Necrotising enterocolitis (NEC) is a progressive disease of the neonatal intestine beginning in the distal ileum and proximal colon and characterised by inflammatory necrosis [1,2]. It typically affects low birth-weight, preterm infants who account for the majority (70–90%) of cases [3–5]. Since the 1960s, advances in medical care have raised the survival rate for preterm infants with increasingly shortened gestation periods, resulting in a concomitant surge in NEC cases. The overall incidence of NEC is generally accepted as ranging from <1% to 5% of neonatal intensive care unit (NICU) admissions, or up to 5 cases per 1,000 live births [4–6]. There is an inverse relationship between NEC and birth-weight, so that very low birth-weight infants (VLBW; <1500 g) carry the greatest burden of disease [4,5,7]. Caplan reported NEC rates for VLBW infants vary greatly across countries, ranging from 1.5% in Japan to 28% in Hong Kong, with racial disparity apparent in VLBW black infants who have an increased risk and greater associated mortality [5,8]. Despite advances in neonatal care, the overall mortality remains high at around 20–30% [3,8-10]. An estimated 20-40% of infants with NEC require surgery, which has a case fatality rate of up to 50%, the smallest, least mature infants having the worst prognosis . Most cases of NEC are sporadic with no clear seasonal distribution, but outbreaks have been documented . Treatment of NEC is mainly supportive with the administration of broad-spectrum antibiotics while surgery is indicated for intestinal perforation or removal of necrotic bowel segments. NEC complications and sequelae include serious neurodevelopmental delay, poor growth, intestinal obstruction due to scarring, short bowel syndrome, and liver failure due to prolonged hyperalimentation [6,9]. The annual financial cost of NEC is considerable and in the USA has been estimated at $500 million to $1 billion .
1.1. Clinical classification
The classification system of Bell has historically proved important in defining three main stages: suspected, definite and advanced NEC . Modifications to Bell’s criteria have provided a more detailed system of clinical staging as shown in Table 1 [12,13]. However, Gordon et al and others have challenged the belief that NEC is a single entity, preferring to view it as an umbrella term for a number of separate diseases with some common features [13,14]. Although relatively uncommon, conditions, which mimic neonatal NEC, such as focal bowel perforation, intussusception, ecchymotic colitis, appendicitis and shigellosis, have been reported and may complicate the clinical diagnosis [14-18].
|Stage I (suspected)||Temperature instability, apnoea, bradycardia||Normal or intestinal dilation; mild ileus||Gastric residuals, occult blood, mild abdominal distension|
|Stage II A (definite)||Temperature instability, apnoea, bradycardia||Intestinal dilation, ileus, focal pneumatosis||Blood in stools, prominent abdominal distension, absent bowel sounds|
|Stage II B (definite)||As above plus mild metabolic acidosis and thrombocytopenia||As II A plus portal vein gas, ascites||Abdominal wall oedema with palpable loops and tenderness|
|Stage III A (advanced)||As stage II B plus mixed acidosis, oligouria, hypotension, coagulopathy||As II B plus worsening ascites||Worsening wall oedema, erythema and induration|
|Stage III B (advanced)||As II A, shock, deterioration in vital signs||As II B plus pneumoperitoneum||Perforated bowel|
1.2. Risk factors
So far, four major risk factors for NEC have been defined with prematurity being the most consistent. At 36 weeks gestation there is a sharp decrease in the incidence of NEC, supporting the concept that gut maturation provides significant protection against development of the disease . Nevertheless, NEC in term and high birth-weight infants is not unknown, although the risk factors appear to be somewhat different [4,6,13]. The introduction of enteral feeds, particularly formula milk, and subsequent colonisation of the neonatal intestinal tract with bacteria are believed to be significant risk factors in the development of NEC [4,6,19]. Not only does formula milk lack the gastrointestinal protective, anti-inflammatory and maturation factors present in breast milk, it can be a source of
