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. Author manuscript; available in PMC: 2022 Jun 2.
Published in final edited form as: J Cell Physiol. 2021 Jun 30;236(12):8148–8159. doi: 10.1002/jcp.30486

1,25-Dihydroxyvitamin D3 and dietary vitamin D reduce inflammation in mice lacking intestinal epithelial cell Rab11a

Sayantani Goswami 1, Juan Flores 1, Iyshwarya Balasubramanian 1, Sheila Bandyopadhyay 1, Ivor Joseph 1, Jared Bianchi-Smak 1, Puneet Dhawan 2, Derya M Mücahit 1, Shiyan Yu 1, Sylvia Christakos 2, Nan Gao 1
PMCID: PMC9161497  NIHMSID: NIHMS1807517  PMID: 34192357

Abstract

A number of studies have examined the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on intestinal inflammation driven by immune cells, while little information is currently available about its impact on inflammation caused by intestinal epithelial cell (IEC) defects. Mice lacking IEC-specific Rab11a a recycling endosome small GTPase resulted in increased epithelial cell production of inflammatory cytokines, notably IL-6 and early onset of enteritis. To determine whether vitamin D supplementation may benefit hosts with epithelial cell-originated mucosal inflammation, we evaluated in vivo effects of injected 1,25(OH)2D3 or dietary supplement of a high dose of vitamin D on the gut phenotypes of IEC-specific Rab11a knockout mice (Rab11aΔIEC). 1,25(OH)2D3 administered at 25 ng, two doses per mouse, by intraperitoneal injection, reduced inflammatory cytokine production in knockout mice compared to vehicle-injected mice. Remarkably, feeding mice with dietary vitamin D supplementation at 20,000 IU/kg spanning fetal and postnatal developmental stages led to improved bodyweights, reduced immune cell infiltration, and decreased inflammatory cytokines. We found that these vitamin D effects were accompanied by decreased NF-κB (p65) in the knockout intestinal epithelia, reduced tissue-resident macrophages, and partial restoration of epithelial morphology. Our study suggests that dietary vitamin D supplementation may prevent and limit intestinal inflammation in hosts with high susceptibility to chronic inflammation.

Keywords: chemokines, cytokines, intestinal inflammation, NF-κB-p65, Rab11a, vitamin D

1 ∣. INTRODUCTION

Chronic inflammation of the intestine can be caused by multiple environmental factors interacting with the genetic makeup of individuals (Benson et al., 2010). Epidemiological studies have indicated an association between vitamin D deficiency and increased risk of chronic intestinal inflammation (Narula & Marshall, 2012). Vitamin D undergoes two successive hydroxylations (by 25-hydroxylase in the liver and 25-hydroxyvitamin D 1α hydroxylase in the kidney) resulting in the formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D (Christakos et al., 2016). 1,25(OH)2D3 binds with high affinity to the vitamin D receptor (VDR), a nuclear receptor that heterodimerizes with the retinoid X receptor and binds to vitamin D response elements in target genes, resulting in activation or inhibition of transcription of inflammation associated genes. (Christakos et al., 2016). Thus, 1,25(OH)2D3/VDR association is critical for intestinal homeostasis.

In addition to its essential role in the maintenance of calcium homeostasis, results from in vivo and in vitro studies indicate multiple immunomodulatory effects of 1,25(OH)2D3. The anti-inflammatory function of 1,25(OH)2D3 has been recognized in autoimmune and chronic inflammatory diseases including diseases of the gastrointestinal tract (Grant et al., 2020; Jalili et al., 2019; Vanherwegen et al., 2017; Yin & Agrawal, 2014). Previous studies have shown that 1,25(OH)2D3 represses the production of inflammatory cytokines including IL-2, IFN-γ, and IL-17 by a transcriptional mechanism mediated by the VDR (Cantorna et al., 2014, Christakos et al., 2016; Jalili et al., 2019; Vanherwegen et al., 2017; Wei & Christakos, 2015). 1,25(OH)2D3 has also been reported to enhance innate immunity by increasing the production of the antimicrobial peptide cathelicidin and the microbial pattern recognition receptor NOD2, an intracellular receptor encoded by a confirmed IBD risk gene (Hampe et al., 2001; T. T. Wang et al., 2010).

