Tespa1 plays a role in the modulation of airway hyperreactivity through the IL-4/STAT6 pathway

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 [15]. 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, USA) 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.

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 min, and the sputum IgE levels were tested using the Human IgE ELISA Kit (EK175-96, MultiSciences, China) as previously described [16]. Samples measurements were obtained at 450 nm by using a SpectraMax Plus 384 microplate reader (MD, USA) and SoftMax Pro software.

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 [13]. 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, USA). 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.

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 [19].

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 [22].

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 1 min, 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].

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 [19].

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 (1:5000). 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 = IA_protein/IA_GAPDH [25].

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 40 μm filter. The cells were cultured in Dulbecco’s modified medium (Gibco Invitrogen, USA) 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 20 ng/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 ℃ 3000 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 ± SD. 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.

To detect the role of Tespa1 in the pathogenesis of asthma patients, we selected sputum from 33 patients with chronic asthma and 36 healthy individuals to test the expression of Tespa1 mRNA and IgE levels. Results showed that compared with the healthy people, the level of Tespa1 mRNA expression of asthma patients was relatively lower, and IgE levels in the sputum of the patients were significantly increased (P < 0.05) (Fig. 1).

To further explore the role of Tespa1 in asthma, we assessed the OVA-induced asthma models of Tespa1^−/− and WT mice. Airway function was assessed by measuring the changes in lung resistance and compliance elicited by acetylcholine inhalation, which induced bronchoconstriction. The airway resistance for each of the four groups was analyzed, and the results are shown in Fig. 2. Airway resistance increased in OVA-primed/challenged mice from the Tespa1^−/− and WT groups compared with the control mice. Airway resistance increased in Tespa1^−/− mouse model compared with the WT mice. Pathology results proved the more evident inflammation in Tespa1^−/− asthma mice than in WT mice. Moreover, PAS dyeing results indicated that the distribution of goblet cells was more intensive in the small bronchial of Tespa1^−/− asthma mice than that in the WT group (Fig. 3a–c).

Further experiments were performed to evaluate the regulatory effect of Tespa1 on mast cells in asthmatic mice. We used toluidine blue staining to evaluate the changes in the number of mast cells in lung tissue, and immunohistochemical staining with tryptase to analyze the changes in mast cell enzyme activity. Five randomly selected digital images were obtained from each lung section using a phase contrast microscope (Olympus BX43, Tokyo, Japan). Two reviewers blinded to the diagnosis counted the numbers of mast cell and tryptase expression score were recorded for each lung section. The results of toluidine blue staining of lung tissues showed that OVA induced an increase in the distribution of mast cells in alveolar parenchyma of mice, and the Tespa1^−/− model mice showed more numerous mast cells compared with that in WT model mice (Fig. 3a, d). The results of immunohistochemical analysis showed that, compared with WT mice, Tespa1^−/− mice had increased expression of tryptase, a marker of mast cell activation (Fig. 3a, e). This might reasonably correlate with an increase of mast cell activity in Tespa1-deficient mice.

To investigate the role of Tespa1 in the JAK1/2-STAT6 pathway, proteins from the lung tissues of OVA-induced asthma models of Tespa1^−/− and WT mice, and control mice were extracted. Six samples were randomly selected from each group, and the expressions of p-JAK1, p-JAK2 and p-STAT6 were detected by Western blot. Due to the large amount of samples, two gels were used for each protein test under the same experimental conditions. Western blot analysis results showed that OVA enhanced p-JAK1, p-JAK2, and p-STAT6 protein expression levels in Tespa1^−/− and WT mice. p-JAK1 and p-STAT6 protein expression levels significantly increased in the Tespa1^−/− mouse model compared with the WT model (Fig. 4a, b). To further verify the effect of Tespa1 on the IL4/STAT6 pathway, levels of IL-4 and IL-13 in BAL of OVA-induced asthma models of Tespa1^−/− and WT mice, and control mice were detected by ELISA. The results showed that the Tespa1^−/− mouse model exhibited increased expressions of IL-4 and IL-13 than the WT mouse model (Fig. 4c). Finally, the changes of p-STAT6 were analyzed by immunofluorescence method to verify the activation of KO on Tespa1 STAT6. And the results showed that OVA could induce the expression of p-STAT6. In Tespa1^−/− mice, the increased expression of p-STAT6 was more remarkable compared with that in WT mice.

To investigate the role of Tespa1 on IL4/STAT6 pathway in mast cells, we stimulated lung mast cells with IL-4 in vitro. Results showed that tryptase expression significantly increased in Tespa1^−/− mice than in the WT after IL-4 stimulation (Fig. 5). In order to further examine the potential mechanisms underlying these processes, primary pulmonary mast cells were derived from the lungs of WT mice, and IL-4 (20 ng/mL) was simultaneously applied for 1 and 4 h. The expression and correlation of Tespa1 and p-STAT6 were detected by immunofluorescence. The co-expression results of p-STAT6 and Tespa1 showed that IL-4 stimulated the increased and decreased expressions of p-STAT6 and Tespa1, respectively (Fig. 6a, b). In addition, a negative correlation was observed between Tespa1 and p-STAT6 in mast cells, as proven by the correlation analysis (r = − 0.7010, Fig. 6c). Furthermore, the relationship between Tespa1and p-STAT6 was detected by co-immunoprecipitation, and the results demonstrated an association between p-STAT6 and Tespa1 (Fig. 6d).