Neutrophil extracellular trap inhibition improves survival in neonatal mouse infectious peritonitis

Abstract

Background Treatment of neonatal peritonitis and sepsis is challenging. Following infection, neutrophils elaborate neutrophil extracellular traps (NETs)—extracellular lattices of decondensed chromatin decorated with antimicrobial proteins. NETs, however, can augment pathogenic inflammation causing collateral damage. We hypothesized that NET inhibition would improve survival in experimental neonatal infectious peritonitis. Methods We induced peritonitis in 7 to 10-day-old mice by intraperitoneal injection with cecal slurry. We targeted NETs by treating mice with neonatal NET-Inhibitory Factor (nNIF), an endogenous NET-inhibitor; Cl-amidine, a PAD4 inhibitor; DNase I, a NET degrading enzyme, or meropenem (an antibiotic). We determined peritoneal NET and cytokine levels and circulating platelet–neutrophil aggregates. Survival from peritonitis was followed for 6 days. Results nNIF, Cl-amidine, and DNase I decreased peritoneal NET formation and inflammatory cytokine levels at 24 h compared to controls. nNIF, Cl-amidine, and DNase I decreased circulating platelet–neutrophil aggregates, and NET-targeting treatments significantly increased survival from infectious peritonitis compared to controls. Finally, nNIF administration significantly improved survival in mice treated with sub-optimal doses of meropenem even when treatment was delayed until 2 h after peritonitis induction. Conclusions NET inhibition improves survival in experimental neonatal infectious peritonitis, suggesting that NETs participate pathogenically in neonatal peritonitis and sepsis. Impact Neutrophil extracellular trap formation participates pathogenically in experimental neonatal infectious peritonitis. NET-targeting strategies improve outcomes in a translational model of neonatal infectious peritonitis. NET inhibition represents a potential target for drug development in neonatal sepsis and infectious peritonitis.

Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial sepsis by inhibiting neutrophil extracellular traps

“…Importantly, our data highlight the potential role of nNIF and NET inhibition as a new treatment approach in the early phase of infection in polymicrobial sepsis in vivo. This study corroborates findings from our recent study in which nNIF treatment before or after initiation of peritonitis inhibited NET formation in a murine model of neonatal polymicrobial peritonitis and endotoxemia resulting from intraperitoneal LPS injection ( 27 ). In the current study, we go a step further and investigate nNIF as an adjuvant therapy in combination with a broad-spectrum antibiotic for the treatment of septic mice.…”

“…This is supported by the fact that nNIF-treated mice displayed a rapid and considerable increase of CFU counts at the site of infection in the peritoneal fluid as well as in the plasma as opposed to the bacteria being removed from circulation by NETs. Of note, we previously demonstrated that neutrophils exposed to nNIF still maintain their ability to generate reactive-oxygen species and phagocytize E. coli bioparticles ( 12 , 27 ). Considering all the data together, we propose that NET inhibition with nNIF effectively blocks NET formation leading to increased bacterial CFUs due to lack of NET-mediated extracellular microbial killing and decreased neutrophil activation.…”

“…An in-house ELISA was used to quantify MPO-DNA complexes in plasma and peritoneal fluid from sham and CLP mice 24 hours after the surgical procedure ( 24 , 27 ). Briefly, after overnight coating with anti-MPO capture antibody in calcium carbonate coating buffer (2 µg/ml; 0400-0002, Bio-Rad) at 4°C, a 96-well plate was blocked with 2.5% bovine serum albumin in PBS for 2 hours at room temperature.…”

Midgut Volvulus Adds a Murine, Neutrophil-Driven Model of Septic Condition to the Experimental Toolbox

“…NETS have been shown to have both protective and detrimental features in sepsis, the latter by induction of thrombosis and multiple organ failure. Yet many key questions regarding their exact role in the pathophysiology of (pediatric) sepsis remain unanswered [11,12].…”

Preterm ETs Are Significantly Reduced Compared with Adults and Partially Reduced Compared with Term Infants

“…A1ATm358, another placental-related modulatory factor found in the perinatal milieu, is no longer active in neonates after birth [ 32 ]. Only recently has it been shown that the group of NET inhibitors improves survival in experimental neonatal infectious peritonitis [ 33 ].…”