Degradation of Extracellular DNA Significantly Ameliorates Necrotizing Enterocolitis Severity in Mice

Klinke, Michaela; Vincent, Deirdre; Trochimiuk, Magdalena; Appl, Birgit; Tiemann, Bastian; Reinshagen, Konrad; Boettcher, Michael

doi:10.1016/j.jss.2018.10.041

Cited by 22 publications

(32 citation statements)

References 40 publications

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“…We 14,15 and others [37][38][39] have shown previously that TLR4 signaling on the intestinal epithelium plays a critical role in NEC development through the development of an inflammatory response and breakdown of the intestinal barrier. Of note, necroptosis in a variety of cells may be induced by TLR4 signaling, [40][41][42] suggesting perhaps that TLR4 activation could be upstream of necroptosis in the pathogenesis of NEC.…”

Section: Necroptosis Is Activated Downstream Of Tlr4 Signaling In Thementioning

confidence: 76%

A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis

Werts

Fulton

Ladd

et al. 2020

Cellular and Molecular Gastroenterology and Hepatology

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Necrotizing enterocolitis is a devastating disease of prematurity characterized by gram-negative bacteria colonization and enterocyte death. We identify a role of Toll-like receptor 4-mediated necroptosis of the intestinal epithelium as a precursor to necrotizing enterocolitis development. BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by Toll-like receptor 4 (TLR4)-dependent intestinal inflammation and enterocyte death. Given that necroptosis is a proinflammatory cell death process that is linked to bacterial signaling, we investigated its potential role in NEC, and the mechanisms involved. METHODS: Human and mouse NEC intestine were analyzed for necroptosis gene expression (ie, RIPK1, RIPK3, and MLKL), and protein activation (phosphorylated RIPK3). To evaluate a potential role for necroptosis in NEC, the effects of genetic (ie, Ripk3 knockout or Mlkl knockout) or pharmacologic (ie, Nec1s) inhibition of intestinal inflammation were assessed in a mouse NEC model, and a possible upstream role of TLR4 was assessed in Tlr4-deficient mice. The NEC-protective effects of human breast milk and its constituent milk oligosaccharides on necroptosis were assessed in a NEC-in-a-dish model, in which mouse intestinal organoids were cultured as either undifferentiated or differentiated epithelium in the presence of NEC bacteria and hypoxia. RESULTS: Necroptosis was activated in the intestines of human and mouse NEC in a TLR4-dependent manner, and was upregulated specifically in differentiated epithelium of the immature ileum. Inhibition of necroptosis genetically and pharmacologically reduced intestinal-epithelial cell death and mucosal inflammation in experimental NEC, and ex vivo in the NEC-in-a-dish system. Strikingly, the addition of human breast milk, or the human milk oligosaccharide 2 fucosyllactose in the ex vivo system, reduced necroptosis and inflammation. CONCLUSIONS: Necroptosis is activated in the intestinal epithelium upon TLR4 signaling and is required for NEC development, and explains in part the protective effects of breast milk.

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Section: Necroptosis Is Activated Downstream Of Tlr4 Signaling In Thementioning

confidence: 76%

A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis

Werts

Fulton

Ladd

et al. 2020

Cellular and Molecular Gastroenterology and Hepatology

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“…Adding to our improved NEC animal induction paradigm, is the fact that maternal separation and hypothermia, two measures to intensify NEC manifestation in murine pups, were not necessary. Hence, this updated NEC model is a simple alteration to the established models and it effectively reduces procedural burdens on subject animals [7][8][9]31 .…”

Section: Discussionmentioning

confidence: 99%

“…This is of interest as NEC is considered a hyperinflammation reaction with neutrophil activity being crucial in its pathogenesis 9,12 . In fact, various studies have documented neutrophils' essential role in NEC pathogenesis, with neutrophil extracellular traps (NETs) being crucial in NEC development 9,13,14 . NETs are web-like DNA structures that neutrophils expel via phagocytosis or during apoptosis (aka NETosis), and are studded with lethal concentrations of antimicrobial proteins and histones, which are able to bind and eliminate microorganisms 15 .…”

mentioning

confidence: 99%

Development of an improved murine model of necrotizing enterocolitis shows the importance of neutrophils in NEC pathogenesis

