Li Ronald H L, Ng Geena, Tablin Fern
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA, United States.
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA, United States.
Vet Immunol Immunopathol. 2017 Dec;193-194:29-37. doi: 10.1016/j.vetimm.2017.10.002. Epub 2017 Oct 20.
Neutrophils release neutrophil extracellular traps (NETs), which are extracellular chromatin decorated with histones and antimicrobial proteins. Although known for antimicrobial properties, overzealous production of NETs (NETosis) may lead to cytotoxicity and multiple organ failure in sepsis. Pathogen-induced NETosis has been extensively studied in mice but its importance in dogs remains largely unknown. This study sought to characterize in vitro NETosis induced by E.coli LPS, including assessing the role of peptidylarginine deiminase (PAD) in canine NETosis. Neutrophils (1×10 cells/ml) from healthy dogs were isolated and treated with 100μg/ml LPS, 100nM phorbol 12-myristate 13-acetate (PMA), or buffer for either 90 or 180min. NETs were assessed using fluorescence microscopy of living neutrophils and immunofluorescent microscopy. Supernatant and cellular debris were purified and cell-free DNA was quantified by spectrophotometry. The role of PAD was assessed by treating LPS- and PMA-activated neutrophils with 50, 100 or 200μM of the PAD inhibitor, Cl-amidine. In vitro NETosis was characterized by co-localization of cell-free DNA, citrullinated histone H3, and myeloperoxidase. LPS stimulation resulted in intracellular citrullination of histone H3. Compared to PMA chemically-induced NETosis, LPS resulted in smaller NETs with less extracellular citrullinated histone H3. Cl-amidine decreased citrullination of histones and NET production in either LPS- or PMA-stimulated neutrophils demonstrating that neutrophil PAD is essential for these cellular processes.
中性粒细胞释放中性粒细胞胞外诱捕网（NETs），它是由组蛋白和抗菌蛋白修饰的细胞外染色质。尽管NETs以抗菌特性而闻名，但过度产生NETs（NETosis）可能导致脓毒症中的细胞毒性和多器官功能衰竭。病原体诱导的NETosis已在小鼠中得到广泛研究，但其在犬类中的重要性仍 largely unknown。本研究旨在表征大肠杆菌脂多糖（LPS）诱导的体外NETosis，包括评估肽基精氨酸脱氨酶（PAD）在犬类NETosis中的作用。从健康犬分离出中性粒细胞（1×10细胞/ml），并用100μg/ml LPS、100nM佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）或缓冲液处理90或180分钟。使用活中性粒细胞的荧光显微镜和免疫荧光显微镜评估NETs。纯化上清液和细胞碎片，并通过分光光度法定量无细胞DNA。通过用50、100或200μM的PAD抑制剂Cl - 脒处理LPS和PMA激活的中性粒细胞来评估PAD的作用。体外NETosis的特征是无细胞DNA、瓜氨酸化组蛋白H3和髓过氧化物酶的共定位。LPS刺激导致组蛋白H3的细胞内瓜氨酸化。与PMA化学诱导的NETosis相比，LPS导致较小的NETs，细胞外瓜氨酸化组蛋白H3较少。Cl - 脒降低了LPS或PMA刺激的中性粒细胞中组蛋白的瓜氨酸化和NET产生，表明中性粒细胞PAD对这些细胞过程至关重要。
