Jagged2-γδT17 is Regulated by Mycobacterium Vaccae in Asthmatic Mice

Background: Mycobacterium vaccae nebulization imparted protective effect against asthma in a mouse model. The Jagged2-γδT17 signal transduction pathway plays an important role in bronchial asthma. However, the effect of M. vaccae nebulization on the Jagged2-γδT17 signal transduction pathway in mouse models of asthma remains unclear. Methods: In total, 30 female C57 mice were randomized to normal control (group a), asthma control (group b), M. vaccae nebulization prevention,and M. vaccae nebulization treatment (group d) groups. Asthma mice models were created using ovalbumin (OVA). The Notch signaling pathway was blocked by DAPT inhibitors. Airway hyperreactivity (AHR) was measured by noninvasive lung function tests. Histopathological analyses using blue-periodic acid Schiff along with hematoxylin and eosin were performed. Immunohistochemistry, immunouorescence, and a Western blotting assay allowed for the detection of lung protein expressions, while spleen expressions of IL-17+γδT+ cytokines were assessed with FLOW cytometry. One-way analysis of variance for within-group comparisons, the least signicant difference t-test or Student-Newman-Keuls test for intergroup comparisons, and the nonparametric rank sum test for analysis of airway inammation scores were used in the study. Results: Asthmatic mice models demonstrated downregulated Notch signaling pathway activation and decreased γδT cells and IL-17 cytokine secretion. There was also increased Jagged2 protein expression which correlated positively with γδT+IL-17+ secretion. In asthmatic mice, the expressions of Jagged2 and γδT17, along with airway inammation and airway reactivity, were all decreased after M. vaccae exposure (p<0.05). Conclusion: The Notch signaling pathway contributed towards asthma initiation and progression by facilitating γδT cells and IL-17 cytokines production. Inhaled M. vaccae led to a signicant decrease in Jagged2 and γδT17 expressions in asthmatic mice, indicating its utility in asthma prevention.


Introduction
Bronchial asthma is a serious global health concern a icting 3 billion people worldwide. The disease imposes a severe burden on health care systems. Currently, there are no effective measures to prevent and treatment the condition of asthma.Studies on the e cacy of immunotherapy during of asthma is at their nascent stage.
Asthma is a chronic in ammatory airway disease that manifests as several different clinical phenotypes, all of which involve a myriad of cells, including mast cells, T lymphocytes, eosinophils, and neutrophils [1] . Current research has been focusing on the immunopathology behind asthma. Of interest is the γδT cell, which appears to be a crucial immune mediator in asthma [2] . The γδT cell response triggers airway hyperreactivity (AHR) as well as airway eosinophilia and Th2 cell recruitment [3] . γδT secretes IL-17, which is a key participant in airway in ammation, AHR, and airway remodeling in asthma [4][5][6][7][8] . Existing research con rms that airway remodeling is modulated by IL-17 through activation of epithelial cells, airway smooth muscle cells, and broblasts [9] . IL-17 promotes airway DC migration and activation, leading to AHR, increased eosinophils, mucus hypersecretion, and decreased IgE levels [10] . Moreover, IL-17-induced autophagy induces bronchial broblasts-mediated brosis [11] . γδT17 cells are γδT cells that produce IL-17 [12] . Compared with other T lymphocytes, γδT secretes more IL-17 cytokines [13] . Investigations have demonstrated that pro-in ammatory cytokines and the proportion of percentage of IL-17 + γδT cells are dampened upon RTA-408 exposure, leading to attenuated in ammation in the airways of asthma mice models stimulated by ozone [14] . Prior research has established the involvement of γδT17 in asthma [15] . Nevertheless, literature linking asthma and γδT17 cells are scarce. Our previous study con rmed the involvement of γδT17 in mice models of OVA-induced asthma [15] . M. vaccae atomization prevents allergic bronchial asthma in mice models through reduction of AHR and airway in ammation [16] . The γδT cells are critical regulators of allergic in ammation, AHR, and airway function in asthmatic mice models [17] . The development of γδT cells is closely related to notch-hes1 and IL-17 production [18] . However, little is known regarding how γδT cells work together with the Notch signaling pathway in asthma pathogenesis. Notch signaling strongly regulates lymphocyte activation and differentiation. The interaction of Notch receptor and ligand may promote cytokine production in addition to Th cell polarization and proliferation [19][20][21] . Notch signaling pathway critically contributes towards asthma initiation. The allergic reaction appears to be mediated by Notch signaling that inhibits Th2 polarization and augments differentiation of Th1 cells, resulting in a proin ammatory Th1/Th2 balance in mice [22] .
Asthma pathogenesis is thought to rely heavily on Notch signaling-induced Th2 cell differentiation [23,24] .
Other signi cant factors in asthma include the upregulation of the Jagged2 factor and subsequent Th2 cell differentiation, a phenomenon that is brought about by CD4T-augmented Th1 cell differentiation [25,26] .
The current investigation utilizes a Notch signal pathway inhibitor to better study the role of Jagged2 in asthma pathogenesis. Experiments were performed on ova-induced asthma mice models, while the DAPT drug was used as a Notch signaling pathway inhibitor to detect changes in markers of in ammation in the γδT17 cells. To study whether Jagged2 is involved in the anti-in ammatory effects of aerosolized M. vaccae in γδT17 cells, different in ammatory responses were compared among the asthma, prevention, and treatment groups.

