3. Vitamin D and the Immune System: Do It Yourself!

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1 3. Vitamin D and the Immune System: Do It Yourself! Liesbeth Viaene, MD Lut Overbergh, PhD Chantal Mathieu, MD, PhD Case 3-1 A 65-year-old female with a history of type 2 diabetes mellitus, coronary artery disease, congestive heart failure, and renal insufficiency presented to her primary care physician for a checkup. She was found to be normocalcemic (9 mg/dl), with a 25-hydroxyvitamin D [25(OH)D] level of 14 ng/dl and a creatinine level of 1.5 mg/dl. After 7 weeks of supplementation with 50,000 IU of vitamin D weekly, she was repleted. Three months later, she presented with forgetfulness and fatigue. Laboratory evaluation was notable for a calcium level of 17.8 mg/dl, an albumin of 3.8 g/dl, and a creatinine level of 2.6 mg/dl. She was treated with aggressive IV hydration, Lasix, and calcitonin, for the presumptive diagnosis of vitamin D intoxication. Her calcium decreased to 13 mg/dl and her renal function and mental status improved. With discontinuation of the hydration, the calcium increased to 14.8 mg/dl and her symptoms recurred. She was treated with intravenous bisphosphonates and her serum calcium level normalized. Her primary care physician undertook an evaluation for the cause of her hypercalcemia. Her parathyroid hormone (PTH) level was undetectable, as was her parathyroid hormone related protein (PTHrP) level. Serum protein electrophoresis (SPEP) was normal and urinary Bence Jones proteins were negative. Thyroid-stimulating hormone (TSH) was 4.2, 25(OH)D was 9 ng/dl, and 1,25-dihydroxyvitamin D [1,25(OH) 2 D] was 64 pg/ml. During the 2-week course of her workup, her symptoms recurred. She was admitted for treatment and an endocrine consultation was obtained. Her history was otherwise non-contributory. She is a former nurse and a former smoker (20 pack-years) and she does not drink alcohol or use recreational drugs. There is no family history of hypercalcemia, but her husband did have hypercalcemia accompanying sarcoidosis. Translational Endocrinology & Metabolism, Volume 2, Number 3,

2 Her medications include glimepiride, metoprolol, Lasix, and Protonix (pantoprazole). She rarely consumes dairy products and does not take calcium supplements, vitamin supplements, or nutritional supplements. On examination, the patient is alert but disoriented to time and place. She looks her stated age, her body mass index (BMI) is 35, and her vital signs are stable. There is no hirsutism or plethora. The thyroid examination is unremarkable and there are no neck masses. Chest is clear, abdominal examination is benign, and there is no peripheral lymphadenopathy. There is no bruising or rash. Gait is unsteady and there is no proximal muscle weakness. Laboratory evaluation is notable for a creatinine level of 2.1 mg/ dl, with an albumin of 3.4 g/dl, and a calcium of 15.9 mg/dl. The patient is treated with aggressive hydration and her symptomatic improvement correlates with a decrease in serum calcium to 13 mg/dl. Her PTH and PTHrP levels are again undetectable. Levels of 25(OH)D are still below normal (10 ng/dl), and 1,25(OH) 2 D is 107 pg/ml, with an angiotensin-converting enzyme level of 103 U/L (normal 9 67). Chest X-ray and CT scan demonstrate emphysema but no interstitial lung disease and no hilar or mediastinal adenopathy. Abdominal CT is notable for fatty liver and cholelithiasis. There is no retroperitoneal adenopathy or splenomegaly. PET scan is unremarkable. Skeletal survey and bone marrow biopsy are also unremarkable. The patient s hypercalcemia resolves with glucocorticoid therapy and she is discharged on 30 mg of prednisone daily. She is tapered to 5 mg daily and remains normocalcemic at 9.2 mg/dl. Repeated attempts to taper her prednisone to 4 mg daily result in recurrence of her confusion and lethargy, accompanied by hypercalcemia in the range of 13.5 to 14 mg/dl. Physical examination and PET scan 4 months later are unremarkable. Seven months after initial presentation, she is found to have a small left anterior neck lymph node. Imaging studies reveal two nodes: 2.4 x 1.4 cm and 1.9 x 1.4 cm. Analysis of lymph node aspirate reveals few atypical lymphocytes with negative flow cytometry. Because of recalcitrant hypercalcemia, the patient undergoes surgical excision of the nodes and is found to have stage 1A nodular sclerosing Hodgkin lymphoma. After completion of induction chemotherapy, she remains normocalcemic and she is off glucocorticoid therapy. 62 Translational Endocrinology & Metabolism: Vitamin D Update

3 Comment Given the onset of severe hypercalcemia after the initiation of vitamin D therapy, vitamin D intoxication was initially suspected, but the recurrence after cessation of vitamin D, with low serum 25(OH) D levels, makes this possibility unlikely. Cancer and primary hyperparathyroidism are the most common causes of hypercalcemia, but primary hyperparathyroidism rarely causes such a high calcium level. Multiple myeloma is also a possibility, given anemia, renal failure, and hypercalcemia. Hypercalcemia of the degree seen in this patient strongly suggests malignancy, particularly in light of the low PTH level. Although several cancers cause hypercalcemia by producing PTHrP (such as squamous cell carcinoma, renal cell carcinoma, and bladder cancer), this patient had a low PTHrP level, suggesting a cancer that causes hypercalcemia by another mechanism. The hypercalcemia observed in patients with multiple myeloma is caused by the release of cytokines that mediate local osteoclast activation of bone resorption. This patient s normal serum and urinary protein electrophoresis and the absence of urinary Bence Jones proteins argue against this diagnosis. In lymphoma, as well as in granulomatous diseases (e.g., tuberculosis, sarcoidosis, and histoplasmosis), ectopic (e.g., non-renal) 1-alpha-hydroxylation of 25(OH)D results in increased levels of 1,25(OH) 2 D. Therefore, this patient s elevated 1,25(OH) 2 D levels in the presence of severe hypercalcemia suggest increased ectopic 1-alpha-hydroxylase activity, since hypercalcemia normally suppresses both PTH and (renal) 1,25(OH) 2 D synthesis. Ectopic 1,25(OH) 2 D production in macrophages does not result in downregulation of 1-alpha-hydroxylase expression. Thus, increased ectopic conversion of 25(OH)D (in tumor cells or in macrophages) is seen with granulomatous diseases (such as sarcoidosis, Wegener granulomatosis, and tuberculosis) and lymphoma. As this case illustrates, differentiating among these possibilities can be difficult. Vitamin D Metabolism In humans, vitamin D is obtained from two sources: either diet or UVmediated synthesis in the epidermal layer of the skin, where UV rays promote photolytic cleavage of 7-dehydrocholesterol into pre-vitamin D 3, which is subsequently converted by a spontaneous thermal isomeriza- Vitamin D and the Immune System: Do It Yourself! 63

