Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
J Innate Immun. 2009;1(6):527-42. doi: 10.1159/000235860. Epub 2009 Aug 27.
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 Listeria monocytogenes-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.
中性粒细胞通过细胞外 DNA 纤维复合物和杀菌蛋白（称为中性粒细胞胞外陷阱（NETs））杀死细菌。NET 的组成和它们使用的杀菌机制尚未完全了解。在这里，我们表明脱氧核糖核酸酶（DNase I）的处理会损害由革兰氏阳性和革兰氏阴性细菌以及佛波酯引起的晚期氧化反应。DNase I 抑制调理后的单核细胞增生李斯特菌刺激的中性粒细胞产生的异鲁米诺依赖性化学发光，并且当吞噬作用被阻断时，DNase 的抑制作用也是明显的，这表明 DNase 抑制了活性氧（ROS）生成的细胞外机制。DNase 的抑制作用与肌动蛋白聚合无关。DNase I 不损害吞噬作用和细胞活力。免疫荧光分析显示髓过氧化物酶存在于 NET 上。此外，在 Rab27a 缺陷中性粒细胞的 NET 中检测到颗粒蛋白，而 Rab27a 缺陷中性粒细胞的胞吐作用不足，这表明胞吐作用和颗粒蛋白在 NET 上的分布是通过独立的机制进行的。还在 NET 上检测到 NADPH 氧化酶亚基，并且 DNase I 的处理和细胞刺激依赖性消除了细胞外陷阱相关 NADPH 氧化酶亚基的检测。体外分析表明 MPO 和 NADPH 氧化酶活性未被 DNase I 直接抑制，这表明其对 ROS 产生的影响取决于 NET 的解体。总之，我们的数据表明，微生物来源的 DNase 对 ROS 产生的抑制作用有助于它们逃避杀伤的能力。
