Vitamin D Regulation of Immune Function: Implications for Bone Loss During Inflammation
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- Bikle, D.D. Clinic Rev Bone Miner Metab (2009) 7: 301. doi:10.1007/s12018-009-9056-4
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Although the best known actions of vitamin D involve its regulation of bone mineral homeostasis, actions critical for a healthy skeleton, vitamin D exerts its influence on many physiologic processes. One of these processes is the immune system. Both the adaptive and innate immune systems are impacted by the active metabolite of vitamin D, 1,25(OH)2D3. In turn, the immune system is now recognized as having a major impact on the skeleton. In this review, I will examine the regulation by 1,25(OH)2D3 of immune function, then examine the evidence for such regulation as potential means of ameliorating the bone loss that accompanies the inflammatory state.
KeywordsVitamin DInnate immunityAdaptive immunityMacrophageKeratinocyteOsteoclast
The potential role for vitamin D and its active metabolite 1,25(OH)2D3 in modulating the immune response has long been recognized since the discovery of vitamin D receptors (VDR) in macrophages, dendritic cells (DC) and activated T and B lymphocytes, the ability of macrophages and DC as well as activated T and B cells to express CYP27B1, the enzyme that produces 1,25(OH)2D3, and the ability of 1,25(OH)2D3 to regulate the proliferation and function of these cells. While these are the key cells mediating the adaptive immune response, 1,25(OH)2D, VDR, and CYP27B1 are also expressed in a large number of epithelial cells, which along with the aforementioned members of the adaptive immune response contribute to host defense by their innate immune response. The totality of the immune response involves both types of responses in complex interactions involving numerous cytokines. The regulation of these different responses and their interactions by 1,25(OH)2D3 is nuanced. In general 1,25(OH)2D3 enhances the innate immune response primarily via its ability to stimulate cathelicidin, an antimicrobial peptide important in defense against invading organisms, whereas it inhibits the adaptive immune response primarily by inhibiting the maturation of DC important for antigen presentation, reducing T cell proliferation, and shifting the balance of T cell differentiation from the Th1 and Th17 pathways to Th2 and Treg pathways. Inflammatory autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (IBD), and psoriasis involve Th17 activation, a cell that expresses RANKL, and so can drive osteoclastogenesis leading to bone loss. The detailed mechanisms are considerably more complex as will be discussed in this review.
Immune Regulation of CYP27B1
Before reviewing the regulation by 1,25(OH)2D3 of immune function, the importance of the ability of cells involved with the immune response to regulate 1,25(OH)2D3 production needs to be underscored. As mentioned above macrophages, DC, T and B lymphocytes all express CYP27B1 [1, 2], but only when these cells are activated. DCs but not T cells also express CYP27A1, the mitochondrial enzyme that produces 25OHD from vitamin D, although both DCs and T cells express CYP2R1 (the microsomal 25-hydroxylase) . However, only DC produce 1,25(OH)2D3 from vitamin D3 suggesting that the CYP2R1 is not functional in the T cells . Furthermore, CYP24A1 expression and activity, the 1,25(OH)2D3 inducible enzyme that catabolizes 25OHD3 and 1,25(OH)2D3, in activated macrophages and DCs is either absent  or blocked [3, 4] removing this feedback control of the 1,25(OH)2D3 produced. Diseases associated with immune activation can and do lead to hypercalcemia and hypercalciuria as a result of increased circulating levels of 1,25(OH)2D3 (review in ). The mechanisms for this lack of feedback control are several. First, the major drivers for CYP27B1 expression and activity in these cells are cytokines, not PTH, and cytokines are not regulated by calcium and phosphate. Second, CYP24A1 induction and/or function in macrophages in response to 1,25(OH)2D3 is blunted. One mechanism appears to involve the expression of a truncated form of CYP24A1, which includes the substrate binding domain but not the mitochondrial targeting sequence. This truncated form is postulated to act as a dominant negative form of CYP24A1, binding 1,25(OH)2D3 within the cytoplasm and preventing its catabolism . A second mechanism involves the ability of STAT-1 (induced by IFNγ) to complex with VDR blocking its ability to bind to and activate the VDRE in the CYP24A1 promoter .
