Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense

Abstract

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.

FIGURE 1. Primary respiratory epithelial cells are primed to convert inactive vitamin D to its active form

A. Cells were cultured in media for 24 hours, harvested and mRNA measured using quantitative Real-time PCR. At baseline hTBE cells express relatively high levels of the activating 1α-hydroxylase (Cyp27B1) and low levels of the inactivating 24-hydroxylase (Cyp24A1) whereas the opposite is true for the cancer cell line, A549 cells. Graph reflects mean relative expression of hydroxylases compared to HPRT and SEM of 3 or more independent experiments. B. hTBE cells were treated with increasing doses of inactive vitamin D (25D₃) and active vitamin D (1,25D₃) was measured in supernatants 24 hours later. hTBE cells convert the inactive 25D3 to the active 1,25D3. When inactive vitamin D is added to media without cells no active vitamin D is detected. Graph reflects mean 1,25D₃ levels and SEM of 3 or more independent experiments. Students T-test, ** p<0.01. C. hTBE cells were cultured in their regular media or A549 media and A549 cells were cultured in their regular media or exposed to Pronase 0.2% and then cultured in hTBE media and baseline expression of Cyp27B1 measured. No change was seen in expression of Cyp27B1 in either cell type. Graph reflects mean relative expression of Cyp27B1 compared to HPRT and SEM of 3 experiments.

FIGURE 2. Locally activated 25D₃ induces vitamin D driven genes to the same extent as exogenous 1,25D₃

A-C, hTBE cells were treated with either active (10^-7M) or inactive vitamin D (10^-7M) for 24 hours. Cells were harvested and Cyp24A1 (A), cathelicidin (B) and CD14 (C) mRNA was measured using quantitative Real-time PCR. There is a significant induction of vitamin D driven genes in cells exposed to either 1,25D₃ or 25D₃ when compared to control. Graphs reflect mean fold increase from control and SEM of three or more independent experiments. Repeated measures ANOVA with Bonferroni’s test for multiple comparisons. Protein levels were analyzed in cell lysates by Western blot (Cyp24A1 and hCAP-18/LL-37) or in supernatants by dot blot (LL-37) or ELISA (CD14) and a similar trend was seen as for the mRNA. Differences from control; * p<0.05, ** p<0.01 and *** p<0.001.

FIGURE 3. Itraconazole, a chemical inhibitor of 1α-hydroxylase (Cyp27B1), reduces conversion of 25D₃ to 1,25D₃ and attenuates up-regulation of vitamin D regulated genes

A-B, hTBE cells were treated with 25D₃ (10^-7M) for 24 hours with or without pretreatment with itraconazole (10^-6M) for 2 hours. A. 1,25D₃ was measured in supernatants. Cells pretreated with itraconazole are significantly less efficient in converting 25D₃ to 1,25D₃. Graph represents mean 1,25D₃ levels and SEM of 4 independent experiments. B. mRNA levels of vitamin D regulated genes were measured using quantitative Real-time PCR. Pretreatment with itraconazole attenuates vitamin D induced transactivation of Cyp24A1, cathelicidin and CD14. Graphs reflect mean fold increase from control and SEM of 3 or more independent experiments. Students T-test, * p<0.05, ** p<0.01.

FIGURE 4. Chromatin modifications augment the effects of 1,25D₃ and 25D₃ on vitamin D regulated genes

hTBE cells were treated with 1,25D₃ (10^-7M) or 25D₃ (10^-7M) for 24 hours with or without the HDAC inhibitor butyrate (2 mM ) and mRNA levels of vitamin D regulated genes were measured using quantitative Real-time PCR. Butyrate significantly increases vitamin D induced up-regulation of cathelicidin and CD14 regardless of which from of vitamin D is used. Graphs reflect mean fold increase from control and SEM of 5 independent experiments. Repeated measures ANOVA with Bonferroni’s test for multiple comparisons. Differences from control; ** p<0.01 and *** p<0.001.

FIGURE 5. Viral RNA increases both the expression of 1α-hydroxylase and conversion of 25D₃ to 1,25D₃ and synergizes with vitamin D to induce the antimicrobial gene cathelicidin

A-B, hTBE cells were treated with the synthetic dsRNA, poly(I:C) (20 μg/ml), with or without 25D₃ (10^-7M), for 24 hours and cells harvested for RNA. mRNA was measured using quantitative Real-time PCR. 1,25D₃ was measured in supernatants. A. Poly(I:C) treated cells have significantly higher expression of 1α-hydroxylase than control cells and convert more 25D₃ to 1,25D₃. B. Poly(I:C) alone does not induce cathelicidin mRNA unless vitamin D is present and then the two synergize. Vitamin D attenuates the poly(I:C) induced induction of IL-8 mRNA. C-D, hTBE cells were infected with RSV (MOI 1) for 24 hours in the presence or absence of 25D₃ (10^-7M) and harvested for RNA. mRNA was measured using quantitative Real-time PCR. 1,25D₃ was measured in supernatants. C. As with poly(I:C) 1α-hydroxylase mRNA is significantly higher in RSV treated cells than in control cells and RSV infected cells are more efficient in converting 25D₃ to 1,25D₃. D. Different from poly(I:C), RSV alone does induce cathelicidin, but like poly(I:C) vitamin D and the virus synergize to induce cathelicidin. Graphs reflect mean and SEM of 4 or more independent experiments. When comparing two groups a paired students T-test was used. For all other comparisons repeated measures ANOVA with Bonferroni’s test for multiple comparisons was applied. Differences from control; ns is non significant, ** p<0.01 and *** p<0.001.