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. 2011 Apr;164(1):72–79. doi: 10.1111/j.1365-2249.2010.04315.x

Regulation of cytokine responses by seasonality of vitamin D status in healthy individuals

A-L Khoo *,†,‡, L Y A Chai †,‡,§, H J P M Koenen *,†, F C G J Sweep , I Joosten *,†, M G Netea †,‡, A J A M van der Ven †,‡
PMCID: PMC3074219  PMID: 21323660

Abstract

The immune modulating capacity of vitamin D3 is well-recognized. Ultra-violet (UV) exposure determines production of vitamin D3in vivo and varies through the course of the year, especially in temperate regions. However, it is not known whether the human innate immune response differs due to seasonality. To validate the seasonal effects of vitamin D3, the effect of 1,25(OH)2D3 on peripheral blood mononuclear cells (PBMC) cytokine response was first determined in vitro. 1,25(OH)2D3 decreased interleukin (IL)-6 and tumour necrosis factor (TNF)-α release by PBMC stimulated with tripalmitoyl-S-glycerylcysteine (Pam3Cys) or lipopolysaccharide (LPS). Subsequently, ex-vivo stimulation studies were performed in 15 healthy volunteers through the course of the four seasons of the year. PBMC were isolated and stimulated with Toll-like receptor (TLR)-2 and TLR-4 ligands Pam3Cys and LPS, respectively. Circulating concentrations of 25(OH)D3 and 1,25(OH)2D3 were higher during summer (P < 0·05) and a down-regulation of TLR-4-mediated IL-1β, IL-6, TNF-α, interferon (IFN)-γ and IL-10 production in summer was observed compared to winter (P < 0·05). The variation in cytokine response upon TLR-2 (Pam3Cys) stimulation was moderate throughout the four seasons. The repressed cytokine production during the summer months could be explained partly by the reduced cell-membrane expression of TLRs. Physiological variation in vitamin D3 status through the four seasons of the year can lead to alteration in the innate immune responses. Elevated vitamin D3 level in vivo is associated with down-regulation of cytokine response through diminished surface expression of pattern recognition receptors.

Keywords: 1,25-dihydroxyvitamin D3; innate immunity; proinflammatory; Toll-like receptors

Introduction

The conventional role of vitamin D3 is that of bone homeostasis and calcium metabolism. Recently, vitamin D3 has been described to exhibit immunomodulatory effects with the uncovering of vitamin D receptors (VDR) expression on activated CD4+ and CD8+ T lymphocytes and antigen-presenting cells (APC) such as monocytes, dendritic cells and macrophages [1–3]. The biologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to influence the differentiation and function of both the innate and adaptive immune cell types and to augment cytokine production differentially [4–6]. 1,25(OH)2D3 has been implicated in various immune-mediated diseases, and investigation into its therapeutic potential in autoimmune diseases and infections are ongoing [7,8].

A major source of vitamin D3 in the body comes from sun exposure [9]. Exposure to ultraviolet (UV) B light (290–315 nm) results in the first step of vitamin D3 biosynthesis, causing 7-dehydrocholestrol to form previtamin D3 in the skin [10]. Previtamin D3 undergoes a spontaneous thermal isomerization into vitamin D3. Vitamin D3 is subsequently hydroxylated into 25-hydroxyvitamin D3 (25(OH)D3) by 25-hydroxylase in the liver. 25(OH)D3 is further converted by 1α-hydroxylase (CYP27B1) in the kidney into the biologically active metabolite, 1,25(OH)2D3. A seasonal variation in vitamin D3 status in temperate climates is well known [11,12]. There has been concern regarding the risk of vitamin D insufficiency among populations residing at higher latitudes where solar radiation during certain periods of the year is inadequate for sufficient cutaneous vitamin D synthesis [13]. Epidemiological studies have implicated seasonality and geographical variation in UV radiation as a contributing factor to the prevalence of autoimmune disorders such as multiple sclerosis (MS), rheumatoid arthritis and insulin-dependent diabetes mellitus [14]. It has been reported that the prevalence of MS rises with increasing latitude [15]. The season of birth has been implicated in MS occurrence and type 1 diabetes [16–18]. Collectively, these data suggest that sun exposure and vitamin D3 levels are candidate risk-modifying factors in certain autoimmune diseases.

