Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL-12 and enhanced IL-10 secretion. We have found that a short treatment with 1,25(OH)(2)D(3) induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. This effect is enhanced by co-administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)(2)D(3) on DCs. Graft acceptance is associated with an increased percentage of CD4(+)CD25(+) regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4(+)CD25(+) cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non-hypercalcemic doses. The enhancement of CD4(+)CD25(+) regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach.     

1,25-Dihyroxyvitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved noncoding sequence region

FOXP3-positive regulatory T (Treg) cells are a unique subset of T cells with immune regulatory properties. Treg cells can be induced from non-Treg CD4(+) T cells (induced Treg [iTreg] cells) by TCR triggering, IL-2, and TGF-β or retinoic acid. 1,25-Dihyroxyvitamin D(3) [1,25(OH)(2)VD(3)] affects the functions of immune cells including T cells. 1,25(OH)(2)VD(3) binds the nuclear VD receptor (VDR) that binds target DNA sequences known as the VD response element (VDRE). Although 1,25(OH)(2)VD(3) can promote FOXP3 expression in CD4(+) T cells with TCR triggering and IL-2, it is unknown whether this effect of 1,25(OH)(2)VD(3) is mediated through direct binding of VDR to the FOXP3 gene without involving other molecules. Also, it is unclear whether FOXP3 expression in 1,25(OH)(2)VD(3)-induced Treg (VD-iTreg) cells is critical for the inhibitory function of these cells. In this study, we demonstrated the presence of VDREs in the intronic conserved noncoding sequence region +1714 to +2554 of the human FOXP3 gene and the enhancement of the FOXP3 promoter activity by such VDREs in response to 1,25(OH)(2)VD(3). Additionally, VD-iTreg cells suppressed the proliferation of target CD4(+) T cells and this activity was dependent on FOXP3 expression. These findings suggest that 1,25(OH)(2)VD(3) can affect human immune responses by regulating FOXP3 expression in CD4(+) T cells through direct VDR binding to the FOXP3 gene, which is essential for inhibitory function of VD-iTreg cells.     

Lithocholic acid down-regulation of NF-kappaB activity through vitamin D receptor in colonic cancer cells

Lithocholic acid (LCA), a secondary bile acid, is a vitamin D receptor (VDR) ligand. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the hormonal form of vitamin D, is involved in the anti-inflammatory action through VDR. Therefore, we hypothesize that LCA acts like 1,25(OH)(2)D(3) to drive anti-inflammatory signals. In present study, we used human colonic cancer cells to assess the role of LCA in regulation of the pro-inflammatory NF-kappaB pathway. We found that LCA treatment increased VDR levels, mimicking the effect of 1,25(OH)(2)D(3). LCA pretreatment inhibited the IL-1beta-induced IkappaBalpha degradation and decreased the NF-kappaB p65 phosphorylation. We also measured the production of IL-8, a well-known NF-kappaB target gene, as a read-out of the biological effect of LCA expression on NF-kappaB pathway. LCA significantly decreased IL-8 secretion induced by IL-1beta. These LCA-induced effects were very similar to those of 1,25(OH)(2)D(3.) Thus, LCA recapitulated the effects of 1,25(OH)(2)D(3) on IL-1beta stimulated cells. Mouse embryonic fibroblast (MEF) cells lacking VDR have intrinsically high NF-kappaB activity. LCA pretreatment was not able to prevent TNFalpha-induced IkappaBalpha degradation in MEF VDR (-/-), whereas LCA stabilized IkappaBalpha in MEF VDR (+/-) cells. Collectively, our data indicated that LCA activated the VDR to block inflammatory signals in colon cells.     

