Four-week-old female BALB/c mice were used as experimental animals (YoungBio, Seongnam, Korea). Each mouse weighed 18–22 g and was maintained under specific pathogen-free conditions. All animal experiments in the present study followed the guidelines and ethics of the Institutional Animal Care and Use Committee of the Biomedical Research Institute of Seoul National University Hospital (16-0106-S1A0[2]).

Mice were divided into 4 groups as follows: group 1 (n = 4) as a negative control group, group 2 (n = 6) as a positive control group, group 3 (n = 6) as a 1,25-dihydroxyvitamin D3 treatment group and group 4 (n = 4) as a sham treatment group. 1,25-dihydroxyvitamin D3 (Bonky® [Yuyu Pharma, Inc., Seoul, Korea] injection 1 μg) and its solvent were acquired from YuYu Pharma, Inc. The main ingredients of the solvent were polysorbate 20 (4.0 mg/mL), NaCl (1.5 mg/mL), sodium L-ascorbate (10.0 mg/mL), disodium edetate (1.11 mg/mL), anhydrous dibasic sodium phosphate (7.5 mg/mL) and monobasic sodium hydrogen phosphate monohydrate (1.84 mg/mL). Allergen sensitization, challenge for the development of the allergic rhinitis murine model, and treatment are summarized in Fig. 1. Briefly, the mice were sensitized by an intraperitoneal injection of 25 μg of ovalbumin (OVA; grade V; Sigma-Aldrich, St. Louis, MO, USA) and 2 mg of aluminum hydroxide on days 0, 7, and 14. The mice were then subjected to intranasal challenges with 100 μg of OVA on 7 consecutive days from days 21 to 27. The negative control mice were intraperitoneally injected and intranasally challenged with phosphate-buffered saline (PBS), instead of OVA following the same schedule until day 27 at which mice were intranasally challenged with OVA. 1, 25-dihydroxyvitamin D3 (20 μL of Bonky® [Yuyu Pharma, Inc.] containing 0.02 μg per mouse) and solvent (20 μL per mouse) were administered by intranasal instillation on days 21 to 27 (3 hours before intranasal OVA challenge) to mice in groups 3 and 4, respectively.

On day 27, after intranasal allergen provocation with 100 μg of OVA, the numbers of sneezing and nose rubbing bouts were counted for 15 minutes to evaluate early allergic responses by blinded observers.

All animal experiments were repeated twice. In both experiments, symptom scores were checked and the mice were then killed 24 hours after the last OVA challenge. Cervical lymph nodes (LNs) (primarily submandibular, 2-3 nodes from each side) were harvested from each mouse and were phyisically dissociated. In the first experiment, nasal tissues were obtained and further homogenized to measure cytokine expression. Serum samples from each mouse were also obtained for further analysis. Cells from the cervical LNs were analyzed by flow cytometry. In the second experiment, nasal tissuses from each mouse were fixed in formaldehyde solution for histological analysis. Cells from the cervical LNs were cultured to measure cytokine release (proliferation assay).

After sacrifice, the head of each mouse was removed. After exposing the nasal cavity, the nasal mucosa in each mouse was carefully taken out by using a curette. Total RNA was isolated from the nasal mucosa using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using a cDNA synthesis kit (Gendepot, Katy, TX, USA). For the analysis of interleukin (IL)-4 (Mm 00445259_m1), IL-5 (Mm 00439646_m1), interferon (IFN)-γ (Mm 99999071_m1), IL-10 (Mm 00439616_m1), IL-13 (Mm 00434204_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Mm 99999915_g1), pre-developed assay reagent kits of primers and probes were purchased from Applied Biosystems (Foster City, CA, USA). The amplification of IL-4, IL-5, IL-10, IL-13, IFN-γ and GAPDH cDNA was performed in MicroAmp optical 96-well reaction plates (Applied Biosystems). The reaction was performed using a QuantStudio™ 3 RT-PCR System (Applied Biosystems). The average transcript levels of genes are normalized to GAPDH expressed as 2^−ΔCT values.

