Effect Of Adoptive γδT Cells And Inhalation Of Nebulized Mycobacterium Vaccae For Injection On Airway Inflammation And Cytokines In Asthmatic Mice

PART I EFFECTS OF γδT CELLS MEDIATED BY INHALATION OF NEBULIZED MYCOBACTERIUM VACCAE FOR INJECTION ON ASTHMATIC MICEOBJECTIVE: To treat asthmatic mice by extracting γδT cells from TCR-β-/-mice treated with nebulized inhalation injection of M.vaccae,and to investigate whether M.vaccae injection can affect asthma by regulating γδT cells.Airway inflammation in mice affects downstream cytokines IL-4 and IFN-γ.METHODS: Forty male Balb/c mice were divided into 4 groups according to random number table: normal group(A),asthma group(B),asthma treatment control group(C)and asthma treatment intervention group(D).(TCR-β-/-mice were divided into control group and treatment intervention group: treatment intervention group once a day,for 5 consecutive days inhalation injection of M.vaccae.The control group was given nebulized saline 10 ml.The spleen and the γδT cell suspension were injected into the BALB/C male mice to obtain the asthma treatment control group(C)and the asthma treatment intervention group(D).)The model group and the treatment control group and the treatment intervention group The mice were all sensitized and challenged with ovalbumin(OVA)to make a mouse bronchial asthma model.Group C and D were challenged with tail vein injection and injected with nebulized saline and nebulized inhalation with M.vaccae to interfere with γδT cells once a day until the end of the challenge.Lung tissue and bronchoalveolar lavage fluid(BALF)were extracted and sacrificed by pulmonary function test.Pathological HE staining was used to observe bronchopulmonary inflammation in lung tissue.The inflammatory cell counts of BALF were centrifuged;the expression levels of IFN-γ and IL-4 in BALF were detected by ELISA;the percentage of IL-4 and IFN-γ in lung tissue mononuclear cells was detected by flow cytometry.RESULTS:The airway responsiveness of the asthma group was higher than that of the normal group.The airway responsiveness of the asthma group was higher than that of the asthma treatment group and the treatment intervention group.Airway inflammatory lesions in the asthma treatment intervention group were alleviated compared with the asthma treatment control group.The ELISA results showed that the level of IL-4 in BALF in asthma group was significantly higher than that in normal group,while the level of IFN-γ was significantly lower.The level of IL-4 in BALF in asthma treatment group and treatment intervention group was higher than that in asthma group.Significantly decreased,while the IFN-γ level was elevated,the difference was statistically significant(p<0.05).The results of flow cytometry showed that the IL-4 secreted by γδT lymphocytes in the asthma group was significantly higher than that in the normal group,and the IFN-γ was significantly decreased,indicating the differentiation characteristics to Th2 cells.The difference was statistically significant(p<0.05).).In the asthma treatment group and the treatment intervention group,the secretion of IL-4 from the γδT cells in the lungs of the mice was significantly reduced compared with the asthma group,and the IFN-γ was significantly increased,indicating the differentiation characteristics of Th1 cells.The difference was statistically significant(p<0.05)CONCLUSION:Inhalation of M.vaccae can alleviate the Th2 dominant immune response induced by OVA sensitization and regulate Th1/Th2 immune imbalance in asthma.Inhalation of M.vaccae for injection by γδT cells affects airway inflammation in asthmatic mice,and its effect is related to the regulation of cytokine secretion.PART II EFFECTS OF ADOPTIVE γδT CELLS ON AIRWAY INFLAMMATION AND CYTOKINES IN ASTHMATIC MICEOBJECTIVE: To discuss the effects of γδT cells on airway inflammation and cytokines IFN-γ,IL-1β,IL-4 and IL-18 in asthmatic mice by using adoptive γδT cells from rat tail vein.METHODS: Divide 24 male Balb/c mice into 3 groups based on the random number table,which are normal control group(Group A),asthma model group(Group B),and asthma treatment control group(Group C).Each group has 8 mice.Create a mice bronchial asthma model(Ovalbumin(OVA)sensitization and challenge).Mice from Group C,after the challenge,was treated with tail vein adoptive γδT cells every day until the challenge was over.Right after examining the pulmonary functions of every mouse of each group by pulmonary function test,extract lung tissue and