Effects of vitamin D on inflammatory and oxidative stress responses of human bronchial epithelial cells exposed to particulate matter

Abstract

Background: Particulate matter (PM) pollutant exposure, which induces oxidative stress and inflammation, and vitamin D insufficiency, which compromises immune regulation, are detrimental in asthma.

Objectives: Mechanistic cell culture experiments were undertaken to ascertain whether vitamin D abrogates PM-induced inflammatory responses of human bronchial epithelial cells (HBECs) through enhancement of antioxidant pathways.

Methods: Transcriptome analysis, PCR and ELISA were undertaken to delineate markers of inflammation and oxidative stress; with comparison of expression in primary HBECs from healthy and asthmatic donors cultured with reference urban PM in the presence/absence of vitamin D.

Results: Transcriptome analysis identified over 500 genes significantly perturbed by PM-stimulation, including multiple pro-inflammatory cytokines. Vitamin D altered expression of a subset of these PM-induced genes, including suppressing IL6. Addition of vitamin D suppressed PM-stimulated IL-6 production, although to significantly greater extent in healthy versus asthmatic donor cultures. Vitamin D also differentially affected PM-stimulated GM-CSF, with suppression in healthy HBECs and enhancement in asthmatic cultures. Vitamin D increased HBEC expression of the antioxidant pathway gene G6PD, increased the ratio of reduced to oxidised glutathione, and in PM-stimulated cultures decreased the formation of 8-isoprostane. Pre-treatment with vitamin D decreased CXCL8 and further decreased IL-6 production in PM-stimulated cultures, an effect abrogated by inhibition of G6PD with DHEA, supporting a role for this pathway in the anti-inflammatory actions of vitamin D.

Conclusions: In a study using HBECs from 18 donors, vitamin D enhanced HBEC antioxidant responses and modulated the immune response to PM, suggesting that vitamin D may protect the airways from pathological pollution-induced inflammation.

Fig 1. Transcription microarray of 24 hour primary human bronchial epithelial cell (HBEC) cultures stimulated with NIST in the presence/absence of vitamin D.

(A) Volcano plot of the 510 genes with ≥ 1.4 fold differential expression comparing 50μg/ml NIST stimulated to unstimulated 24 hour cultures (n = 4), showing fold-change in gene expression in NIST stimulated cultures in the presence vs absence of 100nM 1,25(OH)₂D3 (horizontal axis) plotted again probability of statistical significance for that fold-change (vertical axis). (B) Plot showing microarray results for fold-change in expression of all cytokine genes upon stimulation of HBECs with 50μg/ml NIST (horizontal axis) and fold-change in gene expression upon addition of 100nM 1,25(OH)₂D3 to NIST-stimulated cultures (vertical axis).

Fig 2. Confirmation by qPCR of the effect of NIST and vitamin D on expression of cytokine genes in HBEC cultures.

(A) Gene mRNA expression at 4 hours relative to 18S and the unstimulated Control condition in HBEC cultures stimulated with 50μg/ml NIST in the presence/absence of 100nM 1,25(OH)₂D3. IL6, n = 7; IL8, n = 7; IL24, n = 6; CFS2, n = 8; CXCL10, n = 6; TGFB2, n = 6. (B) mRNA expression at 24 hours. IL6, n = 9; IL8, n = 7; IL24, n = 6; CFS2, n = 8; CXCL10, n = 6 (one outlying replicate excluded); TGFB2, n = 6. All repeated-measures ANOVAs and post-tests with Bonferroni corrections as shown. VC; vehicle control for NIST. Statistical significance as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Fig 3. Effects of 1,25(OH)₂D3 on production of IL-6, CXCL8 and GM-CSF by NIST stimulated HBEC cultures.

(A) Addition of 100nM 1,25(OH)₂D3 reduced production of IL-6 by primary HBEC cultures stimulated for 24 hours with 50μg/ml NIST, but not CXCL8 or GM-CSF. VC: NIST vehicle control. n = 14–17. (B) Fold-increase in production of IL-6, CXCL8, and GM-CSF above that in unstimulated cultures upon stimulation with 50μg/ml NIST in the presence/absence of 1,25(OH)₂D3, by disease status. Two-way ANOVAs with Bonferroni-corrected post-tests. n = 8–9 healthy, n = 7–8 asthmatic. (C) Percentage suppression by 1,25(OH)₂D3 of cytokine production in PM-stimulated cultures of HBECs from healthy donors compared to asthmatic donors. Un-paired t-tests. n = 8–9 healthy, n = 7–8 asthmatic. Statistical significance as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001, ****, p ≤ 0.0001.

