The Role of ICOSL Antibody Intervention in Mouse Model of Neutrophil Asthma


 Background: To investigate the role of ICOSL (Inducible costimulatory molecular ligands) antibody in the mouse model of neutrophilic asthma, and to explore the role of ICOSL in the pathogenesis of Th1/Th2/Th17 imbalance. Methods: 24 Balb/c mice were randomly divided into four groups: C group, N group, my group and G group. The peripheral blood, BALF and lung tissue were collected within 24 hours after the last challenge. Total count and classification of BALF (bronchoalveolar lavage fluid) cells; Detection cytokines of peripheral blood and BALF. Pulmonary tissue section for pathological observation. Results: During seven stimulations,asthma performance in group N was the earliest and obvious and was improved in group I,there was no asthma behavior in group C.The total number of BALF in N group was the highest, in C group was the lowest,(P<0.05).The concentrations of IL-6, IL-13 and IL-17 in peripheral blood and BALF of N group were the highest,(P<0.05).The IFN-γ concentration of C group was higher than that of N and G groups,(P<0.05).HE(hematoxylin-eosin) and PAS(periodic acid-schiff) staining showed that inflammatory infiltration in I group was relative improved than N and G groups.ICOSL immunohistochemical analysis,ICOSL positive cells of alveolar interstitial and airway of N and G groups were more than I group,(P<0.05). Conclusions: After the intervention of ICOSL antibody,the performance of asthma relieved,and airway inflammatory cell infiltration, mucus secretion were reduced.The levels of IL-6, IL-13 and IL-17 in peripheral blood and BALF were partly decreased, and the level of IFN-γwas increased;These results suggest that blocking the ICOS/ICOSL signaling pathway may partially block the development of neutrophilc asthma and provide a new target for the treatment of asthma.


Background
Bronchial asthma (asthma) is one of the most common chronic respiratory diseases in children,consuming huge medical and health resources [1] .Asthma is a chronic in ammatory disease of the airways with various cell and cell components involved in airway in ammation and airway remodeling [2] .The pathogenesis of asthma is complex, and the immunological mechanism plays an important role in the development of asthma.Previously, It has been considered that the classical pathogenesis of asthma is that the imbalance of Th1/Th2 leads to the increase of IgE production, stimulating the proliferation and activation of eosinophils, and then secreting a variety of in ammatory mediators to cause chronic airway in ammation [3][4][5] .However, this imbalance does not explain all types of asthma. Some studies have found that there are no neutrophils in the airways of healthy people, while the number of neutrophils in sputum, BALF and airway tissues of asthmatic patients increased signi cantly,and some patients showed only neutrophils increased, but eosinophils not [6] .
Costimulatory signals and costimulatory molecules were proposed and con rmed by Brestcher and Cohen in 1970 on the basis of the dual-signal theory of T lymphocyte activation [7] .ICOSL molecule is a member of CD28/B7 superfamily [8] ,ICOSL binds to its receptor ICOS to form ICOS/ICOSL signaling pathway.Studies show that ICOS/ICOSL signaling pathway plays an important role in maintaining Tlymphocyte effect and activation of memory cells, T-cell proliferation, lymphokine secretion, immunoglobulin type conversion, and regulating the polarization of Th1, Th2 and Th17 cells [9][10][11][12] .In vitro studies showed that ICOS co-stimulation increased the production of many cytokines such as IL-4, IL-6 and IL-17 in T cells [13][14] .It is speculated that ICOS/ICOSL signaling pathway may play an important role in participating in and aggravating in ammation. In this study, OVA + LPS was used to induce neutrophilc asthma in mice, and ICOSL antibody was used to intervene during induction period to understand the immune mechanism of ICOSL in neutrophilc asthma, so as to nd new ideas for the treatment of severe and refractory asthma with neutrophils in ltration.

2.2.specimens
The mice in each group were anesthetized within 24 hours after the last stimulation (3% chloral hydrate, 0.30 ml/mouse, intraperitoneal injection). The eyeballs were taken for blood 1 ml and placed in EDTA for anticoagulation.The mice were necked off to death. The neck and chest were exposed in supine position and xed on the operating table. Intravenous indwelling needle was used for tracheal intubation. Surgical suture was used to ligate the right lung at the right lung root. The left lung was lavaged with 0.5 ml PBS three times. About 1.5 ml of alveolar lavage uid was recovered (the total recovery rate was not less than 80% as quali ed). The whole blood was centrifuged at 4℃ for 5 min at 2500 r/min. The upper plasma was collected in EP tube. The lavage solution was centrifuged at 4 ℃ for 5 min at 1500 r/min, and then supernatant was taken into EP tube. The supernatants of upper plasma and lavage uid collected after centrifugation were stored at − 80 ℃ for cytokine detection. Cell sediment was added to 100 UL PBS suspension precipitation for total cell count and classi cation count.

