| PART I THE EXPRESSION OF GITRL IN LUNG TISSUES AND DENDRITIC CELLS OF ASTHMATIC MICEOBJECTIVE: To observe the expression of GITRL in lung tissues and dendritic cells of asthmatic mice,and to understand the correlation between GITRL and asthma.METHODS: C57BL/6 mice were randomly divided into normal saline group and house dust mites(HDM)-challenged group.24 hours after the last challenge,the airway hyperresponsiveness was detected by invasive pulmonary function analyzer,and the serum,lung tissues and bronchoalveolar lavage fluid(BALF)were collected.The serum total Ig E content was detected by ELISA kit.The lung inflammation was observed by HE staining.The total number of BALF cells was counted by cell counter,and eosinophil ratio was counted.The expression of GITRL m RNA and protein were tested by Q-PCR,Western blot and immunohistochemistry.GITRL expressed on the surface of dendritic cells was detected by flow cytometry.RESULTS:(1)The invasive lung function and HE staining of lung tissues showed that the airway hyperresponsiveness and lung inflammation in HDM-challenged group were higher than those in control group.ELISA and BALF cells count showed that the serum Ig E content,the total number of BALF cells and the absolute count of eosinophils in HDM-challenged group were higher than those in control group.The above results indicated that the HDM-induced asthma model was successfully established;(2)Q-PCR,Western blot and immunohistochemistry showed that GITRL m RNA and protein in the lung tissues of HDM challenged mice were significantly increased;(3)Flow cytometry showed that the proportion of GITRL on the surface of lung dendritic cells was increased in HDM-challenged mice.CONCLUSION: GITRL m RNA and protein in asthma group were significantly higher than those in the control group,and the expression of GITRL on the surface of dendritic cells was increased,suggesting that GITRL on the surface of dendritic cells may play a role in the pathogenesis of asthma.PART Ⅱ THE EFFECT OF GITRL KNOCKDOWN ON ASTHMA PHENOTYPE AND CD4+ T CELL DIFFERENTIATION IN MICEOBJECTIVE: To observe the effect of GITRL on airway inflammation and airway hyperresponsiveness in asthma,and the effect on the differentiation of CD4+ T cells in lung tissues.METHODS: AAV-GFP and AAV-sh GITRL were constructed and given to mice by nasal route.After 2 weeks of transfection,GITRL knockdown efficiency was detected by immunofluorescence and Western blot.Flow cytometry was used to observe GITRL knockdown on the surface of dendritic cells,and HDM-induced asthma model was established after transfection.After 24 hours of HDM challenge,the airway hyperresponsiveness was detected by the invasive pulmonary function instrument.The serum total Ig E content was detected by ELISA,the inflammatory condition of lung was observed by HE staining and the inflammation score was made.The total BALF cells were counted by the cell counter,and eosinophil ratio was counted.The ratio of Th1,Th2,Treg and Th17 cells in the lung of mice was measured by flow cytometry.RESULTS:(1)Immunofluorescence and Western blot showed that the level of GITRL protein in lung of AAV-sh GITRL group was significantly lower than that of AAV-GFP group.Flow cytometry showed GITRL on lung dendritic cells of AAV-sh GITRL group challenged by HDM was significantly lower than that of AAV-GFP group;(2)Airway hyperresponsiveness and airway inflammation of AAV-sh GITRL group challenged by HDM were significantly lower than that of AAV-GFP group,and the serum Ig E content,BALF cells count and eosinophil absolute count were significantly reduced compared with those of HDM challenged AAV-GFP group;(3)Flow cytometry showed that the proportion of Treg cells in lung of HDM-challenged mice was lower than that of normal saline group,and the proportion of Th2 and Th17 cells increased.However,compared with the AAV-GFP mice challenged by HDM,the proportion of Treg cells in lung of AAV-sh GITRL group challenged by HDM was