Vitamin D Receptor KO Research Articles

Knocking out the Vitamin D Receptor Enhances Malignancy and Decreases Responsiveness to Vitamin D3 Hydroxyderivatives in Human Melanoma Cells.

Simple SummaryActive forms of vitamin D3, including 1,25(OH)2D3, 20(OH)D3 and 1,20(OH)2D3, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while cells with the vitamin D receptor (VDR) silenced showed an increased but not complete resistance to their action. Furthermore, silencing of the VDR in melanoma cells enhanced their proliferation as well as spheroid and colony formation and increased their migration rate. Previous clinicopathological studies have shown an inverse correlation between VDR expression, melanoma progression and poor outcome of the disease. Thus, the expression of VDR is not only necessary for the inhibition of melanoma growth by active forms of vitamin D, but the VDR can also function as a melanoma tumor suppressor gene.Vitamin D3 is not only involved in calcium and phosphate metabolism in humans, but it can also affect proliferation and differentiation of normal and cancer cells, including melanoma. The mechanism of the anti-cancer action of vitamin D3 is not fully understood. The nuclear vitamin D receptor (VDR) is crucial for the phenotypic effects of vitamin D hydroxyderivatives. VDR expression shows an inverse correlation with melanoma progression and poor outcome of the disease. In this study we knocked out the VDR in a human melanoma cell line using CRISPR methodology. This enhanced the proliferation of melanoma cells grown in monolayer culture, spheroids or colonies and their migration. Activated forms of vitamin D, including classical 1,25(OH)2D3, 20(OH)D3 and 1,20(OH)2D3, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while VDR KO cells showed a degree of resistance to their action. These results indicate that expression of VDR is important for the inhibition of melanoma growth induced by activated forms of vitamin D. In conclusion, based on our previous clinicopathological analyses and the current study, we suggest that the VDR can function as a melanoma tumor suppressor gene.

Effects of 1,25 and 24,25 Vitamin D on Corneal Fibroblast VDR and Vitamin D Metabolizing and Catabolizing Enzymes

ABSTRACT Purpose: To investigate the effects of 1,25-Vit D3 and 24,25-Vit D3 on corneal fibroblast expression of the vitamin D-associated enzymes CYP27B1 and CYP24A1 and the roles of the vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3) in these cells. Methods: CYP24A1, CYP27B1, VDR, and Pdia3 expression in corneas was detected using immunohistochemistry. Western blotting was used to measure protein expression in human and mouse fibroblasts, including VDR KO mouse cells, treated with 1,25-Vit D3 (20 nM) and 24,25-Vit D3 (100 nM). The Pdia3 inhibitor LOC14 was used to explore the role of Pdia3 as a Vit D3 receptor in these cells. Results: CYP24A1, CYP27B1, VDR, and Pdia3 were all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 significantly increased VDR expression in human and mouse fibroblasts. 1,25-Vit D3 and 24,25-VitD3 significantly increased CYP24A1 and CYP27B1 expression level in human, VDR WT mouse, and VDR KO mouse corneal fibroblasts. CYP24A1 and CYP27B1 expression was unchanged in VDR KO mouse fibroblasts treated with 1,25-Vit D3 or 24,25-Vit D3 plus LOC14. Human fibroblast VDR, CYP24A1, and CYP27B1 expression were unaffected by LOC14. Conclusions: Vitamin D metabolic enzymes, VDR, and Pdia3 are all expressed in mouse and human corneal fibroblasts. 1,25-Vit D3 modulates fibroblast vitamin D enzymes through both the VDR and Pdia3 pathways in a species-dependent manner. 24,25-Vit D3 can increase expression of fibroblast CYP24A1 and CYP27B1 in the absence of VDR and is likely involved in fibroblast regulation independent of 1,25-Vit D3 or VDR.

