Elastin Drives Cigarette Smoke-induced Autoimmune Responses In A Novel Mouse Model Mimicking Human COPD Characteristics

Chronic obstructive pulmonary disease(COPD)is a common chronic inflammatory airway disease,which is characterized by persistent airway inflammation,mucus hypersecretion,irreversible limitation on pulmonary airflow,and airway remodeling.Cigarette smoke is the most important risk factor of COPD,but the underlying mechanisms have not been fully elucidated,and it is still lack of effective approaches of prevention or treatment to COPD patients.The lack of an appropriate COPD animal model may be one of the main limiting factors to those problems.Lung elastic protein is a kind of insoluble protein,which is formed by highly crosslinked lysine residue condensation.The soluble precursor of elatin(tropoelastin)can be degraded into soluble elastic peptide by neutral granulocyte and macrophage secreted elastases.More and more studies show that the elastin degradation fragments can be detected in COPD patients and smokers,and the levels of elastin specific fragment,cross-linking of desmosome and isodesmosine,have become one of the biological indicators for the diagnosis of COPD.Therefore,it has been proposed that elastic may act as a self-antigen in COPD airway immune response,and that it may mediate the autoimmune reaction and airway inflammation of COPD through the antigen presentation function of lung APC cells.However,at present,there is still no animal experimental evidence todemonstrate the key role of elastin in smoking induced airway inflammation in COPD.Inflammation and immune mechanism of COPD is very complex,and studies have shown that cigarette smoke exposure can activates both the innate and adaptive immunity,and the adaptive immunity mainly depends on the function of T cells.In the local lymph node of lung,antigen presenting cells(APC,including partial lung macrophages and dendritic cells)will ingest antigen and presentto T cells,making them proliferation and differentiation into effector T cells.The effector T cells mainly including cytotoxic T cells(CD8+)and T helper cells(CD4+).Clinical studies have demonstrated the relationship between Tcl and Thl7 cells and COPD,and animal experiments have also confirmed that Thl and Th17 cells play an important role in lung inflammation induced by cigarette smoke.However,there is no clear research and explanation on how these T cells are involved in the process of autoimmunity mechanism of COPD.With the hypothesis that elastin is a self-antigen of COPD,we successfully constructed COPD models,through cigarette smoke exposure combined with short-term elastin challenge to induce an acute inflammation,and cigarette smoke exposure combined with long-term elastin challenge to induce emphysema model.On the basis of those models,we further explored the effects of cigarette smoke,elastin and its polypeptide,APC pulmonary macrophages,and Thl,Thl7 cells in the autoimmune response in COPD,combining the in vivo and in vitro experiments.Part I The establishment of a novel mouse model mimicking human COPD characteristics induced by cigarette smoke combined with elastinObjective:To establish a novel mouse model mimicking human COPD characteristics induced by cigarette smoke combined with elastin.Methods:In order to build the acute inflammation model,C57/6 mice(male,6-8 weeks,8-10/group)were randomly divided into 4 groups:Group-1:Air+NS,Group-2:Air+Eln,Group-3;CS+NS,Group-4:CS+Eln.Group CS+NS and CS+Eln were exposed to cigarette smoke in whole body exposure manner(100 cigarettes/day,5 days/week,a total of 2 weeks),after the interval of 2 weeks,the group Air+Eln and CS+Eln were intratracheal administrated with Eln(50ug/mice/time).After 24h of the last administration,the mice were euthanized and samples were collected.Some mice in each group were sacrificed to analyze brochoalveolarlavage fluid(BALF),including total cell number and different cell counting,to detect mRNA expression of Muc5ac in lung tissue by Real-time PCR,and to measure the protein expression of some cytokines,chemokines and transcriptional factors in BALF and lung homogenate by ELISA.In addition,left lung was inflated by gravity with 4%paraformaldehyde,held at a pressure of 30 cmH20 for 15 minutes and made pathological sections.Tissue samples were stained with haematoxylin and eosin(H&E)to examine peribronchial inflammation and with PAS to examine airway mucus