Recent evidence has linked blood transfusions with subsequent NEC in extremely premature infant [25–30]. The terms TANEC (Transfusion Associated NEC) and TRAGI (Transfusion-Related Acute Gut Injury) have been coined, referring to this association [25,31]. A recent meta-analysis examining evidence for the association concludes that recent transfusion is associated with NEC, and that transfusion-associated NEC has a higher risk of mortality than NEC which was not preceded by transfusion . The reason for the association, and whether it is causal has not been elucidated. Prior to concern about TRAGI, Doppler studies have shown a decrease in neonatal superior mesenteric blood flow during and after blood transfusion . The reason for this is not clear, but blood transfusion appears to have wide-ranging effects on haemodynamics, possibly as a result of changes in the microcirculation. Digestion of food requires a major increase in gut blood supply and it has been suggested that limiting or ceasing milk feeds before, during and after a blood transfusion in susceptible babies may decrease the risk of TRAGI. This has not been subjected to any systematic study and therefore remains speculative. The need for a co-ordinated approach to investigate the association between blood transfusion and NEC has been highlighted by Blau et al and there is an online world registry for TRAGI (www.tragiregistry.com) . Any trial of an intervention for TRAGI prophylaxis would need large numbers, requiring a multicentre approach.
There are other characteristics of the preterm infant favouring the development of NEC. The combination of poor gut motility and under-production of mucous decreases clearance and increases exposure of the epithelium to potentially harmful components of the luminal contents [1,5,19]. Moreover, induction of foetal hypoxia can further reduce postnatal intestinal motility . The lumen contents of preterm infants may be more acidic, due to inadequate digestion/absorption of nutrients and bacterial fermentation of undigested milk, or more toxic, as formula feeding elicits toxic bile acids . Bacterial overgrowth, as indicated by a positive hydrogen breath test, is considered to be a further consequence of delayed transit time and may promote NEC through increasing bacterial translocation from the intestinal lumen into the tissues or through exposure to high concentrations of bacterial antigens [33,34]. More recently, it has been proposed that genetic polymorphisms in the genes encoding the interleukin (IL) 4 binding receptor alpha chain and the chemokine IL-8 may also be a risk factor for NEC . Intrauterine infection is another risk factor for NEC; the microorganisms implicated and proposed causality are discussed in section 2.3.
In the normal course of events, acquisition of the enteric microbiota begins during the birth process through the ingestion of bacteria of maternal origin. Breastfeeding, handling by the mother and exposure to environmental bacteria create further opportunities for gaining new species . Colonisation takes place in the first few days of life and is influenced by a multitude of factors such as mode of delivery, type of feeding (breast milk or formula), gestation age, hospitalisation, the surrounding environs, maternal infection and antibiotic therapy, with mode of delivery and type of feeding considered the most significant . Whereas breast fed infants are regarded as having an enteric microbiota rich in bifidobacteria, a more diverse microbiota, including potentially pathogenic groups such as
2. The role of microbes in NEC
The belief bacteria are crucial for the development of NEC stems from a number of clinical and experimental findings. In two studies totalling over 100 infants, Sántulli et al and Schullinger et al were the first to credibly establish bacterial colonisation of the neonatal intestine was a requirement for this disease [38,39]. NEC does not usually occur immediately post-partum but some days later, when feeding has usually commenced and there is ample opportunity for substantial intestinal colonisation. Early-onset NEC occurs in the first week and it is more often seen in term or near term infants with risk factors such as cardiac disease or severe placental insufficiency, whereas in infants of lower gestational age/birth-weight, NEC is delayed until 13-32 days [40,41]. The reason for this difference is not entirely clear, but it may relate to a difference in pathophysiology, with bowel ischaemia being the predominant factor in early-onset NEC and cytokine priming being the predominant factor in late-onset NEC. The case for bacterial colonisation is further strengthened by the absence of NEC in ischaemic, ileal segments of germ-free rats and in infants who are stillborn [42,43].
