Exacerbation of pulmonary fibrosis following acute lung injury via activin-A production by recruited alveolar macrophages.

Background: Acute respiratory distress syndrome (ARDS) is a complicated pathological cascade process of excessive pulmonary inflammation and alveolar epithelial cell apoptosis that results in respiratory dysfunction and failure. Some cases of ARDS can result in a more severe state of pulmonary fibrosis, referred to as postinjury lung fibrosis. The mortality and incidence rate of ARDS are high, particularly when it leads to continuing alveolar and interstitial fibrosis, which requires urgent treatment and appropriate management. The lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model has been widely implemented for studying ARDS in humans. In our study, we found alterations in the alveolar macrophage (AM) profile in such a mouse model. Specifically, activin-A produced by dominantly recruited AMs (recAMs) was noted to be implicated in the process of post-injury lung fibrosis.

Methods: The ALI animal model in C57BL/6 mice was established via 3.5 mg/kg of LPS intratracheal administration. Single-cell RNA (scRNA) sequencing was used for detailed classification and functional characterization of lung macrophages. Through in vivo experiments, we evaluated the role that activin-A plays in post-injury lung fibrosis in an ALI mouse model using enzyme-linked immunosorbent assay (ELISA), histological staining methods, and immunofluorescence. Through in vitro experiments, we analyzed the effect of activin-A on murine lung epithelial 12 (MLE-12) cells and bone marrow-derived macrophages (BMDMs) using Western blotting (WB), quantitative real-time polymerase chain reaction, RNA sequencing, and immunofluorescence.

Results: Our findings revealed that recAMs replaced tissue-resident alveolar macrophages (TRAMs) as the dominant macrophage population in the setting of ALI. The results of Gene Ontology (GO) analysis suggested that activin-A was associated with wound healing and suppressor of mothers against decapentaplegic (SMAD) protein signaling pathways. Immunofluorescence results revealed that the receptor of activin-A mainly localized to alveolar epithelial cells and macrophages. Subsequently, activin-A was specifically found to drive MLE-12 cells to mesenchymal cell transformation via the transforming growth factor-β (TGF-β)/SMAD signaling. Moreover, the results of transcriptome analysis and WB confirmed that activin-A could enhance the concerted activity of Hippo and TGF-β/SMAD pathways in BMDMs, leading to an increased expression of profibrotic mediator. Moreover, yes-associated protein (YAP) and transcriptional coactivated with PDZ-binding motif (TAZ) proteins were found to drive BMDM activin-A expression, which could generate a positive feedback mechanism that perpetuates fibrosis.

Conclusions: Our findings revealed that activin-A is involved in the pathological mechanisms in post-injury lung fibrosis by promoting epithelial-mesenchymal transition (EMT) and the formation of an underlying profibrotic positive feedback loop in recAMs. Activin-A is thus a potential therapeutic target for developing ALI and ALI-associated pulmonary fibrosis therapeutics.