Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis

Joost Smolders, Mariëlle Thewissen, Evelyn Peelen, Paul Menheere, Jan Willem Cohen Tervaert, Jan Damoiseaux, Raymond Hupperts, Joost Smolders, Mariëlle Thewissen, Evelyn Peelen, Paul Menheere, Jan Willem Cohen Tervaert, Jan Damoiseaux, Raymond Hupperts

Abstract

Background: In several autoimmune diseases, including multiple sclerosis (MS), a compromised regulatory T cell (Treg) function is believed to be critically involved in the disease process. In vitro, the biologically active metabolite of vitamin D has been shown to promote Treg development. A poor vitamin D status has been linked with MS incidence and MS disease activity. In the present study, we assess a potential in vivo correlation between vitamin D status and Treg function in relapsing remitting MS (RRMS) patients.

Methodology/principal findings: Serum levels of 25-hydroxyvitamin D (25(OH)D) were measured in 29 RRMS patients. The number of circulating Tregs was assessed by flow-cytometry, and their functionality was tested in vitro in a CFSE-based proliferation suppression assay. Additionally, the intracellular cytokine profile of T helper cells was determined directly ex-vivo by flow-cytometry. Serum levels of 25(OH)D correlated positively with the ability of Tregs to suppress T cell proliferation (R = 0.590, P = 0.002). No correlation between 25(OH)D levels and the number of Tregs was found. The IFN-gamma/IL-4 ratio (Th1/Th2-balance) was more directed towards IL-4 in patients with favourable 25(OH)D levels (R = -0.435, P = 0.023).

Conclusions/significance: These results show an association of high 25(OH)D levels with an improved Treg function, and with skewing of the Th1/Th2 balance towards Th2. These findings suggest that vitamin D is an important promoter of T cell regulation in vivo in MS patients. It is tempting to speculate that our results may not only hold for MS, but also for other autoimmune diseases. Future intervention studies will show whether modulation of vitamin D status results in modulation of the T cell response and subsequent amelioration of disease activity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Results proliferation suppression assay.
Figure 1. Results proliferation suppression assay.
(A) Representative dot-plot of the sorting protocol. CD25+CD127− cells within the CD4+ lymphogate were sorted as Tregs, CD25− cells were sorted as Tresps. (B) The percentages of suppression which were achieved for the respective Treg/Tresp ratios of the individual patients are shown. The Treg/Tresp ratio at which 50% inhibition of proliferation was achieved is defined as ED50 of suppression. The distribution of ED50 is shown in the rightmost column. The lines show the median values. The closed dots represent the Beta Interferon-treated patients, the open dots the untreated patients.
Figure 2. Correlation between Treg suppressive function…
Figure 2. Correlation between Treg suppressive function and serum 25(OH)D levels.
(A) The correlation between ED50 and serum 25(OH)D levels. (B–E) Correlation between inhibition of suppression and 25(OH)D levels for the individual Treg/Tresp ratios. The Spearman correlation coefficient is shown. The regression line illustrates the direction of the association. The closed dots represent the Beta Interferon-treated patients, the open dots the untreated patients.
Figure 3. Relative and absolute number of…
Figure 3. Relative and absolute number of circulating Tregs.
Within the CD3+CD4+ lymphogate, Tregs were defined as either being (A) CD25+FoxP3+ or being (B) CD25+CD127−. (C) The percentages of Tregs within the CD4+ T cell compartment. (D) Absolute number of circulating Tregs. The lines indicate the median values.
Figure 4. Correlation of the number of…
Figure 4. Correlation of the number of circulating Tregs with serum 25(OH)D levels.
(A–D) Correlation of relative and absolute number of Tregs with 25(OH)D levels. The Pearson correlation coefficient and corresponding regression line are shown.
Figure 5. Correlation of T helper cell…
Figure 5. Correlation of T helper cell subsets with serum 25(OH)D levels.
(A) The percentages of IFN-γ+, IL-4+, IL-17+ and IL-10+ CD4+ T cells. The lines show the median values. (B) The correlation between IFN-γ/IL-4 ratio and serum 25(OH)D levels. The Pearson correlation coefficient and corresponding regression line are shown.

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