Regardless of the initiating factors, pathological changes certainly involve bacteria as the intramural gas produced in pneumatosis intestinalis contains hydrogen of bacterial origin . Demonstration of bacteria and bacterial DNA in the intestinal wall of resected segments from NEC infants supports this finding [45,46]. Epidemiological studies also indicate NEC has an infectious origin as it may occur in clusters of related cases which are amenable to infection control measures . Moreover, prevention of NEC has been achieved through the administration of enteral antibiotics . Bacteraemia and endotoxinaemia are frequent complications of NEC but are more likely to be sequelae rather than the actual cause [49,50]. Historically, there have been many proposals put forward regarding the aetiology of NEC (reviewed by Obladen ) but two main theories have emerged concerning the infectious component:
This theory relies on the existence of a hitherto undiscovered, single bacterial pathogen causing intestinal infection in susceptible infants.
Even in health, many members of the enteric microbiota can be considered to have pathogenic potential. When the balance between pathogenic and commensal species shifts in favour of the former, a chain of events is triggered in susceptible infants resulting in NEC.
2.1. Microbes implicated in the aetiology of NEC
Among the enteric anaerobes,
Other intestinal anaerobic genera have not been fully investigated, probably due to the difficulty of culturing under strict anaerobic conditions. Despite the prevailing view that non-sporing anaerobes are frequently absent in the intestinal tract of preterm infants, a DNA-based study indicated
Coagulase negative staphylococci (CoNS) are commonly found in the stools of NEC infants and have been associated with significant disease [65,66]. Hoy et al noted their presence in duodenal aspirates of VLBW infants . The role of staphylococcal delta(δ)-toxin was examined by Scheifele et al and Scheifele and Bjornson, who believed that toxin positive CoNS were enteropathic [67,68]. δ-toxin, a secreted protein with a detergent-like action, caused significant bowel necrosis in infant rats and was cytotoxic for fibroblasts in vitro. Moreover, it could be detected in the stools of infants colonised with δ-toxin producing CoNS [67,68].
We investigated 25 CoNS isolates from the stools of six NEC and six control infants in Dunedin Hospital NICU. A diagnosis of NEC was made on the basis of clinical indications and pneumatosis intestinalis or peritonitis on X-ray as described previously . CoNS were identified using API ID-32 STAPH strips (bioMérieux). PCR primers for the δ-toxin gene were based on sequence data published by Tegmark et al and PCR conditions were as described by McIntosh [70,71]. Cell-free culture supernatants of the CoNS isolates and a δ-toxin producing
Rejection of the δ-toxin theory does not preclude a role for CoNS
|†δ +||‡CPE||δ +||CPE||δ +||CPE|
Reports citing classical enteric pathogens such as salmonellae and shigellae as causes of NEC or NEC-like conditions are rare [18,90]. Enteric viruses such as norovirus, rotavirus, torovirus, and astrovirus are more frequently implicated. Some investigators consider norovirus to be an emerging pathogen in the NICU, with NEC representing a severe presentation of infection [91,92]. Human astrovirus was reported by Bagci et al to be the cause of NEC in a subgroup of infants and torovirus was found to be more common in NEC compared to control infants by Lodha et al [93,94]. Rotavirus has been demonstrated in the stools of neonates from day 4 of life and its presence is considered a risk factor for NEC . Echovirus type 22, renamed human parechovirus, is also considered to be an enteric pathogen, although causality has not been fully established . Birenbaum et al detected this virus in an outbreak of diarrhoeal illness in a NICU with some patients exhibiting the clinical signs and symptoms of NEC . Rousset et al noted the presence of coronavirus-like particles in gut tissue samples from NEC infants and proposed that secondary proliferation of anaerobic bacteria occurred in the gut wall following viral damage of the intestinal epithelium .
