Tespa1 negatively modulates asthmatic attack is associated with IL-4/STAT-6 pathway of mast cell


 Background Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a critical signaling molecule in thymocyte development. This study aimed to investigate the regulatory effect of Tespa1 on mast cells in the pathogenesis of asthma and its relationship with the interleukin (IL)-4 /signal transducers and activators of transcription 6 (STAT6) signaling pathway. Methods Tespa1 mRNA expression analysis and IgE levels were carried out using the induced sputum of 33 adults with stable asthma and 36 healthy controls. Tespa1- knockout mice ( Tespa1 -/- , KO) and C57BL/6 background (wild-type, WT) mice were sensitized and treated with ovalbumin (OVA) to establish an asthma model. Pathological changes, number and activity of mast cells, and changes in activity of the IL-4 /STAT-6 pathway in lung tissue were detected. The changes of enzyme activity and STAT6 activity after mast cell gene knockout were analyzed in vitro . The changes of enzyme activity and STAT6 activity after mast cell gene knockout were analyzed in vitro . The association between the Tespa1 and P-STAT6 was analyzed by immunoprecipitation method. Results Compared with the healthy controls, Tespa1 expression was decreased, and IgE levels were elevated in the sputum of asthmatic patients. Animal experiments showed that Tespa1 -/- mice exhibited more severe inflammation, higher quantity of goblet cells and mast cells in the bronchium, and greater activity of mast cell tryptase, which is induced by ovalbumin, than WT mice. And IL-4, IL-13, phospho-Janus kinase 1, and P-STAT6 expressions presented a higher increase in the Tespa1 -/- mouse model than in the WT mouse model. Further in vitro studies confirmed that IL-4 could more significantly promote tryptase and P-STAT6 activities in Tespa1 -/- mast cells than their WT counterparts. Correlation analysis results showed a negative correlation between Tespa1 and P-STAT6. Immunoprecipitation results showed a direct link between Tespa1 and P-STAT6. Conclusions Altogether, our results indicate that Tespa1 can negatively regulate mast cell activity, and this event is related to the mast cell IL-4/STAT6 signaling pathway and could be therapeutically exploited to treat asthma attacks.

cytokine shown to be produced by mast cells. IL-4 production in mast cells has been studied in relation to IgE-mediated activation [8,9]. IL-4 is a pluripotent cytokine that plays an important role in inducing allergic Th2-type immune responses [10]. IL-4 activates signal transducers and activators of transcription 6 (STAT6) and induces the expression of IL-4-inducible genes, including class II major histocompatibility molecules, low-a nity IgE receptor, and IL-13 [9,11]. Therefore, IL-4/STAT6 signaling plays a central role in the participation of mast cells in asthma progression.
Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a critical signalling molecule in thymocyte development [12]. Tespa1 is highly expressed in mast cells and is involved in the negative regulation of mast cell activation and mediation of allergic reactions by negatively regulating FcεRImediated signaling [13]. However, the role of Tespa1 in the IL-4/STAT6 signaling pathway, an important pathway for mast cell function and asthma pathogenesis, remains unclear.
In this study, we observed that Tespa1 is associated negatively with the IL-4/Stat-6 signaling pathway in the activation of mast cells, thus providing a new intervention target for mast cells in the pathogenesis of asthma.

Materials And Methods
Detection of Tespa1 mRNA expression in the sputum of asthma patients and healthy controls.
Tespa1 mRNA expression analysis was carried out using the induced sputum of 33 adults with stable asthma and 36 healthy controls. 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 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 ampli ed 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 [14]. 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 [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 [15,16]. Brie y, allergic asthmatic reactions and airway remodelling were induced in the abovementioned mice by using chick ovalbumin (OVA, Sigma, U.S.A). Speci cally, the mice were initially sensitized through intraperitoneal (i.p.) injections of PBS with 25 μg OVA in 1 mg aluminium hydroxide gel (Thermo Scienti c 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 drug was administered 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 [17].
Measurement of IL-4 and IL-13 protein expressions by ELISA Bronchoalveolar lavage uid (BALF) 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 BALF 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.