Results of animal studies have supported a role for vitamin D in the suppression of experimental colitis. Global VDR ablation in mice increased intestinal mucosal damage leading to high mortality in response to dextran sulfate sodium (DSS)-induced colitis. Similarly, the absence of VDR led to spontaneous colitis in Il10−/− mice (Froicu & Cantorna, 2007; Froicu et al., 2003; Kim et al., 2013; Kong et al., 2008). In a CD45RB T cell transfer model, CD4+/CD45RBhigh T cells transferred from Vdr−/− mice induced more severe colitis than wild-type (WT) CD4+/CD454RBhigh T cells, suggesting that the absence of VDR signaling in immune cells contributes to disease pathogenesis (Froicu et al., 2003). Recent studies using intestinal epithelial cell (IEC)-specific disruption of VDR or expression of VDR, have demonstrated the importance of VDR signaling in IECs for the anticolitis effects of vitamin D in animal models of IBD (Liu et al., 2013; Monte et al., 2014; Wei & Christakos, 2015; Wu et al., 2015).

The above studies extensively examined 1,25(OH)2D3 effects on immune cells (such as T cells) originated inflammation, while very little information is available about the effect of vitamin D on inflammation caused by epithelial cell defect. We previously reported that IEC-specific Rab11a knockout mice (Rab11aΔIEC) exhibited chronic intestinal inflammation with increased production of inflammatory cytokines most notably IL-6. Loss of Rab11a increased the abundance of epithelial NF-κB in these knockout mice (Yu et al., 2014). Here, we investigated the acute and chronic effects of 1,25(OH)2D3 and dietary vitamin D on the intestinal phenotypes in Rab11aΔIEC mice. We found that 1,25(OH)2D3 administration to adult mice with pre-existing inflammation reduced inflammatory cytokine production. Dietary supplementation of a high dose of vitamin D to mice spanning fetal and postnatal stages significantly alleviated the inflammatory phenotypes in Rab11aΔIEC. Our results suggest that dietary vitamin D supplementation during pregnancy and lactation may be beneficial for preventing and suppressing chronic intestinal inflammation in hosts at a higher risk for chronic inflammation.

2 ∣. MATERIALS AND METHODS

2.1 ∣. Mice

Rab11aflox and Villin-Cre mice have been described previously (Madison et al., 2002; Yu et al., 2014). Rab11aloxP/+; Villin-Cre males were crossed to Rab11aloxP/loxP females to produce homozygous IEC-specific Rab11a knockout mice (Rab11aΔIEC). Experiments were performed on littermates in an AAALAC-accredited animal facility at Rutgers University and approved by IACUC. The dietary and 1,25(OH)2D3 injection experiments were carried out using two to three independent litters including male and female mice. Previous characterization studies in our lab of the IEC-specific Rab11a knockout mice noted no differences between males and females. Animal numbers were described for each experiment in figure legends.

2.2 ∣. High vitamin D diet feeding

Control diet containing 2000 IU vitamin D/kg diet (TD110799, Teklad diet) or high vitamin D experimental diet containing 20,000 IU vitamin D/kg diet (TD110798, Teklad diet; diets from Envigo) were given to different mating pairs throughout pregnancy, lactation, and continuously given to pups after weaning until the day of the experiment (5 weeks after weaning). Both male and female cage mates were included in the experiments. The bodyweight of individual mice was recorded twice weekly over a span of 5 weeks, starting from the week of weaning to the day they were sacrificed. On the day of the experiment, mice were sacrificed and their intestinal tissue and blood serum were collected. Half of the ileal tissues (1–2 cm upstream of the cecum) were collected for protein and messenger RNA (mRNA) studies, while the rest was used for histology and immunohistochemistry. Data were reported from five to six mice (from different litters) for each treatment group per genotype.

2.3 ∣. Intraperitoneal injection of mice with 1,25(OH)2D3

WT and Rab11aΔIEC mice (adult mice 3 months of age) fed a normal pellet diet were injected with vehicle (propylene glycol) or 1,25(OH)2D3 25 ng on Day 0 and 3. On Day 7, mice were sacrificed and their tissue samples intestinal tissue and blood serum were collected. Ileal tissues were collected for histology and mRNA studies. Data were reported from five to six mice (from different litters) for each treatment group per genotype.

