6A). Image analyses and western blotting further confirmed these findings (Fig. 6B,C). qRT-PCR showed significantly

increased IL-10 mRNA expression in liver MNCs of WT BMC-infused mice, but not in IL-10–deficient BMC-infused mice compared with Tamoxifen controls (Fig. 6D). Moreover, frequencies of Tregs in livers of IL-10–deficient BMC-infused mice were unchanged compared with controls (Fig. 6E,F). These data indicate that infused BMC-derived IL-10 is a key molecule that accounts for the antifibrotic activity observed in this model. Finally, we sought to identify mediators of HSCs that affected expression of IL-10 in BMCs. Because HSCs can produce IL-6, IL-10, and RALDH1-mediated retinoic acid, these factors have been considered as candidate components driving the inflammatory reaction, and expansion and differentiation of Tregs and MDSCs.11, 18-21 Accordingly, we cocultured BMCs with IL-6, IL-10, and RALDH1 gene-depleted HSCs, respectively. In the absence of IL-6 in HSCs, IL-10 expression was significantly increased in both adherent and floating BMCs compared with those of WT BMCs cocultured with WT HSCs (P < 0.05), whereas RALDH1-deficient HSCs did not increase IL-10 expression by BMCs compared with those of WT BMCs cocultured with WT HSCs (Fig. 7A, B). In addition, IL-10–deficient WT HSCs increased IL-10 expression similarly in EGFR inhibitor drugs both adherent

and floating BMCs compared with those of WT BMCs cocultured with WT HSCs (Fig. 7A,B). To reinforce the effect of retinoic acid on IL-10 production by infused BMCs in vivo, we administrated CCl4 to RALDH1-deficient mice for 2 weeks, and these animals were 上海皓元医药股份有限公司 then infused with WT BMCs. Twenty-four hours after infusion of BMCs, fibrosis was not ameliorated (Fig. 7C and Supporting

Fig. 6A). Based on FACS analyses, there were no significant changes in the frequencies of inflammatory cells, such as CD11b+F4/80+ macrophages and CD11b+Gr1+ granulocytes, and Tregs as well in liver (Fig. 7D and Supporting Fig. 6B,C). The beneficial effects of BMC therapy have been investigated recently in mice and humans, yet underlying mechanisms have been overlooked, especially the early effects of BMCs. In the present study, we identify early phase antifibrotic effects of infused BMC in vivo and in vitro, which reflect the interaction between HSCs and BMCs within 24 hours. The mechanisms of liver fibrosis amelioration by infused BMCs are summarized in Fig. 7E. Contrary to the reported long-term effects of BMCs in fibrotic livers of mice and humans,1-3 we have shown that at early time points, infused BMCs ameliorate liver fibrosis without any change in liver injury, hepatocyte regeneration, or albumin production (Fig. 1 and Supporting Fig. 1A), suggesting that there are no effects of bone marrow–derived stem cells within 24 hours after infusion.