The Trojan Horse Hypothesis: Neutrophil Elastase Reprograms Macrophage Function

项目摘要 在囊性纤维化（CF）中，气道先天免疫被破坏。巨噬细胞吞噬和吞噬作用 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（HMGB 1），一种损伤相关的分子模式。虽然CFTR损失 影响巨噬细胞功能，CF气道环境，其以微摩尔浓度的 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和健康受试者的巨噬细胞中。我们认为NE是肺部疾病的生物标志物， CF的进展使巨噬细胞功能从保护性变为促炎性， NE重编程巨噬细胞的机制还不完全清楚。 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如何改变 巨噬细胞功能细胞外NE被巨噬细胞迅速内吞。但不是被 降解的、具有蛋白水解活性的NE定位于细胞质结构域和细胞核， 增加细胞因子表达和巨噬细胞胞外陷阱（MET）释放。因此，NE 像特洛伊木马一样破坏巨噬细胞的功能我们将在小学测试这一假设 来自健康志愿者和CF受试者的人血单核细胞衍生的巨噬细胞。 具体目标如下： 目标1.为了确定NE是否降解HDAC，导致下游靶标的乙酰化，从而导致 TNFα转录上调和HMGB 1释放。 目标二。评价NE蛋白酶活性和/或NE产生的活性氧是否增加 线粒体和核MET的释放。 相关性：本项目的结果将定义NE用于促进 持续的气道炎症，CF发病率和死亡率的主要原因，并确定目标 新的治疗方法来阻止肺部疾病的无情发展。

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.