The advent of molecular techniques has facilitated the detection of viruses and it is likely that future investigations will better define the viruses associated with NEC. However, claims that viruses are
2.2. Diversity and numbers
It is clear many of the bacteria forming part of the enteric microbiota have pathogenic potential and it has been suggested that when the balance between pathogenic and commensal species shifts in favour of the former, a chain of events is triggered in susceptible infants leading to NEC. A number of studies have sought to investigate this abnormal colonisation theory by identifying and quantifying enteric bacteria at the time of NEC presentation and comparing the results with a control group of healthy infants. However, there is evidence the intestinal ecosystem is altered by inflammation while the microbiota is restored after the inflammatory signal is dissipated . This may be described as a ‘chicken and egg’ situation; it is unclear whether changes in the microbiota of NEC infants cause the disease or are a result of the inflammation. Of particular value are prospective investigations, as they have the potential to elucidate the microbiota associated with the initiation of NEC
The early, prospective study of Hoy et al was restricted to culturable bacteria, but nevertheless demonstrated considerable quantitative changes in the faecal microbiota preceding both confirmed and suspected episodes of NEC, with a decline in some species up to 72 hours before clinical onset and the emergence of others, particularly
A recent study encompassing both culture and molecular techniques has strengthened the argument that
A confounding factor is that antibiotic therapy is frequently applied to preterm infants with the possible outcome that the reduced microbial diversity reported to be a feature of NEC could be a consequence of the antibiotic. Tanaka et al demonstrated antibiotic exposure in the pre- or early postnatal period greatly influences the enteric microbiota with arrested growth of beneficial bifidobacteria and overgrowth of
2.3. Significant microorganisms
Despite many attempts, the specific pathogen theory of NEC has not been proven. The strongest candidate organisms, commensal members of the
The abnormal colonisation theory, with its emphasis on community structure rather than specific organisms, has emerged as the most likely explanation for NEC. Possibly some members of the microbiota contribute to health while others increase the likelihood of NEC, with quantitative changes heralding the onset. Even though they have never been proven to be the causative agents,
Several studies challenge the long held belief that the foetus is normally devoid of microorganisms and it now seems likely that colonisation begins before birth and can be detrimental. Intrauterine infection is a major cause of prematurity and is associated with adverse neonatal outcomes [116,117]. According to Gonçalves et al, microorganisms gain access to the amniotic cavity and foetus by four main pathways: (1) ascending from the vagina and cervix; (2) transplacental infection; (3) seeding from the peritoneal cavity via the fallopian tubes; (4) accidental introduction during invasive procedures . The ascending pathway is probably the most common route of infection and a variety of microorganisms have been implicated . PCR-based studies, such as that of DiGiulio et al, indicate microbial invasion of the amniotic cavity is common in the setting of preterm, pre-labour rupture of membranes and is underestimated using standard culture techniques .
The mechanisms underlying the relationship between intrauterine infection, the inflammatory pathway and NEC have not been elucidated but animal experimentation indicates intra-amniotic exposure to lipopolysaccharide or
2.4. Crossing the epithelial barrier
As previously mentioned, motility patterns in the small bowel are poorly developed in the preterm infant, particularly before 28 weeks gestation, with gastrointestinal transit times ranging from 8-96 hours compared to 4-12 hours in adults . Gastric acid production and enterokinase levels are low in the premature infant, which may limit lipid and protein digestion in the small intestine, and together with lowered intestinal motility may be responsible for bacteria having substrate available for growth for longer periods. In 1990, Carrion and Egan investigated supplementing the feeds of premature infants with hydrochloric acid. The results were promising, but this approach has not been widely adopted, and appears not to have been investigated further . It is postulated that bacterial fermentation of substrate (lactose) present in the infant gut damages the mucosa through the production of gas, which increases intraluminal pressure. The ability of some commensal species to ferment lactose is well known but there seems to be no correlation with NEC . The surface of the gastrointestinal tract must allow entry of molecules that are beneficial to the host while at the same time preventing harmful microbes from crossing the barrier. Piena-Spoel et al observed increased intestinal permeability in human neonates with severe NEC, compared with control babies .