1α, 25 Dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor cells (MDSC)

Myeloid derived suppressor cells (MDSC) suppress the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), regulates myeloid cell biology and previous research showed that in mouse models 1,25(OH)2D reduced the tumor level of CD34+ cells, an MDSC precursor, and reduced metastasis. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Vitamin D receptor (VDR) mRNA levels are low in MDSC from bone marrow and spleen but are 20-fold higher in tumor MDSC. At all sites, M-MDSC have 4-fold higher VDR mRNA expression than G-MDSC. Bone marrow MDSC were induced to differentiate in vitro into tumor MDSC-like cells by treating with IFN-γ, IL-13, and GM-CSF for 48 h. This treatment significantly elevated Arg1 and Nos2 levels, activated the T cell-suppressive function of MDSC, increased VDR expression 50-fold, and made the MDSC responsive to 1,25(OH)2D treatment. Importantly, 1,25(OH)2D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70 % and tumor-derived M-MDSC by 30–50 %. Consistent with this finding, VDR deletion (KO) increased T cell suppressive function of in vitro M-MDSC by 30 % and of tumor-derived M-MDSC by 50 % and G-MDSC by 400 %. VDR KO did not alter Nos2 mRNA levels but significantly increased Arg1 mRNA levels in tumor M-MDSC by 100 %. In contrast, 1,25(OH)2D treatment reduced nitric oxide production in both in vitro derived M- and G- MDSC. The major finding of this study is that 1,25(OH)2D signaling through the VDR decreases the immunosuppressive capability of MDSC. Collectively, our data suggest that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells.

Abstract 2364: 1α, 25 dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor sells (MDSC)

Abstract MDSC within the tumor microenvironment are antagonists to immune surveillance and T-cell based immunotherapies because they can suppresses the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active, hormonal form of vitamin D, 1,25(OH)2D, regulates many components of the immune system and previous research shows that in mouse models 1,25(OH)2D reduces the number of CD34+ cells, an MDSC precursor, in tumors and can reduce metastasis when coupled to adoptive immunity. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Messenger RNA levels of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D molecular action, are low in MDSC from marrow and spleen but are 20-fold higher in tumor MDSC. In addition, at all sites the M- subtype cells have 4-fold higher VDR mRNA expression than G- subtype cells. Next, we tested whether MDSC are responsive to changes in 1,25(OH)2D signaling. Bone marrow MDSC were isolated by flow cytometry and induced to differentiate into tumor MDSC-like cells using treatment with a cytokine cocktail of IFN-γ, IL-13, and GM-CSF for up to 72 h. This treatment significantly elevated Arg1 and Nos2 levels (48 h) and activated the ability of the bone marrow MDSC to suppress T cell proliferation in a 16 h assay. 48 h of cytokine treatment also increased VDR expression 100-fold and made the bone marrow MDSC responsive to 1,25(OH)2D treatment i.e. 10 nM for 24 h increased CYP24A1 and VDR mRNA in wild-type (WT) cells. Importantly, 1,25(OH)2D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70% in a 16 h assay. We next examined the impact of VDR deletion on the development of tumor M- and G-MDSC function. WT and VDR KO mice were injected ip with 106 RM-1 cells and tumor MDSC were isolated by FACS 7 days later. As expected, the T cell suppressive function of WT M-MDSC was 4X greater than G-MDSC. However, T cell suppression was significantly greater in both VDR KO M-MDSC (2X vs WT at 1:4 MDSC:T-cell ratio) and G-MDSC (4X vs WT, 1:4 ratio). The major finding of this study is that MDSC are novel targets of 1,25(OH)2D. The effect of 1,25(OH)2D is to decrease the immunosuppressive capability of MDSC. In addition, expression of VDR is necessary to attenuate the immunosuppressive capabilities of MDSC that are recruited to the tumor microenvironment. Collectively, this data suggests that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells. Citation Format: James C. Fleet, Grant N. Burcham, Ryan Calvert, Timothy L. Ratliff. 1α, 25 dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor sells (MDSC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2364.