4 tion into vitamin D 3 (1). Vitamin D and its metabolites are steroids and are bound to a carrier molecule, vitamin D binding protein (DBP), for systemic transport (2). Classic vitamin D metabolism, aimed at producing a regulated amount of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ; calcitriol) in the systemic circulation, occurs in the liver and kidney. In the liver, vitamin D is first hydroxylated at the carbon-25 position by 25-hydroxylases. Several cytochrome P450 (CYP) isoforms have been proposed as candidates for accomplishing the hydroxylation step (including mitochondrial CYP27A1 and microsomal CYP2R1, CYP3A4, Before activation, calcitriol and CYP2J3), but CYP2R1 is believed to be needs two hydroxylation steps, the high-affinity 25-hydroxylase (3). Before a 25-hydroxylation by CYP2R1 vitamin D becomes fully activated, a second hydroxylation occurs in the kidney: the hydroxylation by CYP27B1 in the liver and a final 1-alpha- 1-alpha-hydroxylation (CYP27B1) that generates the bioactive metabolite 1,25(OH) 2 in the kidney. Calcitriol D 3 degradation is accomplished by (calcitriol) (4). The main targets for calcitriol the CYP24A1 enzyme. in calcium metabolism are the parathyroids, intestine, kidney, and bone. Biologically active forms of vitamin D are generally short-lived in target cells. Active vitamin D attenuates/limits its function by inducing rapidly, through the vitamin D receptor (VDR), its own metabolism via 24-hydroxylase (CYP24A1). This single enzyme is responsible for a cascade of sequential metabolic processes that lead to a wide array of products with increasing polarity and eventual loss of hormonal activity. In addition to classic vitamin D metabolism, a parallel, local metabolism in tissues not involved in calcium and bone metabolism has been demonstrated. Indeed, the enzymes responsible for the different (activating and degrading) hydroxylation steps can be found in many normal and malignant tissues (Figure 3-1). This implies that important regulation of vitamin D activity can be achieved locally, independent of circulating levels of calcitriol, but highly dependent on circulating vitamin D or 25-hydroxyvitamin D 3 levels. Local production of calcitriol has been described in skin, prostate, and breast tissue. In the present review, we focus on the possible role of locally produced calcitriol in both innate and acquired immunity and its local production in the immune system. Immune Cells as Targets for Active Vitamin D In the 19 th century it was recognized that dry, warm climates and plenty of sunshine were beneficial for patients suffering from tuberculosis, and this finding was the first clue to a possible role for vitamin D in the immune system. 64 Translational Endocrinology & Metabolism: Vitamin D Update

5 DIET vitamin D 2 /D 3 H 3 C H 3 C H 3 C CH 3 CH 3 OH H OH HO H 7-dehydrocholesterol Pre-vitamin D 3 HO 25-hydroxyvitamin D 3 OH HO OH 1,25-dihydroxyvitamin D 3 1,25-dihydroxyvitamin D 3 Endocrine Actions Intestinal Ca absorption Bone metabolism Renal calcium handling Parathyroid glands Autocrine/Paracrine Actions FIG 3-1. Metabolism of vitamin D 3. Synthesis of vitamin D 3 occurs in the skin, where 7-dehydrocholesterol (7-DHC) is converted into pre-vitamin D 3 in response to UV exposure. Vitamin D 3, obtained from pre-vitamin D 3 in the skin or by intestinal absorption of dietary components, binds to vitamin D-binding protein in the circulation. In the liver, vitamin D 3 is hydroxylated by 25-hydroxylase (CYP2R1). The resulting 25-hydroxyvitamin D 3 (25(OH)D 3 ) is then hydroxylated in the kidney or the extrarenal tissue by 1-alpha-hydroxylase (CYP27B1). Modulation of Innate Immunity Innate immunity is responsible for mounting immediate reactions against foreign invaders like bacteria. Central players are monocytes (differentiating into macrophages) and natural killer (NK) cells. Monocytes are derived from hematopoietic stem cells in the bone marrow. Once released in the circulation, they home to different tissues, where they differentiate into macrophages or myeloid dendritic cells (DCs), depending on environmental signals. Macrophages sense pathogen-associated molecular patterns (PAMPs) of various infectious agents by means of pattern-recognition receptors, such as Toll-like receptors (TLRs). Major functions of macrophages are phagocytosis of pathogens, production of antibacterial Vitamin D and the Immune System: Do It Yourself! 65

