Vitamin D and immune function

Early experiments added calcitriol to peripheral blood mononuclear cells and observed that T-cells in the cultures had decreased proliferation and secreted less IL-2 and interferon-γ⁽ Reference Rigby, Denome and Fanger ^{2 ,} Reference Rigby, Stacy and Fanger ^{3 )}. All T-cell subsets that have been examined express the VDR at a low level and following activation expression of the VDR is up-regulated⁽ Reference Veldman, Cantorna and DeLuca ^{4 )}. Several direct and indirect targets of vitamin D have been identified. Cytokine secretion by Th (T helper) 1 and Th17 cell subsets is inhibited by calcitriol⁽ Reference Bruce, Yu and Ooi ^{5 ,} Reference Cantorna, Yu and Bruce ^{6 )}. Calcitriol- or VDR-deficient T-cells are predisposed to produce IL-17 and interferon-γ⁽ Reference Bruce, Yu and Ooi ^{5 ,} Reference Froicu, Weaver and Wynn ^{7 )}. Conversely, FoxP3+regulatory T (Treg) cells are induced to develop in vitro and in vivo with calcitriol treatment⁽ Reference Gregori, Giarratana and Smiroldo ^{8 ,} Reference Barrat, Cua and Boonstra ^{9 )}. The effects of calcitriol on Th2 cell development and function is less clear with investigators showing inhibition of IL-4 production and induction of IL-4 production using different models and systems⁽ Reference Boonstra, Barrat and Crain ^{10 –} Reference Pichler, Gerstmayr and Szepfalusi ^{12 )}.

VDR knockout (KO) mice have provided a valuable tool for studying the immune system. VDR KO mice have normal numbers of conventional T-cells⁽ Reference Yu and Cantorna ^{13 )}. There are more memory T-cells that are predisposed to develop into Th1 and Th17 cells in VDR KO v. wild-type mice⁽ Reference Bruce, Yu and Ooi ^{5 )}. VDR KO Th2 cells are able to develop normally in vitro ⁽ Reference Mahon, Wittke and Weaver ^{11 ,} Reference Wittke, Chang and Froicu ^{14 )}. Treg cells do not require VDR expression for either development or function⁽ Reference Yu, Bruce and Froicu ^{15 )}. Invariant natural killer T (iNKT) cells require expression of the VDR since they fail to develop in VDR KO mice⁽ Reference Yu and Cantorna ^{16 )}. In addition, the iNKT cells from VDR KO mice are functionally defective and secrete significantly less IL-4 and interferon-γ⁽ Reference Yu and Cantorna ^{16 )}. VDR KO mice have high Th1 and Th17 responses, no change in Th2 or Treg cells and very low iNKT cells.

Vitamin D and multiple sclerosis

MS (multiple sclerosis) is an autoimmune disease where T-cells target the central nervous system. The development of experimental autoimmune encephalomyelitis (EAE; an animal model of MS) results because of a Th17- and Th1-mediated immune attack on the central nervous system⁽ Reference Cantorna ^{17 )}. Other T-cell responses inhibit the development of Th17 and Th1 cells and are therefore important negative regulators of EAE. Negative regulators of EAE include iNKT cells and Treg cells⁽ Reference Matsuda, Mallevaey and Scott-Browne ^{18 )}. Patients with MS have fewer iNKT cells and Treg cells and remission from symptoms is associated with the increased number and function of these cell types⁽ Reference Araki, Kondo and Gumperz ^{19 )}.

Epidemiological data suggest that there may be a link between vitamin D status and MS in human subjects⁽ Reference Sioka, Kyritsis and Fotopoulos ^{20 )}. Low level of circulating vitamin D was linked to increased disability scores in MS patients⁽ Reference van der Mei, Ponsonby and Dwyer ^{21 )}. Both sun exposure and vitamin D supplements during childhood and adolescence were shown to correlate with MS incidence north of the Arctic Circle, and these factors were also linked to time of MS onset⁽ Reference Kampman, Wilsgaard and Mellgren ^{22 ,} Reference McDowell, Amr and Culpepper ^{23 )}. Participants in the nurse's health study who were in the highest quintile of vitamin D intakes had 40% less MS⁽ Reference Munger, Zhang and O'Reilly ^{24 )}. There is evidence for a role of vitamin D in the aetiology and severity of MS in human subjects.

Experimentally vitamin D deficiency accelerates the development of EAE⁽ Reference Cantorna, Hayes and DeLuca ^{25 )}. In addition, calcitriol inhibits EAE and suppression is associated with a reduction in Th1, and Th17 cell responses⁽ Reference Bruce, Yu and Ooi ^{5 ,} Reference Cantorna, Yu and Bruce ^{6 )}. Calcitriol treatment of mice resulted in the increased numbers of Treg cells isolated⁽ Reference Gorman, Judge and Hart ^{26 )}. Recent data also show that calcitriol and vitamin D are positive regulators of iNKT cells⁽ Reference Yu and Cantorna ^{13 ,} Reference Yu and Cantorna ^{16 )}. Together the data suggest that improved vitamin D status would have a beneficial effect on multiple cell types important in the pathology of MS.

