Sub-cytotoxic Effects of Diesel Exhaust Particles and Woodsmoke in an Organotypic Model of the Human Alveolus
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Epithelial cells have historically been the most researched cell type used to represent the human lung in in vitro models of disease; however, recent technology has increased their utility for predicting adverse human health outcomes. Novel complex and sensitive platforms can evaluate the effects of combustion-derived pollutants, including diesel exhaust particulates (DEP) and wood smoke (WS), that contribute to aerosolized particulate matter (PM) known to be responsible for pre-mature morbidities and co-morbidities. Using Transwell inserts, we developed a multicellular model of the alveolar capillary region recapitulated by human lung epithelial cells (H441s), lung fibroblasts (IMR-90s), and lung microvascular endothelial cells (HULECs). As the first line of defense, epithelial cells are plated on the apical surface of a Transwell insert, with fibroblasts on the basolateral surface, and endothelial cells in the underlying well. Direct exposure to DEP and WS in the apical compartment recapitulates direct epithelial cell exposure in the human lung and allows us to quantify changes in each cell type as a result of the epithelial cell response. Epithelial barrier integrity and viability is maintained up to 24 hours after PM exposure. However, changes to endothelial cell gene and protein expression support the presence of cell signaling changes associated with epithelial cell response in the absence of cell death or translocation of PM through the epithelial barrier. Further investigation reveals significant gene expression changes as early as six hours (DEP) or eight hours (WS) after exposure for genes involved in redox cycling (PRDX1, PRDX6, TXN, GSR) and oxidative stress-associated autophagy (SQSTM1). This was confirmed by significant changes to proteins involved in protection against oxidative stress (HMOX-1), glutathione synthesis (GLCM, GCLC), oxidative stress-associated autophagy (SQSTM1), and control of antioxidant expression (NRF2). Continuing work will elucidate specific drivers of DEP and WS toxicity, assess changes in kinase activation, and reveal the specific secreted factors generated by epithelial cells responsible for endothelial cell response. Models like this are necessary to increase the value of in vitro testing as a means of reducing cost and animal use associated with in vivo studies while maintaining the complexity of the lung microenvironment. This abstract does not reflect EPA policy.