The mouse macrophage RAW264.7 cell line was purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) that contained 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) with penicillin (100 U/ml) and streptomycin sulfate (100 µg/ml) at 37°C in an incubator with 5% CO₂. Culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.).

The recombinant Tim-3 overexpression plasmid (pTim-3), empty vector (pScramble) and small interfering (si)Tim-3 were purchased from Promega Corporation (Madison, WI, USA). The sequences of siTim-3 were Sense 5′:GGUGUCUUAAUCCUUAAAU, and Antisense 3′:CCACAGAAUUAGGAAUUUA. The cells were seeded at a density of 1×10⁴ cells/well into a 24-well plate. The cells were then starved overnight by incubating them in DMEM without FBS prior to cell transfection. When the confluence reached 60–70%, the cells were transfected with pTim-3 or siTim-3 (50 pmol) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the protocol of the manufacturer. The time interval between transfection and subsequent experimentation was 24 h. For screening the positive clones, the RAW264.7 cell line with the stable knockdown or Tim-3 overexpression plasmid was cultured in the aforementioned complete medium with 200 and 700 µg/ml G418 respectively, at 37°C for 24 h. According to data from previous studies (26–28), 10⁻⁸ or 10⁻⁷ M 1,25(OH)₂D3 was used to treat the RAW264.7 cells for 3 days. Cell morphology was observed using a laser confocal microscope, with magnifications ×100 and ×200 (Leica TCS SP8; Leica Microsystems, Wetzlar, Germany).

ELISAs were used to measure the concentrations of TNF-α, IL-6, IL-10 and transforming growth factor-β (TGF-β). The kits were Mouse TNF-α Quantikine ELISA Kit (MTA00B, R&D Systems, Inc., Minneapolis, MN, USA), Mouse IL-6 Quantikine ELISA Kit (M6000B, R&D), Mouse IL-10 Quantikine ELISA Kit (M1000B, R&D), and Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA Kit (MB100B, R&D). Following washing, blocking solution was added at 4°C and incubated for 2 h. Next, the primary antibody was added at 4°C and incubated overnight. The secondary antibody was then added and incubated for 1 h at room temperature. Horseradish peroxidase was added dropwise for 30 min at room temperature, and then tetramethylbenzidine was added for 10 min. The absorbance value was measured at 450 nm using a microplate reader (ELX 800; Bio-Tek Instruments, Winooski, VT, USA) and the concentration was calculated following the standard curve.

The expression levels of Tim-3, nitric oxide synthase, inducible (NOS2) and macrophage mannose receptor 1 (COD206) proteins were detected using western blot analysis. Cells were lysed with liquid nitrogen and radioimmunoprecipitation assay lysis buffer (Abmole Bioscience Inc., Houston, TX, USA). Next, the cells were cleaved with 1% phenylmethyl sulfonyl fluoride mixed with phosphatase inhibitors (Abmole Bioscience Inc) for 30 min at 4°C. The supernatant was collected using centrifugation at 13,000 × g at 4°C for 15 min. A standard curve was drawn using the BCA method to determine the protein concentration. Then, proteins (20 µg/lane) were separated using 10% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a Trans-Blot Transfer Slot (Bio-Rad Laboratories, Inc.). The PVDF membranes were then blocked with 5% skimmed milk for 2 h at room temperature. Primary antibodies against Tim-3 (cat. no. ab185703; 1:800; Abcam, Cambridge, MA, USA), NOS2 (cat. no. 14-5920-80; 1:800; eBioscience; Thermo Fisher Scientific, Inc.), CD206 (cat. no. 141703; 1:600; BioLegend, Inc., San Diego, CA, USA) were then added according to the manufacturers' protocols. The samples were agitated at room temperature for 2 h and then incubated at 4°C for 12 h. Rabbit anti-mouse IgG (cat. no. ab99697; 1:9,000 Abcam), mouse anti-rabbit IgG (cat. no. BA1034; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) and goat anti-mouse IgG (cat. no. ab6785; 1:8,000; Abcam) HRP-conjugated secondary antibodies were added and incubated at room temperature for 1.5 h. Chemiluminescence detection was performed using ECL reagent (EMD Millipore, Billerica, MA, USA). The densitometric analysis was performed using Bio-Rad ChemiDoc™ XRS+ System with Image Lab™ Software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