Vitamin D3 constrains estrogen\u2019s effects and influences mammary epithelial organization in 3D cultures

Vitamin D3 (vitD3) and its active metabolite, calcitriol (1,25-(OH)2D3), affect multiple tissue types by interacting with the vitamin D receptor (VDR). Although vitD3 deficiency has been correlated with increased incidence of breast cancer and less favorable outcomes, randomized clinical trials have yet to provide conclusive evidence on the efficacy of vitD3 in preventing or treating breast cancer. Additionally, experimental studies are needed to assess the biological plausibility of these outcomes. The mammary gland of VDR KO mice shows a florid phenotype revealing alterations of developmental processes that are largely regulated by mammotropic hormones. However, most research conducted on vitD3’s effects used 2D cell cultures and supra-physiological doses of vitD3, conditions that spare the microenvironment in which morphogenesis takes place. We investigated the role of vitD3 in mammary epithelial morphogenesis using two 3D culture models. VitD3 interfered with estrogen’s actions on T47D human breast cancer cells in 3D differently at different doses, and recapitulated what is observed in vivo. Also, vitD3 can act autonomously and affected the organization of estrogen-insensitive MCF10A cells in 3D collagen matrix by influencing collagen fiber organization. Thus, vitD3 modulates mammary tissue organization independent of its effects on cell proliferation.

Effects of 1,25 and 24,25 Vitamin D on Corneal Epithelial Proliferation, Migration and Vitamin D Metabolizing and Catabolizing Enzymes

This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.

The role of Pdia3-dependent 1α,25-dihydroxy vitamin D3 signaling on IgE-mediated mast cell activation

Abstract Active 1,25(OH)2 Vitamin D3 (1,25D3) is most commonly known for its role in the regulation of bone metabolism. However, research has now indicated it is a potent immune regulator beyond its calcemic effects. Vitamin D deficiency has been implicated in the pathogenesis of allergic diseases including asthma and atopic dermatitis. 1,25D3 is known to signal through the canonical Vitamin D receptor (VDR) facilitating its anti-inflammatory properties and regulating over 100 genes. However, protein disulfide isomerase family A, member 3 (Pdia3), was recently shown to mediate rapid-membrane responses to 1,25D3. In this study, we show that upon IgE-mediated activation, VDR knockout mast cells display enhanced cytokine production. Additionally, 1,25D3 suppresses cytokine production independently of VDR supporting the possibility of an alternative receptor facilitating the suppressive properties of Vitamin D on mast cells. Pdia3 protein and mRNA levels were upregulated in response to 1,25D3 in both WT and VDR KO mast cells. Upon blocking Pdia3 signaling, VDR KO mast cells lost their suppressive response to Vitamin D. Prostaglandin E2, which is released during Pdia3-dependent 1,25D3 signaling, mimicked Vitamin D effects on WT and VDR KO mast cells. Suppressor of Cytokine Signaling 3 (SOCS3), which is known to be upregulated by PGE2, was induced by 1,25D3 in both WT and VDR KO mast cells. Interestingly, VDR KO mast cells showed an enhanced induction of SOCS3 in comparison to WT. Collectively, our data depict a novel signaling mechanism utilized by mast cells to respond to 1,25D3 that could be explored as a potential therapeutic target for the treatment of allergic and inflammatory diseases.

B cell intrinsic effects of the vitamin D receptor on IgE responses. (IRC10P.417)

Abstract B cells express the vitamin D receptor (VDR). VDR KO mice had higher levels of circulating total IgE than WT mice by 12 wks of age. Mice were sensitized using two OVA/alum injections. VDR KO mice mounted a significantly higher OVA-specific IgE response compared to WT mice. The data also showed that vitamin D deficient mice had higher OVA-specific IgE responses than vitamin D sufficient mice. IL-4, IL-5, IL-2, IFN-γ and IL-17 were not different in OVA re-stimulated spleen and mesenteric lymph nodes (MLN) in WT and VDR KO mice. However, VDR KO spleen and MLN produced significantly less IL-10 than WT. Furthermore B cells and not T cells were the major source of IL-10 in both the WT and VDR KO cultures. Experiments were done using T cell specific-VDR deletion (T-VDR KO), or B cell specific-VDR deletion (B-VDR KO). The serum IgE levels in T-VDR KO mice were comparable to that of WT mice. Conversely, B-VDR KO mice had higher total serum IgE levels than WT mice and, when immunized, produced significantly more OVA-IgE. In our current model, we propose that B cell-derived IL-10 is regulated by the VDR and that autocrine IL-10 is higher in WT mice, therefore inhibiting IgE production to a greater extent in WT than in VDR KO mice. The major finding of this work is that vitamin D receptor expression selectively regulates IgE levels in the serum via a direct effect of the VDR in B cells.

