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

INTRODUCTION: Combined exposures to airborne nanoparticles and acidic gases are plausible during industrial accidents and in military settings involving propellants. Aluminum oxide nanoparticles (Al(2)O(3) 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. This study aimed to investigate early airway responses to repeated apical exposures to Al(2)O(3) NPs, HCl, or their mixture in a human air-liquid interface co-culture model of alveolar epithelial (hAELVi) and fibroblast (MRC-5) cells. METHODS: Cells were exposed daily for four days, and epithelial viability, barrier integrity, and mediator release were assessed. Conditioned media from exposed cultures were applied to naïve fibroblasts to evaluate proliferation and migration responses. RESULTS: Repeated exposures did not induce cytotoxicity, barrier disruption, or increases in canonical pro-fibrotic or pro-inflammatory mediators such as TGF-β1, CTGF, or IL-8. However, conditioned media from exposed epithelial cells consistently triggered fibroblast activation through non-canonical epithelial-mesenchymal signaling pathways. These effects occurred at sub-toxic exposure levels. DISCUSSION: These findings indicate that early sub-lethal co-exposure to Al(2)O(3) NPs and HCl can trigger fibroblast activation independently of classical cytokine induction. This unconventional paracrine response suggests a mechanistic link between early epithelial stress and fibroblast-driven remodeling, underscoring the importance of investigating non-canonical pathways in lung responses to mixed environmental pollutants.