Effects of 1,25-Dihydroxyvitamin D3 on the Inflammatory Responses of Stromal Vascular Cells and Adipocytes from Lean and Obese Mice

Vitamin D status has been implicated in obesity and adipose tissue inflammation. In the present study, we explored the effects of dietary vitamin D supplementation on adipose tissue inflammation and immune cell population, and the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) treatment on pro-inflammatory cytokine production by stromal vascular cells (SVCs) and adipocytes in lean and high-fat diet-induced obese mice. The results show that epididymal fat Mcp-1 and Rantes mRNA levels, which were higher in obese mice compared with lean mice, were significantly down-regulated by vitamin D supplementation. While obese mice had higher numbers of macrophages and natural killer (NK) cells within adipose tissue, these remained unaltered by vitamin D supplementation. In accordance with these in vivo findings, the in vitro 1,25(OH)2D3 treatment decreased IL-6, MCP-1, and IL-1β production by SVCs from obese mice, but not by adipocytes. In addition, 1,25(OH)2D3 treatment significantly decreased Tlr2 expression and increased mRNA levels of Iκba and Dusp1 in SVCs. These findings suggest that vitamin D supplementation attenuates inflammatory response in adipose tissue, especially in SVCs, possibly through inhibiting NF-κB and MAPK signaling pathways in SVCs but not by the inhibition of macrophage infiltration.


Introduction
The prevalence of obesity and related disorders are increasing worldwide. Adipose tissue expansion or fat accumulation in the liver, observed with obesity, activates the pro-inflammatory signaling pathway, which mediates pathogenesis of metabolic disorders, such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) [1][2][3][4]. Hypovitaminosis D is often observed in obese humans [5]. New evidence suggests that low serum vitamin D may cause and exacerbate insulin resistance and NAFLD [6][7][8]. Several in vitro studies have shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment could alleviate inflammatory responses in mouse or human adipocytes [9,10].

Determination of Serum and Adipose Tissue 25(OH)D Levels
Serum 25(OH)D levels were measured by radioimmunoassay (RIA) using a commercial RIA kit (DiaSorin, Stillwater, MN, USA) according to the manufacturer's instruction. The radioactivity was measured with an automatic gamma counter (2470 Wizard2, Perkin Elmer, Shelton, CT, USA). Adipose tissue 25(OH)D levels were measured by using a modification of the original method by Lipkie et al. [32]. A Shimadzu LCMS-8040 triple quadrupole mass spectrometer with a Shimadzu Nexara X ultra-high-performance liquid chromatography (UHPLC; Kyoto, Japan) system was used for the separation of 25(OH)D3 from adipose tissue extracts.

Adipocyte and Stromal Vascular Cell(SVC) Isolation
Visceral fats were cut into small pieces with scissors and put into DMEM (Grand Island, NY, USA) with 1 mg/mL collagenase type 2 (Sigma-Aldrich, St. Louis, MO, USA) and 2% bovine serum albumin (BSA) and were then incubated in a shaking 37 • C water bath (170 cycle/min) for 1 h. Tissue debris were removed using 200 µm nylon cell strainer. After centrifugation (1500 rpm, room temperature (RT), for 5 min), cells floating on the top were transferred to a new tube as adipocytes. The cell pellet at the bottom of the tube was washed with DMEM/10% FBS and resuspended with 1 mL of DMEM/10% FBS and 500 µL of 40% percoll in 10 × PBS. Reconstituted cells were slowly applied to a discontinuous percoll gradient, comprised of 40% and 70% percoll layers and were then centrifuged at 750× g, 20 • C for 20 min (excluding the acceleration and deceleration time) to acquire the immune cell fraction. The layer between the 40% and 70% percoll layers were transferred to a new tube as SVCs and centrifuged in RT at 1500 rpm for 5 min. Both adipocytes and SVCs were washed twice with DMEM/10% FBS and used for flow cytometry (FACS) analysis or cell culture.

