Inflammatory signaling in NEC: role of NFKB and cytokines

Introduction

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.

Initiation of the inflammatory cascade: Bacteria– Lipopolysaccharide- Toll-like receptors

During normal term birth, the neonatal intestine is exposed to bacteria present in the maternal birth canal and the environment, and colonization takes place. Breast milk feedings promotes the development of a rich balanced microflora and the specific growth of probiotic species including bifidobacteria and lactobacillus, which have been shown to have many protective properties for the neonatal intestine. Normal colonization is altered in premature infants which were found to have fewer species but more aggressive bacteria ¹. This is facilitated by prolonged periods of fasting, lack of breast milk, the use of antibiotics and anti-acids ² and the NICU environment. In addition, bacterial overgrowth is common in premature infants, facilitated by immaturities of intestinal digestive and motility functions, the intestinal barrier and the innate immunity. There is evidence that bacteria may play a role in the pathogenesis of acute intestinal injury as germ-free rats do not develop acute intestinal injury after platelet-activating factor (PAF) administration ³. The intestinal microflora found in the early stage of human NEC was associated with a higher proportion of gram-negative bacteria ⁴. In addition to other microbial products, called microbial-associated molecular patterns (MAMPs), gram-negative bacteria contain lipopolysaccharides (LPS or endotoxin) on their outer membranes, which is a potent activator of the host immune response. LPS is a potent activator of the transcription factor nuclear factor-κB (NF-κB)⁵, inducing the production of many cytokines, including IL1, IL6, chemokines and TNF⁶ and of PAF⁷, which all amplify the inflammatory response. Several pieces of evidence suggest that LPS may play a central role in NEC: 1) patients with NEC have been found to have higher levels of LPS in their plasma ⁸; 2) intraperitoneal injection of LPS to rats and mice induces intestinal injury and shock ⁹; 3) LPS is the ligand for toll-like receptor-4 (TLR-4), which has been found to mediate NEC ^10,11.

TLRs are pattern-recognition receptors for MAMPs present on most cells. In humans, 10 different TLRs have been identified to date. The activation of TLR4 has been shown to contribute to NEC ^10,11, while TLR9, a cell receptor for unmethylated CpG dinucleotides originated from bacterial DNA, has been shown to be protective ¹². TLRs and their roles in the pathogenesis of NEC are discussed in chapter 3 of this issue.

MAMPs, generally from commensal bacteria, interact with TLRs located on the intestinal epithelium. This promotes epithelial cell proliferation, IgA production, tight junction integrity and antimicrobial peptide production, which all help to maintain a healthy intestinal barrier¹³. However, when MAMPs interact with TLRs present on underlying lamina propria immune cells, a pro-inflammatory response can be triggered ¹³. The location of these interactions may influence the response: Apical exposure of intestinal epithelial cells (IECs) to CpG-DNA inhibits NF-κB activation, while IEC basolateral exposure leads to its activation ¹⁴. This suggests that those commensal bacteria unable to cross the barrier, thereby remaining on the apical site, elicit a homeostatic, anti-inflammatory response¹³ while invasive bacteria which penetrate the epithelial barrier trigger NF-κB activation and a pro-inflammatory response.

NF-κB

Many pro-inflammatory cytokines, chemokines and leukocyte adhesion molecules ^{15, 16} are upegulated during inflammation via the activation of the transcription factor NF-κB, which consists of five subunits (p50, p65, p52, cRel and RelB). These subunits homo- or heterodimerize to form active NF-κB^{15, 16}. p50-p50 and p50-p65 are the NF-κB dimers mostly found in intestinal tissues ^{17, 18}. NF-κB is constitutively present in the cytoplasm of most cells, in an inactive state, as it is bound to inhibitory proteins IκBs (Fig. 1). When the upstream IKK complex ¹⁵[which consists of two catalytic subunits, IKKα and IKKβ and a regulatory component, NEMO (NF-κB essential modulator)¹⁹] is activated via TLR stimulation, it phosphorylates IκB. IκB is then ubiquitinated and degradated by the 26S proteasome, leaving NF-κB free to translocate to the nucleus and to regulate the gene transcription of many inflammatory mediators^{15, 19}.

Fig. 1.

Signaling events leading to NF-κB activation

NF-κB has been found to be constitutively present at low levels in the intestine of adult rats ¹⁷ and to be activated in a model of acute bowel injury induced by PAF¹⁷. In addition, our lab has shown that the constitutive activation of NF-κB in the intestine appears at 20 days of gestation in fetal rats (total length of gestation: 21 days)²⁰.

