Implications Of Necrotic Clearance Related Molecules In Chronic Obstructive Pulmonary Disease

Objective:In the pathogenesis of chronic obstructive pulmonary disease（COPD）,the patient’s body accumulates a large number of dead cells,which are often accompanied by impaired phagocytosis of full-time and part-time phagocytes and cannot be removed in time,leading to the persistence of inflammation.Calcium-binding protein（S100A8/A9）and filamentous actin（F-actin）are danger-associated molecular patterns（DAMPs）released by dead cells,which binds to pattern recognition receptors（PRR）on the surface of immune cells,such as soluble glycosylated end product receptor（sRAGE）and C-type lectin domain family 9 member A（Clec9A）to activate the corresponding signaling pathway and enhance the intrinsic and adaptive immune response.Meanwhile,mononuclear/macrophage secreted pulmonary activation regulatory chemokine（PARC/CCL18）may be involved in the pathological process of COPD by regulating macrophage polarization.Therefore,the aim of present study was to explore the clinical value of S100A8/A9,sRAGE,Clec9A and CCL18 in the development of chronic obstructive pulmonary disease by detecting the circulating levels of them in COPD and to investigate the correlation with other clinical indicators.Methods:（1）Enzyme-linked immunosorbent assay（ELISA）method was established to detect serum levels of S100A8/A9,sRAGE,Clec9A,CCL18in 203 patients with COPD,41 smoking individuals without COPD（Smoking control group）,and 167 non-smoking healthy subjects（No-smoking control group）April 2018 to March 2019 in respiratory department and medical center of hubei university of traditional Chinese medicine affiliated hospital of integrated traditional Chinese and western medicine.Correlation analysis was conducted between them and the clinical indicators of patients with COPD,such as:FEV₁%in the predicted value,FEV₁/FVC,NEU%,smoking volume（pack-year）.CCL18,Clec9A,S100A8/A9,sRAGE and their combined detection for COPD were analyzed respectively by using the receiver operating characteristic curve（ROC）.（2）The alveolar lavage fluid from 13 patients with COPD,31 patients with acute respiratory tract infection and 10 patients without respiratory system diseases were employed as experiment subiects from May 2019 to June 2019.The proportion of inflammatory cells in alveolar lavage fluid was analyzed by reiss staining,and the expression of Clec9A in alveolar lavage fluid was detected by immunofluorescence.Serum samples of 66patients with acute respiratory tract infection were collected to detecte Clec9A level by ELISA.Clinical microbial culture results of alveolar lavage fluid were collected from all patients.（3）The whole blood of 3 COPD patient and 3 control patient whose gender and age matched were collected to isolate peripheral blood mononuclear cells June 2019 to August 2019.Western blot was used to detect macrophage differentiation indicators of peripheral blood monocytes in COPD patients and control group.Results:（1）Serum S100A8/A9 level of COPD patients increased significantly compared with that of smoking control group and non-smoking control group（P<0.05）;Serum S100A8/A9 level in COPD patients（GOLD I、Ⅱ、Ⅲ、Ⅳ）increased significantly compared with that in non-smoking control group,with statistically significant difference（P<0.05）.The serum sRAGE level of COPD patients decreased significantly compared with smoking control group and non-smoking control group,which has statistical difference（P<0.05）.Serum sRAGE level in COPD patients（GOLD I、Ⅱ、Ⅲ、Ⅳ）decreased significantly compared with non-smoking control group,with statistically significant differences（P<0.05）.Serum S100A8/A9 level was positively correlated with NEU%and smoking amount of patient,and negatively correlated with FEV_l/FVC,FEV_l and sRAGE（P<0.05）.Serum sRAGE level was negatively correlated with NEU%（P<0.05）.In the ROC curve,the area under the curve of S100A8/A9,sRAGE and combined detect-ion were0.922[95%CI（0.897-0.947）],0.926[95%CI（0.899-0.952）],and 0.966[95%CI（0.950-0.983）],respectively.The serum Clec9A levels in patients with COPD（GOLD I、Ⅱ、Ⅲ、Ⅳ）were significantly higher than control group,P<0.05.Ezrin levels（GOLD I、Ⅱ、Ⅲ、Ⅳ）were significantly lower compared with control group,P<0.05.Serum Clec9A levels were positively correlated with Ezrin,P<0.05。In the ROC curve,the area under the curve of Clec9A,Ezrin and combined detection were0.823[95%CI（0.781-0.864）],0.693[95%CI（0.641-0.745）]and 0.847[95%CI（0.808-0.885）],respectively.Circulating levels of CCL18 were higher in patients with COPD than the two control groups（P<0.01）.Notably,the higher level of CCL18 was associated with the severity of COPD in stageⅡtoⅣ.When compared with the non-smoking healthy group,serum concentration of CCL18 in smoking individuals without COPD significantly increased（P<0.001）.In order to further investigate the effect of smoking,we measured serum levels of CCL18 in never-smokers,current smokers,and quitters of COPD patients,and found that serum concentration of CCL18 were significantly higher in patients who were current smokers and quitters than never smoker.Meanwhile,serum levels of CCL18 were markedly higher in patients with COPD who were current smokers than quitters（P<0.0001）.Moreover,the serum levels of CCL18were positively correlated with smoking amount（P<0.0001）,while negatively correlated with FEV₁%in the predicted value and FEV₁/FVC（P<0.0001）.As a clinical diagnostic target,the area under the receiver operating characteristic curve of CCL18 was 0.722（95%CI:0.669-0.775;P<0.001）.（2）Results of alveolar lavage sediment smear:A large number of neutrophils can be seen in alveolar lavage smear of COPD patients and some patients with acute respiratory tract infection under the microscope,macrophages were mainly observed under the microscope in some patients with acute respiratory tract infection.However,due to the changes in cell morphology of alveolar lavage fluid,many cells cannot be accurately judged.Immunofluorescence staining results of alveolar lavage sediment smear:Among the 13 patients with COPD,1 was positive for Clec9A immunofluorescence,14 were positive for 31 patients with acute respiratory tract infection,and 3 were positive for 10 patients without respiratory diseases.Serum Clec9A level of three groups during the same period:serum Clec9A level of COPD patients increased significantly compared with control group and patients with acute respiratory tract infection,with statistically significant difference（P<0.05）（3）The level of arg-1 in COPD patients was slightly higher than that in the control group,but the CCL18 and iNOS bands were not clear,and the changes in their expression levels could not be determined.Conclusion:The serum levels of S100A8/A9 and sRAGE in COPD patients was significantly higher than that of the control subjects,and the level of sRAGE was significantly lower.S100A8/A9 and sRAGE are closely correlated with the degree of airflow limitation and the levels of serum inflammatory mediators,which are expected to be potential biomarkers of COPD.