Flow Cytometric Analysis
For the analysis of SVCs subpopulation, SVCs were resuspended in a FACS-staining buffer (0.09% sodium azide, 1% FBS, 1× PBS based) and 2 × 10 5 cells per sample were incubated with the antibodies at 4 • C for 30 mins. The antibodies used for analysis were purchased from BD Pharmingen (Franklin Lakes, NJ, USA), and specific cell surface markers are shown in Table 2. After staining, cells were washed and resuspended in a FACS-staining buffer with 4% formaldehyde, then analyzed using FACSCalibur II (BD Biosciences, SA, USA) and FlowJo software version 10 (Tree Star Inc., Ashland, OR, USA). Adipocytes were cultured in a 6-well plate (4 × 10 6 cells/well), and SVCs were cultured in a 12-well plate (2 × 10 5 cells/well) with DMEM/10% FBS. To determine the effects of vitamin D, adipocytes and SVCs were incubated with either 1,25(OH)2D3 solution (10 nM, Sigma-Aldrich) in DMEM or 0.1% ethanol in DMEM (vehicle control) for 48 h and were stimulated with or without lipopolysaccharides (LPS ) (100 ng/mL, Sigma-Aldrich) during the last 24 h. Cells were incubated at 37 • C in 5% CO 2 and a 100% humidified atmosphere. After 48 h of incubation, the supernatant was collected for cytokine analysis and cells (SVCs or adipocytes) were collected and stored at −80 • C for RNA or DNA isolation.
To normalize pro-inflammatory cytokine levels for the number of adipocytes, the total DNA from adipocytes was measured using a Quick-DNA™ Miniprep Plus kit (Zymo research, Irvine, CA, USA).

RNA Extraction from SVCs and Real-Time PCR
Total RNA was isolated from SVCs using RNAiso Plus (Takara bio., Shiga, Japan). The RNA quality was determined using agarose gel electrophoresis with the Gel Doc XR system (Bio-Rad Laboratories, Hercules, CA, USA). The total RNA (2 µg) sample was reverse transcribed into cDNA using PrimeScript™ 1st strand cDNA synthesis kit (Takara Bio Inc., Otsu, Shiga, Japan) with a thermal cycler (Applied Biosystems, Foster City, CA, USA). Each PCR reaction mixture contained synthesized cDNA, specific primers of a target gene, with SYBR Premix Ex Taq and ROX reference dye (Takara bio). mRNA levels of Mcp-1, regulated on the activation of a normal T cell expressed and secreted (Rantes), macrophage inflammatory protein-1 gamma (Mip-1γ), Il-6, Il-1β, Tnf-α, and interferon gamma (Ifn-γ) in the adipose tissue, Tlr2, Tlr4, dual specificity protein phosphatases (Dusp)1, Dusp10, nuclear factor of kappa light polypeptide gene enhanced in B-cells inhibitor alpha (Iκbα) in SVCs were determined with real-time quantitative PCR analysis using a StepOneTM Real-time PCR system (Applied Biosystems). The mRNA levels were normalized to the expression of the endogenous control gene (Gapdh) and all values are expressed as relative mRNA levels compared to the average level of the control group using the 2-∆∆CT method. The oligonucleotide sequences of primers are presented in Table 3.

Statistical Analysis
Statistical analysis was performed using SPSS statistical software version 23 (IBM SPSS Statistics, Chicago, IL, USA). For Exp. 1 and 2, two-way ANOVA was used to evaluate the overall effects of the amount of dietary fat (CON or HFD) and dietary vitamin D content (DC or DS). Duncan's multiple range post-hoc test was carried out when the effects of the amount of dietary fat and/or dietary vitamin D content were statistically significant. For Exp. 3, a Student's t-test was conducted for comparing the effects of the amount of dietary fat between the CON and HFD group. In order to evaluate the effects of in vitro 1,25(OH)2D3 treatment on SVCs or adipocytes from the same tissue, a paired t-test was used. All data were represented as means ± SEMs, and p values less than 0.05 were considered statistically significant.

