3.4 PIC Ⅱ promotes CXCL16-expressed M1 macrophages to recruit and activate NK cells
Although PIC Ⅱ was not found to have visibly impact on the dominant amount of NK cells before (Fig. 2E ), the heatmap results inFig. 4A still exhibited that DEGs enriched in the inflammatory cytokines especially for the markers of NK cells, includingIfngr, neural cell adhesion molecule 1 (Ncam1 ), integrin subunit alpha 1 (Itga1 ) and NK cell-mediated target cell killing pathways such asGzmb were changed after PIC Ⅱ administration. Similar results of GESA plots further confirmed that the DEGs enriched in NK cells-mediated immune response were significantly downregulated in the Mdr2-/- group but were increased by PIC Ⅱ administration (Fig. 4B ). Simultaneously, we validated the protein expression of NK cell-mediated cell killing-related targets and proved that PIC Ⅱ slightly increased or maintained the expressions of PERFORIN and GZMB, more importantly, markedly increased the expression of C-X-C motif chemokine receptor 6 (CXCR6, the surface receptor of NK cells) and the apoptosis-inducing factor STAT1 in the Mdr2-/- mice (Fig. 4C and Fig. S4A ). Considering the recruitable feature of NK cells and the characteristic of interaction between immune cells in liver fibrosis, we next construct a co-culture system of macrophages and NK cells to explore whether and how M1 polarized macrophage regulated the activity of NK cells in the Mdr2-/- mice. In brief, we seeded M1-type macrophages induced by IFN plus LPS in the lower cell plate and further plated primary NK cells in the upper transwells (Fig. 4D ). As expected, transwell migration assay also demonstrated that after being treated with PIC Ⅱ, CM secreted by M1-type macrophages (namely CM-M) in different groups promoted the recruitment of NK cells (Fig. 4E ). Likewise, the immunofluorescences co-staining of CD161 (NK1.1) and CXCL16 confirmed that PIC Ⅱ at different dosages increased the expression of CXCL16 in co-cultured NK cells (Fig. 4F and Fig. S4B ), even without significant upregulation of cytotoxicity factors of PERFORIN and GZMB. Previous studies have demonstrated the multiple roles of CXCR6 on HSC activation, fibrogenesis, and proliferation, and the activation of NK might lead to HSCs apoptosis (Ma et al., 2017). Therefore, we further detected whether CXCL16 released from PIC Ⅱ-treated macrophages may recognized by CXCR6 expressed on NK cells. The relative mRNA levels of Cxcr6 both in vivo and invitro were increased after PIC Ⅱ administration in livers of Mdr2-/- mice and co-cultured NK cells (Fig. 4G and 4H ). These results suggested that PIC Ⅱ promoted M1-polarized macrophages to recruit and activate NK cells by the CXCL16-CXCR6 pathway.