Vitamin D receptor expression controls proliferation of naïve CD8+ T cells and development of CD8 mediated gastrointestinal inflammation

BackgroundVitamin D receptor (VDR) deficiency contributes to the development of experimental inflammatory bowel disease (IBD) in several different models. T cells have been shown to express the VDR, and T cells are targets of vitamin D. In this article we determined the effects of VDR expression on CD8+ T cells.ResultsVDR KO CD8+ T cells, but not WT CD8+ T cells, induced colitis in Rag KO recipients. In addition, co-transfer of VDR KO CD8+ T cells with naïve CD4+ T cells accelerated colitis development. The more severe colitis was associated with rapidly proliferating naïve VDR KO CD8+ T cells and increased IFN-γ and IL-17 in the gut. VDR KO CD8+ T cells proliferated in vitro without antigen stimulation and did not downregulate CD62L and upregulate CD44 markers following proliferation that normally occurred in WT CD8+ T cells. The increased proliferation of VDR KO CD8+ cells was due in part to the higher production and response of the VDR KO cells to IL-2.ConclusionsOur data indicate that expression of the VDR is required to prevent replication of quiescent CD8+ T cells. The inability to signal through the VDR resulted in the generation of pathogenic CD8+ T cells from rapidly proliferating cells that contributed to the development of IBD.

Dietary Vitamin D and Vitamin D Receptor Level Modulate Epithelial Cell Proliferation and Apoptosis in the Prostate

Low vitamin D (VD) status may increase prostate cancer risk but experimental evidence for this relationship is modest. We tested whether low VD status or VD receptor (VDR) deletion influences prostate epithelial cell (PEC) biology using intact mice, castrated mice, or castrated mice treated with testosterone propionate (TP, 2.5 mg/kg BW). PEC proliferation (Ki-67 staining) and apoptosis (TUNEL method) were determined in the anterior prostate (AP). In study 1, wild-type (WT) and TgAPT(121) mice (a model of prostate intraepithelial neoplasia) were fed diets with 25, 200 (reference diet), or 10,000 IU VD/kg diet (as vitamin D(3)) prior to castration/repletion. Serum 25 hydroxyvitamin D levels were 26, 78, and 237 nmol/L in the three diet groups, respectively. Castration reduced proliferation and increased apoptosis in the AP while TP reversed these effects. Low VD diet increased proliferation in WT (+82%) and TgAPT(121) (+24%) mice while it suppressed apoptosis in WT (-29%) and TgAPT(121) (-37%) mice. This diet also increased the severity of prostate intraepithelial neoplastic lesions in the AP of intact TgAPT(121) mice. In study 2, mice with PEC-specific VDR deletion (PEC VDR KO) were examined after castration/repletion. TUNEL staining was 60% lower in castrated PEC VDR KO mice compared with castrated WT mice. In castrated mice given TP, Ki-67 staining was 2-fold higher in PEC VDR KO compared with WT mice. Our data show that low diet VDR or VDR deletion provide a prostate environment that is permissive to early procarcinogenic events that enhance prostate cancer risk.

VDR Haploinsufficiency Impacts Body Composition and Skeletal Acquisition in a Gender-Specific Manner

The vitamin D receptor (VDR) is crucial for virtually all of vitamin D's actions and is thought to be ubiquitously expressed. We hypothesized that disruption of one allele of the VDR gene would impact bone development and would have metabolic consequences. Body composition and bone mass (BMD) in VDR heterozygous (VDR HET) mice were compared to those obtained in male and female VDR KO and WT mice at 8weeks of age. Male mice were also evaluated at 16weeks, and bone marrow mesenchymal stem cell (MSC) differentiation was evaluated in VDR female mice. Additionally, female VDR HET and WT mice received intermittent PTH treatment or vehicle (VH) for 4weeks. BMD was determined at baseline and after treatment. MRI was done in vivo at the end of treatment; μCT and bone histomorphometry were performed after killing the animals. VDR HET male mice had normal skeletal development until 16weeks of age but showed significantly less gain in fat mass than WT mice. In contrast, female VDR HET mice showed decreased total-body BMD at age 8weeks but had a normal skeletal response to PTH. MSC differentiation was also impaired in VDR HET female mice. Thus, female VDR HET mice show early impairment in bone acquisition, while male VDR HET mice exhibit a lean phenotype. Our results indicate that the VDR HET mouse is a useful model for studying the metabolic and skeletal impact of decreased vitamin D sensitivity.

