The Role Of B7-H3 In Neutrophilic Asthma Of Children

Part1 Expression and clinical significance of B7-H3 in neutrophilic asthma of childrenObiective: To investigate the expression level of B7-H3 in neutrophilic asthma(NA)of children,and analyze its association with cytokines produced by Th1/Th2/Th17 cells and bronchoalveolar lavage fluid(BALF)neutrophil counts,so as to explore the clinical meaning of B7-H3 in acute exacerbation of neutrophilic asthma.Methods: Forty-two cases of children with bronchial asthma hospitalized in the department of respiration in Children?s hospital of Soochow University from January 2015 to December 2016 were enrolled.BALF were collected from all 42 cases within 5 days after hospitalization,and subjects with asthma were classified into neutrophilic asthma(groupA)(n=20)and non-neutrophilic asthma(groupB)(n=22)according to patients who had a BALF neutrophil count of greater than 48%(95% centile of normal).Sixteen children hospitalized for bronchial foreign bodies were enrolled as control group(groupC).Peripheral blood was collected in all children within 24 hours after hospitalization,the levels of B7-H3 and cytokines(interferon[IFN]-gamma,interleukin[IL]-4,IL-6,IL-8,IL-17,granulocyte-macrophage colony stimulating factor [GM-CSF],matrix metalloprotein[MMP]-9 and neutrophil elastase[NE] were performed by enzyme-linked immunosorbent assay(ELISA)technique.meanwhile,the correlation between B7-H3 and other cytokines,BALF neutrophil counts were obtained.Results: As compared to group C,the levels of B7-H3,IL-4,IL-6,IL-8,IL-17,GM-CSF,MMP-9 and NE in plasma and BALF of group A were significantly elevated(P<0.05);IL-6,IL-8,IL-17 and MMP-9 levels in plasma and BALF of group A were increased compared to group B,while B7-H3,GM-CSF and NE levels of group A was further higher than group B in BALF(P<0.05);B7-H3,IL-4,IL-6,IL-17 and GM-CSF levels of plasma and BALF were raised in group B than group C,and the difference was significant(P<0.05);IFN-gamma levels were significantly lower in group B,and then IFN-gamma levels in both group A and group C were similarly elevated.We found a significant positive correlation between B7-H3 and IFN-gamma,IL-6,IL-8,IL-17 and MMP-9 in plasma and BALF of group A(r>0.3,P<0.05),moreover,B7-H3 levels of group A in BALF correlated with GM-GSF,NE and BALF neutrophil counts significantly(r>0.3,P<0.05);while in group B,B7-H3 levels significantly correlated with IL-4 and IL-17 in plasma and BALF(r>0.3,P<0.05).Conclusion: The levels of B7-H3 and the function of Th17 cells are augmented with neutrophilic asthma acute exacerbation,and B7-H3 significantly correlates with cytokines produced by Th17 cells and BALF neutrophil counts,so B7-H3 which enhances the secretion of Th17 cells may play an important role in the development of airway inflammation and acute phase of neutrophilic asthma in children.Part2 Therapeutic effects of anti-B7-H3 monoclonal antibody in murine model of neutrophilic asthmaObiective:To inquire into the effects of anti-B7-H3 monoclonal antibody(m Ab)in a murine model of neutrophilic asthma.Methods: Twenty-four female,specific-pathogen-free,BALB/c mices(6-8 weeks old)were divided into PBS control group(group A),neutrophilic asthma group(group B),anti-B7-H3 m Ab group(group C),control Ig G group(group D)randomly,with 6 mices in each group.Mices except group A were sensitized by injection of ovalbumin(OVA)plus Lipopolysaccharide(LPS)on days 0,7,14,and then challenged with OVA for a continuous week from days 21.The PBS control group was sensitized by injection of PBS and inhaled PBS in a similar manner.Mices of group C and group D were injected with anti-B7-H3 m Ab and control Ig G on days 0,3,7,10,14.Mices were anaesthetized deeply 24 hours after the last nebulization,and blood samples,bronchoalveolar lavage fluid(BALF)and lung tissues were collected.Total and differential cell counts of BALF were determined by microscope,the levels of cytokines(IFN-gamma,IL-4,IL-5,IL-6,IL-13,IL-17,tumor necrosis factor[TNF]-a,granulocyte colony stimulating factor [G-CSF])in plasma and BALF were assayed by ELISA,lung section were stained with hematoxylin and eosin(H(5)E)and periodic acid-Schiff(PAS)to identify tissue inflammation and mucus production.Immunohistochemistry was used to measure B7-H3 expression level in frozen lung sections of murine.Results: Group B and group D performed breathless,dysphoric and incontinence during atomization,group C with anti-B7-H3 m Ab treatment relieved the asthmatic symptom.We found that group B,group C and group D increased the total BALF numbers,neutrophil counts,eosinophil counts and lymphocyte counts,decreased the macrophages counts when compared with group A(P<0.05),the differences of total BALF numbers,neutrophil counts,eosinophil counts between group C and group B were significantly decreased(P<0.05).With regard to lung infiltration by Th1,Th2 and Th17 cells,the expression levels of IFN-gamma,IL-4,IL-5,IL-6,IL-13,IL-17,TNF-a and G-CSF in plasma and BALF were higher in group B,group C and group D than group A,significant elevated IL-17 and G-CSF levels were presented in plasma and BALF of group B and group D compared to group C(P<0.05),moreover,in group B and group D,the production of IL-6 and TNF-a of BALF were also significantly rised than those in group C.The expression levels of IL-4 in plasma and BALF of group C were significantly increased compared with group B and group D(P<0.05),in addition,the levels of IFN-gamma,IL-5,IL-13 among group B,group C and group D indicated no statistic difference in plasma and BALF.In group B and group D,histological results of lung section showed vast infiltration of inflammatory cells including neutrophils around the bronchioles,and mucus over-production than those in group A,administration of anti-B7-H3 m Ab inhibited the accumulation of neutrophils and mucus over-production in the lung.Compared with group A,levels of B7-H3-positive cells were significantly increased in group B and group D,anti-B7-H3-treated mice showed reduced level of B7-H3-positive cells in the lung tissues as compared with group B and group D(P<0.05).Conclusion: Administration of anti-B7-H3 m Ab can ameliorate the asthmatic syndrome,reduce airway inflammatory cells with a concomitant decrease in neutrophils,eosinophils,mucus production,and down-regulate Th1/Th17 cells,proinflammation cytokines,thus reducing the airway,lung tissue and systematic inflammation,blocking the immunopathogenesis and the development of NA partially.In conclusion,the anti-B7-H3 m Ab may provide a novel therapeutic approach to treatment of neutrophilic asthma.