Clinical Reviews in Bone and Mineral Metabolism

, Volume 7, Issue 4, pp 301–309

Vitamin D Regulation of Immune Function: Implications for Bone Loss During Inflammation

Authors

    • Veterans Affairs Medical Center (111N)
Original Paper

DOI: 10.1007/s12018-009-9056-4

Cite this article as:
Bikle, D.D. Clinic Rev Bone Miner Metab (2009) 7: 301. doi:10.1007/s12018-009-9056-4

Abstract

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.

Keywords

Vitamin DInnate immunityAdaptive immunityMacrophageKeratinocyteOsteoclast

Introduction

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) [2]. However, only DC produce 1,25(OH)2D3 from vitamin D3 suggesting that the CYP2R1 is not functional in the T cells [2]. 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 [2] 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 [5]). 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 [3]. 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 [4].

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 [5]). Epidermal keratinocytes also express CYP27A1 enabling them to produce 1,25(OH)2D3 from endogenous sources of vitamin D3 [6]. UVB radiation, which increases vitamin D and subsequently 1,25(OH)2D3 production in epidermal keratinocytes, suppresses the adaptive immune response mediating contact hypersensitivity [7], while increasing the innate immune response [8]. 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 [7]. I will return to this subsequently. Activation of the innate immune response is due to 1,25(OH)2D3 induced cathelicidin production [8]. 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.

Regulation of CYP27B1 in these cells differs from that of the kidney (Fig. 1). Pulmonary alveolar macrophage production of 1,25(OH)2D3 requires activation by interferon-γ (IFNγ) and tumor necrosis factor-α (TNF), and is inhibited by dexamethasone[9]. The production of 1,25(OH)2D3 by circulating monocytes can be stimulated by IFNγ and other cytokines including TNF, IL-1, and IL-2 [10]. Lipopolysaccharide (LPS) has also been shown to induce CYP27B1 [11]. LPS stimulates through specific toll-like receptors (TLR) in association with the coreceptor CD14, an important trigger of the innate immune response. Such stimulation involves signaling through the JAK/STAT, p38 MAPK, and NFkB pathways, and implicates CEBPβ as a key transcription factor [11]. Like the macrophage, TNF [12] and IFN [13] are potent inducers of CYP27B1 activity in the keratinocyte. In pulmonary epithelial cells double stranded RNA (poly I:C) and the RSV virus, also ligands for specific TLRs, induce CYP27B1 [14], again illustrating the importance of the innate immune response in activating 1,25(OH)2D3 production. Regulation of 1,25(OH)2D3 production in bone cells has received little attention, but given the important role of many of these cytokines in osteoclastogenesis, the role of 1,25(OH)2D3 production by bone cells in this process is worthy of investigation.
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Fig. 1

Comparison of the regulation of CYP27B1 in the kidney to that in the keratinocyte. a CYP27B1 in the kidney is regulated principally by three hormones—PTH, FGF23, and its product 1,25(OH)2D3. PTH stimulates, while FGF23 and 1,25(OH)2D3 inhibit CYP27B1. 1,25(OH)2D3 in turn inhibits PTH production while stimulating that of FGF23. Calcium and phosphate likewise regulate PTH and FGF23 production providing feedback loops that tightly control CYP27B1 activity and maintain normal calcium and phosphate homeostasis. b In the keratinocyte and other extra renal sites of CYP27B1 expression, 1,25(OH)2D3 production is controlled primarily by cytokines such as IFN-γ and TNF-α and activation of toll-like receptors (TLR). Unlike the kidney, 1,25(OH)2D3 regulates its own levels within the cell primarily by induction of CYP24, which catabolizes both the substrate (25OHD3) and product (1,25(OH)2D3) of CYP27B1. In the macrophage, this latter mechanism is lax, and conditions of increased macrophage activation can lead to excess 1,25(OH)2D3 production and hypercalcemia. a Adapted from Fig. 2 in [57]