Altered response of CD4+ T cells to TGF-β and IL-6 in the absence of the vitamin D receptor (88.9)

Abstract Th17 cells play critical roles in autoimmunity. The active form of vitamin D, 1,25(OH)2D3, has been shown to directly regulate T cell function, but the role of vitamin D and vitamin D receptor (VDR) in the differentiation of Th17 cells is not very clear. Induction of Th17 cells using TGF-β and IL-6 resulted in twice as many Th17 cells in VDR KO cultures as WT. The increased frequency of Th17 cells in VDRKO cultures was not a result of higher expression of retinoic acid receptor-related orphan receptor-γT (ROR-γT). 1,25(OH)2D3 deficient CD4+ T cells also overproduced IL-17. Treatment of WT CD4+ T cells with 1,25(OH)2D3 significantly inhibited the generation of Th17 cells. The number of Treg cells in Th0 cultures of CD4+ cells from VDR KO mice was higher, compared to WT. TGF-β addition to the cultures induced Tregs in both the VDR KO and WT cultures, but there were significantly more Tregs in WT than VDR KO cultures. Addition of both TGF-β and IL-6 to WT CD4 cultures caused a 4-fold reduction in Treg cells. The reduction of Tregs by TGF-β and IL-6 in VDR KO cultures was only 2-fold. VDR KO T cells cultured with TGF-β and IL-6 produced significantly more IL-10 than their WT counterparts. Our data suggest the 1,25(OH)2D3 and signaling through the VDR are important regulators of Th17 differentiation. Furthermore, VDR KO T cells have altered responses to TGF-β and IL-6 that resulted in increased numbers of both Th17 and Treg cells.

A requirement for early neonatal exposure to vitamin D for iNKT cells (134.26)

Abstract CD1d-reactive invariant natural killer T (iNKT) cells are a unique subset of lymphocytes, which play important roles in immune regulation. We have shown that the vitamin D receptor (VDR) is required for the development of iNKT cells. Here we determined the effect of the vitamin D ligand (calcitriol) on iNKT cells. Like the VDR KO mice fewer iNKT cells were identified in the calcitriol deficient mice. However, ligand deficiency did not affect the function of the remaining iNKT cells. In addition, calcitirol deficiency did not affect the development of iNKT cells in the thymus, while VDR KO iNKT cells were blocked at the CD44+/NK1.1- stage of development. The 1alpha-hydroxylase (encoded by cyp27B1) is an enzyme required for production of calcitriol. Cyp27B1 KO mice are calcitriol deficient because they cannot convert vitamin D to the active form. Cyp27B1 ko/+ breeders were fed vitamin D deficient diets. At three weeks of age WT and Cyp27B1 KO littermates were weaned and fed diets that contain vitamin D for the next 5 weeks. Analysis of iNKT cells from these mice showed that both the WT and Cyp27B1 KO littermates had very few iNKT cells. In fact the percentage of iNKT cells in the liver of vitamin D deficient mice littermates were very low 1.2-1.8% and lower than in the VDR KO mice (6%). Early exposure to calcitriol in utero is required for iNKT cell development and iNKT cells failed to recover in either WT or Cyp27B1 KO littermates that were calcitriol sufficient from the age of 5-8 wks. The data show that there are differences in the role of the VDR and the vitamin D ligand on iNKT cells. In addition, the data suggest a critical role for calcitriol early either during neonatal development or the first 3 weeks post-birth in inducing iNKT cells.