Epithelia are key players in the initiation of the innate immune response, the first line of defense to invading microorganisms. CYP27B1 expression and activity have been found in most epithelia where they have been sought. These tissues include the prostate, colonic mucosa mammary epithelium, cervical epithelium, lung epithelium, and endometrium, but other cells including bone cells have been reported to produce 1,25(OH)2D3 (review in ). Epidermal keratinocytes also express CYP27A1 enabling them to produce 1,25(OH)2D3 from endogenous sources of vitamin D3 . UVB radiation, which increases vitamin D and subsequently 1,25(OH)2D3 production in epidermal keratinocytes, suppresses the adaptive immune response mediating contact hypersensitivity , while increasing the innate immune response . Suppression of the adaptive immune response is at least partially attributable to 1,25(OH)2D3 induced expression of RANKL in keratinocytes leading to activation of Langerhans cells, and the subsequent induction of Treg . I will return to this subsequently. Activation of the innate immune response is due to 1,25(OH)2D3 induced cathelicidin production . Unlike macrophages, these epithelia also express CYP24A1, which limits the levels of 1,25(OH)2D3 within these tissues such that the 1,25(OH)2D3 produced is likely to play primarily a paracrine or autocrine role in these tissues and not lead to systemic effects on calcium metabolism.
Role of Vitamin D in the Adaptive Immune Response
1,25(OH)2D3 has both direct and indirect effects on regulation of a number of cytokines involved with the immune response (review in ). TNF has a VDRE in its promoter to which the VDR/RXR complex binds. 1,25(OH)2D3 both blocks the activation of NFκB via an increase in IκBα expression and impedes its binding to its response elements in the genes such as IL-8 and IL-12 that it regulates. 1,25(OH)2D3 has also been shown to bring an inhibitor complex containing histone deacetylase 3 (HDAC3) to the promoter of rel B, one of the members of the NFκB family, thus suppressing gene expression. Thus, TNF/NFkB activity is markedly impaired by 1,25(OH)2D3 at multiple levels. In VDR null fibroblasts, NFκB activity is enhanced. Furthermore, 1,25(OH)2D3 suppresses IFNγ, and a negative VDRE has been found in the IFNγ promoter. GM-CSF is regulated by VDR monomers binding to a repressive complex in the promoter of this gene, competing with nuclear factor of T cells 1 (NFAT1) for binding to the promoter.
Clinical Implications of the Inhibition of the Adaptive Immune Response
Inhibition by Vitamin D of Autoimmunity
The ability of 1,25(OH)2D3 to suppress the adaptive immune system appears to be beneficial for a number of conditions in which the immune system is directed at self, i.e., autoimmunity (review in ). In a number of experimental models including inflammatory arthritis, psoriasis, autoimmune diabetes (e.g., NOD mice), systemic lupus erythematosus, experimental allergic encephalitis (a model for multiple sclerosis), IBD, prostatitis, and thyroiditis VDR agonist administration has prevented and/or treated the disease process. As will be discussed later, a number of these conditions are associated with bone loss either directly (e.g., inflammatory arthritis) or indirectly presumably via increased serum levels of inflammatory cytokines. These actions of 1,25(OH)2D3 were originally ascribed to inhibition of Th1 function, but Th17 cells have recently been shown to play important roles in a number of these conditions including psoriasis , experimental colitis , and rheumatoid arthritis  conditions that respond to 1,25(OH)2D3 and its analogs. Although few prospective, randomized, placebo controlled trials in humans have been performed, epidemiologic and case control studies indicate that a number of these diseases in humans are favorably impacted by adequate vitamin D levels. For example, the incidence of multiple sclerosis correlates inversely with 25OHD levels and vitamin D intake, and early studies suggested benefit in the treatment of patients with rheumatoid arthritis and multiple sclerosis with VDR agonists [19, 20]. Children who are vitamin D deficient have a higher risk of developing type 1 diabetes mellitus, and supplementation with vitamin D during early childhood reduces the risk of developing type 1 diabetes (review in ). In VDR null mice myelopoeisis and the composition of lymphoid organs are normal, although a number of abnormalities in the immune response have been found. Some of the abnormalities in macrophage function and T cell proliferation in response to anti-CD3 stimulation in these animals could be reversed by placing the animals on a high calcium diet to normalize serum calcium , indicating the important role of calcium in vitamin D regulated immune function as in skeletal development and maintenance. Other studies have noted an increased number of mature DCs in the lymph nodes of VDR null mice, which would be expected to promote the adaptive immune response . Somewhat surprisingly, RANKL also increases the number and retention of DCs in lymph nodes  suggesting that at least this mechanism is not mediated via the RANKL/RANK system in VDR null mice, which I will discuss at length subsequently. In contrast to these inhibitory actions of 1,25(OH)2D3, Th2 function as indicated by increased IgE stimulated histamine from mast cells is increased in VDR null mice . The IL-10 null mouse model of IBD shows an accelerated disease profile when bred with the VDR null mouse with increased expression of Th1 cytokines . Surprisingly, despite a reduction in natural killer T cells and Treg cells and a decreased number of mature DCs, VDR null mice bred with NOD mice do not show accelerated development of diabetes . Part of the difference in tissue response in VDR null mice may relate to differences in the ability of 1,25(OH)2D3 to alter the homing of T cells to the different tissues . In allergic airway disease (asthma) Th2 cells, not Th1 cells, dominate the inflammatory response. 1,25(OH)2D3 administration to normal mice protected these mice from experimentally induced asthma in one study, blocking eosinophil infiltration, IL-4 production, and limiting histologic evidence of inflammation . However, a study with VDR null mice using a comparable method of inducing asthma showed that lack of VDR also protected the mice from an inflammatory response in their lungs . In an extension of this study, the investigators showed that wildtype (WT) splenocytes were only minimally successful at restoring experimental airway inflammation to VDR null mice, whereas splenocytes from these mice were able to transfer experimental airway inflammation to the unprimed WT host . Thus, the impact of vitamin D signaling on adaptive immunity depends on the specifics of the immune response being evaluated.
