4. Discussion
Liver fibrosis is a global epidemic, characterized by the activated myofibroblasts through secreting ECM proteins and chronic interminable inflammation initiated and exaggerated by macrophages, dendritic cells, NK cells, and neutrophils. In the complex hepatic immune network, macrophages generally initiate adaptive immune inflammatory responses and release or stimulate paracellular cells to release various cytokines, which profoundly affect the activation, senescence, or apoptosis of HSCs (Kisseleva & Brenner, 2021; Tacke & Zimmermann, 2014). In this study, we explored the anti-fibrotic effect of PIC Ⅱ on the liver fibrosis in Mdr2-/- mice and found that PIC Ⅱ might inhibit the activation of HSCs via affecting various immune cells. Specifically, the increase of M1-polarized macrophages was observed in livers of Mdr2-/- mice treated with PIC Ⅱ, accompanied with highly expressed CXCL16 and obviously decreased fibrotic markers (Fig. 1 and 2 ). Although PIC Ⅱ exerted a slightly inhibitory effect on aHSCs, it significantly promotes the function of M1 polarized macrophages without inducing inflammation and facilitates the release of CXCL16 into the extracellular environment (Fig. 3 ). Next, we noticed the changes of NK cells in Mdr2-/- mice and found that NK cells were recruited by CXCL16 released from PIC Ⅱ-treated M1-type macrophages depend on the CXCR6 expressed on its cellular surface (Fig. 4 ). Furthermore, under the stimulation of PIC Ⅱ, the IFN-γ secreted by NK cells activated and phosphorylated the JAK1/TYK2 to promote the nuclear translocation of STAT1 and eventually induce cell death of activated HSCs (Fig. 6 ). In addition, PIC Ⅱ processing an inhibitive effect on neutrophils through blocking the NETs formation and possibly inducing the senescence of neutrophils for eventually preventing the more severe activations of HSCs in fibrotic environment (Fig. 7 ), and the depletion of macrophage will largely counteract this kind of protective effects of PIC Ⅱ (Fig. 8 ).
As the central immune cells in liver fibrosis procedure, macrophages play an important role in both regulating the deposition and breakdown of ECM. Emerging studies have shown that distinct macrophage subsets exert bidirectional roles and contribute to different pathological outcomes of liver fibrosis (Rao et al., 2022; Tacke, 2017). Activated macrophages have reportedly release various cytokines like NF-κB, TGF-β and activated TLRs to aggravate liver fibrosis by increasing the survival of activated HSCs (Rao et al., 2022). Whereas, M1 macrophages are recently treated as cryotherapy targets, which possess anti-liver fibrosis effects by recruiting endogenic macrophages and NK cells to inhibit HSC proliferation through MMPs (MMP2, MMP9, MMP13), IFN-γ and TRAIL(Ma et al., 2017). Our sequencing data showed that PIC Ⅱ markedly increased M1-polarized macrophages and promoted the secretion of several cytokines such as CXCL16 under the fibrotic environment, but exerting a limited direct inhibitory effect on aHSCs (Fig. 2 and3 ). Considering the gene expression of IFN-γ ,Itga1 and Gzmb , and the recruited characteristic of NK cells, we supposed that the CXCL16-positive M1 macrophage may recruit the NK cells and inhibit liver fibrosis in Mdr2-/-mice through CXCR6 that expressed on the surface of NK cells in concert with CXCL16.
The increase in both quantity and activity of NK cells has been acknowledged to provide a salutary effect in alleviating the progression of liver fibrosis (Tsuchida & Friedman, 2017). On one hand, NK cells recruited by macrophages can directly promote HSC apoptosis and ultimately hinder the fibrogenesis through the upregulation of GZMB and PERFORIN (Chigbu et al., 2019; Choi et al., 2021). On the other hand, NK cells can also interfere with the activation pathway of HSCs viaactivating IFN-γ-JAK1/TYK2-STAT1 signaling. Usually, IFN-γ was recognized to binds to its receptors IFNGR1 and IFNGR2 (Guo et al., 2022; Wen et al., 2017), then promote the phosphorylation of JAKs, TYK2 and STAT1. Indeed, JAKs/STATs pathway plays key roles in controlling chronic liver injury progression and liver regeneration. Also, among these STATs, the activation of STAT1 promotes the apoptotic signal in HSCs and eventually limits the development of liver fibrosis. Recently, Alberto and his colleague demonstrated that rilpivirine could ameliorate liver fibrosis by selectively activating the STAT1-dependent apoptosis in HSCs and STAT3-denpendent regeneration in hepatocytes to promote liver reconstruction (Martí-Rodrigo et al., 2020). Likewise, our study reveals that after treated with PIC Ⅱ, NK cells were recruited by CXC16 released from M1 macrophages, activated to induce HSC apoptosis through the activation of JAK1/TAK2-STAT1 following the release of IFN-γ, rather than directly killing HSCs by cytotoxic secretion of Gzmb and perforin (Fig. 5 and 6 ). These results offered a perspective to understand the interaction between the NK cells recruited by macrophages and HSCs in the liver fibrosis.
Interestingly, we also found a trend of neutrophil gene set and NETs formation in Mdr2-/- mouse livers. Previously, NETosis has been more commonly observed in NASH, liver cirrhosis/necrosis, and hepatocellular carcinoma (Scozzi & Gelman, 2023; Wu et al., 2023), but the MPO-citH3 index and the NETs formation results of liver tissue supported the hypothesis that NETs formation also occurs in the fibrotic and inflammatory environment of Mdr2-/- mouse livers. Here, we found the clear relevance between NETs formation and HSCs activation in Mdr2-/- mice and PIC Ⅱ administration could not only inhibited the NETosis but also lessen the collagen formation during this fibrotic process. Interestingly, CXCR4 was often treated as a marker of aged neutrophils, which is a receptor allowing the clearance of neutrophils in the bone marrow (Zhang et al., 2015). Our results also showed that Cxcr4 was upregulated in neutrophils recruited and downregulated in the PIC Ⅱ administration group, which offered a possibility of neutrophils senescence and provided a potential mechanism that PIC Ⅱ works in the liver fibrotic environment. However, the role and pattern of NETs under the crosstalk between M1 macrophages and neutrophils in this study are still uncertain, and further exploration is warranted.
It is also noteworthy that macrophages, as candidates for the treatment of liver fibrosis, have different polarization states and a wide range of plasticity spectra. Currently, the selective macrophage depletion could reduce BDL-induced fibrotic liver injury, which was accompanied by inhibition of lncRNA H19, while the overexpression of H19 could counteract the effects of macrophage depletion in liver fibrosis.(Tian et al., 2021). On the other hand, one fact that cannot be ignored is that CL could not selectively clear a specific subtype of macrophages in liver. Thus, there is another study reported that the depletion of macrophages via CL had no therapeutic effect on bile duct expansion in livers of Anks6-/- mice (Airik et al., 2022). In our study, the depletion of macrophage through CL effectively influenced the release of CXCL16 and partially blocked the anti-hepatic fibrosis effects of PIC Ⅱ on the livers of Mdr2-/-mice (Fig. 8 ). Therefore, a single method of CL depletion may lead to complex and contradictory consequences due to different functions of hepatic macrophages and whether this method can be used for the treatment of liver fibrosis remains to be determined.