Safety and T Cell Modulating Effects of High Dose Vitamin D3 Supplementation in Multiple Sclerosis

Background A poor vitamin D status has been associated with a high disease activity of multiple sclerosis (MS). Recently, we described associations between vitamin D status and peripheral T cell characteristics in relapsing remitting MS (RRMS) patients. In the present study, we studied the effects of high dose vitamin D3 supplementation on safety and T cell related outcome measures. Methodology/Principal Findings Fifteen RRMS patients were supplemented with 20 000 IU/d vitamin D3 for 12 weeks. Vitamin D and calcium metabolism were carefully monitored, and T cell characteristics were studied by flowcytometry. All patients finished the protocol without side-effects, hypercalcaemia, or hypercalciuria. The median vitamin D status increased from 50 nmol/L (31–175) at week 0 to 380 nmol/L (151–535) at week 12 (P<0.001). During the study, 1 patient experienced an exacerbation of MS and was censored from the T cell analysis. The proportions of (naïve and memory) CD4+ Tregs remained unaffected. Although Treg suppressive function improved in several subjects, this effect was not significant in the total cohort (P = 0.143). An increased proportion of IL-10+ CD4+ T cells was found after supplementation (P = 0.021). Additionally, a decrease of the ratio between IFN-γ+ and IL-4+ CD4+ T cells was observed (P = 0.035). Conclusion/Significance Twelve week supplementation of high dose vitamin D3 in RRMS patients was well tolerated and did not induce decompensation of calcium metabolism. The skewing towards an anti-inflammatory cytokine profile supports the evidence on vitamin D as an immune-modulator, and may be used as outcome measure for upcoming randomized placebo-controlled trials. Trial Registration Clinicaltrials.gov NCT00940719


Introduction
Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system, probably of autoimmune origin. It is presumed to arise from a combination of genetic and environmental factors [1]. One of the environmental factors which gained much attention during the last decades, is vitamin D. A poor exposure to either vitamin D or sunlight, its most important inducer, has been associated with a high risk of developing MS [2]. The underlying mechanism has not been unravelled yet, but a central role for the actions of vitamin D on immune regulation has been proposed [3,4]. The biologically active metabolite of vitamin D, 1,25dihydroxyvitamin D (1,25(OH) 2 D), has potent immune modulating properties, both in vitro and in the experimental animal model of MS, Experimental Autoimmune Encephalomyelitis (EAE) [5,6]. In vitro, exposure to 1,25(OH) 2 D inhibits CD4 + T cell proliferation and pro-inflammatory cytokine production (IFN-c and IL-17), and promotes anti-inflammatory cytokine production (IL-4 and IL-10) and acquisition of regulatory T cell (Treg) phenotype [4].
Not only incidence, but also disease activity of MS has been associated with vitamin D status. Disability of MS is inversely correlated with vitamin D status [7,8], but the causality of this association is uncertain [9]. Additionally, a poor vitamin D status has been associated with an increased risk on relapses [7,10,11]. A recent study suggested an increased proportion of relapse free relapsing remitting MS (RRMS) patients in a cohort supplemented with <10 000 IU vitamin D 3 /d for 52 weeks [12]. The immune modulating properties of vitamin D have been proposed to underlie these associations. Supplementation of 1 000 IU vitamin D 3 /d for 6 months induced elevated circulating TGF-b levels in MS patients [13]. We studied the correlation of vitamin D status with T cell regulatory status in a cross-sectional design [14].
Although no correlations with the circulating numbers of CD4 + Treg were found, vitamin D status correlated positively with the suppressive capacity of Treg. Additionally, the ratio between proinflammatory IFN-c + and anti-inflammatory IL-4 + CD4 + T cells was higher in RRMS patients with a poor vitamin D status. These associations were independent from the effects of vitamin D on calcium homeostasis [15].
These clinical observations and experimental data on vitamin D and MS warrant the development of well designed clinical trials to assess the disease and immune modulating effects of vitamin D in MS. The aim of the present study was to explore the safety and the in vivo effects on the peripheral T cell compartment of high dose vitamin D 3 supplementation in MS.

