Vitamin D and 1,25(OH)2D Regulation of T cells

Vitamin D is a direct and indirect regulator of T cells. The mechanisms by which vitamin D directly regulates T cells are reviewed and new primary data on the effects of 1,25 dihydroxyvitamin D (1,25(OH)2D) on human invariant natural killer (iNK)T cells is presented. The in vivo effects of vitamin D on murine T cells include inhibition of T cell proliferation, inhibition of IFN-γ, IL-17 and induction of IL-4. Experiments in mice demonstrate that the effectiveness of 1,25(OH)2D requires NKT cells, IL-10, the IL-10R and IL-4. Comparisons of mouse and human T cells show that 1,25(OH)2D inhibits IL-17 and IFN-γ, and induces T regulatory cells and IL-4. IL-4 was induced by 1,25(OH)2D in mouse and human iNKT cells. Activation for 72h was required for optimal expression of the vitamin D receptor (VDR) in human and mouse T and iNKT cells. In addition, T cells are potential autocrine sources of 1,25(OH)2D but again only 48–72h after activation. Together the data support the late effects of vitamin D on diseases like inflammatory bowel disease and multiple sclerosis where reducing IL-17 and IFN-γ, while inducing IL-4 and IL-10, would be beneficial.


Introduction
Vitamin D is a fat soluble vitamin that is either consumed in the diet or produced in the skin following sunlight exposure of the skin. Vitamin D is inactive and is hydroxylated twice, once in the liver and once in the kidney to make the active form of the vitamin, 1,25 dihydroxyvitamin D (1,25(OH)2D) [1,2]. Production of 1,25(OH)2D in the kidney is tightly regulated by serum calcium, parathyroid hormone and 1,25(OH)2D levels [3]. Vitamin D as 1,25(OH)2D functions by binding to a nuclear vitamin D receptor (VDR) and retinoid X receptor to regulate gene transcription [4]. The classic roles of vitamin D are in the regulation of calcium uptake and homeostasis, bone metabolism, and cell growth and division. The VDR has been identified in many other tissues including the immune system and it is now accepted that 1,25(OH)2D and vitamin D are important immune system regulators [5]. All cells of the immune system have been shown to express the VDR including T cells [6].

T Cells
Several different types of T cells exist to provide defense from a variety of different insults to the body. CD4+ T cells provide help for B cell antibody production and to other cell types to engulf and kill pathogens. T helper (h) cell responses are heterogeneous although it has been shown that specific cytokine patterns are critical for control of susceptibility to infection. Th1 cells are important for the control of intracellular infections with Mycobacterium tuberculosis, Listeria monocytogenes, and a number of viruses [7][8][9]. Th2 cells that produce IL-4, IL-5, and IL-13 are required for host defense against parasitic helminthes [10]. Th17 cells produce IL-17, IL-22, and granulocyte macrophage-colony stimulating factor (GM-CSF) [11] and are essential for resistance to extracellular pathogens [12]. Additional T cells include the innate natural killer (NK)T cells. NKT cells recognize lipid antigens instead of protein antigens and produce large amounts of cytokines early during infection [13]. CD8+ T cells are cytotoxic T cells and are important for killing virally and bacterially infected cells. Each of these T cells are critical for immune protection from infection.
Dysregulation of T cell responses can cause pathology. Immune mediated diseases occur as a result of chronically activated T cells. T regulatory (reg) cells produce IL-10 and serve to inhibit effector T cell responses in an antigen specific and non-specific manner [14]. Deficiency of T reg cells results in multi-organ immune mediated disease [14]. Th1 and/or Th17 cells transfer experimental models of immune mediated diseases such as inflammatory bowel disease and multiple sclerosis. Experimental asthma and allergy models occur in animals that have Th2 cells, and in some cases NKT cells that overproduce IL-13 and/or IL-17 [15]. Control of the T cell response is therefore required for robust elimination of infection and the resolution of inflammation to prevent pathology due to chronic T cell activation.