hypersecretion.Other mice were sacrificed to obtain pulmonary mononuclear cell suspension by PBS perfusion,collagenase type I digestion,grinding and separation and splenocytes suspension by direct grinding.All the cell suspension was applied to detect the frequencies of T cell subpopulations by flowcytometry after PMA and Ionomycin stimulation.In order to build the chronic emphysema model,C57/6 mice(male,6-8 weeks,8-10/group)were randomly divided into 2 groups according to body weight,namely Group-1:Air+NS,Group-2:CS+Eln.Group-2:CS+Eln mice were exposed to cigarette smoke as told before.After 2 weeks,group CS + Eln mice were intratracheal administrated with Elastin(50ug/only/times)for another one day(a total of 15 times).After 24h of the last administration,the mice were euthanized and samples were collected.Some mice in each group were sacrificed to analyze brochoalveolarlavage fluid(BALF),including total cell number and different cell counting.Lung tissue samples were stained with PAS to examine airway mucus hypersecretion,with Masson to examine airway collagen deposition,and with H&E to examine MLI.The expression of apoptosis and necrosis related protein were detected by Western Blot.Some mice of each group were examined the lung function with BUXCO-DFS instrument under mild anesthesia.Results:Cigarette smoke exposure combined with elastin short term administration can induce airway inflammation and high mucus expression.Compared with the control group,the expression of Th1/Th17 related proinflammatory cytokines(IL-6)and chemokines factors(CXCL1,CXCL2)in the supernatant of BALF of CS+Eln mcie were significantly increased,while the Th2 related inflammatory factors(IL-4 and IL-13)expression showed no significantly difference.The protein levels of IFN-y and IL-17A in lung tissue of CS+Eln mice were significantly increased,and the proportion of Th 17 in lung was elevated as determined by flow cytometer.Our data also showed that although elastin combined with Freund's adjuvant sensitization,and then intratracheal administrated with elastin for three times also can induce airway inflammatory,the levels of inflammation is not as high as the acute CE model,and it mainly elevate the proportion of Thl in lung.At the same time,we also tried elastin(abdominal sensitization)+elastin(3 times airway administration)model and CS(smoke)+ smoke(CS)model,all of those models failed to induce airway inflammation.These results suggest that the CE-model is the most consistent model with the characteristics of acute inflammation in COPD model.Then we try to use cigarette smoke exposure combined with long-term administration of elastin method to construct the CE-emphysema model.We found that compared to the control group,the weight of mice in CE group were declined during the in the process of building the model.The mice in CE-group developed airway inflammation and high expression of airway mucus.Pulmonary function test results showed that FEV20(equivalent to the FEV1 index of human lung function)and FEV20/FVC of the mice in CE-group were significantly lower than that of the control group.The MLI of the mice in CE-group were higher than that of the control group.At the same time,the expression of apoptosis related protein(Caspase-3,Caspase-9)and necroapoptosis related protein(RIP,RIP3)in lung tissue of CE-group mice were significantly increased.These results suggest that the cigarette smoke exposure combined with long-term administration of elastin can induce the formation of pulmonary emphysema,and apoptosis and necroapoptosis have taken part in those process and development of COPD.Conclusion:Cigarette smoke exposure combined with short-term(3 days)administration of elastin can successfully simulate acute inflammation of COPD,and cigarette smoke exposure combined with long-term(30 days)administration of elastin can successfully simulate COPD emphysema.Part ? The study of autoimmune mechanism of elastin drives cigarette smoke-induced mouse COPD modelObjective:To explore the effects of cigarette smoke,elastin and its polypeptide,lung APC,pulmonary macrophages,and Thl/Th17 cells in the autoimmune mechanisms in COPD,on the basis of elastin drives cigarette smoke-induced mouse COPD model.Methods:C57/6 mice(male,6-8 weeks,8-10/group)were randomly divided into 2 groups according to body weight,CS group(cigarette smoke exposure group)and Air group(air control group).The cell of BALF was used to