Subjects
Between October -December 2009, 15 RRMS patients were enrolled [16]. Inclusion criteria were age .18 years, disease duration ,6 years, use of Beta Interferon (IFN-b) 1a or 1b, and being relapse free $6 weeks prior to enrolment. Exclusion criteria were current use of drugs associated with an increased susceptibility to hypercalcaemia, treatment with immune modulating/ suppressive drugs other than IFN-b #6 weeks prior to enrolment, pregnancy, hypercalcaemia, serum creatinine .100 mmol/L, and a history of primary hyperparathyroidism, hypercalcaemia, renal dysfunction, cardiac disease, malignancy, or granulomatous disease. Holidays to sunny locations and the use of solariums were not allowed. Continuation of multivitamins containing vitamin D#600 IU mg/day was allowed. Written informed consent was obtained from each subject. The regional ethical committee 'Atrium-Orbis-Zuyd' and the institutional 'Advisory Board on Scientific Research' approved this study. This study is registered at www.clinicaltrials.gov as NCT00940719. The protocol for this trial is available as supporting information; see Protocol S1.

Vitamin D 3 supplementation
To achieve serum 25-hydroxyvitamin D (25(OH)D) levels comparable to the study by Burton et al. within 12 weeks [12], participants were supplemented with 20 000 IU vitamin D 3 /d. An oil-based vitamin D 3 solution in a concentration of 20 000 IU/mL was obtained (Vigantol Oil; Merck Serono, Darmstad, Germany) and patients were instructed to ingest 1 mL each morning [17]. No dose-escalation was incorporated, no dietary restrictions were imposed, and no additional calcium supplementation was provided. Vitamin D 3 solution was provided twice for 6 weeks. At the end of week 6 and 12, residual volume of the vitamin D 3 solution was assessed. Additionally, patients registered their vitamin D 3 intake daily on a calendar. As a safety follow-up, a visit was performed at week 16. To further investigate safety, we added an additional visit at week 24 to the protocol.

Biochemical analysis
At each study visit, calcium, albumin, and 25(OH)D were measured. Additionally, we periodically measured parathyroid hormone (PTH), creatinine, urea, electrolytes and liver function enzymes [alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP)]. Additionally to the original protocol, a urine sample was obtained the morning of the study visits on week 12 and 24, to determine the ratio between the molar concentrations of calcium and creatinine. We measured PTH on the Immunolite 2500analyzer (Siemens Healthcare Diagnostics, Deerfield, IL, USA). Other serum and urine biochemical analytes were measured on the Beckman DXC880i analyzer (Beckman Coulter, Woerden, The Netherlands). A radioimmunoassay kit (Immunodiagnostic Systems, Boldon, UK) was used to measure serum 25(OH)D and 1,25(OH) 2 D concentrations. Toxicity of vitamin D 3 manifests as hypercalcemia (serum total calcium $2.6 mmol/L) or hypercalciuria (urinary calcium to creatinine ratio .1.0 mmol/L x (mmol/L) 21 ) [18].