1,25-Dihydroxyvitamin D3 selectively modulates tolerogenic properties in myeloid but not plasmacytoid dendritic cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)(2)D(3) on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)(2)D(3) up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)(2)D(3) also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)(2)D(3) treatment markedly increased CD4(+) suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)(2)D(3) did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-alpha was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4(+) suppressor T cells was unaffected by 1,25(OH)(2)D(3). Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)(2)D(3) similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)(2)D(3) inhibited NF-kappaB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)(2)D(3) to modulate tolerogenic properties in P-DCs.     

Topically applied 1,25-dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes

The immunomodulatory effects of vitamin D have been described following chronic oral administration to mice or supplementation of cell cultures with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D. In this study, topically applied 1,25(OH)(2)D(3), enhanced the suppressive capacity of CD4(+)CD25(+) cells from the draining lymph nodes. The effects of topical 1,25(OH)(2)D(3) were compared with those of UVB irradiation, which is the environmental factor required for 1,25(OH)(2)D(3) production in skin. CD4(+) cells from the skin-draining lymph nodes (SDLN) of either 1,25(OH)(2)D(3)-treated or UVB-irradiated mice had reduced capacity to proliferate to Ags presented in vitro, and could suppress Ag-specific immune responses upon adoptive transfer into naive mice. This regulation was lost upon removal of CD4(+)CD25(+) cells. Furthermore, purified CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D(3)-treated or UVB-irradiated mice compared with equal numbers of CD4(+)CD25(+) cells from control mice had increased capacity to suppress immune responses in both in vitro and in vivo assay systems. Following the sensitization of recipient mice with OVA, the proportion of CD4(+)Foxp3(+) cells of donor origin significantly increased in recipients of CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D(3)-treated mice, indicating that these regulatory T cells can expand in vivo with antigenic stimulation. These studies suggest that 1,25(OH)(2)D(3) may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.     

The direct action of 1alpha,25(OH)2-vitamin D3 on purified mouse Langerhans cells

The action of vitamin D(3) on Langerhans cells (LCs) is not well understood. Using highly purified murine LCs (>95%), we investigated the direct action of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on their functions. 1,25(OH)(2)D(3) inhibited the expression of cell surface molecules including I-A(d), CD40, CD80, and CD86, leading to impaired ability of LCs to stimulate allogenic T cells in the mixed leukocyte reaction. Furthermore, this reagent inhibited chemotaxis of LCs to CCL21 and their survival. Interestingly, 1,25(OH)(2)D(3) reduced the IL-10 production by LCs, whereas the production of IL-6 and IL-12p40 upon activation by CD40 ligation was enhanced. With regard to inflammatory cytokines and chemokines, 1,25(OH)(2)D(3) upregulated the production of IL-1beta, CCL3, CCL4, and CCL5. The production of Th2-type chemokines, represented by CL17 and CCL22, was inhibited, whereas IFN-gamma-triggered production of Th1-type chemokines, represented by CXCL9, CXCL10, and CXCL11, was augmented. These data indicate that the mode of regulation of cytokine and chemokine production in association with 1,25(OH)(2)D(3) treatment seems to be another characteristic discriminating LCs from classical myeloid dendritic cells.     

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.     

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.     

IL-10 signaling is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) results from an aberrant, neuroantigen-specific, T cell-mediated autoimmune response. Because MS prevalence and severity decrease sharply with increasing sunlight exposure, and sunlight supports vitamin D(3) synthesis, we proposed that vitamin D(3) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) may protect against MS. In support of this hypothesis, 1,25-(OH)(2)D(3) strongly inhibited experimental autoimmune encephalomyelitis (EAE). This inhibition required lymphocytes other than the encephalitogenic T cells. In this study, we tested the hypothesis that 1,25-(OH)(2)D(3) might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D(3) and 1,25-(OH)(2)D(3) strongly inhibited myelin oligodendrocyte peptide (MOG(35-55))-induced EAE in C57BL/6 mice, but completely failed to inhibit EAE in mice with a disrupted IL-10 or IL-10R gene. Thus, a functional IL-10-IL-10R pathway was essential for 1,25-(OH)(2)D(3) to inhibit EAE. The 1,25-(OH)(2)D(3) also failed to inhibit EAE in reciprocal, mixed bone marrow chimeras constructed by transferring IL-10-deficient bone marrow into irradiated wild-type mice and vice versa. Thus, 1,25-(OH)(2)D(3) may be enhancing an anti-inflammatory loop involving hemopoietic cell-produced IL-10 acting on brain parenchymal cells and vice versa. If this interpretation is correct, and humans have a similar bidirectional IL-10-dependent loop, then an IL-10-IL-10R pathway defect could abrogate the anti-inflammatory and neuro-protective functions of sunlight and vitamin D(3). In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D(3) insufficiency, to increase MS risk and severity.     

