| Part Ⅰ impact of Tobacco smoke exposure on expression of mouse lung dendritic cells(CDllc+),Th17 cells and regulatory T cellsObjective:This part tried to explore immune role of dendritic cells in chronic pulmonary inflammatory with COPD via a mouse pattern produced by tobacco smoke exposure,as detailed in impact of tobacco smoke on expressions of mouse lung dendritic cells costimulatory molecules on dendritic cells,CD4+CD25+FOXP3+cells,CD4+IL-17+cells,and CD4+IL-10+ cells along with relevant cytokines.Methods:90 healthy male Balb/c mice at SPF level were randomly assigned into 3 groups,consisting of control-group(A group),4-week of tobacco smoke exposure-group(B group),and 24-week of tobacco smoke exposure-group(C group)and each one having 30 mice.The C group was placed in a self-made glass container for tobacco smoke exposure that was executed with 10 tobaccos on fire for 30 consecutive minutes daily and lasting 5 days every week up to 24 weeks.The A group was put into another self-made glass container with the same size as the box of C group’s and allowed to move freely for 30 minutes continuously per day,covering 5 days a week and 24 weeks.The B group experienced the equal process at the first 20 weeks as the A group did and was operated in such a way at the last 4 weeks as identical as the C group was.All the mice were subjected to the following experiment protocol in batches at the end of 24 weeks.Right lower lobe tissues were fetched to check pathological morphology of both pulmonary parenchyma and small airways with HE stain.Single cells of lung tissue were extracted and among which the proportion of CD11c+expression were detected with flow cytometry.DCs were sorted with magnetic activated cell sorting and examined for pure cell population with flow cytometry that also was applied to detect expression of the attached costimulatory molecules.The proportion of CD4+CD25+FOXP3+,CD4+IL-17+,and CD4+IL-10+expression in single cells of lung were detected with flow cytometry.The expression of CD40,FOXP3+ and RORytmRNA in pulmonary tissue was quantified respectively by Fluorescence quantitative PCR.Concentrations of IL-10,IL-6,IL-12,and IL-17A in peripheral serum and lung tissue were measured by Liquid phase chip technology among those mice exposed to tobacco smoke.Result:Infiltration of inflammatory cells with mild enlargement in partial air sacs were indicated around airway and within alveolar septum among those mice affected by 4-week exposure to tobacco smoke.Besides inflammatory infiltration,24-week exposure to tobacco smoke further developed airway smooth muscle hyperplasia and obviously damaged alveolus.Compared to the control-group,other two tobacco smoke exposure groups revealed significantly increased ratio of CD11c+(P<0.05),distinctively mounting expression of CD40,CD80,and MCH Ⅱ(P<0.05),but in terms of CD86,the 4-week tobacco smoke exposure group showed significantly hign expression(P<0.05)and the 24-weeks group kept stable(P>0.05).The 4-week tobacco smoke exposure group demonstrated significantly higher proportions of CD4+CD25+FOXP3+,CD4+IL-17+(P<0.05).than the control group did.Regardless of significantly increased presence of CD4+IL-17+(P<0.05).as similarly showed in another smoke-affected group,the 24-week tobacco smoke exposure group revealed contrary tends with obviously lower proportions of CD4+CD25+FOXP3+(P<0.05)and CD4+IL-10+(P<0.05)than the control group’s.Expressions of CD40,FOXP3+and ROR RORytmRNA measured by PCR were consistent with that by flow cytometry.Concentrations of IL-6,and IL-17A in peripheral serum,and especially in lung tissue measured by Liquid phase chips were higher in two smoke-affected groups than in the control group.IL-10 exhibited high value in the 4-week tobacco smoke exposure group but decreased in the 24-week tobacco smoke exposure group.Conclusion:(1)short-term tobacco smoke exposure(4-week exposure)promoted expressions of the costimulatory molecules CD40,CD80,CD86 and MHC II,which suggests tobacco exposure can boost activity of dendritic cells.