Exposure Effects Beyond the Epithelial Barrier: Trans-Epithelial Induction of Oxidative Stress by Diesel Exhaust Particulates in Lung Fibroblasts in an Organotypic Human Airway Model

in vitro bronchial epithelial monoculture models have been pivotal in defining the adverse effects of inhaled toxicant exposures; however, they are only representative of one cellular compartment and may not accurately reflect the effects of exposures on other cell types. Lung fibroblasts exist immediately beneath the bronchial epithelial barrier and play a central role in lung structure and function, as well as disease development and progression. We tested the hypothesis that in vitro exposure of a human bronchial epithelial cell barrier to the model oxidant diesel exhaust particulates caused transepithelial oxidative stress in the underlying lung fibroblasts using a human bronchial epithelial cell and lung fibroblast coculture model. We observed that diesel exhaust particulates caused transepithelial oxidative stress in underlying lung fibroblasts as indicated by intracellular accumulation of the reactive oxygen species hydrogen peroxide, oxidation of the cellular antioxidant glutathione, activation of NRF2, and induction of oxidative stress-responsive genes. Further, targeted antioxidant treatment of lung fibroblasts partially mitigated the oxidative stress response gene expression in adjacent human bronchial epithelial cells during diesel exhaust particulate exposure. This indicates that exposure-induced oxidative stress in the airway extends beyond the bronchial epithelial barrier and that lung fibroblasts are both a target and a mediator of the adverse effects of inhaled chemical exposures despite being separated from the inhaled material by an epithelial barrier. These findings illustrate the value of coculture models and suggest that transepithelial exposure effects should be considered in inhalation toxicology research and testing.

Impact/Purpose

This manuscript describes the development and application of a novel organotypic, low-cost, easily adoptable in vitro airway model with scalable throughput. Current in vitro inhalation toxicity testing approaches are challenged by the limited physiological relevance of monoculture models or high-cost of more in vivo-relevant organotypic models (i.e., MatTek and lung-on-a-chip models). Current testing approaches also focus on airway epithelial cells despite lung fibroblasts accounting for approximately 75% of the total interstitial volume of the adult lung. Fibroblasts are an integral component of the airway microenvironment that reside immediately beneath the airway epithelium and mediate airway structure and function, lung disease, and post-injury lung repair. Despite their abundance and role as a target and/or mediator of the effects of inhaled chemical exposure is poorly understood. The findings presented in this manuscript demonstrate that the effects of inhaled oxidant exposure extend beyond the epithelial barrier and may have more substantial adverse impacts on stromal cells, such as fibroblasts. These "trans-epithelial" effects include the induction of redox imbalance, oxidative stress, activation of the master oxidative stress responsive transcription factor NRF2, and induction of oxidative stress responsive gene expression at the RNA and protein levels. Further, we demonstrate that assessing the effects of chemical exposure by measuring gene transcription alone are not representative of changes in the abundance of corresponding protein products and are likely to confound the interpretation and fidelity of transcript/transcriptomics-focused chemical testing approaches. Here we also describe the use of a novel dual fluorescent biosensor for monitoring intracellular reactive oxygen species (ROS) accumulation and glutathione oxidation (indicator of cellular redox state). This new tool allows for the assessment of cellular redox imbalance and the induction of oxidative stress over time using live cell imaging.

Citation

Faber, S., N. McNabb, P. Ariel, E. Aungst, AND S. McCullough. Exposure Effects Beyond the Epithelial Barrier: Trans-Epithelial Induction of Oxidative Stress by Diesel Exhaust Particulates in Lung Fibroblasts in an Organotypic Human Airway Model. Society of Toxicology, RESTON, VA, 177(1):140-155, (2020). [DOI: 10.1093/toxsci/kfaa085]

DOI: Exposure Effects Beyond the Epithelial Barrier: Trans-Epithelial Induction of Oxidative Stress by Diesel Exhaust Particulates in Lung Fibroblasts in an Organotypic Human Airway Model