Dietary Vitamin D Increases Percentages and Function of Regulatory T Cells in the Skin-Draining Lymph Nodes and Suppresses Dermal Inflammation

Skin inflammatory responses in individuals with allergic dermatitis may be suppressed by dietary vitamin D through induction and upregulation of the suppressive activity of regulatory T (TReg) cells. Vitamin D may also promote TReg cell tropism to dermal sites. In the current study, we examined the capacity of dietary vitamin D3 to modulate skin inflammation and the numbers and activity of TReg cells in skin and other sites including lungs, spleen, and blood. In female BALB/c mice, dietary vitamin D3 suppressed the effector phase of a biphasic ear swelling response induced by dinitrofluorobenzene in comparison vitamin D3-deficient female BALB/c mice. Vitamin D3 increased the percentage of TReg (CD3+CD4+CD25+Foxp3+) cells in the skin-draining lymph nodes (SDLN). The suppressive activity of TReg cells in the SDLN, mesenteric lymph nodes, spleen, and blood was upregulated by vitamin D3. However, there was no difference in the expression of the naturally occurring TReg cell marker, neuropilin, nor the expression of CCR4 or CCR10 (skin-tropic chemokine receptors) on TReg cells in skin, SDLN, lungs, and airway-draining lymph nodes. These data suggest that dietary vitamin D3 increased the percentages and suppressive activity of TReg cells in the SDLN, which are poised to suppress dermal inflammation.


Introduction
Vitamin D plays an intrinsic role in shaping innate and adaptive immune responses [1,2]. Vitamin D is produced following skin exposure to ultraviolet B photons of sunlight, resulting in the conversion of the precursor 7-dehydrocholesterol into vitamin D 3 , which can also be acquired through dietary supplementation. The vitamin D-binding protein (VDBP) transports much of this vitamin D 3 into the liver, where a hydroxylation reaction converts vitamin D 3 into 25-hydroxyvitamin D 3 (25(OH)D 3 ). This form of vitamin D 3 is found at nanomolar levels in blood, and because of its relative stability and longer half-life, it is used as a measure of vitamin D sufficiency, with 50 nM currently considered the tipping point for insufficiency by the National Institute of Health [3] (although this remains controversial [4]). In renal proximal tubule epithelial cells, and other cells including disease-activated macrophages (reviewed in [5]), 25(OH)D 3 is converted into the most active vitamin D metabolite, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). It is this form of vitamin D 3 which has the most potent effects on regulating immune responses, with circulating levels in the picomolar range [1,6].
Central to the ability of 1,25(OH) 2 D 3 to modulate immune responses are changes to regulatory T cells (T Reg cells) and dendritic cells (DCs) [7]. Topical (skin) application of 1,25(OH) 2 D 3 enhanced the suppressive capacity [8,9] and proliferative activity [10] of CD4+CD25+Foxp3+ T Reg cells. Stimulation of DCs with bacterial products like lipopolysaccharide or cytokines like transforming growth factor-ß may result in the synthesis of 1,25(OH) 2 D 3 from circulating 25(OH)D 3 , promoting T Reg cell activity (reviewed in [1,2]). The VDBP may also play an important role in this process, whereby high affinity VDBP can prevent conversion of 25(OH)D 3 to 1,25(OH) 2 D 3 by DCs and thus their ability to modulate T Reg cell activity [11]. With the right costimulators, 2 Journal of Immunology Research including interleukin-2, 1,25(OH) 2 D 3 can modulate the suppressive functions of T Reg cells independently of DCs [12].
While the capacity for 1,25(OH) 2 D 3 to regulate adaptive immunity through its effects on T Reg cells and DCs is clear, most studies have used supraphysiological levels of 1,25(OH) 2 D 3 (≥10 nM). During monocyte differentiation into macrophages, increased concentrations of 1,25(OH) 2 D 3 (up to 1 nM) were detected in cell culture media, but this was not observed during monocyte differentiation to DCs [13]. This increased production of 1,25(OH) 2 D 3 could have paracrine effects on colocated DCs [13] and T cells [14]. However, most in vitro studies have used substantially more 1,25(OH) 2 D 3 (≥10 nM) to modulate DC and T cell phenotype and function. T Reg cell numbers and/or their suppressive activity correlate with circulating 25(OH)D 3 levels. This has been observed in patients with pancreatitis [6], multiple sclerosis [15], and asthma [16,17] or those chronically infected with the hepatitis C virus [18]. Supplementation with vitamin D 3 or an analogue increased T Reg cell numbers in healthy individuals (140,000 IU oral vitamin D 3 /month) [19] or patients with undifferentiated connective tissue disease (0.5 g oral alfacalcidol/day) [20]. Other studies report a positive correlation between serum 1,25(OH) 2 D 3 levels (but not 25(OH)D 3 levels) and circulating T Reg cell numbers in patients with multiple sclerosis [21]. A negative correlation between 25(OH)D 3 and T Reg cell numbers has been reported in cord blood [22]. Most studies support a positive relationship between circulating 25(OH)D 3 levels and T Reg cell activity; however, this has not always been associated with improved disease-related outcomes [15,19].
Another intriguing aspect of vitamin D biology includes its ability to modulate the tropism of cells for certain tissues. Tropism for skin has been suggested in some studies, where 1,25(OH) 2 D 3 or an analogue (nM range) increased the expression of the skin-tropic chemokine receptor CCR10 on cultured T cells [14,23]. The 1,25(OH) 2 D 3 analogue TX527 significantly upregulated other skin-homing molecules like CCR4 (but not CLA) on T cells, as well as inflammationhoming molecules (e.g., CCR5, CXCR3, and CXCR6) but downregulated expression of lymph node-homing molecules (CD62L, CCR7, and CXCR4) [23]. Serum 25(OH)D 3 levels are associated with increased expression of the skin-tropic chemokine receptors CCR4 and CLA on circulating T Reg cells from healthy male volunteers [24]. Other studies in HIVinfected participants suggested that serum 25(OH)D 3 was negatively associated with CCR4 expression on circulating T Reg cells. Vitamin D 3 supplementation (25,000 IU/week) of these participants increased CCR4 and CCR10 expression on blood T Reg cells [25]. Collectively, these studies suggest that 1,25(OH) 2 D 3 promotes homing of T Reg cells towards skin or sites of inflammation.
The results of a recent meta-analysis of clinical trials suggest that dietary vitamin D 3 supplementation may reduce symptoms of atopic dermatitis [26], an inflammatory skin disease. In this study, we investigated how the tissue distribution and suppressive function of T Reg cells are regulated by dietary vitamin D 3 . We used a murine model of dietaryinduced vitamin D 3 restriction to induce vitamin D 3 deficiency and compared the effects of dietary vitamin D 3 on T Reg cell function and numbers in various tissues and skin inflammation induced by a hapten.

