Background
Multiple sclerosis (MS) is an inflammatory and neurodegenerative disorder with widespread demyelination and axonal loss within the central nervous system (CNS). The underlying etiology remains undefined although both environmental and genetic factors play a role [
1,
2], resulting in the over-activation of various immune subsets that accumulate in the CNS to produce injury. Familial inheritance, cigarette smoking, viral infection, and ultraviolet (UV) light exposure may all contribute to the risk of MS [
1,
3].
Vitamin D deficiency has previously [
4] and recently been suggested as another contributing factor in the pathogenesis of MS [
5‐
7]. Several studies have reported an inverse association of sunlight exposure, available UV radiation and MS prevalence [
5,
8,
9], implicating vitamin D since UV B radiation (280 to 315 nm) converts 7-dehydrocholesterol to previtamin D3 in the epidermal and dermal layers in humans; previtamin D3 is then converted by a thermal process to vitamin D3 [
10]. Due to the changing angle of declination of the sun, vitamin D insufficiency is common in the winter months in latitudes north of 42 °N latitude [
11]. Therefore, vitamin D is of interest as the biological correlate of available UV radiation, although it has been proposed that other factors could also be involved [
12].
In humans, vitamin D3 undergoes hydroxylation in the liver to produce 25-hydroxyvitamin D3 {25(OH)D3}, the main circulating form of vitamin D. 25(OH)D3 can be further hydroxylated in the liver to 24,25-dihydroxyvitamin D3, or in the kidney to the immunologically active form of vitamin D, 1,25 dihydroxyvitamin D
3 {1,25(OH)2D3} [
10,
13]. Many publications {reviewed in [
13‐
15]} have reported extensively on the immunomodulatory properties of 1,25(OH)2D3. In particular, 1,25(OH)2D3 decreases T cell proliferation, increases the activity and frequency of regulatory T cells, alters the production of specific antibody isotypes, reduces activity of dendritic cells or makes them tolerogenic, and affects tissue-specific lymphocyte homing.
Naïve CD4-positive T helper (Th) cells can differentiate into either pro-inflammatory Th1 and Th17 subsets, or into Th2 subset with anti-inflammatory or regulatory activity [
16,
17]. Vitamin D has been found to elevate Th2 cytokines [
18,
19] and to reduce Th1 cytokine levels [
20,
21]; however, others have also found vitamin D to inhibit Th1 levels without affecting Th2 deviation [
22], or to reduce EAE disease severity without altering Th1 or Th2 levels [
23]. These results emphasize that there needs to be clarity on the activity and mechanism of vitamin D in CD4 Th1/Th2 differentiation. The literature on vitamin D and Th17 cells is still emerging, and vitamin D has been reported to reduce the level of Th17 cytokines in human studies [
24], and to decrease Th17 cells in mice with colitis [
25] or EAE [
26].
Given the uncertain nature of the impact of vitamin D on T cell subsets, and the recent analysis in genetically altered or chimeric mice of the requirement of T cell expression of vitamin D receptors for amelioration of EAE [
27], we have addressed the relationship between vitamin D and Th subsets, focusing on whether 1,25(OH)2D3 acts predominantly through altering one of the Th subsets, and of the attendant mechanisms. We first analysed human and mouse T cells in culture, and then extended to EAE studies. We elucidated a central role for STAT6 in regulating the vitamin D-polarization of Th2 cells to alleviate disease activity.
Methods
Isolation of T Cells
Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy adult volunteers by Ficoll-Hypaque centrifugation [
28]. The PBMCs were washed once with phosphate-buffered saline (PBS) and suspended in serum-free AIM-V medium (Invitrogen Life Technologies, Burlington, Ontario). To activate T cells in the PBMC populations, 96 well round-bottomed plates were coated with 10 or 1000 ng/mL of purified mouse anti-human CD3 (BD Pharmingen, Franklin Lakes, NJ) for a period of 3 h. From previous experiments (data not shown), the coating at 1000 ng/mL of anti-CD3 gives maximal activation of T cells measured by proliferation assays. Since the
in vivo environment in humans is unlikely to lead to the maximal activation of T cells, a submaximal level of activation with 10 ng/ml anti-CD3 was also used in most experiments as both a comparison to maximal activation and to better reflect physiology. This submaximal level of activation may also permit the more sensitive measurement of experimental changes that affect T cell activation.
Human PBMCs were plated at a density of one million cells/mL of anti-CD3 coated 96 well plates (200 μ L/well, 200,000 cells per well). An additional 10 ng/mL of anti-CD28 (BD Pharmingen) was added as a suspension to all cultures, and cells were left for 3 days at 37°C in a 5% humidified CO
2 incubator. In order to promote measurable levels of IL-17, some anti-CD3/CD28 activated cultures were further exposed to IL-23 (20 ng/mL) and IL-1β (10 ng/mL) [
29] Specified sister cultures were further exposed to either 0.1, 1 or 10 nM of 1,25(OH)2D3 (BioMol, Plymouth Meeting, PA). In some experiments, certain PBMC preparations did not receive anti-CD3 or 1,25(OH)2D3, and the floating cells collected 3 days thereafter are referred to as unactivated T cells.
