Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, United Kingdom.
Centre for Cardiovascular and Metabolic Neuroscience, Neuroscience, Physiology and Pharmacology, University College London, London WC1E 6BT, United Kingdom.
EBioMedicine. 2019 Sep;47:457-469. doi: 10.1016/j.ebiom.2019.08.004. Epub 2019 Aug 8.
Neutrophil depletion improves neurologic outcomes in experimental sepsis/brain injury. We hypothesized that neutrophils may exacerbate neuronal injury through the release of neurotoxic quantities of the neurotransmitter glutamate.
Real-time glutamate release by primary human neutrophils was determined using enzymatic biosensors. Bacterial and direct protein-kinase C (Phorbol 12-myristate 13-acetate; PMA) activation of neutrophils in human whole blood, isolated neutrophils or human cell lines were compared in the presence/absence of N-Methyl-d-aspartic acid receptor (NMDAR) antagonists. Bacterial and direct activation of neutrophils from wild-type and transgenic murine neutrophils deficient in NMDAR-scaffolding proteins were compared using flow cytometry (phagocytosis, reactive oxygen species (ROS) generation) and real-time respirometry (oxygen consumption).
Both glutamate and the NMDAR co-agonist d-serine are rapidly released by neutrophils in response to bacterial and PMA-induced activation. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, d-serine and the respiratory burst by activated primary human neutrophils. A highly specific small-molecule inhibitor ZL006 that limits NMDAR-mediated neuronal injury also reduced ROS by activated neutrophils in a murine model of peritonitis, via uncoupling of the NMDAR GluN2B subunit from its' scaffolding protein, postsynaptic density protein-95 (PSD-95). Genetic ablation of PSD-95 reduced ROS production by activated murine neutrophils. Pharmacological blockade of the NMDAR GluN2B subunit reduced primary human neutrophil activation induced by Pseudomonas fluorescens, a glutamate-secreting Gram-negative bacillus closely related to pathogens that cause hospital-acquired infections.
These data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells. FUND: GLA: Academy Medical Sciences/Health Foundation Clinician Scientist. AVG is a Wellcome Trust Senior Research Fellow.
中性粒细胞耗竭可改善实验性脓毒症/脑损伤的神经预后。我们假设中性粒细胞可能通过释放大量神经毒性谷氨酸使神经元损伤恶化。
使用酶生物传感器测定原代人中性粒细胞实时释放的谷氨酸。在存在/不存在 N-甲基-D-天冬氨酸受体（NMDA 受体）拮抗剂的情况下，比较人全血、分离的中性粒细胞或人细胞系中细菌和直接蛋白激酶 C（佛波醇 12-肉豆蔻酸 13-乙酸酯；PMA）激活的中性粒细胞。使用流式细胞术（吞噬作用、活性氧（ROS）生成）和实时呼吸测量法（耗氧量）比较野生型和缺乏 NMDA 支架蛋白的转基因鼠中性粒细胞的细菌和直接激活。
细菌和 PMA 诱导的激活均可使中性粒细胞快速释放谷氨酸和 NMDA 共激动剂 D-丝氨酸。药理学 NMDA 受体阻断可减少激活的原代人中性粒细胞的谷氨酸、D-丝氨酸的自分泌释放和呼吸爆发。一种高度特异性的小分子抑制剂 ZL006 可通过将 NMDA 受体 GluN2B 亚基与其支架蛋白突触后密度蛋白-95（PSD-95）解偶联，减少脓毒症小鼠模型中激活的中性粒细胞的 ROS。PSD-95 基因敲除减少了激活的鼠中性粒细胞的 ROS 产生。药理学阻断 NMDA 受体 GluN2B 亚基可减少与医院获得性感染相关的产生谷氨酸的革兰氏阴性杆菌荧光假单胞菌激活的人中性粒细胞的激活。
这些数据表明，激活的中性粒细胞释放的谷氨酸通过作用于这些细胞表达的 NMDA 受体以自分泌方式增强 ROS 产生。资助：GLA：医学科学院/健康基金会临床科学家。AVG 是威康信托基金会的高级研究员。
