Purpose Activated alveolar macrophages (AMs) secrete extracellular vesicles and particles to mediate the inflammatory response in the acute respiratory distress syndrome (ARDS) although the underlying mechanisms are poorly understood. This study investigated whether secretory autophagosomes (SAPs) from AMs contribute to the inflammation-mediated lung injury of ARDS. Methods We first isolated SAPs from cell culture supernatants of RAW264.7 cells and AMs and quantified Interleukin (IL)-1β levels in SAPs. Next, we employed a lipopolysaccharide (LPS)-induced ARDS model to investigate whether SAP-derived IL-1β could exacerbate lung injury. Finally, we used siRNA to knockdown Rab8a, both in vitro and in vivo, to investigate the effect of Rab8a on SAP secretion and lung injury in ARDS. Results We found that AMs play an important role in ARDS by releasing a novel type of proinflammatory vesicles called SAPs that could exacerbate lung injury. SAPs are characterized as double-membrane vesicles (diameter ~200 nm) with the expression of light chain 3 (LC3). IL-1β in SAPs is the key factor that contributes to the inflammation and lung injury in ARDS. We found that Rab8a is necessary for AMs to release SAPs with IL-1β, and Rab8a knockdown alleviated lung injury in ARDS. Conclusion This study showed the novel finding that SAPs released from AMs play a vital role in ARDS by promoting an inflammatory response and the underlying mechanism was associated with IL-1β secretion.

PurposeAcute respiratory distress syndrome (ARDS) is a prevalent illness in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) assist in ARDS lung injury and the inflammatory process through mechanisms that are unclear. This study investigated the role of AM-derived secretory autophagosomes (SAPs) in lung injury and microRNA (MiR)-199a-3p-regulated inflammation associated with ARDS in vitro and in a murine model.MethodsThe ARDS model in mouse was established by intratracheal LPS lipopolysaccharide (LPS) injection. The agomirs or antagomirs of MiR-199a-3p were injected into the caudal vein to figure out whether MiR-199a-3p could influence ARDS inflammation and lung injury, whereas the mimics or inhibitors of MiR-199a-3p, siRNA of Rab8a, or PAK4 inhibitor were transfected or applied to RAW264.7 cells to evaluate the mechanism of SAP release. Culture supernatants of RAW264.7 cells treated with LPS or bronchoalveolar lavage fluid from mice were collected for the isolation of SAPs.ResultsWe found that MiR-199a-3p was over-expressed in the lungs of ARDS mice. The MiR-199a-3p antagomir alleviated, whereas the MiR-199a-3p agomir exacerbated LPS-induced inflammation in mice by promoting AM-derived SAP secretion. In addition, MiR-199a-3p over-expression exacerbated LPS-induced ARDS via activating Rab8a, and Rab8a silencing significantly suppressed the promoting influence of the MiR-199a-3p mimic on SAP secretion. Furthermore, MiR-199a-3p mimic activated Rab8a by directly inhibiting PAK4 expression.ConclusionThe novel finding of this study is that MiR-199a-3p participated in the regulation of SAP secretion and the inflammatory process via targeting of PAK4/Rab8a, and is a potential therapeutic candidate for ARDS treatment.

Background: Acute respiratory distress syndrome (ARDS) has a high incidence among patients in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) contribute to ARDS-associated lung injury; however, the mechanisms underlying the inflammatory process remain unclear. Purpose: This study investigated the effects and the mechanisms through which AM-derived secretory autophagosomes (SAPs) contribute to ARDS-associated lung injury. Methods: SAPs were isolated from cell culture supernatants of AMs treated or untreated with lipopolysaccharide (LPS) and intratracheally injected to determine whether SAP exacerbated lung injury in mice with ARDS. The SAP proteome was analysed using mass spectrometry. Lung fibroblasts were treated with SAPs and collected for transcriptome sequencing. A transwell assay was conducted to study the chemotactic activity of fibroblasts induced by SAPs. Results: We found that AMs contributed to ARDS-associated lung injury by releasing a novel type of pro-inflammatory vesicles named SAPs, which were characterised as double-membraned vesicles approximately 200 nm in diameter and light-chain-3 expression. Proteomic analysis of SAPs and gene ontology (GO) enrichment analysis revealed that LPS-SAPs contain several differentially expressed proteins involved in effecting an inflammatory response, including interleukin (IL)-1β. We confirmed this result by quantifying IL-1β in SAPs using ELISA. Moreover, administering SAPs from LPS-stimulated macrophages to mice with ARDS exacerbated lung injury, whereas IL-1 receptor antagonist (IL-1RA) pre-treatment significantly ameliorated SAP-induced lung injury. In addition, we conducted transcriptome sequencing of lung fibroblasts treated with SAPs to explore the effect of SAPs on fibroblasts in ARDS, which demonstrated that the expression of several chemokines (CXCL2, CXCL15, and so forth) significantly increased with SAP stimulation. Furthermore, the transwell assay indicated that SAPs could enhance the chemotactic activity of fibroblasts, suggesting that SAPs may exacerbate inflammation in ARDS by promoting neutrophil or monocyte recruitment. Conclusion: This is the first study to show that AM-derived SAPs contribute significantly to ARDS by secreting pro-inflammatory cytokines and promoting inflammatory cell recruitment by activating fibroblasts, which may be beneficial for therapeutic development of ARDS. National Natural Science Foundation of China (Grant numbers 81870066, 81670074, 81930058, 82270083 and 81971888), Six Talent Peaks Project in Jiangsu Province(Grant numbers TD-SWYY-003), and Social Development Specifc Projects of Jiangsu Province [Grant number BE2020786]. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.