Neutrophil Extracellular Traps Induced By Cigarette Smoke Promote Activation Of Plasmacytoid Dendritic Cells And The Effect Of Erythromycin Intervention

PART ?Objective To evaluate the formation of neutrophil extracellular traps(NETs)in the context of chronic inflammation induced by cigarette smoking and explore the role of erythromycin in NETs formation.Methods Male BALB/c mice between the ages of 6 and 8 weeks were randomly divided into two groups: air-control mice or cigarette smoke exposed mice.Polymorphonuclear neutrophils(PMNs)were isolated with mouse peripheral blood neutrophil separation medium from peripheral blood of mice.The formation of NETs in two groups of mice was evaluated by fluorescence microscopy.To assess the NETs-degrading activity of serum from two groups of mice,NETs were induced and exposed to 10% serum from air-control or cigarette smoke exposed mice.NETs degradation was confirmed by fluorescence microscopy.We then investigated whether cigarette smoke extract(CSE)could promote polymorphonuclear neutrophils undergo NETosis and observed the morphological characteristics and composition of NETsinduced by different stimuli by confocal microscopy.Lastly,the role of erythromycin in NETs formation induced by cigarette smoke extract was explored.Results Compared with air-control mice,polymorphonuclear neutrophils(PMN)isolated from chronic cigarette smoke exposed mice were more prone to formation of neutrophil extracellular traps(NETs).Cigarette smoke exposed mice had an impaired function for degrading NETs as compared with air-control mice.Cigarette smoke extract(CSE)directly triggered the formation of neutrophil extracellular traps(NETs)in vitro and polymorphonuclear neutrophils(PMN)isolated from chronic cigarette smoke exposed mice were more sensitive to CSE stimulation.The CSE-induced NETs shared identical property of PMA-induced NETs in morphologic characteristics and in some conserved proteins(Cit H3,NE and MPO)as examined by confocal laser scanning microscope.Erythromycin effectively inhibited the formation of NETs induced by cigarette smoke extract(CSE)in vitro.Conclusion The disturbance of formation and degradation of NETs might contribute to the chronic inflammation in the lung of mice under condition of cigarette smoke exposure.Erythromycin has potential as an anti-inflammatory agent by inhibition of NETs formation induced by cigarette smoking.PART ?Objective To explore the role of cigarette smoke extract(CSE)induced-NETs in the activation of p DCs under condition of chronic cigarette smoke exposure.Methods Male BALB/c mice between the ages of 6 and 8 weeks were randomly divided into two groups: air-control mice or cigarette smoke exposed mice.Morphological changes of mice were observed by HE staining.The qualitative assessment of emphysema was determined by measuring the mean linear intercept(Lm).The expression of Th1 and Th17 cells in peripheral blood,spleens and lungs between air-control and cigarette smoke exposed mice were determined by flow cytometry.p DCs were isolated from spleens of air-control and cigarette smoke exposed mice by magnetic beads and the phenotype of p DCs between two groups were evaluated by flow cytometry.Polymorphonuclear neutrophils(PMN)were separated from peripheral blood of chronic cigarette smoke exposed mice and NETs were induced with cigarette smoke extract(CSE),then NETs were treated with restriction enzyme Alu I or MNase and isolated.Primary p DCs were isolated from air-control mice and treated with CSE-induced NETs.The expression of MHC-? and costimulatory molecules CD40,CD86 was determined by flow cytometry.The concentrations of IFN-?,IL-6 and IL-12/p70 in supernatant were determined by ELISA.Na?ve CD4+T cells were isolated from spleens and co-cultured with primary p DCs from air-control mice or p DCs pre-treated with CSE induced-NETs,the expression of IL-17 and IFN-? were determined by Flow Cytometry.Furthermore,the expression of transcription factor T-bet and ROR-?t were also determined by Flow Cytometry.Results(1)Chronic cigarette smoke exposed mice developed an emphysematous phenotype by showing enlargement of the air spaces and the destruction of the alveolar architecture.The values of mean linear intercept(Lm)in cigarette smoke exposed mice were significantly higher than in air-control mice.(2)The frequencies of Th1 and Th17 cells showing a significant increase in peripheral blood,spleens and lungs of cigarette smoke exposed mice,as compared with air-control mice.(3)The levels of MHC-?and costimulatory molecules CD40 and CD86 on p DCs were significantly increased in cigarette smoke exposed mice,as compared with air-control mice.(4)CSE induced-NETs can promote p DCs maturation and activation by upregulating surface expression of CD40,CD86 and MHC-?.(5)p DCs treated with CSE induced-NETs had a high production of IFN-?,IL-6 and IL-12p70 when compared to un-stimulated p DCs.(6)Na?ve CD4+T cells cocultured with NETs-activated p DCs exhibited predominantly Th1 and Th17 patterns that have a higher levels of IFN-?and IL-17,as compared with cocultured with primary p DCs from air-control mice.(7)Na?ve CD4+T cells cocultured with NETs-activated p DCs had a higher expression of T-bet and ROR-?t,the specific transcription factor of Th1 and Th17 than that cocultured with primary p DCs from air-control mice.Conclusion NETs induced by CSE could promote p DCs maturation and activation,subsequently initiate Th1 and th17 cells-mediated immune responses which might contribute to the enhanced and sustained inflammation in the lung under the context of chronic cigarette smoke exposure.PART ?Objective To identify the phenotypic characteristics of circulating p DCs and explore the role of circulating p DCs in initiating Th1 and Th17 cell-mediate immune responses in patients of COPD.Methods Fifteen healthy smokers and fifteen healthy nonsmoking control subjects were recruited in this study.Thirty-five patients with stable COPD were enrolled at The First Affiliated Hospital of Guangxi Medical University and The Eighth People's Hospital of Nanning.The expression of Th1 and Th17 cells and the phenotype of circulating p DCs in peripheral blood of patients with COPD by using multi-colour flow cytometry.p DCs related-cytokine(IL-6,IL-12 and IL-23)in serum of all subjects were measured by ELISA.Finally,the correlations of p DCs related-cytokine with the expression of Th1 and Th17 cells were also analyzed.Results(1)The frequencies of CD4+T cells was no statistical difference among different groups.However,the frequencies of Th1 and Th17 cells showing a significant increase in peripheral blood of COPD patients,as compared with nonsmokers or healthy smokers.(2)The serum concentrations of IFN-? and IL-17 A were significantly higher in patients with COPD than in nonsmokers or healthy smokers,but there was no detectable alteration between nonsmokers and healthy smokers.(3)The frequencies of circulating p DCs in PBMCs did not show any differ between the three groups.However,the expression of co-stimulatory molecules CD40 and CD86 on p DCs was significantly increased in patients with COPD when compared with healthy smokers and nonsmoking control subjects.Interestingly,the expression of CD40 and CD86 on p DCs was higher in smokers than in nonsmoker.(4)Comparedwith nonsmokers or healthy smokers,the serum levels of IL-6,IL-12 and IL-23 displayed a significant increase in patients with COPD.(5)The serum level of IL-12 correlated positively with the frequencies of Th1 cells in peripheral blood of patients with COPD.The serum levels of IL-6 and IL-23 were correlated positively with Th17 cells in peripheral blood of patients with COPD.Conclusion An enhanced activation phenotype of circulating p DCs might contribute to exaggerated Th1 and Th17 cell responses in circulation of patients with COPD.