1 Vitamin D synthesis and sufficiency

Vitamin D is obtained from limited dietary sources or photochemical and thermal conversion of 7-dehydrocholesterol in skin [1]. ‘Vitamin D’ refers collectively to vitamin D3 (cholecalciferol), derived from metabolism of cholesterol, and related vitamin D2 (ergocalciferol), derived from the fungal steroid ergosterol. In animals, photochemical and thermal conversion of cutaneous 7-dehydrocholesterol occurs in the presence of solar ultraviolet B (~300–325 nm) radiation, which varies with time of the year, latitude, altitude and cloud cover. At 45° latitude (south of many of the major population centers of Europe) near sea level, UVB radiation intensity is insufficient for vitamin D synthesis for ~6 months of the year. This period, known as vitamin D winter, lengthens with increasing distance from the equator [1].

Dietary or cutaneous vitamin D must undergo two modifications to become biologically active. Hepatic hydroxylation catalyzed by CYP27A1, CYP2R1 and possibly other enzymes generates 25-hydroxyvitamin D (25D; refs. [2–5]), which has a half-life of several weeks and represents the major circulating metabolite and measure of vitamin D status. 25D is converted by 1α-hydroxylation catalyzed by CYP27B1 into the hormonally active 1,25-dihydroxyvitamin D (1,25D). The kidney is an important site of 1α-hydroxylation, which is stimulated by the calcium regulatory hormone parathyroid hormone (PTH). 1,25D released into the circulation from the kidney was long considered to be an important endocrine source of hormone. However, research in the last 10 years has revealed that CYP27B1 is widely expressed [6, 7], and, moreover, that 1,25D is produced locally in many tissues for function in an intracrine or paracrine manner. In a negative feedback loop, 1,25D strongly induces expression of the gene encoding CYP24, the enzyme that initiates catabolic degradation by catalyzing hydroxylation of 25D or 1,25D at the 24 position to produce biologically inactive metabolites.

The levels of circulating 25D consistent with vitamin sufficiency are a subject of considerable debate. Vitamin D deficiency and insufficiency are widely defined as circulating 25D levels of less than 20 ng/ml and 20–30 ng/ml, respectively (50 nM and 50–75 nM; refs. [3, 8–10]). Many researchers have long considered that circulating 25D concentrations of 30–32 ng/ml (75–80 nM; refs. [11–13]) or greater represent vitamin D sufficiency, as there is an inverse relationship between 25D levels and circulating PTH until 25D rises above 30 ng/ml, at which point PTH levels bottom-out. However, a recent Institute of Medicine (IOM) report defined insufficiency as circulating 25D levels of <20 ng/ml, but considered levels of 20–50 ng/ml as being ‘normal’, while >50 ng.ml (125 nM) was deemed ‘excessive’ [14]. Based on these definitions, the report concluded (controversially; [15, 16]) that much of the population was not vitamin D deficient or insufficient. Moreover, the report considered that, apart from bone health, there was insufficient experimental evidence for the importance of other physiological actions of vitamin D to inform guidelines for recommended dietary intake, due substantially to a lack of randomized, placebo-controlled clinical trials examining non-bone endpoints. However, as detailed below, studies in the last few years have shown that cells of the immune system are ‘wired’ to synthesize and respond to 1,25D upon detection of an infection. Importantly, clinical evidence from intervention trials for the protective effects of vitamin D sufficiency against infectious diseases is beginning to accumulate. Finally, while vitamin D intoxication does occur, it is rare and not observed until 25D levels of >150 ng/ml (375 nM; [3]). It is characterized by hypercalcemia, which if chronic leads to urinary calculi (renal or bladder stones) and renal failure.

