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

RATIONALE Spatially 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 the spatial heterogeneity of ERK activity in response to pro-inflammatory ligands, and (2) to assess the effects of pharmacological and metabolic regulators on cytokine-mediated SPREADs. METHODS Live-cell ERK biosensor activity and SPREAD events were measured in human bronchial epithelial cell lines (HBE1 and 16HBE) and primary human bronchial epithelial cells (pHBE), 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. MEASUREMENTS AND MAIN RESULTS Pro-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 SPREADs and local STAT3 activation. CONCLUSIONS Pro-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. One Sentence Summary We demonstrate that proinflammatory cytokines cause spatiotemporally organized ERK signaling events called “SPREADs” in human airway epithelial cells, correlating with conditions that disrupt epithelial barrier function. At a Glance Commentary Scientific Knowledge on the Subject: Airway epithelial cells play a critical role in the innate immune response to pro-inflammatory conditions. This response must coordinate the recruitment of adaptive immune cells and permit their trans-epithelial migration. ERK signaling is required for these events and has been shown to modulate cell fate outcomes via temporally dynamic activity. Pro-inflammatory conditions may therefore rely on spatially and temporally specific ERK signaling to coordinate immune responses. However, the occurrence of spatially localized signaling events in the airway epithelium has not been investigated. What This Study Adds to the Field: Combining live-cell ERK biosensors with multiple human airway epithelial models, we demonstrate that pro-inflammatory cytokines induce spatially localized ERK signaling. In addition, we find that the 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.