On a similar note, it is widely perceived that there is a seasonality for influenza as well as viral upper respiratory tract infections (URTI), and wintertime vitamin D insufficiency is said to be an important contributory factor [19–21]. An inverse association between serum 25(OH)D3 levels and incidence of URTI has been demonstrated [21,22]. A significant reduction in the risk of developing viral URTI was attributed to serum 25(OH)D3 levels of more than 30 ng/ml (75 nmol/l). In other studies, this benefit was conveyed by a higher 25(OH)D3 level of 40 ng/ml (100 nmol/l) [20,23]. Currently, clinical studies evaluating the potential benefits of vitamin D supplementation in reducing the occurrence of seasonal influenza in adults has not been conclusive [24,25], although it seems to be protective in children [26]. In a double-blind randomized controlled trial involving school children (aged 6–15 years), supplementation with 1200 IU cholecalciferol daily during winter reduced influenza A infections significantly.

Despite this wealth of data supporting an immunomodulatory role of vitamin D, no information is available on whether the innate immune response of healthy individuals can vary due to physiological variation in vitamin D3 store during different seasons of the year. In this study, we investigated whether seasonal variation in vitamin D3 in the body is associated with differential Toll-like receptor (TLR)-2 and TLR-4-mediated immune response in healthy subjects living in a temperate region. Such a study would enhance our knowledge on the circumstances determining susceptibility to certain diseases as dictated by the immune-modifying effects of vitamin D3in vivo.

Materials and methods

Reagents

TLR-2 ligand lipopeptide (S)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, tripalmitoyl-S-glycerylcysteine (Pam3Cys) was purchased from EMC Microcollections (Tübingen, Germany). TLR-4 ligand lipopolysaccharide (LPS; Esvherichia coli serotype 055:B5) was purchased from Sigma Chemical Co (St Louis, MO, USA). An extra purification step of LPS was performed as described previously [27]. 1,25(OH)2D3 was purchased from Fluka Biochemika, Sigma-Aldrich (Missouri, MI, USA) and dissolved in absolute ethanol. The reagents were all prepared just before commencement of the seasonal study and stored as aliquots at −70°C for single use only. This was conducted to ensure uniformity of the respective stimuli used for the study.

Stimulation assays

The study was approved by the Ethical Committee on Human Experimentation of the Radboud University Nijmegen. Written consent was obtained from all participants to the study. Venous blood was drawn into ethylenediamine tetraacetic acid (EDTA) tubes from healthy subjects for in-vitro experiments. For the ex-vivo study, 15 healthy male volunteers [median age 36 years, range 28–60; mean body mass index (BMI) 22·8 kg/m2, range 20·5–26·2] were recruited and followed-up for 1 year. Venous blood was drawn from the subjects during four consecutive seasons: winter (December–February), spring (March–May), summer (June–August) and autumn (September–November) of 2009. On the rare occasions that any of the participants reported being unwell, the experiment was postponed until 1 week post-recovery.

Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). Cells were washed twice in saline, counted in a Coulter counter and the number adjusted to 5 × 106 cells/ml. A 100 µl volume of PBMC, suspended in culture medium (RPMI-1640 DM; ICN Biomedicals, Costa Mesa, CA, USA) supplemented with 10 µg/ml gentamicin, 10 mM l-glutamine, 10 mM pyruvate and 10% human pooled serum was added to flat-bottomed 96-well plates (Greiner, Alphen a/d Rijn, the Netherlands).

For the in-vitro study to validate the effects of vitamin D3, PBMC were preincubated with 100 nm 1,25(OH)2D3 for 30 min, followed by addition of TLR-2 ligand (Pam3Cys 10 µg/ml), TLR-4 ligand (LPS 10 ng/ml) or RPMI-1640 (as unprimed control). As with the ex-vivo study, PBMC were stimulated with similar concentrations of Pam3Cys and LPS. In addition, other concentrations tested ex vivo include Pam3Cys 1 µg/ml and LPS 1 and 100 ng/ml. Cell cultures were incubated in a 37°C, 95% humidity, 5% CO2 incubator. The culture supernatants were collected after 24 h [or 48 h for interferon (IFN)-γ] of incubation as appropriate and stored at −20°C until cytokine assay.