6 peptides like defensins, and presentation of digested microbial peptides in the context of the major histocompatibility complex II (MHC-II), leading to T-cell activation. There are three major families of antimicrobial peptides in humans: the cathelicidin hcap18 and alpha- and beta-defensins. Calcitriol induces differentiation of monocytes to macrophages (5). Moreover, during differentiation from monocytes to macrophages, cells obtain an enhanced capability to synthesize calcitriol, through increased 1-alpha-hydroxylase expression, in contrast to reduced expression of VDR and 24-hydroxylase (6). CYP27B1 up-regulation is a late phenomenon, allowing macrophages to secrete cytokines that activate and recruit mediators of the adaptive immune system. Calcitriol also enhances the chemotactic and phagocytic capacity of macrophages (7). Furthermore, the antimicrobial actions of calcitriol were recently demonstrated to be mediated via the VDR and to be associated with the up-regulation of the gene for cathelicidin hcap-18. Addition of the active peptide LL37, the active form of cathelicidin cleaved from hcap18, specifically counteracted mycobacterial growth in culture (8). Importantly, TLR activation of monocytes and macrophages (but not DCs) results in up-regulation of CYP27B1 and VDR and leads to the induction of cathelicidin antimicrobial peptide (CAMP) and killing of Mycobacterium tuberculosis (9). The link between vitamin D-triggered antimicrobial activity in monocytes/macrophages and cathelicidin has been confirmed using small interfering RNA (sirna) inhibition of calcitriol-induced CAMP protein production, which resulted in increased mycobacterial growth (10). Initially, hcap was thought to act primarily by disrupting bacterial cell membranes (11). However, recent studies indicate that calcitriol-induced hcap also plays a pivotal role in macrophage autophagy (12). Besides CAMP, the gene encoding the antimicrobial peptide beta-defensin-4 was identified as a direct target for calcitriol (13). However, unlike hcap, induction of beta-defensin-4 gene expression by calcitriol requires simultaneous activation of two nuclear factor kappab (NF-kappaB) sites and the vitamin D response element (VDRE) in the promoter region, through a mechanism involving interleukin (IL)-1 (IL-1) signaling (14). Recently, it has also been demonstrated that calcitriol is a direct and robust inducer of expression of the gene encoding NOD2/CARD15 (IBD1) in cells of monocytic and epithelial origin (15). This gene is involved in the recognition of muramyl dipeptide (MDP), a lysosomal breakdown product of bacterial peptidoglycan common to Gram-negative and Gram-positive bacteria. MDP-induced NOD2 activation stimulates the transcription factor NF-kappaB, which induces expression of beta-defensin-4 (15). Other signaling pathways 66 Translational Endocrinology & Metabolism: Vitamin D Update

7 have also been proposed to participate in the antimycobacterial activities of calcitriol. For example, phosphatidylinositol 3-kinase was found to regulate the antimycobacterial activity of calcitriol by enhancing the generation of reactive oxygen species (ROS) in monocytes and macrophages (16). Also, regulation of inducible nitric oxide synthase (inos) potentially contributes to the antimicrobial effects of calcitriol, but conflicting data have been found: reports of a calcitriol-mediated induction of inos expression in a human macrophage-like cell line are opposed to documented inhibitory actions of calcitriol on this enzyme (17, 18). Strikingly, calcitriol not only promotes the antimicrobial activities of monocytes, but also induces a state of hyporesponsiveness to PAMPs. This effect, which is most prominent after 72 hours, was suggested to be governed by a negative-feedback mechanism, preventing excessive TLR activation and inflammation at a later stage of infection (19). Interestingly, calcitriol was also reported to attenuate the Mycobacterium tuberculosisinduced expression of matrix metalloproteinases (MMP), such as MMP- 7, MMP-9, and MMP-10, by peripheral blood mononuclear cells, while inducing secretion of IL-10 and prostaglandin E2 (PGE2) (20, 21). These findings represent a novel immunomodulatory role for calcitriol in Mycobacterium tuberculosis infection. Calcitriol was also shown to stimulate NK activity through a mechanism involving phosphokinase C and extracellular calcium (22). Modulation of Adaptive Immunity The adaptive immune system is the body s secondary barrier against harm. Adaptive immunity is responsible for a slower response, including immune memory, such as that invoked in defense against viral infections. This arm of the immune system is also responsible for autoimmunity and graft rejection. The central players in adaptive immunity are dendritic cells, as antigen-presenting cells (APCs), and T and B lymphocytes. APCs: Primary Targets for Calcitriol Dendritic cells (DCs) are the main professional APCs and are critical for the initiation of CD4+ T-cell responses. DCs reside in an immature state in the peripheral tissues, where they sample the environment and mediate antigen uptake. When DCs receive a maturation signal, they migrate to the local lymph nodes, where they can provide all the signals necessary for full T-cell activation: presentation of MCH-II-coupled antigen as well as expression of co-stimulatory molecules and secretion of key cytokines, such as IL-12. Vitamin D and the Immune System: Do It Yourself! 67

