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（由申请方提供）：多形核白细胞（PMN）和内皮细胞（EC）在针对入侵病原体的先天免疫应答期间产生的活性氧（ROS）在控制感染部位的PMN捕获和外渗方面具有重要意义。虽然我们已经证明瞬时受体电位melastatin（TRPM）通道2（TRPM 2）参与介导氧化剂诱导的钙离子内流和内皮细胞（EC）内皮屏障完整性的丧失，但TRPM 2通道在ROS诱导的中性粒细胞跨内皮迁移中的功能尚未评估。我们的研究表明，TRPM 2的功能是响应ROS激活感染肺中的血管PMN外渗。因此，我们将测试的假设，即肺微血管PMN的迁移和清除的细菌病原体从感染的肺依赖于内皮TRPM 2的氧化剂的激活。为了检验这一假设，我们将遵循三个具体目标：目标#1将解决内皮细胞-TRPM 2通道在介导细菌感染部位的PMN跨内皮迁移中的需求，从而促进入侵病原体从肺中的清除。所提出的研究将使用表达TRPM 2的内皮细胞限制性缺失的转基因小鼠模型和用TRPM 2crispr/Cas9-gRNA转导的小鼠肺内皮来解决TRPM 2活化在增加的肺PMN迁移机制中的作用。我们将在铜绿假单胞菌肺部感染的小鼠模型中建立TRPM 2在信号转导PMN激活诱导的肺PMN浸润和细菌清除中的功能意义。目的#2将解决PMN激活的TRPM 2是否在细菌感染期间通过在EC表面上分布P-选择并将PMN募集到感染部位而发挥关键作用。具体来说，我们将确定是否TRPM 2调节的Ca 2+进入诱导P-选择素动员EC表面通过激活PKCa信号。目的#3将阐明PMN诱导的内皮细胞TRPM 2活化介导血管内皮间隙形成和PMN跨内皮迁移的信号传导机制。这些研究将确定TRPM 2调节的Ca 2+信号在EC中的作用，通过血管内皮（VE）-钙粘蛋白的磷酸化促进粘附连接复合物的分解，导致内皮细胞-细胞粘附的丧失，从而介导PMN的迁移。总的来说，这些研究将建立TRPM 2在诱导PMN跨内皮迁移中的功能意义，这是宿主防御细菌感染所必需的;因此，操纵TRPM 2在内皮中的功能代表了一种新的靶点，可以有效地提高宿主对肺部革兰氏阴性感染的免疫力。