Germ-free C57BL/6 wild-type (WT) mice were bred and maintained at The Pennsylvania State University (University Park, PA, USA) gnotobiotic animal research facility. The GF isolators and mice are routinely monitored to ensure the GF status of the mice. All mice were fed the autoclaved sterile chow routinely fed to maintain the GF colony. In addition, GF mice have enlarged cecums, which is additional assurance of their GF status on necropsy. At the start of the experiments, mice were 5–8 weeks of age. Male and female mice were used as indicated in the figure legends. Mice were orally supplemented with 5 µg vitamin D₃ in corn oil, or vehicle treated with corn oil, three times weekly. Microbial transplantation was done using the cecal contents of WT mice. For some experiments stool from ASF (Taconic Biosciences, Hudson, NY, USA) colonized mice was used as the source of microbes for transplantation. Infection with pure strains of Citrobacter rodentium used strain ICC169 (gift of Gad Frankel, London School of Medicine and Dentistry, London, United Kingdom, nalidixic acid resistant) cultured in Luria-Bertani (EMD Chemicals, Inc., Gibbstown, NJ, USA) exactly as described (26). The monoclonization with C. rodentium was confirmed by doing gram stains of the cecal contents when the mice were sacrificed. All experimental procedures were approved by the Office of Research Protection’s Institutional Animal Care and Use Committee (Pennsylvania State University).

Mice were injected ip with 6 mg/kg LPS (Sigma-Aldrich, St. Louis, MO, USA) in sterile PBS and blood was collected 6 and 12 h later. For FGF23 neutralization, mice were injected ip with 5 mg/kg rat monocolonal anti-FGF23 or rat isotype control (kindly provided by Amgen, Thousand Oaks, CA, USA) three times weekly for 2 weeks, and sacrificed 24 h after the last injection.

Sample preparation was done as described by Kaufmann et al. (27). 100 µL of pooled 13C₃-vitamin D₃ (100 ng/mL), d₃-25D₃ (100 ng/mL), and d₆-24,25D₃ (50 ng/mL) (Isosciences, King of Prussia, PA, USA) internal standard was added to 50 µL of sample. 50 µL of 0.1 M hydrochloric acid, 50 µL 0.2 M zinc sulfate, and 225 µL of 100% methanol were added to precipitate protein as described (27). Organic extraction was done by adding 350 µL n-hexanes, 350 µL MTBE (methyl tertiary butyl ether, Acros Organics, Geel, Belgium) and collecting the upper organic phase. Derivatization was done by redissolving the dried residue in 30 µL 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD, 0.1 mg/mL in ethyl acetate Santa Cruz Biotechnology, Santa Cruz, CA, USA), drying, and the residue was dissolved in 30 µL 50/50 acetonitrile/water. All other LC–MS/MS solvents and reagents were Optima LC–MS grade (Fisher Scientific, Pittsburgh, PA, USA). The limit of detection was 1 ng/mL each of vitamin D, 25D, and 24,25D. Concentrations of each vitamin D metabolite were determined by calculating the ratio of the integrated peak areas of the metabolite and the relevant internal standard, compared with the values obtained from the standard curve.

Samples (5 µL) were separated by reverse phase HPLC using a Prominence 20 UFLCXR system (Shimadzu, Columbia, MD, USA) with a Waters (Milford, MA, USA) BEH Phenyl column (100 mm × 2.1 mm 1.7 µm particle size) and a flow rate of 250 µL/min. Solvents used were HPLC grade water with 0.1% formic acid and HPLC grade acetonitrile with 0.1% formic acid. The initial conditions were 70% water and 30% acetonitrile, increasing to 50% acetonitrile at 10 min, 90% acetonitrile at 12 min where it was held at 90% acetonitrile until 13 min before returning to the initial conditions. The eluate was delivered into a 5600 (QTOF) TripleTOF using a Duospray™ ion source (AB Sciex, Framingham, MA, USA). The capillary voltage was set at 5.5 kV in positive ion mode with a declustering potential of 80 V. The mass spectrometer was operated with a 250 ms TOF scan from 50 to 950 m/z, and 7 100 ms MS/MS product ion scans (m/z 730.5, 733.5, 746.5, 749.5, 762.5, and 768.5) from 50 to 950 per duty cycle using a collision energy of 45 V with a 30 V spread.

