The Trojan Horse Hypothesis: Neutrophil Elastase Reprograms Macrophage Function

项目总结 在囊性纤维化(CF)中，呼吸道天然免疫被破坏。巨噬细胞吞噬和泡泡吞噬作用 失败，巨噬细胞通过产生过量的细胞因子和释放 高迁移率族蛋白1(HMGB1)，一种损伤相关的分子模式。虽然CFTR的损失 影响巨噬细胞功能，即CF呼吸道环境，以微摩尔浓度为代表 中性粒细胞弹性蛋白酶(NE)是一种压倒性的刺激，激活了强大的促炎反应 在CF和健康人的巨噬细胞中。我们建议去甲肾上腺素是肺部疾病的生物标志物 CFs的研究进展将巨噬细胞的功能从保护功能转变为促炎功能，但 去甲肾上腺素对巨噬细胞重新编程的机制尚不完全清楚。 在这个应用中，我们提出了一个前所未有的假设机制来解释NE是如何改变的 巨噬细胞功能。细胞外去甲肾上腺素被巨噬细胞迅速内吞。但与其说是 降解的、蛋白分解活性的去甲肾上腺素定位于细胞质区域和细胞核，结果 巨噬细胞胞外陷阱(Met)的细胞因子表达和释放增加。因此，东北 像特洛伊木马一样颠覆巨噬细胞的功能。我们将在初选中检验这一假设 来自健康志愿者和CF患者的人血单核细胞来源的巨噬细胞。 具体目标如下： 目的1.确定去甲肾上腺素是否降解HDAC，导致下游靶标乙酰化，从而导致 肿瘤坏死因子α转录上调和HMGB1的释放。 目的2.评估去甲肾上腺素(NE)蛋白水解酶活性和/或去甲肾上腺素(NE)产生的活性氧是否增加 线粒体和核Met的释放。 相关性：该项目的结果将确定NE用来促进 持续的呼吸道炎症是慢性支气管炎发病率和死亡率的主要原因，并确定治疗目标 阻止肺部疾病不断发展的新疗法。

PROJECT SUMMARY In Cystic Fibrosis (CF), airway innate immunity is breached. Macrophage phagocytosis and efferocytosis fail, and macrophages augment airway inflammation by production of excess cytokines and release of High Mobility Group Box 1 (HMGB1), a damage associated molecular pattern. Although loss of CFTR impacts macrophage function, the CF airway milieu, which is typified by micromolar concentrations of neutrophil elastase (NE), is an overwhelming stimulus, activating a robust pro-inflammatory response in macrophages from both CF and healthy subjects. We propose that NE, a biomarker for lung disease progression in CF subverts macrophage function from protective to pro- inflammatory, yet the mechanisms by which NE reprograms the macrophage are not completely understood. In this application, we present an unprecedented hypothetical mechanism to explain how NE alters macrophage function. Extracellular NE is rapidly endocytosed by the macrophage. But instead of being degraded, proteolytically active NE is localized to both cytoplasmic domains and the nucleus, resulting in increased cytokine expression and release of macrophage extracellular traps (METs). Thus, NE functions like a Trojan Horse to subvert macrophage function. We will test this hypothesis in primary human blood monocyte derived macrophages from healthy volunteers and subjects with CF. The Specific Aims follow: Aim 1. To determine whether NE degrades HDACs, resulting in acetylation of downstream targets, leading to transcriptional upregulation of TNFα and release of HMGB1. Aim 2. To evaluate whether NE protease activity and/or NE-generated reactive oxygen species increase release of mitochondrial and nuclear METs. RELEVANCE: Results from this project will define a novel mechanism utilized by NE to promote sustained airway inflammation, the major cause of morbidity and mortality in CF, and identify targets for new therapies to interrupt the relentless progression of lung disease.