Vitamin D regulates inflammatory cytokines and skeletal muscle function, especially in isolated immune cells and various pathophysiological conditions in humans. In the present investigation, we extend those previous reports by providing several original and impactful results obtained from healthy adults during the winter. First, we provide new data identifying serum 25(OH)D and IL-5 concentration decreases. Secondly, supplemental vitamin D at 200 IU/d prevented serum 25(OH)D and IL-5 concentration decreases. Third, supplemental vitamin D at 4000 IU/d induced an immediate increase in serum 25(OH)D concentrations without modulating circulating IL-5. Finally, despite serum 25(OH)D concentrations correlating with muscle strength, supplemental vitamin D did not moderate muscular strength alterations. These unique data indicate that subtle fluctuations in serum 25(OH)D concentrations modulate a TH2-type cytokine, and that muscular strength does not parallel the rapid serum 25(OH)D increase induced by a high-daily dose of supplemental vitamin D in young, reportedly active and healthy adults.
As expected, serum 25 (OH) D concentrations responded disparately to different doses of supplemental vitamin D (Figures and ). Compared to serum 25(OH)D concentrations prior to supplementation, we observed a 15% increase after 7-d, which progressively inflated by ~50% after 28-d of supplemental vitamin D at 4000 IU/d. Thus, in subjects with mean serum 25(OH)D concentrations in the low-30 ng/ml range prior to supplementation, 4000 IU/d of supplemental vitamin D induced a rapid and continuous increase in serum 25(OH)D concentrations during the winter for 28-d. Conversely, in the 200 IU group, serum 25(OH)D concentrations did not change, while serum 25(OH)D concentrations decreased in the placebo group (Figure ; 21-d and 28-d). These are important findings because they establish consistency with previous studies that identify a decrease in serum 25(OH)D concentrations during the winter [1
]. Moreover, our results indicate that at our geographical location, 200 IU/d of supplemental vitamin D plus daily dietary intake of vitamin D is adequate to maintain serum 25(OH)D concentrations and vitamin D sufficiency during the winter, at least in adult non-smokers and during 28-d. However, our data obtained at 200 IU/d of supplemental vitamin D dispute previous findings. Specifically, Barnes et al. [38
] recently demonstrated that 200 IU of daily vitamin D supplementation was ineffective at maintaining serum 25(OH)D concentrations during winter [38
]. The more Northern geographical latitudes (51° and 55°N) and plausibly the longer supplementation duration (22 weeks) in the study by Barnes et al. [38
] could be contributing factors for the discrepancy in maintaining serum 25(OH)D concentration between studies that were conducted at the same reported supplemental dose of vitamin D. This interpretation would further suggest the dose of supplemental vitamin D (plus dietary intake) required to maintain a given serum 25(OH)D concentration increases with increasing Northern latitudes during the winter. Another explanation for the discrepancy between Barnes et al. [38
] and those herein could be differences in dietary vitamin D intake. Unfortunately, neither Barnes et al. [38
] nor do we report vitamin D intake from dietary sources. Future studies are encouraged include dietary sources (and sun exposure) of vitamin D when investigating serum 25(OH)D concentrations at daily doses of supplemental vitamin D that are easily achievable through dietary sources. Finally, it is probable that subjects randomized to the 200 IU group had a lower percentage of body fat compared to those by Barnes et al. [38
]. This could potentially lower the physiological needs of vitamin D in the 200 IU group due to less sequestering to adipose tissue. We speculate that the good vitamin D status reflected by our subjects prior to supplementation is indicative of sun exposure during the preceding summer and fall and daily dietary habits that include vitamin D, such as the consumption of oily fish and vitamin D fortified food sources.