1,25D was measured using a chemiluminescent assay or LC–MS. LIASON XL chemiluminescent assay was performed by Dr. Claudia Zierold (detection limit 5 pg/mL, DiaSorin, Stillwater, MN, USA) (28). For measurement by LC–MS, 1,25D was first concentrated according to kit instructions (Immundiagnostick, Bensheim, Germany) and measured as described earlier using authentic standards (d₃-1,25D₃ and 1,25D₃, Isosciences) 1,25D samples (10 µL) were separated as described earlier with a Waters (Milford, MA, USA) BEH Phenyl column (150 mm × 1.0 mm 1.7 μm particle size) and a flow rate of 90 µL/min. The eluate was delivered either into a 5600 QTOF or a 6500 QTRAP as described earlier, with two 100 ms MS/MS product ion scans (m/z 762.5 and 765.5) from 50 to 950 per duty cycle using a collision energy of 37 V. For the QTRAP the capillary voltage was 5.5 kV in positive ion mode with a declustering potential of 80 V and the collision energy was 38 V. LC–MS/MS using the QTRAP was performed by Dr. Rahul Baghla (detection limit 4 pg/mL, Sciex, Redwood City, CA, USA). Chromatograms, ion spectra, structure, and fragmentation of DMEQ-TAD 4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD) adducts of vitamin D, 25D, 24,25D, and 1,25D examples are shown in Figure S1 in Supplementary Material (27).

Parathyroid hormone (1–84) and FGF23 were measured by ELISA, according to manufacturer’s instructions (Immutopics, San Clemente, CA, USA). Limits of detection were 32 pg/mL PTH and 25 pg/mL FGF23. Serum calcium levels were measured by colorimetric assay using QuantiChrom Calcium Assay Kit (BioAssay Systems, Hayward, CA, USA), according to manufacturer’s instructions.

RNA from kidney, liver, and colon were extracted using TRIzol reagent using manufacturer’s instructions. 4 μg/sample RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Promega, Madison, WI, USA). Mouse HPRT, Cyp2R1, cyp27a1, cyp24a1, cyp27b1, vdr, tnf-α, il-1β, il-6, and ifn-γ mRNA were quantified by real-time PCR using the StepOnePlus real-time PCR system (Thermo Fisher Scientific, Rockford, IL, USA) with StepOnePlus software and BioRad SYBR Green Master Mix (Hercules, CA, USA). Gene expression was determined as relative expression compared with a linear curve based on a gel-extracted standard. Values were normalized to hprt and expressed as a fold change over GF. Primer sequences can be found in Table S1 in Supplementary Material.

Distal colon was fixed in 10% formalin, sectioned, and stained with hematoxylin and eosin (Pennsylvania State University Animal Diagnostic Laboratory). Sections were scored blinded by a board-certified laboratory animal veterinarian with training in pathology (Dr. Mary Kennett) and scored on a scale from 0 to 3 for extent of inflammatory cell infiltrates, and from 0 to 4 for severity of inflammatory cell infiltrates. Values of 0 = normal, 1 = mucosal, 2 = mucosal and submucosal, and 3 = mucosal, submucosal, and transmural for extent of inflammatory cell infiltrates. Values of 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked for severity of inflammatory cell infiltration. Scores were added, resulting in a total inflammation score of 7 for each sample.

Statistical analyses were performed using GraphPad Prism software (GraphPad, La Jolla, CA, USA). One-way ANOVA with Tukey’s post hoc and two-way ANOVA with Bonferroni’s post hoc test were used to compare levels of vitamin D metabolites, PTH, and FGF23 in mice. Two-tailed Student’s t-test was used for some vitamin D metabolite, FGF23, and calcium measurements. For all analyses, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.0001.