Vitamin D Protection of Tissue Transplants
Inhibition of the adaptive immune response may also have benefit in transplantation procedures . In experimental allograft models of the aorta, bone, bone marrow, heart, kidney, liver, pancreatic islets, skin, and small bowel VDR agonists have shown benefit generally in combination with other immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus, and glucocorticoids . Much of the effect could be attributed to a reduction in infiltration of Th1 cells, macrophages and DC into the grafted tissue associated with a reduction in chemokines such as CXCL10, CXCL9, CCL2, and CCL5. CXCL10, the ligand for CXCR3, may be of particular importance for acute rejection in a number of tissues, whereas CXCL9 as well as CXCL10 (both CXCR3 ligands) may be more important for chronic rejection at least in the heart and kidney, respectively. Although there are no prospective trials of the use of VDR agonists in transplant patients, several retrospective studies in patients with renal transplants treated with 1,25(OH)2D3 have suggested benefit with respect to prolonged graft survival and reduced numbers of acute rejection episodes.
Potential Downside of Vitamin D Suppression of Adaptive Immunity
Suppression of the adaptive immune system may not be without a price. Several recent publications have demonstrated that for some infections including Leishmania major  and toxoplasmosis , 1,25(OH)2D3 promotes the infection , while the mouse null for VDR is protected . This may be due at least in part to loss of IFNγ stimulation of ROS and NO production required for macrophage antimicrobial activity . Furthermore, atopic dermatitis, a disease associated with increased Th2 activity , and allergic airway disease, likewise associated with increased Th2 activity, [28–30], may be aggravated by 1,25(OH)2D3 and less severe in animals null for VDR.
Role of Vitamin D in the Innate Immune Response
Although many cells are capable of the innate immune response including bone cells, most studies have focused on the macrophage and the keratinocyte. Vitamin D regulation of the innate immune response in these two cell types is comparable, but differences exist.
The importance of adequate vitamin D nutrition for resistance to infection has long been appreciated but poorly understood. This has been especially true for tuberculosis. Indeed, prior to the development of specific drugs for the treatment of tuberculosis, getting out of the city into fresh air and sunlight was the treatment of choice. In a recent survey of patients with tuberculosis in London , 56% had undetectable 25OHD levels, and an additional 20% had detectable levels but below 9 ng/ml (22 nM). In 1986, Rook et al.  demonstrated that 1,25(OH)2D3 could inhibit the growth of Mycobacterium tuberculosis. The mechanism for this remained unclear until the publication by Liu et al.  of their results in macrophages. They observed that activation of the toll-like receptor, TLR2/1, by a lipoprotein extracted from M. tuberculosis reduced the viability of intracellular M. tuberculosis in human monocytes and macrophages concomitant with increased expression of the VDR and of CYP27B1 in these cells. Killing of M. tuberculosis occurred only when the serum in which the cells were cultured contained adequate levels of 25OHD, the substrate for CYP27B1. This provided clear evidence for the importance of vitamin D nutrition (as manifested by adequate serum levels of 25OHD) in preventing and treating this disease, and demonstrated the critical role for endogenous production of 1,25(OH)2D3 by the macrophage to enable its antimycobacterial capacity. Activation of TLR2/1 or directly treating these cells with 1,25(OH)2D3 induced the antimicrobial peptide cathelicidin, which is toxic for M. tuberculosis. If induction of cathelicidin is blocked as with siRNA, the ability of 1,25(OH)2D3 to enhance the killing of M. tuberculosis is prevented . Furthermore, 1,25(OH)2D3 also induces the production of reactive oxygen species which if blocked likewise prevents the antimyobacterial activity of 1,25(OH)2D3 treated macrophages . The murine cathelicidin gene lacks a known VDR response element in its promoter, and so might not be expected to be induced by 1,25(OH)2D3 in mouse cells, yet 1,25(OH)2D3 stimulates antimycobacterial activity in murine macrophages. Murine macrophages, unlike human macrophages, utilize inducible nitric oxide synthase (iNOS) for their TLR and 1,25(OH)2D3 mediated killing of M. tuberculosis [46, 47].
