CD40 Regulate The Differentiation Of Tc1induced By Dendritic Cells In The Presence Of Cigarette Smoke Exposure

PartⅠ Dendritic cell induce Tc1 cells differentiation by CD40/CD40L pathway in Mice of Cigarette Smoke-exposureObjective:To explore the role of CD40/CD40L pathway on dendritic cells regulation Tc1 cells differentiation in Mouse Models of cigarette smoke-induce emphysema.Methods:C57BL/6J mice and CD40-deficient mice were exposed to smoke or air for up to 6 months.The distribution and differentiation of Tc1 cells in these mice were investigated.Lung dendritic cells distribution and their costimulatory molecules were also investigated.Results:（1）To quantify emphysema,it is recommended to measure the L_m.In C57 mice,a significant increase in Lm（38.16±3.16）was observed when exposure to CS compared with those exposure to airL_m（21.88±1.59）.Additional,in CD40^-/-mice,when exposure to air the L_m（22.24±1.81）were lower compared with those（30.22±1.69）exposure to CS.However,when CD40^-/-smoke-exposed mice were compared with C57 smoke-exposed mice,the L_mwere lower,which indicated that CD40^-/-mice protection against emphysema when exposure to chronic CS.Addition,these was no significant differences between CD40^-/-and C57 when those mice exposured to air..（2）After 24weeks cigarette smoke exposure,lung single-cell were isolated and the CD11c⁺cells were identify by flow cytometry.We found that the percentage of DCs was significant increase of following smoke exposure(0.95±0.32%in C57control vs 1.55±0.26%in C57smoke-exposed;0.93±0.15%in CD40^-/-control vs 1.48±0.44%in CD40^-/-smoke-exposed).Moreover,when compared with C57 control and CD40^-/-control,C57smoke-exposed and CD40^-/-smoke-exposed mice showed a significant increased of the percentage of DCs,but the difference was not found,neither between C57smoke-exposed and CD40^-/-smoke-exposed mice nor between C57 control and CD40^-/-control mice.To detect the effect of cigarette smoke exposure on the maturation of DCs,we investigated the costimulatory molecules such as CD40,CD80 and MHCI on the surface of lung CD11c⁺.Compared with air-exposed mice,the expression of CD40,CD80 and MHCI on lung DCs was higher in smoke-exposed mice.（3）after 24weeks exposure to air or CS.In spleen,there was no significant different of perccentage of CD8⁺T among C57control,CD40^-/-control and CD40^-/-smoke exposed mice.However,when compared with these mice(13.69±2.10%in C57control;11.69±1.99%in CD40^-/-control and 12.04±2.60%in CD40^-/-smoke-exposed),the C57smoke-exposed mice（20.75±5.24）showed a significant increase of percentage of CD8+T cell in spleen.Interesting,the percentage of CD8⁺T was significant increase of following smoke exposure in lung(11.11±2.46%in C57control vs 19.03±3.24%in C57smoke-exposed;12.26±1.86%in CD40^-/-control vs 16.30±1.91%in CD40^-/-smoke-exposed).Addition,when compared with C57control mice,CD40^-/-smoke-exposed showed a significant increase of percentage of CD8+T cell while compared with C57smoke-exposed,there was a significant decrease of that.The percentage of CD8⁺T cell in lung between C57control and CD40^-/-control showed no significant differences.（4）The percentage of Tc1 was significant increase of following smoke exposure in both spleen(14.29±4.19%in C57control vs 28.04±4.77%in C57smoke-exposed;7.07±4.87%in CD40^-/-control vs 17.37±8.76%in CD40^-/-smoke-exposed)and lung(11.10±2.46%in C57control vs 26.47±4.41%in C57smoke-exposed;2.87±1.53%in CD40^-/-control vs 8.55±2.47%in CD40^-/-smoke-exposed).Compared with C57smoke-exposed mice,there was a significant reduced in CD40^-/-control and CD40^-/-smoke-exposed mice.Addition,C57control mice showed a significant increase in the percentage of Tc1 when compared with CD40^-/-control mice in both spleens and lungs.No difference was observed between C57control and CD40^-/-smoke-exposed in spleens and lungs.（5）Consistent with the results of percentage of Tc1 in lung,the expression of T-bet in lung significant increase of following smoke exposure(1.46±0.69 in C57control vs 2.31±0.71 in C57smoke-exposed;0.42±0.17 in CD40^-/-control vs 1.03±0.38 in CD40^-/-smoke-exposed).Compared with CD40^-/-control and CD40^-/-smoke-exposed mice,there was a significant increase in C57smoke-exposed mice.And then,C57control mice showed a significant increase in the expression of T-bet m RNA when compared with CD40^-/-control mice.However,there was no significant difference of expression of T-bet m RNA in lung between C57control and CD40^-/-smoke-exposed mice（6）The concentration of TNF-a in the BLAF was elevated when mice exposed to smoking(14.26±3.37inC57controlvs70.92±25.27in C57smoke-exposed;15.55±4.48 in CD40^-/-control vs 46.08±17.93 in CD40^-/-smoke-exposed).ComparedwiththeC57smoke-exposed,the concentration of CD40^-/-smoke-exposed was lower,but