M-CSF-responsive immortal macrophages are a new model for studying macrophage function. M-CSF-responsive immortal macrophages are a new model for studying macrophage function

We generated non-senescent, macrophage colony stimulating factor (M-CSF) dependent macrophages from murine fetal livers. These fetal liver macrophages (FLM-M) resemble bone marrow macrophage (BMM) morphology and express similar macrophage surface markers as BMM. In comparison to granulocyte monocyte-colony stimulating factor (GM-CSF) derived FLM (FLM-G), FLM-M have higher surface expression of CD11b, CD68, CD14 and CD64, and lower expression of CD11c. BMM, FLM-M and FLM-G all produce TNFα and IL-6 in response to lipopolysaccharide activation. BMM and FLM-M cells have similar phagocytic properties with 99.3% of BMM and FLM-M actively phagocytosing opsonized sheep erythrocytes and a phagocytic efficiency of 0.9. The average number of internalized erythrocytes per cell was higher (13.1±10.0) in BMM than in FLM-M (7.6±2.9). FLM-G had fewer actively phagocytic cells (86%), lower phagocytic efficiency (0.7) and fewer internalized erythrocytes per macrophage (4.2±2.1) than BMM or FLM-M. Principal component analysis and hierarchical clustering demonstrated mRNA expression profiles were more similar between BMM and FLM-M cells than FLM-G. Transcriptome comparison to the ImmGen tissue atlas shows cultured BMM and FLM-M cluster with macrophage progenitors and bone marrow macrophages. In conclusion, FLM-M cell cultures share major morphological, phenotypic and functional properties of BMM except for senescence, and can be an effective substitute to BMM. These new cells will facilitate study of macrophage biology by enabling experiments requiring long-term culture or genetic modifications. Overall design: Comparison of 3 different murine macrophage cell cultures.

本研究从小鼠胎肝中分离培养获得非衰老型、依赖巨噬细胞集落刺激因子（macrophage colony stimulating factor, M-CSF）的巨噬细胞，即胎肝巨噬细胞（fetal liver macrophages, FLM-M）。该类细胞的形态与骨髓来源巨噬细胞（bone marrow macrophage, BMM）相似，且表达与BMM一致的巨噬细胞表面标志物。
与经粒细胞-巨噬细胞集落刺激因子（granulocyte monocyte-colony stimulating factor, GM-CSF）诱导的胎肝巨噬细胞（FLM-G）相比，FLM-M的CD11b、CD68、CD14及CD64表面表达水平更高，而CD11c的表达水平更低。
BMM、FLM-M与FLM-G均可在脂多糖（lipopolysaccharide）激活后分泌肿瘤坏死因子α（TNFα）与白细胞介素6（IL-6）。BMM与FLM-M的吞噬特性相近：99.3%的BMM与FLM-M可主动吞噬调理化绵羊红细胞，吞噬效率为0.9；但每细胞内化红细胞的平均数量，BMM（13.1±10.0）高于FLM-M（7.6±2.9）。相较BMM或FLM-M，FLM-G的主动吞噬细胞占比更低（86%），吞噬效率仅为0.7，且每巨噬细胞内化的红细胞数量更少（4.2±2.1）。
主成分分析（Principal component analysis）与层次聚类（hierarchical clustering）结果显示，BMM与FLM-M的mRNA表达谱相似度高于二者与FLM-G的相似度。
将转录组数据与ImmGen组织图谱（ImmGen tissue atlas）进行比对，可见培养的BMM与FLM-M与巨噬细胞祖细胞及骨髓巨噬细胞聚为一类。
综上，FLM-M细胞培养体系除不具备衰老特性外，在形态、表型与功能层面均与BMM高度相似，可作为BMM的有效替代模型。该新型细胞培养体系可支持长期培养或基因修饰相关实验，有助于推动巨噬细胞生物学研究。
整体实验设计：本研究对3种不同的小鼠巨噬细胞培养体系进行了比较。