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. 2012 Jul 26;16(4):R137.
doi: 10.1186/cc11442.

Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice

Wei Meng, Adnana Paunel-Görgülü, Sascha Flohé, Almuth Hoffmann, Ingo Witte, Colin MacKenzie, Stephan E Baldus, Joachim Windolf, Tim T Lögters

Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice

Wei Meng et al. Crit Care. .

"VSports" Abstract

Introduction: 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. VSports手机版.

Methods: 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 V体育安卓版. 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. .

Results: 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 V体育ios版. 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). .

Conclusions: 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. VSports最新版本.

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Figures

Figure 1
Figure 1
Increased cf-DNA/NETs release and DNase concentration in peripheral blood as well as continuous capability and capacity of NETs release by PMN after CLP. cf-DNA/NETs (A) and DNase (B) levels in serum of mice were determined 6, 24, and 48 hours after CLP or sham operation, that is laparotomy without cecum perforation. Naive mice served as controls. (C) Additionally, cf-DNA/NETs were quantified in the supernatants of freshly isolated PMN from naive, sham and CLP mice, respectively, and further stimulation with 0, 30, 50, and 100 nM PMA for three hours PMA **P < 0.01, ***P < 0.001 versus adjacent time point within one group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus sham; $P < 0.05, $$$P < 0.001 CLP versus naïve. Cf, circulating freely; CLP, cecal ligation and puncture; DNase, desoxyribonuclease; n.d.: not detected; PMA, phorbol-12-myristate-13-acetate; PMN, neutrophils.
Figure 2
Figure 2
Degradation of NETs by rhDNase in vitro. (A) PMN were isolated from bone marrow of naive mice and stimulated with 50 nM PMA. Supernatants were incubated with 0, 0.02, 0.2, 2.0 and 20.0 µg/ml rhDNase. An rhDNase concentration of 2 µg/ml resolves 140 µg/ml of NETs completely. n.d. = not detected. *P < 0.05, ***P < 0.001. (B) Immunofluorescence staining of NETs. The image of unstimulated PMN shows the nuclear localization of DNA (blue fluorescence) and the granular pattern of MPO (green fluorescence; top left). After stimulation morphological changes during NETs formation could be determined with loss of nuclear lobules and granular integrity of MPO (bottom left). Exposure of fixed NETs with 2 µg/ml (top right) and 20 µg/ml (bottom right) rhDNase resulted in the disintegration of NETs with loss of DNA structures. CLP, cecal ligation and puncture; DNase, desoxyribonuclease; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PMN, neutrophils; rh, recombinant human.
Figure 3
Figure 3
Influence of rhDNase treatment on NETs activity, neutrophil and platelet counts. cf-DNA/NETs (A) and DNase (B) levels as well as neutrophil (C) and platelet (D) counts in serum of mice at 6 (n = 5), 24 (n = 7), and 48 (n = 7) hours treated with either 5 mg/kg rhDNase or PBS 1, 4, 7, 10, 21, 24, and 27 hours after CLP. *P < 0.05, **P < 0.01, ***P < 0.001 versus adjacent time point within one group; ###P < 0.001 versus CLP without rhDNase. CLP, cecal ligation and puncture; DNase, desoxyribonuclease; NETs, neutrophil extracellular traps; PBS, phosphate-buffered saline; rh, recombinant human
Figure 4
Figure 4
Influence of NETs depletion on survival after CLP. Survival curve after CLP of mice treated with rhDNase. Mice were injected intraperitoneally with either rhDNase (CLP + rhDNase, n = 20) or PBS (CLP, n = 20) 1, 4, 7, 10, 21, 24 and 27 hours after CLP and were monitored for 6 days or until death. Sham (laparotomy without CLP) groups with (n = 10) and without rhDNase (n = 6) treatment served as further controls. Mortality prevalence of mice treated with rhDNase was lower than in mice treated with PBS 24 hours after CLP (P < 0.01, Fisher's exact test).CLP, cecal ligation and puncture; DNase, desoxyribonuclease; NETs, neutrophil extracellular traps; PBS, phosphate-buffered saline; rh, recombinant human
Figure 5
Figure 5
Enhanced bacterial dissemination without NETs activity. Mice were treated with rhDNase (CLP + rhDNase; n = 44) or PBS (CLP; n = 44), respectively. Bacterial colony forming units (CFU) were recovered 6 hours (n = 8/8 in both groups), 24 hours (n = 7/14 and n = 10/14), and 48 hours (n = 9/22 and n = 11/22) after CLP from the peritoneal cavity, peripheral blood, lung, and liver. Data from two independent experiments are depicted. **P < 0.01, ***P < 0.001 versus adjacent time point; #P < 0.05, ###P < 0.001 versus CLP. CLP, cecal ligation and puncture; DNase, desoxyribonuclease; NETs, neutrophil extracellular traps; PBS, phosphate-buffered saline; rh, recombinant human.
Figure 6
Figure 6
Enhanced IL-6 levels without NETs activity. IL-6 levels in the serum of mice 6 (n = 5), 24 (n = 7), and 48 (n = 7) hours after CLP treated with either 5 mg/kg rhDNase (CLP + rhDNase) or 100 µl PBS (CLP) 1, 4, 7, 10, 21, 24, and 27 hours after CLP. **P < 0.01, ***P < 0.001 versus adjacent time point; ###P < 0.001 versus CLP. CLP, cecal ligation and puncture; DNase, desoxyribonuclease; IL-6, interleukin-6; NETs, neutrophil extracellular traps; PBS, phosphate-buffered saline; rh, recombinant human.
Figure 7
Figure 7
Enhanced neutrophil recruitment into the lung and liver after rhDNase treatment. (A, B) Neutrophil counts in the lung and liver of mice 6 (n = 5), 24 (n = 7), and 48 (n = 7) hours after CLP treated with either 5 mg/kg rhDNase (CLP + rhDNase) or 100 µl PBS (CLP) 1, 4, 7, 10, 21, 24, and 27 hours after CLP. **P < 0.01, ***P < 0.001 versus adjacent time point; #P < 0.05; ###P < 0.001 versus CLP. Chloracetatesterase staining of paraffin sections from the lung and liver of mice 24 hours after CLP treated with either 5 mg/kg rhDNase (CLP + rhDNase) or 100 µl PBS (CLP). Representative sections of the liver (C) and lung (D) are depicted. Scale bar indicated 100 μm. CLP, cecal ligation and puncture; DNase, desoxyribonuclease; NETs, neutrophil extracellular traps; PBS, phosphate-buffered saline; rh, recombinant human.
Figure 8
Figure 8
Histology of liver and lung damage 24 hours after CLP with and without rhDNase treatment. (A) Histology of lung obtained from sham mice. (B) Representative CLP-induced lung damage. (C) Representative lung of CLP mice treated with rhDNase (CLP + rhDNase). (D) Histology of liver obtained from sham mice. (E) Representative CLP-induced liver damage. (F) Representative liver of CLP mice treated with rhDNase (CLP + rhDNase). Sham and CLP mice were sacrificed 24 hours after surgery. Original magnification, ×10. CLP, cecal ligation and puncture; DNase, desoxyribonuclease; rh, recombinant human.
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