Body Weight, Weight Change, WAT Weight, and Food Intake
There was no significant difference in body weight at week 0 among the groups. After 13 weeks, the HFD groups had higher body weight (p < 0.001) and WAT weight (p < 0.001) compared with the CON groups (Table 4, S3, and S4). There was no significant effect of dietary vitamin D supplementation (25,000 IU/kg diet) on WAT weight ( Table 4). The average food intake (g/day) was not affected by dietary fat amount or vitamin D content. In addition, a lower dose of vitamin D supplementation (10,000 IU/kg diet) did not affect either body weight, WAT weight, or food intake (Table S3). Table 4. Body weight, weight gain, body fat, and food intake of mice in the CON-DC, CON-25DS, HFD-DC, and HFD-25DS groups 1,2 .

CON HFD p-Value
Fat Amount

Vitamin D Content Interaction
Body weight at 0 week (g) 22

Serum and Epididymal Adipose Tissue 25(OH)D Levels
Serum 25(OH)D levels were significantly higher in the 25DS groups (25,000 IU/kg diet) when compared with the DC groups (Table 5). When vitamin D was supplemented at the level of 25,000 IU/kg diet, serum 25(OH)D levels were significantly lower in the HFD-25DS group compared with the CON-25DS group. These results were also confirmed with a lower dose of vitamin D supplementation (10,000 IU/kg diet) (Table S3). Epididymal 25(OH)D levels of the 25DS groups were higher than those of the DC groups (3.3-fold, p < 0.001). The amount of dietary fat tended to have a significant effect on epididymal adipose tissue 25(OH)D levels (Table 5).

1-Hydroxylase and Vdr Expression in Epididymal Adipose Tissue
Neither dietary fat amount nor dietary vitamin D content had a significant impact on the mRNA levels of Cyp27b1 and Vdr ( Figure S1).

Subpopulation and the Number of Immune cells of SVCs in Visceral Adipose Tissue
The effects of a high-fat diet and vitamin D supplementation at a dose of 10,000 IU/kg on subpopulations of immune cells in SVCs were determined. The number of each type of immune cell per gram of visceral adipose tissue and total cell numbers are shown in Figure 2 and Table S5. The number of SVCs per g from visceral adipose tissue was significantly higher in the HFD groups than in the CON groups. Numbers of macrophages and NK cells (number/g tissue and total number) in SVCs were significantly higher in the HFD groups compared with the CON groups; however, there was no significant effect of vitamin D supplementation (Figure 2 and Figure S2).  Table S5: total number, p = 0.09).

Production of Pro-Inflammatory Cytokines by SVCs
In order to elucidate whether in vitro 1,25(OH)2D3 treatment (10 nM) had a specific antiinflammatory effect on SVCs from adipose tissue, levels of pro-inflammatory cytokines produced by SVCs from CON and HFD groups were determined (Figure 3). Significantly higher levels of MCP-1 (1.5-fold) and IL-6 (2.9-fold) were produced by LPS-stimulated SVCs from the HFD group compared Numbers of B cells, CD4+ T cells, and CD8+ T cells/g tissue were not different among groups (Figure 2), while the total number of CD4+ T cells was significantly higher in HFD groups than in CON groups (Table S5). The number of CD4+ T cells (numbers/g tissue and total numbers) tended to be lower in the 10DS groups compared with the DC groups ( Figure 2: numbers/g, p = 0.05; Table S5: total number, p = 0.09).

Production of Pro-Inflammatory Cytokines by SVCs
In order to elucidate whether in vitro 1,25(OH)2D3 treatment (10 nM) had a specific anti-inflammatory effect on SVCs from adipose tissue, levels of pro-inflammatory cytokines produced by SVCs from CON and HFD groups were determined (Figure 3). Significantly higher levels of MCP-1 (1.5-fold) and IL-6 (2.9-fold) were produced by LPS-stimulated SVCs from the HFD group compared with the CON group. MCP-1, IL-6, and IL-1β production from SVCs decreased significantly by in vitro 1,25(OH)2D3 treatment (MCP-1: 15% less, IL-6: 12% less, and IL-1β: 34% less) in the HFD group. In the CON group, only IL-6 production was decreased by in vitro 1,25(OH)2D3 treatment (16% less, p = 0.02 by paired t-test). The production of TNF-α by SVCs was not affected by either the high-fat diet or in vitro 1,25(OH)2D3 treatment.