Flow cytometry

Cells were analysed phenotypically by five-colour flow cytometry (Coulter Cytomics FC 500; Beckman Coulter, Fullerton, CA, USA) using Coulter Epics Expo 32 software. PBMC as well as whole blood (after cell lysis) was used for flow cytometry analysis after. Cells were washed with PBS with 0·2% bovine serum albumin (BSA) before being labelled with fluorochrome-conjugated antibodies (mAb). After incubation for 20 min at room temperature in the dark, cells were washed twice to remove unbound antibodies and analysed. For cell surface staining, the following mAbs were used: TLR-2-fluorescein isothiocyanate (FITC) (TL2·1) and TLR-4-phycoerythrin (PE) (HTA125), both from eBioscience (San Diego, CA, USA).

Cytokine measurements

Interleukin (IL)-6, IL-1β, IL-10 and IFN-γ concentrations were measured using commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits (Pelikine Compact; CLB, Amsterdam, the Netherlands) according to the manufacturer's instructions. Human tumour necrosis factor alpha (TNF) was measured by a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA). Detection limits were 8 pg/ml (IL-10 and IFN-γ), 16 pg/ml (IL-6), 20 pg/ml (IL-1β) and 40 pg/ml (TNF-α).

Vitamin D3 measurement

Serum 25(OH)D3 was determined using high-performance liquid chromatography (HPLC) with ultraviolet detection, after prior extraction on small SepPak columns. Tritiated 25(OH)D3, collected from the HPLC system during the passage of the UV peak, was used to correct for procedural losses. Serum 1,25(OH)2D3 was measured using a radioreceptor assay (RRA) with prior extraction and chromatographic prepurification with correction for recovery as described previously [28].

Statistical analysis

Results were pooled and analysed using spss 16·0 statistical software. Data given as means + standard error of the mean (s.e.m.) and Wilcoxon's signed-rank test was used to compare differences between groups (unless otherwise stated). The level of significance was set at P < 0·05.

Results

1,25(OH)2D3 resulted in suppression of proinflammatory cytokine response in vitro

First, we carried out LPS and Pam3Cys stimulation on PBMC with the addition of 100 nM 1,25(OH)2D3. After 24 h, IL-6 secretion was reduced by 53% and 29% upon Pam3Cys and LPS stimulation, respectively, in the cells treated with 1,25(OH)2D3 (Fig. 1a). A significant drop in TNF-α production by 17% (Pam3Cys stimulation) and 35% (LPS stimulation) was also observed with 1,25(OH)2D3 at 24 h. Having demonstrated that 1,25(OH)2D3 had the capacity to modulated IL-6 and TNF-α response in vitro, and with the understanding that physiological vitamin D3 levels in the body may vary through the seasons, we validate these observations in vivo in a cohort of healthy volunteers.

Fig. 1.

Fig. 1

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The effect of vitamin D3 on cytokine responses after in-vitro stimulation. Peripheral blood mononuclear cells (PBMC) were preincubated in the presence or absence (carrier) of 100 nM 1,25(OH)2D3 and thereafter stimulated with 10 µg/ml tripalmitoyl-S-glycerylcysteine (Pam3Cys) or 10 ng/ml lipopolysaccharide (LPS). After 24 h, (a) interleukin (IL)-6 and (b) tumour necrosis factor (TNF)-α were measured in the supernatant. Data show results from five independent experiments performed with cells obtained from different donors. *P < 0·05 compared to respective untreated cell culture without the addition of 1,25(OH)2D3.