Germ-free mice were supplemented with vitamin D for 2 and 4 weeks and vitamin D, 25D, and 24,25D levels were measured in the +D GF mice. Vitamin D levels (P = 0.1, Figure 1A) and 24,25D levels (P = 0.4, Figure 1C) were not different after 2 or 4 weeks of +D supplementation of GF mice. 25D levels were significantly lower at 2 weeks of +D supplementation than after 4 weeks of +D supplementation of GF mice (P = 0.02, Figure 1B). GF mice were vehicle treated or +D supplemented for 4 weeks (Figure 1D) as above. After 2 weeks of +D or vehicle treatment the GF mice were bled and then CN, while maintaining the vehicle and +D treatments (Figures 1E–G). The vitamin D metabolites were then measured in the mice before and after CN (Figures 1E–G). Vitamin D levels were not affected by either the vitamin D supplementation or CN (Figure 1E). The levels of 25D increased significantly in both vehicle and +D treated mice after CN (P < 0.0001, Figure 1F). The increase in 25D seen following CN of vehicle treated mice suggests that the microbiota have additional effects on 25D levels outside of any accumulation seen following the 4-week +D treatment (Figures 1B,F). +D mice had higher serum 25D compared with mice receiving vehicle (P = 0.0005), though the combination of +D and CN resulted in the greatest increase in 25D (Figure 1F). There was not a significant interaction between +D and CN on serum 25D levels (P = 0.07, Figure 1F). There was no change in 24,25D levels between vehicle (12 ng/mL) and +D (13 ng/mL) treated GF mice. CN caused an overall significant increase in 24,25D in vehicle and +D mice (P < 0.0001). There was an effect of +D supplementation on serum 24,25D levels (P = 0.009, Figure 1G). There was a significant interaction between +D and CN on 24,25D levels in the serum of mice (P = 0.002, Figure 1G). +D mice had the highest 25D and 24,25D levels following CN and therefore the rest of the experiments were done using +D treated mice.

The experiments shown in Figure 1 used both male and female mice. Sex was also evaluated separately for effects on vitamin D metabolism (Figure 2). +D males had higher vitamin D levels following CN than females (Figure 2B). Overall, female mice produced more 25D than male mice (Figures 2C,D). In addition, female mice had the biggest increase in both 25D and 24,25D following CN (Figures 2C,D). There were significant effects of CN in both +D male and +D female mice for 25D and 24,25D levels (Figures 2D,F). Females produced more 25D and 24,25D than males (Figures 2C–F). Since sex is an important variable affecting the measurements of vitamin D metabolites, experiments used equal numbers of males and females when possible, or only one sex as indicated in each figure.

mRNA for cyp2R1, cyp27A1, cyp24A1, cyp27B1, and the vdr were measured in GF mice and 2-week CN mice. mRNA for cyp2r1 (P = 0.004) and cyp27a1 (P = 0.04) increased in the liver following CN (Figure S2A in Supplementary Material). In the kidney cyp24a1 was significantly inhibited (P = 0.009, Figure S2B in Supplementary Material) and cyp27b1 was significantly increased following CN (P = 0.03, Figure S2B in Supplementary Material). There was no change in kidney expression of vdr mRNA following CN of GF mice (Figure S2B in Supplementary Material). In the colon, CN resulted in an insignificant induction of cyp24a1 (P = 0.06), no effect on cyp27b1, and significant inhibition of vdr expression (Figure S2C in Supplementary Material). CN resulted in increased mRNA in the liver for Cyp2R1 and Cyp27A1 that corresponded with the increase in 25D following CN. In the kidney, expression of mRNA for cyp24a1 went down at 2 weeks post-CN, even though 24,25D levels were higher in the serum of CN mice suggesting post-transcriptional regulation of cyp24a1. Expression of cyp27b1 was higher in the kidney and VDR was higher in the colon with CN.

+D GF mice were dosed and sacrificed at several different time points following CN to determine the kinetics of the increases in 25D and 24,25D. Early following CN, vitamin D levels decreased and remained low 48 h post-CN (P = 0.005, Figure 3A). The reduced vitamin D was accompanied by reduced levels of 25D (P = 0.007, Figure 3B). 24,25D levels went up early post-CN compared with GF mice, but did not reach significance at 48 h post-CN (P = 0.4, Figure 3C). Vitamin D levels increased significantly at 3 days and were higher at 14-day post-CN than in GF female mice (P < 0.0001, Figure 3D). 25D levels in females spiked at 3 days and remained higher (insignificant) than GF mice at 14-day post-CN (P < 0.0001, Figure 3E). 24,25D levels increased in female (P < 0.0001, Figure 3B) and mixed sexes (P = 0.02) following CN (data not shown), and the increase in 24,25D was significantly higher by 7d of CN (Figure 3F). The more frequent collection of blood needed for the kinetic experiments shown in Figure 3 required sacrificing mice at different time points. The data shown in Figures 1 and 2 utilized the same mice before and after CN. This may account for the discrepancies between the effects of CN on vitamin D and 25D measurements between experiments. Nonetheless, the data are consistent for the increased 24,25D levels as a result of CN. The effects of the microbiota on 24,25D levels were evident by d7 of CN.

