The Function Of Costimulatory Molecule B7-H3 In Regulating Of Th Cell Differentiation And Clinical Significance In Children With Asthma

Background:Bronchial Asthma is one of the most common chronic diseases in children and has dramatically increased in recent decades.Asthma becomes public health problem all over the world and need to work out through interfering in mechanical molecules.The etiology of asthma is very complex and associated with genetic background,environmental factor and individual immunity.The classic theory is that pathogenesis of allergic bronchial asthma focus on unbalance of type 1 T helper?Th1?cell and type 2 T helper?Th2?cell.Thus,it is very important to rectify such unbalance for asthma immunotherapy.The costimulatory molecules not only could regulate immune response of T cells,but also play a key role in T cell differentiation.B7-H3 is one member of the B7family and critical molecule in biologic activity of T cells confirmed by both structure and function studies.However,controversies still exist about the function of regulating Th cells.Recent studies showed that microRNA?miRNA?could regulate relative gene expression during transcription or post-transcription and have great role in inflammatory diseases.These studies support B7-H3 gene is also regulated by mi RNAs.Objectives:Our study try to clarify the mechanism and functions of non-coding RNAs in regulating B7-H3 expression in vivo and vitro and explore the role of B7-H3 in Th cell differentiation.We also analyse the clinical significance of B7-H3 expression in children with asthma and provide a novel therapeutic approach to the treatment of allergic asthma through intervention in cosimulatory molecules.Methods:This study included 21 Chinese children with moderate to severe asthma exacerbation as well as 18 Chinese nonasthmatic control children with selective operation including indirect inguinal hernia,hemangioma,polydactyly from June 2013 to December2015.The diagnosis of asthma and its severity of asthma exacerbation were conducted according to Global Initiative for Asthma guidelines.There was no statistical significance in age between asthma exacerbation and controls.Clinical and laboratory data were collected.Peripheral blood samples were also collected on admission and discharge and cytokines including interferon gamma?IFN-??,IL-4,IL-10,IL-17 and soluble B7-H3?sB7-H3?using enzyme linked immunosorbent assay?ELISA?.Membrane B7-H3?mB7-H3?was detected by flow cytometry.CD4⁺CD45RA⁺native T cells were purified from healthy donors and adjust the concentration to 5×10⁵/well.Then,B7-H3 fusion Ig with different concentration?0.6?g/ml,3?g/ml,and 15?g/ml?was added into wells.After 24h,cells and supernatant were collected to detect typical transcription factors of Th1,Th2,Th17,and regulatory T?Treg?cells including T-bet,GATA-3,ROR?t,and FOXP3 using real time PCR?RT-PCR?.Typical cytokines of Th1,Th2,Th17,and Treg including IFN-?,IL-4,IL-17,TGF-?were detected by ELISA.Gene chip analysis searched the differential expression of miRNAs and bioinformatic analysis predicts the miRNAs associated with B7-H3 gene.Then Real time-PCR?RT-PCR?testified the expression of miRNAs.We constructed chronic viral vector which could overexpress and downregulate mi R-29c in THP-1 cell line and explore the expression of B7-H3.Whether miR-29c could directly combine to B7-H3-3'UTR was comfirmed using luciferase assay.The expression of B7-H3 in 52 cases with asthma and 26healthy controls were also detected by RT-PCR.OVA sensitized asthma mice were constructed and blocking anti-B7-H3 monoclone antibody?mAb?was intraperitoneal injected.Then bronchoalveolar lavage fluid?BALF?was collected for total cell count and classification.Concentrations of IFN-?,IL-4,and IL-17 in BALF were detected by ELISA while lungs were stained with HE,periodic acid-Schiff?PAS?and anti-B7-H3 mAb as a measure of inflammatory cell infitration,mucus production and B7-H3 expression respectively.Results:CD14⁺monocytes highly expressed B7-H3 in