Sae-Khow Kritsanawan, Tachaboon Sasipha, Wright Helen L, Edwards Steven W, Srisawat Nattachai, Leelahavanichkul Asada, Chiewchengchol Direkrit
Translational Research in Inflammation and Immunology Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Excellence Center for Critical Care Nephrology, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand.
J Inflamm Res. 2020 Jun 25;13:263-274. doi: 10.2147/JIR.S252433. eCollection 2020.
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.
This study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis.
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 ) 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 gene.
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.
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.
中性粒细胞功能对于有效抵御细菌感染至关重要，但脓毒症患者的中性粒细胞功能存在缺陷。败血症患者血浆中含量较低的抗坏血酸盐或维生素C，储存于人类中性粒细胞内，对其正常功能至关重要。
本研究旨在确定体外抗坏血酸盐治疗是否能改善脓毒症患者的中性粒细胞功能。
从20例脓毒症患者和20例年龄匹配的健康对照者中分离出人血中性粒细胞。中性粒细胞在有或无抗坏血酸盐（1、5、10、20和40 mM）的情况下孵育长达2小时。使用趋化室评估对趋化因子fMLP的趋化作用。通过流式细胞术测量吞噬作用（摄取pHrodo红色染色物）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并定量中性粒细胞胞外陷阱（NET）的形成。Quantifluor检测NET上清液中双链DNA的量，而定量PCR鉴定基因表达的变化。
脓毒症患者的趋化和吞噬活性降低，但高浓度抗坏血酸盐治疗后增加。脓毒症患者的细胞凋亡增加，但抗坏血酸盐治疗未改变。脓毒症患者中观察到自发NET形成。1 mM抗坏血酸盐可将自发NETosis降低至正常健康中性粒细胞的水平，而高浓度抗坏血酸盐（>10 mM）进一步促进NET形成。
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和预后。暴露于抗坏血酸盐可逆转其中一些功能变化。这些新发现增加了抗坏血酸盐治疗可作为辅助治疗以改善脓毒症期间中性粒细胞功能的可能性。
