Meng Wei, Paunel-Görgülü Adnana, Flohé Sascha, Hoffmann Almuth, Witte Ingo, MacKenzie Colin, Baldus Stephan E, Windolf Joachim, Lögters Tim T
Crit Care. 2012 Jul 26;16(4):R137. doi: 10.1186/cc11442.
Although the formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. This study was performed in order to evaluate the influence of NETs depletion by administration of recombinant human (rh)DNase on bacterial spreading, PMN tissue infiltration and inflammatory response in a mouse model of polymicrobial sepsis.
In a prospective controlled double-armed animal trial, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, mice were treated with rhDNase or phosphate buffered saline, respectively. Survival, colony forming unit (CFU) counts in the peritoneal cavity, lung, liver and blood were determined. PMN and platelet counts, IL-6 and circulating free (cf)-DNA/NETs levels were monitored. PMN infiltration, as well as organ damage, was analyzed histologically in the lungs and liver. Capability and capacity of PMN to form NETs were determined over time.
cf-DNA/NETs were found to be significantly increased 6, 24, and 48 hours after CLP when compared to the levels determined in sham and naïve mice. Peak levels after 24 hours were correlated to enhanced capacity of bone marrow-derived PMN to form NETs after ex vivo stimulation with phorbol-12-myristate-13-acetate at the same time. rhDNase treatment of mice resulted in a significant reduction of cf-DNA/NETs levels 24 hours after CLP (P < 0.001). Although overall survival was not affected by rhDNase treatment, median survival after 24 hours was significantly lower when compared with the CLP group (P < 0.01). In mice receiving rhDNase treatment, CFU counts in the lung (P < 0.001) and peritoneal cavity (P < 0.05), as well as serum IL-6 levels (P < 0.001), were found to be already increased six hours after CLP. Additionally, enhanced PMN infiltration and tissue damage in the lungs and liver were found after 24 hours. In contrast, CFU counts in mice without rhDNase treatment increased later but more strongly 24 hours after CLP (P < 0.001). Similarly, serum IL-6 levels peaked after 24 hours (P < 0.01).
This study shows, for the first time, that depletion of NETs by rhDNase administration impedes the early immune response and aggravates the pathology that follows polymicrobial sepsis in vivo.
尽管在感染和脓毒症期间已检测到中性粒细胞（PMN）胞外诱捕网（NETs）的形成，但其在体内的作用仍不清楚。本研究旨在评估在多微生物脓毒症小鼠模型中，通过给予重组人（rh）脱氧核糖核酸酶（DNase）清除NETs对细菌播散、PMN组织浸润和炎症反应的影响。
在一项前瞻性对照双臂动物试验中，通过盲肠结扎和穿刺（CLP）诱导多微生物脓毒症。CLP后，小鼠分别用rhDNase或磷酸盐缓冲盐水治疗。测定生存率、腹腔、肺、肝和血液中的菌落形成单位（CFU）计数。监测PMN和血小板计数、白细胞介素-6（IL-6）和循环游离（cf）-DNA/NETs水平。对肺和肝进行组织学分析，观察PMN浸润以及器官损伤情况。随时间测定PMN形成NETs的能力。
与假手术组和未处理组小鼠相比，CLP后6、24和48小时发现cf-DNA/NETs显著增加。24小时后的峰值水平与同时用佛波醇-12-肉豆蔻酸酯-13-乙酸盐离体刺激后骨髓来源的PMN形成NETs的能力增强相关。rhDNase治疗小鼠导致CLP后24小时cf-DNA/NETs水平显著降低（P<0.001）。虽然rhDNase治疗对总体生存率没有影响，但与CLP组相比，24小时后的中位生存率显著降低（P<0.01）。在接受rhDNase治疗的小鼠中，CLP后6小时肺（P<0.001）和腹腔（P<0.05）中的CFU计数以及血清IL-6水平（P<0.001）就已升高。此外，24小时后发现肺和肝中PMN浸润增加和组织损伤加重。相比之下，未接受rhDNase治疗的小鼠CFU计数在CLP后24小时升高较晚但更明显（P<0.001）。同样，血清IL-6水平在24小时后达到峰值（P<0.01）。
本研究首次表明，给予rhDNase清除NETs会阻碍早期免疫反应，并加重体内多微生物脓毒症后的病理变化。