A novelty of the present investigation was the IL-5 results obtained with and without supplemental vitamin D (Figure ). In the placebo group, there was a transient IL-5 decrease, while in contrast, IL-5 increased in the 200 IU and remained unchanged in the 4000 IU group. Due to the small sample and effect sizes, it is plausible that 200 IU of supplemental vitamin D maintained as opposed to increased IL-5. This premise is logically harmonious with the data obtained in the 4000 IU group, which in sum suggests that supplemental vitamin D maintains serum 25(OH)D and IL-5 concentrations during the winter. However, consistent with our increase in the 200 IU group, Qi et al. [44
] demonstrated that vitamin D (i.e., 1,25(OH)D) pre-treatment in rats injected-intraperitoneally with lipopolysaccharide (LPS) increased IL-5 gene expression in the spleen [44
]. Similarly, IL-5 production increased in CD4+
cells obtained from mice when incubated in 1,25(OH)D and stimulated with IL-2, phorbol myristate acetate (PMA) and ionomycin [45
]. These results indicate that the active metabolite form of vitamin D increases IL-5. However, results vary. In an experimental mice model of pulmonary eosinophilic inflammation, subcutaneous 1,25(OH)D injections abrogated IL-5 production in bronchoalveolar lavage fluid [46
]. Furthermore, when stimulated with PMA and ionomycin in the presence of 1,25(OH)D, IL-5 production decreased in TH0
-cells and remained unchanged in TH1
- and TH2
]. Regarding no change in IL-5, Yusupov et al. [48
] demonstrated that vitamin D (cholecalciferol) supplementation at 2000 IU/d for 3 months was ineffective at altering circulating IL-5 concentrations in young, healthy adults. Thus, these latter studies potentially conflict with our results at 200 IU but are in agreement with those reported herein at 4000 IU. Collectively, these findings highlight that the cytokine-modulating property of vitamin D could be physiologically dependent on the supplemental dose (and/or environmental factors and dietary intake), the immunological challenge (or lack thereof), experimental model (i.e., humans, rodents, or cell), duration of vitamin D treatment, the timing of data collection, and importantly, the form of vitamin D studied.
Regarding the form of vitamin D, most studies have investigated the influence of 1,25(OH)D on inflammatory cytokines in vitro
, which makes it is difficult to equate or interpret results relative to supplemental D or serum 25(OH)D concentration studies in humans that do not report 1,25(OH)D. Recently, Zhang et al. [49
] demonstrated that 15 ng/ml of serum 25(OH)D was ineffective at suppressing LPS-induced cytokine (i.e., IL-6 and TNF-α) production in human monocytes studied in vitro
. In contrast, 30 ng/ml of serum 25(OH)D significantly inhibited cytokine production and was comparable to that shown at 0.04 ng/ml of 1,25(OH)D [49
]. These paramount findings indicate that serum 25(OH)D concentration, and importantly a specific circulating concentration (i.e., ≥ 30 ng/ml), is of physiological relevance regarding the anti-inflammatory property of vitamin D because its availability in the circulation influences the local tissue production of 1,25(OH)D [49
Vitamin D receptors are located on T-cells and mast cells [50
], and along with eosinophils, are sources of IL-5 [52
]. IL-5 is essential for promoting eosinophil growth, differentiation, survival and activation, and is often expressed with other TH2
-type cytokines, such as IL-4 and IL-13 [53
]. GATA-3 is a transcription factor that promotes IL-5 gene expression and promoter-transactivation in TH2
]. In CD4+
cells derived from mice, GATA-3 message expression increased when incubated with 1,25(OH)D [56
]. In an experimental mouse model of allergic induced-asthma, 1,25(OH)D up-regulated the message expression of GATA-3 [57
]. Thus, we speculate that maintaining serum 25(OH)D concentrations is necessary to sustain substrate availability for the conversion to 1,25(OH)D, which subsequently modulates GATA-3 expression and prevents IL-5 decreases. With that said, it is unclear why IL-5 did not increase in the 4000 IU group, unless there was a transient increase (or decrease) that was not detected with the timing of our blood sampling procedures.
Evidence indicates that several types of hyper-eosinophilic syndromes are mediated by IL-5 [58
]. In patients with hyper-eosinophic syndrome, an anti-IL-5 monoclonal antibody (i.e., mepolizumab) spared corticosteroid use [59
], which is of clinical importance since long-term corticosteroid use is associated with adverse events. Anti-IL-5 treatment also reduced the disposition of extracellular matrix proteins in bronchial biopsies obtained from atopic asthmatic patients [60
], suggesting that neutralizing IL-5 minimizes the repair process following airway injury and eosinophilia. However, the increase in IL-5 could be beneficial. In addition to increasing eosinophils, IL-5 enhances immunoglobulin A (IgA) production [61
], which protects against a variety of viruses and bacterial infections [for review see [55
]]. Recently, Halliday et al. [63
] found that serum 25(OH)D concentrations inversely correlated with frequency of illness in collegiate athletes. Perhaps, maintaining or increasing IL-5, as observed in our supplemental vitamin D groups (Figure ) who also maintained or increased as opposed to decreased serum 25(OH)D concentrations (Figure ), could be beneficial by inducing IgA and the resistance against infectious challenge, such as the influenza virus [64
]. Clearly, this premise warrants additional research.