2 The vitamin D receptor

1,25D binds and activates the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors, which are ligand-activated transcription factors [17, 18]. Much of vitamin D physiology can be explained by the VDR functioning as a regulator of gene transcription, and expression profiling studies, particularly with microarrays, have been a valuable source of insights, particularly into novel aspects of vitamin D physiology [19–21]. The VDR is composed of a highly conserved DNA binding domain, and an α-helical ligand binding domain [18, 22]. In the presence of 1,25D, the VDR heterodimerizes with retinoid X receptors (RXRs), which are required for recognition of cognate vitamin D response elements (VDREs), located in the regulatory regions of 1,25D target genes.

VDREs are composed of direct repeats of PuG(G/T)TCA motifs separated by 3 bp (DR3) or everted repeats with 6 bp spacing (ER6; refs. [18, 23–25]). ER8 motifs are also recognized by the VDR and related retinoic acid receptors, and contribute to regulation by 1,25D of the gene encoding the cytokine interleukin 10, for example [26, 27]. DNA-bound VDR/RXRs sequentially recruit complexes of so-called coregulatory proteins, which stimulate histone modifications, chromatin remodeling, and RNA polymerase II binding necessary for transcriptional initiation [28, 29]. While numerous VDREs have been identified in relatively promoter proximal locations [20], recent work has provided evidence that the DNA-bound VDR can function at distances as great as 75 kb to regulate adjacent target gene transcription [30, 31]. The VDR can also repress transcription in the presence of 1,25D. VDR/RXR heterodimers can displace DNA-bound nuclear factor of activated T cells (NF-AT), thus repressing cytokine gene expression [32]. We recently, found that the ligand-bound VDR repressed transcription of the gene encoding cyclin D2 via its interaction with FoxO transcription factors [33].

3 Overview of vitamin D action in the immune system

Links between vitamin D or solar therapy and treatment of infectious diseases go back millennia. Hippocrates, the father of medicine, apparently used heliotherapy, or exposure to sunlight, to treat phthisis (tuberculosis; TB) [34]. Sun exposure re-emerged as a popular therapy for treatment of TB in pre-antibiotic era, dating from the mid 19th century with the spread of sanitoria. In addition, Niels Finsen won the 1903 Nobel Prize for the demonstration that UV light could treat cutaneous TB (lupus vulgaris). Cod-liver oil, an excellent source of vitamin D (and vitamin A), first described for treatment of chronic rheumatism in the 18th century, was used as early as 1849 to treat TB, and scrofula, a cervical tuberculosis lympadenopathy arising from infection of lymph nodes of the neck by M. tuberculosis (M.tb.) or related mycobacteria [35–37]. Finally, studies in the 1980’s showed that 1,25D inhibits the growth of M.tb. in cultured human macrophages [38], providing the first evidence for direct stimulation by 1,25D of innate immune responses to M.tb. infection.

A number of clinical studies have found associations between either vitamin D deficiency or specific restriction fragment length polymorphisms in the VDR gene, and susceptibility to TB or other infectious diseases, particularly respiratory tract infections of bacterial or viral origin (reviewed in [39]). Two recent intervention trials are notable additions in this regard. Martineau and colleagues investigated the effect of high-dose vitamin D3 on time to sputum culture conversion in patients undergoing antibiotic therapy for pulmonary TB [40]. Four doses of 2.5 mg (100,000 IU) of vitamin D3 (62 patients) or placebo (64 patients) were administered at 14-day intervals. Vitamin D therapy markedly raised mean serum 25D concentrations at 56 day (101.4 nM in the intervention group vs. 22.8 nM in the placebo group). Therapy did not have a statistically significant effect on the time to sputum culture conversion in the study population as a whole 36 day (vitamin D) vs. 43.5 d (placebo) (adjusted hazard ratio 1·39, 95% CI 0·90–2·16; p = 0.14), but it did significantly reduce the time to sputum culture conversion in participants with the VDR tt genotype (8·09, 95% CI 1·36–48·01; p = 0·02) [40]. The authors concluded that vitamin D therapy should benefit at least subset of vitamin D-deficient patients with pulmonary TB and recommended further studies to investigate the apparently selective response in patients with the tt VDR polymorphism [40].

