Beyond cytotoxicity: pollutant mixtures elicit unconventional epithelial-fibroblast signaling in a human lung air-liquid interface co-culture model

Justine Fredoc-Louison¹Maëva Cherrière^1,2Bastien Rival¹Suzanne De Araujo¹Sabine François¹Samir Dekali^1*

• ¹Biomedical Research Institute of the Armed Forces (IRBA), Brétigny-sur-Orge, France
• ²Institut National de l'Environnement Industriel et des Risques, Verneuil-en-Halatte, France

Combined exposures to airborne nanoparticles and acidic gases are plausible during industrial accidents and in military settings involving propellants. Aluminum oxide nanoparticles (Al₂O₃ NPs), widely used in industry and present in propellant formulations, together with hydrogen chloride (HCl), a corrosive combustion by-product, are relevant co-pollutants whose joint impact on lung remodeling remains poorly defined. We used a human air–liquid interface co-culture of alveolar epithelial (hAELVi) and fibroblast (MRC-5) cells to investigate early responses to repeated apical exposures to Al₂O₃ NPs, HCl, or their mixture. Exposures were delivered daily for four days, and epithelial viability, barrier integrity, and mediator release were assessed. Conditioned media from exposed cultures were applied to naïve fibroblasts, and proliferation and migration were monitored in real time. Although exposures did not cause cytotoxicity, barrier disruption, or increases in canonical mediators such as TGF-β1, Connective Tissue Growth Factor (CTGF), or IL-8, combined exposures consistently triggered fibroblast activation through non-canonical epithelial–mesenchymal signaling. conditioned media from co-cultures consistently enhanced fibroblast migration and modulated proliferation, while effects were weaker with monoculture-derived media, underscoring the role of epithelial– mesenchymal crosstalk. Control exposures with TGF-β1, CTGF, lipopolysaccharide, and bleomycin confirmed assay responsiveness. These findings reveal that early, sub-lethal interactions between epithelial and mesenchymal cells can initiate fibroblast migration and proliferation in the absence of cytokine induction, suggesting a mechanistic link between epithelial stress and pro-fibrotic remodeling.These findings show that pollutant mixtures elicit non-canonical, cytokine-independent paracrine cues that prime fibroblasts for migration, highlighting early remodeling signals beyond conventional toxicity endpoints.