chilren with asthma exacerbation compared to healthy controls?P<0.05?.Concentrations of sB7-H3,IL-4,IL-17 obviously increased in chilren with asthma exacerbation compared to healthy controls?P<0.05?while concentration of IFN-?decreased?P<0.05?.No significance was found in expression of IL-10 between groups?P>0.05?.Level of B7-H3 of asthma exacerbation subjects with inhaled corticosteroids?ICS?treatment recently was significantly lower than subjects without ICS treatment?t=2.706;P=0.0136?.Level of sB7-H3 in asthma exacerbation subjects was inversely correlated with level of IFN-??r=-0.644;P=0.002?and FEV₁%predicted?r=0.499;P=0.0212?.Additionally,levels of B7-H3 decreased remarkably after treatment?P=0.003?.B7-H3 fusion Ig chould enhance the expression of GATA-3 and ROR?t in T cells and have a dose dependant pattern in ROR?t expression.Only high concentration of B7-H3fusion Ig?15?g/ml?could enhance the expression of Th2 typical transcription factors GATA-3.Level of IFN-?,IL-4,IL-17A in supernatant increased by B7-H3 fusion Ig and have a dose dependant pattern in both IL-4 and IL-17A?P<0.05?,especially for IL-17A.Only high concentration of B7-H3 fusion Ig?15?g/ml?could enhance the expression of Th1 typical cytokine IFN-??P<0.05?.B7-H3 is the target of mi R-29c searched by gene chips and bioinformatic analysis.Transfection of miR-29c mimic significantly repressed B7-H3-3'UTR reporter activity in wild type THP-1 cells while no significances were found in other groups.It is confirmed that B7-H3 is the target gene of miR-29c.Overexpression or downregulation of miR-29c could decrease or increase B7-H3 expression in THP-1 cells.The level of mi R-29c in children with asthma was significant lower compared to healthy controls?P<0.01?.Inflammatory cells including eosinophils of BALF increased in OVA-induced asthma mice.There was no difference of Characterization in BALF between anti-B7-H3 mAb treatment group at effector phase and IgG treatment group.Meanwhile,total cells and eosinophils significantly decreased in anti-B7-H3 mAb treatment group at induction phase compared to IgG treatment group?P<0.05?.Compared to asthma group,IgG treatment group,anti-B7-H3 mAb treatment group at effector phase,administration of anti-B7-H3mAb at the induction phase significantly inhibited eosinophil infiltration and mucus overproduction in lung tissues.No significance was found in level of IFN-?,IL-4,IL-17between anti-B7-H3 mAb treatment group at effector phase and IgG treatment group?P>0.05?.However,level of IFN-?significantly increased and IL-4,IL-17 decreased in anti-B7-H3 mAb treatment group at the induction phase compared to IgG treatment group?all P<0.05?.Compared to IgG treatment group,total cells and eosinophils significantly increased in B7-H3 fusion Ig treatment group?P<0.05?.Compared to IgG treatment group with TLR2 knock-out,total cells and eosinophils also significantly increased in B7-H3fusion Ig treatment group with TLR2 knock-out?P<0.05?.Administration of B7-H3fusion Ig significantly increased eosinophil infiltration and mucus overproduction in lung tissues.Administration of B7-H3 fusion Ig in TLR2^-/-mice also significantly increased eosinophil infiltration and mucus overproduction in lung tissues.Compared to IgG treatment group,IFN-?and IL-4 concentration in BALF of B7-H3 fusion Ig treatment group significantly increased.Meanwhile,IFN-?,IL-4,and IL-17 concentration in BALF of B7-H3 fusion Ig treatment TLR2^-/-group significant increased compared to IgG treatment TLR2^-/-group?all P<0.05?.Conclusions:B7-H3 plays an important role in pathogenesis of allergic bronchial asthma through promoting Th cell differentiation,especially for Th1,Th2,and Th17polarization.B7-H3 fusion Ig could aggravate immunopathogenesis of asthma,but the specific mechanism is still unclear and do not depend on TLR2 pathway.Meanwhile,admistration of anti-B7-H3 mAb in induction phase could alleviate immunopathogenesis.Therefore,B7-H3 might be a new target and effective biomark for asthma severity estimation.