CD47-SIRPα axis blockade in NASH promotes necroptotic hepatocyte clearance by liver macrophages and decreases hepatic fibrosis. CD47-SIRPα axis blockade in NASH promotes necroptotic hepatocyte clearance by liver macrophages and decreases hepatic fibrosis

Necroptosis contributes to hepatocyte death (HC) in non-alcoholic steatohepatitis (NASH), but the fate and roles of necroptotic hepatocytes (necHCs) in NASH remain unknown. We show here that the accumulation of necHCs is associated with worsening NASH in humans and in mice with diet-induced NASH and that NASH liver showed evidence of impaired necHC clearance by liver macrophages. Further, the "don't-eat-me" ligand CD47 on HCs and its receptor, SIRPα, on liver macrophages, were markedly upregulated in human and mouse NASH. In vitro, anti-CD47 or anti-SIRPα promoted necHC engulfment by primary liver macrophages. In a proof-of-concept mouse model of inducible HC necroptosis, anti-CD47 increased necHC uptake by liver macrophages and inhibited hepatic stellate cell (HSC) activation, which is responsible for liver fibrogenesis. Most importantly, treatment of two mouse models of diet-induced NASH with anti-CD47 or anti-SIRPα increased the uptake of necHC by liver macrophages and decreased HSC activation and liver fibrosis. These findings provide evidence that impaired clearance of necHCs by liver macrophages due to CD47-SIRPα upregulation contributes to fibrotic NASH and suggest therapeutic blockade of the CD47-SIRPα axis as a strategy to decrease the accumulation of necHCs in NASH liver and dampen the progression of hepatic fibrosis. Overall design: Male C57BL/6J mice were fed the Fructose-Palmitate-Cholesterol diet (FPC, Envigo, #TD. 160785 PWD) for 16 weeks to provoke NASH. Between weeks 8 and 16, mice received IgG or anti-CD47 (Bio-X-Cell, #BE0283, 200 µg/mouse) through intraperitoneal injection (n = 7-8 mice/group). Liver tissue were used for RNA-sequencing.

细胞坏死性凋亡（Necroptosis）可促进非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）中的肝细胞死亡（hepatocyte death, HC），但坏死性凋亡肝细胞（necroptotic hepatocytes, necHCs）在NASH中的转归与作用仍未明确。本研究证实，人类及饮食诱导NASH小鼠体内的necHCs蓄积与NASH病情恶化相关，且NASH肝脏存在肝巨噬细胞清除necHCs功能受损的证据。进一步研究发现，人类与小鼠NASH模型中，肝细胞表面的“别吃我”（don't-eat-me）配体CD47及其在肝巨噬细胞上的受体SIRPα均显著上调。体外实验显示，抗CD47或抗SIRPα抗体可促进原代肝巨噬细胞对necHCs的吞噬作用。在可诱导肝细胞坏死性凋亡的概念验证小鼠模型中，抗CD47治疗可增强肝巨噬细胞对necHCs的摄取，并抑制肝星状细胞（hepatic stellate cell, HSC）活化——而后者是肝纤维化发生的关键环节。最为关键的是，采用抗CD47或抗SIRPα治疗两种饮食诱导NASH小鼠模型，可提升肝巨噬细胞对necHCs的摄取能力，同时降低HSC活化程度与肝纤维化程度。上述研究结果证实，CD47-SIRPα通路上调导致肝巨噬细胞清除necHCs功能受损，进而参与纤维化型NASH的发生发展，并提示阻断CD47-SIRPα轴可作为减少NASH肝脏内necHCs蓄积、延缓肝纤维化进展的治疗策略。实验整体设计：将雄性C57BL/6J小鼠喂食果糖-棕榈酸-胆固醇饮食（Fructose-Palmitate-Cholesterol diet, FPC，Envigo公司，货号#TD. 160785 PWD）16周以构建NASH模型。在第8至16周期间，小鼠通过腹腔注射给予IgG或抗CD47抗体（Bio-X-Cell，货号#BE0283，200 μg/只），每组设7-8只小鼠。取肝组织进行RNA测序。