Role of FABP5 in COPD Exacerbations

项目摘要 慢性阻塞性肺疾病（COPD）是美国第三大死因 由于缺乏有效的治疗方法，其全球流行率继续上升。香烟烟雾（CS） 作为COPD最重要的危险因素，反复和持续的感染与疾病明显相关 发病机理（例如，加重）。有趣的是，COPD急性加重是一个风险因素， 急性加重，表明炎症无效解决的恶性循环使个体 未来的情节。因此，识别可能具有复发性加重的患者可以允许早期 实施适当的预防战略，将是治疗的重要新方法。 疾病。我们已经发现脂肪酸结合蛋白5（FABP 5）对于以下问题的解决是重要的： 炎症然而，我们并不完全理解FABP 5在COPD中保护作用背后的机制 加重在这个建议中，我们将重点放在单核吞噬细胞中的FABP 5，因为这些细胞在 炎症消退所需过程的中心，包括凋亡细胞的吞噬 和抗炎性核受体如过氧化物酶体激活受体γ的激活 (PPARγ）。 我们的初步研究表明肺泡巨噬细胞吞噬凋亡细胞受损， FABP 5-/-小鼠中细菌感染后单核细胞募集减少。此外，FABP 5-/-小鼠具有 与野生型（WT）小鼠相比，细菌和病毒诱导的肺部炎症增加。临床 外周血中FABP 5 mRNA和蛋白水平的降低支持了这些发现的重要性 COPD患者的单核细胞（PBMC），尤其是具有支气管炎发作史的患者， 肺炎或COPD恶化，以及在FABP 5基因中发现新的SNP， 病情严重恶化我们假设CS暴露后FABP 5的减少会导致感染- 通过阻止单核肺吞噬细胞重编程诱导肺部炎症，这是必要的 缓解COPD急性加重期的肺部炎症。为了验证这一假设，我们提出了三个具体目标。 在具体目标1下，我们将检验CS损害FABP 5介导的单核肺吞噬细胞的假设 募集和红细胞增多症。使用在单核细胞中补充FABP 5的WT、FABP 5-/-或FABP 5-/-小鼠， 我们将确定FABP 5表达如何影响单核吞噬细胞的募集， 在有或没有CS的情况下感染。我们还将确定FABP 5表达如何影响 单核吞噬细胞吞噬能力后CS和/或感染在体内和体外。根据目标2， 我们将检验FABP 5/PPARγ相互作用对于促进炎症消退是必要的这一假设 并恢复稳态，并确定CS是否损害这种相互作用。我们将操纵FABP 5水平 为了证明单核吞噬细胞中FABP 5的存在对于PPARγ介导的 激活和功能。这些功能包括脂肪酸β-氧化和巨噬细胞交替编程 用于处理被吞没的材料。我们还将确定PPARγ是否增加FABP 5的表达， 使用ChIP的正反馈环、沉默、启动子报告基因测定和诱变。在具体目标3下， 我们将检验新发现的FABP 5 SNPs与COPD急性加重相关的假设， 暴露于CS的肺泡巨噬细胞FABP 5表达减少，吞噬能力降低， 活动我们建议使用我们现有的患者样本分析FABP 5基因和蛋白表达 有或没有新发现的SNP或吸烟者的肺泡巨噬细胞， 表达水平与SNP表达、CS暴露和COPD恶化相关。另外我们 我建议对包含SNPs的不同区域的增强子或抑制子基因进行表征 活性，并使用基于多重CRISPR的增强子干扰来剖析它们的增强子功能。最后， 我们建议观察这些细胞的炎性标记物与促消退标记物以及红细胞增多能力。 这些目标的完成将使人们深入了解政务司司长 抑制FABP 5功能并导致肺组织过度发炎。最终，这项研究的目标是改善 COPD急性加重的临床前诊断和治疗，因此可能具有高度显著的总体 对目前无法治愈的疾病的影响。

PROJECT SUMMARY Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death in the United States and its global prevalence continues to rise due to lack of an effective cure. Although cigarette smoke (CS) is by far the most important risk factor of COPD, repeated and sustained infections are clearly linked to disease pathogenesis (e.g., exacerbations). Interestingly, COPD exacerbations are a risk factor for having more exacerbations, suggesting that a vicious cycle of ineffective resolution of inflammation predisposes individuals for future episodes. Thus, identifying patients likely to have recurrent exacerbations may allow early implementation of appropriate preventive strategies and would be an important new approach in the treatment of the disease. We have discovered that Fatty Acid Binding Protein 5 (FABP5) is important for the resolution of inflammation. However we do not fully comprehend the mechanisms behind FABP5 protective role in COPD exacerbations. In this proposal we will focus on FABP5 in mononuclear phagocytes because these cells are at the center of the processes required for the resolution of inflammation, including engulfment of apoptotic cells and activation of anti-inflammatory nuclear receptors such as Peroxisome Proliferator-Activated Receptor γ (PPARγ). Our preliminary studies indicate impaired apoptotic cell engulfment by alveolar macrophages and reduced monocyte recruitment following bacterial infection in FABP5-/- mice. In addition, FABP5-/- mice have increased bacterial and viral-induced lung inflammation, compared to wild type (WT) mice. The