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

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 speci c 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 emulsi ed in aluminum hydroxide gel on days 0, 7, and 14. The OVA emulsi cation 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 sacri ced for specimen collection using intraperitoneal injection of 10% chloral hydrate (0.1 ml). All experiments were repeated twice.

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.

Histopathological analysis
Lung samples were treated for 24 h with 4% paraformaldehyde, washed, dehydrated with ethanol gradient, and treated with para n. 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 immuno uorescence 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 3,3diaminobenzidine and hematoxylin blue. The control tissue sections and specimens were treated in unison. Protein detection was done via immuno uorescence 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 uorescence microscope (Olympus).

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.

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 lter 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 Cyto x/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 nally being suspended in 200mL PBS. the FlowJo 7.6 software (Becton-Dickinson) was used to perform oc cytometry analysis.

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 signi cant difference (LSD) test for pairwise comparisons between groups. Sample correlations were evaluated using Pearson correlation. Statistical signi cance was determined when P < 0.05. Figure 1 shows that stimulation of 12.5mg/ml, 25mg /ml, and 50mg/ml of methacholine in the asthma group resulted in higher airway resistance in contrast to the control group (P < 0.05). There were also signi cant variability of the sRAW differences between the prevention and treatment groups, as well as in the control and blocking groups (P < 0.01).

Lung histopathological analyses
Lung histopathological analyses of the control group revealed that no abnormalities in bronchial and alveolar morphologies, no tracheal wall thickening, and no epithelial cell proliferation. There were also less in ammatory cells surrounding lung blood vessels and bronchi.
PAS staining of the airway epithelium revealed few goblet cells without mucus exudation. Those in the asthma group demonstrated bronchial lumen stenosis and wall thickening, obvious in ammatory cell in ltration in bronchus and perivascular lung tissue, as well as higher rates of goblet cell proliferation and mucus exudation in contrast to the control groups. Mice in the treatment, prevention, and blocking groups demonstrated much lower rates of in ammatory in ltrates in the bronchial and perivascular cells, as well as fewer amounts of goblet cells and mucus exudation in comparison to the asthma group (Table 1, Figure 2).

Immunohistochemistry, immuno uorescence and western blotting studies
Mice with asthma had more signi cantly elevated Jagged2 expressions in contrast to the control group (P < 0.05). Conversely, the treatment, prevention, and blocking groups revealed statistically signi cant lower Jagged2 expressions in contrast to the asthma group (Fig. 2, P < 0.05).