Production of Pro-Inflammatory Cytokines by Adipocytes
The effects of vitamin D on the production of pro-inflammatory cytokines by adipocytes were determined by in vitro 1,25(OH)2D3 treatment (10 nM). Adipocytes from the HFD group produced higher levels of MCP-1 (2.1-fold, p = 0.02) and IL-6 (5.5-fold, p< 0.001) than those from the CON group ( Figure 4). However, the production of IL-1β was significantly higher in adipocytes from the CON group than those from the HFD group. There was a tendency of decreased MCP-1 production by adipocytes from the HFD group with in vitro 1,25(OH)2D3 treatment (28% less, p = 0.07 by paired t- Figure 3. Production of pro-inflammatory cytokines by SVCs from CON and HFD mice (A) MCP-1 (ng/mL), (B) IL-6 (pg/mL), (C) IL-1β (pg/mL), and (D) TNF-α (pg/mL). SVCs were treated with vehicle (0.1% ethanol) or 1,25(OH)2D3 (10 nM) for 24 h before being stimulated with LPS (0.1 µg/mL) for another 24 hr. Data are presented as mean ± SEM, n = 9~10 for each group. A paired t-test was used to determine the significant effect of in vitro 1,25(OH)2D3 treatment and * indicates a significant difference (p < 0.05). A student's t test was used to determine the significant effect of the amount of dietary fat. CON: 10% kcal fat diet; HFD: 60% kcal fat diet.

Production of Pro-Inflammatory Cytokines by Adipocytes
The effects of vitamin D on the production of pro-inflammatory cytokines by adipocytes were determined by in vitro 1,25(OH)2D3 treatment (10 nM). Adipocytes from the HFD group produced higher levels of MCP-1 (2.1-fold, p = 0.02) and IL-6 (5.5-fold, p < 0.001) than those from the CON group ( Figure 4). However, the production of IL-1β was significantly higher in adipocytes from the CON group than those from the HFD group. There was a tendency of decreased MCP-1 production by adipocytes from the HFD group with in vitro 1,25(OH)2D3 treatment (28% less, p = 0.07 by paired t-test). The in vitro 1,25(OH)2D3 treatment had no significant effect on IL-6, IL-1β, and TNF-α production by adipocytes from the HFD group and MCP-1, IL-6, IL-1β, and TNF-α production by adipocytes from the CON group.

Expression of Genes Involved in Inflammatory Responses in SVCs
The expression of genes involved in inflammatory signaling pathways was determined to see whether a decreased production of pro-inflammatory cytokines by 1,25(OH)2D3 treatment in SVCs could be attributed to the regulation of genes involved in MAPK and NF-κB signaling pathways ( Figure 5). Dusp1 and Dusp10 are phosphatases that inactivate that MAP-kinase and Iκbα is an inhibitor of NF-κB transcription factor. Tlr2 mRNA levels were higher in the HFD group compared with the CON group and significantly decreased by in vitro 1,25(OH)2D3 treatment. Tlr2 expression decreased by 25% in the CON group and by 28% in the HFD group with in vitro 1,25(OH)2D3 treatment. However, in vitro 1,25(OH)2D3 treatment had no significant effect on Tlr4 expression. Dusp1 mRNA levels were not different between the CON and HFD groups. With 1,25(OH)2D3 treatment, Dusp1 expression increased significantly in the CON group (44% higher, p = 0.01 by paired t-test) and tended to be increased in the HFD group (27% higher, p = 0.08 by paired t-test). Dusp10 mRNA levels were significantly lower in the HFD group compared with the CON group. The in vitro 1,25(OH)2D3 treatment had no effect on Dusp10 expression. Iκbα mRNA levels were 27.7% lower (p < 0.001) in the HFD group compared with the CON group and significantly increased by in vitro 1,25(OH)2D3 treatment in the CON (15.3% higher, p = 0.04 by paired t-test) and HFD groups (13.7% Adipocytes were treated with vehicle (0.1% ethanol) or 1,25(OH)2D3 (10 nM) for 24 h before being stimulated with LPS (0.1 µg/mL) for another 24 h. Production of each cytokine level was normalized with total cellular DNA. Data are presented as mean ± SEM, n = 9~10 for each group. A paired t-test was used to determine the significant effects of in vitro 1,25(OH)2D3 treatment and * indicates a significant difference (p < 0.05). A student's t test was used to determine the significant effect of dietary fat amount. CON: 10% kcal fat diet; HFD: 60% kcal fat diet.