2.4 ∣. Treatment of Caco-2 cells with 1,25(OH)2D3

Human colon epithelial cell line, Caco-2, was obtained from American Type Culture Collection (Catalog No. 30-2003) and cultured in Dulbecco's modified Eagle medium (1X), supplemented with 20% fetal bovine serum and 1% penicillin–streptomycin. RAB11A-deficient Caco-2 were prepared using specific short hairpin RNA (shRNA) designed against RAB11A was delivered by lentivirus particles, as described in (Gao & Kaestner, 2010). The efficacy of the knockdown (KD) was confirmed by Western blot analysis as reported in (Yu et al., 2014). These control and shRNA RAB11A-expressing Caco-2 cells were grown into a monolayer and allowed to differentiate for a span of 7 days. These cells were then treated with vehicle, 1 and 10 nM of 1,25(OH)2D3 dissolved in DMSO over a period of 4 h. Cell lysates were extracted for further analysis.

2.5 ∣. Serum samples preparation for calcium measurement

Serum samples were prepared as described previously (Hod et al., 2010). Serum calcium was assayed using a colorimetric assay (Pointe Scientific, Inc.) and was determined by Heartland Laboratories, Ames, Iowa.

2.6 ∣. Reverse-transcriptase quantitative polymerase chain reaction amplification of complementary DNAs

This method was adapted from D'Agostino et al. (2019) and Yu et al. (2014). Briefly, RNA was extracted (tissue or cell lines) using QIA-GEN RNeasy Mini Kit (74106; QIAGEN), as per the manufacturer's instructions. The concentration of RNA extracted was measured (NanoDrop 2000/2000C; Thermo Fisher Scientific) and then reverse-transcribed into complementary DNA (cDNA) using the Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit (K1681; Thermo Fisher Scientific). SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific) were used for the real-time polymerase chain reaction (PCR) reaction. Primers against human and mouse IL-6, IL-8, and β-actin and mouse Il1β, Ifnγ, Cxcl1, Cxcl5, and mouse Hprt, were adapted from (D'Agostino et al., 2019; Yu et al., 2014, 2018). All qRT-PCR reactions were run in replicates of three using Roche Light cycler 480. The PCR cycling conditions are as follows: 10 min preheating at 95°C followed by 45 cycles of denaturing at 95°C for 10 s each, annealing at 60°C for 10 s each and extension at 72°C for 10 s each. Melting curves were analyzed to ensure PCR specificities. The Ct (cycle threshold) values thus obtained were used to calculate ΔCt, that is, Ct values of the gene of interest minus the Ct values of the housekeeping genes (HPRT or β-actin for the mouse and human samples, respectively). The ΔΔCt calculation was then performed (the difference between the ΔCt of the experimental and the reference sample) and relative expression levels of each gene were calculated by the formula 2−ΔΔCt. Prism GraphPad 7.04 (https://www.graphpad.com) was used to represent the expression level of the genes of interest. The Student t-test was used to compare the gene expression levels between two groups. p Values obtained are represented as *(p < 0.05), **(p < 0.01), and ***(p < 0.001).

2.7 ∣. Immunofluorescence staining

Intestinal tissues were fixed with 10% formalin, dehydrated in 70% ethanol, and processed for paraffin embedding. Tissue samples were sliced at 5-μm sections, dewaxed, and subjected to antigen retrieval (0.1 M citric acid, pH 6.0). This was followed by blocking the sample sections in phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 2% BSA, and 2% normal serum for at least 1 h at room temperature. The sections were then incubated with the indicated antibodies overnight at 4°C. The primary antibodies used were as follows: mouse anti-F4/80 (1:200, 123101; BioLegend), rabbit anti-NF-κB p65 (D14E12; 1:200; 8242S; CST), IRF5 (W16007B; 158603; BioLegend), rabbit anti-CD-3 (SP7) (1:200; ab16669; Abcam) and rabbit anti-CD-45 (EP322Y; 1:200; ab40763; Abcam), rabbit anti-phosphohistone H3 (PHH3) (1:400; 369A-1; Millipore Sigma) and mouse anti-E-cadherin (Clone-36; 1:2000; 610181; BD Biosciences). The slides were then washed in cold PBS and incubated with fluorescence-conjugated secondary antibodies for one hour at room temperature. Postincubation with secondary antibodies the slides were again washed with PBS followed by DAPI counter-staining. The slides were then air-dried and mounted with ProLong Gold anti-fade medium. Images were collected by a Zeiss Observer spinning disk confocal microscope and analyzed by AIM software (version 4.2).

