Abstract
ABSTRACTRATIONALESpatially 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.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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