Evelyne van Etten

Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1alpha-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype. The VDR is nearly ubiquitously expressed, and almost all cells respond to 1,25-(OH)(2)D exposure; about 3% of the mouse or human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system, suggesting a more widespread function. VDR-deficient mice, but not vitamin D- or 1alpha-hydroxylase-deficient mice, and man develop total alopecia, indicating that the function of the VDR and its ligand is not fully overlapping. The immune system of VDR- or vitamin D-deficient mice is grossly normal but shows increased sensitivity to autoimmune diseases such as inflammatory bowel disease or type 1 diabetes after exposure to predisposing factors. VDR-deficient mice do not have a spontaneous increase in cancer but are more prone to oncogene- or chemocarcinogen-induced tumors. They also develop high renin hypertension, cardiac hypertrophy, and increased thrombogenicity. Vitamin D deficiency in humans is associated with increased prevalence of diseases, as predicted by the VDR null phenotype. Prospective vitamin D supplementation studies with multiple noncalcemic endpoints are needed to define the benefits of an optimal vitamin D status.

Immunoregulation by 1,25-dihydroxyvitamin D3: Basic concepts

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active metabolite of Vitamin D(3), not only regulates bone and calcium metabolism but also exerts other biological activities, including immunomodulation via the nuclear Vitamin D receptor expressed in antigen-presenting cells and activated T cells. This regulation is mediated through interference with nuclear transcription factors such as NF-AT and NF-kappaB or by direct interaction with Vitamin D responsive elements in the promoter regions of cytokine genes. Dendritic cells (DCs) are primary targets for the immunomodulatory activity of 1,25(OH)(2)D(3), as indicated by inhibited DC differentiation and maturation, leading to down-regulated expression of MHC-II, costimulatory molecules and IL-12. Moreover, 1,25(OH)(2)D(3) enhances IL-10 production and promotes DC apoptosis. Together, these effects of 1,25(OH)(2)D(3) inhibit DC-dependent T cell activation. Immunomodulation by 1,25(OH)(2)D(3) and its analogs in vivo has been demonstrated in different models of autoimmune diseases and transplantation. Moreover, combining analogs with other immunosuppressants leads to synergism in models of autoimmunity and transplantation. The availability of 1,25(OH)(2)D(3) analogs with immunomodulatory activity at non-hypercalcemic doses may allow exploitation of their immunomodulatory effects in a clinical setting of treatment of autoimmune diseases and prevention of allograft rejection.

The vitamin D receptor gene FokI polymorphism: Functional impact on the immune system

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) has important effects on the growth and function of multiple cell types. These pleiotropic effects of 1,25(OH)2D3 are mediated through binding to the vitamin D receptor (VDR). Several polymorphisms of the human VDR gene have been identified, with the FokI polymorphism resulting in VDR proteins with different structures, a long f‐VDR or a shorter F‐VDR. The aim of this study was to investigate the functional consequences of the FokI polymorphism in immune cells. In transfection experiments, the presence of the shorter F‐VDR resulted in higher NF‐κB‐ and NFAT‐driven transcription as well as higher IL‐12p40 promoter‐driven transcription. Marginal differences were observed for AP‐1‐driven transcription, and no differential effects were observed for transactivation of a classical vitamin D‐responsive element. Concordantly, in human monocytes and dendritic cells with a homozygous short FF VDR genotype, expression of IL‐12 (mRNA and protein) was higher than in cells with a long ff VDR genotype. Additionally, lymphocytes with a short FF VDR genotype proliferated more strongly in response to phytohemagglutinin. Together, these data provide the first evidence that the VDR FokI polymorphism affects immune cell behavior, with a more active immune system for the short F‐VDR, thus possibly playing a role in immune‐mediated diseases.

In Vitro and In Vivo Analysis of the Immune System of Vitamin D Receptor Knockout Mice

Immune cells carry receptors for 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3; vitamin D receptor (VDR)] and individuals with severe vitamin D deficiency have immune abnormalities. The aim of this study was to investigate the role of vitamin D in the immune system by studying VDR‐knockout (VDR‐KO) mice. VDR‐KO mice had the same metabolic phenotype as rachitic animals with severe hypocalcemia. Leukocytosis, lymphocyte subset composition in different immune organs, and splenocyte proliferation to several stimuli were normal, except for a lower response to anti‐CD3 stimulation (simulation index [SI] of 13 ± 4 vs. 24 ± 9 in wild‐type mice; p < 0.01). Macrophage chemotaxis was impaired (41 ± 19% vs. 60 ± 18% in wild‐type mice; p < 0.01) but phagocytosis and killing were normal. In vivo rejection of allogeneic (31 ± 12 days vs. 45 ± 26 days of survival in wild‐type mice, NS) or xenogeneic (10 ± 2 days vs. 16 ± 9 days of survival in wild‐type mice, NS) islet grafts was comparable with wild‐type mice. Surprisingly, VDR‐KO mice were protected from low‐dose streptozotocin‐induced diabetes mellitus (LDSDM; 5% vs. 65% in wild‐type mice; p < 0.001). Correcting hypocalcemia by use of lactose‐rich or polyunsaturated fat‐rich diets fully restored the immune abnormalities in vitro and the sensitivity to diabetes in vivo. On the other hand, treatment with 1,25(OH)2D3 protected wild‐type mice against diabetes but did not protect normocalcemic VDR‐KO mice. We conclude that immune defects observed in VDR‐KO mice are an indirect consequence of VDR disruption because they can be restored by calcium homeostasis normalization. This study proves that although 1,25(OH)2D3 is a pharmacologic and probably a physiological immunomodulator, its immune function is redundant. Moreover, we confirm the essential role of calcium in the immune system.

Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants.

BACKGROUND Most immunosuppressants have a narrow margin between efficacy and side effects. A major goal in the development of immunomodulatory strategies is the discovery of combinations of drugs exerting synergistic immunomodulatory effects. The active form of vitamin D, 1,25(OH)2D3, is an immunomodulator that interacts with T cells but mainly targets antigen-presenting cells. We have demonstrated synergism between 1,25(OH)2D3 and cyclosporine, rapamycin, and FK506. The aim of this study was to investigate whether this synergism could be observed with other immunosuppressants (mycophenolate mofetil, leflunomide, and the methylxanthine A802715) and whether analogs of 1,25(OH)2D3 share this synergistic capacity in vivo. METHODS In vitro, the median effect analysis was applied to the inhibition of phytohemagglutinin A-induced lymphocyte proliferation. In vivo, synergism between analogs of 1,25(OH)2D3 and cyclosporine or mycophenolate mofetil was evaluated in experimental autoimmune encephalomyelitis. RESULTS In vitro, all combinations with 1,25(OH)2D3 were synergistic. The strongest synergism was seen with the inhibitors of interleukin 2 secretion, cyclosporine and FK506 (indexes 0.16 and 0.27, respectively). The weakest synergism was observed in combinations using A802715, a second-signal inhibitor (index 0.52), or the nucleotide synthesis inhibitor mycophenolate mofetil (index 0.43). In vivo, analogs of 1,25(OH)2D3 share the in vitro-observed synergism with 1,25(OH)2D3. Moreover, the differences in synergism with different immunomodulators were also present in vivo, where the best synergism was again seen in combination with cyclosporine (up to 100% paralysis protection). CONCLUSIONS These data confirm that 1,25(OH)2D3 and its analogs are potent dose-reducing drugs for other immunomodulators, making them potentially interesting for clinical use in autoimmunity and transplantation.

Human T lymphocytes are direct targets of 1,25-dihydroxyvitamin D3 in the immune system

Besides its actions on minerals and bone, the bioactive vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has important immunomodulatory properties. Within the immune system, dendritic cells represent key targets for this hormone and 1,25(OH)2D3-induced changes in their phenotype and function ultimately affects T lymphocytes. However, the presence of vitamin D receptors (VDR) in activated T cells proposes additional mechanisms for 1,25(OH)2D3 to directly regulate T cell responses. Here, we investigated the expression and kinetics of vitamin D-related genes in human activated T lymphocytes. Different activation stimuli elicited increased VDR- and 1-alpha-hydroxylase expression, with a highly similar kinetic pattern. Addition of 1,25(OH)2D3 effectively triggered VDR signaling, as evidenced by 24-hydroxylase induction, but only when introduced to T lymphocytes expressing high levels of VDR. This enhanced degree of VDR signaling correlated with a stronger inhibition of cytokines (IFN-gamma, IL-10) and modulation of homing receptor expression (CCR10, CLA) in long-term T cell cultures. Importantly, chronic 1,25(OH)2D3-exposure further amplified VDR signaling and the concomitant T cell modulating effects. In conclusion, we validate T cells as direct targets for 1,25(OH)2D3 and provide this optimized in vitro model to improve our understanding of the role of vitamin D as a direct regulator of T cell responses.

Regulation of vitamin D homeostasis: implications for the immune system

Vitamin D homeostasis in the immune system is the focus of this review. The production of both the activating (25- and 1alpha-hydroxylase) and the metabolizing (24-hydroxylase) enzymes by cells of the immune system itself, indicates that 1,25(OH)(2)D(3) can be produced locally in immune reaction sites. Moreover, the strict regulation of these enzymes by immune signals is highly suggestive for an autocrine/paracrine role in the immune system, and opens new treatment possibilities.

Vitamin D and 1,25-dihydroxyvitamin D3 as modulators in the immune system.

Treatment from weaning until old age with 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) prevents diabetes in NOD mice. It is mainly through its actions on dendritic cells (DCs), that 1,25(OH)(2)D(3) changes the function of potentially autoreactive T lymphocytes. In contrast, early life treatment (from 3 to 70 days of age) of NOD mice with vitamin D or 1,25(OH)(2)D(3) did not influence final diabetes incidence at 200 days of age. Also in spontaneous diabetic BB rats, diabetes could not be prevented by early life treatment (from 3 to 50 days of age) with vitamin D (1000 IU per day) or 1,25(OH)(2)D(3) (0.2 microg/kg per day or 1 microg/kg per 2 days). However, when NOD mice were made vitamin D deficient in early life (until 100 days of age), diabetes onset occurred earlier and final incidence was increased. These data further support a role for vitamin D and its metabolites in the pathogenesis of type 1 diabetes in NOD mice.

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.

Analogs of 1α,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D3, 1,25(OH)2D3, is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen‐presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)2D3 are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)2D3 in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19‐nor‐14,20‐bisepi‐23‐yne‐1,25(OH)2D3) is a structural 1,25(OH)2D3 analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother‐molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)2D3 in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin‐stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)2D3 itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases. J. Cell. Biochem. 88: 223–226, 2003.