Redox-sensitive GPCR signaling drives Gq-dependent Ca²⁺ mobilization and cytokine production in human bronchial epithelial cells

Organic dust (OD) from animal production facilities contains a complex mixture of microbial products, metabolites, and particulates that engage airway epithelial signaling pathways. Here we investigated the mechanisms by which OD extract (ODE) activates immune signaling pathways in human bronchial epithelial (hBE) cells. ODE rapidly stimulated reactive oxygen species (ROS) generation and a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]i), consisting of an early transient peak followed by a smaller sustained phase. Antioxidant scavenger pretreatment (glutathione, N-acetylcysteine) markedly attenuated both ROS production and Ca²⁺ mobilization, whereas induction of endogenous antioxidant defenses with bardoxolone abolished the response, indicating redox sensitivity. Pharmacologic inhibition of Gqα with YM-254890 suppressed both phases of the Ca²⁺ response, implicating Gq-coupled receptor activation. Consistent with an autocrine amplification mechanism, selective antagonists of histamine (H1), cysteinyl leukotriene (CysLT1 and CysLT2), leukotriene B4 (BLT1), and prostaglandin receptors (EP1) each reduced ODE-evoked Ca²⁺ mobilization. In parallel, inhibitors of histidine decarboxylase, 5-lipoxygenase, and cyclooxygenases (COX-1/COX-2) attenuated Ca2+ signaling, supporting rapid endogenous ligand production and secretion. Downstream of Ca²⁺ mobilization, ODE activated PKCα/β and PKCδ and induced robust transcription of proinflammatory cytokine and chemokine mRNAs (IL1β, IL6, IL8, IL33, TNFα) within 2 hours of exposure. ELISA confirmed increased secretion of IL-1β, IL-6, IL-8, and IL-33, with differential sensitivity to PKC isoforms and NF-κB inhibition. These findings identify a redox-sensitive GPCR network that amplifies Gq-dependent Ca²⁺ signaling in airway epithelial cells and provides a mechanistic framework for epithelial inflammatory activation following ROS-inducing environmental exposures.