Regulation of neutrophil function through the pleiotropic receptor FPR2/ALX

多效性受体FPR2/ALX对中性粒细胞功能的调节背景和原理：中性粒细胞(PMN)在宿主抵抗感染和组织损伤中发挥核心作用。它们的功能沉默和及时清除对于消解炎症至关重要。在发炎的问题中，PMN会收到多个信号，它们的命运最终将取决于这些信号的平衡。有趣的是，这些信号会聚在特定的受体上。在这些受体中，有甲酰肽受体2/脂氧素受体(FPR2/ALX)，它识别大量结构上不相关的分子，包括从濒死细胞的线粒体释放的促炎N-甲酰化肽，以及促进炎症消退的脂质介质脂素和解决素D1。我们最近在该领域的研究首次证明了一些FPR2/ALX配体之间的层次结构，以调节PMN的激活和凋亡，这是化解炎症的重要控制点。到目前为止，人们对FPR2/ALX识别不同配体并做出不同反应的分子基础知之甚少。这项建议的具体目的是调查：*1.线粒体N甲酰化多肽和促分解脂类介体的相反信号和下游信号通路的FPR2/ALX整合的分子基础.*2.人PMN对促炎和促分解FPR2/ALX配体的反应.*3.不同的FPR2/ALX配体调节PMN功能对自发地消解细菌感染引起的小鼠炎症的结果的影响。*研究方法：*1.我们将使用HEK293以FPR2/ALX为模型细胞，利用生物发光共振能量转移(BRET)技术探索FPR2/ALX的配基偏置，并进行磷酸蛋白质组和磷酸酶谱分析，以识别由促分解脂质介质触发的“停止信号”，以对抗促炎激动剂的作用。*2.我们将评估人类PMN反应(超氧化物形成，我们将研究FPR2/ALX配体对不同的FPR2/ALX配体和潜在的细胞内信号通路的影响。*3.我们将在野生型和fpr2/3-/-(人类FPR2/ALX的同源同源基因)小鼠中研究FPR2/ALX配体对PMN功能和寿命的影响以及细菌杀伤作用。*意义：我预计这项研究将大大促进对FPR2/ALX如何整合控制PMN功能和命运的相反信号的理解，从而影响炎症的结局。

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.***