Vitamin D and asthma

Like MS, asthma is also an immune-mediated disease. Unlike MS, in asthma the pathogenic T-cells are of the Th2 cell and iNKT variety. IL-4, IL-5 and IL-13 are the disease-causing cytokines in asthma pathology⁽ Reference Wills-Karp ^{27 )}. iNKT cells have been shown to be involved in several different experimental models of asthma⁽ Reference Iwamura and Nakayama ^{28 )}. Allergic-induced airway hyperresponsiveness required IL-4 and IL-13 producing iNKT cells⁽ Reference Akbari, Stock and Meyer ^{29 )}. iNKT cell-deficient mice fail to develop experimental allergic asthma⁽ Reference Akbari, Stock and Meyer ^{29 )}. Conversely, Treg cells are important suppressors of asthma development and therapies that induce Treg cells are effective ways to suppress experimental asthma⁽ Reference Akbari, Stock and DeKruyff ^{30 )}.

The role of vitamin D in asthma has been studied by several different groups. There are conflicting data about the role of vitamin D in Th2 and experimental asthma regulation. Calcitriol has been shown to both increase and inhibit IL-4 production from Th2 cells⁽ Reference Boonstra, Barrat and Crain ^{10 –} Reference Pichler, Gerstmayr and Szepfalusi ^{12 )}. Various symptoms of experimental allergic asthma were increased, decreased or not changed with calcitriol treatment⁽ Reference Matheu, Back and Mondoc ^{31 –} Reference Wittke, Weaver and Mahon ^{33 )}. Our data suggest that calcitriol treatment had no effect on experimental asthma development⁽ Reference Wittke, Weaver and Mahon ^{33 )}. VDR KO mice failed to develop experimental allergic asthma but the failure to develop asthma was not because of defective Th2 cells⁽ Reference Wittke, Chang and Froicu ^{14 )}. VDR KO Th2 cells were found to develop normally and to induce asthma when transferred to wild-type mice⁽ Reference Wittke, Chang and Froicu ^{14 )}. VDR KO mice have normal numbers of functional Treg cells⁽ Reference Yu, Bruce and Froicu ^{15 )}. VDR expression was shown to be critical in the lung epithelium⁽ Reference Wittke, Chang and Froicu ^{14 )}. In addition, iNKT cells require the VDR for both development and function. The failure of VDR KO mice to develop experimental asthma is a result of a complex set of factors that include defective iNKT cells and normal functional Treg cells⁽ Reference Wittke, Chang and Froicu ^{14 ,} Reference Yu, Bruce and Froicu ^{15 )}. In addition, there is an immune extrinsic requirement for the VDR in the lung epithelium⁽ Reference Wittke, Chang and Froicu ^{14 )}. The effect of calcitriol on Th2 cells and experimental asthma is harder to dissect but the data suggest that perhaps Th2 responses are less affected by changes in vitamin D than Th1 responses.

Vitamin D regulation of invariant natural killer T-cell function

iNKT cells have two distinct points at which vitamin D and the VDR are required. iNKT cells diverge from conventional T-cells at the CD4/CD8 double-positive (DP) stage (Fig. 1). The iNKT cell precursors rearrange their T-cell receptor and can be stained with CD1d tetramers (bound to ligands including α-galactoceramide). After expressing the invariant T-cell receptor the iNKT cell precursors mature by down-regulating CD24 to become DP^dim/CD24⁻ and then as the iNKT cell precursor diverges from conventional T-cells it undergoes rapid proliferation (Fig. 1). Following proliferation the S0 iNKT cells undergo three additional modifications (S1: CD44⁻; S2: CD44⁺; S3: CD44⁺NK1.1⁺) that result in mature iNKT cells that exit the thymus (Fig. 1).

Fig. 1.

(Colour online) Vitamin D and vitamin D receptor (VDR) targets in invariant natural killer T (iNKT) cell development⁽ Reference Yu and Cantorna ^{13 ,} Reference Yu and Cantorna ^{16 )}. iNKT cells develop in the thymus following several different phenotypic changes. The earliest iNKT cell precursor, DP^dim expresses the invariant T-cell receptor (tetramer⁺) and CD24⁺. The early iNKT cells down-regulate CD24 and diverge from the other CD4/CD8 DP cells that go on to become conventional T-cells. Expression of two transcription factors (Fyn and NF-κB) is important in the movement of iNKT cells from stage (S) 0 to S1. Vitamin D and VDR deficiency affect the number of iNKT cells that rapidly expand and enter the S1 stage in maturation. There is no effect of vitamin D deficiency on the further maturation of iNKT cells. VDR knockout (KO) iNKT cells have an additional block in maturation at the S2 stage and fail to fully develop into mature iNKT cells. T-bet and NF-κB expression is associated with the transition of iNKT cells from S2 to S3. VDR KO iNKT cells express significantly less T-bet than there fully mature S3 wild-type counterparts⁽ Reference Yu and Cantorna ^{16 )}.