extract mRNA and protein,and the expression of MMP-12 was detected by fluorescence quantitative PCR and WB.The expression of elastin in BALF and lung tissue was detected by WB.In some mice of each group,the whole lung APC(sorting by magnetic bead)cells were cultured with spleen CD4+T cells(sorting by magnetic beads).After 3 days of stimulation with elastin in vitro,detect the differentiation of Th cell subsets by flow cytometer.To further validate the important role of Elastin in the CE model,we add a CS+BA4 group(BA4 intervention group,given lOug/mice/kg of BA4 per day before smoking)while constructed the CE-model.All the methods to detect airway inflammation and mucus high expression are as same as we told before.In addition,in some mice of each group,the whole lung APC(sorting by magnetic beads)cells were cultured with spleen CD4+T cells(sorting by magnetic beads).After 3 days of stimulation with elastin in vitro,detect the differentiation of Th cell subsets by flow cytometry.To further verify the important role of Th17 in the CE model,we used IL-17A knockout mice and their littermates to construct the CE-model.All the methods to detect airway inflammation and mucus high expression are as same as we told before.Results:We found that the expression of mRNA and protein level of macrophage specific matrix metalloproteinase MMP-12 in the BALF cells after 2 weeks of CS exposure were significantly higher than those in control group.This suggests that short-term cigarette smoke exposure can activate lung macrophages,thereby increasing the secretion of MMP-12 in the lungs.We also detected the expression of elastin in BALF cells and in lung tissue of mouse with smoking for 2 weeks,which has rapidly increased compared with the control group.Those results indicate that increased secretion of MMP-12 likely leads to the degradation of lung elastic fiber,resulting in increased expression of elastin polypeptide.The result of in vitro culture of T cells of mice with CS exposure for 2 weeks showed that elastin could increase the differentiation of Thl and Th17 cells.These results give direct evidences for the involvement of elastin as an autoimmune antigen in the pathogenesis of COPD.We use elastin neutralizing antibody treated acute CE-model mice,and we found that it could significantly inhibit the airway inflammation induced by CE(such as the total number of BALF cells were decreased significantly,neutrophil percentage and total numberwere relieved,H&E pathology showed infiltration of inflammatory cells in remission,and ELISA results showed that the inflammation factor IL-6 and chemokines CXCL1 expression were significantly decreased)and airway mucus hypersecretion(pathological PAS staining showed decreased expression of mucus).The results of T cells cultured in vitro also showed that BA4 could significantly alleviate the differentiation of Thl and Th17 cells induced by elastin.These results further confirmed that elastin played an important role in the autoimmune process of COPD.We use IL-17 KO and littermate control mice to constructer acute CE-model,and we found that deficient of IL-17A has obvious remissions in CE induced airway inflammation(such as the total number of BALF cells were decreased significantly,neutrophil percentage and total number were relieved,H&E pathology showed infiltration of inflammatory cells was alleviated,ELISA results showed that the inflammation factors IL-6 and CXCL1,CXCL2 expression were significantly dropped)and airway mucus hypersecretion(pathological PAS staining showed decreased expression of mucus).These results further proved that Th17 cells and IL-17A factor were involved in and regulate the autoimmune process of COPD.Conclusion:Our study for the first time provides animal experimental evidences to the hypothesis:cigarette smoke exposure can activate pulmonary macrophagesto produce MMP-12,which increases the degradation of lung elastic fibers and produce free elastin and its polypeptide.Elastin and its polypeptide can be uptaken by lung APC,and then induce Thl and Th17 differentiation,with the increaced secretion of IFN-y and IL-17A.These factors then recruit the inflammatory cells like macrophages and neutrophils,thereby secreting more elastases to produce much more elastin and its polypeptide.The process gradually aggravates airway inflammation and impairs the lung structure and function,which eventually leads to the formation of emphysema.