A recently published randomized double-blind placebo-controlled trial provided strong evidence that vitamin D supplementation would be of benefit in prevention of seasonal influenza A infections [41]. School children in Japan were supplemented with 1,200 IU of vitamin D3 or placebo from December to March. 10.8% of the treatment group (18/167) came down with influenza, whereas 18.6% (31/167) of the placebo group became ill (relative risk, 0.58; 95% CI: 0.34, 0.99; P = 0.04). The reduction was even more prominent in children who had not been taking vitamin D supplements prior to the trial [41]. These results clearly suggest that vitamin D supplementation may be effective in preventing influenza, particularly in vitamin D-deficient children. It will be important to investigate the molecular mechanisms underlying the beneficial effects of therapy on viral infections. It is possible that vitamin D signaling augments innate immune responses to viral infection directly. However, the effects may be indirect. For example, an intriguing animal study suggested that robust innate immune control of intestinal microbiota may influence the severity of pulmonary influenza infections. Mice treated with multiple antibiotics to eliminate commensal intestinal bacteria exhibited strongly reduced T-cell responses to sub-lethal doses of inhaled A/PR8 influenza virus relative to controls, along with increased viral titers [42]. Remarkably, local or distal administration of bacterial lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), a pattern recognition receptor that drives innate immune responses to bacterial infection, attenuated the impairment in antibiotic-treated mice [42]. Given the increasingly well-established roles of vitamin D signaling in anti-bacterial innate immunity in humans (see below), this model study may provide insights into the mechanisms of action by which vitamin D boosts anti-viral immune responses.

4 Vitamin D signaling in T cells

We now know that the VDR and CYP27B1 are expressed in several cell types in the immune system, including in T lymphocytes, neutrophils and antigen presenting cells such as macrophages and dendritic cells, and that 1,25D signaling modulates both innate and adaptive immune responses [43–46]. 1,25D signaling regulates the function and phenotype of dendritic cells, the most potent of the antigen presenting cells. 1,25D enhances dendritic cell tolerogenicity [45], which promotes the production and function of T regulatory (Treg) cells, critical mediators of immune system tolerance. 1,25D also acts directly on T lymphocytes to inhibit their proliferation [43, 46]. 1,25D signaling represses the transcription of genes encoding key T helper 1 (Th1) proinflammatory cytokines, such as interferon-γ (IFN-γ) and interleukins 17 and 21 [46, 47]. T cells treated with 1,25D also acquire the characteristics of Treg cells [47]. Thus, 1,25D signaling directly and indirectly acts to suppress antigen presentation to, and activation and recruitment of Th1 cells, instead favoring a more regulatory T cell phenotype.

The effects of 1,25D on T cell phenotypes would be consistent with the increasing evidence that vitamin D sufficiency acts to suppress T cell-driven autoimmune diseases. Vitamin D or 1,25D analogues are used widely for the treatment of the disfiguring and inflammatory condition psoriasis [48], which is increasingly considered to have a T cell-driven autoimmune component [49, 50]. Several epidemiological studies have reported inverse correlations between circulating 25D levels and risk of other autoimmune conditions such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes [46, 51]. In particular, there are links between vitamin D deficiency in infancy and early childhood and increased risk of type-1 diabetes [46, 52]. While these findings are compelling, several authors have argued (e.g. [51]) that prospective studies are needed to more firmly establish links between vitamin D deficiency and autoimmunity.

Recent work has revealed that 1,25D signaling also regulates T cell antigen receptor function. Engagement of the T cell antigen receptor on naïve human T cells led to p38 MAP kinase-dependent stimulation of VDR expression, which in turn strongly induced expression of phospholipase C-γ1, a cofactor of the classical T cell antigen receptor signaling pathway, within 48 h of initial T cell receptor stimulation [53]. The authors speculated that the 48 h lag might be an evolutionarily conserved mechanism to prevent explosive T cell proliferation in the presence of antigen [53]. If innate immune responses quickly control infection during the lag period, the onset of T cell division would take place in a controlled manner in a relatively uninflammatory microenvironment characterized by limiting antigen concentrations. In the absence of a lag or under conditions of insufficient innate immune response and uncontrolled infection, elevated antigen concentration would produce a more proinflammatory microenvironment and more aggressive T cell proliferation. Given, as detailed below, that 1,25D signaling enhances innate immune responses to infection, the lag in T cell activation would represent a coordinated strategy to decrease the potential for T cell-driven immunopathology.

