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. Author manuscript; available in PMC: 2025 Oct 13.
Published in final edited form as: Am J Physiol Lung Cell Mol Physiol. 2025 Sep 12;329(4):L514–L523. doi: 10.1152/ajplung.00233.2025

Aerosolized Vitamin D Attenuates Ozone-Induced Inflammation and Transcriptional Responses via Membrane Antioxidant Effects in Human Bronchial Epithelial Cells

Kevin D Schichlein 1,2, Syed Masood 1, Hye-Young H Kim 3, Benjamin J Hawley 2, A Ghosh 2, James M Samet 4, Ned A Porter 3, Gregory J Smith 1,5, Ilona Jaspers 1,2,6
PMCID: PMC12515420  NIHMSID: NIHMS2111766  PMID: 40938902

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 pre-treated with vitamin D aerosols apically or as 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 IL-8, FFAR2, COX-2, and NFKB2. Gene set enrichment analysis (GSEA) indicated that vitamin D reversed ozone-driven pathways related to inflammation, oxidative stress, and immune dysfunction. Additionally, vitamin D pre-treatment 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.

Keywords: Ozone, vitamin D, aerosol, lipid oxidation, epithelial cells

Graphical Abstract

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News & Noteworthy

Vitamin D aerosols have the potential to protect against exposure to ozone and other inhaled oxidants and prevent 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.

Introduction

Ozone is a common respiratory irritant formed through photochemical reactions of nitrogen oxides (NOx) and volatile organic compounds (VOC)(1). Exposure to ozone is associated with increased risk of cardiopulmonary and neurological diseases, cancer, and overall mortality. In the respiratory tract, chronic ozone exposure can exacerbate or lead to pathogenesis of lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma, especially in adolescents, through increased airway inflammation, hyperreactivity, decreased pulmonary function, and susceptibility to microbial infection (1).

Numerous factors such as genetics, age, sex, existing respiratory disease, and dietary deficiencies have been found to modify susceptibility to ozone-induced health effects. Recent transcriptomic studies in rodents have identified upregulation of cholesterol biosynthesis pathway genes in the lung following ozone exposure (2-4). Cholesterol is a major component of airway surface liquid and cell membranes and is an important precursor to hormones demonstrated to modulate ozone responses (i.e. corticosteroids and sex steroids) (5-14). Additionally, cholesterol is susceptible to oxidation by ozone, leading to the formation of oxysterols which play a role in the toxicity of ozone (15-21). Our lab recently published an exploratory study examining the effects of acute ozone exposure on derivatives of cholesterol biosynthesis in the lung and systemically in human subjects (21). We found that ozone exposure resulted in significant alterations in cholesterol precursors and derivatives in plasma and sputum from asthmatic and non-asthmatic volunteers. Notably, the primary circulating form of vitamin D, 25-hydroxyvitamin D, was positively correlated with lung function and sputum uteroglobin and negatively correlated with sputum neutrophils, total cell count, and several cytokines in non-asthmatics. As such, we hypothesized that higher vitamin D levels may provide protection against ozone-induced inflammation and lung function decrements.

Vitamin D supplementation has long been proposed as a treatment for pulmonary disorders including COPD, asthma, influenza, COVID-19, and as a protectant against air pollution exposure (22-27). However, despite established links between circulating vitamin D levels and disease status, studies investigating oral vitamin D supplementation for the treatment of lung disease have produced mixed results (22, 25, 26). A recent study of vitamin D-related genes in the airways of COPD patients found that CYP24A1, the enzyme responsible for inactivating the biologically active form of vitamin D, was highly expressed in lung endothelial cells, suggesting that oral vitamin D may be inactivated before reaching the lung lumen (28). This indicates that oral vitamin D may be ineffective in exerting therapeutic or prophylactic effects in the lungs. To address this, we proposed that inhaled vitamin D may provide a more effective route of administration for protecting against ozone and other lung diseases. To do so, we used a novel in vitro aerosol delivery system to evaluate apical vitamin D treatment in primary human airway epithelial cells, mimicking inhaled vitamin D as a protectant against ozone-induced pathological responses. As a benchmark, we compared the effects of apical aerosol delivery to basolateral addition, which served as a model of oral supplementation. Furthermore, we investigated the underlying membrane antioxidant mechanisms mediating vitamin D’s protective effects on human airway cells.

