Activin-A production by recruited alveolar macrophages exacerbates pulmonary fibrosis following acute lung injury
Background: Acute respiratory distress syndrome (ARDS) is a complicated pathological cascade process of excessive pulmonary inflammation and alveolar epithelial cells apoptosis which results in respiratory dysfunction and failure. Some ARDS can result in more severe state of pulmonary fibrosis, referred to as post-injury lung fibrosis. The mortality and incidence rate of ARDS are particularly high when it leads to continuing alveolar and interstitial fibrosis, which requires urgent treatment and appropriate management. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model has been widely implemented for studying ARDS in human. Here, 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 processing of post-injury lung fibrosis. Methods: ALI animal model in C57BL/6 mice was established via 3.5mg/kg LPS intra-tracheal administration. Single-cell RNA (scRNA) sequencing was used for detailed classification and functional characterization of lung macrophages. in vivo experiments, we evaluated the role that activin-A plays in post-injury lung fibrosis in an ALI mouse model using ELISA (Enzyme-linked immunosorbent assay), histological staining methods and immunofluorescence. in vitro experiments, we analyzed the effect of activin-A on MLE-12 cells and bone marrow-derived macrophages (BMDMs) using western blot (WB), qPCR (quantitative real-time PCR), RNA-seq (RNA sequencing) and immunofluorescence. Results: Our findings revealed that recAMs replaced tissue resident alveolar macrophages (TRAMs) as a dominant macrophage population in the setting of ALI. The result of GO (gene ontology) analysis suggested that activin-A was associated with wound healing and SMAD (suppressor of mothers against decapentaplegic) 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 to mesenchymal cell transformation via the TGF-ß (transforming growth factor-ß)/SMAD signaling. Moreover, the results of transcriptome analysis and WB confirmed that activin-A could enhance the concerted activity of Hippo and TGF-ß/SMAD pathways in BMDMs, leading to an increased expression of pro-fibrotic mediator. At the same time, 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. Conclusion: Our findings revealed that activin-A is involved in the pathological mechanisms in post-injury lung fibrosis by promoting EMT (epithelial–mesenchymal transition) 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. Overall design: Mouse lung tissue samples from two ALI and control mice each were minced and digested by collagenase II and trypsin for single cell suspensions.