While physiological activation of NF-κB has a protective role in the intestine, excessive activation may be detrimental and contribute to injury. Breast milk inhibits NF-κB activation by increasing the production of IκBα in IECs ²¹ and probiotics inhibits NF-κB activation through the proteasome ²². Subsequent attenuation of NF-κB activation in IEC may play a protective role against NEC ^{21, 22}. Also, in a murine model of intestinal ischemia-reperfusion, when NF-κB activation was blocked in IEC by IKKβ deletion, the systemic inflammation and multiorgan dysfunction was ameliorated²³. However, in other in vivo models, the activation of IKKβ and NF-κB in IECs has been shown to be protective and to limit intestinal mucosal damage^23–25. Therefore, NF-κB in IECs may play both protective and detrimental roles in the intestine.

NF-κB activation and cytokine production are increased in immature IECs in response to flagellin ²⁶ and in immature inflammatory cells in response to TNF-alpha^{27, 28}, compared to mature cells. Also lower levels of specific IkappaB genes were found in fetal enterocytes compared to adult cells ²⁶.

Taken together, these data suggest that NF-κB activation may be developmentally regulated.

NFκB has been found to be strongly activated in the intestine of newborn rats at birth²⁰. After breast milk feeding this activation is downregulated within 24 hours²⁰. This contrasts with pups exposed to a NEC protocol in whom NFκB remained elevated ²⁰ and endogenous NF-κB inhibitory proteins IκBα and IκBβ were decreased ²⁰. In neonatal rats, TLR-2 staining and NF-κB activation in IEC correlated with NEC severity, IEC apoptosis and impaired proliferation ²⁹. Our laboratory has shown that neonatal rats treated with NEMO-binding domain peptide (NBD peptide), a specific NF-κB inhibitory peptide, were less susceptible to bowel injury and had decreased mortality when exposed to the NEC model ²⁰, suggesting a central role for NF-κB in NEC. NBD was shown to decrease LPS-induced chemokine CXCL2 (or MIP-2) gene expression in IECs³⁰ and LPS-induced interleukin-1 β (IL-1β), IL-6 and TNF-alpha gene expression in macrophages in vitro (J774.1)³¹.

While NF-κB activation is critical for host immunity against pathogens ³², an exaggerated and prolonged NF-κB activation in premature infants may lead to an increased pro-inflammatory response with excessive levels of cytokines, contributing to intestinal tissue injury in NEC ²⁰. Although there have not been any human studies looking at inhibiting cytokines in NEC, many clinical trials have been conducted in IBD: anti-TNF therapies have been shown to make a significant difference to the health-related quality of life of many patients with IBD³³. Anti-IL-12/IL-23, IL-2R and IFN-g have been shown to have limited results in early phase clinical trials, and studies using antibodies against IL-6, IL-6R, IL-13, IL-17, IL-18 and IL-21 are now entering phase I³³.

Interleukin-6 (IL-6)

IL-6 is predominantly generated by activated macrophages, T-cells, and the endothelium, but is also expressed by enterocytes in response to infection³⁴. Its expression is under the control of NF-κB³⁵. IL-6 stimulates the production of acute phase proteins in the liver, B cell proliferation and antibody production. IL-6 levels have been found to be elevated in the plasma and the stools of patients with NEC ³⁶ and to be correlated with the severity of disease ³⁷³⁸. In a study of 62 newborn infants with suspected sepsis or NEC, Interleukin-6 levels were five- to tenfold higher in infants with bacterial sepsis plus NEC at the onset of disease than in infants with bacterial sepsis alone ³⁷. In a study of 60 preterm infants there was a trend to higher levels of IL-6 with a greater degree of NEC³⁸. In an experimental model of NEC induced by Cronobacter sakazakii infection, increased pup serum IL-6 was noted in association with increased enterocyte apoptosis³⁴.

IL8

IL-8 is generally regarded as a pro-inflammatory chemokine predominantly produced by macrophages and endothelial cells. However, exposure to amniotic fluid containing IL-8 has been postulated to be important for promoting intestinal health³⁹. IL-8 is present in significant concentrations in human milk and, when human fetal and adult intestinal cells are treated with rh IL-8 in vitro, it stimulates cell migration, proliferation, and differentiation³⁹. IL-8 is a potent chemoattractant for neutrophils and an angiogenic factor. As with several other proinflammatory cytokines, elevated IL-8 levels have been associated with human NEC⁴⁰⁴¹ and with animal models of intestinal inflammation⁴². Furthermore, high IL-8 levels may correlate with human NEC severity⁴⁰. One recent study reports that IL-8 appears to be a promising biomarker for the extent of intestinal necrosis⁴¹. Cellular maturity may affect the response to bacterial challenge. Indeed, when compared to mature enterocytes, immature fetal intestinal cells have been shown to produce more IL-8 in response to LPS⁴³ and flagellin ²⁶. These developmental differences may predispose the premature intestine to inflammation.

