Ethanol-exposed lung fibroblasts cause airway epithelial barrier dysfunction

Background: Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGF beta 1) 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 TGF beta 1 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 TGF beta 1 receptor type 1 (TGF beta R1) inhibitor (SB431542) or a TGF beta 1 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. TGF beta 1 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 a-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGF beta 1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGF beta 1 activity. Conclusions: Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGF beta 1 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.

Automated summary

Chronic alcohol ingestion induces dysregulated TGF beta 1 and GM-CSF in lung fibroblasts, leading to impaired airway epithelial barrier function. Co-culturing ethanol-exposed lung fibroblasts with airway epithelial cells resulted in decreased transepithelial electrical resistance, reduced expression of tight junction protein ZO-1, and increased expression of collagen type 1A1 and α-smooth muscle actin. These effects were mediated through the activation of TGF beta 1 signaling and suppression of GM-CSF, and could be prevented by blocking TGF beta 1 activity.