3.3 PIC Ⅱ enhances the function of M1-polarized macrophages and promotes the release of chemokine CXCL16
Recently, M1-polarized macrophages were proved to ameliorate the liver fibrosis through modulating immune microenvironment in the carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced liver fibrosis mice (Ma et al., 2017). Hence, we investigated whether and how PIC Ⅱ regulated the genes involved in the function and polarization of macrophages. According to the heatmap shown in Fig. 3A , DEGs enriched in the macrophages-related inflammatory factors and M1-polarized macrophages-related pathways were significantly changed in the Mdr2-/- + PIC Ⅱ group, compared with the Mdr2-/- group. In cluster 1, the expression of genes was upregulated in the Mdr2-/-group and was further decreased in Mdr2-/- + PIC Ⅱ group, such as the spondin 2 (spon2 ) and ccl5 , and other cell adhesion-related genes. Of note, the expression of genes in cluster 3 mainly associated with M1-polarized macrophage was upregulated in the Mdr2-/-+ PIC Ⅱ group, especially genes related to the chemotactic ability of activated M1 macrophages (including Cd80 , Cxcl16 and nitric oxide synthase 2 [Inos ]). Interestingly, cd163in cluster 4 indicated the expansion of phagocytosis accompanying with increased macrophage activity. The consistent GSEA plot results also demonstrated that DEGs enriched in M1-polarized macrophages maker genes were significantly upregulated in the Mdr2-/- + PIC Ⅱ group when compared to the Mdr2-/- group (Fig. 3B ). Subsequently, our qPCR results in Fig. 3C showed that the macrophages in the fibrotic liver of mice appeared not to be polarized into inflammatory state, because PIC Ⅱ did not up-regulate inflammatory markers including il6 andinos, slightly increase Cd163 and decrease the M2-type macrophage maker transglutaminase 2 (Tgm2 ). Meanwhile, the expressions of Mmp13 andMmp2 were significantly upregulated after PIC Ⅱ treatment, consistent with the downregulated Fn1(Fig. 3D ). Interestingly, among those different subsets of changed chemokines listed inFig. 3A , Cxcl16 , Cxcl1 , Cxcr2 andCcl5 were detected by qPCR and cxcl16 showed the clear upregulation in the Mdr2-/- + PIC Ⅱ group when compared with the WT group in Fig. 3D and Fig. S3A . Consistently, PIC Ⅱ markedly enhanced the hepatic expression and intrahepatic distribution of CXCL16 in the liver of Mdr2-/- mice (Fig. 3E , 3F andFig. S3B ). We also confirmed that PIC Ⅱ at different dosages dramatically increased the hepatic level of chemokine CXCL16 in M1-type macrophages induced by the administration of IFN-γ and LPS and promoted its secretion into culture medium (CM) (Fig. 3G-3I andFig. S3C ). Taken together, these results suggested that PIC Ⅱ promoted the M1-polarized macrophages and further triggered the production and secretion of CXCL16 from these macrophages of Mdr2-/- mice. Meanwhile, since the CM of CXCL16 highly expressed M1 macrophages didn’t show a significant inhibitory effect on the HSC activation (Fig. S3D ), we then transfer our attention to find another way to figure out how the M1 macrophages affect the liver fibrosis process.