Isabelle G. De Plaen

Neonatal Necrotizing Enterocolitis: Clinical Considerations and Pathogenetic Concepts

Necrotizing enterocolitis (NEC), a disease affecting predominantly premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. Although several predisposing factors have been identified, such as prematurity, enteral feeding, and infection, its pathogenesis remains elusive. In the past 20 years, we have established several animal models of NEC in rats and found several endogenous mediators, especially platelet-activating factor (PAF), which may play a pivotal role in NEC. Injection of PAF induces intestinal necrosis, and PAF antagonists prevent the bowel injury induced by bacterial endotoxin, hypoxia, or challenge with tumor necrosis factor-a (TNF) plus endotoxin in adult rats. The same is true for lesions induced by hypoxia and enteral feeding in neonatal animals. Human patients with NEC show high levels of PAF and decreased plasma PAF-acetylhydrolase, the enzyme degrading PAF. The initial event in our experimental models of NEC is probably polymorphonuclear leukocyte (PMN) activation and adhesion to venules in the intestine, which initiates a local inflammatory reaction involving proinflammatory mediators including TNF, complement, prostaglandins, and leukotriene C4. Subsequent norepinephrine release and mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. Bacterial products (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and endotoxin synergizes with PAF to amplify the inflammation. Reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final pathway for tissue injury. Protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase, and indigenous probiotics such as Bifidobacteria infantis. The former maintains intestinal perfusion and the integrity of the mucosal barrier, and the latter keep virulent bacteria in check. The development of tissue injury depends on the balance between injurious and protective mechanisms.

Bifidobacteria Stabilize Claudins at Tight Junctions and Prevent Intestinal Barrier Dysfunction in Mouse Necrotizing Enterocolitis

Whether intestinal barrier disruption precedes or is the consequence of intestinal injury in necrotizing enterocolitis (NEC) remains unknown. Using a neonatal mouse NEC model, we examined the changes in intestinal permeability and specific tight-junction (TJ) proteins preceding NEC and asked whether these changes are prevented by administration of Bifidobacterium infantis, a probiotic known to decrease NEC incidence in humans. Compared with dam-fed controls, pups submitted to the NEC protocol developed i) significantly increased intestinal permeability at 12 and 24 hours (as assessed by 70-kDa fluorescein isothiocyanate-dextran transmucosal flux); ii) occludin and claudin 4 internalization at 12 hours (as assessed by immunofluorescence and low-density membrane fraction immunoblotting); iii) increased claudin 2 expression at 6 hours and decreased claudin 4 and 7 expression at 24 hours; and iv) increased claudin 2 protein at 48 hours. Similar results were seen in human NEC, with claudin 2 protein increased. In mice, administration of B. infantis micro-organisms attenuated increases in intestinal permeability, preserved claudin 4 and occludin localization at TJs, and decreased NEC incidence. Thus, an increase in intestinal permeability precedes NEC and is associated with internalization of claudin 4 and occludin. Administration of B. infantis prevents these changes and reduces NEC incidence. The beneficial effect of B. infantis is, at least in part, due to its TJ and barrier-preserving properties.

Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B

We previously reported that neuronal nitric oxide synthase (nNOS) is the predominant NOS in the intestine. Inducible NOS (iNOS), an enzyme involved in the inflammatory response, is regulated by cytokines via the transcriptional factor NF‐κB. We examined a new mechanism of intestinal iNOS regulation with respect to the role of nNOS and its effect on NF‐κB. Young Sprague‐Dawley rats were treated for 4 days with 1) saline, 2) 7‐nitroindazole (7‐NI, specific nNOS inhibitor), 3) 7‐NI + pyrrolidine dithiocarbam‐ate (PDTC, NF‐κB inhibitor), or 4) PDTC. Intestinal iNOS mRNA, NF‐κB activity, and the tissue content of the regulatory IκBα were examined. We found that 7‐NI‐treated animals had higher intestinal NF‐κB (p50‐p65) activity, lower IκBα content, and increased intestinal iNOS mRNA, iNOS protein, and iNOS activity compared with controls. All of these changes were abolished when PDTC was given together with 7‐NI. PDTC alone had no effect. 7‐NI induces a delayed increase in intestinal myeloperoxidase activity (after elevation in NF‐κB and iNOS), which could be abrogated by PDTC. We conclude that in normal rat small intestine, nNOS suppresses the gene expression of iNOS through NF‐κB down‐regulation and that nNOS suppression leads to IκBα degradation, NF‐κB activation, and iNOS expression.—Qu, X.‐w., Wang, H., De Plaen, I. G., Rozenfeld, R. A., Hsueh, W. Neuronal nitric oxide synthase (NOS) regulates the expression of inducible NOS in rat small intestine via modulation of nuclear factor kappa B. FASEB J. 15, 439‐446 (2001)