8 DCs, mainly the myeloid population, were recognized as central targets for calcitriol (23). As already mentioned, DCs themselves are capable of producing calcitriol, whereas they lose VDR expression during maturation, thereby becoming insensitive to calcitriol (24). During DC differentiation, the cells down-regulate the monocyte marker CD14 and up-regulate the DC marker CD1a. Addition of calcitriol completely inhibited the differentiation of CD1a+ DCs, while sustaining the expression of monocyte markers (25, 26). Moreover, activation of VDR signaling pathways also inhibited DC maturation, as evidenced by decreased levels of DC markers, MHC-II, co-stimulatory molecules (CD40, CD80, and CD86), and other maturation-induced surface markers (e.g., CD83) (25 29). Furthermore, calcitriol also modulates DC-derived cytokine and chemokine expression, by Calcitriol-treated DCs have an inhibiting the production of IL-12 and IL-23 immuno-regulatory profile with (major cytokines driving T helper [Th] Th1 a reduced capacity to trigger and Th17 differentiation, respectively), and T-cell proliferation. Moreover, the enhancing the release of IL-10 (a cytokine Th balance shifts towards a Th2 exerting broad-spectrum anti-inflammatory phenotype and the development activities) and the chemokine macrophage of Tregs is favored. inflammatory protein-3alpha (MIP-3alpha) (also known as CCL22, a chemokine involved in the recruitment of C-C chemokine receptor type 4 (CCR4)-expressing regulatory T cells (Tregs) (23, 24, 26 30). Down-regulation of IL-12 results from interference with the NF-kappaB pathway (31). Calcitriol-modulated DCs therefore have a reduced capacity to trigger T-cell proliferation (26, 32). The DC-derived cytokine expression altered by calcitriol changes the Th balance, by limiting inflammatory Th1 and Th17 responses, while skewing the T-cell response toward a Th2-phenotype (26, 29, 33). Importantly, the reduced expression of co-stimulatory molecules and the ability of DCs to produce IL-10 are recognized as tolerogenic features, enabling calcitriol-modulated DCs to favor the development of Tregs with suppressive capacity. Indeed, the ability of VDR agonists to enhance Treg induction in vitro has been observed by different groups (23, 34). In addition, VDR agonists were also shown to enhance the suppressive capacity of Tregs (35). To complicate the picture, microarray analysis by Szeles et al. revealed that the calcitriol-mediated induction of DCs with Treg-inducing capacities results from the autonomous regulation of a set of genes independently from its effects on DC differentiation and maturation (36). Interestingly, Ferreira et al. observed significant differences in the protein profiles of DCs being exposed to a vitamin D analog, TX527, showing major alterations in three specific protein groups, including proteins involved in protein biosynthesis/proteolysis, metabolism, and cytoskeletal structure (37). Such 68 Translational Endocrinology & Metabolism: Vitamin D Update

9 alterations in cytoskeletal proteins not only may contribute to the altered trafficking capacities of calcitriol-modulated DCs toward inflammatory and lymph-node-homing chemokines (28), but also may affect the formation of DC T-cell contacts. Considering the position of DCs at the interface of innate and adaptive immunity, with antigen presentation and T-cell activation as their main functions, modulation of DCs by calcitriol indeed has a major impact on the outcome of T-cell responses. Upon contact with calcitriol, DCs have a reduced capacity to trigger T-cell proliferation, to shift the Th balance toward a Th2 phenotype, and to acquire tolerogenic features favoring the development of Tregs with suppressive capacity. Like DCs, monocytes/macrophages have reduced antigen-presenting and T-cell-stimulatory capacities upon exposure to calcitriol, as demonstrated by the reduced surface expression of MHC-II and co-stimulatory molecules, such as CD40, CD80, and CD86 (7, 38). Furthermore, calcitriol inhibits the expression of inflammatory cytokines in monocytes, including IL-1, IL-6, TNF-alpha, IL-8, and IL-12 (31, 38, 39). Lymphocytes as Direct Targets for Calcitriol When antigens complexed with MHC molecules on the surface of other cells are recognized by the T-cell receptor (TCR), T lymphocytes attack via a variety of cell-mediated adaptive responses. Activation of T cells occurs through the engagement of TCR and co-stimulatory signals. Based on the expression of these co-stimulatory signals, T cells are divided into two subsets, CD4 + and CD8 + T cells. CD8 + T cells are known for their ability to kill virus-infected and malignant cells and therefore are frequently described as cytotoxic T lymphocytes. The majority of CD4 + T cells are categorized as T helper (Th) cells, because they provide help to other immune cells through direct cell cell interactions and the secretion of cytokines. Depending on the signal they receive upon contact with an APC, CD4 + T cells have been shown to differentiate into different subsets, including Th1, Th2, and Th17 cells and adaptive Tregs. Th1 cells are critically involved in the elimination of intracellular pathogens. In contrast, Th2 cells promote humoral immune responses that provide defense against extracellular parasites. Besides Th1 and Th2 cells, the recently discovered Th17 cells constitute an additional T-cell lineage that is involved in host defense against a selection of extracellular bacteria and fungi, mainly at epithelial and mucosal surfaces. Tregs, on the other hand, are indispensable for the maintenance of self-tolerance Vitamin D and the Immune System: Do It Yourself! 69

10 and immune homeostasis, as they paralyze self-antigen-specific T cells that have escaped negative selection in the thymus and also dampen uncontrolled inflammatory responses against foreign antigens. Since VDR expression in T cells is dramatically increased upon T-cell activation, direct actions on T cells are likely to represent an additional or even alternative route for calcitriol to shape T-cell responses. Increased VDR expression can be elicited by various T-cell activation stimuli, including anti-cd3/anti-cd28, as well as by lectin, mitogen, and phytohemagglutinin (PHA), or by triggering more downstream T-cell signaling pathways with PMA/ionomycin (40). Nevertheless, the levels and kinetics of VDR expression seem to vary between the different activation stimuli. In one study, the most physiologically relevant stimulus, anti-cd3/anti-cd28, resulted in elevated VDR levels 8 hours after T-cell activation and levels ultimately peaked 48 hours after activation (40). These findings could provide an explanation for the conflicting results that have been reported regarding the effects of calcitriol on T-cell proliferation (41 44), since the type of activation stimulus was not consistent throughout all the investigations. However, most studies found that calcitriol inhibits the proliferative capacity of human T cells (43 45). Besides the induction of the VDR, T-cell activation was also accompanied by a strong increase in 1-alpha-hydroxylase expression (40). Calcitriol effectively triggered VDR signaling in activated T cells, but introduction of the hormone at a time when VDR is present (maximally at 48 hours after T-cell stimulation) was a prerequisite for efficient induction of 24-hydroxylase, a well-documented VDR target gene that can thus be considered as a read-out for functional activation of VDR-dependent pathways. However, the degree of VDR signaling triggered by calcitriol did not fully reflect the ability of the ligand to interfere with early T-cell cytokine responses, since calcitriol addition at the time of T-cell activation led to an inhibition of cytokine expression similar to that elicited by calcitriol treatment of cells exhibiting maximal VDR levels. This discrepancy needs further research. Possibly, the inability of calcitriol to induce CYP24A1 when it is introduced at basal VDR levels prevents breakdown of the hormone and allows calcitriol to remain intact in the cell culture until activation-induced VDR appears (40). Alternatively, VDR-independent actions of calcitriol could account for this inconsistency (46). Calcitriol also directly alters the cytokine profiles of T cells by inhibiting the production of inflammatory Th1 cytokines, such as IL-2 and interferon (IFN)-gamma, as well as the Th17-derived cytokines IL-17 and IL-21 (33, 41, 47, 48). So far, the direct effects of calcitriol on the production of Th2 cytokines are less clear: some studies show that calcitriol favors the emergence of Th2 cells by up-regulating the 70 Translational Endocrinology & Metabolism: Vitamin D Update