Vitamin D and T Cells
The effects of vitamin D and 1,25(OH)2D as inhibitors of T cells have been well described. There are direct and indirect effects of 1,25(OH)2D on T cells but this review will focus on the direct effects. Since 1983 it has been described that 1,25(OH)2D inhibited T cell proliferation and the secretion of select cytokines after mitogen stimulation [16,17]. Moreover, 1,25(OH)2D directly inhibited IL-2 and IFN-γ transcription [17,18]. More recently 1,25(OH)2D has also been shown to inhibit IL-17 secretion by Th17 cells [19,20]. The effects of 1,25(OH)2D on Th2 cells is more controversial with evidence that 1,25(OH)2D inhibits IL-4 transcriptionally as well as evidence that 1,25(OH)2D upregulates IL-4 in mouse and human T cells [20][21][22][23]. In vitro, 1,25(OH)2D3 treatments induced IL-10 and T regulatory cell development [24]. In addition, 1,25(OH)2D upregulated the gut homing receptor CCR9 and inhibited CXCR3 on T cells potentially changing the homing properties of the Th cells [25]. Vitamin D and 1,25(OH)2D inhibited Th1 and Th17 responses, induced T reg responses, and controlled proliferation and Th cell localization. CD8+ T cells and iNKT cells are also vitamin D targets. In vitro, 1,25(OH)2D treatment inhibited CD8 T cell proliferation and VDR knockout (KO) CD8+ T cells proliferated without antigen stimulation due in part to over-production of IL-2 [26,27]. VDR KO CD8 T cells had altered homing patterns, a reduction in granzyme B production and more rapid contraction in an infection model [28]. Other defects in VDR KO CD8+ T cells included the ability, when transferred to leucopenic hosts, to develop into IL-17 and IFN-γ secreting cells that produced colitis symptoms [26]. In the gastrointestinal tract, CD8αα/TCRαβ T cells important in maintaining homeostasis, required vitamin D to maintain their homeostatic proliferation [29]. Development and function of iNKT cells depends on expression of the VDR and vitamin D [30,31]. In vitro, 1,25(OH)2D inhibited iNKT cell derived IL-17 and induced IL-4 and IL-5 [32]. The requirement for the VDR in the development of iNKT cells was traced to regulation of the survival of maturing iNKT cells in the thymus [31]. Vitamin D controls CD8 proliferation, IL-2 production and the potential to develop into effector T cells that produce IFN-γ, IL-17 and granzyme B. Vitamin D also controls iNKT cell expansion during development. Lastly vitamin D changes early cytokine production by iNKT cells that could shape later T cell responses.
Vitamin D has also been proposed as a regulator of Th2 mediated disease such as allergy and asthma. In vitro, 1,25(OH)2D treatment of T cells has been shown to increase IL-4 secretion by human and mouse Th cells [21,42,43]. IL-13 has been shown to be induced [44] or decreased [45] by 1,25(OH)2D treatment of human T cells. VDR KO Th2 cells made less IL-4 on the C57BL/6 background and less IL-4, IL-5 and IL-13 on the Balb/c background [46]. In addition, VDR KO mice on either the Balb/c or C57BL/6 background failed to develop experimental allergic asthma [46]. However, cell transfer and bone marrow transplantation showed that VDR KO T cells could induce asthma in the WT host and therefore VDR expression in the lung might account for the increased resistance of the VDR KO mice to allergic asthma [47]. iNKT cells also contribute to asthma development and 1,25(OH)2D induced IL-4, and IL-5 from iNKT cells [32,48]. The data on the effects of 1,25(OH)2D treatments of experimental allergic asthma show contradictory results with no effects, worsening of symptoms and symptom amelioration [49][50][51]. The effects of 1,25(OH)2D on T reg cells and IL-10, that also suppress the Th2 response, could explain some of the beneficial effects of vitamin D in experimental asthma [52][53][54].
Overall the data support that vitamin D and 1,25(OH)2D induce production of Th2 cytokines. Other vitamin D targets in the lung, T reg cells and/or other immune cells are likely the explanation for the contradictory results in the experimental allergic asthma models.

