The Role Of ICOSL In Neutrophilic Asthma In Children

Part 1.Expression and clinical significance of the ICOSL of children with neutrophilic asthma.Objective: To study the expression level of ICOSL in peripheral blood and bronchoalveolar lavage fluid of children with neutrophilic asthma,and analyze its correlation with the expression of other cytokines.To explore the abnormal expression of ICOSL in children with neutrophilic asthma and its clinical significance.Methods: A total of 32 asthma patients were collected from the departments of respiratory diseases of Children’s hospital of Soochow University between August 2014 and July 2016,and at the same time,16 after foreign body removal surgery children were enrolled in control group.Peripheral blood was collected within 24 hours after hospitalization and bronchoalveolar lavage fluid was collected when preoperative examination finished.Selected more than fifty percent of neutrophil in bronchoalveolar lavage fluid cell differential as neutrophilic asthma group from thirty-two asthma patients,the rest was asthma group.The level of ICOSL,interleukin-4,interleukin-8,IFN-gamma,neutrophil elastase and matrix metalloprotein-9 in plasma and bronchoalveolar lavage fluid were detected by Enzyme-linked immunosorbent assay.Analyzing the correlation and clinical charateristics between the level of ICOSL and other cytokines.Results: Children with allergic history among neutrophilic asthma group and asthma group were both seventy-five percent.Mean hospitalization of neutrophilic asthma group,11.18±4.29 days,were longer than asthma group,a statistically significant difference.The level of ICOSL,interleukin-4,interleukin-8,neutrophil elastase and matrix metalloprotein-9 of neutrophilic asthma group in plasma were higher than that in asthma group,while IFN-gamma was just the opposite.The level of interleukin-8,interleukin-4 of neutrophilic asthma and asthma group were both higer than control group.In bronchoalveolar lavage fluid,The level of ICOSL,neutrophil elastase and matrix metalloprotein-9 of neutrophilic asthma group was the highest,while the level of IFN-gamma was the lowest in neutrophilic asthma group.The concentration of matrix metalloprotein-9 in asthma group was higer than that in control group,the concentration of neutrophil elastase of asthma group was lower than that in control group,a significant difference statistically.There was of good correlation between the level of ICOSL and interleukin-8 in plasma and bronchoalveolar lavage fluid in children with neutrophilic asthma.(r=0.683,P=0.042),(r=0.700,P=0.036).Conclusion: Asthmatic children include neutrophilic asthma and normal asthma have an immunity imbalance of Th1/Th2/Th17,but this kind of immune imbalance in children with neutrophilic asthma is more serious,the clinical treatment is difficult,the need for hospitalization longer.The expression of ICOSL in neutrophilic asthma children was higer in both plasma and bronchoalveolar lavage fluid than control group.ICOSL may regulate the secretion of interleukin-17 by Th17,enhance the role of interleukin-8 in the recruitment of neutrophils in the airway and the increase of NE and MMP9,which is involved in the development of immune inflammation in neutrophils.Part 2.The effect of ICOSL antibody in murine model of neutrophil asthmaObjective: To investigate the role of ICOSL antibody in the mouse model of neutrophilic asthma,and to explore the role of ICOSL in the pathogenesis of Th1/Th2/Th17 imbalance.Methods:SPF grade 6 week old Balb/c mice were randomly divided into four groups:control group(CON group),neutrophilic asthma group(NEU group),ICOSL antibody intervention group(ICOSL antibody group)and Ig G antibody isotype group(Ig G group).NEU group,ICOSL group and Ig G group were sensitized and challenged by OVA+LPS,CON group was given the same dose of PBS injection and challenged.The ICOSL and Ig G groups were injected intraperitoneally with ICOSL and Ig G antibodies.The peripheral blood,bronchoalveolar lavage fluid and lung tissue were collected within 24 hours after the last challenge.Flow cytometry was used to measure the expression of ICOSL in peripheral blood and lung tissue.Total count and classification of bronchoalveolar lavage fluid cells;Detection cytokines of IFN-gamma,IL-6,IL-13,IL-17 in peripheral blood and bronchoalveolar lavage fluid.Results:There was no behavioral change after challenge in CON group;The first challenge of NEU group,the behavioral changes occurred in the first 5 minutes after stimulation,and the behavioral changes were observed at the beginning of the fifth stimulation;Behavior of ICOSL antibody group changed at the beginning of the third challenge,the behavior of the seventh challenge is not obvious.The behavior of Ig G group was slightly better than that of NEU group,which was more serious than that of ICOSL group.The expression of ICOSL on peripheral blood neutrophil among four groups,the positive rate of NEU group was the highest,ICOSL antibody group was the lowest,the difference between the two groups was statistically significant(P<0.001);There was no significant difference in the expression of ICOSL on neutrophils in lung tissue of four groups.The total number of bronchoalveolar lavage fluid in the NEU group was the highest,in the CON group was the lowest,the difference was statistically significant.The concentrations of IL-6,IL-13 and IL-17 in peripheral blood of NEU group were the highest,and the difference was statistically significant between ICOSL group and CON group(P<0.05).The IFN-gamma concentration of CON group was higher than that of NEU group,ICOSL antibody group and Ig G group,the difference was statistically significant(P<0.05).In bronchoalveolar lavage fluid,IFN-gamma in CON group was significantly higher than NEU group,Ig G group,the difference was statistically significant(P<0.05).The concentration of IL-6,IL-13,IL-17 in CON group was the lowest,in NEU group was the highest,and that in the ICOSL antibody group were lower than those in NEU group after the intervention of the ICOSL antibody,the difference between the two groups was statistically significant(P<0.05).HE and PAS staining showed that inflammatory infiltration in NEU and Ig G group was improved,and ICOSL antibody group was relative improved.ICOSL immunohistochemical analysis,ICOSL positive cells of alveolar interstitial and airway of NEU,Ig G group were significantly more than ICOSL antibody group,the difference was statistically significant(P<0.05).Conclusion: After the intervention of ICOSL antibody in the mice model of neutrophil asthma,the behavioral changes of asthma attack slowed down,and the symptoms were alleviated until the tolerance;Reduce airway inflammatory cell infiltration and mucus secretion;The levels of IL-6,IL-13 and IL-17 in peripheral blood and bronchoalveolar lavage fluid were partly decreased,and the level of IFN-gamma was increased;These results suggest that blocking the ICOS/ICOSL signaling pathway can partially block the development of neutrophil asthma and provide a new target for the treatment of asthma.