Flow cytometry analyses of the floating cells collected after 3 days of the initiation of anti-CD3 treatment indicated that CD3+ T cells constituted approximately 90% of the total cell population (data not shown). Of the CD3 cells, 60% were CD4+ and 40% were CD8+. For the remaining, approximately 8% were CD56+ natural killer cells, approximately 2% were CD19+ B lymphocytes, and less than 1% were CD14+ monocytes. There was no significant difference in the proportion of the various cell subsets between the unactivated, 10 and 1000 ng/mL anti-CD3 activated PBMC populations (data not shown). Since the majority of cells were T cells, henceforth this human culture population is referred to as T cells.
Quantitative Real-Time polymerase chain reaction (qPCR)
For qPCR, T cell RNA was extracted using the RNeasy Mini Kit columns (Qiagen, Mississauga, ON). DNase treatment (M610A) was performed according to the manufacturer's instructions (Promega, Madison, WI). Total RNA extracted was reverse transcribed using Superscript II (Invitrogen). Resulting cDNA was subjected to real-time quantitative PCR using an iCycler (BioRad). Transcripts were quantified by real-time quantitative PCR on the iCycler using RT2 Real Time SYBR Green/Fluorescein PCR Master Mix (SA Biosciences, Frederick, MA). mRNA expression for each gene was calculated using a comparative cycle threshold method, and was normalized to the amount of the reference gene 18S rRNA (expressed as arbitrary units). All PCR primers were purchased from SA Biosciences (18S, PPH05666E; IFNγ, PPH00380B; IL-5, PPH00692A; IL-17, PPH00537B; TBX21/T-bet, PPH00396A/PPM03727A; GATA-3, PPH02143A/PPM05199A; RORC/RORγT, PPH05877A/PPM25095A; STAT6, PPH00760B; Notch 1, PPM04747A).
ELISA
Cytokine production by human and mouse PBMCs was assessed after activation of cells for 72 h. Cytokines in culture supernatants were measured by ELISA according to the manufacturer's protocol. ELISA kits were purchased from Invitrogen. The IL-17 was of the IL-17F form. Data were analysed using a SpectraMax 384 (Molecular Devices Corporation, Sunnyvale, CA) according to the manufacturer's instructions.
Disease induction in mice and EAE analysis
EAE was induced in female C57BL/6 mice (Jackson Laboratories, Bar Harbor, Maine), aged 8-9 weeks, by injecting subcutaneously (s.c.) 50 μg myelin oligodendrocyte glycoprotein (MOG)
35-55 in Complete Freund's Adjuvant (CFA) (Fisher, Michigan USA) supplemented with 4 mg/ml of Mycobacterium tuberculosis on day 0 [
30,
31]. Intraperitoneal (i.p.) pertussis toxin (0.1 μg/200 μl, List Biological labs, Hornby, ON) was administered on days 0 and 2. 1,25(OH)2D3 (100 ng) was given every other day i.p. in 50 μL of DMSO, while 50 μL of DMSO was used as the vehicle control; this method of administering 1,25(OH)2D3 to mice has been reported by others [
18,
19,
26,
32,
33]. Treatment was initiated at the time of MOG immunization. In addition to the wildtype mice, STAT6 -/- knockout (KO) mice on the C57BL/6 background (Jackson Laboratories, Bar Harbour, Maine) were utilized. Animals were assessed daily using a 15-point disease score scale [
30,
31] replacing the more commonly used 5-point scale since the 15-point scale differentiates individual limb disability, rather than grouping both fore- or hind-limbs together. This allows for a more sensitive characterization of disease progression. The 15-point scale is the sum of the disease state for the tail (scored from 0-2) and all 4 limbs (each limb is scored from 0-3). All animals were handled in accordance with the policies outlined by the Canadian Council for Animal Care and the University of Calgary.
Statistical Analysis
Statistical analysis was performed using R version 2.8.1 (The R Foundation for Statistical Computing) and Matlab version 7.7 (The Mathworks, Natick, MA, USA). Statistical differences for cells in culture were addressed using ANOVA with Bonferroni correction for multiple comparisons. Statistical differences between groups of mice in the EAE experiments were evaluated using a nonparametric analysis Mann-Whitney U test. An alpha of 0.05 was selected for statistical significance.
Discussion
Evidence supporting the involvement of vitamin D in the risk of MS include an inverse correlation between MS prevalence and latitude that has been consistently observed [
8,
9,
38], strongly suggesting a latitudinally-related environmental contribution to etiology. Available ultraviolet radiation, inversely correlated to latitude, is responsible for the peripheral conversion of 7-dehydrocholesterol to vitamin D3 in the epidermis. Therefore, vitamin D may be the biological correlate of available ultraviolet radiation conferring disease risk to a population. In MS patients, there is evidence of a seasonality of birth in MS patients (again suggestive of a seasonal environmental factor) [
39], oral vitamin D intake appears to be protective [
7] and vitamin D levels correlate inversely with disability [
40]. In adult [
41] or pediatric [
42] MS populations, incremental increases in serum 25-hydroxyvitamin D3 levels are associated with reduced propensity for relapses. Two small trials have suggested that relapse rates may be reduced in patients taking oral vitamin D supplementation [
43,
44]. More recently, high dose intake (average of 10,000 IU/day) of vitamin D over 1 year in a study of 40 patients, appeared to reduce relapse rate in MS [
45].