Proliferation suppression assay
The CFSE-based proliferation suppression assay was performed as described before [14]. In short, the freshly isolated Tresp were labelled with CFSE (Molecular Probes Invitrogen, Breda, The Netherlands). In a U-bottom 96-wells plate, 2*10 4 Tresps were stimulated with soluble anti-CD3 (2.0 pg/mL, WT32 IgG2a monoclonal antibody, kindly provided by dr. W Tax, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) in the presence of 1*10 5 irradiated accessory cells. Tresp were co-cultured for 5 days with varying amounts of Treg (Treg/ Tresp ratios 0/1, 0.25/1, 0.5/1, 1/1 or 2/1). All conditions were performed in triplo in a final volume of 200 mL. Control conditions were incorporated to validate the assay, including monocultures of un-stimulated Tresp, and of stimulated accessory cells, Treg, and surpluses of Tresp. After culture, cells were stained with anti-CD4-APC and 7AAD (BD Biosciences, #559925), and the CFSE signal of cells in the CD4 + 7AAD 2 lymphogate was analysed by flowcytometry on a FACS Canto II flowcytometer (BD Biosciences). Data were analysed with FACS Diva software version 6.1.2. (BD Biosciences). The amount of proliferation achieved in the 0/1 ratio was set at 0% suppression. The median relative amount of suppression in the Treg/Tresp co-cultures was calculated. By linear interpolation, the inhibitor ratio (Treg/Tresp) ratio at which 40% suppression of proliferation was achieved (IR40) was calculated.

Statistical analysis
Statistical analysis was conducted with SPSS version 15.0 software (SPSS inc., Chicago IL, USA) and figures were constructed with GraphPad Prism 5 software (GraphPad Software inc., La Jolla CA, USA). The median and corresponding range (min -max) are provided for continuous variables. Differences between two related samples (week 0 and 12) were assessed with the Wilcoxon signed-ranks test, the Man Whitney U  test was used for unpaired testing. Paired testing was applied unless indicated otherwise. A p-value ,0.05 was considered statistically significant.

Adverse events and patient compliance
The characteristics of the 15 participants are shown in Table 1. All patients completed the 12 week supplementation protocol without side effects other than increased flatulence (N = 1). Patients reported some beneficial changes, including decreased fatigue (N = 8), decreased headache (N = 2), decrease in eczematous eruptions (N = 1). During the 12 weeks of supplementation, 1 patient developed a relapse of MS, at week 7 of the protocol, which was confirmed by the treating neurologist. This patient was treated with pulsed prednisolone therapy. During the follow-up period, 1 additional patient suffered from an MS relapse (week 19), resulting in a mean annualized relapse rate of 0.

Biochemical assessment
Serum levels of 25(OH)D increased gradually during the study in all patients ( Figure 1A). The median level was 50 nmol/L (31-175) at week 0, and the serum level 25(OH)D of 2 patients exceeded 100 nmol/L (109 and 175 nmol/L, respectively). Remarkably, the patient with the highest serum 25(OH)D level used no vitamin supplements. At week 12, the median 25(OH)D levels was 380 nmol/L (151-535) (P,0.001). Additionally, a rise in serum 1,25(OH) 2 D levels was observed, which stabilized after 4 weeks ( Figure 1B). After 6 weeks of supplementation, a decrease in serum PTH was observed ( Table 2). Serum uncorrected and albumin corrected calcium levels were not significantly elevated, and remained below 2.6 mmol/L in all subjects ( Figure 1C). Additionally, no hypercalciuria (urinary calcium to creatinine ratio .1.0) was observed ( Figure 1D). Liver enzyme tests remained unaffected. However, serum creatinine levels gradually increased during vitamin D 3 supplementation, but remained stable during the follow-up visits ( Table 2).

Regulatory T cell numbers are not affected by vitamin D supplementation
The difference in composition and function of the T cell compartment between week 0 and week 12 was assessed in the 14 patients that remained relapse-free during the first 12 weeks. No difference was observed in the absolute amount of T cells in the circulation [1.37610 6 (0.91-2.39) vs. 1.32610 6 (0.90-2.24) cells/ mL; P = 0.761). PBMC were stained directly ex vivo to determine the proportions of circulating CD4 + Treg cells (Figure 2A). Week 0 and week 12 proportions of CD25 + FoxP3 + and CD25 + CD127 2 CD4 + Treg did not differ significantly ( Figure 2B-C). Proportions of these two Treg phenotypes correlated well (Pearson R = 0.891, P,0.001; ICC = 0.872, P,0.001), and 74.03% (52.09-89.75) of CD25 + CD127 2 Treg was FoxP3 + . The expression of CD45RA was used to stratify Treg for CD45RA + naïve and CD45RA 2 memory Treg (Figure 2A). There was no difference in the circulating proportions of both naïve and memory Treg between week 0 and week 12 ( Figure 2D).