Klinke

Vincent

Trochimiuk

et al. 2020

Sci Rep

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Various research models to induce necrotizing enterocolitis (NEC) in animals exist, yet significant differences in NEC severity between murine animal models and human patients persist. One possible explanation for the difference in severity may be the variance in neutrophil concentration among newborn humans (50-70%) in comparison to neonatal mice (10-25%). However, neutrophil activity has yet to be evaluated in NEC pathogenesis. Thus, the aim of the study was to evaluate the effects of altered neutrophil concentrations in neonatal mice while simultaneously undergoing a NEC induction. A total of 44 neonatal mice were included in this study and 40 were subjected to an established NEC induction paradigm and 4 were assigned a sham group. Of the 40 mice, 30 received granulocytecolony stimulating factor (G-CSF) on a daily basis, while 10 were used as controls (receiving inactivated G-CSF). Mice undergoing G-CSF treatment were further divided into two subgroups: (1) wildtype and (2) ELANE-knockout (KO). ELANE-KO mice are incapable of producing neutrophil elastase (NE) and were used to evaluate the role of neutrophils in NEC. For each of the groups, the following metrics were evaluated: survival, NEC severity, tissue damage, neutrophil count and activation, and NETs formation. An improved murine model of NEC was developed using (1) Lipopolysaccharides and Neocate gavage feeding, (2) hypoxia, and (3) G-CSF administration. The results suggest that the addition of G-CSF resulted in significantly elevated NEC manifestation rates with consequent tissue damage and intestinal inflammation, without affecting overall mortality. Animals without functioning NE (ELANE-KO) appeared to have been protected from NEC development. This study supports the importance of neutrophils in NEC pathogenesis. The optimized NEC induction paradigm, using G-CSF administration, resulted in elevated neutrophil counts, resembling those of neonatal humans. Elevation of neutrophil levels significantly improved NEC disease manifestation by modeling human physiology more accurately than current NEC models. Thus, in the future, murine NEC experiments should include the elevation of neutrophil levels to improve the transition of research findings from mice to humans.

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“…NETs are cytotoxic, induce thrombosis and aggravate inflammation (aka immunothrombosis) (15,16). The involvement of NETs in I/R injury and intestinal hyperinflammation including NEC has been shown in previous studies (6,17,18). This is particularly evident at high neutrophil densities when NETs tend to become matted and form large aggregates endowed with a plethora of enzymatic activities (aka "aggNETs"), which then accelerate the formation of thrombi in blood vessels (19,20).…”

Section: Abbreviationsmentioning

confidence: 64%

Cardiac and Inflammatory Necrotizing Enterocolitis in Newborns Are Not the Same Entity

et al. 2021

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Background: Necrotizing enterocolitis (NEC) is an often-fatal neonatal disease involving intestinal hyperinflammation leading to necrosis. Despite ongoing research, (1) conflicting results and (2) comorbidities of NEC patients make early NEC detection challenging and may complicate therapy development. Most research suggests that NEC pathogenesis is multifactorial, involving a combination of (1) gut prematurity; (2) abnormal bacterial colonization; and (3) ischemia-reperfusion (I/R) injury. As neutrophil extracellular traps (NETs) partially mediate I/R injury and drive inflammation in NEC, we hypothesized that NETs contribute to NEC development; particularly in cardiac patients.Methods: A retrospective analysis of baseline characteristics, clinical signs, laboratory parameters, and imaging was conducted for surgically verified NEC cases over 10 years. Patients were stratified into two groups: (1) prior medically or surgically treated cardiac disease (cardiac NEC) and (2) no cardiac comorbidities (inflammatory NEC). Additionally, histology was reassessed for neutrophil activation and NETs formation.Results: A total of 110 patients (cNEC 43/110 vs. iNEC 67/110) were included in the study, with cNEC neonates being significantly older than iNEC neonates (p = 0.005). While no significant differences were found regarding clinical signs and imaging, laboratory parameters revealed that cNEC patients have significantly increased leucocyte (p = 0.024) and neutrophil (p < 0.001) counts. Both groups also differed in pH value (p = 0.011). Regarding histology: a non-significant increase in staining of myeloperoxidase within the cNEC group could be found in comparison to iNEC samples. Neutrophil elastase (p = 0.012) and citrullinated histone H3 stained (p = 0.041) slides showed a significant markup for neonates diagnosed with cNEC in comparison to neonates with iNEC.Conclusion: The study shows that many standardized methods for diagnosing NEC are rather unspecific. However, differing leucocyte and neutrophil concentrations for iNEC and cNEC may indicate a different pathogenesis and may aid in diagnosis. As we propose that iNEC is grounded rather in sepsis and neutropenia, while cNEC primarily involves I/R injuries, which involves neutrophilia and NETs formation, it is plausible that I/R injury due to interventions for cardiac comorbidities results in pronounced neutrophil activation followed by a hyperinflammation reaction and NEC. However, prospective studies are necessary to validate these findings and to determine the accuracy of the potential diagnostic parameters.

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Degradation of Extracellular DNA Significantly Ameliorates Necrotizing Enterocolitis Severity in Mice

Cited by 22 publications

References 40 publications

A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis

A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis

Development of an improved murine model of necrotizing enterocolitis shows the importance of neutrophils in NEC pathogenesis

Cardiac and Inflammatory Necrotizing Enterocolitis in Newborns Are Not the Same Entity

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