Effects Of β-arrestin-2on Th1/Th2Type Cytokine Secretion Of Splenic CD4~+T Cells Of Allergic Asthmatic Mice And The Mechanism

BackgroundAllergic asthma has been defined as a disease of eosinophil, mast cell and T lymphocyte infiltration as well as increased mucus production, airway hyperreactivity and airway remodeling in the airways. The etiopathogenisis of bronchial asthma is complex. Asthma is a chronic inflammatory airway disease with the characteristics of intermittent and reversible airway obstruction, bronchial hyperresponsiveness, and airway submucosal infiltration of lymphocytes and eosinophils. Regarding the induction of airway inflammation, it was widely recognized that it is a result of immune response in the bronchial airway. However, the underlying mechanism is still not clear and requires further investigation. Many believe that imbalance of two types of T-helper cells, Th1cells and Th2cells, is the underlying cause for asthma. Specifically, the increased activity of Th2cells is a key event in asthma induction. Th1cells and Th2cells are differentiated from a common precursor cells, ThO cells. Transcription factor GATA3regulates ThO cell differentiation in a specific manner. Such regulation is critical in controlling Th1cell and Th2cell activities. IFN-γ and IL-4are typical cytokines for Th1cells and Th2cells.Proteins are actors of all the functions of oganisms in vivo, so the study of diseases must rely on the analysis of the final functional protein. Therefore, the study about the functional protein of asthma plays a irreplaceable role in revealing the pathogenesis of asthma.In recent years, many scientists payed great attention to a protein which was considered to be a potential target for asthma—β-arrestin-2.β-arrestin-2is an intracellular signal regulatory protein, widely involved in G protein-coupled receptors (GPCRs) related signaling pathways, which owns diverse biological functions. Recent studies have found that β-arrestin-2play a positive regulatory role in asthma. β-arrestin-2gene knockout mice may inhibite T cells gathering in airways, airway inflammation and airway obstruction through inhibit T cells chemotaxis to the lung. Airway hyperresponsiveness in asthma is dependent on β-arrestin-2expression in non-hematopoietic stem cell-derived stromal cells. In addition, expression of β-arrestin-2in hematopoietic stem cell-derived cell promotes the formation of airway inflammation and eosinophil migration to lung in asthmatic mice. Therefore, β-arrestin-2is a important regulatory factor in the pathogenesis of allergic asthma.Th1/Th2imbalance is the key factor for asthma pathogenesis, while both Th1and Th2implement their functions by secreting characteristic cytokines. Then, whether β-arrestin-2regulate cytokine productin of T cells? β-arrestin-2is a GPCR regulatory protein which implement functions through regulating GPCRs. Chemokine receptors are a kind of GPCRs which could be regulated by β-arrestin-2to affect cell chemotaxis.T cell expresses some β2ARs on the cell surface which are members of the GPCR family. β2AR on T cell surface regulated T cell cytokine secretion of asthmatic patients. Moreover, previous studies have showed that β-arrestin-2could down regulate β2AR signal transduction pathway by promoting desensitization of P2AR or other way. Therefore,we hypothesize that β-arrestin-2probably indirectly regulate T cell cytokine production through β2AR.RNA interference (RNAi) is a kind of highly conserved gene silencing technology. RNAi technology is the same as gene knockout and gene silencing in functions, but more convenient than gene knockout technology. In this study, we used RNAi to downregulate β-arrestin-2expression of splenic CD4+T cells from mouse models of acute asthma; the characteristic cytokines of Th1/Th2type cytokines IFN-γ, IL-4and IL-5as the evaluation targets; β2AR expressed on splenic CD4+T cells as a bridge, in order to investigate the effect of β-arrestin-2on the cytokine production of splenic CD4+T cells from allergic mouse models. One goal of this