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

Abstract

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 < 0.001). Markers of neutrophil activation and NETosis were significantly increased in animals with NEC and in human samples as compared to controls. Further, prevention of NETosis by protein arginine deiminase (PAD) inhibition in mice significantly reduced mortality, tissue damage, and inflammation in mice induced with NEC. Our results suggest that the hyperinflammation observed in NEC is a NETs-dependent process, as NEC severity was significantly reduced in mice incapable of forming NETs (PAD inhibition) and markers for NEC and NETs correlated positively during the time course of NEC induction. Further, serum surrogate markers of NETosis (such as cfDNA and DNase) appear to predict NEC in neonatal mice. As findings of the mouse NEC model correlate positively with human NEC samples immunohistochemically, the hyperinflammation reaction observed in mice could potentially be applied to human NEC pathogenesis.

MicroRNAs and Neutrophil Activation Markers Predict Venous Thrombosis in Pancreatic Ductal Adenocarcinoma and Distal Extrahepatic Cholangiocarcinoma

“…Hence, in our study we also explored the ability of several markers of neutrophil activation, in order to identify PDAC and DECC patients at high risk of developing VTE during follow-up. To that end, we measured different plasma markers of neutrophil activation following the strategy addressed in previous studies [51][52][53][54][55][56][57]. We have observed an increase in calprotectin and MPO plasma levels in those patients who developed VTE compared with those who did not.…”

“…Different markers of neutrophil activation were measured in every plasma sample obtained during follow-up from the 32 patients studied herein, following the strategy addressed in previous studies [51][52][53][54][55][56][57]. Cell-free DNA (cfDNA; Quant-iT PicoGreen dsDNA kit, Life Technologies, Eugene, OR, USA) and nucleosomes (Cell Death Detection ELISA PLUS kit, Roche, Mannheim, Germany) were measured as markers of the nuclear content of neutrophils released by neutrophils upon NETosis.…”

The emerging role of immunothrombosis in paediatric conditions

“…Data on human histology also showed that immunothrombosis is present in NEC and this can lead to interesting research developments. 30 Elevated circulating cfDNA levels in neonatal plasma have been associated at late-onset sepsis diagnosis and few days before NEC. 31 It is therefore clear that insights into NEC would prove useful for several reasons: (i) it may help develop a treatment for a disease which is still linked to a very high mortality rate; 32 (ii) it may represent a model to further investigate severe immunothrombosis in neonates as most of the interactions between inflammation, immunity and coagulation occur in the gut; (iii) since severe thrombosis of the mesentery is a common finding in NEC, further investigation may determine whether treatment against NETosis can selectively reduce thrombus formation and maintain blood supply to the gut.…”

Markers of NETosis Do Not Predict Neonatal Early Onset Sepsis: A Pilot Study

“…Recent studies evaluating neonatal, and not umbilical cord blood, surrogate markers of NET formation showed an association with sepsis (25,26): elevated circulating cfDNA levels in neonatal plasma have been associated with late-onset sepsis, as well as necrotizing enterocolitis, diagnosis (25). Moreover, neutrophils capable of releasing NETs have also been described as potential sepsis biomarkers in neonates (11,26).…”

“…When released from neutrophils during infection, the proteins and chromatin contained within the NETs form a protective mesh that filters and destroys pathogenic organisms (6). However, NET formation also occurs inappropriately during sterile inflammation resulting in thrombosis, autoimmunity, and tissue damage for instance after ischemia perfusion injuries (9)(10)(11)(12)(13).…”