The intestinal epithelium from the stomach to the rectum is comprised of a single layer of polarised epithelial cells. The main functions of these cells are to absorb nutrients and also to prevent luminal bacteria and other antigens from crossing the intestinal barrier and entering the bloodstream . Extrinsic barriers including gastric acidity, intestinal peristalsis and the mucus layer limit the access and adhesion of bacteria to the epithelial surface. The mucus layer is an organised extracellular matrix containing inorganic salts, non-specific antimicrobials and specific antimicrobial immunoglobulins, water and large glycoproteins (mucins) . Mucins are produced by goblet cells within the crypts of the intestinal epithelium and released either constitutively or in response to infecting organisms . Intrinsic barriers, which include the selectively permeable epithelial cell plasma membrane and the tight junctions that seal the intracellular spaces, block translocation of bacteria and restrict diffusion of macromolecules. Both these barriers are underdeveloped in the premature infant and this coupled with immaturity of the immune or cellular defense mechanisms may result in bacterial translocation leading to the inflammatory cascade resulting in NEC, even without prior injury of the mucosa. This hypothesis is supported by the fact that mice deficient in Muc2 have been shown both to be susceptible to infection and to develop intestinal inflammation. These mice, as well as having a deficiency in mucus production, had increased leakiness in the gut, which allowed microbes (both commensals and pathogens) to transit the mucosa . Bergstrom et al suggest that the epithelium may be subsequently damaged either as a result of bacteria producing high concentrations of toxic metabolites or, alternatively, the presence of the bacteria stimulates recruitment of large numbers of polymorphonucleocytes to the site of infection resulting in epithelial cell death as neutrophils release cytotoxic mediators to control the infection . The blooms of intestinal
Tight junctions and adherens junctions are critical for maintenance of gut permeability and intestinal barrier function [138,139]. Tight junctions form a permeable barrier allowing the passage of fluids and solutes but not the other contents of the intestinal lumen. These junctions are made up of trans-membrane proteins (including occludins, claudins) and junctional adhesion proteins as well as cytoplasmic proteins (zona occludens - ZO-1, ZO-2, ZO-3) . Using an epithelial cell monolayer (Caco2 cells) as in vitro model intestinal barrier, Han et al were able to demonstrate that proinflammatory cytokines interferon -γ, tumor necrosis factor - α and interleukin -1β could affect the expression of occludins and claudins involved in formation of tight junctions . Another study demonstrated that epidermal growth factor prevented the disruption of tight junction proteins in an injury model using Caco-2 monolayers . The importance of occludins and claudins in the formation of functional tight junctions has also been demonstrated in animal models of NEC where a positive correlation between ileal occludin mRNA levels and the progression of ileal injury was noted . Erythropoietin (Epo), a component of human milk, has been suggested to have a physiological role in the developing gut. In vitro studies undertaken by Shiou et al, demonstrated that Epo is able to reverse the effect of IFN - γ and protect ZO-1 expression and barrier function . In a rat model of NEC the same authors demonstrated that oral administration of Epo was able to reduce the incidence of NEC from 45% to 23%. If tight junctions are improperly formed or if they are damaged as a result of cytokine production in response to bacteria interacting with intestinal epithelial cells then bacteria will be able to translocate into the tissue causing some of the typical symptoms observed in NEC e.g. intramural gas. Reduced tight junction complexes have been associated with chronic inflammation in diseases such as ulcerative colitis and Crohn’s disease. In these conditions there are often more bacteria found in association with the epithelium .
