Syndecan 2 Regulates Functions Of Lung Macrophage Through TLR4/MyD88/NF-κB Pathway In COPD With Frequent Exacerbator Phenotype

Objective: Chronic obstructive pulmonary disease(COPD),a complex and highly heterogeneous chronic respiratory disease,has a high morbidity and mortality worldwide,causing a huge burden to society and economy.Due to this high heterogeneity,COPD includes many clinical phenotypes,and it has become the focus of COPD research to explore the internal mechanisms of different phenotypes.Acute exacerbation is a key event in the natural course of COPD,which seriously affects the prognosis and outcome of COPD.Compared with COPD patients with infrequent exacerbator phenotype of COPD,frequent exacerbator phenotype(defined patients have more than twice moderate acute exacerbation a year or have severe exacerbation once a year should be hospitalized)has a worse health spending,rising mortality,and medical insurance,so the COPD frequent exacerbator phenotype calls for our special attention.However,the research on this phenotype is still in its initial stage,and the cellular and molecular mechanisms leading to increased susceptibility of frequent exacerbations are still unclear.Some scholars extracted lung macrophages from the bronchoalveolar lavage fluid of COPD patients with frequent exacerbations and coincubated with lipopolysaccharide(LPS)and interferon-γ(IFN-γ),it was found that the secretion of IL-6 and IL-8 in lung macrophages of these patients were reduced,and the lung macrophages of COPD patients with frequent exacerbator phenotype had more obvious immune response dysfunction to external stimulation.However,the study of this phenotype is still in its infancy,and the cellular and molecular mechanisms that lead to increased susceptibility to frequent acute exacerbations remain unclear.We have found 23 differential proteins,including Syndecan-2(SDC2)in proteomic screening of COPD frequent exacerbator phenotype.SDC2 play a key role in the pathological process of chronic inflammation through multiple ligand interactions,including extracellular matrix,cytokines,chemokines,growth factors,and growth factor receptors.Therefore,based on the previous proteomic findings of COPD frequent exacerbator phenotype,our study further explored the mechanism of SDC2 in the occurrence and development in COPD with frequent exacerbator phenotype through cell experiments in vitro.Research methods:Part I: Study on the expression level of Syndecan 2(SDC2)in COPD frequent exacerbator phenotype1)Based on immunofluorescence and immunohistochemical techniques,the location of SDC2 in human lung tissues and the expression of SDC2 in healthy smoking control group(control)、 COPD patients with infrequent exacerbations group(IFCOPD)and COPD patients with frequent exacerbations phenotype group(FCOPD)were determined.2)Protein expression in lung tissues of the Control group,IFCOPD group and FCOPD group was detected at the protein level through Western Blot.3)q RT-PCR technology was used to detect the m RNA levels in the Control group,IFCOPD group and FCOPD group.4)ELISA was used to verify the presence of SDC2 in serum of COPD patients,and to compare the differences in control group,IFCOPD group and FCOPD group.The accuracy,specificity and sensitivity of the biomarker model were analyzed by ROC curve.Part II: SDC2 regulates the response functions of lung macrophages to LPS stimulation1)Phorbol-12-myristate-13-acetate(PMA)were used to induce THP1 monocytes to differentiate into macrophages.2)Macrophage models with SDC2 overexpression and knockdown were constructed based on plasmid transfection and lentivirus transfection techniques.The expressions of inflammatory factors IL-6,IL-8,IL-1β and TNF-α in supernatant of the macrophage model were detected by ELISA,and the cells were screened by M1 and M2 macrophage through flow cytometry.3)The inflammatory cell models with SDC2 overexpression and knockdown in macrophages were constructed by LPS exposure.ELISA was used to detect the expressions of inflammatory factors IL-6,IL-8,IL-1β and TNF-α in supernatant of cells,and the cells were screened by M1 and M2 macrophage through flow cytometry.Part III: SDC2 regulates macrophage functions through TLR4/MyD88/NF-κB signaling pathway1)An in vitro inflammatory model of macrophages was constructed by lipopolysaccharide(LPS)exposure.The expression levels of SDC2 protein were detected by western blot and the expressions of inflammatory cytokines IL-6,IL-8,IL-1β and TNF-α in supernatant of cells were detected by ELISA.2)Macrophage models with SDC2 overexpression and knockdown were constructed based on plasmid transfection and lentivirus transfection techniques.Western