Methods

Cell Culture

Primary human bronchial epithelial cells (HBECs) were obtained from either the EPA Human Studies Facility or the UNC Marsico Lung Institute Tissue Procurement and Cell Culture Core, following protocols and consenting materials approved by the University of North Carolina School of Medicine Committee on the Protection of the Rights of Human Subjects (#03-1396) approved by the UNC Biomedical IRB and/or by the US Environmental Protection Agency. Cells were cultured in PureCol-coated (Advanced Biomatrix, 5005-100ML) T75 flasks in PneumaCult-Ex Plus Medium (StemCell Technologies, Inc., Cat. No. 05040) supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml; Gibco, Cat. No. 15140-122), and hydrocortisone (96 ng/ml) and passaged at 60-80% confluency. Passage 3 cells were then plated onto 12-well cell culture plates in 12 mm, 0.4-μm permeable supports (CELLTREAT, Cat. No. 05040) coated in human placental type IV collagen (Sigma, Cat. No. C7521-10MG) at >300,000 cells per well. Cells were supplied expansion media in the basolateral and apical compartment until reaching confluence, at which point the apical media was removed and the basolateral media was switched to PneumaCult ALI Growth Medium supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml), hydrocortisone (480 ng/ml; StemCell Technologies, Inc., Cat. No. 07925), and heparin (4 mg/ml; StemCell Technologies, Inc., Cat. No. 07980) to produce an air-liquid interface. Media was replaced 3 times per week, and the apical surface was washed once a week with HBSS + CaCl2, + MgCl2 (HBSS++; Gibco, Cat. No. 14025-092) until the cells exhibited a differentiated, mucociliary phenotype after at least 28 days at ALI. Differentiation and mucociliary phenotype were confirmed through light microscopy evaluation of cilia beating and mucus secretion. Cells were cultured at standard cell culture conditions (37°C, >90% RH, 5% CO2). HBECs were initially screened for ozone responsiveness as characterized by IL-8 secretion (>50% increase over air-exposed controls) (29-31). Donor demographics are provided in Supplementary Table 1.

The bronchial epithelial cell line, 16HBE14o- cells (16HBEs), was utilized for experiments shown in Fig 4B-C. 16HBEs expressing a genetically encoded sensor of glutathione oxidation, reduction-oxidation sensitive green fluorescent protein (roGFP), which have been used to assess cellular oxidation following ozone exposure, were used for the experiment in Fig 4A, and were previously described here (32). Cell lines were negative for Mycoplasma contamination before cryogenic storage and exhibit gene expression characteristic of airway epithelial cells (33). Both 16HBEs and roGFP-16HBEs were cultured in Glutamax MEM (Gibco, Cat. No. 41090036) supplemented with 10% FBS (Sigma-Aldrich, Cat. No. F2442) and penicillin (100 U/ml)/streptomycin (100 ug/ml) until 60-80% confluence in T75 flasks coated with PureCol (30 μg/ml), 0.01% BSA (Sigma-Aldrich, Cat. No. A9647), and 1% human fibronectin (Sigma-Aldrich, Cat. No. F2006) in HBSS++. The cells were then plated on 12- or 24-well permeable supports at over 150,000 cells/cm2 and were supplied complete media in the basolateral and apical compartment until reaching confluence. The cells were then cultured without apical media for at least 24 h prior to use.

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

Figure 4.

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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.

Vitamin D Supplementation

To investigate the utility of vitamin D aerosols as a protectant against ozone exposure, HBECs were treated apically with vitamin D aerosols using our novel On-Plate Aerosol Delivery Array (OPADA; Fig S1). OPADA was used to deliver 10 μL of 10 mM vitamin D in HBSS++ (0.01% EtOH) to the apical surface of each well using an array of four vibrating mesh nebulizers (Tekceleo, Cat. No. P&S T45-M05, ECU Neb x4, HMI P&S MXX) that produce 5-micron particles (Fig 1B). Basolateral pre-treatment was provided in PneumaCult ALI Growth Medium with either vehicle control (0.0001% EtOH) or 100 nM of vitamin D (Fig 1A; Cayman Chemical, Cat. No. 11792). To avoid direct reactions of ozone with vitamin D, apical aerosol pre-treatment was done 24 h before exposure, while basolateral pre-treatment was provided 30 minutes before. Cells were washed once with HBSS++ immediately before both treatments.

