key: cord-326613-253v48i0
authors: Lv, Dandan; Xu, Yiming; Cheng, Hongqiang; Ke, Yuehai; Zhang, Xue; Ying, Kejing
title: A novel cell-based assay for dynamically detecting neutrophil extracellular traps-induced lung epithelial injuries
date: 2020-05-29
journal: Exp Cell Res
DOI: 10.1016/j.yexcr.2020.112101
sha: 
doc_id: 326613
cord_uid: 253v48i0

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.

initiation and progression of ALI/ARDS. Hence, there is a need for a simpler, objective, and quantitative 140 approach for detecting NETs, as well as evaluating lung epithelial disruption caused by NETs. 141 In this study, we describe the optimization and validation of a novel lung epithelial cell-based assay to 142 analyze the damage caused by NETs on lung epithelial barriers using the xCELLigence system. Till now, 143 this label-free, real-time, and dynamically analytic apparatus has been widely used in evaluating Chinese 144 compound medicines 29 or modern pharmaceuticals, 30 monitoring epithelial barrier function, 31 and detecting 145 T cell activation 32 or NK cell killing. 33 The advantages of this system are its high sensitivity, accuracy, effects. Moreover, the protein components of NETs were the prevailing cytotoxic mediators. 154 We compared the consistency of this novel cell-based NETs detection methodology with other common 155 analytical approaches. More encouragingly, the feasibility and practicality of this assay helped to distinguish 156 NETs formation differences between PMNs from severe pneumonia patients and healthy controls, and seem A549 or PMN samples were subjected to washing three times with 0.05 M sodium cacodylate buffer and 206 fixing in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer for 4 h. Secondary fixation with 0.1% 207 osmium tetroxide was performed for 15 min prior to sequential dehydration with increasing concentrations 208 of ethanol. Samples were dried at the critical point using a CO 2 drier, mounted onto an aluminum stub, and 209 sputter-coated with 80/20 gold-palladium. A thin strip of colloidal silver was painted at the sample edge to 210 dissipate sample charging. Samples were imaged with a field emission scanning electron microscope Nova 211 Nano 450 (Thermo FEI). Live-cell imaging 230 This experiment was conducted using a Nikon A1R confocal microscope equipped with phase-contrast 231 microscopy and a temperature-control to maintain incubation at 37°C. PMNs with different treatments 232 (medium, 1 μg/mL LPS, 1 μM DPI, and 1 μg/mL LPS + 1 μM DPI) were re-suspended in RPMI 1640 233 without phenol red containing 5 μM SYTOX Green (S7020, Thermo Fisher). Then, these suspensions were 234 cultured in a 35 mm 4-chamber glass-bottom dish (D35C4-20-1.5-N, Cellvis) (1×10 5 /mL, 500 μL/chamber).

After 30 min, images were randomly taken under low-light illumination at 15 min intervals for a total of 4 h. The procedure used in this study was modified from that described previously. 36-38 PMNs (3 × 10 4 /mL) stimulated by different concentrations of LPS were re-suspended in RPMI 1640 without phenol red 251 containing 5 μM membrane-impermeable DNA dye SYTOX Green (S7020, Thermo Fisher). Then, these 252 suspensions were seeded in a 96-well black plate (WHB-96, WHB) (100 μL/well), usually in quadruplicates, 253 and cultured at 37°C in the dark. Extracellular DNA released from PMNs were quantified by measuring 254 fluorescence values at sequential time points in a microplate fluorescence reader at 488/523 nm.

Following PMNs incubation in a 6-well plate (1 × 10 8 /mL, 1 mL/well), 900 μL culture medium per well was 257 carefully aspirated and 10 μL 10×RIPA buffer (9806S, Cell Signaling Technology) was added into the wells 258 supplemented with EDTA-free protease inhibitor tablets and phosphatase inhibitors (Roche Diagnostics).

After lysis on ice for 5 min, the total PMN lysates were collected and sonicated on ice for 15 s. The protein 260 concentrations were quantified using the BCA assay (Beyotime) before solubilizing in 6×loading buffer, and 

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We would like to thank Ms. Shuangshuang Liu for instructing the application of Nikon A1R confocal microscope throughout the study. Thanks to Editage (www.editage.cn) for English language editing.