Vitamin D 3 diets

Six-week-old C57Bl/6 female mice were fed diets with high (D++), moderate (D+) or no (D−) vitamin D 3 for four weeks. At ten weeks of age, the mice in the three groups were not significantly different in weight (data not shown). D++ mice had significantly higher serum 25(OH)D 3 compared to D+ mice which was in turn higher than D− (Table 1). The serum concentration of activated vitamin D, 1,25(OH) 2 D 3 , was not significantly different between the D++ and D+ groups. As previously observed29, 1,25(OH) 2 D concentrations in the D− mice were lower than in the other groups, though this did not reach statistical significance (p = 0.087). There was no difference in serum calcium levels between the three groups.

Table 1 Serum 25 (OH)D, 1,25(OH) 2 D and calcium levels after 5 weeks on vitamin D diets. Full size table

Vitamin D and DSS-induced colitis

Body weight loss and liquid stools were observed between 2–7 days after commencing the DSS treatment. All DSS-treated mice lost weight compared to pre-DSS measures. The peak percentage weight loss occurred at day 7 (Fig. 2A). At day 6 and 7, the D++ mice lost significantly more weight than D+ mice (p < 0.001), and at day 7 and 8, D− mice lost significantly more weight than D+ mice (p < 0.01) (Fig. 2A,B).

Figure 2 Outcomes of colitis. 6-week old female C57Bl/6 mice were established on diets with high (D++), moderate (D+) or no (D−) vitamin D3, before being treated with DSS for 6 days. Mice were regularly weighed and underwent colonoscopy procedures at regular intervals. (A) The percentage weight loss from baseline to day 10 post-DSS treatment. Comparisons are made to group D+ as the reference group. (B) Weight loss at day 7. (C) Endoscopic severity over time measured by murine endoscopic index of severity (MEICS). Comparisons are made to group D+ as the reference group. n = 35/group for day 6 and 7 assessments, n = 10/group day 14, n = 5/group for days 21 and 35. (D) Serum albumin at day 7, n = 5–10/group. (E) Colonic TNF-α gene expression after 6 days DSS treatment, fold change using the 2−ΔΔT method with TATA-box-binding protein as housekeeping gene, n = 4–5/group. Solid bars for control mice, open bars for DSS mice. Data are shown as mean ± SEM, from two experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Full size image

At the day 6 colonoscopy, control mice in all groups had solid stool, preserved mucosal vascularity, normal colonic translucency with a mean MEICS of 0.6 ± 0.17 (n = 23, range 0–3), consistent with no colitis (Fig. 2C). DSS-treated mice demonstrated endoscopic inflammation with loose stools, loss of intestinal wall translucency and mucosal bleeding with a mean MEICS 4.8 ± 0.2 (n = 102, range 0–10). This was significantly greater than control mice (p < 0.001, 95% CI 3.6–4.7).

At day 6, MEICS was significantly greater in D++ (6.3 ± 0.30, n = 33) than in the D+ (4.1 ± 0.30, n = 34, p < 0.001) and D− (3.9 ± 0.33, n = 35, p < 0.001) groups (Fig. 2C). There was no difference in colitis severity observed in mice from the D+ and D− groups (p = 0.65). At day 14, a higher MEICS was observed in the D++ group compared to the D+ group (p < 0.01), but this difference resolved by day 21 and 35 as recovery was almost complete (Fig. 2C).

The histological grading of colitis at the distal colon on day 14 was greater in mice from the D++ (8.22 ± 2.53, n = 9) than D+ (1.42 ± 0.57, n = 7, p < 0.05) or D− (1.43 ± 0.57, n = 8, p < 0.05) groups. At Day 7 there was a trend for higher inflammation in D++ and D− compared to D+ mice though this did not reach statistical significance (p = 0.25) (Supplementary Fig. 1A,B). Among all groups, there was positive correlation between MEICS and day 7 weight loss (r = 0.60, n = 253, p < 0.001), day 7 histological score (r = 0.51, n = 86, p < 0.001) and day 14 histological score (r = 0.59, n = 56, p < 0.001) (data not shown).

At day 7, the mean serum albumin was less in all groups with colitis (Fig. 2D) than corresponding controls. Among DSS-treated mice, the mean albumin level was significantly lower among the D++, compared to the D+ (p < 0.05) and D− (p < 0.05) group, consistent with a worse colitis seen in the D++ group.

