Data_Sheet_1_Assessment of Alveolar Macrophage Dysfunction Using an in vitro Model of Acute Respiratory Distress Syndrome.docx

Background: Impaired alveolar macrophage (AM) efferocytosis may contribute to acute respiratory distress syndrome (ARDS) pathogenesis; however, studies are limited by the difficulty in obtaining primary AMs from patients with ARDS. Our objective was to determine whether an in vitro model of ARDS can recapitulate the same AM functional defect observed in vivo and be used to further investigate pathophysiological mechanisms.Methods: AMs were isolated from the lung tissue of patients undergoing lobectomy and then treated with pooled bronchoalveolar lavage (BAL) fluid previously collected from patients with ARDS. AM phenotype and effector functions (efferocytosis and phagocytosis) were assessed by flow cytometry. Rac1 gene expression was assessed using quantitative real-time PCR.Results: ARDS BAL treatment of AMs decreased efferocytosis (p = 0.0006) and Rac1 gene expression (p = 0.016); however, bacterial phagocytosis was preserved. Expression of AM efferocytosis receptors MerTK (p = 0.015) and CD206 (p = 0.006) increased, whereas expression of the antiefferocytosis receptor SIRPα decreased following ARDS BAL treatment (p = 0.036). Rho-associated kinase (ROCK) inhibition partially restored AM efferocytosis in an in vitro model of ARDS (p = 0.009).Conclusions: Treatment of lung resection tissue AMs with ARDS BAL fluid induces impairment in efferocytosis similar to that observed in patients with ARDS. However, AM phagocytosis is preserved following ARDS BAL treatment. This specific impairment in AM efferocytosis can be partially restored by inhibition of ROCK. This in vitro model of ARDS is a useful tool to investigate the mechanisms by which the inflammatory alveolar microenvironment of ARDS induces AM dysfunction.

背景：肺泡巨噬细胞（AM）的受损清除功能可能与急性呼吸窘迫综合征（ARDS）的发病机制相关；然而，由于从ARDS患者中获取原始AM的困难，相关研究受到限制。本研究旨在确定ARDS体外模型是否能够重现体内观察到的相同AM功能缺陷，并进一步探究病理生理学机制。方法：从进行肺叶切除术患者的肺组织中分离AM，并使用先前从ARDS患者收集的混合支气管肺泡灌洗（BAL）液进行处理。通过流式细胞术评估AM表型和效应功能（清除功能和吞噬功能）。使用定量实时PCR检测Rac1基因表达。结果：ARDS BAL处理AM后，清除功能（p = 0.0006）和Rac1基因表达（p = 0.016）降低；然而，细菌吞噬功能得以保留。ARDS BAL处理后，AM清除功能受体MerTK（p = 0.015）和CD206（p = 0.006）的表达增加，而抗清除功能受体SIRPα的表达降低（p = 0.036）。通过抑制Rho相关激酶（ROCK），在ARDS体外模型中部分恢复了AM的清除功能（p = 0.009）。结论：ARDS BAL液处理肺切除组织中的AM，可诱导出与ARDS患者相似的清除功能损害。然而，ARDS BAL处理后，AM的吞噬功能得以保留。通过抑制ROCK部分恢复AM的清除功能。此ARDS体外模型是研究ARDS炎症性肺泡微环境诱导AM功能障碍机制的有用工具。