Mice and Diet.
All experiments were performed according to the ethical guidelines of the National Health and Medical Research Council of Australia and with approval from the Telethon Kids Institute Animal Ethics Committee (AEC#229). BALB/c mice were purchased from the Animal Resources Centre, Western Australia. Mice transgenic for the OVA 323-339 -specific (ISQAVHAAHAEINEAGR) T cell receptor (DO11.10) on a BALB/c background were originally purchased from the Jackson Laboratory and bred in-house. Expression of OVA 323-339 -specific T cell receptor on T cells from DO11.10 mice was confirmed as previously described [9]. Female 3-week-old BALB/c or DO11.10 transgenic mice were placed on semipure diets, which were supplemented with vitamin D 3 (2280 IU vitamin D 3 /kg with 1% calcium, SF05-34, Specialty Feeds, Perth, Western Australia) or were not supplemented with vitamin D 3 (0 IU vitamin D 3 /kg with 2% calcium, SF05-033, Specialty Feeds) as previously described [27,28]. At 8 weeks of age, female mice were mated with adult male mice maintained on standard mouse chow (Specialty Feeds, containing 2000 IU vitamin D 3 /kg). Female or male offspring were maintained on the same vitamin D 3replete or vitamin D 3 -deficient diets (as their mothers) for the rest of the experiment. All results shown are for female offspring, unless otherwise stated.

Measurement of Serum 25-Hydroxyvitamin D 3 (25(OH)D 3 )
. Serum 25(OH)D 3 levels were measured in BALB/c mice using IDS EIA ELISA kits (Immunodiagnostic Systems Ltd., Fountain Hills, AZ) as described by the manufacturer (limit of detection was 7 nmol⋅L −1 ). We have previously shown that results from this assay correlate highly ( = 0.99) [29] with a liquid chromatography-tandem mass spectrometry method, which has been certified to a reference measurement procedure developed by the National Institute of Standards and Technology and Ghent University [30,31].

