Abstract
IntroductionAlthough 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.MethodsIn 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.Resultscf-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).ConclusionsThis 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.
Highlights
The formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear
At all time points cf-DNA/NETs values were significantly increased in the cecal ligation and puncture (CLP) group compared to the sham and naive groups, with a maximum at 24 hours after CLP (P < 0.001; Figure 1A)
Degradation of NETs by rhDNase treatment in vivo To investigate the effects of rhDNase treatment on NET activity in vivo, serum values of cf-DNA/NETs and DNase were determined 6, 24 and 48 hours after CLP (Figure 3)
Summary
The formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. Equipped with a full arsenal of antimicrobial proteins at their disposal, PMN have a well described role in phagocytic uptake and intracellular killing [10,11] Besides their ability to eliminate microorganisms by phagocytosis, it has recently been shown that activation of PMN causes the formation of neutrophil extracellular traps (NETs) [12,13,14,15]. This novel mechanism, for the first time described by Brinkmann et al, consists of the release of web-like structures of DNA and proteins that bind, disarm and kill pathogens extracellularly [12]
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