IL-10

In contrast to IL-6 and IL-8, IL-10 is generally considered to be an anti-inflammatory cytokine. IL-10 modulates the innate and adaptive immune response in part by inhibiting cytokine production by macrophages and other antigen presenting cells⁴⁴. Along with several other cytokines, IL-10 is increased in infants with severe NEC ⁴⁰⁴⁵. Several pieces of evidence suggest that this represents a protective mechanism to dampen the intestinal inflammatory response: IL-10 deficient rodents have been shown to develop colitis ⁴⁶. Also, IL-10 knockout mice were found to have more profound histologic intestinal injury in a neonatal NEC model than controls⁴⁷; in these mice, intestinal injury was associated with decreased junctional adhesion molecule-1 and increased inducible nitric oxide synthase (iNOS) expression and with increased enterocyte apoptosis⁴⁷. Intraperitoneal administration of IL-10 has been shown to decrease the severity of intestinal injury⁴⁸ and the level of NO synthesized by inhibiting iNOS expression⁴⁸.

The breast milk of mothers of premature infants with NEC was more likely to lack IL-10 than those without NEC⁴⁹. Indeed, IL-10 levels were not detectable in 86% of breast milk samples obtained from women whose premature infants developed NEC compared to 40% obtained from women whose premature infants did not ⁴⁹. When stimulated with TNF-alpha or LPS, mononuclear leukocytes obtained from premature infants had decreased IL-10 production compared to term infants ⁵⁰. Decreased IL-10 levels may increase the susceptibility of premature infants to inflammation.

IL-11

As with IL-10, IL-11 is another cytokine thought to promote intestinal health. It is produced in the intestine by subepithelial myofibroblasts and downregulates the production of Th1 cytokines such as IL-12 and IFNγ by epithelial cells and lamina propria macrophages⁵¹. It has other functions such as the stimulation of megakaryocyte maturation⁵². In a study of 21 infants with NEC, IL-11 was found to be upregulated and those with elevated IL-11 mRNA appeared to have a decreased risk of pan necrosis⁵³. IL-11 has been shown to prevent mucosal atrophy and to enhances small intestine absorptive function after intestinal resection⁵⁴⁵⁵.

IL-12

IL-12 is secreted by neutrophils, macrophages, dendritic cells and B cells when stimulated with bacteria, viruses and their products. It activated macrophages and Th1 cells, inducing IFNγ. In a small human NEC study, patients with increased IL-12 appeared to have diminished risk of pan necrosis⁵³. However, conflicting data exist on the role of IL-12 in experimental NEC: In one study, IL-12 was found to be elevated in pups exposed to experimental NEC and the increase was correlated with tissue damage ⁵⁶, while in another study, IL-12 was found to be downregulated ⁵⁷. Therefore further investigation is needed to establish the role of IL-12 in NEC.

IL-18

IL-18 is a cytokine produced by macrophages, dendritic cells and IECs, and together with IL-12, induces cell-mediated immunity following activation by MAMPs. It induces the production of IFNγ, IL-1β, IL-8 and TNF-alpha by immune cells. IL-18 production is increased in the ileum of neonatal rats with NEC ^{56, 58} and mice deficient in IL-18 have a decreased incidence of NEC, fewer ileal macrophages and higher level of IkappaB-alpha and beta, suggesting decreased NF-κB activation ^{56, 58}. Infants with NEC were more likely found to have IL-18 polymorphisms ⁵⁹.

TNFα

An increase in TNFα mRNA ⁶⁰ and protein ⁶¹ was detected in intestinal tissues taken from patients with NEC. When examined by in situ hybridization, human NEC tissues were found to have a marked increase in TNF-alpha mRNA in Paneth cells, as well as in infiltrating eosinophils and macrophages ⁶². However, TNFα does not appear consistently elevated in serum samples of patients with NEC^{36, 37, 45, 63}. Increased intestinal TNF-alpha levels were found in hypoxia/reoxygenation-induced bowel injury in neonatal rats ⁶⁴ but not in a neonatal rat NEC model ⁵⁷ and in an acute intestinal injury model (Our lab unpublished data, 2008).