clinical significance of these findings is supported by decreased FABP5 mRNA and protein levels in peripheral blood mononuclear cells (PBMCs) of COPD patients, especially among those with a history of episodes of bronchitis, pneumonia or COPD exacerbations and by the discovery of new SNPs in the FABP5 gene that are associated with severe exacerbations. We hypothesize that decreased FABP5 following CS exposure worsens infection- induced lung inflammation by preventing mononuclear lung phagocyte reprogramming, which is necessary for resolution of lung inflammation in COPD exacerbations. To test this hypothesis, we propose 3 specific aims. Under specific aim 1, we will test the hypothesis that CS impairs FABP5-mediated mononuclear lung phagocytes recruitment and efferocytosis. Using WT, FABP5-/- or FABP5-/- mice complemented with FABP5 in mononuclear phagocytes we will determine how FABP5 expression affects mononuclear phagocyte recruitment in response to infection in the presence or absence of CS. We will also determine how FABP5 expression affects mononuclear phagocyte engulfment capabilities following CS and/or infection in vivo and in vitro. Under aim 2, we will test the hypothesis that FABP5/PPARγ interaction is necessary to promote resolution of inflammation and return to homeostasis and determine whether CS impairs this interaction. We will manipulate FABP5 levels to demonstrate that the presence of FABP5 in mononuclear phagocytes is essential for PPARγ-mediated activation and functions. These functions include fatty acid β-oxidation and macrophage alternative programming for processing of engulfed materials. We will also determine whether PPARγ increases FABP5 expression as a positive feedback loop using ChIP, silencing, promoter reporter assay and mutagenesis. Under specific aim 3, we will test the hypothesis that the newly identified FABP5 SNPs associated with COPD exacerbations and alveolar macrophages exposed to CS have less FABP5 expression, less engulfment capabilities and less PPARγ activity. We propose to analyze FABP5 gene and protein expression using our existing samples from patients with or without the newly identified SNPs or alveolar macrophages from smokers and see whether FABP5 expression level correlates with SNP expression, CS exposure, and COPD exacerbations. Additionally, we propose to characterize the different regions encompassing the SNPs for their enhancer or suppressor gene activity and to dissect their enhancer function using multiplex CRISPR-based enhancer interference. And finally, we propose to look at inflammatory versus pro-resolving markers and efferocytosis capabilities of those cells. The completion of these aims will provide an in-depth understanding of the mechanisms by which CS inhibits FABP5 function and leads to over inflamed lung tissue. Ultimately, the goal of this research is to improve the preclinical diagnosis and treatment of COPD exacerbations, and thus could have a highly significant overall impact on what is currently an incurable disease.