Serum 25(OH)D3 and 1,25(OH)2D3 was increased significantly in summer

We determined the serum levels of 25(OH)D3 and 1,25(OH)2D3 levels in 15 healthy male volunteers through winter (December–February), spring (March–May), summer (June–August) and autumn (September–November). The amount of sunlight in the study region varied with the seasons (Fig. 2a). The total duration of sunlight in a month prior to vitamin D3 levels and cytokine measurement was 103 h and 240 h in winter and summer, respectively. The median 25(OH)D3 level increased steadily from 43 nmol/l in winter to spring and doubled to 89 nmol/l in summer, with a drop again in autumn (Fig. 2b). The median serum 1,25(OH)2D3 levels raised significantly from 219 pmol/l in winter to 237 pmol/l in summer (Fig. 2c).

Fig. 2.

Fig. 2

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Variation in serum vitamin D3 levels throughout the four seasons of the year. (a) Total duration of sunlight in the study region 4 weeks prior to serum vitamin D3 concentration assay (source: the Royal Netherlands Meteorological Institute). Median serum (b) 25(OH)D3 and (c) 1,25(OH)2D3 concentrations of 15 healthy volunteers at different seasons of the year. *P < 0·05 compared to winter.

TLR-4 (LPS)-mediated cytokine response with variation in vitamin D3 levels

Increased serum vitamin D3 level in summer was associated with a significant drop in TNF-α (64%), IL-6 (33%), IL-1β (59%) and IFN-γ (46%) production when compared against winter (Fig. 3a–d). IL-10 production was also reduced by 55% (P < 0·05) during summer compared to winter (Fig. 3e). These observations show that elevated serum vitamin D3 levels are associated with attenuation of the host inflammatory response after engagement of TLR-4.

Fig. 3.

Fig. 3

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Variation in cytokine responses to lipopolysaccharide (LPS) stimulation during the four seasons of the year. Peripheral blood mononuclear cells (PBMC) were isolated from 15 healthy volunteers during each of the four seasons and stimulated with 10 ng/ml LPS. (a) Interleukin (IL)-1β, (b) IL-6, (c) tumour necrosis factor (TNF)-α, (d) interferon (IFN)-γ and (e) IL-10 were measured. Data show results from 15 healthy donors. *P < 0·05 compared among the respective seasons.

TLR-2 (Pam3Cys)-mediated cytokine response with variation in vitamin D3 levels

We also determined the regulation of cytokine production upon Pam3Cys stimulation of PBMC. After 24 h of stimulation, a modest drop in TNF-α and IL-1β production by 27% (P < 0·05) and 22% (P> 0·05), respectively, was seen during summer compared to winter (Fig. 4a and c). The variation in IL-6 and IL-10 (Fig. 4b and d) production was limited throughout the course of the four seasons. The seasonal variation of TLR-2-mediated host cytokine response was more limited compared to TLR-4-mediated responses.

Fig. 4.

Fig. 4

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Variation in cytokine responses to tripalmitoyl-S-glycerylcysteine (Pam3Cys) stimulation during the four seasons of the year. Peripheral blood mononuclear cells (PBMC) were isolated from 15 healthy volunteers during each season and stimulated with 10 µg/ml Pam3Cys. (a) Interleukin (IL)-1β, (b) IL-6 and (c) tumour necrosis factor (TNF)-α and (d) IL-10 were measured. Data show results from 15 healthy donors. *P < 0·05 compared among the respective seasons.

Reduced TLR-2 and TLR-4 expression could explain the suppression of cytokine responses in summer

We have found recently that 1,25(OH)2D3 had the capacity to modulate TLR-2 and TLR-4 expression in vitro[6]. Therefore, we investigated whether the attenuated proinflammatory cytokine responses observed in our ex-vivo experiments was indeed the result of an altered pattern recognition receptors (PRR) expression. Using flow cytometric analysis, we found that the expression of both TLR-2 and TLR-4 on monocytes was reduced during summer when compared to winter (Fig. 5a and b).

Fig. 5.

Fig. 5

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Expression of (a) Toll-like receptor (TLR)-2 and (b) TLR-4 on the cell membrane of monocytes. Peripheral blood mononuclear cells (PBMC) were isolated from 15 healthy volunteers during each season and analysed for the respective markers using flow cytometry as gated on monocytes. Data show results from 15 healthy donors. *P < 0·05 compared to winter.

