Air-Liquid Interface Re-Submersion Alternatively Regulates Stress-Responsive Signaling, Enhances Cytokine Secretion, and Increases Barrier Permeability in Primary Human Bronchial Epithelial Cells#

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 methodologically challenging test agents (e.g., particles and aerosols) 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 common in vitro toxicity endpoints.  To test our hypothesis, we re-submerged differentiated organotypic pHBEC/lung fibroblast ALI co-cultures in basal growth medium and examined global gene expression, epithelial barrier integrity, and pro-inflammatory cytokine secretion.  Re-submersion resulted in the significant alternative regulation of 4038 and 7499 genes in pHBEC at 6 and 24 hours, respectively, with many of the most dysregulated genes being involved in stress-responsive cell signaling and inflammation.  These transcriptional changes were complemented by significant increases 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 testing that requires further characterization.  Abstract does not reflect EPA policy.

Impact/Purpose

Primary bronchial epithelial cell (pHBEC) cultures differentiated under air-liquid interface (ALI) conditions are the most physiologically relevant in vitro model available for regular use in inhalation toxicology research and testing.  The maintenance of ALI conditions is critical to the differentiation of these cultures into in vitro bronchial epithelial tissues and their ability to represent the corresponding tissue in the human respiratory tract.  Exposures to methodologically challenging test agents (e.g., aerosols and particles) are often conducted by re-submerging the differentiated culture in an aqueous solution (e.g., saline or cell culture medium) containing the desired dose.  Despite the relatively common use of re-submersion dosing of differentiated pHBEC ALI cultures, the effect of re-submersion alone on commonly used toxicity endpoints is poorly understood.  Using an organotypic differentiated pHBEC co-culture ALI tissue model, the study described in this abstract examined the effects of re-submersion (in the absence of any test agent) on common in vitro toxicology endpoints.  Re-submersion resulted in significant effects on global gene expression/transcriptional reprogramming, decreased epithelial barrier integrity, and increased pro-inflammatory cytokine secretion.  The magnitude of the effect of re-submersion on these common endpoints increased over time and was greater than that often observed with many known toxicants thus raising the likelihood of false negative test results due to the masking of test agent effects by those of re-submersion alone.  Alternatively, re-submersion up-regulated several stress-responsive cellular pathways, which could result in false positive test results arising from a “two-hit” or synergistic interaction between the effect of re-submersion and that of a test agent.  Overall, these observations suggest that the effects of re-submersion of differentiated pHBEC ALI cultures alone on these endpoints will confound the interpretation of data collected using submerged dosing for in vitro inhaled chemical testing.  

Citation

Mallek, N., E. Martin, L. Dailey, AND S. McCullough. Air-Liquid Interface Re-Submersion Alternatively Regulates Stress-Responsive Signaling, Enhances Cytokine Secretion, and Increases Barrier Permeability in Primary Human Bronchial Epithelial Cells#. 10th Annual Meeting of the American Society of Cellular and Computational Toxicology (ASCCT), Virtual, NC, October 12 - 14, 2021.