Inhibition of Nuclear Factor-κB Ameliorates Bowel Injury and Prolongs Survival in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-κB (NF-κB). It remains unknown, however, whether NF-κB mediates injury in neonatal NEC. We therefore examined the activation status of NF-κB perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-κB is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-κB remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-κB inhibitor protein IκBα and IκBβ at d 2. To determine the importance of elevated NF-κB activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IκB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-κB activity. Our findings therefore lead us to conclude that selective NF-κB inhibition represents a promising therapeutic strategy for NEC.

Intestinal NF-κB is activated, mainly as p50 homodimers, by platelet-activating factor

Abstract NF-κB, a transcription factor, upregulates gene transcription of many inflammatory mediators. Here, we examined the activity of NF-κB in the rat small intestine, and how it may be affected by platelet-activating factor (PAF), an important mediator for intestinal injury and inflammation. Ileal nuclear extracts from sham-operated and PAF (1.5 μg/kg)-injected rats were prepared for the assessment of NF-κB DNA-binding activity, and the identification of NF-κB subunits. The experiment was also performed on neutrophil-depleted rats to examine whether the PAF effect is neutrophil-dependent. Cellular NF-κB was localized by immunohistochemistry. We found that: (a) NF-κB is constitutively active in rat small intestine; (b) PAF at a dose below that causing shock and bowel necrosis enhances DNA-binding activity of NF-κB within 30 min after injection; activated NF-κB contains predominantly p50 subunits; (c) immunohistochemistry showed that PAF induced translocation of p50 into the nucleus of cells of the lamina propria, as well as of the epithelium; and (d) the effect of PAF is abrogated by neutrophil depletion, suggesting a role of neutrophils in NF-κB activation. Our study suggests that NF-κB is weakly active constitutively in the intestine, and inflammatory stimuli such as PAF activate NF-κB and enhance its DNA-binding activity in the intestine, which contains predominantly p50 subunits.

Lipopolysaccharide activates nuclear factor κB in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor

We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor κB (NF‐κB) activation in the intestine and the roles of endogenous platelet‐activating factor (PAF), tumour necrosis factor‐α (TNF) and neutrophils. We also compared the time course of NF‐κB activation in response to PAF and LPS. Ileal nuclear extracts from LPS (8 mg kg−1, IV)‐injected rats were assayed for NF‐κB‐DNA‐binding activity and identification of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti‐TNF antibody, or anti‐neutrophil antiserum. NF‐κB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 μg kg−1, IV) for comparison. LPS activates intestinal NF‐κB, both as p50‐p50 and p50‐p65 dimers within 15 min, and the effect peaks at 2 h. The effect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF‐κB was immunolocalized within epithelial and lamina propria cells. LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti‐TNF antibody, or anti‐neutrophil antiserum (P<0.05). LPS activates intestinal NF‐κB in vivo and neutrophil activation is involved in its action. The LPS effect is mediated by both endogenous PAF and TNF.

Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-κB activation: independence from endogenous TNF-α and platelet-activating factor

CXCL2 (macrophage inflammatory protein‐2 (MIP‐2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet‐activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro‐organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)‐κB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF‐κB‐dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC‐6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF‐ and LPS‐ induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF‐κB inhibitors. NEMO‐binding domain peptide, a specific inhibitor of inhibitor protein κB kinase (IKK) activation, a major upstream kinase mediating NF‐κB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti‐TNF antibodies had no effect on LPS‐induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS‐induced CXCL2 expression is dependent on NF‐κB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.

