Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway

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.Acute lung injury (ALI) and its severe form, ARDS cause significant morbidity and mortality in critically-ill patients.¹ ALI often presents with extensive accumulation of activated inflammatory cells and diffuse alveolar damage (DAD) accompanied by oxidative stress.² Lung epithelial cell damage, a prominent feature of both infectious and non-infectious lung injury, potentially has an important functional role in the pathogenesis of the overwhelming inflammation and vascular leaking involved in ALI/ARDS.^{3, 4} However, it remains incompletely understood how lung inflammation is initiated and propagated during the development of lung injury, particularly by non-infectious stimuli. For example, oxidative stress, such as occurs with the inspiration of a high concentration of oxygen, could lead to reactive oxygen species (ROS) production, inflammasome activation, pro-inflammatory cytokine production, neutrophil influx and lung inflammation,^{5, 6} resulting in severe lung injury and respiratory failure. It has been reported that the deposition of extracellular matrix (ECM) has a role in this process.⁷ Therefore, the cross-talk between damaged epithelial cells and lung inflammation cells during the development of non-infectious lung injury needs to be explored to properly understand the development of ALI/ARDS.Hyperoxia-induced ALI (HALI) is a well-established, non-infectious animal model that mimics human ARDS and has been used extensively by investigators to better understand the pathogenesis of this devastating syndrome.⁸ Oxidative stress, such as occurs with hyperoxia and its derivative ROS, can induce epithelial cell death via apoptosis, autophagic cell death, necrosis and many other pathways.⁹ Prolonged exposure to a high concentration of oxygen is fatal in most animal models, resulting in neutrophil influx and alveolar edema.⁶ However, despite the fact that mouse HALI is a good model of human ARDS, mortality in rodents often results from severe cerebral edema.⁶ Activated alveolar macrophage-released chemokines/cytokines are essential to neutrophil recruitment.⁶ That said, how the oxidative stress specifically activates alveolar macrophages has not been well elucidated. In this study, we used the mouse model of HALI to evaluate the cross-talk between damaged lung epithelial cells and alveolar macrophages during the development of HALI via epithelial cell-derived EVs.For a long time, EVs were considered membrane debris without any specific biological function.¹⁰ Recently, accumulating data have suggested that EVs are in fact crucial mediators of intercellular communications.^{11, 12, 13} EVs are categorized into exosomes, microvesicles and apoptotic bodies based on their origin, size and content.¹⁰ The exosome is 40–120 nm in size and is originated from the endo-lysosomal pathway, intraluminal budding or the fusion of multivesicular bodies with the cell membrane. It is characterized by holding plasma membrane proteins such as the tetraspanin (CD9, CD63, CD81 and so on) and lipid raft proteins (flotillin and caveolin-1).¹⁴ The exosome also contains mRNA and microRNA (miRNA) as well as cytoplasmic and membrane proteins. It is secreted from majority of cells, including macrophages, dendritic cells and epithelial cells among many others. Microvesicles (MVs) are 50–1000 nm in size and are originated from the outward budding of the cell membrane.¹⁰ MVs contain membrane proteins, mRNA, miRNA, non-coding RNAs and cytoplasmic proteins.¹⁰ Apoptotic bodies are significantly larger than exosomes and MVs, averaging 500–2000 nm, and are generated from the surface of apoptotic cells.¹⁰ They are characterized by a large amount of phosphatidylserine, cell organelles, nuclear fractions and certain marker proteins, such as Apaf-1.¹⁰ Both infection and toxic insults have been reported to facilitate the generation of EVs.^{15, 16, 17} EVs are reported to have similar cellular functions as their mother cells.^{10, 18} For instance, resting macrophage-originated MVs exert an anti-inflammatory effect, whereas macrophage-originated MVs are pro-inflammatory after LPS stimulation.¹⁹ Although EVs appear promising candidates for intercellular communication, their roles in lung cells, particularly in the pathogenesis of ALI, have not been reported.We hypothesized that hyperoxia-associated oxidative stress stimulates EV generation in lung epithelial cell and that epithelial cell-derived EVs facilitate the development of inflammatory lung responses after oxidative stress. We further explored the components in epithelial cell-derived EVs after hyperoxia. The underlying mechanisms by which EVs exert their pro-inflammatory effects on alveolar macrophages were also determined. To the best of our knowledge, this is the first study focusing on the role of EVs in the pathogenesis of hyperoxia-induced ALI, the intercellular cross-talk between epithelial cells and alveolar macrophages, as well as the relationship between cell death and pro-inflammatory signals.