Discussion

Much interest has been shown recently in the immunomodulatory capacity of vitamin D3 and the role it plays in health and diseases. While biosynthesis upon UVB exposure serves as the main source of vitamin D3 in the body [9], it is not known if seasonal variation in vitamin D3 store arising from a fluctuating solar exposure can impact the innate immune response. We show for the first time that, relative to winter levels, there was a physiological elevation in vitamin D3 store during summer and this led to down-regulation in proinflammatory cytokine production, particularly when stimulated via the TLR-4-mediated signalling pathway.

25(OH)D3 is the major circulating form of vitamin D3 and its concentration is used commonly as an indicator of vitamin D3 status [29], whereas 1,25(OH)2D3 is the biologically active form of vitamin D3. BMI has been shown to be related inversely to vitamin D3 levels [30]. This confounder has been excluded due to the fact that none of the volunteers in the study was obese (BMI > 30 kg/m2). Here, we found that the difference between winter (December–February) and summer (June–August) 25(OH)D3 levels to be 46 nmol/l. As expected, serum 1,25(OH)2D3 concentrations were also higher in summer compared to winter. The body vitamin D3 levels correlated with the amount of sunlight in the study region. In our cohort of 15 subjects residing at 52°N from the Equator, we were hence able to demonstrate an evident variation of vitamin D3 status throughout the four seasons. Other studies have also demonstrated such a difference in young adults residing along similar latitudes at 40–50°N [11,12,31]. Epidemiological studies have suggested that wintertime vitamin D insufficiency may account for the seasonal variation in incidence of viral respiratory tract infections [19,20,22]. As such, it is most appropriate to look further into how the host immune responses vary during the different seasons.

Looking specifically at TLR-2 and TLR-4-mediated response, as main pattern recognition receptors are involved, we first demonstrated that in-vitro 1,25(OH)2D3-treated cells produced less IL-6 and TNF-α. In line with the modulatory effects of vitamin D3, a general down-regulation of cytokine production was found during the summer months when the serum vitamin D3 levels were elevated, especially when PBMC from the volunteers were challenged with LPS. A fundamental aspect to be taken into account is that the physiological up-regulation of vitamin D3 levels by solar radiation in our study differs from the vitamin D3 doses employed in the in-vivo and in-vitro studies. This could explain why the effect on IL-6 production was less evident with Pam3Cys in our ex-vivo experiment, although there was a 50% reduction in the in-vitro set-up. Conversely, we saw a similar reduction in TNF-α production both in vitro and ex vivo following ligation of TLR-2.

One of the key targets of 1,25(OH)2D3 is the CD4+ T lymphocytes. 1,25(OH)2D3 is known to suppress the secretion of IFN-γ and IL-2, while enhancing IL-4, IL-5 and IL-10 production [32,33]. By inhibiting the secretion of IFN-γ, 1,25(OH)2D3 limits antigen presentation and recruitment of other T cells, thereby down-regulating the proinflammatory response. Conversely, 1,25(OH)2D3 promotes the production of the anti-inflammatory cytokines IL-4 and IL-10, thus shifting the balance towards a T helper type 2 (Th2) or regulatory T cells (Treg) phenotype [34,35]. Of note, a major difference from some of these previous in-vitro and mice studies is that our ex-vivo data show both the proinflammatory and anti-inflammatory cytokines being down-regulated in summer. Again, the concentrations of 1,25(OH)2D3 used in vitro and the actual physiological levels in the body may account for this apparent discordance. In contrast, there have also been recent reports of 1,25(OH)2D3 suppressing both IFN-γ and IL-10 in CD4+ T lymphocytes [36]. In a similar experimental set-up using LPS stimulation, Matilainen et al. pointed out that the impaired IL-10 production induced by 1,25(OH)2D3 was time-dependent and not sustainable. They reported that while IL-10 secretion from monocytse was suppressed by 1,25(OH)2D3 during the initial 8 h, there was a rebound up-regulation at 48 h [37].

