Bone Marrow-Derived Macrophage Contributes to Fibrosing Steatohepatitis Through Activating Hepatic Stellate Cells

Abstract

Background & Aims: The role of macrophages in fibrosing steatohepatitis is largely unclear. We characterized the origin and molecular mechanisms of macrophages and its targeted therapy in fibrosing steatohepatitis. Methods: Fibrosing steatohepatitis was established in Alms1 mutant (foz/foz) and C57BL/6 wildtype mice fed high-fat/high-cholesterol (HFHC) or methionine-and choline-deficient (MCD) diet. Bone marrow transplantation (BMT) was performed to track the macrophage origin in fibrosing steatohepatitis. Macrophages were depleted by liposomal clodronate. Primary macrophages were isolated from bone marrow for adoptive transfer into mice. Results: Macrophage infiltration is significantly induced in two mouse models of fibrosing steatohepatitis and human NASH-fibrosis patients. Bone marrow-derived macrophages (BMMs) contribute to the hepatic macrophage accumulation in experimental fibrosing steatohepatitis. Hepatic BMMs depletion by liposomal clodronate significantly attenuated fibrosing steatohepatitis. This effect was associated with reduced hepatic stellate cell (HSC) activation, collagen deposition and hepatic expression of key pro-fibrotic factors (TIMP1, TIMP2 and TGFβ1) and endoplasmic reticulum (ER) stress markers (GRP78, IRE1α and PDI). Conversely, adoptive transfer of BMMs significantly aggravated fibrosing steatohepatitis. Moreover, macrophage conditioned medium directly promoted the phenotypic transition of primary quiescent HSCs to activated HSCs; enhanced activation and proliferation (P<0.01) but decreased apoptosis (P<0.001) of HSC cell lines (LX-2 and HSC-T6). The effect of BMMs in promoting fibrosing steatohepatitis was mediated by inducing key pro-fibrosis factors and signaling pathways including cytokine/chemokine, TGF-β, and complement cascade by cDNA expression array. Complement 3a receptor (C3aR1) was a predominant effector of macrophage mediated fibrosing steatohepatitis. Knockout C3aR1 in mice blunted the development of fibrosing steatohepatitis. Conclusions: Macrophages recruited from bone marrow promote the progression of fibrosing steatohepatitis through directly activating HSCs. BMMs targeted therapy is a promising approach for fibrosing steatohepatitis. Funding: The project was supported by the RGC-GRF Hong Kong (14106415, 14111216, 14163817); Science and Technology Program Grant Shenzhen (JCYJ20170413161534162); Vice-Chancellor's Discretionary Fund CUHK; Shenzhen Virtual University Park Support Scheme to CUHK Shenzhen Research Institute; CUHK Direct grant. The human liver specimens were provided by Prof. Vincent WS Wong in the Prince of Wales Hospital, the Chinese University of Hong Kong and Prof. Kwan Man in the Queen Mary Hospital, University of Hong Kong, Hong Kong. Competing interests: All authors declare that they have no competing interests. Ethics Approval Statement: All subjects were given written informed consent prior to sample collection and the study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.