For the evaluation of nasal histology, the heads of mice in each group were fixed with 10% formaldehyde solution. The nasal tissues were decalcified with ethylenediaminetetraacetic acid solution, embedded in paraffin, sectioned coronally into 4-μm slices and stained with hematoxylin and eosin (H&E) for the visualization of eosinophils. Under a light microscope (×400 magnification), infiltrating eosinophils were counted in 4 fields of the nasal septal mucosa by a single-blind observer. Eosinophils were morphologically defined by the presence of eosinophilic granules that were stained by H&E and the presence of a 2-lobed nucleus.8

Serum samples from each mouse were obtained at the time of sacrifice. Serum levels of total IgE and OVA-specific IgE, IgG1 and IgG2a were measured by enzyme-linked immunosorbent assay (ELISA). For the analysis of total IgE, 96-well flat-bottom plates were coated with purified rat anti-mouse IgE (#553413; BD Bioscience, San Jose, CA, USA). A purified mouse IgE isotype (#557079; BD Bioscience) was used as a standard. Nonspecific antigen-antibody reactions were blocked with 3% bovine serum albumin. To detect total IgE, horseradish peroxidase (HRP)-conjugated anti-mouse Ig (#554002; BD Bioscience) was added to the wells. For the analysis of OVA-specific IgE, serum samples were added to OVA-coated 96-well flat-bottom plates. After washing, 100 μL of biotin-conjugated rat anti-mouse IgE mAb (#553414; BD Bioscience) was added, followed by the addition of streptavidin-HRP (#554066; BD Bioscience). For the analysis of OVA-specific IgG1 and IgG2a, serum samples were added to OVA-coated 96-well flat-bottom plates. After washing, biotinylated rat anti-mouse IgG1 and IgG2a (#553388 and #553441, respectively; BD Bioscience) were added to each well and incubated. After washing, streptavidin-HRP was added to each well. The reactions were developed using 3,3′,5,5′-tetramethylbenzidine (Moss Inc., Belfast, ME, USA) and terminated by adding 1 N HCl. The absorbance was measured in a microplate reader at 450 nm.

For the analysis, harvested cells from each mouse were distributed into 2 sets and were suspended in 50 μL of cold PBS containing 2% fetal calf serum. The first set of cells was stained with V450-conjugated anti-CD45 (BD Bioscience), BB515-conjugated anti-major histocompatibility complex II (MHCII) (BD Bioscience), phycoerythrin-conjugated anti-CD11c (BD Bioscience) and anaphase-promoting complex (APC)-conjugated anti-CD86 (BD Bioscience) for the detection of activated DCs. The other set of cells was stained with peridinin chlorophyll protein complex-conjugated anti-CD3 (BD Bioscience), fluorescein isothiocyanate-conjugated anti-CD4 (eBiosience, San Diego, CA, USA), APC-conjugated anti-CD25, and phycoerythrin-conjugated anti-Foxp3 for the detection of regulatory T cells. The cells were analyzed by flow cytometry (LSR II; BD Bioscience). The CD11c⁺MHCII^high cells were considered as migratory DCs (Supplementary Fig. S1).9

Cervival LN single-cell suspensions from each mouse were plated in 24-well cell culture plates at a final concentration of 5 × 10⁶ cells/well using Roswell Park Memorial Institute 1,640 containing 10% fetal bovine serum supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA). The cells were incubated in a CO₂ incubator at 37°C for 72 hours and stimulated with OVA for 72 hours. The culture supernatant was collected and stored at −70°C until cytokines were measured. Cytokine levels in the culture supernatant were assayed using a DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. After measuring the absorbance at 450 nm, the concentrations of IL-4, IL-5, IL-10 and IL-13 were determined by interpolation from the respective standard curves; all data are expressed in pg/mL.