5 Vitamin D is an inducer of innate antimicrobial immune responses

The most rapidly evolving area of research into the immunomodulatory properties of vitamin D signaling has been in our understanding of the molecular mechanisms by which vitamin D metabolism and 1,25D signaling are implicated in innate immune responses. As indicated above we have known for over 20 years that 1,25D reduces M.tb. viability in infected macrophages [38]. We have also known since the early ‘90’s that 1,25D strongly induces expression of CD14, a co-receptor critical for recognition of LPS by TLR4 [54].

In 2003, we mapped the positions of promoter-proximal consensus VDREs in the human genome. This in silico screen identified VDREs adjacent to the transcription start-sites of two genes encoding antimicrobial peptides (AMPs) β-defensin 2 (DEFB2/DEFB4/HBD2) and cathelicidin antimicrobial peptide (CAMP/LL37) [55]. AMPs along with various cytokines and chemokines are the first-responders of the innate immune attack against invading pathogens [56–58]. CAMP and some β-defensins also have dual functions as chemoattractants for neutrophils, monocytes and other cellular components of immune responses [56–58]. We found that CAMP expression was strongly stimulated by 1,25D in all cell types examined [55], a finding subsequently borne out in several in vitro and in vivo studies [59–62]. One of the more interesting in vivo studies examined CAMP regulation in the biliary tract, which is normally microbe-free. D’Aldebert and colleagues founds that CAMP expression in biliary epithelial cells was regulated by physiological concentration of bile acids signaling through the VDR and a related nuclear receptor for bile acids, FXR [63]. The potential role of the VDR in this signaling is consistent with previous molecular studies that the VDR can act as a bile acid sensor [64]. Notably, neither the CAMP nor the HBD2 VDRE is conserved in mice, and Gombart and coworkers showed that the CAMP VDRE is embedded in a human/primate specific Alu repeat transposable element [59]. The insertion of the VDRE-containing Alu repeat in the CAMP gene originated in the primate lineage leading to humans, apes, and Old World and New World monkeys [65], which dates the event back 55–60 Ma.

While 1,25D alone was sufficient for strong induction of CAMP expression in cell-based experiments, its effect in isolation on HBD2 expression was modest [54] or not detected [60]. However, 1,25D enhanced 2-fold the strong induction of HBD2 by IL-1β [54]. Subsequent work showed that signaling through TLR1/2 pattern recognition receptors in human monocytes induced IL-1β expression and signaling, and that the combination of IL-1β and 1,25D were required for strong induction of HBD2 expression [66]. IL-1β signaling was very likely mediated by induced binding of the NF-κB transcription factor to tandem binding sites in the HBD2 proximal promoter ([66]; Fig. 1).

Fig. 1
figure 1

Regulation of expression of the gene encoding antimicrobial peptide DEFB2/DEFB4/HBD2 by signaling through pattern recognition receptors and the VDR. As described in the text, the 1,25D-bound VDR can induce expression of the gene encoding pattern recognition receptor NOD2/CARD15 through binding to distal VDREs. NOD2 activated by binding of muramyl dipeptide (MDP) arising from breakdown of bacterial peptidoglycan can activate HBD2 expression through promoter-proximal NF-κB binding sites. Similar autocrine signaling by IL-1β, whose expression is induced by signaling through TLR2/1, also activates HBD2 expression via NF-κB. See text for details

