Microbial sensing through the non-canonical inflammasome modulates airway type 2 immunity.

Introduction: Airway epithelial cells serve as critical sensors of both microbes and allergens, orchestrating immune responses through damage-associated molecular patterns including IL-33. Common aeroallergens induce type 2 inflammation through protease activity and pore-forming mechanisms that trigger epithelial IL-33 secretion and MAPK signaling. While microbial pattern receptors such as caspase-4 (which detects intracellular LPS) similarly generate membrane pores via the non-canonical inflammasome, it remains unknown whether these receptors can engage the same downstream epithelial IL-33 release and MAPK activation pathways. Methods: Using human airway epithelial cell models, we examined caspase-4-dependent pyroptotic signaling downstream of intracellular LPS, including gasdermin D cleavage, IL-33 release, and MAPK-dependent transcriptional responses. We assessed the modulatory effect of protease allergen co-exposure on LPS-induced pyroptosis and interrogated the role of Orai1-mediated calcium signaling Results: Intracellular LPS activated caspase-4-dependent pyroptotic signaling, resulting in gasdermin D cleavage, IL-33 release, and MAPK-dependent transcriptional responses. Protease allergen exposure enhanced LPS-induced pyroptotic responses through Orai1-mediated calcium signaling in vitro. Genetic deletion of caspase-4 attenuated innate type 2 immune responses in the mouse protease allergen challenge model. LPS preparations from different bacterial species demonstrated variable capacity to engage the non-canonical inflammasome. Analysis of human asthma datasets revealed increased airway expression of both caspase-4 and gasdermin D in asthmatic patients relative to healthy controls. Discussion: These findings identify the epithelial non-canonical inflammasome as a pathway capable of linking microbial pattern recognition to IL-33-dependent type 2 responses. This work establishes a mechanistic framework for understanding how bacterial sensing machinery may intersect with allergic inflammation during pathophysiological conditions, and suggests that caspase-4 signaling could represent a therapeutic target in asthma.