Cathelicidiin and CD14 expression in epidermal keratinocytes is also induced by 1,25(OH)2D3 [36, 39, 48]. In these cells butyrate, which by itself has little effect, potentiates the ability of 1,25(OH)2D3 to induce cathelicidin . Keratinocytes treated with 1,25(OH)2D3 are substantially more effective in killing Staphylococcus aureus than are untreated keratinocytes. Wounding the epidermis induces the expression of TLR2 and that of its co-receptor CD14 and cathelicidin . This does not occur in mice lacking CYP27B1 . Unlike macrophages, 1,25(OH)2D3 stimulates TLR2 expression in keratinocytes as well as in the epidermis when applied topically  providing a feed forward loop to amplify the innate immune response. Wounding also increases the expression of CYP27B1, the enzyme that produces 1,25(OH)2D3. This may occur as a result of increased levels of cytokines such as TNF-α and IFN-γ, both of which we have shown stimulate 1,25(OH)2D3 production, as well as by TGF-β and the TLR2 ligand Malp-2 . When the levels of VDR or one of its principal coactivators, SRC3, are reduced using siRNA technology, the ability of 1,25(OH)2D3 to induce cathelicidin and CD14 expression in human keratinocytes is markedly blunted .
Implications for Bone Disease
A second mechanism by which 1,25(OH)2D3 could reduce bone loss during inflammatory states is by inhibition of TNF/NFkB signaling. Although the RANKL/RANK mechanism clearly dominates osteoclastogenesis, TNF/NFkB plays an important synergistic role. When activated by RANKL, RANK promotes NFkB function both by stimulating the degradation of IkB, the cytoplasmic protein that restricts the various NFkB proteins to the cytoplasm, and by stimulating the processing of the NFkB precursor p100 to the active form p52. These actions of RANK require TRAF6. IL-17 also promotes NFkB activation, and Th17 cells produce TNF in addition to IL-17. Mice double negative for two NFkB proteins, p50 and p52, develop osteopetrosis due to lack of osteoclast development (review in ). Similar results were found in mice lacking IKKβ, the enzyme required to phosphorylate IkB leading to its ultimate proteosomal degradation . As previously discussed, 1,25(OH)2D3 is a potent inhibitor of NFkB signaling as well as Th17 differentiation.
Finally, a third mechanism could involve Treg. These cells produce IL-10, which inhibits osteoclastogenesis by reducing NFAT1c expression and its translocation to the nucleus . NFATc1 is the master regulator of osteoclast formation. Treg cells are found in the synovial fluid of patients with rheumatoid arthritis , perhaps in response to the locally produced 1,25(OH)2D3 by the activated macrophages, DC, and lymphocytes, and thus may serve to suppress the inflammation.
The immune system defends the body against microbial invasion by activation of both adaptive and innate mechanisms. The innate immune system is the more primitive system pre built into cells that are on the front line for defense against bacterial and viral invasion, including epithelial cells in the skin, gut, and lung, as well as macrophages and neutrophils. The adaptive immune system provides a more specific response, but takes longer to develop, although once developed provides a powerful response against invading organisms. Vitamin D, via its active metabolite 1,25(OH)2D3, regulates both types of immunity, suppressing adaptive immunity but potentiating the innate immune response. Suppression of the adaptive immune response is useful in combating a variety of autoimmune diseases, and protecting transplanted organs from rejection. Stimulation of the innate immune response at those surfaces exposed to the environment provides a first line of defense against pathogens in the environment. However, it is now apparent these mechanisms also participate in the regulation of bone remodeling, and when activated during inflammatory diseases lead to bone loss. Central to this process is activation of RANKL/RANK signaling leading to osteoclastogenesis and bone resorption. Although 1,25(OH)2D3 stimulates RANKL/RANK signaling in osteoblasts and osteoclasts, during inflammation it is likely that regulation of osteoclastogenesis is dominated by the cytokines produced by other cells that also promote RANKL/RANK signaling and the RANKL expressed by the inflammatory cells that themselves initiate RANKL/RANK signaling. It is at this level that vitamin D is likely to ameliorate the bone loss incurred during inflammatory arthritis.
This work was supported by grants RO1 AR050023 and AR051930 from the National Institutes of Health, a Merit Review from the Department of Veterans Affairs, and grant 07A140 from the American Institute of Cancer Research.