Biphasic Ear Swelling Assay.
A biphasic ear swelling response [32,33] was induced by painting both sides of each ear pinnae with 10 L of 0.05-0.2% 2,4-dinitrofluorobenzene (DNFB, Sigma, St Louis, MO) in acetone using a micrometer to measure ear thickness in a blinded fashion at the indicated times. Results are shown as the change in ear thickness, with baseline measures subtracted from those measured at each time point.

Assessing the Suppressive Capacity of
Cells. We isolated T Reg cells from vitamin D 3 -replete or vitamin D 3deficient DO11.10 mice to test the capacity of dietary vitamin D 3 to modify the suppressive activity of cells located in a number of different immune sites. As the majority of T Reg cells express the OVA 323-339 receptor [9], they will suppress the IL-2-secreting capacity of cocultured OVA 323-339 receptor-specific CD4+ T cells in the presence of antigenpresenting cells and OVA 323-339 peptide, as we have demonstrated previously [9]. CD4+CD25+ cells (≥95% pure, as determined by flow cytometry) were isolated from specified tissues of DO11.10 mice using a CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec) or by cell sorting as previously described [9,34]. Greater than 90% of the purified CD4+CD25+ cells expressed Foxp3 (confirmed by flow cytometry). Peripheral lymph node cells (including SDLN, ADLN, MLN, auricular-draining lymph nodes, and paraaortic lymph nodes) of naïve DO11.10 mice were used as responder cells. These were resuspended in RPMI with 10% FCS and 2 M 2-ME and aliquot into round-bottomed 96well plates at 10 5 cells/0.1 mL/well. CD4+CD25+ cells were added to responder cells at ratios of 1 : 8, 1 : 16, or 1 : 32. OVA 323-339 peptide was added at a final concentration of 1 g/mL. After incubation for 92 h at 37 ∘ C in 5% CO 2 , supernatants were harvested and the concentration of interleukin-2 (IL-2) was determined by ELISA as previously described [9].

Assessing the Ability of Dendritic Cells to Induce
Cells. A single cell suspension of ADLN cells was prepared by physically disaggregating lymph nodes and digesting samples with collagenase IV (1 mg/mL, Worthington) and DNase I (0.1 mg/mL, Sigma) for 30 min at 37 ∘ C. Conventional DCs were enriched from the ADLN cells by removal of CD3+, Thy1.1+ CD19+, GR-1+, and TER-119+ cells using magnetic bead separation as previously described [35]. The remaining cells were then labelled with antibodies specific for CD11c and MHC class II [9] and MHC class II hi CD11c med cells sorted by FACS using the FACSAria (BD Biosciences). MHC class II hi CD11c med cells were incubated with peripheral lymph nodes from naïve DO11.10 (vitamin D-replete) mice (see [9]) at a ratio of 1 : 40 with 1 g/mL OVA 323-339 peptide. DCs were not added to some cultures as controls. Cells were incubated for 62 h at 37 ∘ C and 5% CO 2 , and then CD3+CD4+CD25+Foxp3± cells were identified by flow cytometry using the FACS LSRII, where at least 5,000 Foxp3+ cells were collected. Data were analyzed using FlowJo software.

Statistical Analyses.
Data were compared using an unpaired two-way Student's -test using Prism 5 statistical analysis program for Mac OS X. Differences were considered significant with a value < 0.05. Data are shown throughout as mean ± SEM.

Vitamin D Deficiency Promoted Allergic Dermatitis Responses Measured during a Biphasic Ear Swelling Response.
We investigated the effects of dietary vitamin D on allergic dermatitis responses mimicked by inducing a biphasic ear swelling response. We tested adult female offspring of vitamin D 3 -replete or vitamin D 3 -deficient BALB/c dams, which were maintained on the same diet as their mothers. Serum levels of 25(OH)D 3 were <20 nmol⋅L −1 for offspring fed the vitamin D 3 -deficient diet and >50 nmol⋅L −1 for offspring fed the vitamin D 3 -replete diet (Figure 1(a)). These diets did not significantly alter serum calcium [27,28]. The contact sensitizer DNFB was then used to initiate a biphasic ear swelling response [32,33]. The ears of vitamin D 3 -deficient or vitamin D 3 -replete mice were sensitized with 0.05-0.2% DNFB (in acetone), and ear swelling was recorded over a 3-week period. The ability of dietary vitamin D 3 to suppress ear swelling responses depended on the sensitizing dose of DNFB, where responses to ≤0.1% DNFB were suppressed at 144 h after sensitization, corresponding to the second peak of ear swelling (Figure 1(b)). As expected, the ear swelling response was biphasic, with an initial peak at 3 h and later peak at 168 h after DNFB treatment (Figure 1(c)). Previous studies have shown that this first peak represents an early innate influx of neutrophils and inflammatory cells into ear skin, which may depend on histamine release by mast cells [32], while the second peak is an antigen-specific (DNFB) effector response driven by CD8+ T cells expressing interferon- [33]. Dietary vitamin D 3 significantly suppressed (by 61%) this second "efferent" ear swelling response, which peaked at 168 h after DNFB application in vitamin D 3 -sufficient mice as compared to responses observed in deficient mice (Figure 1(c)).

