Regulation of neutrophil function through the pleiotropic receptor FPR2/ALX

通过多效受体FPR 2/ALX调节中性粒细胞功能 * 背景和原理：* 中性粒细胞（PMN）在宿主防御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炎症的解决至关重要。在发炎的问题中，PMNs会收到多种信号，它们的命运最终取决于这些信号的平衡。有趣的是，这些信号集中在特定的受体上。在这些受体中，甲酰肽受体2/脂氧素受体（FPR 2/ALX）识别异常大量的结构上不相关的分子，包括从垂死细胞的线粒体释放的促炎N-甲酰化肽和脂质介质，脂氧素和消退素D1，其促进炎症的消退。我们最近在该领域的研究首次证明了一些FPR 2/ALX配体之间的层次结构，以调节PMN活化和凋亡，这是解决炎症的重要控制点。迄今为止，关于FPR 2/ALX如何识别和不同地响应各种配体的分子基础知之甚少。本提案的具体目的是调查：**1。FPR 2/ALX整合来自线粒体N-甲酰化肽和促分解脂质介质和下游信号传导途径的相反线索的分子基础。* 2.人PMN对促炎和促消退FPR 2/ALX配体的反应。* 3.通过各种FPR 2/ALX配体调节PMN功能对小鼠中自发解决细菌感染诱导的炎症的结果的影响。研究方法：*1.我们将使用FPR 2/ALX转染的HEK 293作为模型细胞，使用生物发光共振能量转移（BRET）探索FPR 2/ALX的配体偏置，并进行磷酸化蛋白质组和磷酸酶分析，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.我们将通过结合无偏磷酸激酶阵列、免疫印迹和药理学抑制剂来评估人PMN对各种FPR 2/ALX配体的反应（超氧化物形成、脱粒、吞噬作用和细菌杀伤）以及潜在的细胞内信号传导途径。* 3.我们将研究FPR 2/ALX配体对中性粒细胞功能和寿命的相互作用以及对大肠杆菌的杀菌作用。在野生型和fpr 2/3-/-（人FPR 2/ALX的直系同源物）小鼠中，大肠杆菌感染诱发的肺部炎症。重要性：我预计，这项研究将大大促进理解FPR 2/ALX如何整合控制PMN功能和命运的相反线索，从而控制炎症的结果。

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.***