Contact sensitisers activate keratinocytes and induce cytotoxicity via Transient Receptor Potential Ankyrin 1 in allergic contact dermatitis

BACKGROUND AND PURPOSE: Allergic contact dermatitis (ACD) is a frequent inflammatory skin disease with limited therapeutic options. While neuronal Transient Receptor Potential Ankyrin 1 (TRPA1) has been implicated in ACD, the role of keratinocyte TRPA1 remains unclear. We investigated whether allergen binding to keratinocyte TRPA1 drives cytotoxicity, cytokine release and inflammatory amplification. EXPERIMENTAL APPROACH: We combined in vivo oxazolone-induced hypersensitivity in wild-type and Trpa1 knockout mice with pharmacological inhibition by HC-030031, in vitro assays using TRPA1-expressing Chinese hamster ovary (CHO) cells and primary keratinocytes, RNAscope localisation in human and murine skin, cytokine profiling and in silico docking analyses of allergen-TRPA1 interactions. KEY RESULTS: influx, cytotoxicity and IL-1α release, effects absent in Trpa1-deficient cells and inhibited by HC-030031. RNAscope confirmed keratinocyte TRPA1 expression in human and mouse skin. Docking revealed allergen-specific binding modes, where covalent cysteine modification and A-loop stabilisation correlated with potency. Consistent with these observations allergen-induced ROS, which also target the cysteine cluster of TRPA1, may further lower the channel's activation threshold and thereby amplify allergen-driven cytotoxicity. CONCLUSION AND IMPLICATIONS: Keratinocyte TRPA1 integrates direct allergen binding with ROS-mediated sensitisation to drive cytotoxicity and IL-1α release. This dual mechanism helps explain sensitiser potency and positions TRPA1 antagonism as a promising therapeutic approach in ACD, while providing a framework for chemical risk assessment.