3.5 PIC Ⅱ facilitated NK cells to kill activated HSCs dependent on IFN-γ-Jak1/Tyk2-STAT1 pathway
To further elucidate whether NK cells activated by macrophages could kill aHSCs, we then isolated primary NK cells from the spleen of mice, which were pre-treated with IL-2 and/or CM-M with different doses of PIC Ⅱ and were then co-cultured with activated HSCs induced by TGF-β in a 10 : 1 ratio (Fig. 5A ). The purity of NK cells (CD16, CD56, and the staining of CD161 marked the activity of NK cells) isolated from mice have been tested (Fig. S5A and S5B ). As shown inFig. 5B , the cell viability of co-culture cells in the model group and PIC Ⅱ CM-treated group were all significantly decreased. Additionally, we examined the mRNA levels of fibrosis-related targets in co-culture cells and found that with the presence of PIC Ⅱ-treated NK cells, the expression of Acta2 , Col1a1 and Fn1(fibrotic markers mainly expressed in HSCs) were markedly decreased (Fig. 5C ). This result was further refined by flow cytometry analysis stained with both α-SMA antibody and 7-AAD of co-culture cells to distinguish two different types of cells. As shown inFig.5D , compared to the model group, PIC Ⅱ increased the HSC death that marked by both α-SMA and 7-AAD in a dose-dependent manner, according to the clearly separated cell clusters. Meanwhile, immunofluorescence staining results showed that CM-M containing different doses of PIC Ⅱ significantly reduced the expression of a-SMA in co-culture of HSC-NK cells (Fig. 5E ). After we preliminarily confirm that activation and viability of HSCs could be inhibited by NK cells recruited by M1 macrophages, we intended to further explore the underlying mechanisms among these immune cells and HSCs.
Notably, NK cells can suppress aHSC populations via directly killing activated or senescent HSCs. However, the directly cytotoxicity of PERFORIN and GZMB released by NK cells on HSCs have been not upregulated significantly as expected (Fig. S6 andS4A ). On the other hand, the inhibitive effects of NK cells on HSCs are also relied on the IFN-γ-related network. Interestingly, we noticed an obvious upregulation of Ifng after administration of PIC Ⅱ both in Mdr2-/- mouse livers (Fig. 6A ) and in co-culture system of HSC and NK cells (Fig. 6B ). Indeed, the interaction of IFN-γ and its receptor at the cell surface was reported to lead the activation of JAK1 and TYK2, resulting in the phosphorylation and nuclear translocation of STAT1 and directly lead to the transcription of genes involved in cell apoptosis (Martí-Rodrigo et al., 2020). As shown inFig. 6C , compared with the Mdr2-/- mice, we found that the phosphorylation of Jak1, Tyk2 and its downstream factor STAT1 were all upregulated in the mouse liver after treated with different doses of PIC Ⅱ. Notably, phosphorylated STAT1 was mainly translocated in the nucleus after PIC Ⅱ administration, which suggested that NK cells processing an inhibited effect on aHSC mainly mediated by the release of IFN-γ. We also verified the hypothesis through the co-culture system as previously shown in Fig. 5A , Consistently, we noticed that the Ifng level in NK cells was upregulated by PIC Ⅱ (Fig. 6B ), followed by the phosphorylation of Jak1 and Tyk2 and both the phosphorylation and nuclear translocation of STAT1 in the co-cultured HSCs and NK cells (Fig. 6D ). These results suggested that after recruited by M1-macrophages released CXCL16, PIC Ⅱ might facilitate NK cells to kill aHSCs dependent on IFN-γ-Jak1/Tyk2-STAT1 pathway.