Wu Sheng-Yang, Wu-Hsieh Betty A
Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan.
Bio Protoc. 2020 Aug 20;10(16):e3716. doi: 10.21769/BioProtoc.3716.
Fungal pathogen is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill : one is CR3 downstream NADPH oxidase-dependent mechanism that kills opsonized ; the other one is dectin-2-mediated NADPH oxidase-independent neutrophil extracellular trap (NET) that kills unopsonized . Neutrophil killing of opsonized requires phagocytosing the organism and production of reactive oxygen species production (ROS). Most existing protocols that assay for neutrophil killing of requires a washing step after allowing neutrophils to phagocytose the organism. By definition, NET kills organisms extracellularly. Therefore, it is important to skip the washing step and add an optimal ratio of neutrophils and to the wells. To demonstrate the effect of NET, it is necessary to compare killing ability of neutrophils treated with micrococcal nuclease (MNase), an enzyme that digests NET, to that treated with heat-inactivated MNase. MNase is also applied to release NET-bound fungal elements for counting. This protocol can be applied to assay NET killing of other biofilm-forming organisms.
真菌病原体是全球医疗保健相关血流感染的主要原因之一。中性粒细胞是清除感染的主要效应细胞。我们的研究表明，小鼠中性粒细胞利用两种独立机制进行杀伤：一种是补体受体3（CR3）下游烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶依赖性机制，可杀伤被调理的病原体；另一种是树突状细胞相关C型凝集素-2（dectin-2）介导的不依赖NADPH氧化酶的中性粒细胞胞外陷阱（NET），可杀伤未被调理的病原体。中性粒细胞对被调理病原体的杀伤需要吞噬病原体并产生活性氧（ROS）。大多数现有的检测中性粒细胞对病原体杀伤作用的方案在中性粒细胞吞噬病原体后需要进行洗涤步骤。根据定义，NET在细胞外杀伤病原体。因此，跳过洗涤步骤并向孔中加入最佳比例的中性粒细胞和病原体很重要。为了证明NET的作用，有必要比较用微球菌核酸酶（MNase，一种消化NET的酶）处理的中性粒细胞与用热灭活MNase处理的中性粒细胞的杀伤能力。MNase也用于释放与NET结合的真菌成分以便计数。该方案可用于检测NET对其他形成生物膜的生物体的杀伤作用。
