Age, serum 25-hydroxyvitamin D and vitamin D receptor (VDR) expression and function in peripheral blood mononuclear cells

Abstract

The relationship between age, vitamin D status, expression and functionality of the vitamin D receptor (VDR), and key genes in the vitamin D pathway in immune cells is unclear. We enrolled adults 50 to 69 years old (20 subjects) and 70+ (20 subjects) and measured: 1) 25(OH)D levels by liquid chromatography/mass spectrometry; and 2) mRNA expression of VDR, 1α-OHase, 1,25D3-MARRS, TREM-1, cathelicidin, RIG-I, and interferon-β by qRT-PCR. Mean serum 25(OH)D was 30 ± 4 ng/mL and was not associated with age. Baseline expression of VDR, 1α-OHase, 1,25D3-MARRS, TREM-1, and RIG-I also did not differ by age; IFN-β expression, however, was higher in the 70+ year old group. 25(OH)D3- and 1,25(OH)2D3-induced VDR, TREM-1 and cathelicidin expression were similar between age groups, as was LPS-induced expression of VDR and of 1α-OHase. Ligand-induced 1,25D3-MARRS expression was higher in subjects ≥ 70 years. Serum 25(OH)D was inversely associated with LPS-stimulated VDR expression and with baseline or vitamin D-induced TREM-1 expression, adjusting for age, self-rated health, and functional status. In healthy adults ≥ 50 years, the expression and functionality of the VDR, 1α-OHase and key vitamin D pathway genes were not consistently associated with age.

Keywords: 1α-hydroxylase; Gerotarget; PBMC; mRNA expression; vitamin D; vitamin D receptor.

Figure 1. Serum 25(OH)D by age group

The participants were chosen with serum 25(OH)D levels in the sufficient range (mean ± SD, 30.0 ± 4.1. ng/mL) [48]. Serum 25(OH)D was similar for both age groups. The mean of the values within age groups is represented by the horizontal line.

Figure 2. Baseline mRNA expression by age

VDR a., 1α-OHase b., 1,25D₃-MARRS c., TREM-1 d., RIG-I e., IFN-β f. mRNA expression by age group was measured in untreated PBMCs and compared to expression in a calibrator sample (untreated random PBMCs). Gene expression was assessed by RT-qPCR. All assays were done in duplicate and expression was normalized to β-actin. The relative amount of target gene in each sample was estimated using the 2^−ΔΔCT method as described elsewhere [49]. The mean of the values is represented by the horizontal line. Statistical analysis was performed by Student's test (* p <0.05).

Figure 3. Vitamin D-induced mRNA expression by age

The response to vitamin D stimulation by age group was determined by fold change in VDR a., 1aOHase b., 1,25D₃-MARRS c., TREM-1 d. and RIG-I e. mRNA expression after in vitro treatment of PBMCs with active 1,25(OH)₂D₃ or inactive 25(OH)D₃ compared to untreated PBMCs. Cathelicidin f. mRNA expression was measured in vitamin D-treated samples and compared to expression in a calibrator sample (vitamin D treated random PBMCs) due to Cathelicidin mRNA at baseline was below the limit of detection. Gene expression was assessed by RT-qPCR. All assays were done in duplicate and expression was normalized to β-actin. The relative amount of target gene in each sample was estimated using the 2^−ΔΔCT method as described elsewhere [49]. The mean of the values is represented by the horizontal line.u/t: untreated PBMCs; 1,25 VD: PBMCs treated with 1,25(OH)₂D₃; 25VD: PBMCs treated with 25 (OH)D_3. Statistical analysis was performed by ANOVA (* p < 0.05**, p < 0.01, *** p < 0.001).

Figure 4. LPS-stimulated mRNA expression by age

The response to LPS stimulation by age group was determined by fold change in VDR a., 1α-OHase b. and 1,25D₃-MARRS c. mRNA expression after in vitro treatment of PBMCs compared to untreated PBMCs. Gene expression was assessed by RT-qPCR. All assays were done in duplicate and expression was normalized to β-actin. The relative amount of target gene in each sample was estimated using the 2^−ΔΔCT method as described elsewhere [49]. The mean of the values is represented by the horizontal line. u/t: untreated PBMCs; LPS: PBMCs treated with LPS. Statistical analysis was performed by ANOVA (* p < 0.05, **p < 0.01, *** p < 0.001).