There is an increasing appreciation that Vitamin D exerts broad regulatory effects on cells of the adaptive and innate immune system. These include reducing antigen presentation through reducing the activity of dendritic cells or promoting their tolerogenic phenotype, affecting the polarization of monocytoid cells towards an M2 phenotype that produces anti-inflammatory cytokines (unpublished observations), altering B cell function, decreasing chemokine gradients and reducing tissue-specific homing [
13‐
15,
46]. A significant literature in humans also indicates that vitamin D increases the activity of regulatory T cells to prevent the excessive activation of autoreactive T cells [
47,
48]. These broad spectrum effects of vitamin D likely contribute to the apparent benefits of vitamin D in MS.
We sought in this manuscript to evaluate the impact of vitamin D on the polarization of CD4+ T helper subsets. We found a variable and inconsistent response of 1,25(OH)2D3 on generating human Th1 and Th17 subsets, and a predominance in elevating Th2 cells, resulting in the consistent outcome of increased Th2 to Th1 or Th17 ratios. These results were found by measurements of representative cytokines for each subset, and of their transcription factors. Similar results were found for mouse T cells in culture. Extending to studies in vivo, we found that mice treated with 1,25(OH)2D3 had significant generation of Th2 cells in the spleen and lymph nodes detected through GATA-3 upregulation, further emphasizing the predominance of Th2 cells generated through 1,25(OH)2D3 treatment.
In addressing the mechanism by which Th2 cells were generated, our results have highlighted the STAT6 transcription factor upstream of GATA-3, given the correspondence of elevation of STAT6 and GATA-3 in wildtype EAE mice treated with 1,25(OH)2D3, and of the significant loss of effect of 1,25(OH)2D3 in increasing GATA-3 levels in STAT6 null mice. Significantly, the deficiency of STAT6 by using null mice resulted in the inability of 1,25(OH)2D3 to alleviate EAE, and this is linked mechanistically to the failure in STAT6 null mice to elevate GATA-3 levels. Thus, our results have highlighted not only the predominance of generation of Th2 cells by vitamin D, but they have also revealed the intermediary role of 1,25(OH)2D3 in engaging STAT6 to produce Th2 polarization.
Vitamin D has also been reported to lose its efficacy in EAE when mice are deficient in IL-4 (20), IL-10 [
49], Rag-1 [
50], vitamin D receptor [
51] and estrogen receptor signaling [
50]. These results are consistent with our results of the lack of efficacy of 1,25(OH)2D3 in STAT6 null mice since IL-4 and IL-10 are cytokines produced by Th2 cells that require STAT6 for genesis, and the Rag-1 mutation leads to defect in the generation of T cells. A link between estrogen and vitamin D is demonstrated by the findings that estrogen regulates the level of the vitamin D receptor [
50].
There are limitations to our current study that should be considered. The T cell population following 3 days of anti-CD3/CD28 stimulation of human peripheral blood-derived mononuclear cells constitutes approximately 90% purity, so it is possible that the effect of 1,25(OH)2D3 in generating Th2 predominance is indirect through cells that contaminate the T cell cultures. Moreover, our experiments utilize the known biologically active form of vitamin D, 1,25(OH)2D3, rather than the precursors vitamin D3 or 25-hydroxyvitamin D3. The last is the commonly measured form in humans due to its stability compared to 1,25(OH)2D3, so our results could be tempered by the potential differential rates in humans of converting 25-hydroxyvitamin D3 to 1,25(OH)2D3. As well, we did not determine whether other vitamin D metabolites, such as 24,25(OH)2D3, influence T cell function; different vitamin D metabolites can be found in MS patients and have been negatively correlated with outcomes of magnetic resonance imaging [
52].
Another consideration is that while the concentrations of 1,25(OH)2D3 that we used in culture can be achieved in humans that are properly supplemented with vitamin D, it remains to be determined how much 1,25(OH)2D3 is found in mice injected in this study with intraperitoneal 1,25(OH)2D3. Our injection protocol and dose, however, reproduce those used by other groups [
18,
19,
26,
32,
33]. Finally, it is of interest to determine whether 1,25(OH)2D3 would be equally efficacious in mice induced for EAE by active immunization with MOG, as in the current study, compared to mice elicited for EAE through the passive transfer of T cells; in these 2 context, different mechanisms are initially engaged to produce an inflammatory insult to the CNS, and their responses to vitamin D could help discern the key mechanisms for vitamin D in ameliorating EAE.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS performed the majority of the experiments of this manuscript, and wrote the first draft of this manuscript. CS and JW provided technical support, and helped with deriving the results of Figures
1 and
2. VWY supervised this project, and edited and completed the writing of the manuscript. All authors have read and approved the final manuscript.