肝硬化脾-肝区域免疫中脾脏单核-巨噬细胞的表型变化及分子调控

Liver regional immunity is relevant to chronic liver disease. As the largest peripheral immune organ, the spleen has a close relationship with the liver in terms of anatomy and function, and its effect on liver immunity should never be ignored. Under the support of the previous organ regional immunity project, we found that splenic monocytes/macrophages have the capability of promoting the accumulation of M1 macrophages in the liver, and thus contributing to the aggravation of fibrosis; and obtained indirect evidence for the conclusion that the accumulated M1 macrophages in the liver were partially recruited from the spleen. However, there is no direct evidence for the recruitment of spleen mononuclear macrophages by diseased liver; differences in the spleen-derived macrophages and liver resident macrophages, specific mechanisms involved in spleen-derived macrophages mediated liver regional immune regulation and disease progression still need to be studied. To this end, this project intends to use mouse liver cirrhosis model and clinical samples as research objects, determining whether spleen monocytes have been recruited to liver lesion by utilization of spleen transplantation and photo-conversion surgery; uncovering the differences between spleen-derived macrophages and resident macrophages in affecting disease progression by single cell sequencing; studying the impact of spleen-derived macrophages on the various types of liver immune cells; and analyzing the molecular mechanisms by which spleen monocytes are recruited to the liver. These results will fully analyze the liver regional immunity from a new perspective of "mutual control of spleen-liver region immunity" and provide a theoretical basis for the prevention and treatment of liver cirrhosis by “regulating the spleen and treating liver”.

肝区域免疫与慢性肝病密切相关。脾作为最大外周免疫器官、在结构和功能上与肝联系紧密，其对肝区域免疫的影响不可忽视。在前期器官区域免疫培育项目资助下，我们发现脾单核-巨噬细胞（Mφ）能促进肝内M1型Mφ聚集，参与病程进展；并获得了肝内M1型Mφ部分来源于脾的间接证据。然而，病变肝对脾单核Mφ的招募尚缺乏直接证据；募集到肝后分化形成的脾源性Mφ与肝定植Mφ的差异、脾源性Mφ参与肝区域免疫调控的方式和机制及其对肝病进程的影响等亟需研究。为此本项目拟以小鼠肝硬化模型及临床样本为研究对象，利用脾移植、光逆转等方法明确脾单核细胞是否被招募至肝；用单细胞测序等技术明确脾源性Mφ与肝定植Mφ表型及功能差异；研究脾源性Mø对肝各类型免疫细胞功能的影响；同时从多角度解析其被招募至硬化肝组织的分子机制。旨在从“脾-肝区域免疫交互调控”的新角度解析肝的区域免疫，为通过“调脾治肝”防治肝炎肝硬化的新思路提供理论依据。

慢性肝病为常见高发病，肝脏区域免疫与肝病的发生发展密切相关。单核细胞来源的巨噬细胞（monocyte-derived macrophages, MoMFs）是炎症下肝脏巨噬细胞的主要群体，在肝纤维化进展中发挥重要作用。脾作为最大的外周免疫器官、在结构和功能上与肝联系紧密，脾血全部经门静脉回流入肝。临床肝硬化门脉高压时切除脾脏肝病改善；肝纤维化小鼠模型中切除脾脏缓解肝纤维化，且肝脏中MoMFs的聚集与组成发生改变。然而，脾脏是否为病变肝组织提供特定类型的巨噬细胞、进而参与肝病进展调控还不清楚。本项目拟为肝脏募集脾源单核-巨噬细胞提供直接证据，揭示脾源性细胞的特征及其作用机制，主要取得以下研究成果：（1）获得脾脏细胞向肝脏迁移的直接证据。利用单细胞RNA测序技术对分离自纤维化肝脏的脾源性CD11b+细胞进行注释，通过与脾脏、肝脏中总CD11b+细胞合并分析，鉴定出一种CD11b+CD43hiLy6Clo脾单核细胞亚群（sM-1），并发现sM-1表现成熟特征、产生大量活性氧、倾向于向肝脏迁移。这些结果揭示了肝纤维化肝硬化时肝脏对脾脏单核细胞的招募特性，以及被招募至病变肝脏的脾源性单核-巨噬细胞的分子特征。（2）利用拟时序分析及体内外转变实验，阐明了sM-1在肝脏微环境中向巨噬细胞（sM-1—sM-2—sMφ）转变的过程，并明确sMφ通过活化肝星状细胞加剧肝纤维化的作用机制。这一成果首次为脾脏单核-巨噬细胞参与慢性肝病进展提供了直接证据，明确其肝内转变及其通过与肝内细胞相互作用影响肝纤维化进程的机制。（3）研究过程中，构建了模拟体内血流循环的门静脉回流性小鼠脾移植模型，通过CD45.1+/CD45.2+小鼠间脾脏移植，成功实现了对脾脏单核细胞向肝脏迁移的在体追踪；借助KikGR小鼠对脾脏细胞进行特异性光转换，活体成像观察到脾脏细胞从脾脏向肝脏的迁移及其与肝脏细胞的相互作用。这些模型的建立为脾脏细胞追踪及脾脏免疫细胞参与肝脏及其他器官免疫调控的研究提供了新的技术手段。本研究相关标志性成果发表在Hepatology, Theranostics等期刊。