2.3.Data detection
The expressions of IL-6, IFN-γ, IL-13 and IL-17 in peripheral blood and BALF were detected by ELISA. The total number and classi cation of BALF cells were counted under the microscope.In ammatory cell in ltration, airway and alveolar changes and ICOSL expression were detected by periodic acid-Schiff (PAS), hematoxylin-eosin (HE) and immunohistochemical (SABC) staining of lung tissue sections.Image-Pro Plus 6.0 system was used to analyze the results of immunohistochemistry. Ten different visual elds were randomly selected from each slice under 400-fold objective lens. The blank area was used as control. The average gray value of positive visual eld was analyzed by computer. The larger of the relative gray value, the stronger of the dyeing strength.

2.4.Statistical analysis
SPSS18.0 software was used to analysis data, measurements were expressed by (x + SD), rank sum test was used for inter-group comparison, chi-square test was used for comparison of rates, P < 0.05 was the signi cant difference.

Mouse behavioral observation
The behavioral changes of mice after atomization for seven consecutive days are not obvious in C group.In N group,after the rst stimulation for about 5 minutes, the mice can observe sneezing, rapid breathing, irritability, frequent scratching of the neck and skin, and the above performance can occur after the fth stimulation starts to atomize for a few seconds. And the reaction is more obvious, nodding, breathing and incontinence.The above performance was observed in the mice at the third challenge of I group.After the seventh stimulation, nod breathing was relatively insigni cant, and the overall behavioral performance improved compared with the N group.The overall behavioral performance of group G was slightly improved compared with group N, which was more obvious than group I.

BALF cytometry total and cell classi cation
The BALF recovery rate of all mice was greater than 80%, and the average recovery was (87.40 ± 3.37)%. The total number of cells in the BALF of mice was the highest in N group, followed by the lowest in group I and group G, and the lowest in group (P < 0.05). The proportion of neutrophils in BALF was the lowest in group C, which was only 1.30 ± 0.25, and that in group I was 10.46 ± 2.14. The highest in group N and group G were 21.2 ± 4.21 and 20.68 ± 4.61(P < 0.05)( Table 1).

3.3.1.Expression of various cytokines in peripheral blood
The concentration of IFN-γ in peripheral blood of group C was signi cantly higher than that of group N,group I and group G (P < 0.05). There was no signi cant difference between the N group, the I group and the G group. The IL-6 concentration in the peripheral blood of the I group was the lowest, the second was G group and C group, the N group was the highest (P < 0.05).The concentration of IL-13 was signi cantly lower than that in N group, I group and G group (P < 0.05). The concentration of IL-17 of N group was signi cantly higher than that in group C, I and G (P < 0.05). (Table 2). Note: a,compared with group C,P < 0.05; b,compared with group N,P < 0.05; c,compared with group I,P < 0.05.

3.3.2.Expression of various cytokines in BALF
IFN-γ was highest in group C, higher than group N and G, and the difference was statistically signi cant(P < 0.05).The IL-6 level in group C was the lowest, and the difference was statistically signi cant (P < 0.05). The difference between group N and group G was not signi cant.IL-13 was the lowest in group C and the highest in group N. The group I and group G were lower than those in group N(P < 0.05). IL-17 was the highest in N,C, I and G groups were all lower than group N. The difference was statistically signi cant.IL-17 levels after ICOSL antibody intervention were lower than those without intervention, and the difference was statistically signi cant (Table 3). Under HE staining, the tracheal structure, lung tissue and alveolar septum in group C were normal, no obvious in ammatory cell in ltration.The in ltration of neutrophils, lymphocytes and plasma cells was obvious intracheal stenosis, bronchial epithelial edema, shedding and degeneration, peri-tracheal lung tissue and alveolar septum of N and G group.Intravenous mucosal epithelial hyperplasia and in ltration of in ammatory cells around the trachea, lung tissue and alveolar space in group I were better than those in group N and G. (Figs. 1 ~ 8).

3.4.2.PAS staining pathology of lung tissue
Mucus secretion was obvious in N and G groups, and no obvious mucus secretion was observed in group C. There was a certain degree of mucus secretion in group I but improved compared with group N and group G. (Figs. 9 ~ 16).

3.4.3.ICOSL immunohistochemistry in lung tissue
After DAB color development, hematoxylin counterstaining, ICOSL positive expression cells were stained brownish yellow.The expression of ICOSL-positive cells in the alveolar interstitial and airway of N and G groups were signi cantly higher than that of I group. There were no ICOSL-positive cells in the alveolar interstitial and airway of C group(P < 0.05).(Figs. 17 ~ 24, Table 4).