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

Figure 1.

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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.

For supplementation of 16HBEs, basolateral pre-treatment was provided in steroid-reduced starving media (2% charcoal-stripped FBS; Alkali Scientific, Cat. No. FB125) with either vehicle control (0.001% ethanol) or 100 nM of vitamin D (Fig 1C). Charcoal-stripped FBS was used to isolate the effects of vitamin D from other steroid hormones and lipophilic compounds. Apical pre-treatment was delivered in HBSS++ with 10 μM of vitamin D or vehicle control (0.1% ethanol). Both apical and basolateral treatments were provided 24 h before ozone exposure.

Ozone Exposure

Ozone exposure of primary HBECs was conducted at the EPA Human Studies Facility at 0.4 ppm for 4 hrs, as described previously (15, 16). Basolateral supernatant, RNA, and apical wash were collected 4 hrs post-exposure and kept at −80°C for long-term storage. Similar to HBECs, ozone exposure of 16HBEs used 0.4 ppm for 4 hrs and basolateral media was collected for IL-8 quantification. All exposures were conducted at ALI.

RT-qPCR and RNA-seq

Cell lysates were processed for isolation of total RNA using the Pure Link RNA Mini Kit (Life Technologies, Cat. No. 12183025) and checked for purity and concentration with an LVIS plate and CLARIOstar Plus microplate reader (34). For targeted gene expression analysis, RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Cat. No. 4374966) and amplified using TaqMan Universal PCR Master Mix (Life Technologies, Cat. No. 4304437) and TaqMan Probes for CXCL8 (Hs00174103_m1), PTGS2 (Hs00153133_m1), NFKB2 (Hs00174517_m1), FFAR2 (Hs00271142_s1), and ACTB (Life Technologies Cat. No. 4331182 and 4326315E) as a housekeeping gene. Changes in gene expression were determined using the ΔΔCt method.

For RNA-sequencing, purified RNA from primary HBECs (n = 4) was sent to GeneWiz (Azenta Life Sciences). Differentially expressed genes were identified using the limma-voom framework on R, and Gene Set Enrichment Analysis (GSEA) was conducted to evaluate altered pathway (35-37). Pathway level changes were evaluated using GSEA with the Molecular Signatures Database (MSigDB) hallmark gene set for changes to biological processes (38-40).

Oxysterol Extraction and Measurement

Immediately after exposure, 16HBEs at ALI were scraped into 500 μL of HBSS++ and butylated hydroxytoluene (BHT) was added to a final concentration of .005% (w/v). The scraped cells was mixed with 10 μL internal standards (Sterols: 300 μM d7-Chol [cholesterol]; 30 mM d6-Lan [lanosterol], d7-dHLan [dihydrolanosterol], d7-7-DHC [7-dehydrocholesterol], d6-Des [desmosterol], d6-7-DHD [7-dehydrodesmosterol], d7-Lath [lathosterol], d6-DHL [dehydrolathosterol]; Oxysterols: 1 μM of d7-α-epChol [5,6-α epoxycholesterol], d7-7ketoChol [7-ketocholesterol], d7-DHCAO [3,5-dihydroxycholestanone], d7-7-OHChol [7-hydroxycholesterol], d7-24-OHChol [24-hydroxycholesterol], d6-25-OHChol [25-hydroxycholesterol], d5-27-OHChol [27-hydroxycholesterol], and 0.28 μM d7-β-epChol [5,6-β epoxycholesterol]). The sterols and oxysterols were extracted by adding 600 μL Folch solution (2:1 = CHCl3: MeOH) and 400 μL 0.9% NaCl. The CHCl3 layer was collected, dried, and derivatized by addition of 100 μL of a fresh made N,N-dimethylglycine (DMG) solution (Per 1mL of reagent: 20 mg 2-methyl-6-nitrobenzoic acid anhydride, 14 mg DMG, 6 mg DMAP, 0.1mL anhydrous Et3N in 0.9 mL anhydrous CHCl3). After 30 min agitation at room temperature, the resulting solution was dried and reconstituted in 100 μL MeOH. Resuspended samples were analyzed via reverse-phase HPLC using a Waters Acquity UPLC system injecting 10 μL onto the column (Agilent Poroshell EC-C18, 10 cm x 2.1 cm, 1.9 μm) with CH3CN: MeOH:H2O 70:25:5 (0.01% formic acid (v), 1mM NH4OAc) mobile phase at a column temperature 40 °C. The flow rate was 0.4 mL for 12 min then ramped to 0.6 mL with a total run for 17 min. A TSQ Quantum Ultra mass spectrometer (ThermoFisher) was used for MS detections in positive ESI mode, and data were acquired and analyzed with Finnigan Xcalibur software package. Full list of selected reaction monitoring (SRM) of sterols and oxysterols were published previously (41). Final sterol and oxysterol numbers were reported as nmol/mg protein concentration. Total protein concentrations were measured via bicinchoninic acid assay (BCA assay) in the initial cell lysates after homogenization.

