背景:慢性高血糖会影响中性粒细胞功能,导致减少病原体杀死和增加发病率。这种损害与血糖升高直接相关,然而,这对中性粒细胞代谢及其在骨髓中的分化的具体影响尚不清楚,也很难研究。
方法:我们使用高分辨率呼吸测量法来研究静息和活化供体中性粒细胞的代谢,和流式细胞术测量表面CD15和CD11b的表达。然后,我们在标准培养基中使用向嗜中性粒细胞样细胞分化的HL-60细胞,并研究葡萄糖浓度加倍对分化代谢的影响。我们测量了耗氧率(OCR),以及肉碱棕榈酰转移酶1(CPT1)和柠檬酸合酶在中性粒细胞样分化过程中的酶活性。我们比较了表面表型,功能,和在两种葡萄糖浓度下分化的嗜中性粒细胞样细胞的OCR。
结果:富集后3小时,佛波醇12-肉豆蔻酸酯13-乙酸酯刺激后,供体中性粒细胞显示出CD11b和OCR的显着不稳定性。在HL-60中性粒细胞样细胞分化过程中,表面CD15和CD11b表达显着增加,线粒体质量丢失。分化的嗜中性粒细胞样细胞也表现出更高的CD11b表达,并且明显更具吞噬性。在高葡萄糖培养基中,我们测量了中性粒细胞样分化过程中柠檬酸合酶和CPT1活性的降低.
结论:HL-60中性粒细胞样分化概括了人类中性粒细胞分化的已知分子和代谢特征。增加的葡萄糖浓度与高血糖供体嗜中性粒细胞中描述的特征相关,包括增加的CD11b和吞噬作用。我们使用该模型来描述高血糖症中性粒细胞样细胞分化的代谢特征,并首次显示CPT1和柠檬酸合酶活性的下调,独立于线粒体质量。
BACKGROUND: Chronic hyperglycemia affects neutrophil functions, leading to reduced pathogen killing and increased morbidity. This impairment has been directly linked to increased glycemia, however, how this specifically affects neutrophils metabolism and their differentiation in the bone marrow is unclear and difficult to study.
METHODS: We used high-resolution respirometry to investigate the metabolism of resting and activated donor neutrophils, and flow cytometry to measure surface CD15 and CD11b expression. We then used HL-60 cells differentiated towards neutrophil-like cells in standard media and investigated the effect of doubling glucose concentration on differentiation metabolism. We measured the oxygen consumption rate (OCR), and the enzymatic activity of carnitine palmitoyl transferase 1 (CPT1) and citrate synthase during neutrophil-like differentiation. We compared the surface phenotype, functions, and OCR of neutrophil-like cells differentiated under both glucose concentrations.
RESULTS: Donor neutrophils showed significant instability of CD11b and OCR after phorbol 12-myristate 13-acetate stimulation at 3 hours post-enrichment. During HL-60 neutrophil-like cell differentiation, there was a significant increase in surface CD15 and CD11b expression together with the loss of mitochondrial mass. Differentiated neutrophil-like cells also exhibited higher CD11b expression and were significantly more phagocytic. In higher glucose media, we measured a decrease in citrate synthase and CPT1 activities during neutrophil-like differentiation.
CONCLUSIONS: HL-60 neutrophil-like differentiation recapitulated known molecular and metabolic features of human neutrophil differentiation. Increased glucose concentrations correlated with features described in hyperglycemic donor neutrophils including increased CD11b and phagocytosis. We used this model to describe metabolic features of neutrophil-like cell differentiation in hyperglycemia and show for the first time the downregulation of CPT1 and citrate synthase activity, independently of mitochondrial mass.