Wood Smoke Particulate Exposure Induces NRF2-mediated Intercellular Signaling Between Fibroblasts and Epithelial Cells in a Lung Coculture Model
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Wood smoke particulate (WSP) exposure causes respiratory disease including asthma, COPD, infections, and cancer. The number of individuals exposed to WSP is increasing due to climate change and increased development at wildland urban interfaces, yet our knowledge of the cellular and molecular mechanisms leading to adverse respiratory outcomes is not. We hypothesized that exposure to WSP would disrupt the normal function of epithelial cells and engender an oxidative stress and inflammatory response in directly exposed epithelial cells and indirectly exposed fibroblasts. We developed an in vitro organotypic model that combines both airway epithelial cells (16HBEs) and airway fibroblasts (IMR90s). We found that the epithelial cell layer displayed a high electrical resistance, low compound permeability, and polarized tight junction proteins, which recapitulates in vivo physiology. However, after 24 hours of exposure to WSP markers of epithelial barrier integrity decrease, indicating that WSP exposure impacts normal epithelial cell function. We then investigated the effects of WSP exposure on the kinetics of gene expression for targets related to the oxidative-stress response and pro-inflammation in both epithelial cells and fibroblasts as RNA-sequencing indicated a large increase in transcript expression related to these responses. We found the early exposure response (2-6 hours) in epithelial cells involves cytokine induction (IL-8, IL-6, IL-1α, and IL-1β), and the early exposure response in fibroblasts involves redox-sensitive targets (HMOX-1, GCLM, SQSTM-1). The late exposure response (12 and 24 hours) involves enzyme induction (NQO-1 and COX-2) in both cell types. These results indicate a differential exposure response wherein directly exposed epithelial cells undergo an early inflammatory response, and indirectly exposed fibroblasts attempt to mitigate redox stress. To discover the molecular events leading to changes in target expression, we then investigated the role of the NRF2, ERK, and P38 signaling pathways, as these are known to activate oxidative-stress and pro-inflammatory responses. We observed a significant increase in both the stabilization of NRF2 protein and the translocation of NRF2 to the nucleus; however, no significant increase in the phosphorylation of ERK1/2 or p38 was observed, indicating that WSP exposure induces a NRF2-mediated response rather than MAPK-mediated. We then investigated if the NRF2-mediated response could relate to intercellular signaling by inhibiting NRF2 activity solely in fibroblasts, as we determined previously that the fibroblasts had an early redox-sensitive response. We found that NRF2 inhibition in fibroblasts attenuated the induction of redox-sensitive targets, and changed the expression of pro-inflammatory cytokines (IL-8, IL-1α) in fibroblasts. The inhibition of NRF2 in fibroblasts also increased the expression of pro-inflammatory cytokines (IL-8, IL-6, IL-1α, IL-1β) without any change in redox-sensitive targets in epithelial cells, suggesting that the fibroblast NRF2-response mediates the WSP exposure response of epithelial cells. We found that WSP exposure disrupts normal epithelial barrier function, and induces a NRF2-mediated response in both epithelial cells and fibroblasts. Further, the NRF2 response in fibroblasts may modulate the epithelial response to WSP, as loss of NRF2 signaling in fibroblasts increases the induction of pro-inflammatory targets in epithelial cells. These results elucidate the complex multicellular response to WSP exposure in an effort to bridge the gap between environmental exposure and adverse respiratory outcomes. Does not reflect EPA policy.