David M. Guidot

Background Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor‐beta 1 (TGFβ1) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol‐exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM‐CSF and thereby alter airway epithelial barrier function. Methods Human or rat LF were cultured ± ethanol for 2 weeks and then co‐cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co‐culture and on day 5 of co‐culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO‐1 expression and localization. TGFβ1 and GM‐CSF levels in conditioned media from the co‐cultures were quantified by ELISA. Results AEC co‐cultured with ethanol‐exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO‐1 expression, whereas collagen type 1A1 and α‐smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co‐cultures, activated TGFβ1 levels increased and GM‐CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity. Conclusions Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM‐CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.