Vitamin D mediates immunomodulatory functions, and its deficiency has been associated with an increased prevalence of immunological diseases. The supplementation of vitamin D might be therapeutically beneficial, for example, in lupus erythematosus patients. However, its affect on established recall immune responses is undefined.
In all, 32 individuals were randomized in a placebo controlled, double-blind setting, and received vitamin D (daily 2000 IU) for 10 weeks followed by tetanus toxoid (TT) booster immunization.
During vitamin D supplementation the median 25-hydroxyvitamin D serum concentration increased to 80.3 nM, which as expected decreased in the placebo group to 29.1 nM during the ultraviolet-deprived winter months. The TT-specific immunoglobulin G (IgG) boost efficiency was marginal higher in the vitamin D group (P=0.04). The increase of the 25-hydroxyvitamin D levels correlated with the increase of TT–IgG serum concentrations. The induction of specific serum IgA and specific antibody secreting cells was comparable between both groups. Accordingly, the TT-specific and polyclonally triggered T-cell cytokine profiles were stable as well.
Vitamin D supplementation was successful and booster immunization induced efficiently specific antibodies titers.
The vitamin D system, besides its well-described function in calcium homeostasis, is involved in the fine tuning of immunity (Hayes et al., 2003; Holick, 2007; Adams and Hewison, 2008). The biologically active metabolite 1α,25-dihydroxyvitamin D3 (calcitriol) signals after binding to the nuclear vitamin D receptor and transcriptional regulation of target gene expression (Hayes et al., 2003). Calcitriol synthesis is catalyzed enzymatically from the otherwise inert storage metabolite 25-hydroxvitamin D3 (Hayes et al., 2003; Adams and Hewison, 2008). Thus, the precursor serum concentration determines the vitamin D status (Holick, 2007) (Vieth, 2007) and 25-hydroxyvitamin D serum concentrations below 50 nM (20 ng/ml) are considered as insufficient (Holick, 2007). Acquired vitamin D deficiency has been correlated with the prevalence and/or increased severity of autoimmune diseases, but also infectious diseases and cancer (Hayes et al., 2003; Holick, 2007; Adams and Hewison, 2008; Adorini and Penna, 2008). Direct modulation of immune cells by vitamin D is most likely, as different immune cell types can produce and perceive calcitriol including monocytes, dendritic cells, T-helper cells and activated B cells (Hayes et al., 2003; Adorini and Penna, 2008). Previously, we demonstrated that activated human B cells can synthesize calcitriol from its precursor, which mediates enhanced expression of interleukin-10 (IL-10; Heine et al., 2008) and inhibited immunoglobulin E (IgE) production (Heine et al., 2002). In newborns, cord blood levels of 25-hydroxyvitamin D and the ratio of IL-10 to IgE are both higher in infants born in summer compared with winter (Zittermann et al., 2004). Other vitamin D receptor-mediated functions are enhanced innate anti-microbial defense, increased tolerance of adaptive immune reactions and altered lymphocyte trafficking (Hayes et al., 2003; Adams and Hewison, 2008; Adorini and Penna, 2008).
Until now, the safety of vitamin D supplementation was assessed by parameters related to calcium mobilization, reflecting hypercalcaemic toxicity of endogenously synthesized calcitriol (Vieth, 1999) and the no observed adverse event level for daily vitamin D supplementation was set at 2000 IU (50 μg; Vieth, 2007). Still, no data on immunological safety, for example, regarding intact recall responses are available.
Subjects and methods
Study population and design
Individuals were recruited at the Department of Dermatology and Allergy, Charité–Universitätsmedizin, Berlin, Germany. The inclusion criteria were scheduled tetanus/diphtheria immunization because of a previous vaccination that dated back at least 5 years. The exclusion criteria were: history for sarcoidosis, hypercalcemia, creatinine serum concentration >1 mg per 100 ml, nephrolithiasis, any chronic disease and planned exposition to ultraviolet light-including tanning or traveling to countries at lower latitude (<40°). The study was approved by the Ethical Board of the Charité and written informed consent was obtained from all participants.
