CD137 Signaling Induces Macrophage M2 Polarization In Atherosclerosis Through STAT6/PPAR? Pathway

Objective:To investigate whether the CD137 signaling affect the macrophage M2 polarization in atherosclerosis through STAT6/PPAR?pathway.Methods:?1?In vivo experiments:ApoE^-/-male mice aged from 6 to 8 weeks were divided into5 groups,including the control group,agonist-CD137 group,anti-CD137 group,anti-STAT6 group and anti-PPAR?group.HE staining was used to detect the area of atherosclerotic plaque in the aortas of mice,and the expression levels of iNOS and arginase-1,as well as the expression levels of STAT6 and PPAR?of the macrophages in murine aortic plaques were detected by immunofluorescence staining.?2?In vitro experiments:extraction of primary macrophages from the abdominal cavity by inducing aseptic inflammation.Flow cytometry,western blot and ELISA were used to verify the effect of CD137 signal on the polarity transformation of macrophages.STAT6 inhibitor was used to inhibit the expression of STAT6,and western blot was used to verify the inhibition efficiency.PPAR?siRNA was used to interfere with the expression of PPAR?and real-time quantitative?qRT-PCR?was used to verify the interference efficiency.Flow cytometry,western blot and qRT-PCR were used to verify the effect of the STAT6/PPAR?pathway on the polarity transformation of macrophages.?3?In co-culture experiment:macrophages were implanted in the upper compartment,and endothelial cells were implanted in the lower compartment with matrix glue.The experiment was divided into four groups,including the control group,agonist-CD137group,anti-STAT6 group and anti-PPAR?group.The proliferation and migration of endothelial cells were detected by EdU assay and Transwell assay.The tube length and branch numbers of endothelial cells were measured by lumen formation in vitro.Results:?1?HE staining results showed that,compared with the control group,the area of atherosclerotic lesions was larger,the plaque fibrous cap was thinner,and the number of foam cells was more in the agonist-CD137 group.?2?Immunohistochemical results showed that,compared with the control group,the expression of Arginase-1 of macrophages in the atherosclerotic plaques of the agonist-CD137 group was increased,while the expression of iNOS was decreased.The expression of Arginase-1 of macrophages in the plaques of anti-CD137 group was decreased,while the expression of iNOS was increased.?3?Western blot results showed that the expression level of Arginase-1 in macrophages in the agonist-CD137 group was significantly up-regulated compared with the control group,while the expression level of iNOS was significantly decreased.Flow cytometry results showed that the mean fluorescence intensity of CD206 in agonist-CD137 group was higher than that in the control group,while the mean fluorescence intensity of CD86 was lower than that in the control group.ELISA results showed that the secretion level of IL-10 in the agonist-CD137 group was higher than that in the control group,while the secretion level of IL-12p70 was significantly lower than that in the control group.?4?Immunohistochemistry and western blot results showed that the expression levels of p-STAT6 and PPAR?of macrophages in the agonist-CD137 group were significantly up-regulated compared with the control group,but significantly decreased compared with the anti-CD137 group.In addition,the expression levels of p-STAT6 and PPAR?of macrophages in the anti-STAT6 group were significantly lower than those in the agonist-CD137 group.?5?Flow cytometry results showed that the expression level of CD206 in macrophages in the anti-STAT6 group and the anti-PPAR?group was significantly lower than that in the agonist-CD137 group.qRT-PCR results showed that the expression levels of FIZZ1,ARG1 and MRC1 of macrophages in the anti-STAT6group and the anti-PPAR?group were decreased significantly compared with the agonist-CD137 group.?6?The results of EdU and Transwell experiments respectively showed that the proliferation and migration of endothelial cells in the agonist-CD137 group increased significantly compared with the control group,while the proliferation and migration in the anti-STAT6 group and the anti-PPAR?group decreased significantly compared with the agonist-CD137 group.?7?The results of lumen formation experiments of endothelial cells showed that the length and the tube numbers of endothelial cells in the agonist-CD137 group increased significantly compared with the control group,while the length and the tube numbers of endothelial cells in the anti-STAT6 group and the anti-PPAR?group decreased significantly compared with the agonist-CD137 group.Conclusion:CD137 signaling regulates the macrophage M2 polarization in atherosclerosis by activating the STAT6/PPAR?pathway,and indirectly affects the the proangiogenic features of M2 macrophages.