IN VITRO MODEL DEVELOPMENT FOR INHALATION TOXICOLOGY RESEARCH AND THEIR APPLICABILITY FOR NOVEL INSIGHTS INTO THE CELLULAR AND MOLECULAR EXPOSURE RESPONSE OF WOOD SMOKE PARTICULATE MATTER EXPOSURE

Wood smoke particulate matter (WSP) exposure causes respiratory diseases including asthma, COPD, and cancer. The number of individuals exposed to WSP is increasing worldwide due to climate change and increased development at wildland-urban interfaces, yet our knowledge of the cellular and molecular mechanisms leading to adverse outcomes is not. Here, we established both a primary cell-based and cell line-based epithelial cell and fibroblast cell coculture model to investigate the cellular and molecular effects of WSP exposure. We established that common dosing paradigms for air liquid interface (ALI) cultures were improper for investigating the effects of WSP exposure, but that a cell line-based model would be appropriate for determining the response to exposure. We further investigated how the liquid application dosing of the ALI model had the potential to bias toxicity testing as in vivo models of inhalation toxicity are being phased out in favor of in vitro alternatives. We identified global transcriptome changes, activation of stress-responsive signaling pathways, the secretion of pro-inflammatory cytokines and growth factors, as well as decreases in epithelial cell barrier functionality. These responses from liquid application dosing of ALI cultures preclude the replacement of in vivo models for their ALI in vitro alternatives, and may contribute toward the discrepancy of our WSP exposure results and those of the established research literature. Using our cell line model, we established that WSP exposure induced a NRF2-mediated response in both directly exposed epithelial cells and trans-epithelial exposed fibroblast cells. We identified a difference in mRNA and protein expression kinetics for NRF2-dependent targets between epithelial cells and fibroblast cells. We also observed a difference in mRNA induction kinetics for pro-inflammatory cytokines in epithelial cells and fibroblasts. Further, when NRF2 was inhibited in fibroblasts, we observed changes in the mRNA induction of pro-inflammatory targets in epithelial cells. We further observed a decrease in the secretion of pro-inflammatory cytokines when fibroblast NRF2 activity was lost, thus indicating a novel form of fibroblast NRF2-mediated intercellular communication. These results highlight the challenges of working with in vitro models for inhalational toxicology research, and the importance of lung fibroblasts in coordinating toxicity for inhaled particulates.