Catalytic Site Inhibitors of Group 1 Allergens Prevent Toll-like Receptor (TLR)4- and TLR1/2-Dependent Innate Responses in Keratinocytes and Airway Epithelial Cells Exposed to House Dust Mite Allergenic Extract

Abstract

Dysregulated immune responses to domestic allergens are underlying events in atopic dermatitis (AD), an inflammatory disease of humans and domestic companion animals characterized by itching and eczema. The house dust mite (HDM) allergome, notably the cysteine protease group 1 allergens, is an important trigger of AD. This protease activity is implicated in innate mechanisms which both initiate and reinforce allergic sensitization, prompting interest in the design of protease inhibitors as a novel allergy therapy. We examined pyruvamide chemotype protease inhibitors on intracellular reactive oxidant species (ROS) production induced by HDM allergen extracts in HaCaT keratinocytes and identified promising topical and orally bioavailable candidates. We then explored the wider signaling network affected by this allergen inhibition in keratinocytes and airway epithelial cells (AECs). Optimized pyruvamides with different properties (viz. neutral vs charged, cell-impermeant molecules) inhibited ROS generation evoked by HDM allergen extract, but biochemical potency against Der p 1 per se was not a direct indicator of cellular efficacy. ROS production was stimulated by Der p 1 through canonical activation of protease-activated receptor 1 which propagated the activation signal through a network involving ATP, purinoceptors, transient receptor potential channels, nitric oxide formation, and the ligation of Toll-like receptor (TLR) 4 and TLR 1/2 heterodimers by endogenous activators. These data reveal that inhibition of a single allergen in HDM allergenic extracts prevents an extensive signaling network which is coupled to the redox control of keratinocytes and AECs. These data underscore the exciting possibility that allergic responses can be inhibited at source.