Study On The Regulation Of Macrophage Polarization And Autophagy By Cxcl10(IP-10)/Cxcr3 And Its Function

Inflammation is a response to tissue damage induced by various stimuli such as trauma,infection or transformed cells.Also,inflammation is essential for immune defense,but excessive inflammation can develop into inflammatory diseases Macrophages play an important role in the inflammatory response and show a high degree of phenotype plasticity,which can be classified into the classic activation of Ml macrophage with pro-inflammatory function and the alternative activation of M2 macrophage with anti-inflammatory function.Therefore,the balance between M1 macrophage and M2 macrophage regulates the physiological inflammatory response,and disrupting this balance will lead to inflammatory disorder.Autophagy is a process of lysosome-dependent degradation,which widely exists in eukaryotic cells.During autophagy,intracellular long-lived protein and damaged organelles are transfered into autolysosome and degraded.Autophagy involves in a variety of cellular processes including cell survival,cell homeostasis,stress reaction,innate immune,acquired immune and cancer.Macrophages play an active role in host defense and tissue homeostasis maintenance,which requires a delicate balance between the proinflammatory and immunomodulatory functions of macrophages.Therefore,the interaction between the inflammatory response mediated by macrophage polarization and the production of amino acids,cholesterol and other nutrients in the lysosomes regulated by macrophage autophagy is particularly important in ensuring an appropriate response to environmental stimuliCxcl10(IP-10)mainly induces the chemotaxis of monocytes and macrophages Combining with its receptor Cxcr3,IP-10 activates and recruits monocytes and macrophages into the inflammatory site to participate in immune cell migration,activation and differentiation,inflammation,hematopoiesis and angiogenesis.And,IP-10 also plays an important role in infection,autoimmune and tumor diseases Currently,the Cxcl10(IP-10)/Cxcr3 axis is not clear in terms of mediating macrophage polarization and function.Therefore,this study aims to explore how the Cxcl10(IP-10)/Cxcr3 axis affects polarization and autophagy in macrophage(1)The Cxcl10(IP-10)/Cxcr3 axis regulated macrophage polarization.In this study,we obtained the consistent with the literature describing the experimental results IFN-? increased the mRNA expression of iNOS,Il-1?,Il-6,Il-12?,and Tnf-?,and IL-10 up-regulated the mRNA expression of Argl,Cd163,and Mrcl.In addition,this study also confirmed that Mmp9 is a marker of M2 polarization in macrophages.In this study,IP-10 did not affect the expression of iNOS,Il-1?,Il-6,Il-12? in mRNA level.However,Arg1,Cd163,Mrcl,Mmp9,Vegfa and Pdcd1 mRNA expression were increased treated with IP-10.ELISA results showed that IFN-y induced the expression of Tnf-a,and IL-10 and IP-10 did not affect the secretion of Tnf-a.This suggests that IP-10 induced M2 polarization in macrophages.In this study,siRNA Cxcr3 enhanced Il-1?,Tnf-? mRNA expression,but Arg1,Cd163,Mrcl,Mmp9 and Vegfa mRNA expression were reduced.Cxcr3 inhibitor AMG487 could also induce Il-1?,Tnf-?mRNA expression,but inhibited Arg1,Cd163,Mrcl,Mmp9,Vegfa and Pdcd1 mRNA expression.This indicated that siRNA Cxcr3 and AMG487 could reverse macrophage phenotype from M2 to M1.(2)The Cxcl10(IP-10)/Cxcr3 axis regulated macrophage autophagy.In this study,IP-10 not only up-regulated the mRNA expression of Arg1,Mmp9 and Pdcd1,but also enhanced the protein expression of Argl,Mmp9 and Pdcdl.Therefore,Arg1,Mmp9 and Pdcdl were not only used as markers of macrophage polarization,but also possibly involved in the regulation of inflammatory response and cell metabolism However,cell stress,nutrition,hunger and so on can promote cell autophagy.Western Blot results showed that IP-10 increased the expression of autophagy marker Atg5-Atg12 complex,Lc3-?,p62,Lampl.This suggested that IP-10 could promote macrophage autophagy.This study also found that Cxcr3 inhibitor AMG487 could inhibit Lc3-?,p62,Lampl expression.Therefore,the Cxcl10(IP-10)/Cxcr3 axis mediated macrophage autophagy regulation.Further,Western Blot results showed that there were no differences in Lc3-? and p62 protein expression in cells treated with IL-10.However,IP-10 could collaborate IL-10 to up-regulate Lc3-? and p62 protein expression.Therefore,this suggested that IP-10 activated autophagy in M2 macrophage.(3)The Cxcl10(IP-10)/Cxcr3 axis mediated macrophage polarization and autophagy via JNK1 signaling pathway.In this study,it was found that Cxcl10(IP-10)/Cxcr3 mediated the expression of JNK1 in macrophages.QPCR results showed that JNK1 inhibitor SP600125 inhibited the mRNA expression of Il-1?,Il-6,and Tnf-a,but activated the mRNA expression of Arg1,Cd163,Mrcl,Vegfa and Mmp9.Western Blot showed that SP600125 could also up-regulate the protein expression of Mmp9.In addition,SP600125 could also synergistically inhibit the expression of Il-1? and enhance the expression of Mrcl and Mmp9 in mRNA level.This study further showed that JNK1 inhibitor SP600125 did not change Lc3-?,p62,Lampl protein expression,but SP600125 could cooperate with IP-10 to enhance Lc3-?,p62,and Lamp1 expression.Therefore,the Cxcl10(IP-10)/Cxcr3 axis mediated the regulation of macrophage polarization and autophagy through JNK1 signaling pathway.(4)Macrophage polarization interacted with autophagy.Further,we found that siRNA Lamp1 could suppress Atg5-Atg12 complex,Lc3-?,p62 protein expression in IP-10 treated cells.Besides,siRNA Lamp1 transfection showed a trend of increased Il-1?,and Tnf-? mRNA expression,but no significant difference was shown in Mrcl and Mmp9 mRNA expression.However,siRNA Lamp1 collaborated IP-10 to promote Il-1?,Tnf-? mRNA expression.In contrast,siRNA Lamp1 inhibited the mRNA expression of Mrc1 and Mmp9 under IP-10 treatment.In addition,siRNA Lampl also inhibited Argl protein expression in macrophages treated with IP-10.Therefore,it was suggested that autophagy might regulate macrophage polarization,but IP-10 mediated the interaction between macrophage polarization and autophagy through JNK1/Lamp1 signaling pathway.(5)The Cxcl10(IP-10)/Cxcr3 axis affected angiogenesis.As is reported,chemokines largely orchestrate the tissue repair process that is involved in overlapping phases of hemostasis and inflammation,proliferation and angiogenesis,and remodeling of the nascent tissue after following wounding.In this study,macrophage conditioned medium treated with IP-10 and Cxcr3 inhibitor AMG487 was collected and used to continue to culture scratched b.End.3 cells.The results showed that each group could promote wound healing in different degrees compared with the control group.The healing degree was most obvious in IP-10 treatment group.Although AMG487 promoted cell migration,the healing degree was lower than that of IP-10 treatment group.In conclusion,the above results suggested that IP-10 might regulate macrophage autophagy through Cxcr3-mediated signaling pathway,and subsequently regulate macrophage polarization.The regulation of autophagy and polarization of macrophages by IP-10 might eventually affect the repair of vascular endothelial cells damaged by inflammation.