First, IFN-γ production by NK cells was significantly enhanced when cocultured with early activated D4 HSCs, yet compared

with D4 Lumacaftor clinical trial HSCs, IFN-γ production was lower when cocultured with intermediately activated D8 HSCs (Fig. 4). Second, western blotting and RT-PCR analyses showed that TGF-β1 mRNA and protein expression were highly induced in HSCs from advanced liver fibrosis (Figs. 3C-F) and intermediately activated D8 HSCs (Fig. 4D). Third, blocking TGF-β with a neutralizing antibody increased NK cell killing and restored IFN-γ production of NK cells (Fig. 4E,F), whereas treatment with TGF-β decreased NK cell cytotoxicity (Supporting Fig. 4). Finally, TGF-β is known to inhibit NK cell–mediated selleck inhibitor cytotoxicity and cytokine production.7, 17, 21 Taken together, TGF-β likely plays an important role in inhibiting the antifibrotic effect of NK cells, and resistance of intermediately activated HSCs to NK cell killing is likely mediated by the overproduction

of TGF-β. In addition to HSCs known as one of the major sources for TGF-β production,9, 10 Kupffer cells also play an important role in producing TGF-β during liver fibrogenesis.22 Future studies are required to determine whether Kupffer cells can also negatively regulate NK cell functions through production of TGF-β in advanced liver fibrosis. In addition MCE to resistant to NK cell killing, HSCs isolated from advanced fibrosis liver or intermediately activated D8 HSCs are also less responsive to IFN-γ stimulation (Fig. 3F and Supporting Figs. 3 and 6). The reduced responsiveness of these cells to IFN-γ stimulation is likely due to the increased expression of SOCS1 (Figs. 3F and 5B-D), because SOCS1 is known to be a key mediator in suppressing IFN-γ signaling.23 This conclusion was supported by the fact that IFN-γ inhibition of cell proliferation and activation of STAT1 were

restored in D8-cultured IFN-γ−/− SOCS1−/− HSCs compared with D8-cultured IFN-γ−/−SOCS1+/+ HSCs (Fig. 5E,F). Further experiments suggest that up-regulation of SOCS1 in D8 HSCs is due to RA production during HSC activation. Quiescent HSCs store approximately 80% of the body retinols, which are released or metabolized into Ralds by alcohol dehydrogenase and subsequently RA by retinaldehyde dehydrogenases during HCS activation.8, 24, 25 An increase of RA and a decrease of retinol content have been reported in CCl4 and thioacetamide-induced fibrotic livers of rats, and in cultured HSCs.8, 26 In the current study, we demonstrate that inhibition of retinol metabolism by 4-MP–reduced expression of SOCS1 and subsequently increased the IFN-γ activation of STAT1 signaling in HSCs (Fig. 6), suggesting a role of retinol metabolites in the induction of SOCS1 in HSCs.