Regulation of vitamin D metabolism following disruption of the microbiota using broad spectrum antibiotics

Abstract

Vitamin D, 25hydroxyvitamin D (25D), and 24,25dihydroxyvitamin D (24,25D) were measured before and after broad spectrum antibiotic (Abx) treatment for 2 wks. Abx treatments increased 25D and 24,25D levels suggesting that the microbiota or Abx were altering vitamin D metabolism. Increased 25D, but not 24,25D, following Abx treatments were found to be dependent on toll like receptor signaling. Conversely, the effects of Abx on 24,25D levels required that the vitamin D receptor (VDR) be expressed in tissues outside of the hematopoietic system (kidney) and not the immune system. Fibroblast growth factor (FGF)23 increased following Abx treatment and the effect of Abx treatment on FGF23 (like the effect on 24,25D) was not present in VDR knockout (KO) mice. The Abx mediated increase in 24,25D was due to changes to the endocrine regulation of vitamin D metabolism. Conversely, 25D levels went up with Abx treatment of the VDR KO mice. Host sensing of microbial signals regulates the levels of 25D in the host.

Keywords: Antibiotics; Microbiota; Vitamin D Metabolism; Vitamin D receptor.

Figure 1. Abx treatments induced vitamin D metabolism

A) Experimental design for Abx treatment and Ctl or +D supplementation. B) Vitamin D, 25D, and 24,25D levels from serum of control (Ctl) and vitamin D supplemented (+D) mice, before and after 2 wks of Abx. The results are from one representative of 3 independent experiments. Values are the mean ± SEM of n=4 mice per group. 2-way ANOVA with Bonferroni post-hoc test was used to test significance. C) Water treated D+ mice after 2 and 4 wks of vitamin D supplementation. Values are the mean ± SEM of n=5 mice per group, mixed sex. Paired t-test was used to test significance. *P<0.05, **P<0.01, ***P<0.001

Figure 2. Sex effects of +D supplementation and Abx

A) Vitamin D, B) 25D, and C) 24,25D levels from serum of +D male and female mice, before and after Abx treatment. Values are the mean ± SEM of a total of n= 8 males or n=5 females, and data from 3 independent experiments. 2-way ANOVA with Bonferroni post-hoc test was used to test significance. *P<0.05, **P<0.01, ***P<0.001

Figure 3. The effects of single Abx and direct effects of Abx treatment of GF mice on vitamin D metabolites

Vitamin D, 25D, 24,25D levels from serum of +D mice, before and after 2 wks of A) metronidazole (M) or B) vancomycin (V) or C) +D WT GF mice before and after Abx treatment. Values are the mean ± SEM of n=8 mice per group, 4 males and 4 females (A and B) or (C) n=12 mice per group, n=9 males and n=3 females. C is the pooled data from two independent experiments. Paired t-test was used to test significance. *P<0.05, **P<0.01, ***P<0.001

Figure 4. The VDR is necessary for Abx-induced production of 24,25D

A) Vitamin D, B) 25D and C) 24,25D levels from serum of WT and VDR KO mice. Values are the mean ± SEM of n=11–14 mice per group, 15 males and 10 females, and 2 pooled independent experiments. D) 24,25D levels from serum of WT and Rag KO. Values are the mean ± SEM of n=5 mice per group, 4 males and 6 females. E) 24,25D levels from serum of WT/WT and WT/VDR KO. Values are the mean ± SEM of n=4 recipient male mice per group. 2-way ANOVA with Bonferroni post-hoc test was used to test significance. *P<0.05, **P<0.01, ***P<0.001

Figure 5. WT levels of 24,25D in Cyp27B1 KO mice following Abx treatment

A) 25D and 24,25D levels from Cyp27B1 KO and WT mice. Values are the mean ± SEM of n=12–14 mice per group, 16 males and 10 females combined from 3 independent experiments. B) 25D and 24,25D levels from +D WT and MyD88 KO mice. Values are the mean ± SEM of n=5–6 mice per group, 5 males and 6 females. 2-way ANOVA with Bonferroni post-hoc test was used to test significance. *P<0.05, **P<0.01, ***P<0.001

Figure 6. Abx treatments affect PTH, FGF23, and TNF-α but not serum calcium

A) Ca levels in WT mice after Abx treatment B) PTH and FGF23 levels from WT, Cyp27B1 KO and VDR KO mice. Values are the mean ± SEM of n=5–10 mice per group and data from 2 independent experiments. C) Expression of tnf-α, il-1β, il-6, and ifn-γ in the colon, n=5–8 per group, and data from 2 independent experiments. Student’s t-test was used to test significance. *P<0.05, **P<0.01, ***P<0.001

Figure 7. Abx mediated effects on 25D and 24,25D occur rapidly

A) 25D, 24,25D and FGF23 levels from +D mice after d0, d3, d7, and d14 of Abx treatment. B) Inflammation and cumulative histology scores of distal colons from mice d0, d3, d7, and d14 after Abx treatment. Values are the mean ± SEM of n=3 females per time point. Kruskal-Wallis test with Dunn’s multiple comparisons test was used to test significance.