2.1 Experimental materials
Reagent OVA; Phorbol myristate ester (PMA) (Sigma); Ionomycin (sigma); aluminum hydroxide gel (Pearce, USA); M. vaccae f.u.22.5 μg injection (Anhui Zhifei Longkema Biopharmaceutical Co., Ltd.); DATP (Selleck), immunohistochemistry, immunofluorescence, and Western blotting antibody Jagged2 (CST); Flow cytometry antibody percp-cy5-5cd3, APC γδT、PE-IL-17(eBioscience, USA); Flow cytometry antibody monemycin, fixed / membrane breaking solution (eBioscience, USA). Microplate reader (Bio Rad, United States), ultrasonic atomizer wh-2000 (Guangdong Yuehua Medical Equipment, China), pathological image analysis system (Leica, Germany), 5810R high-speed cryogenic centrifuge (Eppendorf, Germany), FACS canto II flowmeter (BD), and a self-made atomization inhaler were used.
2.2 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 approval was gained from the Animal Ethics Committee of Guangxi Medical University (Protocol number: 201711033). We using Chloral hydrate anesthesia and all efforts were made to minimize suffering
2.3 Animals
Healthy male C57 mice (about 6 weeks old weighing approximately 20g each) were supplied by the Animal Experimental Center of Guangxi Medical University and reared under specific pathogen-free conditions. Animals were allowed free access to food and water and were kept at appropriate room temperatures and humidity. A total of 5 mice groups (n=6 per group) were formed: treatment group (OVA + M. vaccae), prevention group (M. vaccae + OVA), blocking group (DAPT + OVA), asthma group (OVA), and the control group. 25mg OVA was emulsified in aluminum hydroxide gel on days 0, 7, and 14. The OVA emulsification was then diluted on days 21-28 and soaked in phosphate buffered saline (PBS) for 30 minutes prior to exposing the concoction to the mice. Mice in the blocking group were treated for 30 min with aerosolized DAPT (0.3 mg/kg) prior to OVA exposure. In the prevention and treatment groups, mice were treated with vaccinia aerosolized with 10mL of normal saline at days 21-28 and 28-35, respectively. Normal saline in place of all the above treatments was used for the control group. After the experiments, mice were sacrificed for specimen collection using intraperitoneal injection of 10% chloral hydrate (0.1 ml). All experiments were repeated twice.
2.4 Airway hyperreactivity in mice
Mice were stimulated by methacholine and placed in a testing cubicle equipped with a ventilation switch that was able to detect the special airway resistance (sRaw) value. Different concentrations of methacholine were used (6.25, 12.5, 25 and 50 mg/ml), with sRAW value compared against the value gained upon PBS stimulation.
2.5 Histopathological analysis
Lung samples were treated for 24 h with 4% paraformaldehyde, washed, dehydrated with ethanol gradient, and treated with paraffin. 4mm-thick slices were treated with hematoxylin & eosin as well as periodic acid-Schiff. An optical microscope (Olympus, Japan) was used to visualize the sections.
2.6 Immunohistochemical and immunofluorescence studies
4mm sections were dewaxed in xylene and rehydrated in graded alcohol. 3% hydrogen peroxide was used to block endogenous reactions. Tissue sections were then boiled for 10 min in 10 mm citrate buffer (pH 6.0) for antigen recovery, before being cultured in 5% goat serum albumin followed by anti-human jagged2 antibody (1:2000; D4y1r, CST). Non-immune serum instead of a primary antibody was used in the negative control group. Samples underwent another 60 min incubation with secondary antibody bound to horseradish peroxide at room temperature. The tissues were then stained with 3,3-diaminobenzidine and hematoxylin blue. The control tissue sections and specimens were treated in unison. Protein detection was done via immunofluorescence and observed under an optical microscope (Olympus).
For Jagged2 staining, 2mm sections of air-dried, frozen lung tissue were treated for 20 min with cold methanol before being air-dried again. Lung tissues were then cultured for 10 min with PBS supplemented with 20% fetal bovine serum (FCS) before being permeabilized with 30 ml of 1.5% H2O2. PBS was then used to rinse the sections twice before they were incubated for another 10 min with avidin D solution. Biotin solution was used to block endogenous peroxidase, rinsed, and cultured overnight at 4℃ with rabbit anti-mouse Jagged2 antibody (1:1500; D4y1r, CST). The following morning, sections were rinsed twice with PBS and exposed for 2 h to goat anti-mouse IgG secondary antibody in the dark. All sections were then observed under a fluorescence microscope (Olympus).
2.7 Western blotting
Lung tissue was homogenized on ice with 10 ml RIPA buffer containing PMSF. Samples were mixed with 5s protein sample buffer at a 4:1 ratio, boiled and denatured, and separated by a 10% SDS-PAGE gel. The protein strips were charged onto the membrane and sealed with 10% BSA at 4°C for 1 hour. Proteins were detected with anti-GAPDH (1:5000) and anti-Jagged2 (1:1500) antibodies overnight at 4°C, washed once with TBST, then re-incubated with secondary antibody (1:800) for another hour. The positive bands were analyzed by Licor Odyssey software.
2.8 Flow cytometry
Mouse lung tissue was digested with collagase IV-containing RPMI 1640 media at 37℃ (2 mL, 2.5g/L) (Gibco, USA). The partially digested lung and spleen tissue particles were ground with a 200mm mesh filter and centrifuged at 250g at 4℃ for 5min. The supernatant was discarded and the granules were cultured in red cell lysis buffer for 4 min in the dark before being centrifuged for another 5 min at 250 g and 4℃. The supernatant was discarded, and the granules were rinsed with PBS. Cells were isolated in an incubator of 5% CO2 and 37℃ for 72 hours. Retained granules containing lung mononuclear cells were re-suspended in RPMI 1640 media supplemented with 0.2% monensin, 1 mg/l ionomycin, 25 mg/l PMA, and 10% fetal bovine serum at a concentration of 106 cells/ml. The cell suspension was cultured for another 4 h at 5%CO2 and 37℃ before being centrifuged for another 5 min at 250 g and 4℃. The pellets were incubated in the dark for 30 min at 4°C with the APC anti-anti-γδT17 antibody and the percp-CY5-5 anti-CD3 antibody. PBS was then used to rinse the cells, before they were resuspended in Cytofix/Cytoperm solution (Becton Dickinson, USA). The cell suspension was then left in the dark for 20 min at 4 °C. Samples were then rinsed, incubated for 30 min with PE anti-IL-17 antibody, rinsed again with PBS twice before finally being suspended in 200mL PBS. the FlowJo 7.6 software (Becton-Dickinson) was used to perform floc cytometry analysis.
2.9 Statistical methods
SPSS22.0 software (IBM, USA) was used for statistical analysis, and Prism 5.0 software (GraphPad, USA) was used to generate graphs. Data were reported as mean ±SE. Analysis of variance was utilized to evaluate the differences between groups, followed by the post-hoc Fischer minimum significant difference (LSD) test for pairwise comparisons between groups. Sample correlations were evaluated using Pearson correlation. Statistical significance was determined when P < 0.05.