Role of Vitamin D in the Adaptive Immune Response

The adaptive immune response is initiated by cells specialized in antigen presentation, DC and macrophages in particular, activating the cells responsible for subsequent antigen recognition, T and B lymphocytes (Fig. 2). These cells are capable of a wide repertoire of responses that ultimately determine the nature and duration of the immune response. Activation of T and B cells occurs after a priming period in tissues of the body, e.g., lymph nodes, distant from the site of the initial exposure to the antigenic substance, and is marked by proliferation of the activated T and B cells accompanied by post translational modifications of immunoglobulin production that enable the cellular response to adapt specifically to the antigen presented. Importantly, the type of T cell activated, CD4 or CD8, or within the helper T cell class Th1, Th2, Th17, Treg, and subtle variations of those, is dependent on the context of the antigen presented by which cell and in what environment. Systemic factors such as vitamin D influence this process. Vitamin D in general exerts an inhibitory action on the adaptive immune system. 1,25(OH)2D3 decreases the maturation of DC as marked by inhibited expression of the costimulatory molecules HLA-DR, CD40, CD80, and CD86, decreasing their ability to present antigen and so activate T cells [15]. Furthermore, by suppressing IL-12 production, important for Th1 development, and IL-23 and IL-6 production important for Th17 development and function, 1,25(OH)2D3 inhibits the development of Th1 cells capable of producing IFN-γ and IL-2, and Th17 cells producing IL-17 [16]. These actions prevent further antigen presentation to and recruitment of T lymphocytes (role of IFN-γ), and T lymphocyte proliferation (role of IL-2). Furthermore, suppression of IL-12 increases the development of Th2 cells leading to increased IL-4, IL-5, and IL-13 production, which further suppresses Th1 development shifting the balance to a Th2 cell phenotype. Treatment of DCs with 1,25(OH)2D3 can also induce CD4+/CD25+ regulatory T cells (Treg) cells [17] as shown by increased FoxP3 expression, critical for Treg development [16]. These cells produce IL-10, which suppresses the development of the other Th subclasses. Treg are critical for the induction of immune tolerance [18]. In addition, 1,25(OH)2D3 alters the homing of properties of T cells for example by inducing expression of CCR10, the receptor for CCL27, a keratinocyte specific cytokine, while suppressing that of CCR9, a gut homing receptor [2]. The actions of 1,25(OH)2D3 on B cells have received less attention, but recent studies have demonstrated a reduction in proliferation, maturation to plasma cells and immunoglobulin production [1].
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Fig. 2

1,25(OH)2D3 regulates adaptive immunity. CYP27B1 activity in either the macrophage or keratinocyte is increased by cytokines. The 1,25(OH)2D3 produced then serves to inhibit the adaptive response by suppressing Th1 and Th17 proliferation and function while promoting Th2 and Treg functions. This figure is adapted from Fig. 3 in [57]

1,25(OH)2D3 has both direct and indirect effects on regulation of a number of cytokines involved with the immune response (review in [19]). 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 [20]). 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 [21], experimental colitis [16], and rheumatoid arthritis [21] 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 [15]). 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 [22], 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 [23]. Somewhat surprisingly, RANKL also increases the number and retention of DCs in lymph nodes [24] 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 [25]. 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 [26]. 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 [27]. 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 [2]. 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 [28]. 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 [29]. 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 [30]. 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 [31]. 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 [31]. 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 [32] and toxoplasmosis [33], 1,25(OH)2D3 promotes the infection [33], while the mouse null for VDR is protected [32]. This may be due at least in part to loss of IFNγ stimulation of ROS and NO production required for macrophage antimicrobial activity [32]. Furthermore, atopic dermatitis, a disease associated with increased Th2 activity [34], and allergic airway disease, likewise associated with increased Th2 activity, [2830], 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

