Bhagirath Vinai C, Dwivedi Dhruva J, Liaw Patricia C
From the Departments of *Medical Sciences †Medicine, McMaster University; and the ‡Thrombosis and Atherosclerosis Research Institute, Hamilton, Ontario, Canada.
Shock. 2015 Sep;44(3):265-71. doi: 10.1097/SHK.0000000000000397.
Cell-free DNA (CFDNA) is elevated in sepsis and correlates with mortality. This DNA may come from nuclear, mitochondrial, or bacterial sources. Cell-free DNA from all three sources may play a pathogenic role in sepsis via activation of coagulation through the contact pathway, whereas CpG motifs on bacterial and mitochondrial DNA may additionally stimulate inflammatory responses via Toll-like receptor 9. This study elucidates the relative effects of nuclear, mitochondrial, and bacterial DNA on inflammatory and procoagulant pathways with relevance to sepsis.
DNA was extracted from plasma of septic patients and control subjects, and nuclear and mitochondrial CFDNA concentrations were measured by quantitative polymerase chain reaction. Viability of primary cultured human neutrophils was measured by flow cytometry for phosphatidyl serine exposure and cell permeability to propidium iodide. Continuous thrombin generation was measured with a fluorogenic substrate (Technothrombin, Vienna, Austria). Interleukin 6 secretion was measured by enzyme-linked immunosorbent assay. Platelet activation was measured by flow cytometry for P-selectin and activated αIIbβ3.
Mitochondrial DNA and nuclear DNA were elevated in plasma from septic patients compared with control subjects. Both mitochondrial and bacterial DNA prolonged neutrophil viability. Bacterial DNA increased neutrophil interleukin 6 secretion, but mitochondrial and nuclear DNA did not. Nuclear, mitochondrial, and bacterial DNA increased thrombin generation in platelet-poor plasma to a similar degree in a FXI- and FXII-dependent manner, indicating dependence on the intrinsic pathway of coagulation. Independently of coagulation, DNA from all three sources was capable of causing activation of platelet integrin αIIbβ3.
Cell-free DNA from nuclear, mitochondrial, and bacterial sources have varying proinflammatory effects, although all three have similar procoagulant and platelet-stimulating potential. The pathophysiological effects of CFDNA in sepsis may vary with the source of DNA.
脓毒症患者的游离DNA（CFDNA）水平升高，且与死亡率相关。这种DNA可能来源于细胞核、线粒体或细菌。来自这三种来源的游离DNA可能通过接触途径激活凝血，从而在脓毒症中发挥致病作用，而细菌和线粒体DNA上的CpG基序可能通过Toll样受体9额外刺激炎症反应。本研究阐明了细胞核、线粒体和细菌DNA对与脓毒症相关的炎症和促凝途径的相对影响。
从脓毒症患者和对照受试者的血浆中提取DNA，通过定量聚合酶链反应测量细胞核和线粒体CFDNA浓度。通过流式细胞术检测磷脂酰丝氨酸暴露和碘化丙啶细胞通透性，以测量原代培养的人中性粒细胞的活力。使用荧光底物（Technothrombin，奥地利维也纳）测量连续凝血酶生成。通过酶联免疫吸附测定法测量白细胞介素6分泌。通过流式细胞术检测P-选择素和活化的αIIbβ3，以测量血小板活化。
与对照受试者相比，脓毒症患者血浆中的线粒体DNA和细胞核DNA升高。线粒体DNA和细菌DNA均延长了中性粒细胞的活力。细菌DNA增加了中性粒细胞白细胞介素6的分泌，但线粒体DNA和细胞核DNA没有。细胞核、线粒体和细菌DNA以类似程度增加了无血小板血浆中的凝血酶生成，且依赖于FXI和FXII，表明依赖于内源性凝血途径。独立于凝血作用，来自所有三种来源的DNA都能够激活血小板整合素αIIbβ3。
来自细胞核、线粒体和细菌来源的游离DNA具有不同的促炎作用，尽管这三种都具有相似的促凝和血小板刺激潜力。CFDNA在脓毒症中的病理生理作用可能因DNA来源而异。