Live Cell Measurement of Oxidative Stress

Investigation of upstream oxidative events was evaluated through live-cell imaging of 16HBEs as described by us before (32, 42). Pre-treatment was conducted as done previously; however, immediately before exposure, cells were transferred to a black, glass-bottom plate and equilibrated in Locke’s Buffer for 1 h.

For analysis of lipid peroxidation, the cells were then incubated with 10 μM of BODIPY 581/591 C11 (Invitrogen, Cat. No. D3861) in Locke’s Buffer in both the apical and basolateral compartments for an additional h. After, cells were washed twice with HBSS++ and Locke’s Buffer was added to the basolateral compartment prior to analysis. For analysis of glutathione oxidation, roGFP-16HBEs were similarly washed twice before a 2-h incubation in Locke’s Buffer. Ozone exposure was conducted at 1 ppm for 1 h, after which, the cells were immediately analyzed using a CLARIOstar Plus microplate reader using the following parameters: BODIPY 581/591 C11 (Ex/Em 478-20/520-20) or roGFP (Ex/Em 405-15/523-20 and 480-15/523-20). BODIPY 581/591 C11 fluorescence was quantified as percent increase over air-exposed controls while roGFP-16HBEs were normalized to their baseline fluorescence pre-exposure and a positive control (1 mM H2O2) (32).

Data Analysis

Data were analyzed using a matched one-way ANOVA with Bonferroni post hoc test and/or paired two-tailed t-tests. Analyses and bar graphs were conducted and generated using GraphPad Prism 10 (GraphPad Software, San Diego, CA). RNA-seq analysis was conducted in R. Heatmaps, GSEA plots, and multi-dimensional scaling plot were generated using the pheatmap and ggplot2 R packages (43, 44).

Results

Effects of Basolateral Vitamin D Supplementation on Transcriptional Responses to Ozone

To evaluate the protective effects of vitamin D against ozone exposure, well-differentiated HBECs from 4 donors were exposed to a sub-cytotoxic dose of ozone that elicits a robust inflammatory response (Fig S2). Transcriptomic analysis was conducted to investigate the effects of vitamin D on ozone-induced transcriptional responses and to identify potential mechanisms mediating vitamin D’s effects. Through unbiased clustering of differentially expressed genes and dimensional reduction of normalized counts, we found that vitamin D pre-treatment reversed transcriptional changes associated with ozone exposure, clustering with air controls (Fig 2A-B). Ozone-exposed cells exhibited a unique transcriptional profile with upregulation of genes and pathways related to inflammation, oxidative stress, and immune dysfunction (CXCL8, PTGS2, NFKB2, IL1A), and pre-treatment with vitamin D reduced the enrichment of these pathways (Fig 2A-D). Comparison of the air control group to the ozone exposure with vitamin D pre-treatment group produced no significantly enriched pathways, suggesting that vitamin D treatment reverses ozone-induced transcriptional changes in HBECs.