11 expression of the Th2-specific transcription factors GATA-3 and c-maf and concomitant cytokines, including IL-4, whereas other studies contradicted these findings (47 49). A vitamin D analog, TX527, was found to inhibit Th1, Th17, as well as Th2 reactivity of cells (45). At the level of Treg induction by calcitriol, the involvement of tolerogenic DCs does not seem to be a prerequisite, as it was shown that calcitriol, either alone or in combination with dexamethasone, could induce IL-10- producing Tregs in an APC-free in vitro system (41, 50). In this respect, Baeke et al. Calcitriol also directly (without found that TX527 triggered the emergence the interaction with altered of a functional active CD4 + CD25 high APCs) influences T cell behavior. CD127 low Treg phenotype and selectively lt inhibits proliferative capacity induced IL-10 expression within the CD4 + of T cells and favors the T-cell subset (45). Interestingly, Tregs development of active Tregs. induced by calcitriol and dexamethasone expressed high levels of TLR9, and ligand-dependent activation of this receptor abrogated their suppressive capacity, possibly allowing the induced Treg function to be silenced when infectious agents have to be cleared (51). Recently, naive human T cells were shown to have very low expression of VDR and PLC-gammaL. TCR signals through the alternative p38 MAPK pathway induced VDR expression. VDR binds calcitriol and activated the gene encoding PLC-gammaL 48 hours after initial TCR signaling. PLC-gammaL has a central role in classic TCR signaling and T-cell activation (52, 53), leading to much greater proliferation and cytokine production by primed T cells compared to naive T cells (54). The lag phase between antigen recognition and antigen-specific T-cell division imposed by the vitamin D VDR interaction might allow the innate immune system the opportunity to quickly control infection and to diminish the risk of unwanted immunopathology (55). B lymphocytes are responsible for the production and release of highly specific antibodies. Although calcitriol s modulation of Th responses inevitably affects the B-cell compartment, B cells are directly targeted by calcitriol as well. Exposing B cells to calcitriol inhibits their proliferation, inhibits plasmacell differentiation and immunoglobulin secretion (IgG and IgM), inhibits memory B-cell generation, and induces B-cell apoptosis (56). Recently, calcitriol was put forward as an important regulator of lymphocyte trafficking. Active calcitriol imprints activated T cells and terminally differentiating B cells with skin-homing properties via induction of the skin-homing receptor CCR10 (57, 58). In contrast, another study revealed that calcitriol inhibits T-cell surface expression of cutaneous Vitamin D and the Immune System: Do It Yourself! 71

12 lymphocyte-associated antigen (CLA), another receptor directing T cells to the skin. Furthermore, Baeke et al. observed that TX527 profoundly altered the migratory signature of human T cells, not only affecting their skin-homing properties, but also inducing a homing receptor profile that would favor migration to inflammatory sites (40, 45). Vitamin D Metabolism in the Immune System For many years the presence of CYP27B1 in immune cells was suspected, because clinicians were familiar with the findings described in the case presented above. With uncontrolled proliferation or hyperactivation of immune cells (as in lymphoma or sarcoidosis), hypercalcemia, linked to increased levels of calcitriol produced by locally activated immune cells, was indeed frequently observed. The presence of CYP27B1 has been demonstrated in many immune cell types and it is clear that the gene and protein are the same as in the kidney, but that regulation is quite different in the different systems (59). Whereas in the kidney CYP27B1 is tightly regulated by PTH, the hormone does not seem to play a role in the regulation of CYP27B1 expression in immune cells like monocytes/macrophages. In contrast, CYP27B1 expression in immune cells is regulated mainly by immune signals (60). CYP27B1 regulation has been extensively studied in monocytes/macrophages, where it is strongly up-regulated by IFN-gamma, lipopolysaccharide (LPS) (TLR4-ligand), and TLR2/1-complex ligands (9, 61, 62). CYP27B1 is synergistically induced by IFN-gamma in combination with TLR4-ligand (LPS) or phorbol myristate acetate (PMA). This induction was shown to require JAK-STAT, NF-kappaB, and p38 MAPK pathways. In addition, phosphorylation of CCAAT/ enhancer binding protein-beta (C/EBPbeta) by members of the p38 MAPK pathway, as well as direct binding of C/EBPbeta to its recognition sites in the CYP27B1 promotor, are necessary to enable this immune-stimulated up-regulation (61 66). Time course analysis revealed that up-regulation of 1-alpha-hydroxylase during monocyte activation and differentiation is a late phenomenon, preceded by the up-regulation of activating macrophage products such as IL-1 and TNF-alpha (62). This profile of transcription suggests that calcitriol is produced by activated macrophages only after they have had the opportunity to secrete the activating cytokines, allowing for recruitment and activation of other immune cells, such as T and B lymphocytes. This system may act as a negative-feedback loop in order to tone down inflammation (62). CYP27B1 gene expression is also up-regulated by ligands of the TLR2/1 complex on monocytes, including the 19 kda lipoprotein of Mycobacterium tuberculosis (9), indicating that both Gram-pos- 72 Translational Endocrinology & Metabolism: Vitamin D Update

