Interestingly, in HBV-tg mice, depletion of NK cells before CCl4 administration had no effect on HSCs activation but depletion of NKT and NK cells together decreased HSCs activation by examining transcription of α-SMA (Fig. 7C), indicating NKT cells possibly play a critical role in the HSC activation. On the contrary in C57BL/6 mice, depletion LY294002 of NK or NKT cells, both increased the transcription of α-SMA (Fig. 7C), which was similarly documented previously.20-22 These results suggest that NKT cells play a critical role in HSCs overactivation and liver fibrosis only in HBV-tg mice, but both NK and NKT cells are antifibrotic in C57BL/6 mice. To further demonstrate the role of NKT cells in HBV-related liver fibrosis, we

adoptively transferred the purified liver NKT cells from C57BL/6 or HBV-tg mice to Rag1−/− mice and then treated the cellular-adoptively transferred Rag1−/− mice with CCl4. It was noted that the α-SMA expression was increased (Fig. 7D), along with more inflammatory cells in the liver (Supporting Information Fig. 4), if the

transferred NKT cells were derived from HBV-tg mice but not from C57BL/6 mice, indicating NKT cells from HBV-tg mice might exert a function to activate HSCs in liver fibrosis. These results LDK378 raise the possibility that more inflammation exists in HBV-tg mice-derived NKT cell-transferred Rag1−/− mice, which may initiate the activation of the stellate cells. Because CD1d expression by antigen-presenting cells is required for CD1d-restricted NKT cell activation, we blocked CD1d-NKT cell recognition by injecting anti-CD1d antibody before CCl4 injection. We observed that the HSC activation was reduced in CD1d antibody-pretreated HBV-tg mice (Fig. 7E). We also found that the transcription levels of TIMP1, one of the representative fibrotic genes, correlated with the change of α-SMA in NKT cell-depleted HBV-tg mice (Supporting

Information Fig. 5A), HBV-tg mice-derived liver NKT cell-transferred Rag1−/− mice (Supporting PAK5 Information Fig. 5B), and anti-CD1d mAb-treated HBV-tg mice (Supporting Information Fig. 5C). Taken together, these data suggest that NKT cells from HBV-tg mice aggravate the HSC activation to cause liver fibrosis. NKT cells are well known for their strong and rapid production of cytokines. We observed that the transcriptional expression of IL-4, IL-13, and IFN-γ were significantly higher in the livers of HBV-tg mice after CCl4 injection (Fig. 8A). Moreover, the absolute number of NKT cells increased much more in HBV-tg mice after CCl4 injection at 6, 12, and 24 hours, respectively (Fig. 8B), along with significantly more increase in the number of IL-4-, IL-13-, or IFN-γ-secreting NKT cells in HBV-tg mice after CCl4 treatment than that of C57BL/6 mice (Fig. 8C). In the HSC and NKT cell coculture experiments, we found that neutralizing antibodies against IL-4 and IL-13 could attenuate the activation of HSC, but not the one against IFN-γ (Fig.