2.8 ∣. Quantification of histology

Staining was carried out on two independent sections of paraffin-embedded ileum samples for each mouse. The number of positive cells per crypt (or crypt-villus axis based on the target protein) were counted from a total of 20–50 crypts imaged from at least five different fields. The measurements were then plotted in Graphpad Prism to show intra- and interanimal data variations. NF-κB (p65) staining intensity was analyzed by ImageJ for signals within regions of interest (ROI) drawn manually along the crypt-villus axis. The mean gray values of the p65 signal were quantified from five to eight different fields per animal and plotted using GraphPad Prism. Animal numbers used in each experiment were described in the individual figure legends.

2.9 ∣. Western blot

Tissues obtained from control and Rab11a-deficient mice were washed in cold PBS and lysed with NP-40 nondenaturing lysis buffer and sonicated for 10 s three times (Microson XL2000). The lysis buffer composition and concentration were prepared as per D'Agostino et al. (2019). The lysates thus obtained were spun down at max speed in a table-top refrigerated microcentrifuge for 10 min and the supernatant was collected. Protein concentrations of the lysate were determined using the Bradford assay. Twenty micrograms of total protein were loaded and run onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. Proteins from gels were transferred onto the nitrocellulose membranes using wet transfer methods. Membranes thus obtained were then blocked with 5% milk in TBS with 0.1% Tween-20 for 1 h at room temperature followed by overnight incubation at 4°C with the primary antibodies. The primary antibodies used were as follows: rabbit anti-NF-κB p65 (D14E12; 1:1000; 8242S; CST), and mouse anti-β-actin (1:2000; SC47778; Santa Cruz). Postprimary antibody incubation, the membranes were washed in TBS with 0.1% Tween-20 and incubated with either anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody for 1 h. The membranes were finally washed and developed using ECL (RPN2209; standard GE Healthcare) or ECL prime (RPN2232; GE Healthcare) detection solution and developed in the darkroom using chemiluminescent sensitive X-ray films.

2.10 ∣. ChIP assay

Procedures for chromatin preparation and precipitation have been described previously (Joshi et al., 2011). Briefly, freshly isolated mouse intestinal mucosa from the intestinal segment was crosslinked in 1% formaldehyde, washed, lysed, and the chromatin pellet was sonicated to generate 200–1000 bp fragments. The lysates were diluted into ChIP dilution buffer and immunoprecipitation was performed using p65 antibody (D14E12 from Cell Signaling Technology), as well as immunoglobulin G control. qPCR was performed using specific genomic primers flanking the NF-κB sites on mouse IL-6 promoters, that is, −73 to −64 (Baccam et al., 2003). Primers containing the NF-κB site: 5′-TCGATGCTAAACGACGTCAC-3′ and 5′-CTATCGTTCTTGGTGGGCTC-3′ (primers from Integrated DNA Technologies).

2.11 ∣. Quantification and statistical analysis

One-way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) test was used to evaluate all the in vivo experiments with four different groups, except for the bodyweight curve, which was analyzed by two-way ANOVA with Tukey's HSD test. All analyses were conducted using Prism GraphPad 7.04 (https://www.graphpad.com). Animal numbers were specified in the figure legend for each genotype per condition. For Caco-2 assays, three independent cell culture experiments were carried out and each was done in three technical replicates for qPCR analysis. Bar graphs are presented as mean ± SEM. p Values are labeled on individual graphs and reported on the graphs with p < 0.05 considered as statistically significant.

3 ∣. RESULTS

3.1 ∣. 1,25(OH)2D3 treatment attenuates intestinal inflammation in Rab11aΔIEC mice

Rab11aΔIEC mice progressively developed blunted and branched intestinal villi, hyperplastic crypts, and marked immune cell infiltration with increased inflammatory cytokines and chemokines (D'Agostino et al., 2019; Yu et al., 2014). The phenotype was the most prominent in the ileum. Three-month-old WT and Rab11aΔIEC mice were treated with intraperitoneal (IP) injection of 25 ng of 1,25(OH)2D3 on Days 0 and 3. Intestinal tissues were harvested on Day 7 (ileum shown in Figure 1a). No change in bodyweight was observed in both groups of mice during the course of the treatment. Also, no change in serum calcium was observed after 1,25(OH)2D3 treatment compared to control, in Rab11aΔIEC mice (Rab11aΔIEC mice, 9.6 ± 0.4 mg/dl; 1,25(OH)2D3-treated Rab11aΔIEC mice, 9.5 ± 0.3mg/dl, p > 0.5). 1,25(OH)2D3 treatment partially restored the morphology of blunted and branched villi of the Rab11aΔIEC mouse intestine toward normal villous architecture (Figure 1b). We estimate a reduction in approximately 50% of ileal epithelia to 5% in 1,25(OH)2D3-treated mice. A significant reduction in macrophage infiltration (identified by F4/80) and crypt cell proliferation (identified by pHH3) in subepithelial layers of the Rab11aΔIEC mice was also observed after 1,25(OH)2D3 treatment (Figure 1c,d for F4/80 and Figure 1g,h for pHH3, respectively). Proinflammatory macrophages identified by IRF-5 (Krausgruber et al., 2011), showed a significant reduction in 1,25(OH)2D3-treated Rab11aΔIEC mice compared to vehicle-treated counterparts (Figure S1A,B).