We attempted to study the mechanisms responsible for the down-regulation of the proinflammatory cytokine response during summer. An explanation could be that 1,25(OH)2D3 induces down-regulation of TLR expression, and this may account for the attenuated response after stimulation with TLR-2 and TLR-4 ligands [6,38]. Indeed, we found a reduced surface expression of TLR-2 and TLR-4 associated with elevated vitamin D3 stores during summer which persisted throughout autumn. Through modification of TLR-2 and TLR-4 expression, 1,25(OH)2D3 had the propensity to limit cytokine response following ligation of the receptors. Similarly, Sadeghi et al. has demonstrated in vitro that 1,25(OH)2D3 down-regulates TLR-2 and TLR-4 mRNA and protein expressions on monocytes, and this resulted in an impaired TNF-α production upon LPS challenge [38]. Furthermore, they concluded that this effect was VDR-dependent by showing that the down-regulation of TLR expression was reversed by a vitamin D receptor antagonist. However, we note from our ex-vivo experiments that the reduction of TLR-2 expression during summer was accompanied by a corresponding drop in TNF-α production following stimulation with Pam3Cys, but this trend was less evident for IL-6.

A still unresolved question arising from ongoing bench and clinical research has been the serum vitamin D3 concentrations needed to elicit an optimal immune response [39]. The present results also suggest that there may be differences in the intracellular pathways leading to TNF-α and IL-6 production [40], and these pathways may be modulated differentially by vitamin D3 concentration in vivo. Nevertheless, overall it seems remarkable here that our observations streaming from the physiological changes in vitamin D3 levels show similar trends correlating TLR expression and cytokine production compared to those from an in-vitro setting.

In the present study, we assessed a homogeneous study population (healthy, adult males with normal BMI) to establish if and how host cytokine response vary with seasons. Unique to previous in-vitro and in-vivo studies examining the role of vitamin D3, our data showed how innate immune responses can be influenced by the physiological variation of serum vitamin D3 levels during the four seasons of the year. It would be prudent to validate these observations in a larger and more diverse population cohort to identify any possible differences in cytokine responses among the extreme of ages and different genders. None the less, with these results in mind, it is tempting to hypothesize whether response to vaccination programmes could be different when carried out during winter or summer. A recent study conducted in a small cohort of prostate cancer patients suggested that there was a trend towards a better serological response rate against among patients with higher serum 25(OH)D3 levels [41]. However, the 25(OH)D3 level delineating such a difference was not reported. Conversely, a clinical trial in healthy volunteers showed that co-administration of 1,25(OH)2D3 with influenza vaccine did not affect serological response against H1N1, H3N2 or influenza B antigens [42]. Further research is needed to validate this hypothesis, as this would have far-reaching implications for implementation of preventive health policies. These data may also provide better insight into previous epidemiological findings regarding the prevalence of autoimmune diseases and infections, which have been attributed to seasonal variation in vitamin D3 status. In a step further, the season of birth has been implicated with the occurrence of MS and insulin-dependent diabetes mellitus [16–18]. In addition to that, higher serum 25(OH)D3 levels have been associated with lower relapse risk in MS, occurring in a dose-dependent linear fashion [43]. Hypothetically, a heightened proinflammatory response in utero during wintertime may have far-reaching implications on eventual development of MS and insulin-dependent diabetes mellitus. Further studies are warranted to validate this by looking at cytokine responses during pregnancy across the four seasons and assessing the therapeutic benefits of vitamin D supplementation during pregnancy.

In conclusion, we have demonstrated for the first time that variations in innate immune response exist throughout the four seasons of the year. In summer, elevated serum vitamin D3 levels are associated with an attenuated cytokine-producing capacity attributable to a suppressed expression of TLR-2 and TLR-4. This harbours potentially important implications for our understanding of disease epidemiology and implementation of vitamin D3 supplementation in temperate regions during winter.

Acknowledgments

The authors are grateful to Mr André Brandt for his technical assistance. This work is supported by The Nutricia Research Foundation, the Netherlands. L. C. was supported by the Health Manpower Development Plan (HMDP) Fellowship, Ministry of Health, Singapore and the International Fellowship, Agency for Science, Technology and Research (A*STAR)/National Medical Research Council (NMRC), Singapore. M. G. N. was supported by a Vici grant of the Netherlands Organization for Scientific Research.

Disclosure

None.

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