title: Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants.
abstract: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH).
  
  
  
  BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry.
  
  
  
  CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release.
  
  
  
  Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment.
authors.affiliation: Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands; Department of Respiratory Medicine, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: b.benedikter@maastrichtuniversity.nl.
authors.forename: Birke J
authors.initials: BJ
authors.lastname: Benedikter
authors.affiliation: Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: c.volgers@maastrichtuniversity.nl.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Volgers
authors.affiliation: Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: pascallevaneijck@gmail.com.
authors.forename: Pascalle H
authors.initials: PH
authors.lastname: van Eijck
authors.affiliation: Department of Respiratory Medicine, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: e.wouters@mumc.nl.
authors.forename: Emiel F M
authors.initials: EFM
authors.lastname: Wouters
authors.affiliation: Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands; Department of Medical Microbiology & Infection Control, VU University Medical Center, Van der Boechorststraat 7, 1081BT Amsterdam, The Netherlands. Electronic address: paul.savelkoul@mumc.nl.
authors.forename: Paul H M
authors.initials: PHM
authors.lastname: Savelkoul
authors.affiliation: Department of Respiratory Medicine, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: n.reynaert@maastrichtuniversity.nl.
authors.forename: Niki L
authors.initials: NL
authors.lastname: Reynaert
authors.affiliation: Department of Pharmacology and Toxicology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Electronic address: g.haenen@maastrichtuniversity.nl.
authors.forename: Guido R M M
authors.initials: GRMM
authors.lastname: Haenen
authors.affiliation: Department of Respiratory Medicine, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: g.rohde@mumc.nl.
authors.forename: Gernot G U
authors.initials: GGU
authors.lastname: Rohde
authors.affiliation: Department of Pharmacology and Toxicology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands. Electronic address: a.weseler@maastrichtuniversity.nl.
authors.forename: Antje R
authors.initials: AR
authors.lastname: Weseler
authors.affiliation: Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address: f.stassen@maastrichtuniversity.nl.
authors.forename: Frank R M
authors.initials: FRM
authors.lastname: Stassen
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000171:Acrolein / Q000378:metabolism*; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D000073865:Cigarette Smoking / Q000009:adverse effects / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D000067128:Extracellular Vesicles / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D020545:Respiratory Mucosa / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D060225:Tetraspanin 28 / Q000378:metabolism; D060149:Tetraspanin 30 / Q000378:metabolism
chemical_list: D000975:Antioxidants; D013438:Sulfhydryl Compounds; D060225:Tetraspanin 28; D060149:Tetraspanin 30; D000171:Acrolein; D000111:Acetylcysteine
keywords: Acrolein; Airway epithelial cells; COPD; Chronic inflammation; DTNB; EV; Extracellular vesicles; Lung; Oxidative stress