FIGURE 1.

FIGURE 1

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1,25-(OH)2D3 administration reduces intestinal inflammation in Rab11aΔIEC mice. (a) WT (n = 6) and Rab11aΔIEC mice (n = 6) were injected intraperitoneally with vehicle (propylene glycol) or 25 ng of 1,25(OH)2D3 twice over a span of 7 days. Bodyweights were recorded. Mice were sacrificed 7 days after the first injection. Intestinal tissues were collected for molecular and phenotypic analysis. The ileum is shown in all figures. (b) Hematoxylin and eosin staining of ileum of WT and Rab11aΔIEC mice injected with either vehicle or 1,25(OH)2D3. Scale bar = 100 μm. (c) Immunostaining for F4/80 (in red) and DAPI (in blue). Scale bar = 100 μm. (d) Quantification of the number of F4/80 positive cells. n = 6 mice for each group. Data represent littermates from independent litters. (e) Immunostaining for CD3 (in green), E-cadherin (in red), and DAPI (in blue). Scale bar = 100 μm. (f) Quantification of the number of CD3-positive cells. (g) Immunostaining for pHH3 (in green), E-cadherin (in red), and DAPI (in blue). (h) Quantification of the number of pHH3 positive cells per crypt. Data were reported from ileal sections of six mice for each group

Rab11aΔIEC intestines exhibit a significant increase in the total number of the immune cell population. 1,25(OH)2D3 administration to the Rab11aΔIEC mice resulted in a significant decrease in the number of CD45 positive cells (Figure S1C,D) and CD3-positive T cells populations (Figure 1e,f) in the subepithelial layers, compared to their control counterparts.

1,25(OH)2D3 administration to the Rab11aΔIEC mice also resulted in a significant decrease in proinflammatory cytokines, IL-6, IL-1β, IFN-γ, and chemokines CXCL-1 and CXCL-5 in the ileum (Figure 2a). Suppression of inflammatory cytokines, IL-6 and IL-8, by 1,25(OH)2D3 was also observed using RAB11A-KD Caco-2 cells (Figure 2b), suggesting that the observed effect by 1,25(OH)2D3 was likely mediated by epithelial cell-intrinsic mechanism.

FIGURE 2.

FIGURE 2

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1,25-(OH)2D3 injection reduces inflammatory cytokines in Rab11aΔIEC mouse ileum. (a) Quantitative RT-PCR analysis of cytokines (Il-6, Il-1β, and IFN-1γ) and chemokines (CXCL-1, CXCL-5) in the ileum of WT and Rab11aΔIEC mice treated by vehicle or 1,25(OH)2D3. Five to six mouse ileal RNA samples were used for each group. (b) Quantitative RT-PCR analysis of cytokines in control and RAB11A-KD Caco-2 cells treated by vehicle or 1,25(OH)2D3. Data represent three independent experiments. (c) Immunostaining for NF-κB-p65 (in green), E-cadherin (in red), and DAPI (in blue) in WT and Rab11aΔIEC ileal tissues. Scale bar = 100 μm. (d) Quantification of p65 abundance along the crypt-villi axis. Data represent six mice for each group. (e and f) Ileal lysates obtained from WT and Rab11aΔIEC littermates injected with vehicle or 1,25(OH)2D3 were analyzed for NF-κB (p65) via immunoblotting. β-Actin was used as a loading control. Data were quantified from littermates of n = 2 for each group

We previously identified abnormal elevation of NF-κB as a requirement, in part, for the cytokine production and inflammation observed in the Rab11a-deficient intestinal epithelia (Yu et al., 2014). Chromatin immunoprecipitation of p65 at mouse Il6 promoter region using ileal epithelia showed approximately 1.75-fold enrichment in Rab11a-deficient epithelia than WT epithelia (Figure S2A). 1,25(OH)2D3 treatment resulted in a decrease in NF-κB (p65) abundance in the IECs (Figure 2c; quantified in Figure 2d). In addition, total ileal lysates obtained from Rab11aΔIEC mouse injected with 1,25(OH)2D3 exhibited a decrease in the NF-κB (p65) abundance (Figure 2e and quantified in Figure 2f), suggesting that the reduced production of proinflammatory cytokines such as Il6 were potentially attributable to the reduced NF-κB abundance by 1,25(OH)2D3 treatment.

