Breaking the Barrier: Using Novel In Vitro Organotypic Airway Trans-Epithelial Exposure Model to Study the Depth and Dynamics of Inhaled Chemical Toxicity
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Inhaled chemical testing and research have focused heavily on airway epithelial cells; however, there are a diverse range of other lung cell types within the cellular microenvironment whose role as targets and/or mediators of exposure effects are poorly understood. While one such cell type, the lung fibroblast, occupies a large percentage of the respiratory tract tissue volume and plays a critical role in regulating tissue homeostasis, we know very little about its role as a target and/or mediator of adverse exposure effects. To address this knowledge gap, we developed in vitro organotypic models that recapitulate in vivo exposure of the airway microenvironment by incorporating a functional human bronchial epithelial cell (HBEC) barrier to separate human lung fibroblasts (HLF) from the inhaled exposure material. The model design allows also for the assessment of direct and trans-epithelial effects of toxicant exposure in the HBEC and HLF, respectively, in parallel to provide insight into the roles that these different cell types within the airway microenvironment play in the effects of inhaled chemical exposures. Using this model, we identified the molecular dynamics of direct and trans-epithelial oxidative stress and induction of a pro-inflammatory state including intracellular ROS accumulation, glutathione oxidation, cellular signaling pathway activation, NRF2 signaling, and gene expression at the RNA and protein levels. Trans-epithelial DEP and WS exposures caused oxidative stress as well as oxidative stress-responsive and pro-inflammatory gene expression in HLF. The effects of trans-epithelial DEP and WS exposure on fibroblasts were similar to, or greater than, those observed in adjacent directly exposed HBEC. We also determined that pre-treatment of HLF with antioxidants augments the oxidative stress response in both HLF and HBEC. Our findings indicate that inhaled oxidants have trans-epithelial effects on HLF, suggest that HLF are more susceptible to oxidative injury than HBEC, and demonstrate that HLF mediate the effects of direct inhaled oxidant exposure in adjacent HBEC. These observations illustrate the prospective benefit of improving the physiological relevance of in vitro models and examining the potential for trans-epithelial exposure effects in inhaled chemical research and testing. Does not reflect EPA policy.