13 itive (TLR4) and Gram-negative (TLR2) bacteria are able to promote local synthesis of calcitriol. The pathways associated with CYP27B1 induction by TLR ligands alone remain unclear, although recent studies using monocytes treated with TLR2 ligand 19 kda lipoprotein have introduced the cytokine IL-15 as an intermediary in localized synthesis of calcitriol (67). CYP27B1 expression in macrophages and DCs is not suppressed by calcitriol, which may explain the massive local production of calcitriol by disease-associated macrophages in patients with granulomatous diseases. In DCs, CYP27B1 levels increase during their maturation. Thus, as DCs integrate pro-inflammatory signals inducing their maturation, they simultaneously enhance CYP27B1 expression and thus calcitriol production. The activation and cooperative interaction of p38 MAPK and NF-kappaB are crucial for DC maturation. Furthermore, these factors are also essential for CYP27B1 expression, because inhibition of these pathways prevented both terminal differentiation and CYP27B1 up-regulation in DCs (68). As in other cells of the immune system, CYP27B1 expression in T lymphocytes is controlled by immune signals. Expression of CYP27B1 is enhanced upon T-cell activation. Interestingly, the expression pattern of CYP27B1 in long-term T-cell cultures closely mimicked the kinetics of VDR expression upon T-cell activation (40), supporting the hypothesis that not only APCs, but also activated T cells, are able to convert 25(OH)D 3 into calcitriol (44, 58). Most immune cells not only express CYP27B1, but also possess the machinery to perform the first hydroxylation step necessary for vitamin D activation, namely, one of the enzymes responsible for 25-hydroxylation. In activated T cells, CYP2R1 expression was demonstrated, and in DCs both CYP27A1 expression and CYP2R1 expression were found (58). Activated T cells generate calcitriol from 25(OH)D 3 but not from vitamin D 3 itself, suggesting that CYP2R1 is not sufficiently expressed or functional to mediate efficient 25-hydroxylation of vitamin D 3 in T cells. DC T-cell co-cultures, as well as DCs alone, however, were shown to be able to convert vitamin D 3 to the fully active form 1,25-dihydroxyvitamin D 3 (calcitriol). Finally, the enzyme responsible for degradation of calcitriol is also present in most immune cells. CYP24A1 catalyses 24-hydroxylation and subsequent inactivation of 25(OH)D 3 and calcitriol. In most tissues, its expression is down-regulated by PTH and up-regulated by phosphate (69, 70). Two vitamin D response elements (VDRE) are present in the promoter of the 24-hydroxylase gene, making 24-hydroxylase highly inducible by calcitriol. In addition, a CCAAT/enhancer binding protein-beta (C/EBPbeta) recognition site has been identified in the CYP24A1 gene promoter. C/EBPbeta, which is by itself up-regulated by calcitriol and VDR, thus cooperates in 24-hydroxylase up-regulation by calcitriol (71). This negative-feedback loop probably serves Vitamin D and the Immune System: Do It Yourself! 73

14 as an internal rescue to avoid excessive calcitriol levels and signaling. In immune cells like monocytes/macrophages and DCs, however, the induction of CYP24A1 by calcitriol depends on the differentiation/maturation stage of the cells. Undifferentiated monocytes are highly susceptible to calcitriol-mediated 24-hydroxylase induction, whereas differentiated/activated macrophages are resistant. The latter is due to an interplay between IFNgamma-mediated and calcitriol-mediated effects: signal transducer and activator of transcription-1alpha (STAT-1alpha), a transcription factor involved in IFN-gamma signaling, interacts with the DNA-binding domain of the VDR, thereby prohibiting binding of the calcitriol/vdr/retinoid X receptor (RXR)- complex to the CYP24A1 promoter and preventing calcitriol-mediated induction of the enzyme (72, 73). Secondly, monocytes/macrophages also express a truncated form of the 24-hydroxylase protein in which the N-terminal mitochondrial-targeting sequence is spliced out (74). Despite being metabolically inactive, the 24-hydroxylase splice variant retains its steroid-binding pocket and is therefore still able to bind substrates like calcitriol or 25(OH)D 3. Furthermore, molecular modeling suggests that truncation of 24-hydroxylase switches substrate preference from calcitriol to 25(OH)D 3 (75). Therefore, the splice variant may act primarily as a decoy to limit the availability of 25(OH)D 3 to other enzymes, notably the 1-alpha-hydroxylase. Remarkably, 24-hydroxylase expression in DCs was only observed when the cells underwent their differentiation process in the presence of calcitriol (68). In addition to local production of calcitriol, the immune system also regulates the local effects of calcitriol by regulating the levels of VDR expression. VDR expression in some immune cells is controlled by immune signals. Whereas naive T cells display only very low VDR levels, the receptor is abundantly present upon T-cell activation (40, 76). By contrast, differentiation of monocytes into either macrophages or DCs is accompanied by a decrease in VDR expression, making these cells less sensitive to calcitriol when they mature (6, 68). Together, the high abundance of receptors for active vitamin D throughout the immune system and their regulation by immune signals argue for an important role for this hormone as a modulator of immune responses. Vitamin D in the Immune System: Proposed Model The importance of vitamin D in the immune system is reflected by the presence in the immune system of the whole machinery necessary for vitamin D activation, with different regulatory mechanisms than in the organs related to calcium and bone metabolism, as well as by the tightly regulated presence of VDR in most immune cells. 74 Translational Endocrinology & Metabolism: Vitamin D Update