3.2 ∣. High dose dietary vitamin D alleviates intestinal inflammation in Rab11aΔIEC mice

WT and Rab11aΔIEC mice were either fed the control diet (2000 IU/kg diet) or the high vitamin D3 diet (20,000 IU/kg diet). Female mice were fed throughout pregnancy and lactation. The pups born to these mice were kept on the diet 5 weeks after weaning. These animals were sacrificed after 5 weeks and intestinal tissues were collected (Figure 3a). The tissues were then assessed for the effects of high dietary vitamin D by histological features and intestinal inflammation. The Rab11aΔIEC mice have previously been reported to exhibit lower bodyweight compared to their WT counterparts (Yu et al., 2014). The high vitamin D3 diet restored the bodyweight of Rab11aΔIEC mice by comparing it to Rab11aΔIEC mice on the control diet (Figure 3b). However, no change in serum calcium was noted in the Rab11aΔIEC mice fed the high vitamin D3 diet compared to Rab11aΔIEC mice fed the control diet (9.8 ± 0.3 vs. 9.6 ± 0.4 mg/dl, respectively) (p > 0.5). The high vitamin D3 diet also restored the morphology of blunted and branched villi of the Rab11a-deficient intestine towards normal intestinal villus (ileum shown in Figure 3c).

FIGURE 3.

FIGURE 3

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High vitamin D3 diet reduces the inflammatory phenotype in Rab11aΔIEC mice. (a) Mating pairs were fed by a control diet (2000 IU/kg) or high vitamin D3 diet (20,000 IU/kg), and gave birth to WT and Rab11aΔIEC mice. All mice were kept on the same diet over a span of 5 weeks. Mice were sacrificed and intestinal tissues collected. The bodyweight of individual mice was recorded twice weekly. (b) Changes of bodyweights of Rab11aΔIEC mice on high vitamin D3 diet compared to control diet. A two-way analysis of variance with Tukey's honestly significant difference test was used to determine statistical significance. (c) Hematoxylin and eosin staining of WT and Rab11aΔIEC mouse ileum on control or high vitamin D3 diet. Scale bar = 100 μm. (d) Immunostaining for F4/80 (in red) and DAPI (in blue). Scale bar = 100 μm. (e) Quantification of the number of F4/80 positive cells per crypt-villus axis. n = 6 mice for each group. (f) Immunostaining for CD3 (in green), E-cadherin (in red), and DAPI (in blue). Scale bar = 100 μm. (g) Quantification of the number of CD3-positive cells. n = 6 mice for each group. (h) Immunostaining for pHH3 (in green), E-cadherin (in red), and DAPI (in blue). Scale bar = 50 μm. (i) Quantification of the number of pHH3 positive cells per crypt. n = 6 for each group

To determine if the histological changes observed in high vitamin D supplemented cohorts correlated with the decreased inflammation in Rab11aΔIEC mice, intestines were immunostained for F4/80 (macrophages), IRF-5 (proinflammatory macrophage subtype), CD45 (total immune cells), and CD3 (T cells) to quantify infiltrating immune cell populations. High vitamin D diet-fed mice had decreased infiltrating proinflammatory macrophage, total immune cells, and T cells compared to mice fed by control diet (Figure 3d,e for F4/80; Figure 3f,g for CD3; Figure S1E,F for IRF-5; and Figure S1G,H for CD45). In addition, crypt hyperplasia, assessed by immunostaining for pHH3, was significantly reduced in the mice fed the high vitamin D diet (Figure 3h,i). Thus, high dietary vitamin D supplements reduced the immune cell numbers in Rab11aΔIEC mouse intestines.

qPCR revealed a significant reduction in IL-6, IL-1β, IFN-γ, and the chemokine CXCL-1, CXCL-5 in the Rab11aΔIEC mice fed high vitamin D compared to mice fed the control diet (Figure 4a). Rab11aΔIEC mice on the control diet exhibited increased NF-κB (p65) abundance in epithelia. In contrast, Rab11aΔIEC mice on high vitamin D3 diet decreased the overall abundance of NF-κB (p65) (Figure 4b,c) and events of p65 nuclear positivity (Figure S2B). A similar decrease in NF-κB was observed with ileal lysates obtained from high dose vitamin D fed Rab11aΔIEC mice (Figure 4d,e). These findings suggest beneficial effects of dietary vitamin D intervention that appeared to protect against the onset of chronic intestinal inflammation.

