Interaction with the immune system is one of the most well-established nonclassic effects of vitamin D. For many years this was considered to be a manifestation of granulomatous diseases such sarcoidosis, in which synthesis of active 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) is known to be dysregulated. However, recent reports have supported a role for 1,25(OH) 2 D 3 in mediating normal function of the innate and adaptive immune systems. Crucially, these effects seem to be mediated via localized autocrine or paracrine synthesis of 1,25(OH) 2 D 3 from precursor 25-hydroxyvitamin D 3 , the main circulating metabolite of vitamin D. The ability of vitamin D to influence normal human immunity is highly dependent on the vitamin D status of individuals, and may lead to aberrant response to infection or autoimmunity in those who are lacking vitamin D. The potential health significance of this has been underlined by increasing awareness of impaired vitamin D status in populations across the globe. This article describes some of the recent developments with respect to vitamin D and the immune system, and possible clinical implications.
Nonclassic actions of vitamin D were first recognized 30 years ago when receptors for active 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) were detected in various neoplastic cells lines. Other studies immediately following this showed that binding of 1,25(OH) 2 D 3 to the vitamin D receptor (VDR) promoted antiproliferative and prodifferentiation responses in cancer cells, highlighting an entirely new facet of vitamin D action. The spectrum of nonclassic responses to vitamin D was then extended to include actions on cells from the immune system. This interaction was further endorsed by the observation that some patients with the granulomatous disease sarcoidosis present with increased circulating levels of 1,25(OH) 2 D 3 and associated hypercalcemia. In these patients the high serum level of 1,25(OH) 2 D 3 is caused by increased activity of the enzyme 25-hydroxyvitamin D-1α-hydrooxylase (1α-hydroxylase). However, in contrast to normal subjects in whom 1α-hydroxylase is classically localized in the kidney, the increased synthesis of 1,25(OH) 2 D 3 in patients with sarcoidosis involves 1α-hydroxylase activity in disease-associated macrophages. Thus, it was concluded that the immune system had the potential to synthesize 1,25(OH) 2 D 3 and elicit autocrine or paracrine responses from immune cells expressing the VDR.
Despite these early advances, the precise nature of the interaction between vitamin D and the immune system remained unresolved for many years. Some pieces of the puzzle were easier to complete than others. For example, it became evident that dysregulation of 1,25(OH) 2 D 3 was not restricted to sarcoidosis but was a common feature of many granulomatous disorders and some forms of cancer. Likewise, at least in vitro, it was possible to potently regulate a range of immune cell functions using 1,25(OH) 2 D 3 or its synthetic analogs. However, the key remaining question was whether or not vitamin D could act as a physiologic regulator of normal immune responses. Answers to this question began to appear about 5 years ago and new information on the fundamental nature of vitamin D sufficiency/insufficiency has provided a fresh perspective on nonclassic actions of vitamin D. As a consequence, there is now a much broader acceptance that vitamin D plays an active role in regulating specific facets of human immunity. Details of this are reviewed and the possible effect of vitamin D insufficiency and vitamin D supplementation on normal immune function and human disease are discussed in this article.
Vitamin D and innate immunity
Macrophages, Vitamin D, and Cathelicidin
Consistent with the earlier seminal observations of extrarenal 1α-hydroxylase activity in patients with sarcoidosis, the effects of vitamin D on macrophage function have been central to many of the new observations implicating vitamin D in the regulation of immune responses. In common with natural killer cells (NK) and cytotoxic T lymphocytes (cytotoxic T cells), macrophages, and their monocyte precursors play a central role in initial nonspecific immune responses to pathogenic organisms or tissue damage, so-called cell-mediated immunity. Their role is to phagocytose pathogens or cell debris and then eliminate or assimilate the resulting waste material. In addition, macrophages can interface with the adaptive immune system by using phagocytic material for antigen presentation to T lymphocytes (T cells).
