Detection of Tespa1 mRNA expression in the sputum of asthma patients and healthy controls.
33 patients with physician diagnosed asthma and 36 healthy adults without asthma were enrolled for this study. All subjects were non-smokers, and those on maintenance oral corticosteroid therapy were excluded . All subjects were recruited from the Respiratory Clinic of Lishui People’s Hospital, Lishui, China. The Global Initiative for Asthma guidelines (www.ginasthma.org) were used by a respiratory medicine specialist to diagnose asthma. Total RNA was extracted from the induced sputum samples asthmatic patients and healthy controls by using the TRIzol reagent (Invitrogen, U.S.A) according to the manufacturer’s instructions. All sputum samples were processed with RNAprotect cell reagent and phosphate-buffered saline (PBS) according to the manufacturer’s instructions. An IQ SYBR Green SuperMix polymerase chain reaction (PCR) array kit was purchased from Bio-Rad (USA). Two micrograms of extracted RNA was converted to cDNA by Moloney murine leukemia virus reverse transcriptase (Fermentas, CAN), which was used according to the manufacturer’s instructions. The cDNA was amplified using the following forward and reverse primers: forward: 5′-CAACCATCCAACT GATGTGCC-3′ and reverse: 5′- TCCAACACAA CTTGGTCCAAA′; for β-actin, forward: 5′- TGACGTGGACATCCGCAAAG-3′ and reverse: 5′-CTGGAAGGTG GACAGCGAGG-3′. The human β-actin housekeeping gene was used as an internal control. The primers were designed and synthesized at Shanghai Generay Biotech (Shanghai, China). The reaction was evaluated using a CFX Connect Real-Time PCR system (Bio-Rad, USA). The relative expression levels of the mRNA in each sample were calculated by normalizing the threshold cycle (Ct) value to the Ct value of the β-actin housekeeping gene by using the 2-ΔΔCt method. The mRNA expression levels were expressed in arbitrary units.
IgE enzyme-linked immunosorbent assay（ELISA）
The sputum was collected in a plastic container, and homogenized by adding an equal volume of 1% dithiothreitol for 30 min at room temperature. After incubation, the supernatant was centrifuged at 2000 r/min for 10 minutes, and the sputum IgE levels were tested using the Human IgE ELISA Kit (EK175 - 96, MultiSciences, China) as previously described . Samples measurements were obtained at 450 nm by using a SpectraMax Plus 384 microplate reader (MD, U.S.A) and SoftMax Pro software.
Animal and mouse models of acute asthma
Tespa1-knockout mice (Tespa1-/-, KO) and C57BL/6 background (wild-type, WT) mice were generated via homologous recombination-mediated gene targeting at the Shanghai Research Center for Model Organisms as previously described . The mice were housed in a temperature-controlled room under a 12 h dark/light cycle and were allowed access to food and water ad libitum. This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and its protocol was approved by the Animal Research Ethics Board of the Lishui University (Lishui, Zhejiang Province, China. Permit Number: 0901-2018).
Tespa1-/- mice and WT mice were divided into two groups (15 mice per group), namely, the control and asthma groups. The asthma mouse model was established using a traditional protocol [17, 18]. Briefly, allergic asthmatic reactions and airway remodelling were induced in the abovementioned mice by using chick ovalbumin (OVA, Sigma, U.S.A). Specifically, the mice were initially sensitized through intraperitoneal (i.p.) injections of PBS with 25 μg OVA in 1 mg aluminium hydroxide gel (Thermo Scientific Inc., Germany) and in 0.2 mL PBS, at pH 7.4, on days 0, 7, and 14. The mice were subsequently randomized into groups that were repeatedly administered nebulized with 5% OVA in PBS or PBS alone by using an ultra-sonic nebulizer with an aerosol chamber (Yuyue Medical Equipment & Supply Co., Ltd., Shanghai, China) on days 15 to 42. The mice were given aerosolized OVA for 30 min each day for 28 consecutive days. The mice in the control group were i.p. and atomized with equal volume of PBS.
Measurement of airway resistance
On day 42, nine mice from each group were anesthetized via i.p. injections of 300 mL pentobarbital sodium (60 mg/kg) before undergoing tracheostomy tube insertion. Airway resistance and compliance measurements were performed using a FinePointe RC system (Buxco Research Systems, Wilmington, NC). The mice were subsequently challenged with aerosolized PBS (baseline) acetylcholine treatment doses of 0, 1, 2, 4, 8, and 16 mg/mL. Average compliance values were recorded during a 3 min period following each challenge .
Measurement of IL-4 and IL-13 protein expressions by ELISA
Broncio Alveolar Lavage (BAL) was centrifuged at 1,000 × g for 5 min at 4 °C. After centrifugation, the IL-4 and IL-13 protein expression levels in the BAL supernatant were measured using a sandwich ELISA kit (70-EK204, 70-EK213, MultiSciences, China) according to the manufacturer’s instructions. Samples measurements were obtained at 450 nm by using a SpectraMax Plus 384 microplate reader (MD, U.S.A) and SoftMax Pro software.
For the histological evaluation of mouse lung tissue specimens, we fixed the left lung of each mouse in 10% buffered formalin. The fragments were then dehydrated, cleared, and embedded in paraffin. The whole lung was serially sectioned (3 - 4 μm -thick), stained with H&E for pathological analysis, and stained with periodic acid-Schiff (PAS) for goblet cell detection. The degree of peribronchial and perivascular inflammation was evaluated according to a subjective scale ranging from 0 to 4 [20, 21]. The degree of cell infiltration in the above tissues was scored as follows: 0, no cells; 1, a few cells; 2, a ring of cells with a depth of one cell; 3, a ring of cells with a depth of two to four cells; 4, a ring of cells with a depth of more than four cells. Reticular basement membrane thickness was measured by image analysis of multiple randomly selected tissue sections, with each of section comprising 30 analysis points, by using an Olympus software microscope system. Repeat measurement error was assessed by performing multiple measurements on a single membrane area in four subjects, as previously described .