15 Considering the timing of the expression of the enzymes involved in calcitriol production and the timing of VDR expression, it is clear that local calcitriol secretion will follow activation of the immune system by a couple of hours (Figure 3-2). One can envisage that when an immune challenge happens, e.g., a viral infection, macrophages and DCs will P A M P Antimicrobial response Chemotaxis Phagocytosis Cathelicidin INNATE IMMUNITY Defensin β4 25(OH)D 3 Reactive oxygen species IL-12 1αOHase IL-6 TNF-α 1αOHase 25(OH)D 3 IL-23 TLR2/1 Mo TLR4 Mo/M IL-10 CCL22 DC CD80/86 MHC II CD40 target tissue Nº Tc IL-1 IL-6 TNF-α M 1,25(OH) 2 D 3 IFN-γ Th1 Th1 IL-2 Th1 IL-17 Th17 Th17 TCR CD40L CD28 CD4+T IL-4 IL-5 IL-13 IL-10 TGF-β Th2 Treg ADAPTIVE IMMUNITY Th2 Th2 Treg FIG 3-2. The immunomodulatory effects of 1,25-dihydroxyvitamin D 3. Production of calcitriol (1,25-dihydroxyvitamin D 3 ; 1,25(OH) 2 D 3 ) in macrophages starts 24 hours after initial monocyte stimulation by PAMPs. Calcitriol targets different components of the innate and adaptive immune systems. Calcitriol stimulates innate immune responses by enhancing the chemotactic and phagocytotic functions of macrophages as well as the production of antimicrobial proteins such as cathelicidin. On the other hand, calcitriol also modulates adaptive immunity. At the level of the APC (like the DC), calcitriol inhibits the surface expression of MHC-II-complexed antigen and of co-stimulatory molecules, in addition to production of the cytokines IL-12 and IL-23, thereby indirectly shifting the polarization of T cells from a Th1 and Th17 phenotype toward a Th2 phenotype. In addition, calcitriol directly affects T-cell responses by inhibiting the production of Th1 cytokines (IL-2 and IFN-gamma) and Th17 cytokines (IL-17 and IL-21) and by stimulating Th2 cytokine production (IL-4). Moreover, calcitriol favors Treg cell development via modulation of DCs and by directly targeting T cells. Finally, calcitriol blocks plasma cell differentiation, IgG and IgM production, and B-cell proliferation. Mo/Ma: monocyte/macrophage, 1alphaOHase: 1-alpha-hydroyxlase Vitamin D and the Immune System: Do It Yourself! 75

16 first become activated, with secretion of proteins and peptides necessary for combating the attack, as well as expression of co-stimulatory molecules and cytokines/chemokines necessary for attracting and activating other immune system cells. After some time, CYP27B1 levels will be high enough to lead to calcitriol production (provided levels of circulating 25(OH)D 3 are sufficient), which will provide feedback to the macrophages and DCs themselves, thus altering their function (improving chemotaxis, phagocytosis, and microbial killing and decreasing antigen presentation). Once the other partners of the immune system have arrived (T lymphocytes, B lymphocytes), they will be activated and also start to express VDR. After (several rounds of) activation, these cells will also become sensitive to the surrounding calcitriol they will even make some themselves leading to altered behavior (toward tolerogenic rather than activated profiles). Feedback in all these immune cells by calcitriol itself will switch on 24-hydroxylase, thus leading to degradation of calcitriol and limitation of its effect. In normal circumstances, this is a tightly regulated system, but in certain circumstances, it can go wrong. For example, the system cannot work when not enough vitamin D (or 25(OH)D 3 ) is circulating, as in vitamin D deficiency. When no precursor is present, the immune cells cannot make calcitriol, potentially causing a missing link in the control of immune reactions. The other extreme where the system can go wrong is when feedback loops malfunction. The activated CYP27B1 enzyme does not respond to feedback by calcitriol in macrophages, in particular in the presence of IFN-gamma. Because IFN-gamma also inhibits CYP24A1, inflammatory processes leading to hyperactivation of macrophages and local overproduction of IFN-gamma can lead to local overproduction of calcitriol with systemic spillover, as in the patient described above. Therapy consists of elimination of the overwhelming macrophage activation and IFN-gamma production, typically by high doses of corticosteroids. Clinical Evidence for a Role of Vitamin D as Modulator of the Immune System For many years, a relation between UV exposure and immune defense has been described, with both a direct (immune suppressive) effect of UV in the short term as well as the effects of UV exposure on repletion of vitamin D reserves being involved. Vitamin D sufficiency or deficiency has important effects on defense against bacteria and viruses, with Mycobacterium tuberculosis being the best-known example. A higher 76 Translational Endocrinology & Metabolism: Vitamin D Update