The ifng gene is essential for vdr gene expression and vitamin d3-mediated reduction of the pathogenic T cell burden in the central nervous system in experimental autoimmune encephalomyelitis, a multiple sclerosis model

Compelling evidence suggests that vitamin D(3) insufficiency may contribute causally to multiple sclerosis (MS) risk. Experimental autoimmune encephalomyelitis (EAE) research firmly supports this hypothesis. Vitamin D(3) supports 1,25-dihydroxyvitamin D(3) (1,25-[OH](2)D(3)) synthesis in the CNS, initiating biological processes that reduce pathogenic CD4(+) T cell longevity. MS is prevalent in Sardinia despite high ambient UV irradiation, challenging the vitamin D-MS hypothesis. Sardinian MS patients frequently carry a low Ifng expresser allele, suggesting that inadequate IFN-γ may undermine vitamin D(3)-mediated inhibition of demyelinating disease. Testing this hypothesis, we found vitamin D(3) failed to inhibit EAE in female Ifng knockout (GKO) mice, unlike wild-type mice. The two strains did not differ in Cyp27b1 and Cyp24a1 gene expression, implying equivalent vitamin D(3) metabolism in the CNS. The 1,25-(OH)(2)D(3) inhibited EAE in both strains, but 2-fold more 1,25-(OH)(2)D(3) was needed in GKO mice, causing hypercalcemic toxicity. Unexpectedly, GKO mice had very low Vdr gene expression in the CNS. Injecting IFN-γ intracranially into adult mice did not increase Vdr gene expression. Correlating with low Vdr expression, GKO mice had more numerous pathogenic Th1 and Th17 cells in the CNS, and 1,25-(OH)(2)D(3) reduced these cells in GKO and wild-type mice without altering Foxp3(+) regulatory T cells. Thus, the Ifng gene was needed for CNS Vdr gene expression and vitamin D(3)-dependent mechanisms that inhibit EAE. Individuals with inadequate Ifng expression may have increased MS risk despite high ambient UV irradiation because of low Vdr gene expression and a high encephalitogenic T cell burden in the CNS.     

Vitamin D signaling in immune-mediated disorders: Evolving insights and therapeutic opportunities

1,25(OH)(2)D(3), the active form of vitamin D, is a central player in calcium and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. The widespread presence of the vitamin D receptor (VDR) in the immune system and the expression of the enzymes responsible for the synthesis of the active 1,25(OH)(2)D(3) regulated by specific immune signals, even suggest a paracrine immunomodulatory role for 1,25(OH)(2)D(3). Additionally, the different molecular mechanisms used by 1,25(OH)(2)D(3) to exert its immunomodulatory effects prove of a broad action radius for this compound. Both, the effects of vitamin D deficiency and/or absence of the VDR as well as intervention with pharmacological doses of 1,25(OH)(2)D(3) or one of its less-calcemic analogs, affects immune system behavior in different animal models of immune-mediated disorders, such as type 1 diabetes. This review aims to summarize the data as they stand at the present time on the role of vitamin D in the pathogenesis of immune-mediated disorders, with special focus on type 1 diabetes, and on the therapeutic opportunities for vitamin D in the prevention and treatment of this autoimmune disease in mouse models and humans.