Expression of Genes Involved in Inflammatory Responses in SVCs
The expression of genes involved in inflammatory signaling pathways was determined to see whether a decreased production of pro-inflammatory cytokines by 1,25(OH)2D3 treatment in SVCs could be attributed to the regulation of genes involved in MAPK and NF-κB signaling pathways ( Figure 5). Dusp1 and Dusp10 are phosphatases that inactivate that MAP-kinase and Iκbα is an inhibitor of NF-κB transcription factor. Tlr2 mRNA levels were higher in the HFD group compared with the CON group and significantly decreased by in vitro 1,25(OH)2D3 treatment. Tlr2 expression decreased by 25% in the CON group and by 28% in the HFD group with in vitro 1,25(OH)2D3 treatment. However, in vitro 1,25(OH)2D3 treatment had no significant effect on Tlr4 expression. Dusp1 mRNA levels were not different between the CON and HFD groups. With 1,25(OH)2D3 treatment, Dusp1 expression increased significantly in the CON group (44% higher, p = 0.01 by paired t-test) and tended to be increased in the HFD group (27% higher, p = 0.08 by paired t-test). Dusp10 mRNA levels were significantly lower in the HFD group compared with the CON group. The in vitro 1,25(OH)2D3 treatment had no effect on Dusp10 expression. Iκbα mRNA levels were 27.7% lower (p < 0.001) in the HFD group compared with the CON group and significantly increased by in vitro 1,25(OH)2D3 treatment in the CON (15.3% higher, p = 0.04 by paired t-test) and HFD groups (13.7% higher, p = 0.01 by paired t-test).

Discussion
In this study, dietary vitamin D supplementation down-regulated the gene expression of proinflammatory cytokines in adipose tissue despite no difference in macrophage infiltration into the adipose tissue. The anti-inflammatory effects of vitamin D on adipose tissue might be due to the action of 1,25(OH)2D3 on inflammatory signaling in SVCs, since in vitro treatment of 1,25(OH)2D3 decreased pro-inflammatory cytokine production from SVCs possibly through regulating Dusp1 and Iκbα.
Obesity has been known to increase the infiltration of immune cells into adipose tissue and the secretion of pro-inflammatory cytokines [11,38], which results in chronic adipose tissue inflammation. In the current study, the HFD groups had higher numbers of SVCs in adipose tissue (numbers/g tissue) compared with the CON groups, especially the numbers of macrophages and NK cells (per g tissue), which were 3 and 2 times higher, respectively, in the HFD groups. This is consistent with the reports that the numbers of macrophages as well as NK cells were higher in the