higher than C57control.Addition,the concentration between C57control and CD40^-/-control was no different.Conclusion:Cigarette smoke exposure increased the percentage of lung dendritic cells and then induced the mature of these cells to promote Tc1 cells differentiation.The CD40/CD40L pathway plays an important role in Tc1 cells differentiation independent of help of CD4+T cells and blocking the pathway can protect from emphysema when mice exposure to CS.Part Ⅱ Dendritic cell induce Tc1 cells differentiation by CD40/CD40 L pathway in Mice of Cigarette Smoke-exposureObjective:To demonstrate the mechanism of CD40 regulate the differentiation of Tc1 in the presence of CSE.Methods:m DCs were obtained from bone marrow-derived DCs（BMDC）as detailed previously by modifying.Briefly,Ficoll-Hypaque gradient centrifugation was used to isolated the mononuclear cells from bone marrow.Subsequently,a density of 1x106 of mononuclear cells were plated in six-well plates and cultured in with RPMI-1640 which contained GM-CSF（40ng/ml）and IL-4（10ng/ml）.（Pepro Tech London,UK）At day 2 and 4,6,one-half of the old medium was discarded and then the same volume fresh medium was added into the plates.At day 7,the cells were harvested and washed,and then were cultured in the presence or not of CSE（0.3%）for 12 h.After that,the culture supernatant was collected to analysis the IL-12p70 by ELISA.And then the cells were washed and were co-cultured with allogenic naive CD8+T cells at a ratio（1x106T cells /2x105DCs）for 24 h in 24-well plates.The naive CD8+T cells were isolated by the negative select（life,USA）.The Tc1 differentiation was tested by flow cytometry.Results:Although we found that CD40 is required for Tc1 responses to cigarette smoke exposure in vivo,However,many cells can express CD40,such as dendritic cell,B cell and macrophage cell,so which cell plays an important role in Tc1 differentiation is unknown.Addition,whether DCs induce Tc1 differentiation through CD40/CD40 L independent of the help of CD4+T or not also be unknown.Interesting,We found that CSE can significantlyup-regulated the expression of CD40（9.25±4.13% in untreated m DCs vs29.29±4.97% in CSE-treated m DCs）and also increased the secretion of IL-12/p70（312.49±38.91pg/ml）in untreated m DCs vs（1204.80±12.99pg/ml）in CSE-treated m DCs.However,these effects were not detected CD40^-/-DC（data was not shown）.Compared CD8+T cells（13.56±2.95%）,and the DCs co-cultured control group（15.45±5.22%）,the CSE-pretreated DCs co-culture group（22.49±4.11%）shown a significantly increased in the expression of IFN-γ.Addition,the CSE-pretreated DCs co-culture group also have a higher expression of IFN-γ,when compared with the CD40^-/-DCs co-cultured control group（14.05±1.22%）and CSE-pretreated CD40^-/-DCs co-culture group（15.83±1.95%）.However,there was no significantly difference among CD8+T cells control,CD40^-/-DCs co-cultured control group and CSE-pretreated CD40^-/-DCs co-culture group.Conclusion: In the presence of CSE,m DC can upregulate the expression of CD40 and induce CD8+T cell differentiation into Tc1 cell by IL-12,which was secretion by m DC.Part Ⅲ The percentage of Myeloid DCs（% of leukocytes）and the expression of CD40 in the peripheral blood from COPD patientsObjective: To observe the percentage of Myeloid DCs（% of leukocytes）and the expression of CD40 in the peripheral blood from COPD patients.Methods: 12 never-smokers,10 smokers and 16 patients with the diagnosis of COPD were included in the study.4-5ml of peripheral blood of each case was collected with empty stomach.The blood was used to analysis of the percentage of dendritic cells and the expression of costimulatory molecules CD40.These case of lung function were also analysis,espectially the FEV1/% pred.Results:Characteristics of the participants are detailed in Table 1.There were no differences in age and gender distribution between the groups.There was no difference in the number of pack years（PY）between the group of asymptomatic smokers and the group of patients with COPD.Patients with COPD displayed significantly worse lung function parameters,as compared to both control groups.Although the percentages of m DCs in blood were significantly higher in never-smokers than in smokers and patients with COPD,there were no difference between smokers and patients with COPD.In addition,Patients with COPD displayed a significantly increased expression of CD40（Figure 3C）on m DCs,as compared with never-smoking controls.However,the expression of this markers did not differ between smokers and patients with COPD.Moveover,smokers also displayed a significantly increased expression of CD40,as compared with never-smoking controls.Conclusion :Smoking exposure can decrease the percentage of Myeloid DCs（% of leukocytes）but upregulated the expression of CD40.