Study On The Functional Changes And Mechanism Of Dendritic Cells In Smoker With Acute Exacerbation Of Chronic Obstructive Pulmonary Disease

PART 1 Effect of Smoking on Activation of Myeloid Dendritic Cells in Peripheral Blood of Patients with Acute Exacerbation of Chronic Obstructive Pulmonary DiseaseObjective:The acute exacerbation of chronic obstructive pulmonary disease(COPD)is difficult to control.Smoking is an independent risk factor for acute exacerbation of COPD(AECOPD).Whether its occurrence is related to changes in dendritic cell activation is still unclear.This part of the study focuses on the effects of continuous smoking on the activation of peripheral blood myeloid dendritic cells(mDC)in patients with AECOPD.Methods:Eight AECOPD patients in the current smoker group who smoked more than 10 cigarettes per day after diagnosis of COPD in the outpatient department and inpatient department of the First Affiliated Hospital of Guangxi Medical University from June 2019 to June 2020 and eight AECOPD patients in the former smoker group who had quit smoking for more than 1 year were collected,and peripheral blood was extracted for detection of mDC activation and inflammatory factors.Flow cytometry was used to detect the phenotypic characteristics(CD40,CD80 and CD86)of mDC activation in the peripheral blood of the subjects in each group.Enzyme linked immunosorbent assay(ELISA)was used to detect the level of IL-6 in plasma.Results:(1)There was no statistically significant difference between the general clinical data of AECOPD patients in the smoking group and AECOPD patients in the smoking cessation group(p>0.05).(2)Compared with the former smokers AECOPD patients,the percentage of CD40~+mDC in the peripheral blood of the current smokers AECOPD patients had no change,and the difference was not statistically significant(p>0.05),while the percentage of CD80~+mDC and CD86~+mDC statistically reduced(p<0.05).(3)There was no statistically significant difference in the plasma IL-6 level between the current smokers AECOPD patients and the former smokers AECOPD patients(p>0.05).Conclusion:Continuous cigarette smoke exposure suppressed the positive expression of CD80~+mDC and CD86~+mDC in peripheral blood of AECOPD patients.There was no difference in IL-6 levels between AECOPD patients with continuous cigarette smoke exposure and AECOPD patients with smoking cessation.PART 2 Cigarette Smoke Affects LPS and poly(I:C)-Induced Myeloid Dendritic Cell Activation and the Downstream Pathways of TLR2,TLR3 and TLR4 and the Secretion of Inflammatory FactorsObjective: In the first part of the study,it was concluded that smoking inhibits the activation of mDC in the peripheral blood of patients with AECOPD.In this part,by studying cigarette smoke extract(CSE)on LPS and poly(I:C)-induced activation of myeloid dendritic cells and on TLR2,TLR3 and TLR4 downstream pathways and the effects of inflammatory cytokine secretion,to explore possible mechanisms.Methods: Mononuclear cells in C57/BL mouse bone marrow cells were obtained using mouse bone marrow lymphocyte separation fluid.The mononuclear cells contained granulocyte-macrophage colony stimulating factor(GM-CSF)and interleukin 4(IL-4).After induction in the medium for 6 days,high-purity immature myeloid dendritic cells(BMDC)can be obtained,and the purity is checked by flow cytometry.Then use 0.1% cigarette smoke extract(CSE),0.2% CSE and 0.3% CSE to treat BMDC respectively,and observe the activity and activation degree of BMDC by inverted phase contrast microscope observation,trypan blue staining experiment and flow cytometry.Selected the most suitable intervention CSE concentration.Next,BMDCs were randomly divided into blank control group,CSE group,LPS group,poly(I:C)group,CSE+LPS group and CSE+poly(I:C)group.Finally,flow cytometry was used to detect the positive expression of BMDC surface activation phenotype,TLR2,TLR4 and intracellular TLR3 in each group.Real-time fluorescence quantitative PCR(qRT-PCR)were detected the mRNA transcriptions of TLR3, TRIF,and p65 which in the key factor of TLR-nuclear κB(NF-κB)pathway.ELISA was used to detect the content of interferon α(IFNα)and interleukin 6(IL-6)in the cell supernatant.Results:(1)The purity of BMDC induced by the above method is greater than 85%.(2)Compared with the blank control,the cell activity rate of 0.2% and 0.3% CSE intervention was reduced.In the comparison of activation degree,only with 0.3% CSE intervention CD86 positive expression were increased,the difference was statistically significant(p<0.05),There were no difference among the other groups(p> 0.05).Based on the above results,this study used 0.1% CSE as the concentration for subsequent experiments.0.1% CSE can inhibit BMDC activation induced by LPS and poly(I:C),the difference is statistically significant(p<0.05).(4)0.1% CSE alone can inhibit TLR2 and promote the positive expression of TLR4 on BMDC,the difference is statistically significant(p<0.05).LPS can promote the expression of TLR2 on BMDC(p<0.05),but has no effect on the expression of TLR4(p>0.05),and poly(I:C)has no effect on the expression of TLR2 and TLR4.TLR3 is low expressed in BMDC induced by GM-CSF.CSE inhibited the upregulation of TLR2 on BMDC surface by LPS(p<0.05),and promoted the upregulation of TLR4 on BMDC surface by LPS(p<0.05).(5)qRT-PCR results showed that CSE inhibited the relative expression levels of TLR3 mRNA,TRIF mRNA and p65 mRNA induced by LPS and poly(I:C)(p<0.05).(6)CSE inhibited LPS and poly(I:C)-induced IFNα and IL-6 produced by BMDC.Conclusion: LPS and poly(I: C)can induce BMDC secrete IFNα and IL-6 and activation.TLR2 and TLR4 changed opposite when exposed to CSE.CSE may inhibit LPS and poly(I: C)-induced BMDC activation and IFNα and IL-6 secretion by down-regulating the expression of TLR2 and TLR3 and the downstream key factors TRIF and p65.