Correlation analysis of Jagged2 and cdT17 cells in mice spleen tissue
Mice of the asthma model group had increased proportions of γδT17 cells (26.7 ± 3.7%) in contrast to the control group (8.1 ± 0.5%). The number of γδT17 cells was 9.0 ± 1.0% in the DAPT + OVA, 10.8 ± 1.4% in the M. vaccae + OVA, and 11.0 ± 0.9% in the OVA + M. vaccae groups. Compared with the asthma group, the IL-17 + γδT ratio of T + cells was low (Fig. 3, P < 0.05). The IL-17 + γδT percentage of T + cells correlated positively with Jagged2 protein expression in lung tissue (r = 0.63, P < 0.001).

Discussion
Asthma involves several yet-to-be determined signal pathways. Previous studies have supported the Notch signaling pathway as a cornerstone of cell development, differentiation, proliferation, and survival [27,28] . Speci cally,δligands are involved in Th1 cell development, while Jagged2 ligands are closely in Th2 cell development. Many studies in asthma show that the Notch signaling pathway stimulates Th2 cell differentiation [20,21,29,30] . Notch guides Th1 and Th2 cell differentiation while enabling T cell signal transduction [22,31] . Tu et al. highlights that asthma is modulated primarily through Th2 cell immunity [28] . The central function of the Notch signaling pathway makes it an ideal therapeutic target in modulating Th2 cell immunity in asthmatic patients [32] . Data shows that jagged2 is involved in immune regulation in allergic airway in ammation [12,32] . Studies have also shown that allergic asthma can be alleviated by inhibiting IL-4R α-Stat6 and the Jagged1/Jagged2-Notch1/Notch2 signaling pathways in mice [11] . On the other hand, IL-17 exerts signi cant in uence in activating and recruiting neutrophils, enhancing in ammatory cell in ltration, participating in airway remodeling, and promoting airway hypertrophy [33][34][35][36] . γδT cells support the Th1/Th2 imbalance, implicating γδT cells in asthma development [3,37,38] . γδT17 cells contribute towards the in ammatory response by producing proin ammatory cytokines and augmenting in ammatory messenger secretion.
Previous investigations have underscored the importance of γδT17 in asthma pathogenesis [39] . Dysregulated Th1/Th2 cell proportions among the γδT cell population is implicated in asthma development in animal models. Aerosol inhalation of M. Vaccae corrects the Th1/Th2 imbalance in γδT cells in asthma, ahus alleviating lung in ammation [5] . It was also demonstrated that M. vaccae inhalation is hypothesized to be able to prevent mice allergic bronchial asthma by suppressing in ammation and AHR [16] . γδT17 cells participate in the in ammatory response by inducing in ammatory cytokine and messenger production and release. The current investigation establishes an asthma mouse model and evaluated the degree of airway reactivity, lung pathological manifestations, lung protein expression, and CD3 + γδT proportion of T + IL-17 + cells. Compared to the control group, airway reactivity, airway in ammation, IL-17, as well as γδT expression of T+ and jagged2 protein in the asthma group lung specimens increased signi cantly. We noted a positive correlation between Jagged2 protein and CD3 + γδT+IL-17+ cell populations. The Notch signaling pathway may regulate airway reactivity and airway in ammation in asthmatic mice through its receptor, Jagged2. Jagged2 and γδT17 cell secretion appear to be closely related in asthma pathogenesis. M. Vaccae appears to regulate the Notch / jagged2 signal pathway in alleviating asthma, representing a potential therapeutic candidate in this common respiratory condition.

Declarations ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Approval was gained from the Animal Ethics Committee of Guangxi Medical University.
CONSENT FOR PUBLICATION manuscript is approved by all authors for publication.

AVAILABILITY OF DATA AND MATERAIL
Data set for the proteomic inventory and quantitative analysis, FLOW cytometry, and in ammatory responseof mice during asthma below "attach les",inclede Figures and table.

COMPETING INTERESTS
The authors declare no con ict of interest.    The percentage of γδT in T17 cells of CD3+T cells was determined. Data is presented in terms of mean ± S.E. relative percentage of the control group (n = 6). T17 cells correlated positively with γδ and Jagged2 cells (r = 0.63, P < 0.05). **P<0.01 vs control group; $P<0.05, &P<0.05, ##P<0.01 vs. asthma group (ANOVA).

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