The innate immune response involves the activation of toll-like receptors (TLRs) in polymorphonuclear cells, monocytes and macrophages as well as in a number of epithelial cells including those of the epidermis, gingival, intestine, vagina, bladder and lungs (review in [35], Fig. 3). There are 10 functional TLRs in human cells (of 11 known mammalian TLRs). TLRs are an extended family of host noncatalytic transmembrane pathogen-recognition receptors that interact with specific membrane patterns shed by infectious agents that trigger the innate immune response in the host. A number of these TLRs signal through adapter molecules such as myeloid differentiation factor-88 (MyD88) and the TIR-domain containing adapter inducing IFN-β (TRIF). MyD88 signaling includes translocation of NFkB to the nucleus, leading to the production and secretion of a number of inflammatory cytokines. TRIF signaling leads to the activation of interferon regulatory factor-3 (IRF-3) and the induction of type 1 interferons such as IFNβ. MyD88 mediates signaling from TLRs 2, 4, 5, 7 and 9, whereas TRIF mediates signaling from TLR 3 and 4. TLR1/2, TLR4, TLR5, TLR2/6 respond to bacterial ligands, whereas TLR3, TLR7, and TLR 8 respond to viral ligands. The TLR response to fungi is less well defined. CD14 serves as a coreceptor for a number of these TLRs. Activation of TLRs leads to the induction of antimicrobial peptides and reactive oxygen species, which kill the organism. Among those antimicrobial peptides is cathelicidin. Cathelicidin plays a number of roles in the innate immune response. The precursor protein, hCAP18, must be cleaved to its major peptide LL-37 to be active. In addition to its antimicrobial properties, LL-37 can stimulate the release of cytokines such as IL-6 and IL-10 through G protein coupled receptors, and IL-18 through ERK/P38 pathways, stimulate the EGF receptor leading to activation of STAT1 and 3, induce the chemotaxis of neutrophils, monocytes, macrophages, and T cells into the skin, and promote keratinocyte proliferation and migration [36]. The expression of this antimicrobial peptide is induced by 1,25(OH)2D3 in both myeloid and epithelial cells [37, 38]. In addition, 1,25(OH)2D3 induces the coreceptor CD14 in keratinocytes [39]. Stimulation of TLR2 by an antimicrobial peptide in macrophages [40] or stimulation of TLR2 in keratinocytes by wounding the epidermis [39] results in increased expression of CYP27B1, which in the presence of adequate substrate (25OHD) stimulates the expression of cathelicidin. Lack of substrate (25OHD) or lack of CYP27B1 blunts the ability of these cells to respond to a challenge with respect to cathelicidin and/or CD14 production [3840]. In diseases such as atopic dermatitis, the production of cathelicidin and other antimicrobial peptides (AMPs) is reduced, predisposing these patients to microbial superinfections [41]. Th2 cytokines such as IL-4 and 13 suppress the induction of AMPs [42]. Since 1,25(OH)2D3 stimulates the differentiation of Th2 cells, in this disease 1,25(OH)2D3 administration may be harmful. An important role of these AMPs besides their antimicrobial properties is to help link the innate and adaptive immune response.
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Fig. 3

1,25(OH)2D3 regulates innate immunity. CYP27B1 and the VDR in either the macrophage or keratinocyte are induced by activation of TLR by foreign proteins such as the lipopeptide of M. tuberculosis. The 1,25(OH)2D3 produced from either endogenous or exogenous 25OHD3 promotes innate immunity by increasing cathelicidin expression, which kills the invading microorganism. This figure is adapted from Fig. 3 in [57]

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.

Macrophages

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 [43], 56% had undetectable 25OHD levels, and an additional 20% had detectable levels but below 9 ng/ml (22 nM). In 1986, Rook et al. [44] 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. [40] 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 [45]. 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 [46]. 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].

Keratinocytes

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 [48]. 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 [39]. This does not occur in mice lacking CYP27B1 [39]. Unlike macrophages, 1,25(OH)2D3 stimulates TLR2 expression in keratinocytes as well as in the epidermis when applied topically [39] 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 [39]. 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 [48].

