Air-Liquid Interface Re-Submersion Alternatively Regulates Stress-Responsive Signaling, Enhances Cytokine Secretion, and Increases Barrier Permeability in Primary Human Bronchial Epithelial Cells
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The in vivo functions of the bronchial epithelium can be recapitulated in vitro through the differentiation of primary human bronchial epithelial cells (pHBEC) under air-liquid interface (ALI) culture conditions. pHBEC ALI models have rapidly gained popularity for in vitro chemical testing and research; however, there are practical challenges facing the delivery of many test agents to ALI cultures. These limitations have led to the common practice of suspending test agents in aqueous solution and dosing by re-submersion of differentiated ALI cultures. Given the physiological characteristics that develop during pHBEC ALI differentiation, we hypothesized that re-submersion alone would have a significant effect on in vitro toxicity endpoints. To test our hypothesis, we re-submerged differentiated pHBEC ALI cultures in basal growth medium and examined global gene expression, stress-responsive signaling pathway phosphorylation, pro-inflammatory cytokine secretion, and epithelial barrier integrity. Re-submersion resulted in the significant alternative regulation of 4170 and 10269 genes at 6 and 24 hours, respectively, with many of the most dysregulated genes being involved in stress-responsive cell signaling and inflammation. Western blot analysis indicated a significant increase in the phosphorylation of the stress-responsive signaling pathways ERK1/2, p38, and p65. The transcriptional changes were further complemented by significant increases in the secretion of the pro-inflammatory cytokines IL-8, IL-6, IL-1β, and TNFα. We also observed a progressive decrease in trans-epithelial electrical resistance and increase in small molecule permeability. Cumulatively, our findings indicate that the use of re-submersion approaches to test agent dosing in ALI cultures is likely to be a substantial confounding factor in chemical toxicity testing that requires further characterization. Does not reflect EPA policy.