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

Figure 2.

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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.

Protective Effects of Basolateral and Apical Vitamin D on Inflammatory Response

As shown previously in human in vitro and in vivo studies, ozone exposure caused a significant increase in IL-8 secretion compared to air-exposed controls (Fig 3A). The addition of vitamin D pre-treatment was able to reduce the secretion of IL-8 induced by ozone exposure compared to ozone-exposed cells, to levels equivalent to the air-exposed controls. Additionally, vitamin D treatment alone, without ozone exposure, did not induce IL-8 secretion.

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

Figure 3.

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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.

Although basolateral vitamin D provided protection against ozone exposure in this model, HBECs alone do not recapitulate the potential inability of oral and systemic vitamin D to reach the airway epithelium. As such, we decided to investigate vitamin D inhalation as a prophylactic strategy against ozone exposure (28). To do so, well-differentiated HBECs from 3 donors were exposed using the same exposure paradigm following aerosol pre-treatment with vitamin D on the apical surface at 10 μM, an equimolar dose to the basolateral treatment, for 24 h (Fig 3B). Specifically, vitamin D was delivered in an aqueous solution through aerosolization by vibrating mesh nebulizers, depositing 10 μL onto the cells. We first confirmed the ability of aerosolized vitamin D to attenuate ozone-induced IL-8 secretion, as seen with basolateral treatment. Vitamin D aerosol pre-treatment produced a similar attenuation of IL-8, returning the levels to that of the air-exposed group. To confirm that vitamin D treatment via aerosol could similarly attenuate ozone-induced transcriptional responses, we chose several genes found to be differentially expressed by ozone exposure and attenuated with basolateral treatment through our previous RNA-sequencing data. These genes were then analyzed through RT-qPCR in our aerosol-treated samples (Fig 3C). Of the four genes chosen, all exhibited the same patterns of induction and attenuation previously seen with basolateral treatment.

The Effects of Vitamin D on Bronchial Epithelial Oxidative Responses

Given the nearly complete reversal of ozone-induced inflammatory responses and lack of transcriptional changes caused by vitamin D alone, we hypothesized that vitamin D may be acting as a membrane antioxidant, potentially reducing the formation of lipid ozonation products. To determine if vitamin D supplementation could modulate ozone-induced oxidative stress responses in airway cells, we first utilized roGFP-16HBEs to evaluate changes in cellular glutathione redox potential. As shown in prior experiments, ozone led to an increase in glutathione oxidation, which decreased by roughly 30% with both apical and basolateral vitamin D pre-treatment (Fig 4A). For analysis of lipid peroxidation, we utilized a fluorescent probe BODIPY 581/591 C11. As shown previously, ozone produced a robust oxidative response characterized by an increase in lipid peroxidation and glutathione oxidation (32, 42). Pre-treatment with vitamin D, both in the basolateral and apical compartment, was able to reduce these oxidative responses (Fig 4B).

It has previously been shown by our group and others that ozone’s toxicity is mediated through the formation of lipid ozonation products including oxysterols, cholesterol ozonation products. To evaluate the effects of vitamin D on ozone-induced oxysterol and ozonide production, we exposed 16HBEs to ozone at various doses and cell culture conditions. Similar to our previous studies, exposure of 16HBEs increased levels of an ozone-derived oxysterol, β-epoxycholesterol (15, 16). Notably, both basolateral and apical aerosol vitamin D pre-treatment reduced formation of β-epoxycholesterol (Fig 4C).