It is known that signals from the bacteria colonising the gut after birth play a role in maturation of physiological, anatomical and biochemical functions of the intestinal epithelial barrier . Comparisons between conventional and gnotobiotic animals have demonstrated that the microbiota is involved in development, maintenance and repair of the intestinal mucosa [139,145]. As outlined previously, colonisation of the gut in neonates is influenced by gestation, postnatal age, environmental factors such as diet and the rearing environment, and administration of antibiotics. Cilieborg et al conclude that genetically determined gut characteristics (structure, function, immunity) and the time and mode of birth are the most crucial factors in the development of the enteric microbiota, which may be of a beneficial or harmful nature in terms of mucosal integrity .
The mechanisms by which classical enteric pathogens cross the intestinal epithelial barrier have been carefully studied as they provide putative targets for the prevention and treatment of infectious diarrhoeal diseases. Adherence is generally the beginning of the colonisation process leading to infection and, for invasive pathogens such as
There is potential for bacterial cytotoxins to assist translocation of bacteria in the gut lumen through induction of enterocyte death. In a pilot study, we investigated 53
|Number of isolates||3||30||4||16|
|Toxin titre range||4–16||8–16|
3. Maturity of the gut and the innate immune response
As described in the previous section, the extrinsic barriers in the premature gut are not fully developed. Goblet cells, found throughout the intestine, are responsible for the secretion of mucus that protects the intestinal lining as well as providing some protections and nutrients for the bacteria that colonise it. There are both secretory mucins (Muc2) and membrane bound mucins (Muc3), which are co-secreted with trefoil factors (TFFs) . The intestine can respond to injury by increasing mucin production. Resident microbes in the gut can also induce an increase in mucin production . Mucins have been implicated in cellular signalling by virtue of the fact that they are able to develop binding sites for lectins, adhesion molecules, cytokines and chemokines. In the immature gut the coverage of mucin is scanty which may facilitate bacterial adherence to the epithelial cells. Paneth cells in the small intestine are able to secrete a wide spectrum of antimicrobial peptides against bacteria, fungi and viruses. Microfold (M) cells in the intestine sample the intestinal environment and deliver antigens to more specialised lymphoid tissue. Their role in disease in the premature neonate is unknown. However, impaired production of both MUC2 and TFF3 has been reported in clinical and experimental cases of NEC . In a rat model of NEC Khailova et al demonstrated that when rats were given a probiotic bacterial strain
The innate immune system of the intestinal epithelium barrier has to be able to distinguish commensal bacteria from pathogens. Pattern recognition receptors on the intestinal epithelial barrier (transmembrane Toll-like receptors and intracellular nucleotide binding oligomerisation domain –like (NOD) receptors) have to be able to recognise microbial ligands (lipopolysaccharide, flagellin, lipotechoic acid, peptidoglycans and formylated peptides) known as microbial-associated molecular patterns (MAMPs). Depending on how the signal is perceived a number of responses can be generated - with commensal bacteria a protective response; with pathogenic bacteria an inflammatory response; or it can be a response that triggers apoptosis . Commensal bacteria can dampen TLR-mediated inflammatory signals. The nuclear factor kappa B (NFκB) transcriptional control pathway has both anti-inflammatory and pro-inflammatory roles dependent on the microbial signal received . Once the MAMP has bound to its respective TLR, the TLR triggers recruitment of the myeloid differentiation primary-response gene 88 (
The microbiota, mucin and antibacterial products such as defensins and immunoglobulins help protect the host against pathogens. In infants with NEC who have a poorly developed gut with little mucin production and an abnormal microbiota compared to full term healthy infants, the potential for bacteria to cross the intestinal barrier and initiate inflammatory disease is much greater. Once we have a better understanding of the microbial-mucosal signalling components of inflammatory pathways and the regulation of these by commensal bacteria we may be able to find new ways of preventing NEC in premature infants. Alternatively, or as well as, damage to the intestinal mucosa by enteric viruses, harmful members of the gut microbiota, acidic or toxic luminal contents are likely to facilitate translocation. Injury due to hypoxic-ischaemic events in late-onset NEC is generally considered to be transient and unlikely to directly induce translocation. However, there may be subtle effects and when severe, it could be a permissive factor .