RT-qPCR was performed to determine the mRNA levels of Tim-3, NOS2 and COD206. The cells were triturated and lysed by TRIzol^® (Thermo Fisher Scientific, Inc.) at 0°C for 5 min. RNA was then extracted using CCl₃ (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) and dissolved in diethyl pyrocarbonate-treated water (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). RNA concentration was measured using a UV NanoDrop One Microvolume UV–Vis spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Reverse transcription assays were performed on RNA samples to synthesize cDNA using a reverse transcription kit (Takara Biotechnology, Co., Ltd., Dalian, China). The samples were incubated at 37°C for 15 min, followed by heat inactivation at 85°C for 15 sec. RT-qPCR experiments were performed using the SYBR Prellix Ex Taq™ Real-Time PCR kit (Takara Biotechnology, Co., Ltd.). qPCR was performed by activating the DNA polymerase at 95°C for 5 min, which was followed by 40 cycles of a two-step PCR (at 95°C for 10 sec and at 60°C for 30 sec) with a final extension step at 75°C for 10 min. Samples were then held at 4°C. β-actin was used as an internal control. All primers were purchased from Genewiz, Inc., (Suzhou, Jiangsu China), and are summarized in Table I. The 2^−ΔΔCq method was used to quantify the gene expression levels (29).

All experimental data are presented as means ± standard deviation. Statistical analysis was performed using SPSS 20 (IBM Corp., Armonk, NY, USA). The one-way analysis of variance followed by Tukey's multiple comparison post-hoc test was performed to analyze the differences among experimental groups. P<0.05 was considered to indicate a statistically significant difference.

The viable RAW264.7 macrophages were normal at magnifications, ×100 and ×200 (Fig. 1).

It was identified that the levels of TNF-α and IL-6 secreted by RAW264.7 cells were decreased in the presence of 1,25(OH)₂D3. However, the levels of IL-10 and TGF-β were increased (P<0.01; Fig. 2A-D). These differences were the most significant at concentrations of 10⁻⁷ M (P<0.05).

To investigate the mechanism of how 1,25 (OH)₂D3 affects the expression levels of inflammatory factors, levels of Tim-3, and NOS2 and COD206, the markers of M1 and M2, were examined. The results indicated that the expression level of Tim-3 was gradually increased in the 10⁻⁸ M and 10⁻⁷ M groups. However, whilst 1,25 (OH)₂D3 did not affect the expressions of the NOS2 and COD206, it decreased NOS2 mRNA levels (P<0.05; Fig. 2E-K). This observation indicated that 1,25(OH)₂D3 promoted Tim-3 expression in a dose-dependent manner.

Following silencing of the Tim-3 gene, the levels of TNF-α and IL-6 secreted by RAW264.7 cells were upregulated, while the levels of IL-10 and TGF-β were downregulated (P<0.01; Fig. 3A-D). Following the decrease of the Tim-3 gene expression, the mRNA and protein expression levels of NOS2 were observed increased compared with that of the NC and control groups, while the expression levels of CD206 mRNA and protein were significantly decreased (P<0.01; Fig. 3E-K). This phenomenon suggested that silence of Tim-3 gene promoted the polarization of macrophages to M1.

The effects of the overexpression of Tim-3 on cytokines were also examined. It was identified that the levels of TNF-α and IL-6 in the pTim-3 group were decreased compared with those in the NC group, and that the levels of IL-10 and TGF-β were increased compared with those in the NC group (P<0.01; Fig. 4A-D). When Tim-3 was overexpressed, the mRNA and protein expression levels of NOS2 were decreased, while the expression levels of CD206 mRNA and protein were increased (P<0.01; Fig. 4E-K). This suggested that the overexpression of Tim-3 promoted the polarization of macrophages to M2.

To additionally explore the effect of 1,25(OH)2D3 on the polarization of macrophages, RAW264.7 cells were cultured in the presence of 10⁻⁷ M 1,25(OH)₂D3. The expression levels of inflammatory factors, and M1 and M2 marker proteins NOS2 and CD206, were detected.

It was identified that 1,25(OH)2D3 downregulated the levels of TNF-α and IL-6 and upregulated the level of TGF-β in siTim-3 cells. In addition, 1,25(OH)₂D3 promoted the expression of Tim-3 and increased the expression levels of CD206 protein in siTim-3 cells. However, 1,25(OH)₂D3 decreased the expression levels of NOS2. For RAW264.7 cells with stable overexpression of Tim-3, 1,25(OH)2D3 was observed to additionally decrease TNF-α and IL-6 levels and increase IL-10 and TGF-β levels, compared to the Tim-3 overexpression group. In addition, 1,25 (OH)₂D3 increased the level of Tim-3 and the expression levels of CD206 in RAW264.7 cell with stable expression of Tim-3 gene (Fig. 5A-K). This indicated that 1,25(OH)₂D3 promoted the polarization of macrophages to M2 and inhibited polarization of macrophages to M1 via upregulating the expression of Tim-3.