Implications for Bone Disease

Inflammatory conditions not only in bone but elsewhere in the body lead to bone loss. In general, this is due to stimulation of osteoclastogenesis, the cell that resorbs bone, although treatment of such diseases with agents such as glucocorticoids also leads to suppression of bone formation (Fig. 4). Central to the stimulation of osteoclast formation and activation is the RANKL/RANK system. This signaling mechanism is the key means by which osteoblasts (expressing RANKL) stimulate osteoclast (expressing RANK) formation and activation. These are members of the super family of TNF ligands and receptors (RANK stands for Receptor Activator of NFkB, and RANKL is its Ligand). Osteoprotegerin (OPG) is a decoy receptor for RANKL, and is a potent inhibitor of RANKL/RANK signaling. Mice null for either RANK or RANKL fail to produce osteoclasts, and develop osteopetrotic bone, whereas mice null for OPG develop osteoporosis [2830]. 1,25(OH)2D3 stimulates RANKL expression in osteoblasts. Thus, one might expect 1,25(OH)2D3 to make the bone loss connected with inflammatory arthritis worse. However, RANKL is expressed in a wide variety of cells including keratinocytes, mammary gland epithelia, heart, skeletal muscle, lung, stomach, placenta, thyroid, brain and, most importantly for this discussion, lymphocytes (review in [49]). Similarly, RANK is also widely expressed, being found in DC including Langerhans cells, skeletal muscle, thymus, liver, colon, small intestine, adrenal glands, as well as osteoclasts (review in [49]). RANK was originally cloned as a factor enhancing DC survival in addition to its role in osteoclast formation. In fact, deletion of RANK or RANKL leads not only to osteopetrotic bone but to the absence of lymph nodes and loss of the B cell follicles in the spleen [50]. The ability of 1,25(OH)2D3 to stimulate RANKL/RANK signaling in these non bone cells is not known. However, it has been hypothesized that 1,25(OH)2D3 plays a role in immune suppression following UV radiation of the skin by inducing RANKL in keratinocytes, which would stimulate the Langerhans cells to induce Treg formation [7]. More to the point a number of cytokines (e.g., IL-1, IL-11, IL-17, TNFα) also stimulate RANKL, and their influence on RANKL production by lymphocytes may be key to the mechanism by which inflammation leads to bone resorption. Th17 is now appreciated as the major T cell subclass producing RANKL and activating osteoclasts (Th1 and Th2 are actually inhibitory) [21]. Furthermore, as mentioned above, IL-17, the product of Th17, is a potent inducer of RANKL in synovial fibroblasts and osteoblasts [51]. Since 1,25(OH)2D3 suppresses Th17 production, this could explain why 1,25(OH)2D3 would more likely suppress the bone loss in these conditions rather than promote it. The inhibition of bone resorption by Th1 cells is related to their elaboration of IFNγ, which leads to the degradation of TRAF6, a key intermediary in RANK signaling in osteoclasts [52]. In that 1,25(OH)2D3 suppresses IFNγ production, this mechanism might be expected to counter the suppression of bone loss by 1,25(OH)2D3. On the other hand, Th2 cells also inhibit osteoclastogenesis, in this case via their elaboration of IL-4 and IL-13, which are associated with increased OPG production and suppression of RANK/RANK signaling [53]. 1,25(OH)2D3 promotes Th2 differentiation.
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Fig. 4

1,25(OH)2D3 regulation of inflammatory bone disease. RANKL/RANK signaling is central to the mechanism by which inflammation activates osteoclastogenesis leading to bone resorption. However, NFkB plays an important synergistic role. Under normal circumstances this process is controlled by osteoblasts, which express RANKL and stimulate the differentiation and function of osteoclasts expressing RANK. 1,25(OH)2D3 stimulates this process by inducing RANKL in osteoblasts. However, during inflammation activated T cells and their cytokines dominate this process. Dendritic cells can induce the differentiation of undifferentiated CD4 T cells into a number of different cell types each with different impacts on osteoclastogenesis. Shown in this figure are three types of T cells. Th17 elaborate IL-17, which promotes RANKL expression in osteoblasts, and TNFa, which activates NFkB in osteoclasts. Th17 also express RANKL, which like the RANKL in osteoblasts induces osteoclastogenesis. Th2 elaborate IL-4 and IL-13, which not only inhibit RANKL expression in osteoblasts but also stimulate OPG production, an inhibitor of RANKL/RANK interaction. Thus, Th2 cells suppress osteoclastogenesis. Treg produce IL-10, which blocks the expression and nuclear translocation of NFAT1c, the master transcription factor for osteoclast differentiation. 1,25(OH)2D3 has an overall dampening effect on osteoclastogenesis by these immune processes. In particular, 1,25(OH)2D3 inhibits the maturation and effectiveness of DC for antigen presentation. Moreover, of the subsets of T cells produced, 1,25(OH)2D3 decreases Th17 production while increasing that of both Th2 and Treg. 1,25(OH)2D3 also inhibits TNFα production and NFkB activation. The origin of 1,25(OH)2D3 within an inflammatory locus is likely to come from the activated DC and T cells themselves, although osteoblasts may also contribute

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 [49]). Similar results were found in mice lacking IKKβ, the enzyme required to phosphorylate IkB leading to its ultimate proteosomal degradation [54]. 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 [55]. NFATc1 is the master regulator of osteoclast formation. Treg cells are found in the synovial fluid of patients with rheumatoid arthritis [56], 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.

Conclusion

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.

Acknowledgments

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.

Copyright information

© Humana Press Inc. 2009