Neutrophil Extracellular Traps Induced By Cigarette Smoke Promote The Activation Of Myeloid Dendritic Cells And The Observation Of The Inhibitory Effect Of Erythromycin

PART ⅠObjective: This part of study is aimed to investigate the expression and critical role of neutrophil extracellular snare(NETs)in sputum and circulating dendritic cells in patients with stable chronic obstructive pulmonary disease(COPD).Methods: NETs in sputum were detected by Picogreen.The circulating Th1,Th17 and CD40 and CD 86 of myeloid dendritic cells(mDCs)of patients with COPD were assessed by flow cytometry.Results: Thirty-two patients with COPD and thirty-two healthy controls(sixteen current smokers and sixteen non-smokers)were recruited from the First Affiliated Hospital of Guangxi Medical University between May 2017 and May2018.NETs were abundantly produced in the sputum of COPD patients.The levels of NET-DNA in the sputum of COPD patients were correlated with the Th1 response,the activation of mDCs and airflow limitation.However,NETs insputum was not correlated with age,smoking pack years or body mass index.Conclusion: The NETs in sputum of patients with COPD were associated with airflow limitation,which can be served as one of the biomarkers for the severity of COPD.Sputum NETs in COPD patients may be associated with activation of mDCs and Th1 cell responses.The amount of NETs in airways under stimulation of smoking may reflect the susceptibility to COPD disease in each individual.PART ⅡObjective: This part of study is aimed to observe the effects of CSE induced neutrophil extracellular traps(NETs)on the activation of dendritic cells(mDCs).Methods: Monocytes isolated from blood of healthy volunteer were induced to dendritic cells(MoDCs)by 1000U/ml GM-CSF and 500U/ml IL-4 for 6 days.Neutrophils were isolated from blood of COPD patients.NETs were generated by CSE induction,and were isolated by Alu I enzyme digestion.MoDCs was stimulated by NETs induced by CSE in vitro for 15 h,and the expressions of CD40,CD86 and HLA-DR were detected by flow cytometry.The IL-1β,IL-12 and TNF-α in supernatants of MoDCs were detected by ELISA.Na?ve CD4~+T lymphocytes in human peripheral blood were isolated by immunomagnetic bead selection.Na?ve CD4~+T lymphocytes were co-cultured with MoDCs stimulated by NETs for 4 days.The differentiation of na?ve CD4~+T lymphocytes was detected by flow cytometry.Results: The CD40,CD86 and HLA-DR on the surface of MoDCs were upregulated by NETs stimulation.The IL-1β,IL-12 and TNF-α in supernatants of MoDCs stimulated by NETs were markedly increased.Na?ve CD4~+T lymphocytes co-cultured with MoDCs stimulated by NETs promoted the differentiation of Th1 and Th17.Conclusion: The stimulation of NETs induced by CSE can promote MoDCs IL-1β,IL-12 and TNF-α production,and promote MoDCs maturation and activation.MoDCs stimulated by NETs induced by CSE can promote the differentiation of na?ve CD4~+T lymphocytes into Th1 and Th17.PART ⅢObjective: This part of study is aimed to investigate the mechanism of erythromycin inhibiting neutrophil extracellular snare(NETs)induced by Cigarette smoke extract(CSE).Methods: Blood was obtained from 10 patients with stable chronic obstructive pulmonary disease(COPD).The neutrophils were isolated and purified using Histopaque 1119+Percoll methods.Effects of CSE on NETs formation and the effect of erythromycin/DPI on NETs,NET-associated NE and NET-associated MPO were investigated.The changes of NET-DNA in the supernatant were detected by Picogreen fluorescence quantitative method,and the production and release of NET-related neutrophils elastase(NE)and myeloperoxidase(MPO)were detected by N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide substrate chromometry and ELISA.The neutrophils from another 6 patients with COPD were isolated and purified using Histopaque 1119+Percoll methods.Effects of CSE on reactive oxygen species(ROS)production and the effect of erythromycin/DPI on intracellular ROS production were determined by2′-,7′-dichlorofluorescein diacetate(DCFH-DA)staining and quantitative analysis of mean fluorescence intensity(MFI)by flow cytometry.Results: CSE stimulation can increase NETs generation and the release of NET-related NE and MPO.DPI can inhibit CSE-induced NETs.Erythromycin reduced the production of NET-DNA and decreased NET-associated NE and MPO under CSE stimulation.In addition,CSE stimulation could increase the levels of ROS production in PMNs,which could be suppressed by DPI and erythromycin.Conclusion: Cigarette smoke exposure may trigger NETs in a ROS-dependent way.Erythromycin may reduce the production of NETs by inhibiting NADPH oxidase-dependent ROS responses.Erythromycin may also affect NETs formation by inhibiting NE and MPO.PART ⅣObjective: This part of study is aimed to observe the effects of erythromycin on the neutrophil extracellular net(NETs)and activation of dendritic cells to the pulmonary inflammation in mice.Methods: Thirty wild-type mice were randomly divided into cigarette smoke exposure group,cigarette smoke exposure + erythromycin intervention group and air exposure control group.The cigarette smoke exposure group and the cigarette smoke exposure + erythromycin intervention group were exposed to cigarette smoke for 24 weeks.The cigarette smoke exposure + erythromycin intervention group was given erythromycin(100mg/kg/d)by gavage from the end of the 12 th week to the end of the 24 th week.The air exposure control group was given air exposure for 24 weeks.On the 24 th week,the mice were euthanased.BALF was obtained to detect NET-DNA by Picogreen,and NET-associated NE,MPO,Histone 3 and CITH3 by ELISA.The spleen and right lung were prepared into single-cell suspension and the ratio of dendritic cells to Th1 and Th17 was determined by flow cytometry.The left lung was stained with pathological HE and the mean interlining interval(MLI)was calculated.Results: The NETs,soluble NE,MPO,Histone 3 and CITH3 in BALF were lower in the erythromycin intervention group than in the cigarette smoke exposure group.At the same time,in the erythromycin intervention group,the co-stimulatory molecules CD40 and CD86 of the myeloid dendritic cells in the lungs and the proportion of Th1 and Th17 in lungs and spleens of mice were decreased.In addition,the erythromycin intervention group showed decreased MLI.MLI was positively correlated with the level of NETs,the expression of CD40 and CD86 on dendritic cells,and the proportion of Th1 and Th17.Conclusion: Erythromycin can inhibit the NETs in mice,and may reduce smoking-related lung inflammation by reducing the production of NETs and negatively regulating the activation of dendritic cells.