Spatiotemporal Clusters of ERK Activity Coordinate Cytokine-induced Inflammatory Responses in Human Airway Epithelial Cells

Abstract

RATIONALESpatially coordinated ERK signaling events ("SPREADs") transmit radially from a central point to adjacent cells via secreted ligands for EGFR and other receptors. SPREADs maintain homeostasis in non-pulmonary epithelia, but it is unknown whether they play a role in the airway epithelium or are dysregulated in inflammatory disease. OBJECTIVES(1) To characterize spatiotemporal ERK activity in response to pro-inflammatory ligands, and (2) to assess pharmacological and metabolic regulation of cytokine-mediated SPREADs. METHODSSPREADs were measured by live-cell ERK biosensors in human bronchial epithelial cell lines (HBE1 and 16HBE) and primary human bronchial epithelial (pHBE) cells, in both submerged and biphasic Air-Liquid Interface (ALI) culture conditions (i.e., differentiated cells). Cells were exposed to pro-inflammatory cytokines relevant to asthma and chronic obstructive pulmonary disease (COPD), and to pharmacological treatments (gefitinib, tocilizumab, hydrocortisone) and metabolic modulators (insulin, 2-deoxyglucose) to probe the airway epithelial mechanisms of SPREADs. Phospho-STAT3 immunofluorescence was used to measure localized inflammatory responses to IL-6. RESULTSPro-inflammatory cytokines significantly increased the frequency of SPREADs. Notably, differentiated pHBE cells display increased SPREAD frequency that coincides with airway epithelial barrier breakdown. SPREADs correlate with IL-6 peptide secretion and localized pSTAT3. Hydrocortisone, inhibitors of receptor signaling, and suppression of metabolic function decreased SPREAD occurrence. CONCLUSIONSPro-inflammatory cytokines modulate SPREADs in human airway epithelial cells via both secreted EGFR and IL6R ligands. SPREADs correlate with changes in epithelial barrier permeability, implying a role for spatiotemporal ERK signaling in barrier homeostasis and dysfunction during inflammation. The involvement of SPREADs in airway inflammation suggests a novel signaling mechanism that could be exploited clinically to supplement corticosteroid treatment for asthma and COPD. Brief SummaryCombining live-cell ERK biosensors with multiple human airway epithelial models, we demonstrate that pro-inflammatory cytokines cause spatiotemporally organized ERK signaling events called "SPREADs", correlating with conditions that disrupt epithelial barrier function. Additionally, common anti-inflammatory treatments tocilizumab, gefitinib, and hydrocortisone suppress cytokine-induced SPREADs. These findings suggest that localized ERK signaling coordinates the innate immune response via spatially restricted cytokine release and regulation of airway barrier permeability.