The present study was approved by The Ethics Committees of The People's Hospital of Shanxi Province. A total of 33 specific pathogen-free (SPF) male Wistar rats (age, 6–8 weeks; weight, 180–200 g) were obtained from The Laboratory Animal Center of Hangzhou Hibio Technology Co., Ltd. The animals were acclimatized to laboratory conditions (23°C, 12-h light/dark cycle, 50% humidity and ad libitum access to food and water) for 2 weeks prior to experimentation. Animals were sacrificed by intravenous injection of pentobarbital sodium (150 mg/kg) and animal death was confirmed by lack of reflexes, heartbeat and breathing. Specific tissues were collected for experimentation. Bone marrow was collected from the femurs and tibiae of Wistar rats as previously described (19). The intact femurs and tibiae were kept in 70% ethanol for 30 min, and then rinsed with fresh RPMI medium (Gibco; Thermo Fisher Scientific, Inc.). The extremities of the femurs and tibiae were removed with scissors, and the bone marrow was flushed using 5–10 ml medium with a 19-gauge syringe. A lysis buffer containing ammonium chloride (KHCO₃ 1.0 g/l; NH₄CL 8.3 g/l; EDTA-Na₂ 0.037 g/l) was used to lyse red blood cells and the samples were washed twice to isolate the bone marrow cells. Bone marrow cells were resuspended in RPMI medium supplemented with recombinant rat IL-4 (10 ng/ml; PeproTech, Inc.) and recombinant rat granulocyte monocyte colony-stimulating factor (GM-CSF; 10 ng/ml; PeproTech, Inc.), and cultured at a density of 1×10⁶ cells/ml in six-well plates with 3 ml culture medium/well at 37°C in a 5% CO₂ humidified incubator. Fresh culture medium and cytokines were added at day 3. Cell aggregates attached to the dish surface were observed between day 3 and 4. The culture medium was removed and replaced by fresh medium with GM-CSF at day 5 and 6.

The phenotype of the cultured dendritic cells was examined by investigating the protein expression levels of α E2 integrin (OX62) and MHC-II, as assessed by flow cytometry (Fig. S1), conducted as follows: One well of a 6-well plate was centrifuged at 300 × g for 5 min at 37°C, and the cell pellet was collected and resuspended in 5X volume of washing solution (0.01 M PBS). The cells were rinsed twice and resuspended in 200 µl of staining buffer (cat. no. 420201; BioLegend, Inc.) at a concentration of 1×10⁶ cells/ml. The cells were equally divided into two 1.5-ml centrifuge tubes; one tube was an experimental group, whereas the other was a blank group. Phycoerythrin (PE)-conjugated anti-MHC class II (1:50; cat. no. 12-0920-82; Invitrogen; Thermo Fisher Scientific, Inc.) and PE-conjugated anti-Ox62 (1:50; cat. no. 12-1030-82; Invitrogen; Thermo Fisher Scientific, Inc.) antibodies were added separately and incubated for 30 min at room temperature in the dark; the blank group for each assay was incubated without primary antibody. MHC-II and OX62 were directly detected using an Accuri C6 flow cytometer (BD Biosciences); the blank group was used to determine the negative region. The test was repeated three times.

The sequences of the shRNA targeting CCR7 and the control sequences were as follows: shRNA-CCR7, 5′-TGGATCTTTGGTGCCTACCTGTGTA-3′; control shRNA, 5′-TTCTCCGAACGTGTCACGTAA-3′. shRNA-CCR7 and control shRNA was inserted into a pHBAd-U6-GFP backbone, which was packaged into an adenovirus. The 293A cells were transfected with the shCCR7 plasmid, and the adenovirus was harvested. The plasmid, shRNA and adenovirus were all supplied by Hangzhou Hibio Technology Co., Ltd. As control, an empty vector was used, and the infection efficiency was assessed by western blot analysis (Fig. S2). Then, 1×10⁶ imDCs were suspended in 1 ml adenoviral supernatant (MOI=50) with 1% FBS (Invitrogen; Thermo Fisher Scientific, Inc.), 10 ng/ml GM-CSF and 10 ng/ml IL-4 at 37°C for 72 h. Subsequently, cells were centrifuged at 300 × g at 37°C for 2 h. After infection, imDCs were washed twice in PBS and incubated with RPMI-1640 medium containing 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4 at 37°C with 5% CO₂. The cells were harvested for injection 48 h after infection.