blot and q RT-PCR technology were used to detect the protein and m RNA expressions of TLR4,MyD88 and NF-κB p65.The nuclear translocation of NF-κB p65 was observed by immunofluorescence technique.3)TLR4/MyD88 blocker was administered to observe the nuclear translocation of NF-κB p65 and the expression of inflammatory factors IL-6 and IL-8 in supernatant.Results:Part I: Study on the expression level of Syndecan 2(SDC2)in the COPD frequent exacerbator phenotype1)Based on immunofluorescence and immunohistochemistry,It was confirmed that SDC2 was mainly expressed in pulmonary macrophages and extracellular matrix.2)Compared with the IFCOPD group,the protein expression of SDC2 in FCOPD group decreased based on Western Blot.3)Compared with the IFCOPD group,the expression level of SDC2 m RNA in FCOPD patients were increased through q RT-PCR.4)SDC2 was found in human serum samples through ELISA.Compared with the IFCOPD group,the expression of SDC2 was decreased in the FCOPD group.The AUC was 0.65(p=0.09),SDC2 is not suitable for the identification of COPD subtypes as biomarker.Part II: SDC2 regulates the response functions of lung macrophages to LPS stimulation1)Macrophage models with SDC2 overexpression and knockdown were successfully constructed based on plasmid transfection and lentivirus transfection technology.Compared with SDC2 knockdown group,SDC2 over-expression macrophages were polarized to M1 type,and the expressions of inflammatory cytokines IL-6,IL-8,IL-1β and TNF-α in supernant of SDC2 overexpression macrophages were increased,while the polarization of SDC2 knockdown group did not change.2)LPS exposure was used to successfully construct an in vitro inflammatory model of SDC2 over-expression and in macrophages.Compared with the empty vector control group and SDC2 knockdown group,macrophages in the SDC2 over-expression group showed increased M1 polarization after LPS stimulation,while macrophages in the SDC2 knockout group showed decreased M1 polarization.The expressions of inflammatory cytokines IL-6,IL-8,IL-1β and TNF-α in supernatant of SDC2 overexpressed macrophages were significantly increased,while the secretion of inflammatory cytokines in SDC2 knockdown macrophages was significantly decreased.The secretion of inflammatory cytokines IL-6 and IL-8 in SDC2 knockdown macrophages stimulated by LPS at high concentration(1ug/m L)was significantly lower than that in SDC2 overexpression group stimulated by LPS at low concentration(0.01ug/m L).Part III: SDC2 regulates macrophage response functions through TLR4/MyD88/NF-κB signaling pathway1)LPS can induce SDC2 expression in macrophages.With the increase of LPS concentration,the expression of SDC2 protein increased,and the expressions of inflammatory cytokines IL-6,IL-8,IL-1β and TNF-α increased.2)The expression of TLR4 and MyD88 protein and m RNA levels increased in SDC2 over-expression group,decreased in SDC2 knockdown group.The nuclear shift of NF-κB p65 moving from cytoplasm to nucleus was were observed by immunofluorescence when SDC2 over-expression.3)TLR4/MyD88 blocker inhibited nuclear translocation of NF-κB p65 and decreased expression of inflammatory cytokines IL-6 and IL-8 in supernatant.Conclusion:1.SDC2 was observed in macrophages and extracellular matrix of human lung tissues,and the expression of SDC2 protein was decreased in COPD patients with frequent exacerbations of COPD.The expression level of SDC2 decreased in serum of patients with frequent exacerbations of COPD,but SDC2 was not suitable to be a biomarker for the identification of COPD subtypes according to ROC working curve analysis.2.SDC2 can activate macrophage polarization to M1 type and promote macrophage to secrete inflammatory factors such as IL-6,IL-8,IL-1β and TNF-α.Knockdown SDC2 did not activate macrophage polarization.SDC2 can enhance macrophages polarize to M1 type and increase the secretion of inflammatory cytokines IL-6,IL-8,IL-1β and TNF-α when macrophages are stimulated by LPS,while SDC2 knockdown can reduce this response to LPS.3.LPS stimulation induced the expression of SDC2 protein in macrophages.TLR4 and MyD88 protein and m RNA levels of overexpressed SDC2 macrophages increased,while TLR4 and MyD88 protein and m RNA levels of SDC2 knockdown macrophages decreased,indicating that SDC2 may be the upstream molecule of TLR4/MyD88.SDC2 induced macrophage NF-κB p65 to migrate from cytoplasm to nucleus and activated NF-κB pathway.Inhibition of TLR4/MyD88 can inhibit NF-κB p65 nuclear migration induced by SDC2,and decrease the inflammatory factors’ secretion of IL-6 and IL-8 in macrophages.SDC2 can initiate and regulate the function of macrophages by regulating TLR4/MyD88/NF-κB pathway.