Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis

Abstract

Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis.

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation

“…The NRPs provided an early survival advantage in this model ( Figure 9A), suggesting that NETs are agents of inflammatory damage in the absence of pathogens, and a consequent requirement for their containment and elimination. nNIF also improved mortality in the CLP model of polymicrobial sepsis ( Figure 9, B and C), supporting existing evidence that NETs are effectors of collateral vascular and tissue injury in this experimental syndrome (53). The results in both models suggest that NET generation, like other neutrophil effector functions (54), has evolved to contain and eliminate pathogens, but can also injure the host if it is activated by pathologic inflammatory signals in the absence of infection or by microbes in an uncontrolled fashion.…”

“…Mice treated with nNIF had lower clinical illness scores (52) at 24 hours and significantly increased survival at 144 hours after CLP compared with nNIF-SCRtreated animals ( Figure 9, B and C). Together, these experiments ( Figure 8 and Figure 9) demonstrate that nNIF and CRISPP inhibit NET formation in vivo and provide initial evidence that they have beneficial effects in models of systemic sterile inflammation and infection in which NET formation may influence tissue injury and mortality (9,13,14,53).…”

“…This may be due to differences in pharmacokinetics or half-lives of nNIF and CRISPP under these conditions. We also performed similar experiments using the cecal ligation and puncture (CLP) model of polymicrobial sepsis (51)(52)(53). Mice treated with nNIF had lower clinical illness scores (52) at 24 hours and significantly increased survival at 144 hours after CLP compared with nNIF-SCRtreated animals ( Figure 9, B and C).…”

SOCS1 is a negative regulator of metabolic reprogramming during sepsis

“…MPO activity in lung homogenates was determined 18 hours after CLP, as previously described (35)(36)(37)(38)82). Results are expressed as units of MPO activity per gram tissue.…”

Neutrophil extracellular traps as the main source of eDNA

“…The mechanism of NETosis is presented in Figure 1. In neutrophils, bacterial stimuli through the Raf-MEK-ERK signalling pathway induce histone citrullination mediated by peptidylarginine deiminase (PAD), which results in chromatin decondensation, disruption of cellular membrane, and release of DNA with associated proteins, including histones, into the extracellular environment, thus entrapping pathogens [7][8][9][10][11]. The exact mechanism of the NETosis process is still under investigation; nevertheless, there are some data indicating that the formation of neutrophil networks is mainly dependent on production of reactive oxygen species (ROS), which are generated by the NADPH oxidase Nox2, and are required for translocation of neutrophil elastase (NE) from the azurophilic (AZ) granules to the nucleus, resulting in decondensation of chromatin, which is subsequently promoted by myeloperoxidase (MPO) [12].…”