Increased Percentages of Cells Were Observed in the Skin-Draining Lymph Nodes of Vitamin D 3 -Replete Mice.
We have previously published that topically applied 1,25(OH) 2 D 3 increased the capacity of T Reg cells to suppress contact hypersensitivity responses initiated by DNFB [9]. To examine the effects of dietary vitamin D 3 on T Reg cells, their percentages in various tissues were measured in naïve female mice prior to sensitization with DNFB. The percentages of CD3+CD4+CD25+Foxp3+ T Reg cells in the skin, SDLN, lung, ADLN, MLN, spleen, and blood were determined by flow cytometry (Figure 2 = 6/treatment) of vitamin D 3 -replete mice in comparison to vitamin D 3 -deficient mice, but there was no difference in the percentages of these cells in the skin, lungs, MLN, spleen, or blood (Figure 2(b)). There was also a trend ( = 0.08, Student's -test) for increased Foxp3 expression (by 16%) on CD3+CD4+CD25+Foxp3+ cells from the SDLN of vitamin D 3 -replete mice, when geometric mean fluorescence intensity was compared (1053 ± 43 (vitD+); 910 ± 63 (vitD−); = 6/treatment, data from cells collected in Figure 2(b)). There was no difference in the expression of Foxp3 on CD3+CD4+CD25+Foxp3+ cells from the other tissues (data not shown). With the exception of blood, CD3+CD4+CD25+Foxp3− T "effector" cell (T Eff ) (Figure 2 1.7 ± 0.1 × 10 7 /mouse (vitD+); 39% reduction; = 6/treatment) were isolated from vitamin D 3 -supplemented mice (Figure 3(a)). These effects were in the opposite direction to those of dietary vitamin D 3 on T Reg cell percentages in the SDLN. There was no difference in the numbers of cells isolated from other tissues (Figure 3(a); data not shown for skin and lung). There was a trend for fewer CD4+ T Reg cell numbers in the SDLN (1.5 ± 0.2 × 10 6 /mouse (vitD−); 0.9 ± 0.1 × 10 6 /mouse (vitD+)); 40% reduction; = 6/treatment) of vitamin D 3 -supplemented mice in comparison to vitamin D 3 -deficient mice (Figure 3(b)). Similarly, numbers of CD3+CD4+CD25+ Foxp3− T Eff cells were significantly reduced in the SDLN of mice fed a vitamin D 3 -supplemented diet (3.8 ± 0.2 × 10 5 /mouse (vitD−); 1.8 ± 0.2 × 10 5 /mouse (vitD+); 53% reduction; = 6/treatment) (Figure 3(c)). There was no effect of dietary vitamin D 3 on the total cell numbers or numbers of T Reg or T Eff cells identified in the SDLN, ADLN, or blood of male mice (data not shown). These data suggest that while the proportions of CD4+ T Reg cells increased in the SDLN with dietary vitamin D 3 , this significant increase did not persist when cell numbers were considered, as significantly fewer SDLN cells were isolated from mice fed a diet containing vitamin D 3 .

