The Role Of CD163/TWEAK/NF-κB Pathway On M1 And M2 Macrophages

Objective:The role of macrophages in different microenvironments is not fully understood.In this study,we compared the differential expression of NF-kappa B in M1 and M2 macrophages by CD163/TWEAK/Fn14 pathway.Methods:APOE-/-mice peritoneal macrophages were extracted and differentiated into M1(CD68+/iNOS+/CD163-)and M2(CD68+/CD163+/iNOS-)macrophages.The following studies were carried out after confirmation by laser confocal microscopy exogenous recombinant CD 163 and TWEAK proteins were added into M1 and M2 macrophages,respectively,and siCD163,siTWEAK and their isotypes were used as controls.The expression of CD 163,Tweak and Fn14 proteins and their possible downstream inflammatory factor NF-kappa B were compared and analyzed by Western blotting to determine whether CD 163 could affect the level of NF-kappa B by regulating TWEAK/Fn 14 pathway.Results:①Laser confocal microscopy confirmed that macrophages extracted from abdominal cavity were M1 type(CD68+/iNOS+/CD 163-),which could be induced by dexamethasone and transformed into M2 type(CD68+/CD163+/iNOS-).②Compared with M1+isoCD163 group(0.7786+0.05344),the protein expression level of TWEAK in M1+CD163 group(0.5036+0.03109)was significantly lower(P=0.004);compared with the control group,when silenced or stimulated by TWEAK,the protein level of Fn 14 remained unchanged;③In M2 macrophages,the level of CD 163 in M2+isoCD163 group was significantly higher(P=0.004).In M2+siCD163 group,the level of CD 163 significantly decreased(P=0.0010).Compared with M2+isoCD163 group(0.9436+0.05592),the level of TWEAK protein expression in M2+CD163 group(0.6670+0.03413)was significantly decreased(P=0.0055),but CD163 may not directly regulate the expression of Fn 14 protein(M2+isoCD 163 vs M2+CD163,P=0.08),but after silencing CD 163,the expression of TWEAK protein in M2+CD163 group was significantly decreased(P=0.0055);④After incubated by TWEAK or siTWEAK to M2 macrophages,the protein expression levels of CD 163 and Fnl4 did not change when compared with the control group,indicating that TWEAK did not regulate the expression of CD 163 and Fn 14;⑤Compared with the control group,the level of NF-kappa B did not change significantly whether stimulated by CD 163 or silenced by CD 163 in M1 cells.Compared with isoTWEAK group(0.6777 ± 0.07908),the level of NF-kappa B in siTWEAK group(0.4015±0.05787)was significantly decreased(P±0.0304);⑥In M2 macrophages,by siCD 163 incubation(1.509±0.1246)in M2 macrophages,as compared with isoCD 163 group(0.9861 ±0.0444),the level of NF-kappa B expression was significantly increased(P=0.0075).When incubated by TWEAK or siTWEAK,NF-kappa B protein level was significantly changed.⑦Stimulating M1 macrophages with CD 163,siTWEAK,CD163+siTWEAK and isoTWEAK respectively showed that TWEAK could reverse down-regulate CD 163 protein level.Compared with Ml+isoTWEAK group,the expression of NF-kappa B in M1+CD163 group and M1+CD163+siTWEAK group were significantly decreased,but there was no difference in the expression of NF-kappa B between M1+CD163 group and M1+CD163+siTWEAK group.⑧M2 macrophages were incubated with M2+oxLDL and then stimulated with CD163,which significantly decreased the protein expression of TWEAK and NF-kappa B.When M2 macrophages were incubated with M2+oxLDL and then stimulated with TWEAK,the level of CD 163 decreased,while CD 163 increased in M2+oxLDL+siTWEAK group,and TWEAK significantly increased the expression of NF-kappa B in M2+oxLDL cells.Conclusion:①TWEAK level protein was significantly decreased by exogenous CD 163 incubation in M1 macrophages;Fn14 did not change when exogenous TWEAK stimulated M1 macrophages,so Fn14 did not participate in CD163/TWEAK regulation pathway in M1 macrophages.siTWEAK could down-regulate NF-kappa B expression in M1 macrophage,so TWEAK could directly regulate the expression of NF-kappa B,while CD163 did not directly regulate the expression of NF-kappa B.②The protein levels of CD 163 and Fn14 remained unchanged regardless of whether they were stimulated by TWEAK or silenced by dexamethasone-induced CD 163-positive M2 macrophages,suggesting that M2 may have a natural anti-TWEAK-induced inflammatory response.③After siCD163 of M2 macrophages,the protein level of NF-kappa B increased significantly,and TWEAK could positively regulate the expression of NF-kappa B(when TWEAK was added or TWEAK was silenced,the protein level of NF-kappa B increased or decreased significantly).④ In the microenvironment of atherosclerosis,CD 163 can regulate the levels of TWEAK and NF-kappa B,which may play an anti-atherosclerosis role.Moreover,CD 163 can only regulate the expression of NF-kappa B through TWEAK,but not directly act on NF-kappa B.In conclusion,CD163/TWEAK/NF-kappa B pathway can regulate the biological activity of M1 and M2 macrophages,and provide new therapeutic targets for atherosclerosis.