Main reagents and instruments
Ovalbumin (OVA) was obtained from Sigma-Aldrich (St. Louis, MO), aluminum hydroxide gel from Thermo Fisher Scientific (Waltham, MA), and M. vaccae injections from Anhui Chi dragon coma Biological Pharmaceutical Co. The general SP detection kit and the 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogenic reagent kit were obtained from Beijing Jinqiao Biological Technology Co., Ltd. (Beijing, China). TGF-β1 antibody, TβR1 antibody, Smad1 antibody, and Smad7 antibody were obtained from Abcam (Cambridge, United Kingdom).
Other materials and equipment used were type IV collagenase, mouse viscera lymphocyte separation solution (Haoyang, Tianjing, China), phorbol 12-myristate 13-acetate (PMA)/lonomycin mixture, fetal bovine serum, FIX&PERM Kit, PE-CY5-anti-mouse CD3, FITC-IgG1, FITC-anti-mouseγδTCR, PE-IgG1, PE-anti-mouse-IL-13, PE-anti-mouse-LAP, glycogen staining kit, Wright stain, hematoxylin–eosin (HE) staining kit, cytometer, electron microscope, high-speed cryogenic centrifuge, ultrasonic atomizer WH-2000 (Yuehua medical instrument factory, Guangdong, China), atomized inhalation box (self-made), carbon dioxide incubator, lung function machine (Buxco, USA), 10% chloral hydrate, normal saline, 10% formaldehyde, and phosphate-buffered solution (PBS).
Ethics statement
The study was performed in accordance with the guide for the care and use of laboratory animals of the National Institutes of Health, and was approved by the Guangxi Medical University Animal Care and Use Committee (Protocol number: 20131002). All surgeries were performed under pentobarbital anesthesia and all efforts were made to minimize suffering.
Establishment of the mouse bronchial asthma model
Twenty-four 8–10-week-old pathogen-free female Balb/c mice (20–25 g) were provided by the Medical Animal Center of Guangdong Province (Guangdong, China). The mice were randomly divided into 3 groups, namely, normal group (group A), asthmatic model group (group B), and M. vaccae nebulization group (group C). Both groups B and C were sensitized with OVA and Group A with PBS. The mice in group C were nebulized with M. vaccae before the asthmatic models were established according to our former research [19]. The mice in group B were sensitized by OVA intraperitoneal injection and nebulization (overall 200 μL PBS mixed with 25 μg OVA, 1 mg aluminum hydroxide gel, and PBS liquid were injected intraperitoneally into each mouse on day 1, 8, and 15). On day 22, 24, 26, 28, and 30, the mice were nebulized with 20 mL 1% OVA fluid). OVA was replaced with PBS fluid in group A. The mice in group C were nebulized with 22.50 μg M. vaccae mixed with 20 mL PBS fluid once a day for 5 consecutive days (Fig. 1). Airway responsiveness was evaluated in all the mice, 24 h after the last nebulization.
Airway responsiveness measurement
Airway responsiveness was measured using a noninvasive lung function machine (Fine-Pointe™ NAM system TBL4500, Buxco, Wilmington, NC, USA) after the last stimulation with OVA. After calibration and 5 min of adaptation, the mice were nebulized with 20 μL PBS and methacholine (Mch; Sigma-Aldrich) at concentrations of 6.25, 12.5, and 25 mg/mL, respectively, for 30 s each. Data were recorded for 3 min, and the mice were allowed to recover for 4 min. The results were automatically analyzed after the experiment ended. The airway responsiveness presented as specific airway resistance (sRaw) (Fig. 2).
Specimen collection
The mice were anesthetized using an intraperitoneal injection of 1% pentobarbital (50 mg/kg body weight), dehematized through their eyeballs, and fixed on their backs. The lungs were lavaged using 500 μL iced PBS thrice, and the bronchoalveolar lavage fluid (BALF) was collected in Eppendorf tubes on ice. The recovery of BALF was > 80%. The BALF was centrifuged to identify cytokines in the supernatant. The right upper lobes were fixed in formalin for histology and the left lobes were cut into pieces and digested with 0.1% type IV collagenase (Sigma-Aldrich) for 45 min to produce a single cell suspension. Lymphocytes in the single cell suspension were separated using mouse tissue lymphocyte separation liquid according to the operating instructions for cytometry analysis.
Bronchoalveolar lavage fluid cell count and cell classification
The BALF was centrifuged for 10 min at 4 °C and 1500 r/min, the supernatant was discarded, and the sediment resuspended and mixed in 100 μL PBS. Of this, 30 μL was used for cell counts in the cell counting chamber, 30 μL was smeared for Wright’s staining, and 200 inflammatory cells were counted under an oil microscope. Cells were classified on the basis of morphological and staining characteristics.