Discussion

Vitamin D supplementation has been proposed as a treatment for respiratory diseases and infections, and most recently, to protect against unhealthy exposure to air pollution and other inhaled toxicants (45-58). We have previously demonstrated a link between circulating vitamin D levels and pulmonary response to acute ozone exposure (21). Despite these studies linking circulating vitamin D status, alterations in VDR expression, or vitamin D metabolizing enzymes with lung diseases such as COPD, asthma, and cystic fibrosis, studies investigating oral supplementation of vitamin D as a therapeutic intervention have produced inconclusive results (22-28, 59-71). Here, we are expanding our previous study linking increased circulating vitamin D with reduced ozone-induced pulmonary inflammation and a) examined effects of vitamin D on ozone-induced inflammation in airway epithelial cells, b) evaluated whether nebulized apical administration of vitamin D could protect against ozone, and c) determined potential cellular mechanisms mediating the protective effects of vitamin D (21). To do so, we utilized primary human bronchial epithelial cells cultured at an air-liquid interface to model the airway epithelium and evaluated inhaled delivery of vitamin D through aerosolization in conjunction with a well-characterized in vitro model of ozone exposure. Our data show that nebulized apical delivery of vitamin D effectively reduces ozone-induced inflammation and markers of oxidative stress and as such inhaled vitamin D supplementation should be further investigated as a protectant against ozone exposure and may have potential future applications to protect against other air pollutants, oxidant injury, or respiratory disease (21). To the best of our knowledge, this is the first study causally linking vitamin D supplementation as protective against pollutants in human airway cells and the first to investigate vitamin D inhalation as a prophylactic strategy for environmental lung disease.

The respiratory immune response to ozone is characterized by an increase in IL-8 secretion leading to neutrophil chemotaxis (15, 29, 30, 72, 73). As such, IL-8 has been found to be a biomarker of ozone-induced airway inflammation in human exposure studies. Therefore, the in vitro secretion of IL-8 can serve as a marker of the ozone-induced inflammatory response. Similar to previous studies, ozone exposure led to an increase in IL-8 secretion which was attenuated with vitamin D pre-treatment in either the basolateral or apical compartments. Control cells exposed to conditioned and filtered air but treated with vitamin D did not exhibit any changes in gene expression or IL-8 secretion. Additionally, we observed reductions in ozone-induced gene expression with vitamin D pre-treatment to PTGS2/COX2, NFKB2, and CXCL8, all of which play a role in the inflammatory and oxidative stress response. Interestingly, we also observed increases in expression of FFAR2, a G protein-coupled receptor that binds to short-chain fatty acids, which may be linked to breakdown and oxidation of membrane fatty acids. Overall, this leads us to conclude that in this model, vitamin D exhibits protective effects without any unwanted inflammatory responses.

To investigate upstream oxidative responses to ozone, a commonly used bronchial epithelial cell line, 16HBEs, was cultured at an ALI. Our group has previously shown that 16HBEs at ALI produce similar responses to ozone as primary airway cells such as increased inflammatory cytokine secretion and the production of lipid ozonation products such as oxysterols (15, 16, 19). Other studies have also demonstrated that 16HBEs exposed to ozone exhibit increased glutathione oxidation and lipid peroxidation; however, these studies were carried out at sub-confluent, submerged conditions (42). We validated these findings at ALI and showed that vitamin D pre-treatment reduced ozone-induced lipid peroxidation and glutathione oxidation. Additionally, we found that vitamin D attenuated the formation of β-epoxycholesterol, an oxysterol formed through ozonation of cholesterol found in cell membranes and airway surface liquid. Following the cascade mechanism of ozone toxicity theorized by Pryor et al., we propose that vitamin D in the cell membrane protects against ozone exposure by preventing oxysterol formation and the subsequent inflammatory response (74-85). As our group has previously shown that oxysterols mediate ozone-induced responses, decreased oxysterol formation from vitamin D treatment is likely responsible for the attenuated downstream inflammatory response. While further research is needed, we theorize that because vitamin D and cholesterol are structurally similar, due to their shared biosynthesis pathway and common precursor (7-DHC), vitamin D modulates the ability of ozone to oxidize cholesterol. This could potentially be through vitamin D altering the distribution of cholesterol within the membrane (and subsequently reducing exposure of cholesterol to ozone) or being preferentially oxidized by ozone instead of cholesterol.

The majority of research on vitamin D has focused on its biologically active form, 1,25-dihydroxyvitamin D (calcitriol), rather than the primary form of vitamin D (cholecalciferol). Few studies have looked at the non-genomic effects of vitamin D (i.e., not mediated by calcitriol though the vitamin D receptor [VDR]) (86, 87). Non-VDR mediated effects include antioxidant effects (membrane antioxidant and inhibition of lipid oxidation) and altered inflammatory signaling (modulation of lipid raft signaling or formation) (88, 89). The proposed antioxidant properties of vitamin D are innovative and build upon previous research of membrane oxidants/antioxidants (EPA, DHA, vitamin E) in the lung (32, 42, 90, 91). The findings reported here on the potential membrane antioxidant properties of vitamin D have implications for both lung diseases and extra-pulmonary pathologies and present a novel, under-studied mechanism and route of delivery of vitamin D.

