巨噬细胞亚群和功能异质性在肝纤维化中的作用、分子机制和干预研究

Hepatic macrophages can exert dual function in the progression and regression of liver fibrosis by either promoting or abrogating the excessive deposition of extracellular matrix, which have been proposed as potential targets in combating fibrosis. Recent experimental studies reveal the breakthrough progression on macrophages heterogeneity that have been presented by the origin of macrophages subsets and macrophages function. However, the detailed molecular mechanisms of regulation on macrophages heterogeneity during liver fibrosis are not well understood. Using macrophage-specific RBP-J deficient mice, our previous studies have shown that Notch signaling blockade is required for macrophages to ameliorate hepatic fibrosis by inhibiting NF-κB activation through cylindromatosis (CYLD). Moreover, the transplantation of cultured macrophages, either bone marrow derived macrophages or Kupffer cells, into mice suffering from liver fibrosis can obviously impede the progression of liver fibrosis, suggesting that Notch signaling could regulate different origin of macrophage subsets and distinctive activation of macrophages involved in liver fibrosis. In the current project, based on Notch, we intend to clarify the molecular and cellular role of heterogeneity of hepatic macrophages subsets and macrophages activation in liver fibrosis by using several fate mapping mice and genetic modified mice. Furthermore, we will verify the role of macrophages heterogeneity in patients with liver fibrosis by using clinical tissue samples, and try to develop some possible macrophages-based interventional strategies for liver fibrosis therapy in near future These studies are expected to uncover the new molecule mechanisms on regulation of macrophages heterogeneity in liver fibrosis, and provide new strategies and targets for the treatment of liver fibrosis. Therefore, our studies are of important theoretical significance and potential application value.

肝巨噬细胞在肝纤维化进展和消退中发挥重要调控作用，是极具潜力的治疗靶点。近来巨噬细胞亚群来源和活化模式的异质性研究获突破性进展，但不同亚群和活化模式的巨噬细胞在肝纤维化中的作用及机制仍不清楚。我们利用巨噬细胞特异性RBP-J剔除小鼠发现： Notch-CYLD信号通过抑制NF-κB减轻肝纤维化；给肝纤维化小鼠回输培养的M1型巨噬细胞或Kupffer细胞可有效减轻肝纤维化。提示：Notch信号可能调控不同来源或活化模式的肝巨噬细胞参与肝纤维化的进展和消退。本课题拟以Notch信号为切入点，利用细胞命运示踪和基因修饰小鼠，结合肝纤维化模型，从分子、细胞、动物和临床标本水平回答不同亚群和活化模式的肝巨噬细胞在肝纤维化中的作用及其调控的分子机制，并建立基于巨噬细胞的肝纤维化干预方案。本研究有望揭示巨噬细胞亚群和功能异质性在肝纤维化中的作用和机制，为肝纤维化治疗提供新策略，具有重要的理论和实践意义。

肝巨噬细胞（Mφ）在肝纤维化进展和消退中发挥重要调控作用，是极具潜力的治疗靶点。近来Mφ亚群来源和活化模式的异质性研究获突破性进展，但其在肝纤维化中的作用及机制仍不清楚。本课题中，我们通过成功建立示踪来源不同Mφ：即卵黄囊来源组织定居巨噬细胞（KC）和骨髓单核来源巨噬细胞（BMDM）的遗传修饰小鼠以及相应的特异阻断/激活Notch信号的遗传修饰小鼠，结合肝纤维化模型发现：在纤维化进展期，髓系细胞激活Notch信号可通过调控CEBP/α抑制Ly6chi向Ly6clo的Mφ转变而加重肝纤维化；在肝纤维化消融期，组织定居KC阻断Notch信号可通过促进KC细胞增殖而加快消融。以类似思路将研究分别拓展到肾和肺纤维化中，发现：在输尿管结扎引发的肾纤维化中，髓系细胞阻断Notch信号通过调控CCR2+Mφ的浸润而减轻肾纤维化；在博来霉素诱导的肺纤维化中，髓系细胞阻断Notch信号则通过调控ly6clo AMs的增多而减轻肺纤维化。这些研究高度提示：阻断髓系细胞Notch信号活化可能是减轻组织纤维化的共同策略，具有重要的临床转化意义。. 研究显示M1型Mφ具有抗纤维化作用，而M2型具有促纤维化作用。我们将M0、M1和M2型Mφ分别回输给CCL4诱导的中度和重度纤维化小鼠，发现回输M1型Mφ通过改变肝脏免疫微环境而减轻小鼠肝纤维化。研究发表后，受到同行高度关注并发表评述认为我们的研究为利用更加精准的Mφ亚群治疗炎症、纤维化和肿瘤带来新的希望。之后，对M1型Mφ在纤维化肝脏中维持M1表型的机制发现： Notch信号可通过下游一系列microRNAs，如miR-125a、miR-148和miR-99b等，调控M1型Mφ维持其活化模式。. 由于肝纤维化持续进展将发展为肝硬化甚至肝癌，我们进而研究了Mφ异质性在肝癌进展中的作用和机制，发现Notch或Wnt/β-catenin信号调控不同来源肿瘤相关巨噬细胞（TAMs）极化和功能而参与肝癌进展。靶向TAMs递送miR-99b-5p通过重塑TAMs表型而抑制肝癌生长。. 总之，我们的研究明确和揭示了Notch信号调控Mφ亚群在肝纤维化进展/消融中的作用和机制，建立了输入特定激活模式的Mφ治疗肝纤维化/肝硬化的新思路，以及Mφ靶向miRNA递送系统，为肝癌治疗提供了新技术。