In contrast to our hypothesis, supplemental vitamin D did not increase muscular strength (Figure and ) or power (Figure and ) despite serum 25(OH)D concentrations increasing by ~50% in the 4000 IU group (Figure ). This finding extends previous reports [65
] suggesting that supplemental vitamin D does not improve muscular strength. However, previous reports, and including the results here, are not consistent with the majority of the data. There could be several reasons for the inconsistencies. First, vitamin D insufficiency or deficiency results in muscular weakness [7
]. Compromised muscle strength correlated with serum 25(OH) concentrations in vitamin D deficient adolescent girls [7
]. In vitamin D deficient elderly and stroke and osteomalacia patients, supplemental vitamin D improved muscle strength concurrently with serum 25(OH)D concentrations [15
]. Furthermore, increasing serum 25(OH)D concentrations from ~9 to 16 ng/ml improved muscle strength and function; further but less pronounced strength and function improvements occurred from ~16 to 38 ng/ml in elderly [11
], thereby indicating a diminished return in muscle strength with increasing serum 25(OH)D concentrations. In the present investigation, serum 25(OH)D concentrations were ≈ 32 ng/ml for all subjects prior to supplementing (Figure ), and therefore, subjects were vitamin D sufficient prior to supplementing. Evidence supporting vitamin D sufficiency is also provided by the PTH and calcium concentrations (Table ), which were within normal clinical reference ranges prior to and following supplementation and despite 25(OH)D decreases in the placebo group. Based on the existing literature and the data presented here, individuals with vitamin D insufficiency or deficiency could be more prone to muscular strength improvements with increasing serum 25(OH)D concentrations than those who are already vitamin D sufficient [68
]. This theory would explain why muscle strength did not improve despite an increase in serum 25(OH)D concentrations following vitamin D supplementation at 4000 IU/d in subjects who were already vitamin D sufficient.
Another probable explanation for the inconsistencies in the literature is that there are few prospective, randomized studies investigating the influence of supplemental vitamin D on serum 25(OH)D concentrations and muscular strength. The majority of the evidence identifying the beneficial influence of vitamin D on muscular strength or physical performance is correlative [9
] or between groups demarcated on circulating 25(OH)D concentrations [13
]. From these data, authors have concluded that increasing circulating 25(OH)D concentrations improves muscular strength or physical performance. Finally, our study was conducted in young adults (men and women). Studies identifying the positive influence of supplemental vitamin D on muscular-based outcomes have been conducted in elderly [10
], in patients with diverse diseases or illnesses [16
] or in experimental animals [70
]. It is plausible that healthy lifestyles in young adults mask the influence of supplemental vitamin D on muscular strength [72
]. Although few arguments exist refuting the influence of vitamin D on muscular strength, future randomized trials incorporating various vitamin D dosing regimens that increase serum 25(OH)D concentrations differentially are required to confirm causation in young adults.
Study limitations include: first, a rather short-intervention phase. Future studies are encouraged to conduct longer durations of supplemental interventions when examining the influence of vitamin D on inflammatory cytokines and muscular strength. Second, the level of physical activity was not stringently examined. Recording activities and activity intensities and volumes performed during the preceding seasons both indoor and outdoor is recommended. Third, this study consisted of thirty subjects total (n
= 10/group). Future studies investigating the influence of different doses of supplemental vitamin D on inflammatory cytokines and muscle strength are encouraged to include a larger sample size. Fourth, our data collection was limited to weekly blood draws and emerging evidence is suggesting that the cytokine modulating property of vitamin D could be time sensitive [34
]. Thus, future studies should consider the temporal cytokine response mediated by vitamin D, especially at higher doses of supplemental vitamin D that increase serum 25(OH)D concentrations rapidly. Next, there was variability across time in the cytokine data. To account for this variability statistically, we performed a rank transformation to achieve normality and equal variance. Finally, serum 25(OH)D, inflammatory cytokines and muscular strength might respond differently in diverse populations or conditions, such as in obese, smokers, elderly or critically ill. Thus, extrapolating the present findings to other populations is not recommended.
In summary, low and high doses of supplemental vitamin D prevented serum 25(OH)D and IL-5 decreases in vitamin D sufficient adults during the winter. These impactful findings defy current belief by suggesting that a low dose of supplemental vitamin D (plus dietary intake) that is easily attainable through dietary sources maintains serum 25(OH)D and IL-5 concentrations.
Additionally, despite concentrations correlating with muscle strength, our shocking data reveal that muscular strength does not parallel the increase in serum 25(OH)D concentrations induced by supplemental vitamin D at 4000 IU/d. We conclude that maintaining serum 25(OH)D concentration during the winter prevents a TH2-type cytokine decrease, which could be influential in protecting against viral and bacterial infections; and apparently in reportedly healthy and vitamin D sufficient adults, that a further increase in serum 25(OH)D mediated by a high dose of supplemental vitamin D does not improve muscular strength.