innate immune response against invading pathogens are of major importance in governing PMN capture and extravasation at a site of infection. Although we have demonstrated the involvement of the transient receptor potential melastatin (TRPM) channel 2 (TRPM2) in mediating oxidant-induced calcium entry and loss of the endothelial barrier integrity in endothelial cells (ECs), the functio of the TRPM2 channel in ROS-induced trans endothelial migration of PMNs have not been assessed. Our studies suggest that TRPM2 functions in response to ROS to activate vascular PMN extravasation in infected lungs. We will thus test the hypothesis that the increase in lung micro vessel PMN transmigration and clearance of the bacterial pathogens from the infected lungs depend on the activation of endothelial TRPM2 by oxidants. To test this hypothesis we will follow the three specific aims: Aim #1 will address the requirement of the endothelial cell-TRPM2 channel in mediating transendothelial migration of PMNs at sites of bacterial infection, thereby promoting clearance of the invading pathogen from the lung. The proposed studies will use a transgenic mouse model expressing endothelial cell- restricted deletion of TRPM2 and mouse lung endothelia transduced with TRPM2 crispr/Cas9-gRNA to address the role of TRPM2 activation in the mechanism of increased lung PMN transmigration. We will establish the functional significance of TRPM2 in signaling PMN activation-induced lung PMN infiltration and bacterial clearance in a murine model of Pseudomonas aeruginosa pulmonary infection. Aim #2 will address whether PMN-activated TRPM2 plays a critical role during bacterial infection by distributing P-selecting on the EC surface, and recruiting PMNs to the site of infection. Specifically, we will determine whether TRPM2-regulated Ca2+ entry induces P-selectin mobilization to EC surface through activation of PKCa signaling. Aim #3 will address the signaling mechanisms by which PMN-induced activation of TRPM2 in ECs mediates formation of vascular interendothelial gaps and PMN transendothelial migration. The studies will establish the role of TRPM2-regulated Ca2+ signaling in ECs in promoting disassembly of Adherens Junctions complexes through phosphorylation of the vascular endothelial (VE)-cadherin, resulting in loss of endothelial cell-cell adhesion and thereby mediating PMN transmigration. Overall, these studies will establish the functional significance of TRPM2 in the induction of PMN transendothelial migration, which is necessary for host defense against bacterial infections; therefore, manipulating TRPM2 function in the endothelium represents a novel target to mount effective host's immunity against pulmonary gram-negative infection.

 描述(由申请人提供)：在对入侵病原体的天然免疫反应中，由多形核白细胞(PMN)和内皮细胞(ECs)产生的活性氧物种(ROS)在控制PMN在感染部位的捕获和外渗方面起着重要作用。尽管我们已经证明了瞬时受体电位褪黑素(TRPM)通道2(TRPM2)参与了氧化剂诱导的内皮细胞(ECs)内钙内流和内皮屏障完整性的丧失，但TRPM2通道在ROS诱导的PMN跨内皮细胞迁移中的作用尚未得到评估。我们的研究表明，TRPM2的功能是响应ROS，激活感染肺中血管PMN的外渗。因此，我们将检验这样一种假设，即肺微血管PMN迁移的增加和细菌病原体从感染肺中的清除依赖于氧化剂激活内皮细胞TRPM2。为了验证这一假设，我们将遵循三个具体目标：目标1将解决内皮细胞-TRPM2通道在介导中性粒细胞在细菌感染部位的跨内皮迁移中的需求，从而促进侵入肺部的病原体的清除。这项研究将使用表达内皮细胞限制性缺失TRPM2的转基因小鼠模型和转导TRPM2 CRISPR/Cas9-gRNA的小鼠肺内皮细胞来研究TRPM2激活在增加肺PMN迁移的机制中的作用。我们将在铜绿假单胞菌肺部感染的小鼠模型中建立TRPM2在信号转导PMN激活诱导的肺PMN渗透和细菌清除中的功能意义。目的#2通过将P-选择素分布在EC表面，并将PMN重新招募到感染部位，探讨PMN激活的TRPM2是否在细菌感染过程中发挥关键作用。具体地说，我们将确定TRPM2调节的钙离子内流是否通过激活PKCA信号诱导P-选择素动员到EC表面。目的#3将阐述PMN诱导内皮细胞激活TRPM2介导血管内皮细胞间隙的形成和PMN跨内皮细胞迁移的信号机制。这些研究将确定TRPM2调节的内皮细胞内钙信号在促进血管内皮细胞(VE)-钙粘附素磷酸化，促进黏附连接复合体的分解，导致内皮细胞-细胞黏附丧失，从而介导PMN迁移中的作用。总体而言，这些研究将确立TRPM2在诱导PMN跨内皮细胞迁移中的功能意义，这是宿主抵御细菌感染所必需的；因此，调控内皮细胞中的TRPM2功能是建立有效宿主对肺部革兰氏阴性菌感染免疫的新靶点。