Aerosolized vitamin D attenuates ozone-induced inflammation and transcriptional responses via membrane antioxidant effects in human bronchial epithelial cells

Abstract

Ozone exposure increases the risk of infection, worsens lung diseases, and causes systemic health issues. As ambient ozone levels continue to rise globally, effective interventions are needed to reduce these harmful effects. Vitamin D, known for its anti-inflammatory properties, has been inversely linked to various lung conditions, including ozone-induced airway inflammation and reduced lung function. However, oral vitamin D supplementation has shown inconsistent results, possibly due to poor delivery to lung tissues. This study explores a novel approach using vitamin D aerosols to counter ozone-induced damage in primary human bronchial epithelial cells. Cells were pretreated with vitamin D aerosols apically or as a bulk addition basolaterally before ozone exposure at the air-liquid interface. Both treatment routes significantly reduced the ozone-induced secretion of the inflammatory cytokine IL-8. Furthermore, vitamin D suppressed the ozone-induced expression of inflammation- and oxidative stress-related genes, including CXCL8, FFAR2, PTGS2 (COX-2), and NFKB2. Gene set enrichment analysis indicated that vitamin D reversed ozone-driven pathways related to inflammation, oxidative stress, and immune dysfunction. In addition, vitamin D pretreatment reduced lipid peroxidation, glutathione oxidation, and formation of ozone-derived oxysterols, suggesting a membrane antioxidant effect. These findings support vitamin D's potential as a protective agent against inhaled oxidants and highlight inhaled delivery as a promising therapeutic strategy for treating lung diseases.NEW & NOTEWORTHY Vitamin D aerosols have the potential to protect against exposure to ozone and other inhaled oxidants and prevent the development and exacerbation of lung disease. Here, we show that aerosolized vitamin D treatment decreased ozone-induced oxidative stress and inflammatory responses, as well as decreased production of an oxysterol, β-epoxycholesterol, indicating vitamin D may act as a membrane antioxidant in the airway epithelium.

Keywords: aerosol; epithelial cells; lipid oxidation; ozone; vitamin D.

Figure 1.. Experimental design of ozone exposure and vitamin D treatments.

A) For investigation of basolateral pre-treatment, HBECs were pre-treated with vitamin D 30 minutes before ozone exposure in the basolateral media, followed by transcriptomic and IL-8 secretion analyses. B) For investigation of aerosolized pre-treatment, HBECs were pre-treated with vitamin D 24 h before ozone exposure through apical delivery, followed by confirmatory RT-qPCR and IL-8 secretion analyses. C) Mechanistic investigations were conducted in 16HBEs or roGFP-16HBEs at ALI which were pre-treated either apically via aerosol or in the basolateral media 24 hrs prior to ozone exposure.

Figure 2.. Ozone-induced transcriptional responses are reversed with vitamin D treatment.

HBECs were pre-treated with vitamin D 30 minutes before ozone exposure in the basolateral media, then analyzed using RNA-sequencing. A) Heatmap of normalized counts of significantly differentially expressed genes from ozone compared to air control. B) Multi-dimensional scaling plot of normalized counts. C-D) Gene Set Enrichment Analysis (GSEA) using the Hallmarks gene sets. Significance was determined by a false discovery rate (FDR) less than 0.05. A negative normalized enrichment score (NES) indicates downregulation by treatment/exposure.

Figure 3.. Ozone-induced IL-8 secretion is attenuated with basolateral and aerosolized vitamin D treatment.

Primary HBECs were pre-treated with A) basolateral vitamin D 30 minutes before ozone exposure, or B) aerosolized vitamin D 24 h before ozone exposure and analyzed for IL-8 secretion. C) Gene expression was analyzed the apical aerosol pre-treated HBECs. Genes were chosen based on the RNA-sequencing results from Figure 2 which exhibited differential expression from ozone and reversal with basolateral vitamin D pre-treatment. Mean ± SD (n=3-4 donors). Matched one way ANOVA test with Bonferroni post hoc test.

Figure 4.. Basolateral and aerosolized vitamin D reduce ozone-induced oxidative response.

16HBEs or roGFP-16HBEs were pre-treated with vitamin D 24 h before ozone exposure and were analyzed immediately after for A) glutathione oxidation, B) lipid peroxidation, and C) formation of β-epoxycholesterol. Mean ± SD (n=3 passages). Matched one way ANOVA test with Bonferroni post hoc test.