17 susceptibility to tuberculosis is seen in individuals with relatively low serum vitamin D levels, including the elderly, uremic patients, and dark-skinned people (77). A meta-analysis of seven observational studies noted a reduced risk of active tuberculosis in those with the highest levels of calcitriol (OR: 0.68; 95% CI ) (78). Associations between low 25(OH)D 3 levels and frequency of ordinary respiratory tract infections have been demonstrated in Finnish and American populations (79). In observational data from NHANES III, persons with 25(OH)D 3 values lower than 10 ng/ml were more likely to have had a recent upper respiratory tract infection than those with higher 25(OH)D 3 in all four seasons of the year. This association was even stronger in those with asthma or chronic obstructive pulmonary disease (80). Cross-sectional comparisons of vitamin D levels in HIV-infected patients compared to HIV-uninfected controls showed significantly lower levels of calcitriol in HIV-infected patients (81, 82). Calcitriol levels were lowest in symptomatic patients, independent of the presence of opportunistic infections (82). In addition, a positive correlation was observed between serum calcitriol and CD4 cell count (81, 82). However, differences in serum 25(OH)D 3 levels between HIV-infected patients and controls were not consistent between studies (81 83). A prospective analysis revealed that low vitamin D levels at baseline were significantly associated with increased risk of HIV disease progression and severe anemia in HIV-infected Tanzanian women (84). Women in the highest quintile of vitamin D also had a significantly lower risk of all-cause mortality; no association was observed between vitamin D status and AIDS-related mortality or T-cell count. The proposed relation between circulating vitamin D levels and immune defense is further confirmed by experimental studies investigating the consequences of impaired vitamin D signaling on immune function. Disease progression following infection with Mycobacterium tuberculosis was severely aggravated when mice were rendered vitamin D deficient (85). Lack of vitamin D resulted in impaired macrophage functions, with defective chemotaxis, phagocytosis, respiratory burst capacity, and proinflammatory cytokine production, all of which are essential for macrophage antimicrobial activity (86, 87). To understand the effects of vitamin D status on the adaptive immune system, and a possible role in the onset of autoimmune diseases in humans, we are mainly dependent on epidemiological data linking latitude (and thus UV exposure and vitamin D status) or vitamin D status itself to the prevalence of diseases. Different epidemiological studies report an inverse correlation between vitamin D status and the incidence of autoimmune Vitamin D and the Immune System: Do It Yourself! 77

18 diseases, such as type 1 diabetes (T1D), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), and rheumatoid arthritis (RA) (88 92). For example, a considerable percentage of the population living in more northern latitudes of the Northern hemisphere (and thus receiving less UV radiation) is vitamin D-deficient, and the deficiency positively correlates with higher incidences of autoimmune diseases. Also, like the seasonal variation in serum levels of 25(OH)D 3, the onset and exacerbation of different autoimmune diseases have been documented to vary with seasonality. Furthermore, patients suffering from different autoimmune diseases, such as MS, SLE, RA, and T1D, display lower serum 25(OH)D 3 levels than healthy individuals. In the context of type 1 diabetes, a Finnish birth cohort study revealed a threefold increase in disease incidence in individuals who were vitamin D-deficient during early life (93). In animal models of various autoimmune diseases, vitamin D deficiency profoundly affects disease incidence and severity [reviewed in (92)]. For example, in non-obese diabetic (NOD) mice (a mouse model spontaneously developing T1D with a pathogenesis similar to the human disease), vitamin D deficiency during early life resulted in more aggressive disease manifestations and a higher incidence (87). Administration of 1,000 IU of regular vitamin D 3 (intraperitoneally) in early life to vitamin D-sufficient NOD mice did not prevent diabetes development, although pancreatic insulin content was higher in treated mice than in controls (94). Possibly, higher doses, different routes of administration, or a longer time-frame of supplementation with regular vitamin D may be required to effect diabetes prevention. Therapeutic Implications Protective effects of vitamin D supplementation have been described for colds and influenza (95). Several case series have reported that daily doses of 625 mcg to 2.5 mg of vitamin D improve patients response to antimicrobial treatment for pulmonary tuberculosis (96). Randomized controlled trials investigating doses of up to 125 mcg/day of vitamin D or equivalent in active tuberculosis have shown no clinical benefit (97 101). Although promising results were obtained in a few clinical trials, there is currently a lack of non-biased, large-cohort studies that support the proposed benefits of vitamin D supplementation for optimal immune function. Small sample sizes, short follow-up duration, and a lack of control groups constitute major limitations of the reported studies. In addition, different doses of vitamin D have been employed, and the initial vitamin D status of the individuals included was not always 78 Translational Endocrinology & Metabolism: Vitamin D Update

19 known, making it unclear whether the administered vitamin D supplements restored existing deficiencies or augmented circulating vitamin D levels in already sufficient individuals. Supplementation studies have also been conducted in the context of autoimmune diseases. With regard to T1D, distinct studies have found that supplementation with regular vitamin D in early life is associated with a lower risk of disease onset. In 1999, the results of a largescale study sponsored by the European Community were published: the Concerted Action on the Epidemiology and Prevention of Diabetes showed a 33% reduction of T1D in children who received vitamin D supplementation early in life (102). In accordance with these results, Hypponen et al. also found that the risk of T1D development was significantly reduced when high doses of vitamin D supplementation (up to 2,000 IU/day) were given during infancy (93). Furthermore, a metaanalysis of data from four case-control studies and one cohort study supports the beneficial effects of vitamin D in T1D prevention, since infants receiving vitamin D supplementation showed a 29% reduction in disease onset (103). Over all, these studies suggest that vitamin D-mediated diabetes protection may be dose-dependent, with individuals receiving higher amounts of vitamin D having a lower risk of developing T1D. On the other hand, some studies did not find a correlation between T1D prevention and vitamin D supplementation. In Norway, intake of cod-liver oil by children younger than 1 year old did not have significant effects on T1D prevention, although there was a tendency for a negative association between cod liver oil intake and diabetes development (104). More recently, a study in Sweden with 1- to 2.5-year-old children who received vitamin D supplementation could not find a correlation between supplementation and development of diabetes-related autoantibodies (105). However, despite the fact that some studies failed to show an association between the reduction of T1D risk and vitamin D supplementation during infancy, none of them found any association with an increased risk. Conclusion Vitamin D appears to have a physiological role in immune regulation. In vitro as well as in vivo data on the detrimental effects of vitamin D deficiency on immune function support this concept. When considering vitamin D levels and supplementation policies, the immunologic role of vitamin D should be taken into account. Vitamin D and the Immune System: Do It Yourself! 79