Department of Respiratory Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310016, China.
Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, China.
Exp Cell Res. 2020 Sep 15;394(2):112101. doi: 10.1016/j.yexcr.2020.112101. Epub 2020 May 29.
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are common lung disorders characterized by alveolar-capillary barrier disruption and dyspnea, which can cause substantial morbidity and mortality. Currently, a cluster of acute respiratory illnesses, known as novel coronavirus (2019-nCoV)-infected pneumonia (NCIP), which allegedly originally occurred in Wuhan, China, has increased rapidly worldwide. The critically ill patients with ARDS have high mortality in subjects with comorbidities. Previously, the excessive recruitment and activation of neutrophils (polymorphonuclear leukocytes [PMNs]), accompanied by neutrophil extracellular traps (NETs) formation were reported being implicated in the pathogenesis of ALI/ARDS. However, the direct visualization of lung epithelial injuries caused by NETs, and the qualitative and quantitative evaluations of this damage are still lacking. Additionally, those already reported methods are limited for their neglect of the pathological role exerted by NETs and focusing only on the morphological features of NETosis. Therefore, we established a cell-based assay for detecting NETs during lung epithelial cells-neutrophils co-culture using the xCELLigence system, a recognized real-time, dynamic, label-free, sensitive, and high-throughput apparatus. Our results demonstrated that lung epithelial injuries, reflected by declines in cell index (CI) values, could be induced by lipopolysaccharide (LPS)-activated PMNs, or NETs in a time and dose-dependent manner. NETs generation was verified to be the major contributor to the cytotoxicity of activated PMNs; protein components of NETs were the prevailing cytotoxic mediators. Moreover, this cell-based assay identified that PMNs from severe pneumonia patients had a high NETs formative potential. Additionally, acetylsalicylic acid (ASA) and acetaminophen (APAP) were discovered alleviating NETs formation. Thus, this study not only presents a new methodology for detecting the pathophysiologic role of NETs but also lays down a foundation for exploring therapeutic interventions in an effort to cure ALI/ARDS in the clinical setting of severe pneumonia, including the emerging of NCIP.
急性肺损伤（ALI）及其更严重的形式，急性呼吸窘迫综合征（ARDS）是常见的肺部疾病，其特征为肺泡毛细血管屏障破坏和呼吸困难，可导致大量发病率和死亡率。目前，一组称为新型冠状病毒（2019-nCoV）感染性肺炎（NCIP）的急性呼吸道疾病，据称最初发生在中国武汉，已在全球迅速增加。患有 ARDS 的重病患者在患有合并症的患者中死亡率很高。以前，据报道，中性粒细胞（多形核白细胞[PMN]）的过度募集和激活，伴随着中性粒细胞细胞外陷阱（NETs）的形成，与 ALI/ARDS 的发病机制有关。然而，仍然缺乏对 NETs 引起的肺上皮损伤的直接观察，以及对这种损伤的定性和定量评估。此外，已经报道的方法仅限于其忽视 NETs 发挥的病理作用，而只关注 NETosis 的形态特征。因此，我们使用 xCELLigence 系统建立了一种在肺上皮细胞-中性粒细胞共培养中检测 NETs 的基于细胞的测定方法，该系统是一种公认的实时、动态、无标记、敏感和高通量的仪器。我们的结果表明，肺上皮损伤，反映为细胞指数（CI）值下降，可以由脂多糖（LPS）激活的 PMN 或 NETs 以时间和剂量依赖性方式诱导。NETs 的产生被证明是激活的 PMN 的细胞毒性的主要贡献者；NETs 的蛋白成分是主要的细胞毒性介质。此外，这种基于细胞的测定方法确定来自严重肺炎患者的 PMN 具有高 NETs 形成潜力。此外，发现乙酰水杨酸（ASA）和对乙酰氨基酚（APAP）可减轻 NETs 的形成。因此，本研究不仅提出了一种新的方法来检测 NETs 的病理生理作用，还为探索治疗干预措施奠定了基础，以期在严重肺炎的临床环境中治愈 ALI/ARDS，包括新型冠状病毒的出现。