Individuals were randomly assigned in a double-blind manner to the vitamin D group receiving orally 2000 IU vitamin D3 oil per day (50 μg, n=20) or to the placebo group (equal volume neutral oil; n=12). The daily calcium intake of 1200 mg by each participant was guaranteed by supplementation. Compliance was assessed by determining the 25-hydroxyvitamin D serum levels and consumed study medication amount at the end of the supplementation period of 10 weeks. An intramuscular booster immunization with a combined tetanus/diphtheria toxoid vaccine (Td-Merieux, Sanofi Pasteur MSD, Strasbourg, France) was performed after 9-weeks-supplementation period.
The 25-hydroxyvitamin D was measured by enzyme immunoassay (Immundiagnostik AG, Bensheim, Germany). TT-specific IgG and IgA were measured using an enzyme immunoassay from serially diluted sera (DRG Diagnostics, Marburg, Germany). A long-term protective immunity (5–10 years) is assumed >1.0 IU/ml anti-tetanus IgG. TT-specific IgA concentrations were validated by serially dilution of strongly positive sera, and units were calculated on the basis of the absorption at 405 nm. Tetanus-specific IgE was determined using the ImmunoCAP system (Phadia, Freiburg, Germany). All other parameters were determined by the Charité routine laboratory facility.
Peripheral T and B lymphocytes were quantified from whole blood after surface staining using TruCount beads and fluorochrome-conjugated monoclonal antibodies against CD4 or CD8 and CD19, CD20, CD27 (all from BD, Heidelberg, Germany) and CD38 (Beckman Coulter, Krefeld, Germany), respectively. Tetanus-specific B cells and plasmablasts were stained from isolated peripheral blood mononuclear cells after density gradient centrifugation (lymphocyte separation medium, PAA, Cölbe, Germany) exactly 7 days after TT immunization by intracellular binding to the fluorescence labeled recombinant C-fragment of tetanus toxin (TT), as described (Odendahl et al., 2005; Mei et al., 2008) following CD38, CD3 and CD14 surface staining. T-cell activation was monitored following stimulation of whole blood with 2 μg/ml Staphylococcus enterotoxin B (Sigma Dreieich, Germany) in the presence of 1 μg/ml Brefeldin A (Sigma; Frentsch et al., 2005). Flow cytometric analyses were performed using a LSRII cytometer equipped with an additional ultraviolet laser or FACS Canto II and DivaSoft operation system (all from BD) and FlowJo7 software (TreeStar, Ashland, OR, USA).
TT-induced cytokine release
The TT-specific T-cell cytokine profile was monitored before and exactly 7 days after immunization, at the peak of TT-specific T-cell circulation (De Rosa et al., 2004). Isolated peripheral blood mononuclear cells (4 × 105 cells in 200 μl) were cultured in RPMI1640 containing 10% fetal calf serum as described (Heine et al., 2002), in 96-well round bottom culture plates in the presence or absence of 0.5 μg/ml S. enterotoxin B (Sigma) or 1% TT (Tetanus-Merieux, Sanofi Pasteur MSD, Strasbourg, France) in triplicates. After 24 h, the supernatants were stored at −80 °C until analysis using the Th1/Th2 cytokine CBA kit (BD), which allows the simultaneous quantification of secreted IL-2, IL-4, IL-5, IL-10, tumor necrosis factor-α (all sensitivity thresholds <3 pg/ml) and interferon-γ (IFN-γ; <8 pg/ml).
Statistical evaluations and graphic display were performed with Prism software (GraphPad Software Inc., CA, USA) and SPSS15.0 (SPSS Inc., Chicago, IL, USA). Normal distribution was tested by the Kolmogorov–Smirnov test and the D′Agostino and Pearson's omnibus normality test. Normally distributed values were tested by the Student t-test and the mean±s.d. are shown. Whitney–Mann U-test or Wilcoxon test was applied for non-normally distributed values for independent or dependent parameters, respectively, and median and interquartile ranges are shown.
