Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE) a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of E. coli bio-particles. We performed LC-MS/MS proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and down-regulated 211 proteins. NE released not only cell surface proteins into the media but also released cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors (including LRP1), 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/ secretome included proteins from other macrophage cellular domains suggesting that NE may globally regulate macrophage structure and function.

中文翻译：

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中性粒细胞弹性蛋白酶调节的巨噬细胞 Sheddome/Secretome 和吞噬功能衰竭

囊性纤维化 (CF) 患者的巨噬细胞吞噬作用和胞吞作用存在缺陷。一些报告表明，中性粒细胞弹性蛋白酶 (NE) 是 CF 气道中的一种主要炎症蛋白酶，会损害巨噬细胞的吞噬功能。迄今为止，NE 受损的巨噬细胞吞噬功能归因于细胞表面受体或调理素的裂解。我们应用了一种无偏见的蛋白质组学方法来确定可能调节吞噬功能的 NE 蛋白酶活性的其他潜在巨噬细胞靶标。使用鼠巨噬细胞系、RAW 264.7、人血单核细胞衍生的巨噬细胞和来自 Cftr-null 和野生型同窝小鼠的原代肺泡巨噬细胞，我们证明了 NE 暴露阻止了大肠杆菌生物颗粒的吞噬作用。我们对来自 RAW264 的条件培养基进行了 LC-MS/MS 蛋白质组学分析。7 用活性 NE 或非活性（煮沸）NE 作为对照处理。在条件培养基中鉴定出的 840 种蛋白质中，活性 NE 上调了 142 种蛋白质，下调了 211 种蛋白质。NE 不仅将细胞表面蛋白释放到培养基中，而且还释放在条件培养基中检测到的细胞骨架、线粒体、细胞质和核蛋白。至少有 32 种蛋白质与吞噬过程相关，包括 11 种吞噬受体（包括 LRP1）、7 种与吞噬杯形成相关的蛋白质以及 14 种参与吞噬成熟（包括钙蛋白酶 2）和吞噬溶酶体形成的蛋白质。NE 对调节吞噬作用和吞噬溶酶体形成的所有阶段所需的蛋白质组有广泛的影响。此外，