NotI and PstI restriction sites were added to the ends of the CCR7 coding sequence, which was cloned into a pDC316-MCMV-ZsGreen backbone, which was packaged into an adenovirus. The recombinant adenoviral vector containing the rat CCR7 gene was prepared as previously described by Hibio Technology Co., Ltd. (20), and the centrifugal enhancement method was used to increase the infection efficiency (21). Empty plasmid vector was used as negative control. Viral packaging was verified by the detection of green fluorescent protein via fluorescence microscopy (data not shown). Subsequently, 1×10⁶ imDCs were suspended with 1% FBS, 10 ng/ml GM-CSF and 10 ng/ml IL-4, adding the adenovirus at MOI=50, and cells were subsequently centrifuged at 300 × g at 37°C for 2 h. After infection, the imDCs were washed twice in PBS and incubated with RPMI-1640 medium containing 10% FCS supplemented with 10 ng/ml GM-CSF and 10 ng/ml IL-4 at 37°C with 5% CO₂. The cells were harvested for injection 48 h after transfection. The infection efficiency of CCR7 was determined by western blot analysis (Fig. S3).

Healthy SPF male Wistar rats were used to establish an animal model of allergic asthma, according to a modified previously described protocol (22). Rats were immunized by intraperitoneal injection of 100 µg ovalbumin (OVA; Sigma-Aldrich; Merck KGaA) adsorbed into 400 µg aluminium hydroxide in 0.2 ml sterile saline (0.9%) on day 1 and 8. After 2 weeks, rats were exposed to aerosol spray containing 1% OVA in 0.9% sterile saline for 30 min every day, 6 days every week, for a total of 8 weeks. The experimental rats were divided into three groups (n=10 in each group) as follows: i) Control (Con) group, rats injected with 2×10⁵ cultured wild-type imDCs; ii) CCR7 overexpression (Over) group, rats injected with 2×10⁵ cultured imDCs infected with adenoviral particles overexpressing CCR7; and iii) CCR7 interference (Sh) group, containing rats injected with 2×10⁵ cultured imDCs infected with adenoviral particles encoding a shRNA targeting CCR7. imDCs were injected via the tail vein at day 1, 10 and 18.

For immunohistochemical staining, lung tissues were fixed at room temperature for 5 days using 4% formaldehyde solution, embedded in paraffin and sectioned (4-µm), dewaxed in 100% xylene (twice in total, 20 min each time), rehydrated in a graded alcohol series (100% for 5 min, 95% for 5 min, 80% for 5 min), and incubated with 0.3% H₂O₂ in methanol to block the endogenous peroxidase activity for 15 min at 37°C. Mounted sections were boiled in 10 mM citrate solution (pH 6.0) for 20 min for antigen retrieval, and incubated overnight at 4°C with the following primary antibodies: Anti-rat CCR7 (1:50; cat. no. bs-1305R; Bioss, Inc.) and anti-rat OX62 (1:50; cat. no. sc-53085; Santa Cruz Biotechnology, Inc.). Horseradish peroxidase-conjugated secondary antibody (1:25; cat. no. PV-6001; Beijing Zhongshan Golen Bridge Biotechnology Co., Ltd.; OriGene Technologies, Inc.) was incubated with the sections at 37°C for 30 min. A two-step technique (SuperPicture Third Generation IHC Detection kit; Invitrogen; Thermo Fisher Scientific, Inc.) was used to visualize the stained samples, and 0.1 mg/ml 3,3′-diaminobenzidine diluted in a 0.02% H₂O₂ solution (Vector Laboratories, Inc.) was used as chromogen for 2 min at room temperature. The tissue sections were counterstained with hematoxylin (1 g/ml, 2 min) and eosin (5 g/l, 30 sec) at room temperature, and observed using a light microscope (magnifications, ×100 and ×400; Olympus Corporation).

Lung tissues were homogenized in RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.) and the protein concentration was determined using the bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Total protein was separated by SDS-PAGE on 10% gels. In total, 30 µg protein was loaded in each lane. Proteins were then transferred to PVDF membranes (EMD Millipore). Membranes were blocked for 2 h at room temperature in 5% non-fat dry milk and incubated with a primary antibody anti-rat CCR7 (1:1,000; cat. no. ab32527; Abcam) or anti-OX62 (1:1,000; cat. no. bs-1274R; Bioss, Inc.) at 4°C overnight. Membranes were then incubated with the appropriate horseradish peroxidase-labeled anti-mouse [1:1,000; cat. no. GAM007; MultiSciences (Lianke) Biotech Co., Ltd.] or anti-rabbit IgG [1:1,000; cat. no. GAR0072; MultiSciences (Lianke) Biotech Co., Ltd.] secondary antibodies at room temperature for 50 min. Anti-β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology Inc.) or GAPDH (1:1,000; cat. no. sc-32233; Santa Cruz Biotechnology Inc.) was used as the loading control. Bands were visualized by autoradiography (ChemiDoc XR+System; Bio-Rad Laboratories, Inc.) and quantified by densitometry (ImageJ V1.46; National Institutes of Health). The results were normalized to GAPDH or to β-actin. All experiments were performed in triplicate.

