Study On The Correlation Between Cav-1 And Th17、Treg And Their Cytokines In Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease(COPD)is a common disease that can be prevented and treated,characterized by persistent respiratory symptoms and airflow limitation,and disease often develops progressively associated with airway and / or alveoli abnormalities caused by significant exposure to toxic particles or gases.The main symptoms include shortness of breath and cough with sputum production.Its acute exacerbations and comorbidities affect the severity of the disease.Globally,as of 2010,COPD affected approximately 329 million people(4.8% of the population),resulted in an estimated economic cost of $2.1 trillion.and will be the third leading cause of mortality worldwide in 2020.It seriously affects the quality of life of patients,and brings a heavy financial burden to the family and society.Previous studies have focused on airway,pulmonary and pulmonary vascular inflammatory responses.In recent years,studies have shown that COPD patients continue to progress in the pulmonary inflammatory response after smoking,macrophages,neutrophils,CD4+T,CD8+T lymphocytes are still in the local infiltration,indicating that COPD may be an autoimmune diseases,the incidence may be related to the regulation of the immune system disorders.Therefore,understanding the mechanism of COPD may provide new ideas for future treatment and intervention of COPD.Although the immunopathogenesis of COPD remains unclear,current evidence suggests multiple immune cells(e.g.,CD4+IL-17+T-helper(Th17),CD4+Foxp3+ regulatory T(Treg)cells)and inflammatory cytokines(e.g.,Caveolin-1,transforming growth factor(TGF)-β,interleukin(IL)-17)are involved in the development of COPD.Acute and chronic infection with bacteria and viruses contribute to the inflammation and activate lymphocytes in COPD.Accumulating evidence indicates that two subsets of CD4+ T cells(i.e.,Th17 and Treg cells)play a pivotal role in the immunopathogenesis of inflammatory diseases,such as COPD,asthma,and idiopathic pulmonary fibrosis.Th17 cells account for pro-inflammatory immune responses but damaging tissue,and Treg cells control inflammatory immune responses but hindering antibacterial immunity.An imbalance between Treg and Th17 cells has recently been observed in COPD and mustard lung disease.caveolae,omega-shaped invaginations of the cell plasma membrane 10-100 nm wide.In the normal cell,the main functions of caveolae(little caves)are to regulate fundamental cellular processes including transcytosis,signal transduction,endocytosis andintracellular cholesterol transport.Caveolins are structural and functional proteins of the caveolae,As regulators of caveoli,the caveolins have broad importance in human physiology and pathology.There are a total of three caveolin proteins in humans coded for by the genes Cav-1,Cav-2 and Cav-3.Cav-1 is the most highly expressed and best marker of caveolae.As a hot topic,cav-1 has been implicated in the fibrosis and smooth muscle function in the respiratory tract.Recent works have discovered that Cav-1 plays the potential roles in regulating capillary leakage of lung,pulmonary edema,and lung injury by modulating the acute inflammatory response.The expression of Cav-1 was detected in macrophages,dendritic cells(DCs),lymphocytes,peripheral blood mononuclear cells(PBMCs),and serum.Garrean found that Cav-1 inhibits nitric oxide synthase expression,reduces NO production,further activates NF-k B expression in lipopolysaccharide-induced airway inflammation,promotes airway inflammation;Hu et al.demonstrated the reduced Cav-1 expression ameliorated polymorphonuclear neutrophils(PMN)activation-induced lung inflammation and vascular injury.Interestingly,the expression of Cav-1 was drastically decreased in peripheral blood monocytes or serum from patients with asthma or COPD with pulmonary hypertension.Th17 and Treg cells interact with each other during development,and there is an important plasticity between Th17 and Treg cells.These characteristics indicate that Th17/Treg balance plays an important role in the development of inflammatory diseases and disease outcomes with animal models and human.An imbalance between Treg and Th17 cells has recently been observed in COPD and mustard lung disease.In one study by Anne Sch?nleet al.,the frequency of CD4+Foxp3+ Treg was increased in Cav-1-deficient CD4+T cells in the context of TGF-β.Thus,we hypothesize that Cav-1 may promote process of COPD via regulating the imbalance of Th17/Treg ratio.In this study,we examined the plasma levels of Cav-1 and the frequencies of circulating Th17 and Treg cells,and evaluated the relationship between the expression of Cav-1 and the frequency of Treg or Th17 as well as Th17/Treg ratioinpatients with COPD.OBJECTIVETh17 and Treg cells have opposite roles in the development of autoimmune and inflammatory diseases.The imbalance of Th17/Treg cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease.Caveolin-1 has been regarded as a potential critical regulatory protein in pathological mechanisms of chronic inflammatory respiratory diseases.Therefore,We examined the expressions of plasma Cav-1 and circulating Th17,Treg cells,and the related cytokines in patients with COPD and investigated whether the loss of Cav-1 is involved in the homeostasis of Th17/Treg cells in COPD.MATERIALS AND METHODSBetween August 2014 and July 2015,50 subjects with COPD and 23 smokers with normal lung function as healthy control(HC)subjects from the Department of Respiratory Medicine,the Affiliated Hospital of Qingdao University(Qingdao,China)were enrolled in this study.All COPD patients were diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease(GOLD)guidelines.Fifty subjects with COPD were subdivided into 2 groups: 26 patients with acute exacerbation COPD(AECOPD)diagnosed on the basis of COPD exacerbation criteria described previously and 24 patients with clinically stable