Vitamin D Regulation of Mouse versus Human T Cells
Much of the work describing the basic mechanisms of vitamin D, the VDR and 1,25(OH)2D on T cells in vivo have been done in mice. These in vivo experiments are difficult to replicate in humans. However, since the goal is to use mice to model the effects of vitamin D and 1,25(OH)2D in humans, it is important to determine which of the effects of vitamin D in murine T cells can also be observed in human T cells. It should be noted however, that much of the work using human T cells is done with peripheral blood mononuclear cells (PBMC). In the mouse the T cells studied come from different tissues (usually not the blood) and the functions of the T cells depend to a large extent on where they are located.
The early work utilized human PBMC to demonstrate that T cells expressed the VDR and were vitamin D targets. Human CD8 and CD4 T cells were inhibited from proliferating in the presence of 1,25(OH)2D [18,27,55]. In addition, 1,25(OH)2D inhibited IL-2, IFN-γ, and IL-17 in human and mouse T cells [21,42,43,56]. Freshly isolated PBMC were stimulated with CD3 and CD28 antibodies or αGalCer in the presence of 0-50nM 1,25(OH)2D. Confirming the literature, our experiments also showed 1,25(OH)2D inhibited IFN-γ and T cell proliferation and induced IL-4 production from PBMC stimulated with CD3/CD28 (data not shown). Activation of both human and mouse T cells induced expression of the VDR and it took 48-72h to induce VDR protein in the T cells [6,21,57]. Human Th1, Th2 and Th17 cells expressed similar and high amounts of the VDR protein 72 hours after activation [57]. The amount of 1,25(OH)2D addition to activated T cells protected the VDR protein from proteasomal degradation and 1,25(OH)2D has been shown to stabilize VDR protein in other cell types as well [57,58]. In addition, activation of mouse CD8+ T cells and human CD4+ T cells for 48-72 hours induced expression of the vitamin D 1-alpha hydroxylase (Cyp27B1) suggesting that T cells might be able to locally produce 1,25(OH)2D [59,60]. In human PBMC 1,25(OH)2D induced the expression of IL-4 when added in vitro and 1,25(OH)2D induced human T reg development and IL-10 production [42,43,61]. Collectively the effects of vitamin D, production of Cyp27B1 and 1,25(OH)2D on mouse T cells in vitro reflect the effects of 1,25(OH)2D on human T cells from the PBMC.
PBMC are readily accessible sources of human immune cells including iNKT cells. However, the frequencies of iNKT cells (CD1d tetramer+/CD3+) in the PBMC is very low and ranged from 0.008%-0.292% of the cells (data not shown). α-Galactoceramide (GalCer), an iNKT cell specific ligand, was used to stimulate freshly isolated human PBMC. IFN-γ was inhibited by 10 and 50 nM of 1,25(OH)2D addition to αGalCer stimulated PBMC (Figure 1). IL-4 went up with 50nM but not 10 nM 1,25(OH)2D (Figure 1). Cultures were set up to expand and purify the iNKT cells. The ability to expand iNKT cells from some donors was low (11-29 fold expansion) while iNKT cell expansion from 3 individuals was high (123-596 fold expansion, Figure 2). Adding 10 and 50 nM 1,25(OH)2D at the start of the 12 day culture reduced the iNKT cells that could be recovered from the cultures (data not shown). Cell lines were generated utilizing magnetic bead purified iNKT cells (95% CD1d tetramer+/CD3+) for experiments. Resting iNKT cell lines had very low expression of the VDR that was upregulated with 48h of αGalCer and irradiated PBMC incubations (Figure 3). There was not an effect of 1,25(OH)2D on the proliferation of three different human iNKT cell lines (data not shown). The data we provide here suggest that there are important differences between the effects of 1,25(OH)2D on tissue iNKT cells in the mouse and PBMC iNKT cells in the human. Human iNKT cells, like T cells, optimally express the VDR after several days of activation. Induction of IL-4 by 1,25(OH)2D in murine and human iNKT cells was the same. The reason for the difference between the effects of 1,25(OH)2D on mouse and human iNKT cells might be a species difference, the location of the cells, the low frequencies of the iNKT cells in the freshly isolated PBMC or changes that occur in vitro when culturing and expanding human iNKT cells.

Conclusions
T cells become vitamin D targets by expressing the VDR and inducing autocrine 1,25(OH)2D following activation. The 1,25(OH)2D inhibits murine and human T cells from proliferating, producing IFN-γ, and IL-17 while inducing IL-4. In animal models these effects correspond to the vitamin D mediated inhibition of experimental immune mediated diseases caused by Th1 and Th17 cells. Further, 1,25(OH)2D induced IL-4 in both mouse and human iNKT cells. The effects of 1,25(OH)2D on proliferation and IFN-γ production in mouse and human iNKT cells was different. Overall the effects of vitamin D on both iNKT cells and T cells seem to require 48-72 h of activation and suggest that vitamin D is important late during the T cell response. Together the data suggest an important role for vitamin D and 1,25(OH)2D in regulating T cells to limit immune mediated diseases where IL-17 and IFN-γ, are pathogenic.