Gene expression of TNF-α in colon tissue by RT-PCR at day 7, was increased in all DSS-treated groups compared to controls. This was again greatest in the D++ group compared to D− (6.5 ± 3.1 vs 2.2 ± 0.36 fold, p < 0.05) with a trend to be greater than that measured in the D+ mice (4.1 ± 0.11, p = 0.08) (Fig. 2D).

Serum cytokines TNF-α, IFN-γ, IL-10, IL-6, IL-12p40 and IL-1β at day 7 were greater among all DSS mice compared to control mice (p < 0.05 for all cytokines, data not shown). When comparisons are stratified by vitamin D groups, the DSS group had higher levels than controls though this was not always statistically significant (Supplementary Fig. 2). IL-12p40 was highest among D++ compared to D− groups (p < 0.01), but similar changes were not seen with other cytokines (Supplementary Fig. 2).

25(OH)D, 1,25(OH) 2 D and VDBP concentrations

At day 7, serum 25(OH)D reduced by greater than 60% among DSS mice from the D++ group (Fig. 3A). This difference remained at day 35 (p = 0.053, n = 5). At day 7 a similar but smaller reduction in serum 25(OH)D levels was observed in the D+ group (Fig. 3B). Differences were not seen at the later time points. In mice from the D− group, given that 25(OH)D levels were already low at baseline, no reduction in 25(OH)D was detectable (Fig. 3C).

Figure 3 Vitamin D and vitamin D binding protein levels. Serum concentrations of 25(OH)D 3 at day 7, 14 and 35 among DSS-treated mice and controls, (A) D++, (B) D+, (C) D−, n = 7–8/group at day 7 and day 14, n = 5/group at day 35. (D) 1,25(OH) 2 D and (E). Vitamin D binding protein concentrations in serum at day 7, n = 3–5/group. Solid bars for control mice, open bars for DSS mice. Values are expressed as mean ± SEM, from at least two experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Full size image

As the large decrease in 25(OH)D may have been due to an increase in its conversion to 1,25(OH) 2 D, changes in the levels of 1,25(OH) 2 D were investigated. A significant decrease at day 7 in 1,25(OH) 2 D concentrations (relative to control mice), was detected in both the D++ and D+ groups suggesting that increased conversion was not the cause of 25(OH)D reductions (p < 0.001) (Fig. 3D).

As 25(OH)D, and to a lesser extent 1,25(OH) 2 D, are mostly bound to the VDBP, we questioned if the large decrease in both these vitamin D metabolites was due to a loss of VDBP. As described previously (Fig. 2D), serum albumin dropped with colitis. Surprisingly, the VDBP levels, measured by ELISA, increased with colitis in all groups and this was statistically significant in the D++ and D− groups (Fig. 3E). The increase in VDBP with DSS colitis was also seen when VDBP was measured by radial immunodiffusion, though this was significant among the D+ and D− group and there was a trend to significance among the D++ group (p = 0.076) (Supplementary Fig. 1C). These data suggest that the induction of colitis increases circulating VDBP levels.

Kidney CYP24A1 gene expression in DSS colitis

In an attempt to explain the reduced 25(OH)D and 1,25(OH) 2 D at day 7 post-DSS treatment, changes in the level of expression of mRNA of enzymes involved in vitamin D metabolism were explored. Neither liver CYP2R1 nor kidney CYP27B1 mRNA levels changed significantly with the induction of colitis (Fig. 4A,B). There was 5.5 ± 1.3 fold more CYP24A1 mRNA in the kidneys of DSS-treated mice on D+ diets compared to their corresponding control group (p < 0.01) (Fig. 4C). Similarly, kidney CYP24A1 mRNA was expressed 4.3 ± 0.6 fold more among DSS-treated mice on D− diets compared to corresponding controls (p < 0.001). Kidney CYP24A1 was up-regulated 4.5 ± 0.9 fold among D++ controls compared to D+ controls (p < 0.05) as an appropriate homeostatic mechanism, but there was no further increase with the induction of colitis. Thus, increased kidney metabolism may help to explain the reduced 25(OH)D and 1,25(OH) 2 D at day 7 in the D+ group, though a yet to be identified mechanism must exist to explain the drop in the D++ group.