bronchoalveolar lavage fluid(BALF).Pathological H&E stain was used to observe bronchopulmonary inflammation in lung tissue.The inflammatory cell count of BALF was centrifuged;Using enzyme linked immunosorbent assay(ELISA)to examine IL-1β,IL-18,IL-4 and IFN-γ expressionlevels inmice from each group.Using flow cytometry(FCM)to examine the percentage of lung tissue mononuclear IL-4 and IFN-γin γδT cells.RESULTS: The airway responsiveness of the asthma group was higher than that of the normal control group and asthma treatment group.Airway inflammatory lesions in mice from the asthma treatment group were alleviated compared to that from the asthma group.The ELISA result shoed that IFN-γ level in BALF of mice from the asthma group was significantly lower than that of the normal control group,but IL-1β,IL-18,and IL-4 levels were significantly higher;IFN-γ level in BALF of mice from the asthma treatment group was significantly higher than that from the asthma group,but IL-1β,IL-18,and IL-4 levels were significantly lower,which were statistically significant(P<0.05).The FCM result shoed that IFN-γin lungsof mice from the asthma group,secreted by γδT lymphocytes,was significantly lower than that of the normal control group,IL-4 was significantly higher,showing the characteristics of differentiation toward TH2 cells,and the difference is statistically significant(P<0.05).IFN-γof mice from the asthma treatment group,secreted by γδT lymphocytes,was significantly higher than that from the asthma group,and IL-4 was significantly lower,showing the characteristics of differentiation toward TH1 cells,and the difference is statistically significant(P<0.05).CONCLUSION: Rat tail vein adoptive γδT cells can alleviate airway inflammation in asthmatic mice,lowering IL-1β 、 IL-4 and IL-18 levels,increasing IFN-γ levels,as well as reducing the TH2 dominant immune response caused by OVA sensitization.PARTⅢ EFFECTS OF INHALATION OF NEBULIZED MYCOBACTERIUM VACCAE FOR INJECTION ON NLRP3 INFLAMMASOME,IL-1Β AND IL-18 IN ASTHMATIC MICEOBJECTIVE:To observe the expression level of the NLRP3 inflammatory corpuscles in peripheral blood mononuclear cells in asth-matic mice,as well as the level change in the serum downstream factors,interleukin-1β(IL-1β)and interleukin-18(IL-18)after inhalingnebulized Mycobacterium vaccae for injection,to discuss its impact on the NLRP3 inflammasome,IL-1β and IL-18 in asthmatic mice.METHODS: Divide 40 male Balb/c mice into 4 groups based on the random number table,which are general control group(Gro-up A),asthma model group(Group B),asthma treatment control gr-oup(Group C)and treatment intervention group(Group D).Createa mice bronchial asthma model(Ovalbumin(OVA)sensitization and challenge).Group C,after the challenge,was given an inhalation of nebulized Mycobacterium vaccae for injection,every day.Group D,before the challenge,was given the inhalation of nebulized Mycobacterium vaccae for injection,every day.Right after examining the pulmonary functions of every mouse of each group bypulmonary function test,extract lung tissue and bronchoalveolar lavage fluid(BALF).Pathological H&E stain was used to observe bronchopulmonary inflammation in lung tissue.The inflammatory cell count of BALF was centrifuged;Using the real-time quantitative polymerase chain reaction(PCR)to examine NLRP3 in mononuclear cells in peripheral blood of mice from each group.Using enzyme linked im-munosorbent assay(ELISA)to examine serum IL-1β and IL-18 leve-ls inmice from each group.RESULTS: The airway responsiveness for mice of the asthma group was higher than that of the normal control group,as well as that of the asthma treatment group and intervention group.NLRP3 in the mononuclear cells,IL-1β and IL-18 in the serum in periphe-ral blood of mice from the asthma group are higher than that of mice from the normal control gr oup(P<0.01);NLRP3 in the mononuclear cells,serum IL-1β and IL-18 in peripheral blood of mice fro-m the asthma group are higher than that of mice from both asthma treatment and intervention groups(P<0.05).CONCLUSION: The levels of NLRP3 inflammasomes as well as the downstream factors in IL-1β and IL-18 in peripheral blood of asthmatic mice are elevated,and NLRP3 inflammasomes pathwaymight promote the pathogenesis of asthma.The inhalation of nebuli-zed Mycobacterium vaccae for injection can alleviate the airway inflammation in bronchial asthmatic mice,the effect of which is associated with the regulation of NLRP3 inflammasomes as well as serum IL-1β and IL-18.