Conventionalization of GF mice increased 24,25D levels significantly across multiple experiments (Figures 1–3). ASF, which contains eight commensal organisms, was used to colonize GF mice (29, 30). Colonization of +D GF mice with ASF significantly increased vitamin D, 25D, and 24,25D levels in the serum (Figure 4). Vitamin D increased with ASF colonization at 2 weeks (P = 0.0006 Figure 4A). Increased vitamin D levels with CN have been seen in some experiments (Figure 3D) but not all (Figure 1). ASF colonization for 2 weeks increased 25D levels significantly (P < 0.0001, Figure 4B) and increased 24,25D levels significantly (P = 0.003, Figure 4C).

+D GF mice were infected with C. rodentium, a murine enteric pathogen. C. rodentium infection of WT mice is cleared within 4 weeks of colonization, while in GF mice the infection is not cleared although the mice no longer exhibit symptoms [inflammation or diarrhea (31)]. Vitamin D levels went down in GF mice monocolonized with C. rodentium for 2 weeks and then back up after 4 weeks of monocolonization (Figure 5A). The changes in vitamin D over time following monocolonization with C. rodentium were not significant (P = 0.08, Figure 5A). There was a decrease in 25D at 2 weeks post-colonization with C. rodentium that mirrored the decline in vitamin D at this time point (P < 0.0001, Figure 5B). 25D levels increased significantly at 4 weeks of monocolonization compared with the levels in the GF mice before C. rodentium infection (P < 0.0001, Figure 5B). There was an insignificant increase in 24,25D at 2 weeks and a significant increase at 4 weeks of monocolonization with C. rodentium (P < 0.001, Figure 5C). Monocolonization with C. rodentium for 2 weeks decreased 25D levels and had no effect on 24,25D levels. The effects of C. rodentium monocolonization for 4 weeks were to increase 25D and 24,25D levels significantly compared with GF mice before infection (Figure 5).

Germ-free mice were hypocalcemic and had high levels of FGF23 and low levels of 1,25D (Figures 6A–C). PTH levels were not significantly different (data not shown). Calcium levels were low in the serum of mice 3-day post-CN and then were significantly higher at 1-week post-CN (P < 0.0001, Figure 6A). FGF23 levels went down 3-day post-CN and remained lower than GF values at 1- and 2-week post-CN (P = 0.007, Figure 6B). 1,25D levels were extremely low in GF and 3-day post-CN mice and in some experiments remained below or at detection levels (<5 pg/mL, Figure 6C; Figure S3 in Supplementary Material). 1,25D levels increased and were detectable in all mice by 1 week after CN (Figure S3 in Supplementary Material). At 2-week post-CN mice had significantly higher 1,25D levels than GF mice (P = 0.007, Figure 6C). FGF23 blocking antibodies were administered to +D GF mice. Blocking FGF23 in +D GF mice had no effect on vitamin D or 25D levels (Figures 6D,E) but significantly increased 1,25D (P = 0.02, Figure 6F) and 24,25D levels (P = 0.03, Figure 6G). Either CN or blocking FGF23 in GF mice induced 1,25D and 24,25D levels.

The colon length of GF mice decreased at day 3 of CN and did not change significantly thereafter (P = 0.005, Figure 7A). Histopathology sections showed an increase in inflammation in the colon (P = 0.03, Figure 7B). Histology scores were higher at 1 week of CN and remained unchanged at 2 weeks compared with GF histology scores (Figure 7B). mRNA for tnf-α increased significantly after day 1 and was highest at day 2 of CN (P < 0.0001, Figure 7C). By day 3 of CN tnf-α mRNA went down to the same level present in GF mice (Figure 7C). mRNA in the colon for il-1β, and il-6 did not change significantly with CN (data not shown). ifn-γ mRNA was higher but not significantly higher with time post-CN (P = 0.08, Figure 7D). CN resulted in early (day 1–day 2 post-CN) expression of tnf-α mRNA in the colon and shortening of the colon compared with GF mice.

Sterile inflammation was induced in +D GF mice following injection with LPS. There was no significant change in vitamin D after LPS injection (Figure 8A) There was no effect of LPS injection on 25D levels (Figure 8B). 24,25D decreased significantly after LPS injection, with the largest decrease 6 h after injection (P = 0.03, Figure 8C). Sterile inflammation reduced 24,25D levels in GF mice.