Pulmonary histopathological examination and inflammation scores
The lung tissue was fixed in 10% formaldehyde solution for 12 h. After gradient alcohol dehydration, dewaxing with xylene, and paraffin embedding, the slice was sectioned with a slice thickness of 3 µm. Both HE staining and periodic-acid Schiff (PAS) staining were performed to observe the alveolar structure, infiltration of inflammatory cells around the airway, coloration of the airway epithelium, distribution of goblet cells, and secretion of mucus. The inflammation score was evaluated based on the inflammatory cell infiltration around the airway [no infiltration (0 points); a little (1 point); more (2 points); a large number, less than a group (3 points); a large number of groups (4 points)] and presence of airway goblet cells [no (0 score); < 25% (1 score); 25% to 50% (2 scores); 50% to 75% (3 scores); > 75% (4 scores)].
Expression of TGF-β1, TβR1, Smad1, and Smad7 protein in the lung tissue
Immunohistochemistry was used to detect TGF-β1, TβR1, Smad1, and Smad7 protein as follows: (1) preparation of lung tissue sections: the lung tissue was fixed for 12–24 h in 10% formaldehyde solution, treated with gradient alcohol dehydration, dewaxed with xylene, and embedded in paraffin. A slice of transverse section was obtained with a thickness of 3 µm. (2) immunohistochemical detection: the lung tissue slice was roasted in a thermostat at 60 °C for 3 h, then dewaxed with xylene, and washed with alcohol and water. After dewaxing, the lung tissue was repaired with citric acid solution at a high temperature and pressure for 2 min, incubated for 15 min in 3% H2O2 for blocking the endogenous peroxidase, closed for 10 min with goat serum to reduce non-specific staining, and incubated overnight in an appropriate concentration of the first antibody at 4 °C. The next morning, the slice was reheated in a 37 °C constant temperature box for 30 min and soaked completely with PBS. Then, the secondary antibody was added after removing the PBS solution.next, it was incubated with horseradish peroxidase at room temperature for 15 min and rinsed with PBS. After being incubated at room temperature for 10 min, freshly prepared DAB (20:1:1) was applied for coloring. Every sample was washed with PBS to terminate coloring, and finally the samples were colored with hematoxylin for 40 s. Next, water was dehydrated with 80, 90 and 100% alcohol. After the neutral gum was sealed, it was clustered and photographed with a microscope. Each slice was photographed under the microscope for 400 times of non-repetition. The images were analyzed using the Image-Pro Plus 6 software. The average integral light density (IOD mean) was measured.
Estimation of the percentage of lung gamma delta T cells and detection of IL-13 and TGF-β 1 positive gamma delta T cells
(1) Single cell suspension of lung tissue preparation: the right lung was cut and crushed, then digested with type IV collagenase and filtered using a 200-mesh (aperture diameter = 0.08 mm) sieve. Lymphocytes were isolated using mouse viscera lymphocyte separation solution. (2) Lymphocyte activation: the single cell suspension of lung tissue was suspended in PBS solution. The concentration of the cells was regulated at 1 × 109/L. The total amount was 300 μL. Addition of 1.2 μL PMA/Ionomycin/BFA/Monensin mixture was performed and the solution was mixed. This solution was incubated at 37 °C and 5% CO2 in an incubator for 4 h. (3) Estimation of the percentage of lung gamma delta T cells and IL-13, and detection of TGF- β1 positive gamma delta T cells: The specimens were divided into three tubes as A, B and C, each containing 100 μL. PE-CY5-anti-mouse CD3 10 μL was added to all the tubes. FITC-IgG1, 20 μL, was added to tube A and FITC-anti-mouse –γδTCR, 20 μL, was added to tubes B and C. These were then incubated for 15 min at room temperature. Next, 100 μL FIX & PERM Reagent A was added to the three tubes and incubated for 15 min in the dark at room temperature. Then 4 mL PBS with 5% FBS was added and centrifuged at 300×g and the supernatant was discarded. FIX & PERM Reagent B, 100 μL was added to all tubes. PE-IgG1, 20 μL, was added to tube A. PE-anti-mouse-IL-13, 5 μL, and PE-anti- mouse-LAP, 5 μL, were added to both tubes B and C. After vortexing for 2 s, they were incubated for 20 min at room temperature. PBS 4 mL with 5% FBS was added to each tube, and centrifuged at 300×g. The supernatant was discarded. Lastly, 200 μL 0.1% polyoxymethylene was added, placed at 4 °C in the dark, and analyzed using flow cytometry within 24 h.
Statistical analysis
The data were expressed as mean ± standard deviation (\( \bar{x} \) ± s) and were analyzed using the SPSS 16.0 software. One-way analysis of variance was used to analyze the comparison within groups and the least significant difference t-test or Student–Newman–Keuls test was used for comparison of quantitative data between groups. The nonparametric rank sum test was used to analyze the semi-quantitative data of the airway inflammation scores. P < 0.01 or P < 0.05 was considered statistically significant.
