BACKGROUND There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control) and 2) that serum nuclease activities are increased as well. METHODS Following ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 μg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis. RESULTS In contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils was decreased by 46.3% (4.3% ± 1.8 SD vs. 8.2% ± 2.9, p ≤ 0.0001) and 48.1% (4.9% ± 2.5 vs. 9.4% ± 5.2, p = 0.002) after 2 and 4 h compared to volunteers. mtDNA further decreased NET formation in neutrophils from septic patients (4.7% ± 1.2 to 2.8% ± 0,8; p = 0.03), but did not alter NET formation in neutrophils from volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3 ± 2 vs 5 ± 0, median and ICR, p = 0.0001) compared to volunteers. CONCLUSIONS Unstimulated NET formation and nuclease activity are decreased in septic patients. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. TRIAL REGISTRATION DRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.

中文翻译：

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败血病患者中性粒细胞胞外陷阱的形成和核酸酶活性。

背景技术关于败血症中性粒细胞胞外陷阱（NET）的形成和NET降解核酸酶活性的改变是否知之甚少。因此，我们测试了以下假设：1）在没有刺激的情况下以及与线粒体DNA（mtDNA），损伤相关分子模式或佛波醇12-肉豆蔻酸酯13-结合后，与健康志愿者相比，脓毒症患者中性粒细胞的NET形成增加了醋酸盐（PMA;阳性对照）和2）血清核酸酶活性也增加。方法经伦理委员会批准，我们在该前瞻性观察性试验中纳入了18名败血症患者和27名志愿者。抽血并在无刺激的情况下并在与mtDNA（10μg/孔）或PMA（25 nmol）孵育后体外分析中性粒细胞形成的NET。此外，使用凝胶电泳评估血清核酸酶活性。结果与我们的假设相反，在败血症患者中，中性粒细胞的未刺激净释放降低了46.3％（4.3％±1.8 SD vs. 8.2％±2.9，p≤0.0001）和48.1％（4.9％±2.5 vs. 9.4％）与志愿者相比，在2小时和4小时后±±5.2，p = 0.002）。mtDNA进一步降低了败血病患者中性粒细胞的NET形成（4.7％±1.2至2.8％±0.8； p = 0.03），但并未改变志愿者中性粒细胞的NET形成。值得注意的是，使用PMA作为阳性对照，我们确保中性粒细胞仍然能够形成NET，败血症患者的NET形成增加至73.2％（±29.6），志愿者的NET形成增加至91.7％（±7.1）（p = 0.22）。此外，我们发现败血病患者的血清核酸酶活性（范围：0-6）降低了39.6％（3±2 vs 5±0，中位数和ICR，p = 0）。0001）与志愿者相比。结论败血病患者的未刺激的NET形成和核酸酶活性降低。mtDNA可以进一步减少败血症中NET的形成。因此，尽管体内核酸酶活性降低，来自败血病患者的中性粒细胞在体外显示出减少的NET形成。TRIAL REGISTRATION DRKS00007694，德国临床试验数据库（DRKS）。追溯注册2015年2月6日。