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.

Public Health Relevance

Oxidant generation by activated PMNs and endothelial cells is a critical factor that mediates PMN transendothelial migration in lungs, which is necessary for host defense against pulmonary bacterial infections. The proposed studies will address how activation of the endothelial TRPM2 by these oxidants mediates increase in lung PMN transmigration, and will identify for the first time an important, physiologically-relevant functio for the oxidant-sensitive TRPM2 channel in the mechanism of PMN-induced lung inflammation and host defense's response against gram-negative infection. Establishing such a role for TRPM2 may provide a useful therapeutic strategy for improving host's immunity against pulmonary gram-negative infection.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL122157-02
Application #
9069040
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Sarkar, Rita
Project Start
2015-05-15
Project End
2019-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Pharmacology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Mittal, Manish; Nepal, Saroj; Tsukasaki, Yoshikazu et al. (2017) Response by Mittal et al to Letter Regarding Article, ""Neutrophil Activation of Endothelial Cell-Expressed TRPM2 Mediates Transendothelial Neutrophil Migration and Vascular Injury"". Circ Res 121:e87
Soni, Dheeraj; Regmi, Sushil C; Wang, Dong-Mei et al. (2017) Pyk2 phosphorylation of VE-PTP downstream of STIM1-induced Ca2+ entry regulates disassembly of adherens junctions. Am J Physiol Lung Cell Mol Physiol 312:L1003-L1017
Mittal, Manish; Nepal, Saroj; Tsukasaki, Yoshikazu et al. (2017) Neutrophil Activation of Endothelial Cell-Expressed TRPM2 Mediates Transendothelial Neutrophil Migration and Vascular Injury. Circ Res 121:1081-1091
Wang, Gang; Cao, Luyang; Liu, Xiaowen et al. (2016) Oxidant Sensing by TRPM2 Inhibits Neutrophil Migration and Mitigates Inflammation. Dev Cell 38:453-62
Mittal, Manish; Tiruppathi, Chinnaswamy; Nepal, Saroj et al. (2016) TNF?-stimulated gene-6 (TSG6) activates macrophage phenotype transition to prevent inflammatory lung injury. Proc Natl Acad Sci U S A 113:E8151-E8158
Mittal, Manish; Urao, Norifumi; Hecquet, Claudie M et al. (2015) Novel role of reactive oxygen species-activated Trp melastatin channel-2 in mediating angiogenesis and postischemic neovascularization. Arterioscler Thromb Vasc Biol 35:877-87