NEC is likely a NETs dependent process and markers of NETosis are predictive of NEC in mice and humans

Necrotizing enterocolitis (NEC) is one of the most devastating diseases affecting premature and mature infants. It is hypothesized that NEC is the result of neutrophils’ active role in hyperinflammation after bacterial gut colonization, through their nuclear DNA release and formation of neutrophil extracellular traps (NETs) to combat pathogens. The aim of this study was to evaluate the importance of NETs in NEC pathogenesis, as well as to identify and validate markers of NETosis to predict NEC. NEC was induced in mice by gavage feeding of Neocate and lipopolysaccharide, followed by ten minutes of hypoxia (5% O2) q12h for five days, starting on day four postpartum (p.p.). The interrelation of NEC and neutrophils, including NETs, was assessed macroscopically (i.e. NEC score, SYTOX Orange), microscopically (i.e. Chiu score, citrullinated histone H3, neutrophil elastase), and in blood samples (i.e. cell-free DNA (cfDNA), DNase). In order to determine the exact role of NETs in NEC pathogenesis, a protein arginine deiminase (PAD) inhibition model was established (preventing NETs formation in mice) by injecting BB-Cl-amidine once daily, starting on day one p.p. Additionally, human intestinal samples of diagnostically verified NEC were analyzed. In total, 76 mice were analyzed in the experiment. Serum cfDNA correlated positively with NEC manifestation, as measured by macroscopic NEC score (r = 0.53, p = 0.001), and microscopic evaluation with Chiu score (r = 0.56, p