The degree of goblet cell hyperplasia in the airway epithelium was quantified in accordance with the following five-point system: 0, no goblet cells; 1, <25% of the cells in the epithelium are hyperplasic; 2, 25%–50% of the cells in the epithelium are hyperplastic; 3, 50%–75% of the cells in the epithelium are hyperplastic; and 4, >75% of the cells in the epithelium are hyperplastic. Five randomly distributed left lung airway sections were analyzed in each mouse, and the average score was calculated by summing the scores from the five fields.
Mast cells were stained with toluidine blue. The sections were stained with 1% toluidine blue (Sigma-Aldrich) solution in 1% sodium chloride with diluted 1:10 for 10 min, washed for 1min, differentiated with 0.5% glacial acetic acid for several seconds, and washed for 5 min. Mast cells were identified by metachromatic staining of their granules. The stained slides were all quantified under identical light microscope conditions, in terms of magnification, gain, camera position, and background illumination [23, 24].
Immunofluorescence and horseradish peroxidase (HRP)-diaminobenzidine immunohistology
For immunofluorescent staining, sections of lungs or cells were incubated with the antibody against mast cell tryptase (ab2378, Abcam, UK), Tespa1 (R1309-16, HuaAn Biotechnology, China), p-STAT6 (ab263947, Abcam, UK) and DAPI (4ʹ,6-diamidino-2-phenylindole, Life Technologies,), and images were obtained by using a confocal laser scanning microscope (LSM 880, Zeiss). The protein expression levels were analyzed using Image J.1.44 software.
For immunohistochemical staining, the slides were incubated with 3% H2O2 for 10 min after dewaxing, and then washed with PBS for 5 min at room temperature. Antigen retrieval was performed in citrate buffer (pH 6.0) by microwave heating, and blocking was performed with 10% non-immune goat serum for 30 min after cooling. The slides were incubated with an antibody against mast cell tryptase (ab2378, Abcam, UK) overnight at 4 ℃. After rinsing with PBS, the sections were incubated with the HRP-conjugated secondary antibody (Maixin, Fuzhou, China) for 30 min at room temperature. Hematoxylin was applied as a counterstain. Eight fields were randomly selected for the quantification of positive cells in every sample, as previously described .
Western blot analysis
Proteins of lungs tissues were extracted with RIPA buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid disodium, 10% glycerol, 2% Triton X-100, and a protein inhibitor mixture [Beyotime Biotechnology, Shanghai, China]). For immunoblot analysis, 30 μg solubilized protein was loaded and resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis on 8% –15% gels. 6 samples were randomly selected from each group for Western blot detection. To detect the expression of one protein, 24 samples were divided into two gels, and the experiment was carried out simultaneously under same conditions. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skim milk in PBS/Tween-20 for 1 h, and incubated with primary antibodies targeting phospho-Janus kinase 1 (p-JAK1) (D7N4Z , Cell signaling Technology), p-JAK2 (3771, Cell signaling Technology), p-STAT6 (ab263947, Abcam) and GAPDH (MAB5465, MultiSciences Biotech) overnight at 4 °C. Membranes were then washed and incubated with secondary HRP-labelled anti-rabbit/anti-mouse antibodies (1:5,000). Chemiluminescent images of the blots were captured using a ChemiDoc System. Image J software was used to calculate the integrated absorbance (IA) of the identified bands, and the expression of each protein was calculated using the following formula: Relative protein expression = IAprotein/IA GAPDH .
Primary pulmonary mast cell culture and treatment
Primary pulmonary mast cells were derived from the lungs of 4-week-old mice as previously described [26, 27]. In brief, lung samples were cut into pieces, dissociated by collagenase (50 U/mL in Hanks’ balanced salt solution), and filtered through a 40μm filter. The cells were cultured in Dulbecco’s modified medium (Gibco Invitrogen, U.S.A) containing 10% fetal bovine serum (Gibco). Mast cells were confirmed via flow cytometric analysis of surface markers, CD117 (553869; BD, CA) and FcεRI (11-5898; eBioscience, CA). The cells were seeded in six-well plates at a density of 1 × 106 cells/mL, and 20ng /mL IL-4 (R&D Systems) was simultaneously applied for 1 and 4 h. Then the cells were collected for testing [28, 29].
Immunoblotting was performed as previously described [30, 31]. The cells were harvested in a cell lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.5% Triton X-100, 10 mM dithiothreitol, and protease/phosphatase inhibitor cocktails). Cell extracts were then incubated with 2 μg p-STAT6 antibody and 20 μL LProteinA/G at 4℃ for 3 h. After the immunoprecipitation reaction, the samples were centrifuged at 4℃ 3,000 rpm for 5 min, and then wash thrice. Finally, the proteins were eluted with elution buffer and boiled for 5 min prior to Western blot analysis. The PVDF membranes were blocked with a solution containing primary antibodies against Tespa1. A control lacking the primary antibodies was incubated with anti-mouse IgG.
The data are reported as the mean ± S.D. Statistical significance was determined by ANOVA followed by Tukey’s correction for multiple comparisons or Student’s two-tailed t test for independent means. Non-parametric analyses were performed using Kruskal-Wallis one-way analysis. Pearson correlation (r) was used for correlation analysis. All analyses were performed using SPSS 11.0 for Windows (SPSS) software. P values less than 0.05 were considered statistically significant.