FIGURE 4.

FIGURE 4

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High vitamin D3 diet reduces inflammatory cytokines in Rab11aΔIEC mouse ileum. (a) Quantitative RT-PCR analysis of cytokines (Il-6, Il-1β, and IFN-1γ) and chemokines (CXCL-1, CXCL-5). Each group represents five to six ileal RNA samples. (b) Immunostaining for NF-κB (p65) (in green), E-cadherin (in red), and DAPI (in blue). Scale bar = 100 μm. (c) Quantification of the NF-κB (p65) abundance per crypt-villi axis. n = 6 mice for each group. (d and e) Ileal lysates were obtained from WT or Rab11aΔIEC mice on control or high vitamin D3 diets and were analyzed for p65 via immunoblotting. β-Actin was used as a loading control. Data were quantified from three samples of each group of littermate mice

4 ∣. DISCUSSION

Using the IEC-specific Rab11a knockout mice with intestinal inflammation driven by epithelial cell abnormality, we show that either 1,25(OH)2D3 or dietary vitamin D provides protection against disease progression. 1,25(OH)2D3 or increased dietary vitamin D restored the morphology of blunted and branched villi of the Rab11aΔIEC mouse towards normal villus architecture. The improved histology was correlated with decreased macrophage infiltration and decreased crypt hyperplasia. The protective effects were mediated, at least in part, through inhibition of intestinal proinflammatory cytokines and reduced NF-κB abundance in intestinal epithelia. Together, these data suggest that dietary vitamin D and 1,25(OH)2D3 are effective at decreasing epithelial inflammatory responses and that vitamin D supplementation may be useful in the prevention of chronic inflammation by limiting inflammatory cytokine production and ameliorating the disease.

Previously, vitamin D has been established as an anti-inflammatory agent, however, most studies noted that administration of metabolically active 1,25(OH)2D3 led to the development of hypercalcemia (Cantorna, 2010; Peterson & Heffernan, 2008). However, Rowling et al. (2007) previously reported that long-term treatment of C57BL/6 with a 20,000 IU/kg vitamin D diet did not result in hypercalcemia. In our study of a model of chronic and progressive intestinal inflammation, we found that vitamin D feeding (20,000 IU/kg diet) spanning the fetal and postnatal stages significantly prevented progression of intestinal inflammation and restored most of the intestinal epithelial defects towards normal villus architecture without alterations in serum calcium. Our data suggest the importance of vitamin D nutrition in inflammation prevention and that vitamin D3 supplements may have utility in the therapy of intestinal inflammation. However, future studies are needed to examine the effects of prenatal high vitamin D supplementation versus postnatal supplementation on protection against intestinal inflammation. It is plausible that maternal or early childhood dietary intervention of individuals with a predisposition to chronic inflammation may be as beneficial as treating an established and advanced disease of adulthood.

There is a paucity of studies showing that dietary vitamin D can suppress intestinal inflammation in animal models and variable results have been reported (Glenn et al., 2014; Hummel et al., 2013; Larmonier et al., 2013; Meeker et al., 2014). For example, in previous studies using the Il10 KO mice (feeding on weaning until 3 months of age), a maximum dose of 5000 IU vitamin D/kg diet had no effect on the severity of colitis (Glenn et al., 2014; Larmonier et al., 2013). However, Smad3−/− mice infected with Helicobacter bilis (which develop transient colitis) and fed 5000 IU/kg beginning before infection showed suppression of the inflammatory response and decreased disease score without alterations in serum calcium (Meeker et al., 2014). The variable responses to dietary vitamin D may be due to the different model systems used for studying intestinal inflammation and will reflect different pathogenic mechanisms.

