title: Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.
abstract: Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after hyperoxia exposure. These circulating EVs also activated systemic macrophages other than the alveolar ones. Collectively, the results show that hyperoxia-induced, lung epithelial cell-derived and caspase-3 enriched EVs activate macrophages and mediate the inflammatory lung responses involved in lung injury.
authors.affiliation: Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
authors.forename: H-G
authors.initials: HG
authors.lastname: Moon
authors.affiliation: Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
authors.forename: H S
authors.initials: HS
authors.lastname: Choi
authors.affiliation: Division of Pulmonary and Critical Care, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Jin
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D004847:Epithelial Cells / Q000378:metabolism*; D000067128:Extracellular Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D018496:Hyperoxia / Q000150:complications / Q000473:pathology; D007249:Inflammation / Q000150:complications / Q000473:pathology*; D008168:Lung / Q000166:cytology*; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D044967:Serum / Q000378:metabolism; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: C515854:Rock1 protein, mouse; D054460:rho-Associated Kinases; D053148:Caspase 3