CD4 + T cell cytokine profile shifts from pro-to antiinflammatory
The cytokine profile of the CD4 + T cells was assessed with an intracellular FACS-staining ( Figure 4A). Cytokines of interest were IFN-c (Th1 cytokine), IL-17 (Th17 cytokine), IL-4 (Th2 cytokine) and IL-10 (regulatory cytokine). The efficacy of stimulation, as evaluated by the expression level of the activation marker CD69, was comparable between week 0 and 12 (median MFI = 91 030  Figure 4B).

Discussion
In this study, the effects of high dose vitamin D 3 supplementation on safety and T cell parameters in patients with RRMS were assessed. We observed that supplementation of 20 000 IU/d vitamin D 3 for 12 weeks was without negative side-effects and without decompensation of calcium metabolism (i.e. hypercalcaemia and hypercalciuria). The number of Treg remained unaffected, as well as the numbers of Treg memory and naïve subtypes. Results regarding Treg suppressive capacity were inconclusive. However, a shift was observed in the cytokine profiles of CD4 + T cells, with an increased circulating proportion of IL-10 + CD4 + T cells, and a decreased ratio of Th1/Th2 cells (IFN-c + /IL-4 + CD4 + T cell ratio). These results further add to the notion that vitamin D is a physiological immune modulator in vivo. Monitoring the cytokine profile of CD4+ T cells could be a valuable tool to assess the immunological efficacy of vitamin D 3 in randomized controlled clinical trials on vitamin D supplementation in patients with MS.
This study confirmed that supplementation of high doses vitamin D 3 does not induce a decompensation of calcium metabolism in patients with MS [12,18]. Without a dose-escalation supplementation scheme, we did not detect hypercalcaemia or hypercalciuria. The slight non-significant increase in serum calcium and phosphorus most likely reflects a correction of vitamin D insufficiency in this cohort. A gradual increase in serum creatinine levels was observed during vitamin D 3 supplementation, which however remained below 110 mmol/L in all patients. The dramatic complication of a hypercalcaemia associated decrease of glomerular filtration rate (GFR) is unlikely, since neither hypercalcaemia nor hypercalciuria was detected. Earlier dose-escalation trials supplementing up to 280 000 IU/w for 28 weeks and up to 40 000 IU for 52 weeks did not report either significant elevations of serum creatinine levels, or calcifications in the kidneys [12,18]. Interestingly, however, earlier reports described a rise in serum creatinine which was not accompanied by a decreased GFR in patients treated with 1,25(OH) 2 D or 1a(OH)D [22]. Physiologically, 25(OH)D is filtrated in complex with vitamin D binding protein (DBP) by the glomeruli and re-absorbed by proximal tubular epithelial cells [23]. Interestingly, creatinine is being secreted in the proximal renal tubuli as well. We postulate that a massive increase of 25(OH)D-DBP complex reabsorbtion competes with tubular creatinine excretion and hereby increases serum creatinine levels. Although we believe that the increased serum creatinine levels do not reflect a decreased GFR, future studies on massive dose vitamin D supplementation should be cautious towards patients with an impaired kidney function. On the whole, biochemical data show the absence of vitamin D 3 toxicity in participants of this study.
We found no effect of vitamin D supplementation on the number of Treg in the circulation. In MS, the function rather than the number of Treg has been reported to be impaired [24,25], and to relate with serum 25(OH)D levels [14]. Additionally, other immune modulating therapies also have been shown to improve Treg function rather than Treg numbers [26,27]. An expansion of naïve Treg has been described in Glatiramer Acetate treatment [26], and a decrease of memory Treg cells in IFN-b treatment [27]. Therefore, we also assessed these Treg subsets, but found no Previously, we observed a correlation of Treg suppressive capacity with serum 25(OH)D levels [14]. Although Treg suppressive function was improved in several patients