Discussion
With intensive study on airway in ammation mechanism of bronchial asthma, it has been found in recent years that a large number of neutrophils exist in bronchial biopsy and induced sputum of some patients with severe asthma and acute exacerbation of asthma, but there is no typical eosinophil in ltration [15~17] .Neutrophils are the last differentiated cells with the shortest life span in blood cells and the most important in ammatory cells in body. Normally, neutrophils start their spontaneous apoptotic process from the beginning of maturation.Active damage and repair mechanisms may exist in the respiratory tract during the onset of asthma, and many mediators produced by neutrophils play an important role in this process.Studies have shown that the levels of IL-6, IL-8 and IL-17 in peripheral blood or BALF are signi cantly increased during acute attack of asthma, and these cytokines can inhibit the apoptosis of neutrophils and promote their aggregation into in ammatory airways [18~20] .ICOSL molecule is the main member of CD28/B7 superfamily [8] . Because of its importance in T lymphocyte activation, polarization of Th1/Th2 subgroup and immunoglobulin homology transformation, ICOSL molecule has attracted much attention and become a research hotspot in immunology.In order to observe the pathophysiological effects of ICOSL in mice with neutrophilic asthma, the model of neutrophilic asthma was established by OVA + LPS, and ICOSL antibody was added to the model during induction period. Through the study of behavioral manifestations, characteristic cytokine levels of Th1, Th2 and Th17 cells and pathological changes of lung tissue to understand the role of ICOSL antibody intervention in neutrophilic asthma, and to deduce the pathophysiological role of ICOSL in neutrophilic asthma.In this experiment, the mice with neutrophilic asthma showed obvious sneezing, accelerated breathing, restlessness and frequent scratching of the face and neck skin. Lung histopathology showed obvious in ltration of neutrophils in lung tissue, alveolar interstitium and around the o cial cavity, hypersecretion of mucus in the o cial cavity, tracheal stenosis and so on, which indicated that the model of neutrophilic asthma in mice was successfully established.
During seven stimulations,asthma performance in group N was the earliest and obvious and was improved in group I,there was no asthma behavior in group C.In group N, the performance of asthma appeared earliest.The behavioral changes of mice after ICOSL intervention were delayed.,The overall clinical manifestations of group I were milder than those of group N.These results suggest that ICOSL antibody intervention before stimulation in mice with neutrophilic asthma can slow down the behavioral manifestation of asthma and alleviate the symptoms of asthma until the mice are tolerant to stimulation.
It is inferred that ICOSL is involved in the occurrence, development and acute attack of neutrophilic asthma.
Current studies on ICOSL are mostly focused on in ammatory diseases, autoimmune diseases and tumors. ICOSL is highly expressed in in ammatory diseases and is closely related to the severity of in ammation [21] .The role of ICOSL in asthma is relatively rare, and the conclusions are inconsistent.In Matsic's study, ICOS + T cells transfected with OVA-speci c T cell receptor were transplanted into OVAsensitized BALB/c mice,observed that lymphocytes, macrophages, neutrophils and eosinophils were signi cantly increased in bronchial lavage uid [22] .However, Akbari used allergen-induced asthma model in mice to study the role of ICOS/ICOSL signaling pathway. It was found that the co-stimulation of ICOS induced the production of regulatory T (Treg) cells. Treg cells depended on the high level of ICOSL expressed by pulmonary dendritic cells. Treg cells could inhibit the function of antigen-speci c T cells and the formation of AHR [23~24] .Lung histopathology showed that there were obvious in ltration of in ammatory cells and neutrophils and hypersecretion of airway mucus in group N and group G. After intervention with ICOSL antibody, the in ltration of in ammatory cells and mucus secretion in airway could be alleviated, indicating that ICOSL was involved in the pathological damage process of respiratory tract in neutrophilic asthma.Compared with group C, the percentage of total cells and neutrophils in BALF in group N was signi cantly higher than that in group C. The levels of cytokines IL-6, IL-13 and IL-17 in peripheral blood and BALF in group N were signi cantly higher than those in group C, and IFN-γwas decreased.The levels of IL-6, IL-13 and IL-17 decreased and IFN-γincreased after ICOSL antibody intervention compared with group N, but there were still differences compared with group C, suggesting that ICOSL antibody could partially alleviate the pathological and immune in ammation process of neutrophilic asthma.ICOSL antibodies were given to mice at 0, 3, 7, 10 and 14, respectively. The irritation time of mice was delayed compared with that of neutrophilic asthma group. No obvious nodding breathing was observed after continuous atomization for one week. The overall symptoms were improved, the in ltration of in ammatory cells in lung tissue and airway was alleviated, and the secretion of IL-6, IL-13 and IL-17 was also reduced. The aggregation of neutrophils in lung tissue was relatively reduced.All of these prove that ICOSL is a positive regulator, which can synergistically stimulate T cell proliferation and promote the secretion of various cytokines and chemokines.

Conclusions
To sum up, ICOSL antibody intervention before stimulation delayed the onset of asthma and alleviated the symptoms.ICOSL intervention can reduce in ammatory cell in ltration and mucus secretion in airway, partly reduce IL-6, IL-13, IL-17 levels in peripheral blood and BALF, and increase IFN-γlevels.These results suggest that blocking ICOS/ICOSL signaling pathway may partially block the development of neutrophilic asthma and may provide a new target for the treatment of asthma.