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.