TNF-alpha has been shown to cause a marked loss of mucus-containing goblet cells in immature mice ⁶⁵. TNF-alpha causes apoptosis in IECs by inducing mitochondrial ROS (reactive oxygen species) production and by activating the JNK/p38 signaling pathway ⁶¹.

While a role of TNF-alpha in inflammatory bowel disease has been clearly established ⁶⁶, the evidence for TNFα implication in the pathogenesis of NEC is less certain. Anti-TNFα therapy has been shown in some studies to decrease the degree of experimental NEC^{67, 68}. However, we did not identify similar findings within our own experimental models when we compared rats treated with anti-TNF antibodies versus control immunoglobulin (2008, unpublished data).

IFN gamma

While interferon gamma (or type II interferon)(IFNγ) is mostly released by T cells and natural killer cells, it is also produced by B cells, NK-T cells, dendritic cells and macrophages ⁶⁹.

Early during infection, macrophages produced IFNγ in response to IL-12 and IL-18 stimulation ⁶⁹. IFNγ activates several signaling pathways, including STAT1, PI-3 kinase/Akt and MAPKs which control the expression of more than 500 genes known to regulate apoptosis, proliferation, leukocyte migration and epithelial permeability ⁶⁹.

IFNγ synthesis is inhibited by IL-4, IL-10, TGFβ and glucocorticoids⁶⁹.

A role for IFNγ in NEC has been suggested by the evidence that knock-out mice are protected against experimental NEC and have improved epithelial cell restitution compared to wild-type controls when exposed to the NEC model⁷⁰. IFNγ was found to inhibit enterocyte migration by reversibly displacing connexin 43 from lipid rafts⁷¹.

PAF

Platelet-activating factor (PAF), an endogenous phospholipid mediator is released by many cells, including platelets, neutrophils, mast cells, eosinophils, macrophages and endothelial cells. Bacteria such as Escherichia coli and Helicobacterpylori have also been found to produce PAF ^72–75. The receptor for PAF (PAF-R) is a G protein-coupled receptor which is mainly expressed in the ileum, but also abundantly present in the jejunum and the spleen ⁷⁶. PAF-R is not only present on inflammatory cells including neutrophils, macrophages and eosinophils ^{76, 77} but also on epithelial ⁷⁷ and endothelial cells⁷⁸. In vivo, free PAF is rapidly degraded by PAF-acetylhydrolase (PAF-AH), a PAF degrading enzyme. PAF-R activation leads to the activation of several transduction pathways including the activation of NF-κB¹⁷ and of PI3kinase/Akt⁷⁹, and the production of endogenous PAF (by phospholipase A2 (PLA2))⁸⁰, oxygen radicals by xanthine oxidase ⁸¹ and TNF-alpha⁸² which cause lasting effects in vivo. Several pieces of evidence suggest an important role for PAF in NEC: 1) Circulating PAF levels⁸³ are increased and plasma PAF-acetylhydrolase⁸⁴ levels are decreased in infants with NEC compared to age-matched controls; 2) human milk contains PAF-AH, the enzyme degrading PAF⁸⁵; 3) experimental NEC is prevented by PAF-receptor antagonists in a neonatal rat model⁸⁶ and in a piglet model⁸⁷; 4) intravenous infusion of recombinant PAF-AH prevents NEC in rats^{88, 89}; 5) PAF-AH knock-out mice are more susceptible to NEC when exposed to the NEC protocol⁹⁰, although they have a decreased mortality; and 5) the ileum, the site of predilection of NEC, has the highest amounts of PAF-R⁷⁶. PAF also plays a role in the intestinal injury induced by hypoxia/reperfusion⁹¹, TNF-alpha⁹² and LPS⁹³. Exogenous administration of PAF causes systemic hypotension, increased vascular permeability, hemoconcentration, and tissue necrosis⁹⁴ which is limited to the intestine and predominates in the small intestine (frequently the ileum) as seen in NEC. This is why PAF administration has been used as a model of acute bowel necrosis and NEC⁹⁴. PAF-induced bowel injury is worsened by the intraperitoneal administration of LPS⁹. Furthermore, PAF has been shown to mediate LPS⁹⁵ and hypoxia-induced bowel necrosis⁹⁵. We have shown that the intravenous injection of PAF, even at a dose below that which causes bowel necrosis¹⁷, causes a very rapid activation of NF-κB in the intestine (within 5 minutes, peaking at 30 minutes), and induces the gene expression of PLA2⁸⁰, TNF-alpha⁸², NF-κB p50 precursor p105⁹⁶, PAF-R⁷⁶ and TLR4 in IECs⁹⁷ and the release of leukotriene C4⁹⁸. PAF plays an important role in the allergic and inflammatory response causing neutrophil and platelet aggregation, systemic and mesenteric vasodilation and increased vascular permeability⁹⁹.