After the animal model was successfully established (8 weeks), the animals were anesthetized and the lungs were exposed. The right principal bronchus was occluded using a hemostat clamp. A total of 4 ml saline was used for the bronchoalveolar lavage in the left lung, and 3–4 ml of fluid was collected after flushing twice. The alveolar lavage fluid was centrifuged at 300 × g for 10 min at room temperature, and the supernatant was used for ELISA. Next, 0.5 ml saline was added to 0.5 ml BALF containing sediment and mixed. Then, the samples were smeared and the cells were counted using the Wright-Giemsa staining technique [Wright's dyeing powder (1 g), Jimsa dyeing powder (0.5 g), 20 min incubation, room temperature] and observed with a CX21 light microscope (Olympus Corporation). Cells were counted under a microscope using a hemocytometer and a counting place (25×16 grid cells); the number of cells in each corner of the plate was calculated.

After the animal model was successfully established, blood (4 ml) was extracted from the vena cava, anticoagulated with heparin and centrifuged (500 × g for 10 min at room temperature), and serum was collected. ELISA kits were used for measuring IFN-γ (cat. no. EK0374), IL-4 (cat. no. EK0406), IL-12 (cat. no. A01152-2), IL-10 (cat. no. EK0418), TGF-β (cat. no. EK0514; all Boster Biological Technology) and IgE (cat. no. E-EL-R0517c; Elabscience Biotechnology, Inc.) concentrations in serum and BALF supernatant, according to the manufacturer's protocol.

All assays were repeated three times. The data are presented as the mean ± SD. Statistical analysis was performed using SPSS 11.5 software (SPSS, Inc.). One-way ANOVA was performed to compare multiple groups, followed by Student-Newman-Keuls-Q post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Immunohistochemical analysis suggested that the protein expression level of CCR7 was increased in the airways of rats in the CCR7-overexpressing group (Fig. 1). In addition, the cell membranes were stained yellow and brown, indicating the localization of CCR7 in the peribronchial stroma (Fig. 1C). Compared with the control group, the protein expression of CCR7 was significantly higher in the airways of rats in the overexpression adenovirus-infected (Over) group and lower in the shRNA adenovirus-infected (Sh) group (P<0.01; Fig. 1B). The protein expression levels of CCR7 in various groups detected by western blotting was consistent with the immunohistochemical results (Fig. 1A).

HE staining suggested the presence of numerous inflammatory cells in the lung tissues in the control group (Fig. 2). Additionally, the number of infiltrated inflammatory cells, including lymphocytes, eosinophils and neutrophils (as determined by cell morphology following H&E staining), was increased in the airway and lung tissues in the Over group compared with the Con group, and the airway wall was markedly thicker in the CCR7 overexpression group (Fig. S4). In the Sh group, the bronchiole structure was normal and the number of inflammatory cells infiltrating the lung tissue was reduced.

OX62 was used a marker for DC detection in the airways as it is specifically expressed by DCs (23). The CCR7 Over group exhibited higher expression levels of OX62 compared with the Con and Sh groups (Fig. 3). In addition, the protein expression level of OX62 was lower in the CCR7 interference group compared with the control group (P<0.05).

The numbers of leukocytes (P<0.01), neutrophils (P<0.05) and lymphocytes (P<0.01) were higher in the CCR7 Over group compared with the Con group and the Sh group (Fig. 4). Conversely, the expression of eosinophils was not affected by CCR7 overexpression. The present data suggested that CCR7 expression could affect the number and types of immune cells recruited to the airways and infiltrating the lung tissues.

Compared with the control group, the protein expression levels of IL-12, IL-4, IFN-γ and IgE, in both the BALF supernatant (Fig. 5) and serum (Fig. 6), were increased in the CCR7 Over group compared with the Con and the Sh groups (P<0.01). Compared with the control group, the protein expression levels of IL-10 and TGF-β in the BALF supernatant (Fig. 5) and serum (Fig. 6) were significantly lower in the CCR7 Over group, and higher in the Sh group (P<0.01).