Suppressive effects of bone marrow cells and natural killer cells on activated hepatic stellate cells via hepatic immune modulation

Abstract

Part I. $CD11b^{+}Gr1^{+}$ Bone Marrow Cells Ameliorate Liver Fibrosis by Producing Interleukin-10 in Mice Recent clinical trials and animal models demonstrated that infusion of bone marrow cells (BMCs) is an effective therapy for liver cirrhosis, but the underlying mechanism is still unclear. Here, we investigated the early effects of BMC infusion and identified the subsets of BMC showing antifibrotic effects in mice with two weeks of carbon tetrachloride-induced liver fibrosis. In addition, an interaction between BMCs and activated hepatic stellate cells (HSCs) were investigated. Within 24 hours, infused BMCs were in close contact with activated HSCs, which was associated with reduced liver fibrosis, enhanced hepatic expression of interleukin (IL)-10, expanded Tregs but decreased macrophage infiltration in the liver. Moreover, major subsets of infused BMCs, which produced IL-10, were $CD11b^{+}Gr1^{high}F4/80^{-}$ and $CD11b^{+}Gr1^{+}F4/80^{+}$ BMCs in the fibrotic liver. In addition, we found that BMC-derived IL-10 is mandatory for anti-fibrotic effects of BMC infusion, because IL-10-deficient ($IL-10^{-/-}$) BMCs failed to reproduce these effects in the fibrotic livers. In vitro, $CD11b^{+}Gr1^{high}F4/80^{-}$ and $CD11b^{+}Gr1^{+}F4/80^{+}$ BMCs expressed more IL-10 after co-culture with activated HSCs. In contrast, the expression of collagen and $\alpha$ -smooth muscle actin in HSCs was suppressed by them. Indeed, these effects were either enhanced or abrogated, respectively, when BMCs co-cultured with $IL-6^{-/-}$ and retinaldehyde dehydrogenase 1 $(RALDH1)^{-/-}$ HSCs. Similar to murine data, human BMCs expressed more IL-10 after co-culturing with human HSC lines (LX-2 or hTERT). In conclusion, activated HSCs increase IL-10 expression in infused BMCs ($CD11b^{+}Gr1^{high}F4/80^{-}$ and $CD11b^{+}Gr1^{+}F4/80^{+}$), which in turn ameliorates liver fibrosis. Our findings could enhance the design of BMC therapy for liver fibrosis.

Part II. BMC infusion with TLR3 Agonist Poly I:C Reverse Advanced Stages of Liver Fibrosis in Mice. Immunotherapy based on activation of innate immunity (interferon-$\gamma$ [IFN-$\gamma$] and natural killer [NK] cells) has been investigated to treat the patients with liver fibrosis as well as cancer and viral diseases. However, in patients with chronic liver fibrosis or cirrhosis, this immunotherapy failed to show treatment effects due to the resistance of activated hepatic stellate cells (HSCs) to IFN-$\gamma$ /NK cells treatment. Therefore, we infused bone marrow cells (BMCs) prior to treatment of polyinsinic-polycytidylic acid (poly I:C), which is a potent activator of NK cells, in advanced liver fibrosis induced by a 10-week carbon tetrachloride ($CCl_4$) challenge to overcome the resistance of activated HSC to IFN-$\gamma$ /NK cells. Poly I:C treatment followed by BMC infusion significantly reduced liver fibrosis and $\alpha$ -smooth muscle actin $(\alpha -SMA)^{+}$ activated HSCs compared with poly I:C treatment alone and BMC infusion alone. In vitro experiment, BMCs enhanced 8-day cultured (intermediately-activated) HSCs killng by NK cells via upragulation of death receptors, Fas and tumor necrosis factor receptor type 1 (TNFR1), in the HSCs, although BMCs directly reduced NK cell cytotoxicity via interleukin (IL)-10. Moreover, the upregulation of TNFR1 in activated HSCs by BMCs was confirmed in vivo. The direct effects of BMCs to NK cells reducing those cytotoxicity contributed to reduce poly I:C-related toxicities in vivo. Our findings provide novel therapeutic way to overcome the resistance of intermediately-activated HSCs to IFN-$\gamma$ and poly I:C treatment for patients with chronic liver fibrosis or cirrhosis.