Exposure of airway epithelial cells to cigarette smoke extract causes the release of extracellular vesicles via thiol-reactive carbonyl compounds

Introduction: Secreted extracellular vesicles (EVs) participate in multiple processes by transferring lipids, proteins and RNA between cells. Yet, their contribution to chronic inflammation in the lungs is largely unexplored. We determined if exposure of airway epithelial cells (AEC) to cigarette smoke extract (CSE) results in release of EVs. Using the CSE components H₂O₂ and acrolein, we determined the respective contributions of reactive oxygen species and thiol-reactive carbonyls to CSE-induced EV release.

Methods: AEC were exposed for 6, 24 or 48h to different concentrations of CSE, H₂O₂ or acrolein, in the presence or absence of the thiol-group bearing antioxidant N-acetylcysteine (NAC). Relative levels of CD63+CD81+ EVs in conditioned media were measured by bead-coupled flow cytometry. Glutathione disulfide (GSSG) and reduced glutathione (GSH) were assessed using a GSSG reductase cycling assay.

Results: CSE induced EV release in a time- and concentration-dependent manner up to 2.3-fold after 24h exposure to 1.5% CSE. Direct incubation of CSE with GSH resulted in complete GSH oxidation, demonstrating direct thiol reactivity of CSE. In cell culture, the enhanced EV release was paralleled by 3.1 and 5.8 fold increases in GSSG and GSH, respectively, indicating oxidative cell stress. NAC prevented CSE-induced EV-release, likely by scavenging thiol-reactive components of CSE. Similar to CSE, acrolein, but not H₂O₂, induced EV release in a NAC-reversible manner. Conclusion AEC release an increased quantity of EVs when exposed to CSE. This is likely mediated by thiol-reactive compounds like acrolein. Experiments are ongoing to explore whether the CSE-induced EVs exert pro-inflammatory effects relevant to the pathogenesis of COPD.