Activation of innate immunity (natural killer [NK] cell/interferon‐γ [IFN‐γ]) has been shown to play an important role in antiviral and antitumor defenses as well as antifibrogenesis. However, little is known about the regulation of innate immunity during chronic liver injury. Here, we compared the functions of NK cells in early and advanced liver fibrosis induced by a 2‐week or a 10‐week carbon tetrachloride (CCl4) challenge, respectively. Injection of polyinosinic‐polycytidylic acid (poly I:C) or IFN‐γ induced NK cell activation and NK cell killing of hepatic stellate cells (HSCs) in the 2‐week CCl4 model. Such activation was diminished in the 10‐week CCl4 model. Consistent with these findings, the inhibitory effect of poly I:C and IFN‐γ on liver fibrosis was markedly reduced in the 10‐week versus the 2‐week CCl4 model. In vitro coculture experiments demonstrated that 4‐day cultured (early activated) HSCs induce NK cell activation via an NK group 2 member D/retinoic acid–induced early gene 1–dependent mechanism. Such activation was reduced when cocultured with 8‐day cultured (intermediately activated) HSCs due to the production of transforming growth factor‐β (TGF‐β) by HSCs. Moreover, early activated HSCs were sensitive, whereas intermediately activated HSCs were resistant to IFN‐γ–mediated inhibition of cell proliferation, likely due to elevated expression of suppressor of cytokine signaling 1 (SOCS1). Disruption of the SOCS1 gene restored the IFN‐γ inhibition of cell proliferation in intermediately activated HSCs. Production of retinol metabolites by HSCs contributed to SOCS1 induction and subsequently inhibited IFN‐γ signaling and functioning, whereas production of TGF‐β by HSCs inhibited NK cell function and cytotoxicity against HSCs. Conclusion: The antifibrogenic effects of NK cell/IFN‐γ are suppressed during advanced liver injury, which is likely due to increased production of TGF‐β and expression of SOCS1 in intermediately activated HSCs. (HEPATOLOGY 2011;)

The important roles of retinols and their metabolites have recently been emphasized in the interactions between hepatic stellate cells (HSCs) and natural killer (NK) cells. Nevertheless, the expression and role of retinol metabolizing enzyme in both cell types have yet to be clarified. Thus, we investigated the expression of retinol metabolizing enzyme and its role in liver fibrosis. Among several retinol metabolizing enzymes, only alcohol dehydrogenase (ADH) 3 expression was detected in isolated HSCs and NK cells, whereas hepatocytes express all of them. In vitro treatment with 4-methylpyrazole (4-MP), abroad ADH inhibitor, or depletion of the ADH3 gene downregulated collagen and transforming growth factor-β1 (TGF-β1) gene expression, but did not affect α-smooth muscle actin gene expression in cultured HSCs. Additionally, in vitro, treatments with retinol suppressed NK cell activities, whereas inhibition of ADH3 enhanced interferon-γ (IFN-γ) production and cytotoxicity of NK cells against HSCs. In vivo, genetic depletion of the ADH3 gene ameliorated bile duct ligation- and carbon tetrachloride-induced liver fibrosis, in which a higher number of apoptotic HSCs and an enhanced activation of NK cells were detected. Freshly isolated HSCs from ADH3-deficient mice showed reduced expression of collagen and TGF-β1, but enhanced expression of IFN-γ was detected in NK cells from these mice compared with those of control mice. Using reciprocal bone marrow transplantation of wild-type and ADH3-deficient mice, we demonstrated that ADH3 deficiency in both HSCs and NK cells contributed to the suppressed liver fibrosis. Conclusion ADH3 plays important roles in promoting liver fibrosis by enhancing HSC activation and inhibiting NK cell activity, and could be used as a potential therapeutic target for the treatment of liver fibrosis.

Clinical trials and animal models suggest that infusion of bone marrow cells (BMC) is effective therapy for liver fibrosis, but the underlying mechanisms are obscure, especially those associated with early effects of BMC. Here, we analyzed the early impact of BMC infusion and identified the subsets of BMC showing antifibrotic effects in mice with carbon tetrachloride-induced liver fibrosis. An interaction between BMC and activated hepatic stellate cells (HSCs) was investigated using in vitro co-culturing system. Within 24 hours, infused BMC were in close contact with activated HSCs, which was associated with reduced liver fibrosis, enhanced hepatic expression of interleukin (IL)-10, expanded regulatory T cells but decreased macrophage infiltration in the liver at 24 hours after BMC infusion. In contrast, IL-10-deficient (IL-10−/−) BMC failed to reproduce these effects in the fibrotic livers. Intriguingly, in isolated cells, CD11b+Gr1highF4/80− and CD11b+Gr1+F4/80+ BMC expressed more IL-10 after co-culturing with activated HSCs, leading to suppressed expression of collagen and α-smooth muscle actin in HSCs. Moreover, these effects were either enhanced or abrogated, respectively, when BMC were co-cultured with IL-6−/− and retinaldehyde dehydrogenase 1−/− HSCs. Similar to murine data, human BMC expressed more IL-10 after co-culturing with human HSC lines (LX-2 or hTERT), and serum IL-10 levels were significantly elevated in patients with liver cirrhosis after autologous BMC infusion. Conclusion Activated HSCs increase IL-10 expression in BMC (CD11b+Gr1highF4/80− and CD11b+Gr1+F4/80+ cells), which in turn ameliorates liver fibrosis. Our findings could enhance the design of BMC therapy for liver fibrosis.

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