The Role Of Lung-resident Memory T Cells In Allergic Asthma

Objective: Tissue-resident memory T cells(TRM)is a new sub-group of the memory T cells.After to be activated by antigen,the naive T cells will migrate to a variety of organs which are not peripheral Lymphoid organs and settle down to differentiate into specific memory T cells in situ.The TRM can response rapidly when the same antigen is encountered in the organization and its protective immunity function is independent of circular the memory T cells.At present,TRM has been found in the skin,lungs,mucous membrane of the vagina,brain and even in lymphoid tissue.The phenotype of CD8+TRM cells is CD69+CD103+,and the phenotype of CD4+TRM cells is CD69+CD11a+.Allergic asthma is a common,complex and polygenic control of chronic respiratory disease,which is characterized by chronic airway inflammation and nonspecific airway hyper-responsiveness.A variety of inflammatory cells,such as eosinophils,mast cells,T lymphocytes,neutrophils,macrophages and airway epithelial cells,and inflammatory factors are involved in the inflammatory response of allergic asthma.Its clinical symptoms are characterized by recurrent attacks of wheezing,shortness of breath,chest tightness and cough.At present,nearly 300 million people around the world suffer from allergic asthma,there are about 30 million patients in China,and its incidence is still increasing by nearly 50% per decade,serious harms to people's health.Allergic asthma belongs to the category of secondary immune response,and allergens can induce immune response and form specific TRM,existed in the lungs for a long time.The pulmonary TRM which may play an important role in the pathogenesis of allergic asthma will be activated quickly when allergens enter the respiratory tract again.The present study take advantages of allergic asthma model in mice to investigate the role of pulmonary TRM in the pathogenesis of allergic asthma.Methods:1 Establishment of allergic asthma model in mice1.1 Sensitization and inspirationSix week old female BALB/c mice were randomly divided into two groups: Control group and asthma model group.Asthma model group: Each mouse was injected with 200?l OVA sensitization liquid intraperitoneally on 0 day and 14 day.And each mouse was anesthetized with 100-200?l 1% pentobarbital sodium and inhaled 50?l 0.4%OVA inspiration liquid intranasally for 5 days since 35 days of the experiment.Control group: Mice were treated with PBS as above methods.1.2 Detection of IgE in serumWe collected the blood of mouse eyes and separated the serum at two time points respectively: the third week after the last sensitization and 24 hours after the last inspiration.Then the level of OVA specific antibody IgE was detected by ELISA.1.3 Detection of lung airway resistance and dynamic lung compliance by airway hyper-responsiveness test1.4 The mice were sacrificed to observe inflammatory infiltration and airway mucus secretion in lung tissue by HE staining and PAS staining respectively.1.5 The concentration of cytokine including IFN-?,IL-4,IL-17,IL-1?,MCP-1 and MIP-1?.in supernatant of bronchoalveolar lavage fluid and lung homogenate was examined by ELISA1.6 Counting the different inflammatory cells in bronchoalveolar lavage fluid by Wright-Giemsa staining1.7 the mRNA expression of IL-5,IL-10,IL-13,IFN-?,IL-1?,MCP-1,MIP-1?,RANTES,Eotaxin and GRO-? in lung tissue was examined by Real-time PCR2 Analysis of the TRM in OVA-sensitized mouseMice were splited and sensitized as the same as 1.1.The number of memory cells in lung and spleen was detected by Flow Cytometry respectively at the third week after the last sensitization.3 Analysis of the role of TRM in allergic asthma by injecting adoptive transfer memory T cells to mice and inhibiting TRM using inhibitor FTY7203.1 Six week old female BALB/c mice were randomly divided into five groupsAdoptive transfer of lung PBMC group: Sensitization method was the same as 1.1.Lung mononuclear cells of two mice were collected and injected into normal mouse by tail intravenous injection on the thirty-fifth day of experiment.And each adoptive transfer mouse was anesthetized with 100-200?l 1% pentobarbital sodium and inhaled 50?l 0.4%OVA inspiration liquid intranasally for 5 days after one week.Adoptive transfer of lung TRM cells group: Sensitization method was the same as 1.1.Lung TRM cells of two mice were collected and injected into normal mouse by tail intravenous injection on the thirty-fifth day of experiment.And each adoptive transfer mouse was anesthetized with 100-200?l 1% pentobarbital sodium and inhaled 50?l 0.4%OVA inspiration liquid intranasally for 5 days after one week.Adoptive transfer of spleen memory T cells group: Sensitization method was the same as 1.1.Spleen memory cells of two mice were collected and injected into normal mouse by tail intravenous injection on the thirty-fifth day of experiment.And each adoptive transfer mouse was anesthetized with 100-200?l 1% pentobarbital sodium and inhaled 50?l 0.4%OVA inspiration liquid intranasally for 5 days after one week.Only challenged group: The mice was anesthetized with 100-200?l 1% pentobarbital sodium and inhaled 50?l 0.4%OVA inspiration liquid intranasally for 5 days since 42 day of experiment.Immunosuppressor group: Each mouse was injected with 200?l OVA sensitization liquid and 10?l FTY720 immunosuppressor diluent intraperitoneally on 0 day and 14 day.Then each mouse was anesthetized with 100-200?l 1% pentobarbital sodium and inhaled 50?l 0.4%OVA inspiration liquid intranasally for 5 days at the third week after the last sensitization.3.2 The analysis and detection were the same as 1.3-1.7.Res?lts:1 Asthma model of mice was successfully