Discussion
In this study, dietary vitamin D supplementation down-regulated the gene expression of pro-inflammatory cytokines in adipose tissue despite no difference in macrophage infiltration into the adipose tissue. The anti-inflammatory effects of vitamin D on adipose tissue might be due to the action of 1,25(OH)2D3 on inflammatory signaling in SVCs, since in vitro treatment of 1,25(OH)2D3 decreased pro-inflammatory cytokine production from SVCs possibly through regulating Dusp1 and Iκbα.
Obesity has been known to increase the infiltration of immune cells into adipose tissue and the secretion of pro-inflammatory cytokines [11,38], which results in chronic adipose tissue inflammation. In the current study, the HFD groups had higher numbers of SVCs in adipose tissue (numbers/g tissue) compared with the CON groups, especially the numbers of macrophages and NK cells (per g tissue), which were 3 and 2 times higher, respectively, in the HFD groups. This is consistent with the reports that the numbers of macrophages as well as NK cells were higher in the obese mice that were fed 45% or 60% kcal fat for 12 weeks compared with lean mice [2,39]. Furthermore, SVCs and adipocytes from obese mice produced higher levels of MCP-1 and IL-6, respectively, than those from lean mice. These results confirm that HFD-induced obesity results in inflammatory responses in adipose tissue with increased immune cell numbers in adipose tissue and the up-regulated synthesis of inflammatory cytokines from both adipocytes and SVCs.
In vitro 1,25(OH)2D3 treatment decreased MCP-1, IL-6, and IL-1β production by SVCs from obese mice (MCP-1: 15%, IL-6: 12%, and IL-1β: 34% decreased production); however, no significant difference was observed in the production of these cytokines by adipocytes with 1,25(OH)2D3 treatment. In obesity, the MCP-1 secreted by both hypertrophic adipocytes and M1 macrophages has been reported to accelerate macrophage infiltration into the adipose tissue and increase adipose tissue-derived IL-6 and IL-1β levels [11,40,41]. Elevated levels of IL-6 and IL-1β in adipose tissue have been reported to contribute to the risk of type 2 diabetes [42][43][44]. Although both adipocytes and non-adipocytes secrete these inflammatory cytokines, non-adipocytes (SVCs) produce 8~20 times more inflammatory cytokines, including MCP-1, IL-6, IL-1β, and TNF-α, compared with adipocytes, indicating that the majority of inflammatory cytokine released are by non-adipocytes [45]. Therefore, despite the lack of significant effects of 1,25(OH)2D on adipocytes, our results suggest that vitamin D could contribute to the alleviation of adipose tissue inflammation by reducing the production of inflammatory cytokines from SVCs.
The effects of 1,25(OH)2D on adipose tissue inflammation have been investigated by other researchers [10,27,31,46,47]; however, contradictory results have been reported regarding its effects on adipocytes, and few studies have been conducted to examine the effect of vitamin D on SVCs. Sun et al. [31,48] reported that MCP-1 and IL-6 productions were increased by treatments with 10 nM of 1,25(OH)2D3 in 3T3-L1 cells [10,26] and differentiated human adipocytes [9], while others have shown that the production of IL-6, MCP-1, and IL-1β were reduced by 1,25(OH)2D3 in 3T3-L1 or differentiated human adipocyte; however, the cause of conflicting results were not delineated. Using 3T3-L1 and human adipocytes, Marcotorchino et al. [10] showed that vitamin D (100 nM of 1,25(OH)2D3) reduced IL-6, MCP-1, and IL-1B production and inactivated NF-κB by inducing IκBa. Gao et al. [27] reported the anti-inflammatory effect of vitamin D on SVCs by showing decreased MCP-1 and IL-6 levels with 10 or 100 nM of 1,25(OH)2D3 treatment on human pre-adipocytes. Although pre-adipocytes are one of the components of SVCs and have been shown to be involved in adipose tissue inflammation [49,50], major contributors to the adipose inflammation are immune cells, especially M1-like macrophages, which release a substantial amount of inflammatory cytokines [13]. In this study, 47%~50% of SVCs from HFD-induced obese mice were immune cells (CD45+ cells), of which more than 60% were macrophages. Since LPS can induce M1 polarization, the anti-inflammatory effects of vitamin D on SVCs in this study is likely to be due to its effect on immune cells in SVCs.
The activation of TLR signaling upon recognition of LPS or fatty acids released from lipolysis could induce pro-inflammatory cytokine production in adipose tissue through the NF-κB and MAPKs signaling pathways [14,20]. Increased levels of TLR2 protein have been reported in adipose tissues from obese or diabetic humans [23], and the expression of Tlr2 mRNA was greater in HFD-induced obese mice [27]. In this study, Tlr2 mRNA levels in SVCs were significantly higher (117% higher) in the HFD group compared with the CON group and were decreased by 1,25(OH)2D3 treatment in both the HFD and CON groups. TLR2 antisense-treated mice were insulin sensitive and resistant to chronic inflammation when fed a HFD for 8 weeks [51]. Therefore, down-regulated Tlr2 expression by 1,25(OH)2D3 could have contributed to the alleviation of inflammatory signaling. In addition, both Iκbα and Dusp1 mRNA levels were increased by 1,25(OH)2D3 treatment. Iκbα is the inhibitory subunit of NF-κB activation since it blocks the binding of NF-κB transcription factors to DNA [52] and Dusp1, which is known as the MAPK phosphatase 1 (MKP1) and plays a key role in the dephosphorylation and inactivation of MAPK in mammalian cells [53]; thus, elevated expression of Iκbα and Dusp1 suggests that NF-κB and MAPK signaling pathways might be inhibited by 1,25(OH)2D3 treatment. Cohen-Lahav et al. [54] reported that 1,25(OH)2D3 treatment (100 nM) could reduce NF-κB activation by elevating IκBα levels in murine macrophage. Additionally, Zhang et al. [47] demonstrated that the anti-inflammatory effect of vitamin D in a bone marrow-derived macrophage is MKP1 dependent, by showing that mice lacking MKP-1 were unable to reduce inflammatory cytokines upon vitamin D treatment. Therefore, decreased Tlr2 mRNA levels and inflammatory cytokine production by 1,25(OH)2D3 treatment in SVCs in this study might be partially due to up-regulated Iκbα and Dusp1 levels.
Overall, dietary vitamin D supplementation (25,000 IU/kg diet) resulted in lower mRNA levels of inflammatory cytokines (Mcp-1, Rantes, Il-6, and Il-1β) in adipose tissue by increasing adipose tissue 25(OH)D levels. However, the number of macrophages infiltrated into adipose tissue was not significantly changed by dietary vitamin D supplementation (10,000 IU vitamin D3/kg diet). Although the amount of dietary vitamin D supplemented was different between two experiments, the average serum 25(OH)D level in the 10,000 IU/kg diet-supplemented group (88.7 ng/mL) was not lower than its level in the 25,000 IU/kg diet-supplemented group (74.9 ng/mL); thus, even if vitamin D supplementation was at the 25,000 IU vitamin D3/kg diet level, it is likely that significant changes in immune cell numbers would not be observed. Dietary vitamin D might have reduced adipose tissue inflammation by regulating inflammatory gene expression directly without a significant reduction of macrophage infiltration; the anti-inflammatory effects of in vitro 1,25(OH)2D treatment on SVCs isolated from obese mice provides the mechanisms behind the in vivo effects of vitamin D.

