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

Deirdre Vincent,Birgit Appl,Robert Bergholz,Michaela Klinke,Magdalena Trochimiuk,Michael Boettcher,Bastian Tiemann,Konrad Reinshagen,Georg Eschenburg

doi:10.1038/s41598-018-31087-0

Deirdre Vincent, Birgit Appl + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-31087-0

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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.

Highlights

Necrotizing enterocolitis (NEC) is considered one of the most devastating diseases affecting premature and term-born infants, as its manifestation often leads to necrosis of the small intestine, which results in mortality rates of up to 30% in very low birth weight (VLBW) infants[1]
Neutrophils eliminate pathogens via: (1) phagocytosis and (2) through the production of web-like DNA structures that are coated with histones and proteolytic enzymes, which are known as NETs28
These neutrophil extracellular traps (NETs) may be the key to understanding NEC pathogenesis

Summary

Introduction

Necrotizing enterocolitis (NEC) is considered one of the most devastating diseases affecting premature and term-born infants, as its manifestation often leads to necrosis of the small intestine, which results in mortality rates of up to 30% in very low birth weight (VLBW) infants[1]. One of the main mechanism supporting this theory, on a cellular level, is the activity of the intestinal epithelial cell toll-like receptor 4 (TLR4), which exhibits a higher expression in the preterm intestine than in the intestine of term-born infants This upregulation of TLR4 leads to increased mucosal injury, as: (1) apoptosis of enterocytes becomes accelerated and (2) the rate of healing is reduced through impaired intestinal restoration and proliferation[3]. Numerous studies, including clinical trials, have strengthened the case for TLR4′s role in the pathogenesis of NEC One such example is the protective effect of breastfeeding on NEC development, in that it inhibits the signaling cascade that leads to TLR4 activation[3,5]. Neutrophils combat pathogens using three main mechanisms: (1) phagocytosis, (2) degranulation, and (3) formation of neutrophil extracellular traps (NETs)[10]

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