The data are presented as mean ± standard error mean. The Mann-Whitney U test was used to compare results between the negative and positive control groups and between the treatment and positive control groups. P values of <0.05 were considered statistically significant. Statistical analysis was performed using SPSS 22.0 software (IBM Co., Armonk, NY, USA).

Fig. 2. shows symptom scores for each group after nasal challange with OVA. In the mice of group 2 (positive control), the mean numbers of sneezing and nose rubbing bouts were significantly higher than those of group 1 (negative control) (P < 0.001 and P = 0.001, respectively). The mice of group 3 (treatment group) showed significantly lower numbers of sneezing and nose rubbing bouts than those of groups 2 and 4 (P < 0.001 and P = 0.001, respectively). There was no signficiant difference in the mean numbers of sneezing of nose rubbing bouts between groups 2 and 4.

Fig. 3. shows eosinophil infiltration in the nasal mucosa for each group. Significantly higher number of eosinophils infiltrating the nasal mucosa per high-power field was observed in group 2 than in group 1 (P = 0.004). The mean number of eosinophils infiltrating the nasal mucosa per high-power field was significantly lower in group 3 than in groups 2 (P = 0.002) and 4 (P = 0.004). However, no significant difference was observed between groups 2 and 4.

Fig. 4. shows cytokine expression in the nasal tissue. Compared to group 1 mice, significantly increased mRNA levels of IL-4 (P = 0.010), IL-5 (P = 0.010), IL-10 (P = 0.010), IL-13 (P = 0.010), and IFN- γ (P = 0.019) were observed in the nasal tissues of group 2 mice. The IL-4 and IL-13 mRNA levels were significantly decreased in group 3 compared to groups 2 (P = 0.010 and P = 0.038, respectively) and 4 (P = 0.029, both). The IL-5 expression in group 3 tended to follow the same pattern; however, the results were not significant, compared to groups 2 and 4. The mRNA levels of IFN-γ and IL-10 in group 3 did not significantly differ from those in groups 2 and 4. No significant difference was observed in the mRNA levels of IL-4, IL-5, IL-10, IL-13 or IFN-γ between groups 2 and 4.

CD86 expression among CD11c⁺MHC^high cells from cervical LNs was examined. Group 2 showed significantly increased CD86 expression among CD11c⁺MHC^high cells compared to group 1 (P = 0.038). Although the number of CD86⁺ cells among CD11c⁺MHC^high cells was not significantly different among groups 2, 3 and 4 (data not shown), mean fluorescence intensity (MFI) showed that CD86 expression was significantly lower in group 3 than in groups 2 (P = 0.041) and 4 (P = 0.010). The CD86 expression in group 3 did not significantly differ from that in group 1. Similarly, CD86 expression did not significantly differ between groups 2 and 4. The number of CD4⁺CD25⁺Foxp3⁺ cells tended to be higher in group 3 than in groups 2 and 4; however, the differences were not significant (Fig. 5). The MFI of Foxp3 expressed by CD4⁺CD25⁺ cells did not significantly differ from that expressed by most of the cells (≥90%) (data not shown).

To investigate whether the decreased CD86 expression in cervical LNs down-regulates T-cell activity, we measured the level of cytokines in the culture supernantants (Fig. 6). Levels of IL-4 (P = 0.016), IL-5 (P = 0.008), IL-10 (P = 0.008), and IL-13 (P = 0.008) were significanlty higher in group 2 than in group 1. IL-4 level was significantly lower in group 3 than in groups 2 (P = 0.029) and 4 (P = 0.029). Levels of IL-5, IL-10, and IL-13 in group 3 showed a similar pattern; however, the differences were not significant when compared to groups 2 and 4.

Serum total IgE and OVA-specific IgE, IgG1, and IgG2a levels were significantly higher In group 2 than in group 1 (P = 0.010) (Fig. 7). However, no significant differences were observed among groups 2, 3 and 4.