Conclusions
In conclusion, dietary vitamin D supplementation attenuated inflammatory responses in adipose tissue from obese mice, and this might be attributed to the specific anti-inflammatory effect of 1,25(OH)2D3 in SVCs. Vitamin D supplementation did not result in reduced numbers of macrophages and NK cells in adipose tissue, but, 1,25(OH)2D3 treatment inhibited NF-κB and MAPK signaling in SVCs by increasing the expression of Iκbα and Dusp1. As a result, 1,25(OH)2D3 reduced the production of pro-inflammatory cytokines, which are increased by obesity. These findings suggest that obese people with chronic inflammation may benefit from vitamin D supplementation, however, clinical intervention studies to confirm these findings are needed.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/2/364/s1, Table S1: Composition of the experimental diets (Exp. 2), Table S2: Composition of the experimental diets (Exp. 3), Table S3. Body weight, weight gain, body fat, food intake, and serum 25(OH)D level of mice in the CON-DC, CON-10DS, HFD-DC and HFD-10DS groups, Table S4. Body weight, weight gain, body fat, and food intake of mice in the CON and HFD groups (Exp. 3), Table S5. Visceral fat tissue weights (g) and total cell numbers of Stromal vascular cell(SVC)s, CD45+ cells, macrophages, NK cells, B cells, CD4+ T cells, and CD8+ T cells in visceral adipose tissue, Figure S1: The mRNA levels of Cyp27b1 and Vdr in epididymal adipose tissue, Figure S2  Acknowledgments: In this section you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).

Conflicts of Interest:
The authors declare no conflict of interest.