established1.1 Ig E: Compared with the control group which failed to detect IgE,the titer of OVA specific antibody IgE in the serum of asthma model group increased significantly.1.2 Lung tissue pathological changes in mice: The HE staining showed that mice of control group had integrated alveolar wall,had a few inflammatory cells around alveoli and had no bodiness of bronchial wall.While mice of asthma model group got opposite results.On the other hand,the PAS staining showed that there was no mucus in mice of control group,while there existed much mucus in mice of asthma model group.1.3 The results of airway hyper-responsiveness test: Compared with the control group,the RI of asthma model group increased significantly and Cdyn decreased prominently2 TRM was induced by OVA sensitization: Compared with the control group,the number of OVA specific TRM in lung tissue and TCM in spleen of asthma model group increased significantly.3 Results of detection of six groups3.1 IgE: There was no production of IgE in control group and there was no significant difference of IgE among sensitization group,asthma group and Immunosuppressor group.But compared with control group,IgE of above three groups increased significantly.Compared with control group,titer of Ig E of Only challenged group,adoptive transfer of lung PBMC group,adoptive transfer of lung TRM cells group and adoptive transfer of spleen memory T cells group all increased significantly.And there was no difference among them.3.2 Results of airway hyper-responsiveness showed that mice of asthma model group,Adoptive transfer of lung PBMC group were all induced to airway hyper-responsiveness,but asthma model group was more obvious;We can't detect obvious airway responsiveness change in mice of Immunosuppressor group and Adoptive transfer of lung TRM cells group when using low-dose Mch.The results indicated that TRM participated in airway hyper-responsiveness.3.3 Pathological changes of lung tissue in mice3.3.1 The HE staining showed that mice of control group had integrated alveolar wall,had a few inflammatory cells around alveoli and had no bodiness on bronchial wall.While mice of asthma model group had broken lung tissues,had a large of inflammatory cells around alveoli and bronchia,had bodiness on bronchial wall and also had narrow luminal.Compared with control group,there were not many inflammatory cells in Adoptive transfer of lung PBMC group,Adoptive transfer of lung TRM cells group,but had obvious bodiness on bronchial wall.Compared with asthma model group,inflammatory cells infiltration of Immunosuppressor group were distributed around bronchus.3.3.2 PAS staining showed that there was no mucous in control group and only challenged group,but there were a little mucous in Adoptive transfer of lung PBMC group and Adoptive transfer of lung TRM cells group.While there were a lot of mucous in asthma model group and Immunosuppressor group.3.4 Counting the different inflammatory cells in bronchoalveolar lavage by Wright-Giemsa staining.Compared with control group,the total number of white blood cells,the proportions of eosinophils,neutrophils and lymphocytes in asthma model group and Immunosuppressor group increased significantly,while the proportion of macrophages decreased significantly.Compared with asthma model group,the proportions of eosinophils in Immunosuppressor group increased significantly,while the proportion of lymphocytes decreased significantly.The total number of white blood cells,the proportions of eosinophils and lymphocytes in Adoptive transfer of lung PBMC group also increased significantly compared with control group.3.5 The concentration of cytokine including IFN-?,IL-4,IL-17,IL-1?,MCP-1 and MIP-1? in bronchoalveolar lavage fluid and lung homogenate was detected by ELISACompared with control group,concentration of IL-4,IL-1?,MCP-1 and MIP-1? in asthma model group increased significantly.Compared with asthma model group,concentration of IL-4 and MCP-1 in Immunosuppressor group increased significantly.While only the concentration of IFN-? in asthma model group increased significantly compared with all other groups.The concentration of IL-1? of lung homogenate in Immunosuppressor group decreased significantly compared with asthma model group.3.6 the mRNA expressions of IL-5,IL-10,IL-13,IFN-?,IL-1?,MCP-1,MIP-1?,RANTES,Eotaxin and GRO-? of lung tissue was detected by Real-time PCR techniqueCompared with control group,the mRNA expressions of IL-5,IL-10,IL-13,IL-1?,MCP-1,MIP-1?,RANTES,Eotaxin and GRO-? in asthma model group and Immunosuppressor group increased significantly.And compared with asthma model group,the mRNA expressions of IL-5,IL-10,IL-13,MCP-1,RANTES,Eotaxin and GRO-? in Immunosuppressor group also increased significantly.Compared with control group,the mRNA expression of IFN-? in asthma model group increased significantly,the mRNA expressions of IFN-? in Immunosuppressor group,Adoptive transfer of lung PBMC group,Adoptive transfer of lung TRM cells group,Adoptive transfer of spleen memory T cells group and Only challenged group decreased significantly.All the above results indicated that TRM played an important role in the balance between Th1 and Th2 cells.Conclusion:1 Establishment the OVA-induced mouse allergic asthma model successfully.2 TRM in lung of model mice was detected successfully.3 TRM of lung cells participated in the airway hyper-responsiveness of allergic asthma,but other cells such as mononuclear cells of lung aslo played an important role in it.4 TRM had an influence on the balance between Th1 and Th2 cells in allergic asthma.The reduction of TRM induced the dominant Th2 immune response and resulted more serious inflammation in lung and airway.