For many years, the key action of vitamin D on macrophages was believed to be its ability to stimulate differentiation of precursor monocytes to more mature phagocytic macrophages. This concept was supported by observations showing differential expression of VDR and 1α-hydroxylase during the differentiation of human monocytes macrophages. The latter report also emphasized early studies showing that normal human macrophages were able to synthesize 1,25(OH) 2 D 3 when stimulated with interferon gamma (IFNγ). Localized activation of vitamin D, coupled with expression of endogenous VDR was strongly suggestive of an autocrine or intracrine system for vitamin D action in normal monocytes/macrophages.
However, confirmation of such a mechanism was only obtained in 2006 when Liu and colleagues carried out DNA array analyses to define innate immunity genes that were specifically modulated in monocytes by Mycobacterium tuberculosis . In a seminal investigation both the VDR and the gene for 1α-hydroxylase (CYP27B1) were shown to be induced following activation of the principal pathogen recognition receptor for M tuberculosis , toll-like receptor 2/1 (TLR2/1). Subsequent experiments confirmed that precursor 25-hydroxyvitamin D 3 (25OHD 3 ) was able to induce intracrine VDR responses in monocytes that had been treated with a TLR2/1 activator. In particular, the TLR2/1-25OHD 3 combination stimulated expression of the antibacterial protein cathelicidin, so that vitamin D was able to promote monocyte killing of M tuberculosis . Notably, the ability to promote expression of the antibacterial protein following a TLR2/1 challenge was directly influenced by the 25OHD 3 status of the donor serum used for monocyte culture. More recently, the authors have shown that vitamin D supplementation in vivo can also enhance TLR2/1-induced cathelicidin expression. Cathelicidin was identified several years ago as a target for transcriptional regulation by 1,25(OH) 2 D 3 -liganded VDR, in that its gene promoter contains a functional vitamin D response element (VDRE). This VDRE occurs within a small interchangeable nuclear element (SINE) sequence which only seems to be present in the cathelicidin gene promoter of higher primates, suggesting that vitamin D regulation of this facet of innate immunity is a relatively recent evolutionary development.
Recent reports have underlined the importance of cathelicidin as a target for vitamin D but also suggest that this mechanism may be more complex than initially believed. As yet, the precise signal system by which TLR activation induces expression of VDR and 1α-hydroxylase remains unclear. Promoter-reporter analysis of the events involved in transcriptional regulation of CYP27B1 suggest that TLR4-mediated induction of the enzyme involves JAK-STAT, MAP kinase and nuclear factor kappaB (NF-κB) pathways, and that these synergize with IFNγ-mediated induction of CYP27B1. However, other studies have proposed that TLR2/1 induction of 1α-hydroxylase occurs indirectly as a consequence of TLR2/1-induced interleukin (IL)-15, which is a potent inducer of CYP27B1 and 1α-hydroxylase activity. In a similar fashion, IL-17A has been shown to enhance 1,25(OH) 2 D 3 -mediated induction of cathelicidin, although this response does not seem to involve transcriptional regulation of 1α-hydroxylase or increased VDR sensitivity. One pathway that has been poorly studied in this regard concerns the enzyme 24-hydroxylase, which is conventionally considered to function by inactivating 1,25(OH) 2 D 3 . The gene for 24-hydroxylase (CYP24) is potently induced by 25OHD 3 following TLR2/1 activation of monocytes but, as yet, it is unclear whether this involves the nonmetabolic splice variant form of CYP24 known to be expressed by macrophages.
Regulation of the antibacterial protein by 1,25(OH) 2 D 3 has been described for a wide variety of cell types other than macrophages, including keratinocytes, lung epithelial cells, myeloid cell lines, and placental trophoblasts. In some cases, this seems to involve an intracrine response similar to that reported for monocytes. However, the mechanisms controlling local synthesis of 1,25(OH) 2 D 3 in these cells vary considerably. In keratinocytes, low baseline expression of 1α-hydroxylase is enhanced following epidermal wounding by transforming growth factor beta (TGFβ). The resulting increase in 1,25(OH) 2 D 3 concentration up-regulates expression of TLR2 and TLR4 by keratinocytes, thereby priming these cells for further innate immune responses to pathogens or tissue damage. By contrast, in trophoblasts, induction of cathelicidin and subsequent bacterial killing by 25OHD 3 seems to be caused by constitutive 1α-hydroxylase activity, which is not further enhanced by TLR activation. The latter may be a result of the rapid nonimmune induction of 1α-hydroxylase and VDR that occurs within the placenta during early gestation.