Efficient increase of 25-hydroxyvitamin D serum concentrations by daily intake of 2000 IU cholecalciferol
At study entry, the distribution of age, gender and body mass index (Supplementary Table 1) and vitamin D status did not differ between the two groups (Supplementary Table 2; Figure 1b). The median 25-hydroxyvitamin D concentrations significantly increased on daily intake of 2000 IU (50 μg) vitamin D from 28.4 to 80.3 nM after 10 weeks (P=0.0001, Figure 1b). As expected, in the placebo group the median 25-hydroxyvitamin D serum concentration decreased by 13.1 nM to 29.1 nM after the study period (P=0.0007). Consequently, the 25-hydroxyvitamin D serum concentrations were strongly different between both groups after the supplementation period (P=0.0001). During 10 weeks of daily intake of 2000 IU vitamin D (cholecalciferol), the calcium, phosphate and IgA, IgG and IgM serum concentrations were not altered in any individual (Supplementary Table 2). The differential blood count was comparable in both groups, except the absolute monocyte numbers, which decreased in the placebo group (−0.04/nl) and remained constant in the vitamin D group (P=0.04, Supplementary Table 2). Adverse events during the study period occurred in both groups in comparable frequencies (vitamin D group: 30.0% common colds, n=6; placebo: 33.3% common colds, n=4, data not shown). No treatment-related adverse event occurred during the study.
Efficient immunization triggered TT-specific antibody induction on vitamin D supplementation
Before immunization, the TT-specific IgG concentrations were comparable between both groups (Figure 1b). After 9 weeks of supplementation, a tetanus/diphtheria toxoid immunization was performed intramuscularly and the TT-specific IgG concentrations were strongly boosted by 29.8 IU±14.8 on vitamin D intake and 21.0 IU±13.3 with placebo (both intragroup changes P<0.0001). The TT-specific IgG boost efficiency was marginally higher in the vitamin D group (P=0.04). The baseline and immunization-induced TT-specific IgA serum concentrations were independent of vitamin D or placebo treatment (P=0.70; data not shown). In this study, TT-specific IgE was present in nine participants and comparable changes on immunization were observed in both groups (vitamin D group: 3 up, 3 down; placebo 2 up, 1 down; data not shown). In addition, circulating TT-specific antibody secreting plasmablasts (Figure 1c) were analyzed exactly 7 days after immunization, as these cells are only present in the peripheral blood in a very limited time frame (Odendahl et al., 2005). As shown in Figure 1d, most recombinant C-fragment of TT-specific plasmablasts were of IgG isotype in both groups (intergroup difference P=0.57) followed by recombinant C-fragment of TT-specific IgA plasmablasts (P=0.53). The numbers of recombinant C-fragment of TT-specific plasmablasts and relative frequency of all plasmablasts showed no statistically significant differences in both groups (P=0.51–0.26, data not shown). The frequencies of peripheral naive B cells (CD27−CD19+), memory B cells (CD27+CD19+) or terminally differentiated plasmablasts (CD27highCD20−CD38highCD19+) were comparable between the vitamin D or placebo group (data not shown). Interestingly, a significant correlation between the increased 25-hydroxyvitamin D concentration and TT–IgG serum concentration was observed (P=0.042, R=0.351, Figure 2).
Stable TT-induced cytokine secretion by peripheral immune cells
Whether the TT-specific and/or polyclonal cytokine response is modulated on vitamin D intake was analyzed from ex vivo stimulated peripheral blood cells before and exactly 7 days after immunization, at the peak of specific T cell circulation (De Rosa et al., 2004). Unstimulated peripheral blood mononuclear cells served as negative control and no significant spontaneous cytokine release was observed, otherwise the individual data set was excluded because of the subclinical concomitant infection (vitamin D group: n=5; placebo n=4). As positive control and reference for the circulating T-cell pool polyclonal T-cell stimulation by S. enterotoxin B resulted in the activation CD4+ T-helper cells in the vitamin D or placebo group in comparable frequencies (4.7±0.8 or 3.9±0.5% of all lymphocytes, data not shown) as determined by CD154 upregulation (Frentsch et al., 2005). These data were paralleled by S. enterotoxin B-induced IFN-γ, tumor necrosis factor-α, IL-10, IL-2, IL-4 and IL-5 secretion (intergroup differences P-value range 0.84–0.11, Table 1). TT stimulation of peripheral blood mononuclear cells for 24 h before and at 1 week after immunization induced IL-2, IL-10, IFN-γ, or tumor necrosis factor-α secretion, and no significant differences between both groups regarding the respective TT-induced cytokine secretion were observed (P-values 0.76–0.11, Table 1). TT-induced IL-4 or IL-5 secretion was below the detection threshold in all samples (data not shown).