after 12 weeks of vitamin D 3 supplementation, this improvement was not statistically significant. The size of the cohort assessed is most likely too small to detect a possible effect in this complex assay. Alternatively, one could speculate from our previous work that only in the patients with the poorest vitamin D statuses, an improved suppression can be expected [14]. Although vitamin D statuses at week 0 were lower in the 9 improving patients when compared to the 4 non-improving patients, the difference was not significant. Additionally, Treg suppressive function is not impaired in all RRMS patients, and shows a substantial overlap with healthy individuals [25][26][27][28]. Therefore, it can be speculated that an adequate Treg function cannot be improved further on vitamin D 3 supplementation. Indeed, the patients of whom Treg suppressive function improved tended to have a poorer suppression at week 0. Lastly, the genetic background of an individual may affect the immunological response to vitamin D 3 supplementation [29]. Altogether, the data collected in this study regarding Treg suppressive function remain inconclusive.
A significant effect on the composition of the T helper cell compartment was detected. The ratio of IFN-c + /IL-4 + CD4 + T cells represents the balance between pro-inflammatory Th1 and anti-inflammatory Th2 cells, and is regarded an important variable in autoimmune diseases [30]. During supplementation, this ratio changed towards a less pro-inflammatory profile. The effect on absolute proportions of IFN-c + and IL-4 + CD4 + T cells may be too small to be detected in this small cohort. This is in accordance with previous cross-sectional findings [14]. Interestingly, we detected also more IL-10 + CD4 + T cells after supplementation. The source of IL10 + cells in the CD4 + T cell compartment may be the T regulatory cell type 1 (Tr1) [31]. Alternatively, IL-10 has also been proposed to be a typical Th2 cytokine. However, we measured no double producers of IL-10 and the (other) typical Th2 cytokine IL-4 (data not shown). Therefore, we speculate that the rise in IL-10 + CD4 + T cells might reflect expansion of inducible regulatory Tr1 cells. The finding of an altered profile of pro-and anti-inflammatory CD4 + T cells conforms to observations in experimental studies [5] and further supports the assessment of vitamin D 3 as a natural immune modulator in MS [3].
Although the present study did not comprise a placebo-group, it delivered valuable new information. We earlier performed a crosssectional study, in which serum 25(OH)D levels correlated with peripheral T cell homeostasis [14]. However, the cross-sectional design did not allow statements on causality, since physical exercise and UV exposure per se could be the real underlying mediators [32]. Our current study revealed 25(OH)D as a good causal candidate. Ultimately, however, placebo controlled studies should demonstrate that vitamin D 3 is an immune modulator in vivo. Regarding these upcoming trials, it is also of interest that we included patients treated with immune modulating drugs (IFN-b). Interestingly, therapy with 1,25(OH) 2 D showed synergistic effects with IFN-b in an EAE model of MS [33]. Upcoming clinical trials will most likely assess add-on effects of vitamin D 3 on current immune modulating drugs. Results from the present study suggest that it is worthwhile to combine immune-modulating drugs like IFN-b with vitamin D 3 in order to further modulate the immune system in a for MS beneficial way.
In conclusion, we showed in a cohort of RRMS patients that supplementation of high doses vitamin D 3 did not result in a decompensated calcium metabolism. Additionally, vitamin D 3 appeared to skew the CD4 + T cell compartment to a more pronounced anti-inflammatory state. Herewith, we confirmed that the peripheral CD4 + T cell compartment cytokine profile is a potential outcome measure in upcoming randomized controlled clinical trials. Ultimately, these trials should demonstrate whether vitamin D 3 is an immune and disease modulating compound in MS.