Although most of the studies on vitamin D-mediated innate immunity have focused on the role of 1,25(OH) 2 D 3 -bound VDR as a pivotal transcriptional regulator of cathelicidin, it is also important to recognize that other ligands may interact with the VDR. For example, recent studies of biliary epithelial cells have shown that cathelicidin expression can be induced in a VDR-dependent fashion by bile salts. This provides a mechanism for maintaining biliary sterility, although additive effects of 1,25(OH) 2 D 3 also highlight a novel therapeutic application for vitamin D in the treatment of primary biliary cirrhosis. Conversely, other compounds may act to disrupt normal 1,25(OH) 2 D 3 -VDR-mediated immunity. The polycyclic aromatic hydrocarbon benzo[ a ]pyrene, a prominent product of cigarette smoking, has been shown to attenuate vitamin D-mediated induction of macrophage cathelcidin in a VDR-dependent fashion by stimulating expression of 24-hydroxylase and vitamin D catabolism. The precise mechanism by which this occurs has yet to be determined but these data suggest that some toxic compounds are actively detrimental to vitamin D-mediated immunity.
The observations described earlier show clearly that vitamin D is a potent stimulator of mechanisms associated with pathogen elimination. In subsequent sections the clinical importance of this with respect to vitamin D insufficiency and immune-related diseases is discussed in more detail. However, 1 key question that immediately arises from the current observations is why there is a need to involve the vitamin D system in the TLR-induction of innate immunity. As previously described, VDR-mediated transcriptional regulation of cathelicidin is a relatively recent evolutionary change and was presumably advantageous when primates (including early Homo sapiens ) were exposed to abundant sunlight, thereby priming high serum levels of vitamin D. Other benefits of incorporating vitamin D into innate immune regulation include the fact that it is associated with key feedback control pathways. As already mentioned, vitamin D has its own catabolic enzyme in the form of 24-hydroxylase, which sensitively attenuates responses to 1,25(OH) 2 D 3 and, in the case of the CYP24 splice variant, may also attenuate synthesis of this vitamin D metabolite. However, vitamin D may also provide feedback regulation of immune activation pathways in that 1,25(OH) 2 D 3 has been shown to potently down-regulate expression of monocyte TLR2 and TLR4, thereby suppressing inflammatory responses that are normally activated by these receptors. Thus, by using CYP24 and TLR regulatory mechanisms, vitamin D may help to promote appropriate innate immune responses while preventing an over elaboration of innate immune responses and the tissue damage frequently associated with this.
Dendritic Cells and Antigen Presentation
In addition to the phagocytic acquisition and elimination of pathogens and cell debris, innate immunity also involves the presentation of resultant antigen to cells involved in the adaptive arm of the immune system ( Fig. 1 ). Although several cells are able to do this, the most well-recognized group of professional antigen-presenting cells (APCs) are dendritic cells (DCs). Expression of VDR by purified tissue DCs was first reported in 1987. Subsequent studies using populations of DCs isolated from skin (Langerhans cells) provided evidence that 1,25(OH) 2 D 3 could act to attenuate antigen presentation. However, it was not until the later advent of in vitro monocyte-derived DC models that the effects of vitamin D metabolites on antigen presentation were fully elucidated. In 2000, parallel studies by the Adorini and Kumar groups showed that 1,25(OH) 2 D 3 and its synthetic analogs inhibited the maturation of monocyte-derived DCs, thereby suppressing their capacity to present antigen to T cells. Based on these observations, it was proposed that vitamin D could act to promote tolerance and this was endorsed by studies of pancreatic islet transplantation in which lower rejection rates were observed in 1,25(OH) 2 D 3 -treated mice. Crucially this response to 1,25(OH) 2 D 3 appeared to be caused by decreased DC maturation and concomitant enhancement of suppressor or regulatory T cells (Treg). Further studies have underlined the importance of Treg generation as part of the interaction between vitamin D and the immune system, and this is discussed in greater detail in later sections of this article.