Previous studies noted that suppression of Th1 and Th17 cells and induction of regulatory T cells and Th2 cells are responsible for the protective effect of 1,25(OH)2D3 in experimental colitis (Cantorna et al., 2019). However beneficial effects of 1,25(OH)2D3 on gut inflammation have been noted in T cell-deficient mice, indicating the importance of other pathways for the anticolitis effects of vitamin D (Axelsson et al., 1996). In our model loss of Rab11a specifically in IECs predisposed animals to chronic intestinal inflammation (Yu et al., 2014).

F. Wang et al. (2017) did address the potential importance of vitamin D signaling during DSS-induced inflammation by studying colon-epithelial cell-specific VDR deletion. However, very little information is currently available with regard to the molecular contribution of intestinal epithelial VDR to suppression of the disease progression. In our study, we observed a significant reduction in the expression of proinflammatory cytokines in the ileum in 1,25(OH)2D3 and high dose vitamin D treated Rab11aΔIEC mice. Interestingly, as analyzed by Western blot and immunohistochemistry, both 1,25(OH)2D3 administration and high dose dietary vitamin D was capable of reducing the elevated expression of NF-κB (p65) in Rab11aΔIEC mouse intestine. These findings suggest that vitamin D and 1,25(OH)2D3 mediate the repression of the proinflammatory cytokines and chemokines in the intestine at least in part by interfering with the activity of NF-κB (a key regulator of the inflammatory response). Previous studies using different cell types (monocyte and macrophage-like cells, pancreatic islets, and fibroblasts) have reported that 1,25(OH)2D3 or 1,25(OH)2D3 analog downregulates vitamin D target genes (including IL-6, IL-8, IL-12, and proinflammatory chemokines) by affecting NF-κB-mediated upregulation of these cytokines and chemokines. Mechanisms include blocking NF-κB DNA binding and arresting p65 nuclear translocation (D'Ambrosio et al., 1998; Giarratana et al., 2004; Harant et al., 1998). It has also been reported that VDR suppresses NF-κB activation of IL-6 upregulation by directly interacting with IKKβ protein (IκB kinase β) thus blocking NF-κB activation (Chen et al., 2013). A primary effect of 1,25(OH)2D3/VDR binding on gene activity may also be involved in inhibitory, protective effects of vitamin D. Further studies are needed to determine the exact pathways modulated by vitamin D in IECs and whether differential anti-inflammatory actions may be observed in additional cell types in Rab11aΔIEC mice.

In addition, although our findings support an effect of vitamin D and 1,25(OH)2D3 on epithelial cell-intrinsic cytokine/chemokine production, differential anti-inflammatory actions of vitamin D may be observed in additional cell types. Elucidation of cellular and molecular targets of vitamin D in different cell types in the Rab11aΔIEC mouse remains an area of future investigation. In addition to suppression of the inflammatory response, we found that 1,25(OH)2D3 and vitamin D treatment of Rab11aΔIEC mouse results in a significant reduction in crypt cell proliferation. Previous studies noted an important role for vitamin D in intestinal homeostatic regeneration (Peregrina et al., 2015). Also, studies using human colon crypt organoids derived from colorectal cancer patients showed that 1,25(OH)2D3 induces a differentiated phenotype and can reduce cell proliferation (Fernández-Barral et al., 2020). Thus, in addition to its anti-inflammatory effects, as noted by our findings, vitamin D and 1,25(OH)2D3 may reduce intestinal inflammation by regulating cell proliferation.

In summary, our findings show that acute administration of 1,25(OH)2D3 or prolonged dietary vitamin D supplementation can both suppress intestinal inflammation in a model carrying epithelial intrinsic defects. Vitamin D supplementation may be useful in preventing and limiting diseases in inflammatory susceptible individuals.

Supplementary Material

SUPPLEMENTARY FIGURE

ACKNOWLEDGMENTS

This study was supported by the National Institute of Health (NIH) Grants R01DK102934, R01AT010243, R01DK119198, NSF/DBI grant 1952823, a Rutgers University-Newark (RU-N) Chancellor's IMRT award to Nan Gao; R01DK112365 to Sylvia Christakos; NJCCR fellowship (DFHS16PPC038) to Sayantani Goswami; NJCCR fellowship (DCHS19PPC038) to Juan Flores and NJCCR fellowship (DFHS17PPC036) to Sheila Bandyopadhyay (currently supported by an NIH F31 DK121428).

Footnotes

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

SUPPORTING INFORMATION

Additional Supporting Information may be found online in the supporting information tab for this article.

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