Lung histology
For the histological evaluation of mouse lung tissue specimens, we xed the left lung of each mouse in 10% buffered formalin. The fragments were then dehydrated, cleared, and embedded in para n. 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 in ammation was evaluated according to a subjective scale ranging from 0 to 4 [18,19]. The degree of cell in ltration 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 [20].
The degree of goblet cell hyperplasia in the airway epithelium was quanti ed in accordance with the following ve-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 ve elds.
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 identi ed by metachromatic staining of their granules. The stained slides were all quanti ed under identical light microscope conditions, in terms of magni cation, gain, camera position, and background illumination [21,22].
For immunohistochemical staining, the slides were incubated with 3% H 2 O 2 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 HRPconjugated secondary antibody (Maixin, Fuzhou, China) for 30 min at room temperature. Hematoxylin was applied as a counterstain. Eight elds were randomly selected for the quanti cation of positive cells in every sample, as previously described [17]. (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 identi ed bands, and the expression of each protein was calculated using the following formula: Relative protein expression = IA protein /IA β-actin [23].
Primary pulmonary mast cell culture and treatment Primary pulmonary mast cells were derived from the lungs of 4-week-old mice as previously described [24,25]. In brief, lung samples were cut into pieces, dissociated by collagenase (50 U/mL in Hanks' balanced salt solution), and ltered through a 40μm lter. The cells were cultured in Dulbecco's modi ed medium (Gibco Invitrogen, U.S.A) containing 10% fetal bovine serum (Gibco). Mast cells were con rmed via ow 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 × 10 6 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 [26,27].

Co-immunoprecipitation
Immunoblotting was performed as previously described [28,29]. The cells were harvested in a cell lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 0.5% Triton X-100, 10 mM dithiothreitol, and protease/phosphatase inhibitor cocktails). Cell extracts were then incubated with 2 mg P-STAT6 antibody and 20 mg 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.

Statistical analysis
The data are reported as the mean ± S.D. Statistical signi cance was determined by ANOVA followed by Tukey's correction for multiple comparisons or Student's two-tailed t test for independent means. Nonparametric 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 signi cant.

Results
The expression level of Tespa1 mRNA in the sputum of asthmatic patients was lower and IgE level was higher than that of healthy controls 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 signi cantly increased (P < 0.05) (Fig. 1).

OVA-induced asthma is more serious in Tespa1 -/mice than in WT mice
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 in ammation 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, 2B, 2C).

KO of Tespa1 enhanced lung mast cell activity
The results of toluidine blue staining of lung tissues showed that OVA induced an increase in the distribution of mast cells in the lungs of mice, and the Tespa1 -/model mice showed more numerous compared with that in WT mice. Immunohistochemical analysis results showed that OVA could induce the increase in mast cell tryptase, a marker of mast cell activity, and this increase was more notable in Tespa1 -/mice than in WT mice (Figs. 3A, 3D, and 3E).
KO of Tespa1 can reduce the activity of STAT6 pathway in asthma mice Immuno uorescence results showed that OVA could induce the expression of P-STAT6. In Tespa1 KO mice, the increased expression of P-STAT6 was more remarkable compared with that in WT mice. ELISA was used to detect the changes in IL-4 and IL-13 levels to further verify the changes in the IL4/STAT6 signaling pathway. The results showed that the Tespa1 -/mouse model exhibited increased expressions of IL-4 and IL-13 than the WT mouse model. 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 signi cantly increased in the Tespa1 -/mouse model compared with the WT model (Fig. 4).
Mast cell activity and IL4/STAT6 pathway are closely related to Tespa1 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 activity signi cantly increased in Tespa1 -/mice than in the WT after IL-4 stimulation (Fig. 5). Moreover, co-expression results of P-STAT6 and Tespa1 showed that IL-4 stimulated the increased and decreased expressions of P-STAT6 and Tespa1, respectively. In addition, a negative correlation was observed between Tespa1 and P-STAT6 in mast cells, as proven by the correlation analysis (P < 0.01). Furthermore, immunoprecipitation results showed that P-STAT 6 and Tespa1 are closely linked (Figs. 6).

