INSERM UMRS-1149, Faculté de Médecine Xavier Bichat, Paris, France.
CNRS ERL-8252, Centre de Recherche sur l'Inflammation, Paris, France.
Gut. 2018 Aug;67(8):1505-1516. doi: 10.1136/gutjnl-2016-313443. Epub 2017 Jun 10.
Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients' susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients' neutrophils and blood.
Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR.
The impaired superoxide production by patients' neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 /NOX2 and p22 ), its cytosolic partner p47 but not p67 . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients' plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients' neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients' neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production.
Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.
肝硬化通过其抗菌超氧化物生成系统 NADPH 氧化酶 (NOX2) 下调吞噬细胞的氧化剂产生，增加患者感染的易感性和死亡率。为了探索解释易感性的新生化参数，我们研究了严重酒精性肝硬化患者中性粒细胞中 NOX2 和伙伴的表达，并提供了一种恢复患者中性粒细胞和血液中超氧化物产生能力的新方法。
从失代偿性酒精性肝硬化患者中分离中性粒细胞。用细菌衍生肽 fMet-Leu-Phe 刺激纯化的中性粒细胞或全血后，评估 NOX2 活性。通过 Western blot 分析、流式细胞术和逆转录-PCR 研究 NOX2 及其伙伴的表达。
患者中性粒细胞产生的超氧化物减少与 NADPH 氧化酶催化核心黄素细胞色素-b558 (gp91 /NOX2 和 p22 )、其胞质伴侣 p47 但不是 p67 的严重表达缺陷有关。NOX2 表达在健康中性粒细胞脱颗粒时用 fMet-Leu-Phe 刺激迅速通过涉及弹性蛋白酶释放的蛋白降解而减少，或在患者血浆中高度存在。有趣的是，用 Toll 样受体 7/8 (TLR7/8) 激动剂治疗患者的中性粒细胞和全血可逆转缺乏超氧化物的产生。这种治疗通过涉及哺乳动物雷帕霉素靶蛋白 (mTOR) 的过程刺激 NOX2 的快速转录和翻译，mTOR 的表达在患者的中性粒细胞中也不足。TLR4 激动剂脂多糖也增加了 NOX2 的表达，但只有适度改善活性氧的产生。
酒精性肝硬化中性粒细胞氧化剂产生的损害与 NOX2 降解和 mTOR 依赖性翻译机制缺陷有关。通过 TLR7/8 激活可以逆转 NOX2 耗竭，并可能用于恢复免疫功能低下患者的抗菌反应。
