| Background:The main pathological changes of bronchial asthma are edema of airway mucosa,infiltration of eosinophils and lymphocytes in different degrees.Its mechanism is very complex,involving many aspects such as heredity,environmental and immune regulation,in which immune abnormality or immune imbalance plays an important role in the development of the disease.The dominant state of Th2 cells,airway hyperresponsiveness and tissue remodeling constitute the three characteristic pathological changes of asthma.The polarization of Th2 cells in asthma is related to many factors.However,the causal relationship between this airway inflammatory damage and Th1 / Th2 cell imbalance and its possible mechanism is not clear.Further studies on the cellular and molecular basis of inducing Th2 cell polarization and maintaining Th2 cell dominance are required for better understanding of the immunopathological mechanism of asthma,providing a basis for correcting the immune imbalance as well as development of precisive diagnosis or treatment.Objective:Airway epithelial cells are the initial site of exogenous stimulation.The structural and functional changes of airway epithelial cells are closely related to the occurrence of asthma and Th2 cell-mediated inflammation.The purpose of this study is to analyze the initiating factors of Th2 cell polarization in asthma,and to explore the advantages of IL-9 formed under the condition of immune imbalance and its influence on Th2 response related immune cells.Methods:1.Source and feeding of experimental animals:BALB/c mice,female,6-8 weeks old,weighing 18-20 g,were purchased from the medical comparison center of Yangzhou University.They were randomly divided into two groups,5 mice in each group,and fed in the sterile environment of the animal experimental center of Jiangsu University.2.The OVA-induced asthma mice model was constructed and the asthmatic attack of the model mice was observed,such as the symptoms of rapid breathing,even nodding breathing,bristle,purple lips,forelimb retraction and lifting,slow response,etc.,the pathological analysis of lung inflammation was carried out by HE staining,the BALF was collected for cell count;the eosinophils in peripheral blood were detected by HE staining,the level of Ig E in lung tissue or serum was analyzed by immunohistochemistry and ELISA.3.The qRT-PCR or ELISA was used to analyze the expression level of Rac1 and IL-33 in peripheral blood or lung tissue of asthmatic mice,exogenous Rac1 or Rac1-si RNA was used to transfect respiratory epithelial cells and macrophages,the effect of Rac1 on the apoptosis of respiratory epithelial cells and the function of phagocytes were analyzed in vitro to explore the relationship between Rac1,formation of airway hyperresponsiveness,type II immune response and the occurrence of asthma.4.The expression of cytokines such as IL-9,IL-5,IL-13,IL-33 and transcription factors including ROR?,PU.1,GATA3 in the lung,spleen and peripheral blood of asthmatic mice were analyzed by flow cytometry,immunohistochemistry,ELISA and qRT-PCR.The cell ratio of ILC2 s,Th2,Th9 and the expression level of related cytokines or transcription factors in response to Th2 were observed.The correlation between Th2 response and the expression of cytokines or transcription factors was analyzed in order to understand the factors that maintain the dominant differentiation of Th2 cells in asthma.5.Flow cytometry,immunohistochemistry and qRT-PCR were used to detect the IL-9 and Th9 cells in the lung tissue and BALF of asthmatic mice.6.The effects of IL-9 on the differentiation of BMDMs were analyzed by cell culture in vitro.The effects of IL-9 on the differentiation of Th0 cells into Th2 cells were observed by flow cytometry,immunohistochemistry and qRT-PCR.Results:1.Expression of Rac1 and its effect on apoptosis and clearance of apoptosis cells in airway epithelial cells(1)Animal test results show that the expression level of Rac1 was significantly decreased(P < 0.005 compared with control),and IL-33 expression was significantly increased(P < 0.005 in peripheral blood and P < 0.001 in lung,compared to the control).(2)It was found that the transfection of Rac1 si RNA or the treatment of NSC23766 could promote curcumin induced apoptosis of airway epithelial cells and weaken the phagocytic ability of phagocytes(P < 0.005,compared to untreated group).(3)Compared with the control group,the migration ability of the airway epithelial cells transfected with the exogenous Rac1 gene was significantly enhanced(P <0.001),while the migration ability was significantly reduced in the airway epithelial cells transfected with Rac1 si RNA and treated with NSC23766(P < 0.005).In addition,the expression of CD80,CD86 and MHC-II in RAW264.7 cells were upregulated by Rac1 inhibitor(P < 0.001).2.Th2 cells may be an intermediate for Th0 to differentiate into Th9 cells In vitro study,the results showed that the optimal conditions for Th0 cells to differentiate into Th9 cells were: 5 ng/ml TGF-? and 30 ng/ml IL-4 for 5 days,under which Th0 cells could first differentiate into Th2 cells(about 3 days)and then into Th9 cells(day 5).The results of qRT-PCR also showed that the expression of Smad3,Smad4 and IRF4 in Th9 cells induced by TGF-? and IL-4 were significantly higher than that in the control group(P < 0.05).3.Th9,ILC2 s and their related cytokine receptors in the lung of asthmatic mice(1)Compared to the control group,the expression of IL-9 producing cells(Th9)and ILC2 s in the lung tissue of the asthmatic mice model were increased significantly(P< 0.05;P < 0.01),and the expression of ROR?,PU.1,IL-5R,IL-9R,IL-13 R and Fc?R in the lung tissue were also significantly higher than that in the control mice(P< 0.0001).(2)There was a positive correlation between the expression of ILC2 s related receptor and Th9 related cytokines(ROR? and IL-9: r = 0.6883,P = 0.0278;IL-5R and IL-9: r = 0.7646,P = 0.0100;IL-13 R and IL-9: r = 0.8112,r = 0.0044).(3)The expression of IL-5R?,IL-13 R and IL-9R in inflammatory lung tissue of asthmatic mice were positively correlated with serum Ig E(r = 0.8522,P = 0.0017;r= 0.8211,P = 0.0036;r = 0.8926,P = 0.0005).4.Effect of IL-9 on macrophage differentiation and polarizationThe results showed that bone marrow cells expressed high level of IL-9R,while BMDMs expressed very low level of IL-9R(almost no expression),the difference was significant(P < 0.005).The stimulation of IL-9 had no direct effect on the differentiation and activity of BMDMs,neither on the polarization to M2 nor M1 macrophages.Conclusion:The down-regulation of Rac1 expression is related to airway hyper-responsiveness,and promotes the expression of IL-33 to form the basis of ILC2 s polarization.ILC2 s,Th9 cells and their characteristic cytokines IL-4,IL-13 and IL-9 play a role in maintaining the dominant differentiation of Th2 cells and participate in the immunopathological mechanism of asthma. |
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