COPD(SCOPD)for >3 months prior to inclusion in this study.we measured the expression of plasma Cav-1、IL-17、TGF-β by ELISA and the frequencies of circulating CD4+CD25+Foxp3+Treg and CD4+IL-17+Th17 cells by FCM in patients with stable COPD(SCOPD)and acute exacerbation COPD(AECOPD)as well as healthy control(HC)subjects.To investigate the relationship between the plasma levels of cav-1 and the frequencies of circulating Tregs、Th17 and Tregs/Th17 in different stage of COPD patient.PBMCs from patients with COPD were transfected with Cav-1-plasmid or Cav-1-si RNA and then exposed to 10% CSE for 24 h,and then measured the expression of plasma IL-17 、 TGF-β by ELISA and the frequencies of circulating CD4+CD25+Foxp3+Treg and CD4+IL-17+Th17 cells by FCM.Finally we evalucated relationship between the plasma levels of cav-1 and the frequencies of circulating Tregs,Th17,IL-17 and TGF-β in COPD patient.RESULT 1.Clinical Data AnalysisThree groups of subjects were compared in terms of demographics and clinical features: No significant difference was ditected between groups in the mean age,gender and body mass index(BMI).Gender inequality is mainly due to the prevalence of smokers and COPD in Chinese among men than in women.Lung function was measured using forced expiratory volume in one second(FEV1)and forced vital capacity(FVC).Analysis of variance identified significant differences in FEV1,FEV1/FVC,and FEV1/predicted value,with group HC having significantly higher values for each than groups AECOPD and SCOPD(P﹤0.01),compared with SCOPD,AECOPD group had lower FEV1(P ﹤ 0.05),and group AECOPD had significantly lower FEV1/FVC,FEV1/pred and higher RV/TLC than group SCOPD(P﹤0.01).2.The Plasma Levels of Cav-1 and the Frequencies of Circulating Tregs and Th17 in COPD PatientsIn order to investigate the potential association between the Cav-1 levels and the frequency of Treg or Th17 cells,we measured the expression of plasma Cav-1 by ELISA and the frequencies of circulating CD4+CD25+Foxp3+Treg and CD4+IL-17+Th17 cells by FCM in patients with stable COPD(SCOPD)and acute exacerbation COPD(AECOPD)as well as healthy control(HC)subjects.ELISA analysis indicated that the Cav-1 levels were significantly low in COPD patients compared to HC group.The Cav-1 levels were significantly lower in patients with SCOPD than those in AECOPD.Alongside reduced Cav-1 levels,the frequency of Treg was significantly elevated in SCOPD group compared with AECOPD and HC group.While the frequency of Th17 cells was significantly increased in AECOPD group compared with SCOPD and HC group.Interestingly,the percentage of Treg was significantly lower in AECOPD patients than HC group.For the expression of Th17 cells,there was no significant difference between SCOPD and HC group.Th17/Treg ratio presents one of the important indicators in diseases development showing the balance between the immunosuppressive and pro-inflammatory subpopulations of CD4+T cells.patients with AECOPD had the highest Th17/Treg imbalance ratio.Interestingly,the Th17/Treg ratio was significantly declined in patients with SCOPD compared to the HC group.These data revealed that Th17/Treg ratio is obviously worsened in patients with COPD.3.The Levels of Circulating TGF-β1 and IL-17 in Patients with COPDAugmented immunosuppression in patients with SCOPD was reflected by distinct increments in the plasma levels of Treg-produced immunosuppressive cytokine TGF-β1.In contrast,the peripheral blood levels of Th17-generated pro-inflammatory cytokine IL-17 were significantly increased in patients with AECOPD.These findings were consistent with the frequencies of circulating Treg and Th17 cells.4.Correlations of Cav-1 Levels with the Frequencies of Treg and Th17 Cells and the Th17/Treg RatioWe observed that the levels of Cav-1 did not show any correlation with the frequencies of Treg and Th17 cells and the ratio of Th17/Treg in HC group.Strikingly,the reduced levels of Cav-1 in patients with COPD displayed an inverse correlation with the frequency of Treg and a positive relationship with the frequency of Th17 and the ration of Th17/Treg cells,suggesting that levels of Cav-1 may play an important role in the imbalance of Th17/Treg ratio in patients with COPD.5.Changes in Levels of Cav-1 Affect the Frequencies of Treg and Th17 CellsTo assess whether alteration of Cav-1 expression affects the proportions of Treg and Th17 cells,PBMCs from patients with COPD were transfected with Cav-1-plasmid or Cav-1-si RNA and then exposed to 10 % CSE for 24 h as described in BMATERIALS AND METHODS section.FCM analysis showed that the overexpression of Cav-1 resulted in the reduction of Treg frequency and the increase in Th17 frequency,whereas downregulation of expression of Cav-1 promoted the elevation of Treg cells and declined the expression of Th17 cells.The expression of TGF-β and IL-17 was consistent with the expression of Treg and Th17.CONCLUSION 1.Our findings suggested that Th17/Treg-mediated distributions were involved in the progression of chronic lung injury of COPD patients.Imblance of Th17/Treg and opposite effect were found,which can be used as a marker of COPD progression.2.Our study demonstrated that the reduction of plasma Cav-1 levels decreaced dramatically.3.The reduction of plasma Cav-1 levels acted to elevate frequency of circulating Treg and reduce frequency of Th17 cells in patients with COPD.4.The levels of Cav-1 in patients with COPD displayed an inverse correlation with the frequency of Treg、the level of TGF-β and a positive relationship with the frequency of Th17、the level of IL-17 and the ratio of Th17/Treg cells.5.These results collectively provide the first evidence indicating that Cav-1 is involved in the imbalance of Th17/Treg ratio and thereby disrupting inflammatory immune reactions occurring in the lung of COPD patients.Further studies are needed to evaluate the detailed mechanisms of Cav-1 promoting the imbalance of Th17/Treg ratio in patients with COPD.