up regulated,and the ratio of Th2 and Th17 cells decreased significantly.There was no significant difference in Th1 cells in each group.CONCLUSION: Th1/Th2 and Th17/Treg cells in the lung of asthmatic mice are in imbalance.Knockdown of GITRL in lung and dendritic cells can significantly improve airway hyperresponsiveness and pulmonary inflammation in asthmatic mice,which may be related to regulating the differentiation of CD4+ T cells and restoring the balance of Th1/Th2 and Th17/Treg cells.PART Ⅲ EFFECT OF GITRL EXPRESSED BY BMDCS ON CD4+ T CELL DIFFERENTIATIONOBJECTIVE: To observe the role of GITRL expressed by BMDCs on the differentiation of CD4+ T cells in vitro.METHODS:(1)BMDCs were isolated and cultured in vitro,and the purity was identified by flow cytometry.BMDCs were stimulated with PBS or HDM for 24 hours,and GITRL expression on the surface of BMDCs was observed by Western blot,immunofluorescence and flow cytometry;(2)BMDCs were transfected with lentivirus LV-GFP and LV-sh GITRL,and the efficiency of transfection was observed by fluorescence microscope and flow cytometry,and the knockdown effect of GITRL was verified by Western blot,Q-PCR and immunofluorescence;(3)BMDCs were co cultured with splenocytes under the stimulation of PBS or HDM.The proportion of Th1,Th2,Treg and Th17 cells was observed by flow cytometry,and Foxp3,GATA3,RORγt and T-bet m RNA were detected by Q-PCR;(4)Spleen cells were stimulated by anti-CD3 or anti-CD3 and GITRL for 48 hours respectively.The ratio of Th1,Th2,Treg and Th17 cells was detected by flow cytometry,and Foxp3,GATA3,RORγt and T-bet m RNA was detected by Q-PCR.RESULTS:(1)The purity of BMDCs cultured in vitro was more than80%.Western blot,immunofluorescence and flow cytometry results showed that GITRL expression on the surface of BMDCs stimulated by HDM was significantly higher than that of PBS group;(2)Fluorescence microscopy and flow cytometry results showed that the proportion of GFP positive cells after lentivirus transfection was about 50%.Western blot,Q-PCR and immunofluorescence showed that compared with LV-GFP group,the protein and m RNA levels of GITRL in LV-sh GITRL group decreased by about 50%;(3)Flow cytometry showed that compared with PBS treatment group,the proportion of Treg cells decreased significantly in HDM treatment group,while the proportion of Th2 and Th17 cells increased significantly.However,compared with LV-GFP group,the proportion of Treg cells in LV-sh GITRL group was significantly increased,while the proportion of Th2 and Th17 cells was significantly decreased.There was no significant difference in the percentage of Th1 cells in each group.Q-PCR showed that compared with PBS treatment group,Foxp3 m RNA was significantly decreased,while GATA3 and RORγt m RNA were significantly increased in HDM treatment group.Knockdown of GITRL significantly increased Foxp3 m RNA and decreased GATA3 and RORγt m RNA.There was no statistically significant difference in T-bet m RNA among the groups;(4)Flow cytometry showed that compared with the cells treated with anti-CD3 alone,the percntage of Treg cells in the cells stimulated by anti-CD3 and GITRL decreased significantly,the percntage of Th2 and Th17 cells increased,and the percntage of Th1 cells had no significant difference.Q-PCR showed that under GITRL stimulation,the expression of Foxp3 m RNA was significantly decreased,while the expression of GATA3 and RORγt m RNA was increased,and T-bet m RNA had no significant change.CONCLUSION: HDM stimulation increases the expression of GITRL on the surface of BMDCs;GITRL on the surface of BMDCs may regulate the differentiation of CD4+ T cells and promote the imbalance of Th1 / Th2 and Th17/Treg cells;Knockdown of GITRL on the surface of BMDCs can partially restore the balance of Th1/Th2 and Th17/Treg cells.In vitro GITRL stimulation inhibited Treg cell differentiation and promoted Th2 and Th17 cells differentiation. |
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