Background: Neutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function.
Objective: This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis.
Patients and Methods: Human blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene.
Results: Chemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (> 10mM) further promoted NET formation.
Conclusion: Dysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.



中文翻译：

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脓毒症患者的中性粒细胞功能缺陷主要通过体外抗坏血酸培养来恢复。

背景：中性粒细胞功能对于有效防御细菌感染至关重要，但在脓毒症患者中存在缺陷。败血症患者血浆中含量较低的抗坏血酸盐或维生素 C 储存在人类中性粒细胞中，对其正常功能至关重要。
目的：本研究旨在确定体外抗坏血酸治疗是否能改善脓毒症患者的中性粒细胞功能。
患者和方法：从 20 名败血症患者和 20 名健康年龄匹配的对照中分离出人类血液中性粒细胞。中性粒细胞在有或没有抗坏血酸盐（1、5、10、20 和 40 mM）的情况下孵育长达 2 小时。使用趋化室响应趋化剂 fMLP 评估趋化性。吞噬作用（摄取 pHrodo 红染色通过流式细胞术测量金黄色葡萄球菌）和细胞凋亡（膜联蛋白-V/碘化丙啶染色）。使用 DAPI、抗髓过氧化物酶和抗中性粒细胞弹性蛋白酶免疫荧光染色检测和量化中性粒细胞胞外陷阱 (NET) 的形成。Quantifluor 检测了 NET 上清液中 dsDNA 的含量，而定量 PCR 检测了PADI4基因表达的变化。
结果：脓毒症患者的趋化和吞噬活性降低，但在高浓度抗坏血酸治疗后增加。脓毒症患者的细胞凋亡增加，但抗坏血酸治疗没有改变。在脓毒症患者中观察到自发的 NET 形成。1mM 抗坏血酸盐的量将自发性 NETosis 降低至正常、健康的中性粒细胞，而高浓度的抗坏血酸盐 (> 10mM) 进一步促进 NET 形成。
结论：在脓毒症患者中观察到中性粒细胞功能失调，这可能导致疾病病理学和结果。接触抗坏血酸可能会逆转其中一些功能变化。这些新发现提高了抗坏血酸治疗可用作辅助疗法的可能性，该辅助疗法可改善脓毒症期间的中性粒细胞功能。