Failure of T cell homing, reduced CD4/CD8αα intraepithelial lymphocytes, and inflammation in the gut of vitamin D receptor KO mice

Specific pathogen-free IL-10 KO mice failed to develop inflammatory bowel disease (IBD), whereas IL-10/vitamin D receptor (VDR) double KO mice developed fulminating IBD. WT CD4 T cells inhibited experimental IBD, while VDR KO CD4 T cells failed to suppress IBD. VDR KO mice had normal numbers and functions of regulatory T cells. The percentages of IL-17- and IFN-gamma-secreting T cells in the gut of mice reconstituted with WT and VDR KO CD4 T cells were also not different. Instead, there were twice as many CD8alphaalpha intraepithelial lymphocytes (IEL) in mice that were reconstituted with WT CD4 T cells than in mice reconstituted with VDR KO CD4 T cells. Furthermore, VDR KO mice had reduced numbers of CD8alphaalpha IEL, absent CD4/CD8alphaalpha populations, and as a result low IL-10 production in the IEL. The lack of CD8alphaalpha IEL was due in part to decreased CCR9 expression on T cells that resulted in the failure of the VDR KO T cells to home to the small intestine. We conclude that the VDR mediates T cell homing to the gut and as a result the VDR KO mouse has reduced numbers of CD8alphaalpha IEL with low levels of IL-10 leading to increased inflammatory response to the normally harmless commensal flora.

The vitamin D receptor is required for iNKT cell development

CD1d-reactive natural killer T (NKT) cells with an invariant T cell receptor Valpha14 rearrangement are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance, and host defense against pathogens. Vitamin D is a nutrient/hormone that has been shown to regulate conventional T cell responses but not T cell development. The data show that expression of the vitamin D receptor (VDR) is required for normal development and function of iNKT cells. The iNKT cells from VDR KO mice are intrinsically defective and lack T-bet expression. VDR KO iNKT cells fail to express NK1.1, although they express normal levels of CD122. Extrinsic factors that impact iNKT cell development and function in VDR KO mice include a failure of the liver to support homeostatic proliferation and reduced thymic expression of CD1d and other factors important for optimal antigen presentation in the thymus. In addition, VDR KO iNKT cells were intrinsically defective even when WT antigen-presenting cells were used to stimulate them.

Vitamin D receptor expression is required for the normal development and function of iNKT cells

CD1d‐reactive natural killer T (NKT) cells with an invariant T cell receptor Vα14 rearrangement are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. Vitamin D is a potent regulator of conventional T cell development and function but there is no information on whether vitamin D regulates NKT cells. Expression of the vitamin D receptor (VDR) is required for normal development and function of iNKT cells. The iNKT cells from VDR KO mice are intrinsically defective and lack T‐bet expression that result in a population of immature NK1.1− iNKT cells that express normal levels of CD122. Bone marrow transplantation studies showed that non‐hematopoetic cell expression of the VDR was essential for intact iNKT cell responses. Extrinsic factors that impact iNKT cell development and function in the VDR KO mice included a failure of the liver to support homeostatic proliferation and reduced thymic expression of CD1d and other factors important for optimal antigen presentation in the thymus. VDR deficiency resulted in reduced thymic CD1d expression that impaired the ability of thymocytes but not peripheral DC to act as antigen presenting cells. The defects in VDR KO iNKT cells stem from both intrinsic and extrinsic requirement for VDR expression.

Vitamin D receptor expression by the lung micro-environment is required for maximal induction of lung inflammation

Mice lacking the vitamin D receptor (VDR) are resistant to airway inflammation. Pathogenic immune cells capable of transferring experimental airway inflammation to wildtype (WT) mice are present and primed in the VDR KO mice. Furthermore, the VDR KO immune cells homed to the WT lung in sufficient numbers to induce symptoms of asthma. Conversely, WT splenocytes, Th2 cells and hematopoetic cells induced some symptoms of experimental asthma when transferred to VDR KO mice, but the severity was less than that seen in the WT controls. Interestingly, experimentally induced vitamin D deficiency failed to mirror the VDR KO phenotype suggesting there might be a difference between absence of the ligand and VDR deficiency. Lipopolysaccharide (LPS) induced inflammation in the lungs of VDR KO mice was also less than in WT mice. Together the data suggest that vitamin D and the VDR are important regulators of inflammation in the lung and that in the absence of the VDR the lung environment, independent of immune cells, is less responsive to environmental challenges.

Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury

BackgroundThe active form of vitamin D (1,25(OH)2D3) has been shown to inhibit development of inflammatory bowel disease (IBD) in IL-10 KO mice. Here, the role of the vitamin D receptor (VDR) and 1,25(OH)2D3 in acute experimental IBD was probed.ResultsVDR KO mice were extremely sensitive to dextran sodium sulfate (DSS) and there was increased mortality of the VDR KO mice at doses of DSS that only caused a mild form of colitis in wildtype (WT) mice. DSS colitis in the VDR KO mice was accompanied by high colonic expression of TNF-α, IL-1 α, IL-1β, IL-12, IFN-γ, IL-10, MIP-1α and KC. DSS concentrations as low as 0.5% were enough to induce bleeding, ulceration and weight loss in VDR KO mice. VDR KO mice failed to recover following the removal of DSS, while WT mice showed signs of recovery within 5 days of DSS removal. The early mortality of DSS treated VDR KO mice was likely due to perforation of the bowel and resulting endotoxemia. VDR KO mice were hyper-responsive to exogenously injected LPS and cultures of the peritoneal exudates of moribund DSS treated VDR KO mice were positive for bacterial growth. 1,25(OH)2D3 in the diet or rectally decreased the severity and extent of DSS-induced inflammation in WT mice.ConclusionThe data point to a critical role for the VDR and 1,25(OH)2D3 in control of innate immunity and the response of the colon to chemical injury.

Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism

FGF23 suppresses both serum phosphate and 1,25-dihydroxyvitamin D [1,25D] levels in vivo. Because 1,25D itself is a potent regulator of phosphate metabolism, it has remained unclear whether FGF23-induced changes in phosphate metabolism were caused by a 1,25D-independent mechanism. To address this issue, we intravenously administered recombinant FGF23 to vitamin D receptor (VDR) null (KO) mice as a rapid bolus injection and evaluated the early effects of FGF23. Administration of recombinant FGF23 further decreased the serum phosphate level in VDR KO mice, accompanied by a reduction in renal sodium-phosphate cotransporter type IIa (NaPi2a) protein abundance and a reduced renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alphaOHase) mRNA level. Thus FGF23-induced changes in NaPi2a and 1alphaOHase expression are independent of the 1,25D/VDR system. However, 24-hydroxylase (24OHase) mRNA expression remained undetectable by the treatment with FGF23. We also analyzed the regulatory mechanism for FGF23 expression. The serum FGF23 level was almost undetectable in VDR KO mice, whereas dietary calcium supplementation significantly increased circulatory levels of FGF23 and its mRNA abundance in bone. This finding indicates that calcium is another determinant of FGF23 production that occurs independently of the VDR-mediated mechanism. In contrast, dietary phosphate supplementation failed to induce FGF23 expression in the absence of VDR, whereas marked elevation in circulatory FGF23 was observed in wild-type mice fed with a high-phosphate diet. Taken together, FGF23 works, at least in part, in a VDR-independent manner, and FGF23 production is also regulated by multiple mechanisms involving VDR-independent pathways.

A crucial role for the vitamin D receptor in experimental inflammatory bowel diseases.

The active form of vitamin D (1,25D3) suppressed the development of animal models of human autoimmune diseases including experimental inflammatory bowel disease (IBD). The vitamin D receptor (VDR) is required for all known biologic effects of vitamin D. Here we show that VDR deficiency (knockout, KO) resulted in severe inflammation of the gastrointestinal tract in two different experimental models of IBD. In the CD45RB transfer model of IBD, CD4+/CD45RBhigh T cells from VDR KO mice induced more severe colitis than wild-type CD4+/CD45RBhigh T cells. The second model of IBD used was the spontaneous colitis that develops in IL-10 KO mice. VDR/IL-10 double KO mice developed accelerated IBD and 100% mortality by 8 wk of age. At 8 wk of age, all of the VDR and IL-10 single KO mice were healthy. Rectal bleeding was observed in every VDR/IL-10 KO mouse. Splenocytes from the VDR/IL-10 double KO mice cells transferred IBD symptoms. The severe IBD in VDR/IL-10 double KO mice is a result of the immune system and not a result of altered calcium homeostasis, or gastrointestinal tract function. The data establishes an essential role for VDR signaling in the regulation of inflammation in the gastrointestinal tract.