The Suppressive Activity of Cells Was Enhanced by Dietary Vitamin D 3 in Most Immune Tissues but Not the Airway-Draining Lymph
Nodes. An in vitro assay was used to test if dietary vitamin D 3 altered the suppressive function of T Reg cells in comparison to those from vitamin D 3 -deficient mice. Purified CD4+CD25+(Foxp3+) cells were cocultured with responder lymph node cells from DO11.10 mice and OVA 323-339 peptide for 92 h. Responder CD4+ T cells expressing the OVA 323-339 TCR proliferate and produce cytokines like IL-2 in response to presentation of the OVA 323-339 peptide by antigen-presenting cells. We assessed IL-2 levels as a measure of the proliferative capacity of responder cells, where cocultured T Reg cells significantly suppressed supernatant levels of IL-2 in a dose-dependent manner (Figure 4). T Reg cells produce very low levels of IL-2 when stimulated in vitro. These levels are up to 10 times less than CD4+ T Eff responder cells [12]. T Reg cells therefore do not significantly contribute towards the pool of IL-2 in coculture assays. CD4+CD25+(Foxp3+) cells from the SDLN (Figure 4(a)), MLN (Figure 4(c)), spleen (Figure 4(d)), and blood (Figure 4(e)) of vitamin D 3 -replete mice had increased capacity to suppress IL-2 production by cocultured responder cells. There was no significant difference in suppressive function observed for CD4+CD25+(Foxp3+) cells from the ADLN (Figure 4(b)) of vitamin D 3 -replete or vitamin D 3 -deficient mice. The results reported in Figure 4 were for suppressive activities of CD4+CD25+(Foxp3+) cells from female mice; however, similar results were obtained for cells from male mice (data not shown). We were not technically able to assess the suppressive activity of T Reg cells in the skin or lungs as their numbers were too infrequent for efficient isolation. These data suggest that dietary vitamin D 3 is required for the optimal activity of T Reg cells at various immune sites throughout the body, with the exception of the ADLN.

Vitamin D 3 Did Not Induce
Cells in the Periphery. Surface expression of neuropilin can be used to identify naturally occurring T Reg cells [36]. We examined the expression of neuropilin on T Reg cells from the skin, SDLN, lungs, or ADLN of vitamin D 3 -replete or vitamin D 3 -deficient mice and observed no difference in the expression of this molecule ( Figure 5(a)). These results suggest that dietary vitamin D 3 did not favour the induction of new T Reg cells in the SDLN. The observed reduction in the percentage of T Reg cells in the ADLN of mice fed a vitamin D 3 -containing diet was a surprising result. A lack of difference in neuropilin expression on T Reg cell from the ADLN of vitamin D 3 -replete and vitamin D 3 -deficient mice suggested that dietary vitamin D 3 did not prevent the induction of new T Reg cells ( Figure 5(a)). However, to confirm this observation, we then tested whether there was a functional difference between DCs from vitamin D 3 -deficient and vitamin D 3 -replete mice, as DCs are required for the induction of new T Reg cells in the periphery [37]. Conventional MHC class II hi CD11c med DCs were sorted ( Figure 5(b)) from the ADLN of vitamin D 3 -replete and vitamin D 3 -deficient mice and cocultured with lymph node cells from naïve DO11.10 mice and OVA peptide. The percentage of CD4+CD25+Foxp3± cells was determined after 62 h of coculture ( Figure 5(c)). CD4+CD25+Foxp3− T Eff cell and CD4+CD25+Foxp3+ T Reg cell percentages were increased (>2-fold) by the presence of DCs in the cocultures ( Figures  5(c) and 5(d)). However, there was no effect of dietary vitamin D 3 on the ability of ADLN DCs to regulate T Reg cell percentages ( Figure 5(d)). These results suggest that dietary vitamin D 3 did not impair the induction of new T Reg cells in the ADLN.

Dietary Vitamin D 3 Did Not Affect the Expression of CCR4 or CCR10 on
Cells in the SDLN or ADLN. Dietary vitamin D 3 could induce the migration of T Reg cells to augment the percentages of these cells in the SDLN, facilitated by the expression of chemokine receptors. There was no difference in the expression of the skin-homing receptors CCR4 or CCR10 on T Reg cells in the skin, SDLN, lungs, or ADLN of vitamin D-replete or vitamin D-deficient mice ( Figures  6(a) and 6(b)). Significant levels of CCR4 (Figure 6(a)) were detected on T Reg cells in skin and SDLN with less expression on cells from the lung and ADLN, while similar levels of CCR10 (Figure 6(b)) were identified on T Reg cells from these tissues. While dietary vitamin D 3 promoted T Reg cell accumulation in the SDLN, there was no difference in the expression of CCR4 and CCR10 skin-homing receptors once cells entered the SDLN.

