Supplemental Files for: Aerosolized Vitamin D Attenuates Ozone-Induced Inflammation and Transcriptional Responses Through Membrane Antioxidant Effects in Human Bronchial Epithelial Cells

Ozone exposure can lead to increased risk of infection, exacerbation or pathogenesis of lung disease, and a number of systemic health effects. As worldwide ambient ozone levels continue to cause adverse health effects, there is a need for prophylactic interventions to mitigate the harmful health effects of ozone. Vitamin D has well-established anti-inflammatory properties and circulating levels have been inversely associated with numerous lung pathologies including ozone-induced airway inflammatory responses and decrements in lung function. Interestingly, studies on oral supplementation to treat lung disease have produced mixed results, potentially due to unfavorable pharmacokinetics in the pulmonary endothelium leading to failure of circulating vitamin D to reach the airways. Here, we tested a novel intervention using vitamin D aerosols to attenuate ozone-induced pathological responses in primary human bronchial epithelial cells. We exposed cells at an air-liquid-interface to ozone following vitamin D pre-treatment and found that both basolateral and apical aerosol pretreatment with vitamin D reduced the ozone-induced secretion of the pro-inflammatory cytokine, IL-8. Similarly, ozone-induced upregulation of inflammation- and oxidative stress-related genes such as IL-8, FFAR2, COX-2, and NFKB2 were downregulated with both basolateral and apical aerosol vitamin D treatment and gene set enrichment analysis (GSEA) revealed that vitamin D treatment reversed ozone-induced increases in inflammation, oxidative stress, and immune dysfunction pathways. In addition, vitamin D pre-treatment attenuated lipid peroxidation, glutathione oxidation, and formation of ozone-derived oxysterols, indicating that vitamin D may act as a membrane antioxidant. These results provide support for the use of vitamin D as a protectant against inhaled oxidants and support the need for further investigation into inhaled vitamin D as a novel route of delivery for treating lung pathologies.