VDR KO mice have fewer iNKT cells. The iNKT cells that remain in VDR KO mice are blocked at the stage just before they fully develop and exit the thymus (Fig. 1)⁽ Reference Bendelac, Savage and Teyton ^{34 )}. Most of the iNKT cells in the VDR KO mouse are blocked at S2 and the immature iNKT cells produce less cytokines than their wild-type counterparts⁽ Reference Yu and Cantorna ^{16 )}. The iNKT cells in vitamin D-deficient mice are fewer than those from vitamin D-sufficient mice⁽ Reference Yu and Cantorna ^{13 )}. Unlike the result from the VDR KO mice, vitamin D-deficient iNKT cells are functionally normal and the frequency of iNKT cells in S2 and S3 stages of maturation are similar to the frequencies in vitamin D-sufficient mice⁽ Reference Yu and Cantorna ^{13 )}. The expansion defect in vitamin D-deficient and VDR KO mice was a result of the increased apoptosis of early DP^dim CD24⁺ iNKT cells (Fig. 1)⁽ Reference Yu and Cantorna ^{13 )}. In the absence of vitamin D and the VDR fewer iNKT cells are produced (Fig. 1)⁽ Reference Yu and Cantorna ^{16 )}. In addition, the VDR is required for the full maturation of the iNKT cells (Fig. 1)⁽ Reference Yu and Cantorna ^{16 )}. There is one pathway in iNKT cell development that is regulated by both vitamin D and the VDR; which is the expansion and proliferation of early iNKT cell precursors. In addition, expression of the VDR also affects the last stage in iNKT cell maturation.

Vitamin D status is affected by season. Furthermore, Tsang et al. showed that children born in the summer started out with high levels of calcidiol that went down to low levels 6 months later in winter⁽ Reference Namgung, Mimouni and Campaigne ^{35 ,} Reference Namgung, Tsang and Specker ^{36 )}. Conversely, children born in winter started out with low levels of calcidiol that increased 6 months later in summer⁽ Reference Namgung, Mimouni and Campaigne ^{35 ,} Reference Namgung, Tsang and Specker ^{36 )}. We used mice to model these changes in calcidiol levels and looked at the effect of changing levels of vitamin D on iNKT cell numbers. The offspring from vitamin D-deficient breeders was maintained vitamin D-deficient throughout life and at 8 weeks the mice had very few iNKT cells compared with vitamin D-sufficient mice (Fig. 2)⁽ Reference Yu and Cantorna ^{13 )}. A series of experiments were carried out to supplement vitamin D or calcitriol between the age of 3 and 8 weeks. Vitamin D had no effect on iNKT cell numbers when given from age 3 to 8 weeks (Fig. 2)⁽ Reference Yu and Cantorna ^{13 )}. Conversely, calcitriol increased the numbers of iNKT cells but not to the level found in vitamin D-sufficient mice (Fig. 2)⁽ Reference Yu and Cantorna ^{13 )}. Earlier treatment with calcitriol given at birth and through 8 weeks of age also failed to recover iNKT cell numbers to those in vitamin D-sufficient mice (Fig. 2)⁽ Reference Yu and Cantorna ^{13 )}. Treating breeders and offspring with calcitriol throughout gestation resulted in the same numbers of iNKT cells as vitamin D-sufficient mice (Fig. 2)⁽ Reference Yu and Cantorna ^{13 )}. Vitamin D is required early in utero for normal iNKT cell numbers to develop in mice. There is a gestational effect of vitamin D on early iNKT cell precursors that cannot be recovered later with vitamin D or calcitriol treatment. Early changes in vitamin D status can affect immune function.

Fig. 2.

(Colour online) Gestational effects of vitamin D deficiency on invariant natural killer T (iNKT) cells⁽ Reference Yu and Cantorna ^{13 )}. Vitamin D-sufficient, vitamin D-deficient or calcitriol-supplemented diets were fed to mice during three different windows of time: pregnancy, lactation (0–3 weeks), following weaning (3–8 weeks of age). The vitamin D-sufficient or calcitriol-treated throughout mice had the highest numbers of iNKT cells. Vitamin D-deficient throughout or switching to D sufficient diets from 3 to 8 weeks had the fewest iNKT cells. Supplementing D-deficient mice with calcitriol from 3 to 8 weeks or during lactation until 8 weeks increased iNKT cell numbers somewhat but not to the level found in the vitamin D-sufficient mice.