study is to elucidate the effect of β-arrestin-2on IFN-γ, IL-4and IL-5secretion of splenic CD4+T cells from mouse model of acute asthma. Furthermore, the other goal of our study is to detect the effect of β-arrestin-2on expression of P2AR and the effect on IFN-γ, IL-4and IL-5secretion regulated by β-arrestin-2of splenic CD4+T cells from mouse model of acute asthma. Through this study, we hope to find out the effect of β-arrestin-2on cytokine production of splenic CD4+T cells of acute asthmatic mice and open new avenues and provide further experimental evidence for therapeutic treatment of asthma. Part I Establish mouse model of acute asthma and confirm Reliability of the modelObjective To establish mouse models of acute allergic asthma and confirm the reliability of the models with modified evaluation criterion.Methods28SPF female BABL/c mice were randomly divided into normal control group and asthma group, with20mice in asthma group and8mice in normal control group. The asthma model was established by sensitization with intraperitoneal injection of OVA and aerosol challenge with repeated inhalation of OVA in the asthma group. The control group received PBS as the substitution of OVA. After24hours of the last inhalation, evaluation items include:(1) asthmatic symptoms;(2) the changes in airway response were determined by lung resistance (RL) and Lung dynamic compliance (Cdyn) stimulated by Methacholine (Mch);(3) lung tissue sections were stained for general pathology;(4) total cell counts, cell counts of neutrophils, eosinophils and lymphocytes of bronchoalveolar lavage fluid (BALF) were measured;(5) the cell counts of splenic CD4+T cells were detected;(6) the levels of IL-4concentration of BALF and splenic CD4+T cell culture supernatant were examined.Results(1) Asthma symptoms were more severe in asthma group, compared with normal control group; (2) The dose-response curves for RL was obviously shifted upward in asthma group compared to normal control group (P<0.01); while the dose-response curves for Cdyn was significantly shifted downward in asthma group compared to normal control group (P<0.01);(3) There were more extensive inflammatory cells infiltration around the bronchi and blood vessels in asthma group, compared with normal control group (P<0.01);(4) Total cell counts, cell counts of neutrophils, eosinophils and lymphocytes of BALF significantly increased in asthmatic mice than those of normal control group (respectively P<0.01);(5) The total cell counts of splenic CD4+T cells is obviously increased in asthmatic group than those of normal control group (P<0.01);(6) In either splenic CD4+T lymphocyte culture supernatant or BALF, the levels of IL-4concentration were significantly elevated in asthma group compared with normal control group (respectively P<0.01).Conclusions(1) The established acute mouse model of allegic asthma was successfully confirmed. Part Ⅱ Effect of β-arrestin-2on Thl/Th2type cytokine secretion of splenic CD4+T cells from mouse model of acute asthmaObjective To investigate the change of β-arrestin-2expression in mouse model of acute asthma, and to elucidate the effect of β-arrestin-2on Th1/Th2type cytokine (IFN-γ, IL-4, IL-5) secretion of splenic CD4+T cells from mouse models of acute asthma.Methods (1) Grinding spleen of asthmatic group and normal group into splenic mononuclear cell suspension. After remove red blood cells from the suspension, CD4+T cells were isolated from splenic mononuclear cells with immuno-magnetic beads, and then were cultivated in RPMI-1640with10%Fetal Bovine Serum.(2) First determine the levels of P-arrestin-2protein and mRNA by Western blot and Real-time PCR.(3) Splenic CD4+T cells from mouse model of acute asthma were transfected with three chemosynthesis β-arrestin-2siRNA sequences and negative control sequences. The best efficient siRNA-ARRB2was chosed by detection of β-arrestin-2expression by RT-PCR.(4) β-arrestin-2protein of transfected group and negative control group were measured by Western blot.