Nitric oxide (NO)

NO is a free radical that is implicated in many homeostatic and pathologic processes. It has been implicated in regulation of the vascular tone, inflammation, neurotransmission and tissue restitution and repair¹⁰⁰. NO is formed by the action of nitric oxide synthase (NOS) on oxygen and arginine. There are three isoforms of NOS, each encoded by a separate gene; endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS)(Fig 2). eNOS and nNOS are typically expressed at low (picomolar) constitutive levels. iNOS is not expressed under normal conditions. When iNOS is upreguated, it generates high concentrations on NO (micromolar). NO is soluble in both lipids and water, and is able to diffuse rapidly to neighboring tissues and cells without reliance on channels or transporters. At low levels NO appears to be effectively cleared, however at increased concentrations it reacts with a superoxide anion producing peroxynitrite, a highly toxic oxidation intermediate¹⁰¹. Peroxynitrite disrupts protein conformations and alters cellular process and reactions¹⁰². Although low levels of NO may be beneficial¹⁰¹, by increasing intestinal water absorption¹⁰³ and controlling intestinal barrier permeability¹⁰⁴, high levels appear clearly detrimental increasing enterocyte apoptosis ¹⁰⁵ and impairing cell proliferation ¹⁰⁶(Fig 2). NO released by activated macrophages has been found to inhibit enterocyte migration ¹⁰⁷. NO mediates dendritic cell apoptosis ¹⁰⁸, while protecting against apoptosis in other cell types ¹⁰⁹.

Fig. 2.

Role of nitric oxide in vivo

iNOS was first implicated in the pathogenesis of NEC, after elevated levels of iNOS induced NO were found in the IEC of resected intestinal samples taken from infants undergoing bowel resection secondary to NEC¹¹⁰. Animal studies also have shown that iNOS is upregulated in a neonatal rat NEC model ⁵⁷ and that iNOS mediates LPS-induced increase in intestinal permeability¹¹¹. In the intestine of neonatal rats fed with formula eNOS and nNOS were downregulated while iNOS was upregulated when compared with dam fed controls ¹¹² and in piglets, formula feeding downregulated eNOS ¹¹³. When L-NAME, a NO synthase inhibitor, was administered intravenously in a neonatal piglet model of NEC, it worsened the intestinal injury while L-arginine, a NO synthase substrate, attenuates intestinal injury ¹¹⁴. Under physiological conditions, we found that nNOS suppresses iNOS gene expression in the small intestine of rats through NF-κB downregulation ¹¹⁵. When nNOS was inhibited, IκB alpha was degradated, NF-κB was activated, and iNOS expression induced ¹¹⁵. In a mouse model of DSS colitis, eNOS and iNOS were found to be detrimental while nNOS was found to be beneficial ¹¹⁶. However, in other studies using a similar model, eNOS was found to be protective against intestinal inflammation ^{117, 118}.

ROS

Under physiological conditions, when bacteria interact with intestinal epithelial cells, reactive oxygen species are reversibly produced which, among other functions, modulate neddylation of cullin-1, leading to downregulation of the proteasome pathway and subsequent NF-κB pathway suppression^{119, 120} and beta-catenin stabilization^{119, 120} leading to increased intestinal cell proliferation¹²¹. However, during inflammation, reactive oxygen species (ROS) may be a major effector of PAF and many other cytokines. The xanthine oxidase/dehydrogenase system is one of the main producers of ROS in the intestine. Indeed, the enzyme xanthine dehydrogenase is constitutively expressed in large amounts in the intestinal villous epithelium ¹²² and catalyzes the transformation of xanthine into uric acid (Xanthine+H2O+NAD→ Uric acid+NADH+H+). However, during ischemia, xanthine dehydrogenase is converted into xanthine oxidase, which leads to the transformation of xanthine into uric acid and the production of superoxide (Xanthine+H2O+O2→ Uric acid+2O2−.+2H+).

