Regulation of neutrophil function through the pleiotropic receptor FPR2/ALX

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Regulation of neutrophil function through the pleiotropic receptor FPR2/ALX*******Background and Rationale:****Neutrophil granulocytes (PMNs) play a central role in host defense to infection and tissue injury. Their functional silencing and timely removal are essential for the resolution of inflammation. In inflamed issues, PMNs receive multiple cues and their fate would ultimately depend on the balance of these signals. Intriguingly, these signals converge on select receptors. Among these receptors is the formyl-peptide receptor 2/lipoxin receptor (FPR2/ALX) that recognizes an unusually large number of structurally unrelated molecules, including pro-inflammatory N-formylated peptides released from mitochondria of dying cells and the lipid mediators, lipoxins and resolvin D1, which promote resolution of inflammation. Our recent studies in the field are the first to demonstrate the hierarchy among some FPR2/ALX ligands to regulate PMN activation and apoptosis, important control points in resolving inflammation. To date, little is known about the molecular basis how FPR2/ALX recognizes and differently responds to various ligands. The specific aims of this proposal are to investigate:*******1. The molecular basis of FPR2/ALX integration of opposing cues from mitochondrial N-formylated peptides and pro-resolving lipid mediators and downstream signalling pathways.****2. Human PMN responses to pro-inflammatory and pro-resolving FPR2/ALX ligands.****3. The impact of modulating PMN functions by various FPR2/ALX ligands on the outcome of spontaneously resolving bacterial infection-induced inflammation in mice.*******Research Approach:****1. We will use HEK293 transfected with FPR2/ALX as model cells to explore ligand biasing of FPR2/ALX using bioluminescence resonance energy transfer (BRET) and to perform phosphoproteome and phosphatase profiling to identify “stop signals” triggered by pro-resolving lipid mediators to counter the actions of pro-inflammatory agonists.****2. We will assess human PMN responses (superoxide formation, degranulation, phagocytosis and bacterial killing) to various FPR2/ALX ligands and the underlying intracellular signalling pathways by combining unbiased phosphokinase arrays, immunoblotting and pharmacological inhibitors.****3. We will study the interplay among FPR2/ALX ligands on PMN function and life span and bacterial killing in E. coli infection-evoked lung inflammation in wild type and fpr2/3-/- (the orthologues of human FPR2/ALX) mice.*******Significance: I anticipate that this study will significantly advance understanding how FPR2/ALX integrates opposing cues that govern the function and fate of PMNs, and consequently the outcome of inflammation.***