Discussion
Tespa1 plays an important role in T cell development and negatively regulates Fc εR1-mediated mast cell activation and allergic reactions [13,30]. The regulation of Tespa1 on mast cells is related to the negative regulation of the IL-4/STAT6 signaling pathway. This study provides a new perspective into the mechanism of asthma progression.
The analysis of Tespa1 mRNA expression in clinical samples including normal individual and asthmatic patients, showed that Tespa1 gene expression was lower, and IgE levels are higher in asthmatic patients, suggesting a relationship between Tespa1 and asthma. Tespa1 −/− mice were used for further in vivo experiments to extensively study the relationship between Tespa1 and the incidence of asthma. Results showed that Tespa1 −/− mice experienced more evident OVA -induced asthma attacks, which were manifested as increased airway resistance, increased in ammation and increased goblet cells. Toluidine blue staining showed that KO Tespa1 gene could increase the number of mast cells. Immunohistochemistry indicated that the activity of tryptase, a marker of mast cell activity [31,32], is more reactive in Tespa1 −/− asthmatic lungs than in WT mice, suggesting that Tespa1 could negatively regulate the aggregation and activation of mast cells. Hence, Tespa1 is associated with the proliferation and activation of mast cells in asthmatic mice. To further determine the role of Tespa1 in mast cells, we performed in vitro studies and observed that tryptase activity induced by IL-4 in Tespa1 −/− mast cells was evidently enhanced compared with in WT cells, indicating that Tespa1 caused a certain inhibitory effect on mast cell activity.
The increased number and activity of mast cells mainly cause asthma, which involves many signaling pathways, in which the role of IL-4 /STAT6 pathway in mast high sensitivity has been studied [33,34].
The abnormality of STAT6 is closely related to allergic diseases. Moreover STAT6 −/− and IL-4 −/− mice yield similar phenotypes, and cannot induce Th2 production, nor produce normal amounts of IgE, or even CD23, and IL-4 receptor subunit alpha [35][36][37]. Moreover, the hyperactivity of the IL-4 /STAT6 pathway is associated with asthma and chronic obstructive pulmonary disease, and the transcriptional inhibitors of this pathway are potential targets for the prevention and treatment of diseases caused by the hyperactivity of the IL-4 /STAT6 pathway [38,39].
To study the effect of Tespa1 on STAT6 pathway, we detected the changes in IL-4 and IL-13 in the BALF of asthmatic mouse model, and the results showed that IL-4 and IL-13 in Tespa1 −/− mice signi cantly increased compared with those in the WT asthma group. And level of P-STAT6 expression also increased, further indicating the negative regulatory effect of Tespa1 on the STAT6 signaling pathway. The STAT6 signaling pathway can be regulated by IL-4 /JAK1 and IL13/JAK2 [33,40]. Western blot analysis results of lung tissues showed that in Tespa1 −/− asthma mice, the phosphorylation level of JAK1 increased, but the increase in P-JAK2 level was negligible, indicating that Tespa1 mainly regulates the IL-4 /JAK1 signaling pathway. Experiments showed that tryptase activity increased in Tespa1 −/− mast cell compared to with that WT in vitro, indicating that Tespa1 could negatively regulate the activation of mast cells. In addition, after IL-4 stimulation, the expression of P-STAT6 increased, whereas that of Tespa1 decreased. Thus, a negative correlation exists between P-STAT6 and Tespa1. When further exploring the relationship between Tespa1 and STAT6, immunoprecipitation results showed that Tespa1 and STAT6 directly interacted with each other.

Conclusion
In this study, experiments have con rmed that Tespa1 negatively regulates mast cells during asthma, and this event is related to the IL-4 /STAT6 signaling pathway. Thus, Tespa1 may be a potential target for asthma prevention and treatment. However, two de ciencies were identi ed. First, the exact mechanism of Tespa1 in regulating STAT6 still needs further study. Second, Tespa1 expression was also weakened after STAT6 signal activation. Hence, whether a cross-talk network exists between Tespa1 and IL-4 /STAT6 signaling pathway is unclear.

Declarations Ethical Approval and Consent to participate
The use of the specimens was in accordance with the Declaration of Helsinki. This study was approved by the Animal Research Ethics Board of the Lishui University (Lishui, Zhejiang Province, China. Permit Number: 0901-2018).   Effect of Tespa1 on acetylcholine-induced airway hyperresponsiveness (AHR) in asthmatic mice Airway resistance measurements were performed using a FinePointe RC system. The mice were challenged with aerosolized PBS (baseline) before treatment with acetylcholine at the following ascending doses: 0, 1, 2, 4, 8, and 16 mg/mL. KO: Tespa1-/-mice; WT: wild-type mice. The data are expressed as mean ± SD (n= 9 per group). **, P<0.01 compared with the WT control group; #, P<0.01 compared with the WT model group; ##, P<0.01 compared with the WT model group.