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Oxidative Stress and Pro-Inflammation Beyond the Barrier: Inhaled Chemical Exposures Alter Stromal and Epithelial Cellular Dynamics within the Airway Microenvironment
Citation:
Faber, S., S. McCullough, AND N. McNabb. Oxidative Stress and Pro-Inflammation Beyond the Barrier: Inhaled Chemical Exposures Alter Stromal and Epithelial Cellular Dynamics within the Airway Microenvironment. 2019 Society of Toxicology Annual Meeting, Baltimore, MD, March 10 - 14, 2019.
Impact/Purpose:
Presentation of the development and use of an organotypic in vitro model of the airway microenvironment to identify novel inhaled toxicant exposure effects and identify underlying molecular mechanisms to inform the development of multi-dimensional adverse outcome pathways.
Description:
Oxidative stress and release of pro-inflammatory mediators are well established signaling events following inhaled chemical exposure; however, our understanding of the distinct cellular and molecular pathways involved in inhalation toxicity remain limited due to studies centered around a single airway cell type (i.e., epithelial cells), and lack of physiologically-relevant studies on diverse cell types beyond the epithelial barrier. Utilizing an in vitro organotypic model that recapitulates the “trans-epithelial” nature of analogous exposures in vivo, we investigated the effect of the model toxicant (diesel exhaust particulates (DEP)) on human bronchial epithelial cell (HBEC) and lung fibroblast (HLF) oxidative stress and pro-inflammatory signaling. We hypothesized that trans-epithelial exposure to DEP drives oxidative stress/pro-inflammatory response through an imbalance of oxidant/antioxidant gene expression and alternative cellular signaling pathway activation within the airway microenvironment. Temporal gene expression analysis demonstrated peak HMOX-1 response at 6 hours in HBEC and HLF, while IL-8 peaked at 4 hours in HBEC and 10 hours in HLF, by which time HMOX-1 had declined substantially. Genes involved in glutathione homeostasis and H2O2 signaling (NQO1, TRX1, PTGS2 and GCLM1) were alternatively regulated in response to DEP exposure. Live-cell imaging with a dual fluorescent biosensor for glutathione oxidation and intracellular H2O2 accumulation (roGFP2/HyPER red) showed that treatment with DEP led to an increase in overall oxidation in both cell types, which was attenuated by pre-treatment with the free radical scavenger, N-acetylcysteine (NAC). Upregulation of antioxidant response and pro-inflammatory genes in our studies suggested a role for key stress-response pathways (MAPK, NF-B and NRF2) in DEP-dependent oxidative stress/pro-inflammatory signaling. The kinetics of MAPK, NF-B and NRF2 pathway activation by DEP exposure were determined via time-course (2-24 hours) immunoblotting and densitometry analysis; confirming DEP-mediated and time-dependent phosphorylation of key cellular targets. This study is the first to characterize the dynamics of oxidative stress/pro-inflammatory signaling within the airway microenvironment following trans-epithelial chemical exposure and provides novel insight for the development of therapeutic and/or preventative interventions to reduce adverse effects of inhaled chemical exposure.