Dietary Vitamin D 3 May Promote Dermal Tolerance to
Reduce Skin Inflammation. Our findings of the immunosuppressive effects of dietary vitamin D 3 in controlling DNFBinduced skin inflammation reiterate emerging data from clinical trials, which suggest that vitamin D 3 supplementation reduces symptoms of allergic dermatitis [26]. Similar suppressive effects of dietary vitamin D 3 have been observed in other animal models that used haptens to induce skin inflammation [38]. In other studies, hypocalcaemia induced by vitamin D 3 deficiency may have impaired hapten-induced ear swelling responses [39]. The vitamin D 3 -containing diets used in our studies and those of others [38] were enriched with calcium to prevent hypocalcemia [27,28]. While clinical trials suggest that maintaining optimal serum levels of 25(OH)D through dietary vitamin D 3 supplementation (or safe sun exposure) reduces symptoms of atopic dermatitis [26], we are still uncertain of the optimal levels of 25(OH)D needed to limit atopic dermatitis. It is also important to note that some studies have observed no significant effect of vitamin D 3 supplementation [40], with suggestions that genetic or other population-based factors (e.g., initial circulating 25(OH)D levels) or the supplementation regimen (e.g., dose, schedule of treatment, and type of vitamin D) may have limited the efficacy of the vitamin D treatment.

Dietary Vitamin D 3 Increased the Percentages and Activity of
Cells in the SDLN. We observed a greater suppressive activity of CD4+CD25+(Foxp3+) cells isolated from the SDLN, MLN, spleen, and blood of mice fed the vitamin D-containing diet, suggesting a systemic effect of dietary vitamin D on T Reg cell activity. It is important to note that we examined T Reg cell numbers and function prior to sensitization with DNFB, and so these findings are independent of skin inflammation induced by the irritant. These observations were accompanied by a lack of effect of dietary vitamin D on the suppressive activity of cells from the ADLN. This curious observation is difficult to explain but may represent a site from which T Reg cells actively migrate (to the SDLN). We did not observe increased expression of skintropic chemokine receptors CCR4 or CCR10 on T Reg cells from any of the tested sites (skin, SDLN, lung, and ADLN) of vitamin D 3 -fed mice, but it may be that these molecules are upregulated during transition (in blood) between locations, which could be a focus of future studies. Crosstalk between the immune reactions initiated in the skin and  airways is illustrated by the "atopic march" concept, where allergic responses in the skin affect immunity in the airways. Indeed, hapten-induced skin inflammation can worsen signs of allergic airway disease in mice [41]. We suggest that dietary vitamin D 3 may prevent the "atopic march" by promoting T Reg cell accumulation and activity in the SDLNs. The lack of difference in neuropilin levels suggests that vitamin D 3 may increase SDLN T Reg cell accumulation through migration.  [42]. We did not see any difference in the percentages of T Reg cells in the lungs of mice fed a vitamin D 3 -replete or vitamin D 3 -deficient diet and did not assess the percentages of these cells in the trachea or bronchoalveolar lavage fluid. Furthermore, reduced percentages of T Reg cells were observed in the ADLN of mice fed a vitamin D 3 -replete diet. In addition, it is possible that, upon respiratory stimulation with allergen, T Reg cell percentages could increase in the lungs of vitamin D 3 -replete mice. In other studies, Mann et al. (2015) found that more CD4+ cells stimulated with 1,25(OH) 2 D (100 nM) in vitro expressed neuropilin compared to control cells [43]. It is therefore possible that new T Reg cells may be generated under conditions of highly concentrated 1,25(OH) 2 D 3 .

Modelling Skin Inflammation.
We induced a biphasic cutaneous skin reaction using the contact sensitizer DNFB to examine the effects of dietary vitamin D 3 on skin inflammation. A humanized mouse model would have been an interesting alternative means of comparing these treatments through xenotransplantation of human skin [44] or bioengineered human skin equivalents [45] onto immunodeficient mice. These models can induce some (but not all) clinical manifestations of atopic dermatitis, particularly acute lesions [45]. While such models would improve our understanding of the effects of dietary vitamin D 3 in humanized settings, we were more interested in the capacity of dietary vitamin D 3 to modulate T Reg cell proportions and function in certain tissues (e.g., skin or lungs) prior to the onset of inflammation.

Conclusion
Our studies suggest that dietary vitamin D 3 increased the percentages and suppressive function of T Reg cells in the SDLN and that these cells are poised to suppress dermal inflammation. These studies support the notion that maintaining adequate serum 25(OH)D through dietary vitamin D 3 supplementation or safe sun exposure is important to reduce the severity of allergic dermatitis and other inflammatory skin conditions. Skin-draining lymph nodes VDBP: Vitamin D-binding protein.