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The NF-kB binding sites in the HBD2 promoter represent an important hub of innate immune signaling. We recently found that the 1,25D-activated VDR directly induces expression of the gene encoding pattern recognition receptor NOD2/CARD15 (nucleotide oligomerization domain protein 2/caspase recruitment domain-containing protein 15) [67]. NOD2 is member of a class of pattern recognition receptors that is structurally distinct from the TLRs. It is an intracellular protein that is activated by muramyl dipeptide (MDP), a lysosomal breakdown product of bacterial peptidoglycan. 1,25D strongly induced NOD2 expression in human myeloid and epithelial cells via VDR binding to at least two distal high-affinity VDREs. Similar to IL-1β, NOD2 signaling induces NF-κB function and enhances HBD2 expression [68] (Fig. 1). We found that the combination of pretreatment with 1,25D to induce NOD2, followed by addition of MDP synergistically induced HBD2 expression [67].

The induction of the NOD2-HBD2 innate immune pathway by 1,25D is significant for several reasons. Importantly, attenuated or disrupted expression of NOD2 or HBD2 is associated with an enhanced risk of development of Crohn’s disease (CD), a chronic inflammatory condition [69, 70]. While often mislabeled as an autoimmune disease [71], CD likely arises from a defect in innate immune handling of intestinal bacterial load, which leads to intestinal inflammation in the absence of autoimmunity [72, 73]. The epithelial lining of the colon is a site of strong CYP27B1 expression [15], and vitamin D deficiency is associated with active disease, although this may arise through defective intestinal absorption. Rates of CD apparently increase with increasing latitude in Europe and North America [74, 75], suggestive of a contribution of vitamin D insufficiency/deficiency, although data concerning seasonal variations in disease relapse rates are conflicting [76–78]. There are also polymorphisms in the VDR gene that correlate with susceptibility to CD [79]. The direct and indirect regulation by 1,25D of the NOD2-HBD2 innate immune pathway strongly suggests that 1,25D deficiency or insufficiency may indeed contribute to the pathogenesis of CD, and provides further evidence that 1,25D signaling is important for optimal innate immune responses.

6 Intracrine 1,25D signaling driven by immune system regulation of CYP27B1 expression

Rather than acting predominantly as a renal endocrine hormone, it is likely that most of the physiological actions of 1,25D are mediated by locally produced hormone acting in an intracrine or paracrine manner. Nowhere is this truer than in cell types critical for innate immune responses. Several studies have documented the role of 1,25D in inducing monocytic differentiation [80]. Notably, both VDR expression and the capacity of monocyte-derived macrophages and dendritic cells to produce 1,25D from 25D are developmentally regulated [80–82]. The pathophysiological consequences of excessive macrophage 1,25D biosynthesis are manifested in sarcoidosis, a granulomatous inflammatory disease, in which elevated 1,25D can lead to hypercalcemia in extreme cases [83].

The last few years have seen a number of interesting reports detailing the immune signaling pathways leading to the induction of CYP27B1 expression, particularly in macrophages. Expression profiling studies of human macrophages revealed that activation of TLR1/2 toll-like receptor heterodimers with 19 kDa lipopeptide stimulated expression of genes encoding CYP27B1 and VDR ([60]; Fig. 2). Other work showed that LPS signaling through TLR4 receptors also induced CYP27B1 expression [84], our unpublished results), consistent with correlations between expression levels of TLR4 and CYP27B1 [85, 86]. In human macrophages cultured in human serum, downstream VDR-driven induction of CAMP expression observed upon TLR2/1 stimulation was strongly dependent on serum 25D concentrations [60]; responses were markedly attenuated in macrophages cultured in serum from vitamin D-deficient individuals. Importantly, VDR-regulated gene expression could be restored by supplementation with 25D. Notably, consistent with other studies [87, 88], serum levels of 25D in African Americans were approximately one half those of Caucasian Americans [60], a difference likely arising from reduced UVB-induced cutaneous synthesis of vitamin D3 in darker African-American skin. Subsequent work showed that IL-15 expression induced by TLR1/2 signaling was required for CYP27B1 induction [89] (Fig. 2). These findings also provided a mechanistic basis for the previously observed increased expression of CYP27B1 expression during macrophage development, as IL-15 is an inducer of monocyte-macrophage differentiation. This work is important because it reveals that naïve macrophages acquire the capacity to respond to circulating 25D upon detection of a microbial infection, and suggests that downstream VDR-driven gene expression is proportional to the levels of 25D throughout the physiological range.