(5) Then the best siRNA-ARRB2was transfected into splenic CD4+T cells from asthma group, and transfected cells were stimulated by ConA and PMA for24hours. The levels of IFN-γ, IL-4and IL-5were measured by ELISA;(6) GATA3protein and mRNA were determined by Western blot and Real-time PCR.Results(1) The expression of (3-arrestin-2mRNA and protein in splenic CD4+T cells from asthma group significantly elevated compared to normal control group (respectively P<0.01);(2) siRNA-ARRB2-1123had the best silencing effect among all siRNA sequences (P<0.01);(3) The GATA3protein and mRNA expression of splenic CD4+T cells from transfected asthma group elevated compared to non-transfected asthma group (respectively P<0.01);(4) IFN-γ, IL-4and IL-5secration of CD4+T lymphocytes had no significant difference between transfected asthmatic group and non-transfected asthmatic group.Conclusions(1) β-arrestin-2had no direct effect on IFN-γ, IL-4and IL-5secration of splenic CD4+T cells from mouse model of acute asthma;(2) β-arrestin-2may enhance GATA3expression of splenic CD4+T cells from mouse model of acute asthma, but had no direct effect on IFN-γ, IL-4and IL-5secration. PartⅢ Effect of β-arrestin-2On the secration of Thl/Th2type cytokines of splenic CD4+T cells from mouse model of acute asthma through β2ARObjective To investigate the effect of (3-arrestin-2on Th1/Th2type cytokine (IFN-γ, IL-4, IL-5) secration from splenic CD4+T cells through β2AR, and whether it is related to GATA3.Methods The best siRNA-ARRB2were transfected to splenic CD4+T cells from mouse model of acute asthma.(1) Surface-expression of β2AR of splenic CD4+T cells from normal mice, mouse model of acute asthma and siRNA-ARRB2transfected asthmatic group were detected by FCM (Flow cytometry).(2) Cell-surface β2AR expression of CD4+T cells in transfected asthma group and non-transfected asthma group were measured by FCM after stimulated by ConA and PMA for24hours and stimulated by terbutaline for the last hour.(3) The expression of GATA3mRNA was measured by Real-time PCR, and GATA3protein by Western blot after stimulated by ConA and PMA as well as terbutaline for24hours;(4) IFN-γ, IL-4and IL-5from splenic CD4+T cells were determined by ELISA after stimulated by ConA and PMA as well as terbutaline for24hours.Results(1) Surface-expression of β2AR of CD4+T lymphocytes from asthmatic group significantly decreased than that of normal group (P<0.01);(2) After siRNA-ARRB2transfection, surface-expression of β2AR of CD4+T lymphocytes from asthmatic group didn’t change.(3) After terbutaline stimulation:1) After terbutaline stimulation, the expression of (32AR of splenic CD4+T cells from mouse model of acute asthma decreased (P<0.05); IFN-γ secretion of splenic CD4+T cells from mouse model of acute asthma significantly decreased (P<0.05); IL-4、IL-5and GATA3didn’t change;2) After terbutaline stimulation, β2AR of splenic CD4+T cells from transfected asthma group was expressed at significantly higher levels compared to non-transfected asthma group (P<0.01); IL-4and IL-5secretion of splenic CD4+T cells from transfected asthma group significantly decreased compared to non-transfected asthma group (respectively P<0.05); GATA3mRNA of splenic CD4+T cells from transfected asthma group decreased compared to non-transfected asthma group (P<0.01); after terbutaline stimulation, GATA3protein of splenic CD4+T cells from transfected asthma group decreased compared to non-transfected asthma group (P<0.01).Conclusions (1) β2AR of splenic CD4+T cells of mouse model of acute asthma may be defective;(2) β-arrestin-2didn’t regulate the expression of β2AR of splenic CD4+T cells in mouse model of acute asthma;(3) β-arrestin-2could enhance β2AR downexpression of splenic CD4+T cells in mouse model of acute asthma induced by β2AR agonist;(4) β-arrestin-2could promote Th2type cytokine production of splenic CD4+T cells from mouse model of acute asthma, probably through P2AR;(5) The effect that β-arrestin-2could probably promote Th2type cytokine production of splenic CD4+T cells from mouse model of acute asthma through GAT A3was depend on β2AR.