Fig 4. Comparison of expression of vitamin D axis genes between HBECs from healthy and asthmatic donors.

(A) Induction of CYP24A1 by NIST with 100nM 1,25(OH)₂D3, relative to the unstimulated control condition, in 4 hour and 24 hour HBEC cultures. H, Healthy donor HBECs; A, Asthmatic donor HBEC cultures. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 7; 24hr asthmatic, n = 6.(B) Expression of CYP24A1 as measured by qPCR relative to 18S in 4 hour and 24 hour HBEC cultures stimulated with 50μg/ml NIST and 100nM 1,25(OH)₂D3. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 7–8; 24hr asthmatic, n = 6. (C) Induction of CAMP by NIST with 100nM 1,25(OH)₂D3, relative to the unstimulated control condition, in 4 hour and 24 hour HBEC cultures. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 5; 24hr asthmatic, n = 6. (D) Expression of CAMP as measured by qPCR relative to 18S in 4 hour and 24 hour HBEC cultures stimulated with 50μg/ml NIST and 100nM 1,25(OH)₂D3. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 7; 24hr asthmatic, n = 6.

Fig 5. Effect of 25(OH)D3 on antioxidant responses in primary HBEC cultures.

(A) Ratio of reduced (GSH) to oxidised (GSSG) glutathione in 24 hour cultures of HBECs treated with/without 100nM 25(OH)D3; n = 6. (B) Fold-increase in 8-isoprostane levels in culture supernatants from primary HBECs cultured with 50μg/ml NIST with/without 100nM 25(OH)D3 for 24 hours, compared to VC control cultures; ratio paired t-test, n = 8. Statistical significance as follows: *, p ≤ 0.05.

Fig 6. Effect of antioxidant enzyme-inducing sulforaphane on production of IL-6, CXCL8 and GM-CSF by NIST-stimulation HBECs.

Cytokines produced by primary HBECs in submerged cultures for 24 hours stimulated with NIST at 50μg/ml and/or sulforaphane at 3μM. Friedman’s tests with Dunn’s multiple comparisons tests; n = 4. Statistical significance as follows: **, p ≤ 0.01.

Fig 7. Capacity of vitamin D to enhance primary HBEC expression of G6PD.

(A) Expression of G6PD in 4 hour cultures of primary HBECs stimulated with 50μg/ml NIST with/without 100nM 1,25(OH)₂D3 (n = 6). (B) Expression of G6PD in 24 hour cultures of primary HBECs stimulated with 50ug/ml NIST with/without 100nM 1,25(OH)₂D3 (n = 6) or a concentration series of 25(OH)D3, n = 5. (C) Expression of G6PD as measured by qPCR relative to 18S in unstimulated 4 hour and 24 hour HBEC cultures. H, Healthy donor HBECs; A, Asthmatic donor HBEC cultures. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 8; 24hr asthmatic, n = 6. (D) Induction of G6PD by 50μg/ml NIST in the presence / absence of 100nM 1,25(OH)₂D3 in 4 hour and 24 hour cultures of HBECs from healthy and asthmatic donors. 4hr healthy, n = 5; 4hr asthmatic, n = 5; 24hr healthy, n = 6; 24hr asthmatic, n = 6. Statistical significance as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Fig 8. Effect of vitamin D pre-treatment of HBEC cultures on suppression of NIST induced IL-6 and CXCL8.

(A) Percentage suppression of cytokine production in 50μg/ml NIST stimulated 24 hour cultures by concurrent and additional 24 hour pre-treatment with 100nM 25(OH)D3 compared to concurrent only; n = 7–9 (3–4 Healthy, 4–5 Asthmatic). Two-tailed paired t-tests. (B) Percentage suppression of cytokine production by 100nM 25(OH)D3 concurrent and pre-treatment in 50μg/ml NIST stimulated HBEC cultures, in the absence or presence of the G6PD inhibitor DHEA at 100μM; n = 7–9 (2–3 Healthy, 5–6 Asthmatic). Two-tailed paired t-tests. Statistical significance as follows: *, p ≤ 0.05; **, p ≤ 0.01.