In vitro assessment of respiratory sensitization potential and allergic asthma: A miRNA-based approach

Occupational exposure to respiratory sensitizers is a major cause of allergic asthma. Although miRNAs are increasingly linked to asthma pathogenesis, their specific role in allergic asthma remains poorly defined. This study aims to assess a human-based 3D in vitro model to investigate the possible role of miRNAs in allergic asthma. Calu-3 cells cultured in ALI were exposed to three respiratory sensitizers: hexamethylen diisocyanate (HDI), ammonium hexachloroplatinate (HClPt), and trimellitic anhydride (TMA) using VITROCELL® Cloud Alpha 6 System. In addition, 2,4-dinitrochlorobenzene (DNCB) as skin sensitizer and the irritant sodium dodecyl sulphate (SDS) were tested. Cytotoxicity was assessed by MTT and LDH assays, while epithelial barrier integrity was evaluated by TEER. MiRNA profiling was performed, followed by targeted analysis. Cytokine release (IFN-γ, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17, IL-18, IL-33, MCP-1, MIP-1α, TGF-β and TSLP) was quantified, and miRNAs involvement was investigated using miRNA mimics and inhibitors. HMOX gene expression was assessed. MiRNA profiling revealed several miRNAs consistently downregulated across all respiratory sensitizers, including miR-26b-5p, miR-34a-5p, miR-125b-5p, miR-148a-3p, and miR-200a-3p. HClPt induced a significant increase in IL-6 release and HMOX expression, whereas HDI and TMA caused a significant reduction. To understand the possible link, specific miRNA mimic and inhibitor conditions were used. Results indicate that miR-18b-5p, miR-135b-5p and let-7a-5p regulated IL-6 release and HMOX expression. Respiratory sensitizers from different chemical classes were able to induce distinct miRNA-dependent regulatory mechanisms affecting IL-6 and HMOX expression. These findings support the relevance of human cell-based in vitro models for investigating allergic asthma.