Figure 4 Kidney Cyp24A1 gene expression is upregulated with DSS colitis. Female C57Bl/6 mice were established on three vitamin D diets for 4 weeks before treatment with DSS. On day 7 mice were sacrificed with livers and kidneys harvested to determine (A) Liver Cyp2R1, (B) Kidney Cyp 27B1, and (C). Kidney Cyp24A1 gene expression. mRNA gene expression by qtPCR was calculated using the 2−ΔΔCT method with eEF1α as the housekeeping gene, n = 5–10/group. Solid bars for control mice, open bars for DSS mice. Values are expressed as mean ± SEM, from at least two experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Full size image

UV radiation-induced 25(OH)D and colitis

The reduced circulating levels of 25(OH)D and 1,25(OH) 2 D observed in mice with DSS-induced colitis could be caused by decreased intestinal absorption of vitamin D. If so, then 25(OH)D derived from skin exposed to UVB radiation should not fall with inflammation. To test this, mice fed vitamin D-deficient diets for 4 weeks were treated with daily UV radiation (1 kJ/m2) for 4 days followed by biweekly UV (1 kJ/m2) for the remainder of the experiment (D−UV+). After the 4 days of UV pretreatment, mice were treated with DSS for a further 6 days.

After 4 doses of UV irradiation, serum levels of 25(OH)D in mice from the D−UV+ treatment was 58.0 ± 2.49 nmol/L compared to 4.8 ± 0.15 nmol/L among D− mice without UV treatment (D−UV−) (n = 4/group, p < 0.001), Fig. 5A. The 25(OH)D concentrations among D−UV+ mice were similar to the D+ without UV treatment (D+UV−).

Figure 5 Serum levels of 25(OH)D 3 in mice treated with and without UV radiation. The shaved dorsal surfaces of female C57Bl/6 mice on vitamin D deficient diets (D−) were irradiated with 1 kJ/m2 ultraviolet radiation daily for 4 days before undergoing DSS treatment. (A) 25(OH)D 3 levels 24 h after four doses of UVB irradiation. (B) 25(OH)D levels after 6 days of DSS treatment in a separate group of mice. Solid bars control mice, open bars DSS mice. Values are expressed as mean ± SEM, from two experiments. n = 4/group day 0 and n = 7–8/group day 7. **P < 0.01, ***P < 0.001. Full size image

After 6 days of DSS treatment, there was no significant difference in endoscopic severity of colitis between vitamin D-deficient mice (D−) exposed (UV+) or not exposed (UV−) to UV radiation (MEICS 4.3 ± 0.45 vs 3.9 ± 0.33, n = 35/gp, p = 0.42), nor was there a difference compared to D+UV− mice (MEICS 4.3 ± 0.45 vs 4.12 ± 0.45, n = 35/group, p = 0.64), Supplementary Fig. 1D. By day 7, the 25(OH)D (Fig. 5B) and 1,25(OH) 2 D concentrations (not shown) were significantly lower among UV-irradiated vitamin D-deficient, DSS-treated mice as compared to corresponding controls. Thus, these data suggest that the drop in circulating 25(OH)D in mice where vitamin D is acquired only through irradiation of the skin, cannot be due to malabsorption.

Effect of vitamin D on faecal microbiota

Microbiota analysis was performed on 42 faecal samples, comprised of 5 samples from each of the vitamin D groups among controls and DSS mice at day 7 and 4 samples per group among controls at day 35. One control mouse was considered an outlier and excluded from further analyses (Supplementary Fig. 3A).

Effect of Vitamin D on faecal microbiota from control, non-DSS mice

There were no differences seen in α-diversity as measured by species richness, evenness or Shannon’s diversity in day 7 samples collected from plain water-treated control mice, and the result was reproducible for day 35 samples (Fig. 6A, Supplementary Fig. 3B,C). Similarly, no significant differences in β-diversity were noted between day 7 and day 35 samples from control mice (data not shown). Further analysis was carried out only on day 7 samples.