ROS activates the intestinal mitochondrial apoptotic signaling pathway during oxidative stress ¹²³ and leads to IEC apoptosis via the activation of p38 MAPK ¹²⁴. Xanthine oxidase and superoxide have been found to mediate intestinal reperfusion injury ¹²⁵ and PAF-induced bowel necrosis ⁸¹, as allopurinol, a xanthine oxidase inhibitor, has been shown to be protective in these models. Together, these studies suggest a central role for XO and ROS in the pathogenesis of intestinal injury.

Role of immune cells

Several evidences suggest that neutrophils may play an important role in NEC: 1) Neutropenic rats are protected against PAF-induced bowel injury ¹²⁶ and have decreased intestinal NF-κB activation ¹⁷; 2) P-selectin deficient mice ¹²⁷ are protected against PAF-induced bowel injury, as are mice treated with antibody against beta-2 integrin ¹²⁸ (P-selectin and beta-2 integrin are adhesion molecules necessary for neutrophils to roll and adhere to the endothelium); 3) mice treated with antibodies against CXCL2, a major chemokine for neutrophils, have decreased systemic inflammation and are protected against PAF-induced acute intestinal injury ¹²⁹. However, when NEC was induced by Cronobacter sakazakii infection, PMN and macrophages from the lamina propria were responsible for C. sakazaki killing and clearance, and PMN and macrophage depletion exacerbated cytokine production and bowel injury¹³⁰. In this model, C. sakazakii induced epithelial damage was secondary to dendritic cells (DCs) recruitment into the intestine ¹³¹ with subsequent TGF-β production, iNOS production, apoptosis and epithelial cell damage ¹³¹. Other investigators have found that, in utero, intestinal macrophages progressively acquire a non-inflammatory profile due to increased mucosal TGF-β (2) production and macrophages with a hyper-inflammatory phenotype reside in the premature intestine, increasing its susceptibility to injury¹³². Activated macrophages have been found to inhibit enterocyte gap junction formation and IEC restitution via the release of nitric oxide¹⁰⁷.

Systemic inflammation

In NEC, the inflammatory response is not limited to the intestine, but liver and brain inflammation might be present. A study done on neonatal rats suggests that the liver may amplify the inflammation during NEC ¹³³. Indeed, the number of Kupffer cells (KC) and the level of hepatic IL-18 and TNF-alpha were found to be elevated in experimental NEC and to correlate with the severity of the intestinal damage ¹³³. When KC were inhibited with gadolinium chloride, the levels of TNF-alpha found in the intestinal lumen of rats with NEC were significantly decreased¹³³.

It is recognized that high cytokine levels in preterm infants may contribute to neurologic injury, and poor long-term outcomes¹³⁴. In a study of 84 very low birth weight infants in a neonatal intensive care unit (NICU), 22% developed NEC. White matter injury was found 2.2 times more frequently in those infants with NEC¹³⁵. Higher plasma levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNFα) were found in those infants. Whether the cytokines exhibit a direct effect on the oligodendrocytes remains inconclusive.

SUMMARY – CURRENT HYPOTHESIS

SUMMARY - Current Hypothesis

(Fig 3)

Fig. 3.

Schematic representation of the pathogenetic events leading to NEC

While we don’t clearly understand NEC pathogenesis, studies have helped elaborate the current hypothesis, which is that NEC results from a local intestinal inflammation initiated by perinatal stress. Upon introduction of feedings, intestinal bacteria proliferate, favored by the immaturity of the neonatal mucosal innate immune system. Intestinal microbes and their products (MAMP) adhere to the epithelium, breach the immature and fragile intestinal mucosal barrier and activate NF-κB in lamina propria immunocytes, causing them to secrete pro-inflammatory mediators, chemokines (CXCL2), cytokines (TNF, IL), prostanoids, platelet-activating factor, and nitric oxide. (We hypothesized that the bowel injury in NEC results from inappropriately elevated and prolonged NF-κB activity in inflammatory cells). These inflammatory agents attract further inflammatory cells, in particular neutrophils, induce the production of reactive oxygen species, and inflict further damage to the intestinal barrier resulting in increased bacterial translocation, intestinal epithelial damage, impaired epithelial cell restitution, apoptosis and mucosal necrosis. Thus, a vicious cycle characteristic of severe NEC is created by bacterial invasion, immune activation, uncontrolled inflammation with production of reactive oxygen and nitrogen species, vasoconstriction followed by ischemia-reperfusion injury, gut barrier failure, intestinal necrosis, sepsis and shock.