背景:中性粒细胞是具有必需的抗微生物效应子功能和短寿命的粒细胞。在感染或无菌炎症期间,紧急粒细胞生成导致未成熟的中性粒细胞从骨髓中释放,用于增加循环中性粒细胞计数。稳定状态和紧急粒细胞生成尚未完全了解,部分原因是缺乏遗传上适合的中性粒细胞发育模型。
方法:我们优化了从CD34+造血祖细胞离体产生人嗜中性粒细胞的方法。使用流式细胞术,我们在表型上比较了培养的中性粒细胞与来自经历紧急粒细胞生成的供体的天然中性粒细胞,和来自非攻击供体的稳态中性粒细胞。我们对培养的嗜中性粒细胞进行功能和蛋白质组学表征,并建立祖细胞的基因组编辑。
结果:我们获得了离体培养的中性粒细胞的高产量,其表型类似于在紧急粒细胞生成期间释放到循环中的未成熟中性粒细胞。培养的中性粒细胞具有相似的ROS产生和细菌杀伤率,但改变了脱颗粒,与从外周血分离的成熟嗜中性粒细胞相比,细胞因子释放和抗真菌活性。这些差异可能是由于颗粒蛋白的不完全合成,蛋白质组学分析表明。
结论:离体培养的中性粒细胞可通过前体的基因组编辑进行遗传处理,并为研究未成熟中性粒细胞的特性和行为提供了强大的模型系统。
BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency
granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency
granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development.
METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency
granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors.
RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency
granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis.
CONCLUSIONS: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.