Figure 6 Microbial composition of faecal samples from control mice. Faecal pellets were collected control mice from each of the vitamin D dietary groups (D++, D+ and D−). (A) Comparison of day 7 and day 35 species richness for samples from control mice (measured by chao1). (B) Day 7 Shannon’s diversity (H′). (C) Day 7, Relative abundance (%) of OTUs that correlated with serum vitamin D levels among controls using distance based linear modeling (DistLM) analysis. P-values calculated by PERMANOVA on Euclidean distance resemblance matrices generated from square root transformed relative abundance from each OTUs. n = 5/group for day 7 analyses, n = 4/group at day 35. *P < 0.05. Full size image

Comparisons between vitamin D groups found increasing vitamin D doses did not affect species richness as measured by chao1 among control mice (Fig. 6A), but it did reduce Shannon’s diversity between D++ controls compared to D− controls (Fig. 6B). No significant difference was noted between D− and D+ control groups; however, PERMANOVA analysis, a measure of global β-diversity, confirmed significant differences between D− and D++ controls (p = 0.012, t = 1.68, Permutations = 126) and D+ and D++ controls (P = 0.01, t = 1.76, Permutations = 126).

To examine the effect of vitamin D grouping on individual taxa, linear discriminant analysis (LEfSe) was performed. Forty microbial taxa at all taxonomic levels were found to be significantly different between the three vitamin D groups, of which 37 showed strong associations (linear discriminant analysis score > 3) (Supplementary Table Ia).

To determine the effect of measured serum 25(OH)D 3 levels on individual taxa, correlation analysis was measured using distance based linear modelling (DistLM) analysis between serum 25(OH)D3 levels (Euclidean distance resemblance matrix) and relative abundances of microbial taxa. This identified a significant correlation with four taxa (>0.1% average relative abundance) which included: Paulidibacter|OTU46 (Pseudo-F: 4.6, P = 0.04, Df: 26); Bacteroidales|S24-7|OTU58 (Pseudo-F: 6.7, P = 0.02, Df: 26); Sutterella|OTU174 (Pseudo-F: 5.1, P = 0.038, Df: 26); and Coprococcus|OTU118 (Pseudo-F: 4.8, P = 0.02, Df: 26). To further inform our 25(OH)D3 correlation analyses and establish the response of these four taxa to vitamin D intake, the relative abundance of these four taxa across each vitamin D diet group were plotted (Fig. 6C). The relative abundance of Paulidibacter|OTU46, Bacteroidales|S24-7|OTU58, and Sutterella|OTU174 increased with vitamin D intake, while Coprococcus|OTU118 decreased.

Effect of DSS colitis on faecal microbiota

Treatment with DSS reduced the number of operational taxonomic units (OTUs) within samples analysed at day 7 from the D− (P = 0.09) and D+ (P = 0.04) but not D++ group relative to D− controls (not shown), but there was no significant difference between the DSS groups (Fig. 7A). DSS did not affect other measures of α-diversity, in particular species evenness and Shannon’s diversity (Fig. 7B, Supplementary Fig. 3D). However, DSS had a significant impact on overall microbial composition (β-diversity) at day 7 (Fig. 7C). Further, 111 microbial taxa at all taxonomic levels were found to be differentially abundant between controls and DSS mice using LEfSe analysis (Supplementary Table Ib). There was enrichment with DSS of disease-associated Proteobacteria and a reduction in taxa belonging to Firmicutes.

Figure 7 Microbial composition of faecal samples at Day 7 from DSS-treated mice. (A) Species richness (measured by chao1), n = 5/group. (B) Shannon’s diversity (H′), n = 5/group. (C) Non-metric multidimensional scaling (NMDS) plot of the Bray-Curtis resemblance matrix following square-root transformation of relative abundance data showing the impact of DSS on the overall microbial composition, confirmed by pair-wise PERMANOVA (Control vs DSS): t = 3.34, p = 0.01, permutations = 999, n = 15/group (with and without nesting for vitamin D subgrouping). (D) Relative abundance (%) of Sutterella OTU174 between control and DSS mice (LDA 4.27, P < 0.0001), p-value derived from Linear discriminant analysis effect size (LEfSe), n = 15/group. (E) NMDS plot of the Bray-Curtis resemblance matrix following square-root transformation of relative abundance data demonstrating a significant shift of the control D++ microbial composition (green) towards that of the DSS group, n = 5/group. ***P < 0.001. Full size image

Notably, Sutterella OTU174 increased in relative abundance in DSS mice as compared to controls (LDA score: 4.27, p < 0.0001) (Fig. 7D, Supplementary Table Ia). This is relevant given similar rise of Sutterella in non-DSS treated controls from the D++ group, Fig. 6C, suggesting a shift in faecal microbiome of D++ controls to that of DSS mice. Further, when examining the overall microbiome composition there is a clear shift for D++ mice towards that of DSS mice (Fig. 7E).