In this study, we show that the daily intake of 2000 IU vitamin D over 10 weeks was well tolerated, and sufficient to restore the vitamin D status as determined by a median 25-hydroxvitamin D concentration of 80.3 nM, which is considered as sufficient for optimal vitamin D function (Holick, 2007; Adams and Hewison, 2008). The TT boost immunization is fully operational on vitamin D intake with regard to TT-specific immunoglobulin induction and cytokine expression and more importantly, is not negatively altered in a placebo-controlled setting. In contrast, the data indicate that induction of TT-specific IgG was marginally more efficient in vitamin D compared with the placebo group (P=0.04). These data are in line with previous studies (Ivanov et al., 2006; Enioutina et al., 1999, 2008; Van der Stede et al., 2001) and attributed most likely to induction of specific IgG1, which is the dominant subclass induced by the vaccination used herein (>92% of the circulating specific plasmablasts; Frolich et al., 2010). That vitamin D supplementation did not alter the specific IgG subclass composition is also suggested by our data that vaccination-induced boosted anti-tetanus antibodies of IgA or IgE isotype was independent of vitamin D supplementation, although common cytokines were involved, and further supported by previous studies in animals (Van der Stede et al., 2001). However, our data should be validated by investigation of a larger study cohort, because of the limited group size.
The reports on vitamin D-mediated modulation of T-helper cell polarization in mice and human are heterogeneous (Hayes et al., 2003) and include altered Th1/IFN-γ (Daniel et al., 2005), Th2/IL-4 (Hayes et al., 2003), but also tolerogenic responses (Schleithoff et al., 2006; Adorini and Penna, 2008). In this study, we observed a stable recall T-helper cell response on vitamin D regarding the secretion of TT-specific or polyclonally induced IL-2, IL-4, IL-5, IL-10, IFN-γ or tumor necrosis factor-α compared with placebo. Interestingly, the blood monocytes counts were higher in the vitamin D group after the supplementation period, compared with placebo. As differentiation of monocytes/macrophages from progenitor cells is promoted by calcitriol (Hayes et al., 2003; Adams and Hewison, 2008), our findings indicate that supplemented vitamin D metabolized to calcitriol modulated cellular immunity. However, whether and how vitamin D-induced monocytes contributed to the enhanced TT–IgG boost efficiency, for example, by altered antigen presentation and IgG promoting costimulation of T and B lymphocytes, is not known yet.
An advantage in our study setting during the winter months was the lack of ultraviolet-derived vitamin D synthesis, allowing a correlation between vitamin D provided by supplementation with biological effects. As prerequisite, a high prevalence vitamin D deficiency in December was observed in this study and confirms a recent epidemiological report from Germany (Hintzpeter et al., 2008). As vitamin D supplementation was efficient, differing averagely <2% from a calculation by Heaney et al. (2003), and the 25-hydroxyvitamin D serum levels further decreased in the placebo group during the study period, strongly different 25-hydroxyvitamin D serum concentrations between both groups were observed (P=0.0001). In further placebo-controlled studies during the winter months, the efficacy of immunological functions of endogenously produced calcitriol following vitamin D supplementation can be examined in different disease settings, for example, allergy or autoimmunity.
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We thank Dr Helmut Orawa and Ramona Scheuffele (both Institut für Biometrie und klinische Epidemiologie, Charité Campus Mitte) for statistical assistance and Dennis Ernst for outstanding technical assistance. This work was supported by the Deutsche Forschungsgesellschaft (DFG–SFB650/TP5 and TP16) and by the Charité–Universitätsmedizin Berlin. GH and GD were supported by the DFG–SFB650 TP5 and HM by the DFG–SFB650/TP16.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on European Journal of Clinical Nutrition website
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Heine, G., Drozdenko, G., Lahl, A. et al. Efficient tetanus toxoid immunization on vitamin D supplementation. Eur J Clin Nutr 65, 329–334 (2011). https://doi.org/10.1038/ejcn.2010.276
- vitamin D
- tetanus toxoid
- humoral recall response
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