(2)long-term tobacco smoke exposure(24-week exposure)elevated level of dendritic cells,strengthened expression of CD40 and led to abnormal expressions of Th 17,regulatory T cells as well as relevant cytokines,which result in worsened inflammation and damage of lung.Part Ⅱ Impact of CD40-CD40L pathway on myeloid dendritic cells inducing to differentiate CD4+T cells into Th17 and regulatory T cells in a mouse model established by Tobacco Smoke ExposureObjective:This part tried to explore impact of smoking and CD40-CD40L pathway on role of myeloid dendritic cells in T lymphocyte differentiation,as detailed in influence of duration of tobacco smoke exposure towards expression of costimulatory molecules attached to mouse myeloid dendritic cells;and affection of myeloid dendritic cells on T lymphocytes differentiating into Th17 cells and regulatory T cells by blocking up CD40-CD40L pathwayMethods:Myeloid monocytes were prepared with lymphocyte separation medium from all the 90 mice among the control group,the 4-week tobacco smoke exposure group and the 24-week tobacco smoke exposure group,all that had been developed in Part I.A combination of Recombinant Murine GM-CSF(rm-CSF,40ng/ml)and Recombinant mouse IL-4 was applied in vitro to induce myeloid monocytes to differentiate into dendritic cells(DCs).DCs were sorted and purified with CD 11 c+magnetic activated cell sorting,then detected for expression of costimulatory molecules attached with flow cytometry and proliferation activity with MTT colorimetric method.CD4+T cells were sorted with magnetic activated cell sorting from mice spleens of the control group.Myeloid DCs deriving from all the three groups were co-cultivated with CD4+T cells and the co-cultivation combining DCs and CD4+T cells was regrouped to the following 6 subgroups marked with capital letters,A1-subgroup referring to DCs of the control group being co-cultivated with CD4+T cells,A2-subgroup to A1-subgroup plus CD40 antagonist,B1-subgroup to DCs of the 4-week tobacco smoke exposure group being co-fostered with CD4+T cells,B2-subgroup to B1-subgroup plus CD40 antagonist,C1-subgroup to DCs of the 24-week tobacco smoke exposure group being co-laid with CD4+T cells,and C2-subgroup to Ci-subgroup plus CD40 antagonist.After 24-hour cultivation in serum-free medium,all the six subgroups collected fostered cells and detected proportions of CD4+CD25+FOXP3+,CD4+IL-17+,CD4+IL-10+cells with flow cytometiy as well as concentrations of IL-10,IL-6 and IL-17A in cell supernatant with Liquid phase chip technology.Result:Both two tobacco smoke exposure groups indicated significantly increasing expressions of costimulatory molecules attached to mouse myeloid dendritic cells,CD40(P<0.05)than the control group did.DCs proliferation activity didn’t show significant difference between the control group and the 4-week tobacco smoke exposure group,but 72-hour DCs proliferation activity decreased in the 24-week tobacco smoke exposure group.Compared to the control group,B1-subgroup in the 4-week tobacco smoke exposure group indicated significantly increased proportion of CD4+IL-17+cells(P<0.05)but decreased proportions of CD4+CD25+FOXP3+cells(P<0.05);B2-subgroup revealed rising proportions of CD4+CD25+FOXP3+cells,but obviously reductive proportion of CD4+IL-17+cells(P<0.05)in the presence of CD40 antagonist;Ci-subgroup in the 24-week tobacco smoke exposure group exhibited significantly high proportion of CD4+IL-17+cells(P<0.05)that was lessened significantly(P<0.05)by supplement of CD40 antagonist in C2-subgroup that also showed increased proportion of CD4+IL-10+cells(P<0.05).Both B2-subgroup and C2-subgroup revealed significantly higher concentration of IL-10 but obviously reductive level of IL-6 and IL-17A(P<0.05).Conclusion:(1)Both 4-week and 24-week tobacco smoke exposure promoted expressions of the costimulatory molecules CD40 attached to mouse myeloid dendritic cells and differentiation of T lymphocytes into Th17 cells;(2)Blocking up CD40 cell signal channel can impede the differentiation from T lymphocyte into Th17 cells,which suggest CD40 signal channel significantly impact on the sigh of T cell differentiating into Th17 cell on the mouse model exposed by tobacco smoke... |
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