A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed pulmonary macrophages’ role as modulators of the fibrotic processes. The proportion, phenotype, and function of alveolar (AM) and interstitial macrophages (IM) at the early stages of bleomycin-induced pulmonary fibrosis have not been clearly described. In this way, our study aimed to characterize these macrophage populations and investigate the effect on fibroblast activation. C57BL/6 mice were intratracheally injected with bleomycin and were sacrificed at day 3, 5, and 7 for the performance of flow cytometry and fluorescent-activated cell sorting analysis for protein and gene expression quantification. After bleomycin administration, the proportion of IM was significantly higher than that of AM, which showed a decay during the inflammatory phase, and peaked at day 7. At day 7 of the inflammatory phase, AM started shifting their phenotype from M1-like towards M2, while IM showed a M2-like phenotype. Conditioned medium derived from IM sorted at day 7 induced fibroblast activation and differentiation in myofibroblasts in vitro. Our findings indicate that IM are the largest macrophage population at the early stages of experimental pulmonary fibrosis and are secreted mediators able to activate fibroblasts, pointing to macrophage modulation as a potential therapeutic strategy to restrain progressive fibrosing lung disorders.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury that currently lacks effective clinical treatments. Evidence highlights the potential role of glycogen synthase kinase-3 (GSK-3) inhibition in mitigating severe inflammation. The inhibition of GSK-3α/β by CHIR99021 promoted fetal lung progenitor proliferation and maturation of alveolar epithelial cells (AECs). The precise impact of CHIR99021 in lung repair and regeneration during acute lung injury (ALI) remains unexplored. This study intends to elucidate the influence of CHIR99021 on AEC behaviour during the peak of the inflammatory phase of ALI and, after its attenuation, during the repair and regeneration stage. Furthermore, a long-term evaluation was conducted post CHIR99021 treatment at a late phase of the disease. Our results disclosed the role of GSK-3α/β inhibition in promoting AECI and AECII proliferation. Later administration of CHIR99021 during ALI progression contributed to the transdifferentiation of AECII into AECI and an AECI/AECII increase, suggesting its contribution to the renewal of the alveolar epithelial population and lung regeneration. This effect was confirmed to be maintained histologically in the long term. These findings underscore the potential of targeted therapies that modulate GSK-3α/β inhibition, offering innovative approaches for managing acute lung diseases, mostly in later stages where no treatment is available.