Supplementary Material for: DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils

Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized <i>Listeria monocytogenes</i>-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.

中性粒细胞（Neutrophils）可通过由DNA纤维与杀菌蛋白构成的胞外复合物——中性粒细胞胞外陷阱（neutrophil extracellular traps, NETs）——杀灭细菌。目前学界对NET的组成成分及其介导的杀菌机制尚未完全阐明。本研究显示，脱氧核糖核酸酶I（DNase I）处理可削弱革兰氏阳性菌（Gram-positive）、革兰氏阴性菌（Gram-negative）以及佛波酯所诱导的晚期氧化应答。经调理的单核细胞增生李斯特菌（Listeria monocytogenes）刺激中性粒细胞所产生的异鲁米诺依赖化学发光可被DNase I抑制；且在吞噬作用被阻断的情况下，DNase I的抑制效应依然显著，这表明DNase I可抑制活性氧（ROS）生成的胞外途径。DNase I的抑制效应不依赖于肌动蛋白聚合，且不会对吞噬作用及细胞活力造成损伤。免疫荧光分析显示，髓过氧化物酶（myeloperoxidase）可存在于NET表面。此外，在存在胞吐缺陷的Rab27a缺陷型中性粒细胞所形成的NET中仍可检测到颗粒蛋白，这提示胞吐作用与NET上的颗粒蛋白分布是通过互不依赖的机制完成的。NET表面还可检测到烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidase）的亚基；且DNase I处理可消除与胞外陷阱结合的NADPH氧化酶亚基的检测信号，且该信号依赖于细胞的活化刺激。体外实验证实，DNase I不会直接抑制MPO及NADPH氧化酶的活性，这提示其对ROS生成的影响依赖于NET的解离。综上，本研究数据表明，微生物来源的DNase可通过抑制ROS生成，助力其逃逸宿主的杀伤作用。