Our group has previously shown that 16HBEs and HBECs respond differently to ozone, such as the increased sensitivity of 16HBEs to ozone as seen through increased oxysterol formation (15). This presents a limitation for this study and is most likely due to the presence of different cell types, differences in media composition, and altered metabolism and transport in HBECs compared to 16HBEs. A recent study from Lester et al. highlighted the effect of different HBEC media on responses to ozone, showing that certain media can dampen inflammatory and oxidative responses (92). The differential responses of 16HBEs and HBECs to vitamin D has also been found in previous studies of vitamin D in airway epithelial cells and highlights the difficulty of studying vitamin D in airway epithelial cells. Future work should be aware of the methodological challenges of in vitro vitamin D treatment and past studies using cell lines may need further validation in primary cells or in vivo experiments. Notably, during preliminary experiments, we found that 1,25-dihydroxyvitamin D did not exert any protective effects against ozone exposure at various timepoints before exposure and with both routes of delivery, potentially due to pharmacokinetic differences between calcitriol and cholecalciferol (Fig S3). This negative finding is contrary to previous studies investigating vitamin D treatment with air pollution exposure, although many of these studies were conducted in cell lines or submerged primary cell (45-52, 54, 56, 58, 93).

The use of in vitro models (primary airway cells at ALI and 16HBEs) presents a limitation for the study of vitamin D in the respiratory system. As mentioned previously, oral vitamin D has unfavorable pharmacokinetics for treating the airways, potentially due to increased metabolism in the pulmonary vasculature (28). The cell culture models used in this study are unable to fully recapitulate oral supplementation due to the lack of endothelial and immune cells, hepatic and renal metabolism, and transport by vitamin D binding protein (DBP) and endocytosis via megalin/cubilin (94). Future studies should utilize more complex in vitro (epithelial-endothelial co-culture) or in vivo models and investigate other ozone exposure and vitamin D treatment patterns.

Conclusions

Overall, we demonstrate that inhaled vitamin D has utility as a protectant against exposure to ozone, an oxidant pollutant, through reducing lipid peroxidation and oxidative responses as well as preventing the formation of oxysterols. Inhaled delivery of vitamin D should be further characterized in in vivo models of oxidant pollutant exposure and in the context of other oxidant-mediated lung injuries and environmental lung diseases. Additionally, our work highlights the need to further characterize the utility of antioxidants, vitamins, and nutraceuticals as inhaled therapeutics as a potentially more efficacious alternative to oral supplementation, especially in the context of lung disease and air pollution exposure (95).

Supplementary Material

Supplemental figures and tables are available on the UNC CEMALB Dataverse (https://doi.org/10.15139/S3/CNB8QC).

Acknowledgements

Thank you to Edward R. Pennington from the EPA Human Studies Facility and the UNC MLI Tissue Procurement and Cell Culture Core for proving the cells used in this study. Graphical abstract and Fig 1 were created by K.D.S. in BioRender. Schichlein, K. (2025) https://BioRender.com/7yoc358; Schichlein, K. (2025) https://BioRender.com/lpb4o24.

Grants

This work was supported by funding from the National Institute of Environmental Health Sciences (NIEHS; Grant Nos. 5T32ES007126 and 5R01ES028269) and a pilot project from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)(Grant No. P30DK056350).

Footnotes

Disclosures

No conflicts of interest, financial or otherwise, are declared by the authors.

Data Availability

RNA-seq, R code for RNA-seq analysis, and oxysterol data are available on the CEMALB Dataverse (https://doi.org/10.15139/S3/CNB8QC).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

RNA-seq, R code for RNA-seq analysis, and oxysterol data are available on the CEMALB Dataverse (https://doi.org/10.15139/S3/CNB8QC).

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