Endotoxin, but not platelet-activating factor, activates nuclear factor-κB and increases IκBα and IκBβ turnover in enterocytes

Bacterial endotoxin (lipopolysaccharide; LPS) and platelet‐activating factor (PAF) are important triggers of bowel inflammation and injury. We have previously shown that LPS activates the transcription factor nuclear factor (NF)‐κB in the intestine, which up‐regulates many pro‐inflammatory genes. This effect partly depends on neutrophils and endogenous PAF. However, whether LPS and PAF directly activate NF‐κB in enterocytes remains controversial. In this study, we first investigated the effect of LPS and PAF on NF‐κB activation in IEC‐6 (a non‐transformed rat small intestinal crypt cell line) cells, by electrophoresis mobility shift assay and supershift, and found that LPS, but not PAF, activates NF‐κB mostly as p50–p65 heterodimers. The effect was slower than tumour necrosis factor (TNF). Both LPS and TNF induce the expression of the NF‐κB‐dependent gene inducible nitric oxide synthase (iNOS), which occurs subsequent to NF‐κB activation. We then examined the effect of LPS and TNF on the inhibitory molecules IκBα and IκBβ. We found that TNF causes rapid degradation of IκBα and IκBβ. In contrast, LPS did not change the levels of IκBα and IκBβ up to 4 hr (by Western blot). However, in the presence of cycloheximide, there was a slow reduction of IκBα and IκBβ, which disappeared almost completely at 4 hr. These observations suggest that LPS causes slow degradation and synthesis of IκBα and IκBβ and therefore activates NF‐κΒ via at least two mechanisms: initially, through an IκB‐independent mechanism, and later, via an increased turnover of the inhibitor IκB. NF‐κΒ activation precedes the gene expression of iNOS (assayed by reverse transcription–polymerase chain reaction), suggesting that LPS up‐regulates iNOS via this transcription factor.

Inflammatory Signaling in Necrotizing Enterocolitis

The pathogenesis of necrotizing enterocolitis (NEC) is complex and its speed of progression is variable. To gain understanding of the disease, researchers have examined tissues resected from patients with NEC; however, as these are obtained at late stages of the disease, they do not yield clues about the early pathogenic events leading to NEC. Therefore, animal models are used and have helped identify a role for several mediators of the inflammatory network in NEC. In this article, we discuss the evidence for the role of these inflammatory mediators and conclude with a current unifying hypothesis regarding NEC pathogenesis.

Inflammatory signaling in NEC: Role of NF-κB, cytokines and other inflammatory mediators.

The pathogenesis of necrotizing enterocolitis (NEC) is complex and the exact etiology remains unknown. Clinically, the presentation and progression of NEC is variable and often difficult to predict. The majority of affected infants (>90%) are premature, and the following factors may play a role in NEC pathogenesis: 1) The immaturity of the intestinal barrier, the mucus layer, decreased Immunoglobulin A (IgA) and defensins; 2) an abnormal intestinal capillary blood flow due do the incomplete development of the intestinal microvasculature or due to the immature regulation of its vascular tone, causing insufficient substrate and O2 delivery to the intestinal epithelial cells; 3) abnormal bacterial colonization of the enteric tract triggering a mucosal pro-inflammatory response; and 4) immaturity of the immune system preventing normal control and killing of microbes, allowing them to penetrate the epithelium. Concomitantly, this immature immune system mounts an excessive production of inflammatory mediators which cause the recruitment of inflammatory cells such as neutrophils and subsequent tissue injury and necrosis. In order to investigate the pathogenesis of NEC, correlative studies have been conducted measuring different inflammatory mediators such as cytokines in the plasma or in the tissues resected from patients with NEC. However, these tissues are obtained at late stages of the disease when these are commonly necrotic, and therefore may not yield information about the early pathogenic events leading to NEC. As mechanistic studies obviously cannot be conducted in humans, animal models have been used. Studies on rats and mice have contributed to the discovery of several potentially important inflammatory mediators in the pathogenesis of NEC. In this chapter, the current evidence for the role of these inflammatory mediators is presented and a current unifying hypothesis regarding NEC pathogenesis is proposed.