Fig. 2
figure 2

Regulation of vitamin D metabolism and intracrine signaling by cytokine and pattern recognition receptors. Signaling through TLR2/1 induces expression of IL-15, which in turn induces expression of CYP27B1. CYP27B1 expression can also be induced by signaling through TLR4. Elevated intracrine production of 1,25D activates the VDR, which induces expression of genes encoding AMPs such as CAMP, pattern recognition receptor NOD2/CARD15, or TLR4 coreceptor CD14

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More recent studies have provided evidence that T cell cytokines can influence TLR-regulated production of CYP27B1 in macrophages. IFN-γ, a cytokine secreted by proinflammatory Th1 cells, enhanced TLR2/1-dependent induction of 1,25D target genes CAMP and HDB2 expression in macrophages in vitamin D sufficient serum. In contrast, IL-17, secreted by the T helper subtype Th17, had no effect, whereas the Th2 cytokine IL-4 strongly suppressed the effect of TLR2/1 stimulation on CAMP and HBD2 expression [90]. Remarkably, IL-4 enhanced TLR2/1-stimulated expression of the CYP27B1 and VDR genes, as did IFN-γ. However, studies of bioconversion of 25D in cytokine-treated cells suggested that, while 1α-hydroxylation was enhanced in IFN-γ-treated cells, 25D was preferentially converted to 24,25D in the presence of IL-4. Gene ablation studies showed that the effect of IL-4 on 25D metabolism was abolished by siRNA-mediated knockdown of CYP24, although there was no apparent effect of IL-4 on CYP24 mRNA or protein expression. The authors speculated that the elevated levels of 24-hydroxylation induced by IL-4 may have arisen from enhanced delivery of 25D to the mitochondrial compartment containing CYP24 [90]. No matter what the mechanism by which IL-4 regulates CYP24 function, the paper clearly showed that T-cell cytokines can (differentially) control TLR-stimulated vitamin D metabolism and 1,25D-driven innate immune antimicrobial responses in macrophages.

7 Concluding remarks

The above has highlighted many of the exciting recent advances in our understanding of how the immune system regulates vitamin D metabolism and how, in turn, 1,25D modulates immune system function. In particular, vitamin D has emerged as a key inducer of human innate immune responses to microbial infection, and the next few years will certainly see more important findings in this area. It should be kept in mind, however, that research into the role of vitamin D signaling in controlling innate immunity may be complicated by the fact that many of the responses may be human- (or human/primate)-specific and that animal models such as mice may not recapitulate many of the vitamin D-dependent molecular-genetic mechanisms seen in humans. Unlike adaptive immunity, which is found in vertebrates only, innate immune responses are present in a vast array of plant and animal species [91, 92]. However, many details and mechanisms of regulation of innate immune function are species-specific. For example, TLR-driven induction of AMP expression in mouse macrophages is mediated by NO. However, this does not appear to be conserved in humans, where 1,25D signaling appears to play a key role as an intermediate [60]. Similarly, the promoter-proximal VDREs in the HBD2 and CAMP genes are not conserved in mice [55, 59, 65]. Indeed, the arrays of genes encoding AMPs vary considerably between species [93, 94], as does the conservation of certain cytokines and chemokines, and numbers of TLRs [95]. Therefore, mouse models, which have been powerful tools in furthering our understanding of the calcium homeostatic functions of vitamin D, may not be as appropriate for dissecting the roles of vitamin D signaling in immune system regulation.