4. The inflammatory response
The initial step in any infection is the adherence of a microorganism to a host surface. As previously discussed, this binding may trigger a number of host responses such as chemokine and cytokine release, alterations in intracellular signalling pathways and induction of apoptosis. Cytokines play an important role in the regulation of inflammation. In cases of NEC several cytokines have been identified as being associated with the disease. A number of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-18 and TNF-α), an anti-inflammatory cytokine (IL-10) and platelet activating factor (PAF) have been associated with NEC pathogenesis and neonatal sepsis in both infants and in animal models of NEC [157–159].
Pro-inflammatory cytokines can cause increased production of nitric oxide which is known to modulate various physiological processes including inflammation [88,141,]. Nitric oxide is produced from arginine by nitric oxide synthases of which there are three isoforms one of which is inducible (iNOS). This inducible isoform is expressed at high levels during inflammation and is activated by cytokines such as gamma interferon and by bacterial lipopolysaccharide [88,160]. Nitric oxide may cause damage either directly or through its toxic intermediate, ONOO- resulting in a direct cytopathic effect on the cells (apoptosis) and inhibiting enterocyte proliferation and migration so that the intestinal mucosa cannot repair itself . iNOS knockout mice have been shown to be more susceptible to infection with a number of microorganisms including
Platelet activating factor (PAF) has also been implicated in cases of NEC. This pro-inflammatory lipid mediator has been associated with intestinal mucosal injury and bowel necrosis in animal models of NEC and neonates with NEC [162,163]. A two-step enzymatic process is used to produce PAF . Experimental animal model research has suggested that both PAF and intestinal bacteria are required to cause NEC as PAF alone cannot induce experimental NEC in a rodent model in the absence of the intestinal microflora [164,165]. PAF is released in response to hypoxia, infection or local injury and Soliman et al hypothesise that this then results in up-regulation of TLR4 in the intestinal epithelium allowing excessive bacterial activation of the intestinal inflammatory response . Another group has also demonstrated that when PAF degrading enzyme is given in association with enteral feeding in a rat model of NEC that initiation of NEC is prevented .
IL-10 plays a protective role in the pathogenesis of NEC. Using wild type and IL-10 knockout mice Emami et al have demonstrated that in IL-10 deficient mice there is more evidence of epithelial apoptosis and dissociation of tight junctions compared to wild type animals . In addition, when IL-10 knockout mouse pups were treated with IL-10 or phosphate buffered saline, pups given IL-10 had a greater rate of survival than the PBS treated pups and improved intestinal villus architecture. IL-10 can suppress the secretion of pro-inflammatory cytokines such as IL-2, TNF-α and gamma interferon. This cytokine is found in human breast milk and has been postulated to be one of the protective factors preventing the development of NEC. In addition, IL-10 can suppress expression of iNOS at the mucosal level in macrophages . As described above, high levels of iNOS have been associated with cases of NEC. Lee and Chau have demonstrated that the enzyme heme-oxygenase -1 is induced by IL-10 and that this enzyme is required to mediate the action of IL-10 both in vitro and in vivo. When an HO inhibitor was given to mice, IL-10 mediated protection against LPS-induced septic shock was decreased . IL-10 protection appears to be the result of down regulation of iNOS expression leading to less damage of the intestinal surface. However, if levels of HO-1 are reduced or absent IL-10 is not able to control iNOS expression. Further evidence for the role of NO in NEC has been provided by Ford et al who examined levels of inflammatory cytokines and NO in samples of intestine obtained from infants undergoing surgical resection for NEC and who demonstrated that NO was produced in large amounts by enterocytes in the intestinal wall leading to apoptosis of enterocytes in apical villi through peroxynitrite formation .
4.1. Signaling pathways
One of the newer approaches to understanding the pathology of NEC has been at a molecular level and examines the relationship between the intestinal epithelium and commensal bacteria. This research has identified a class of bacterial receptors known as Toll-like receptors (TLRs), in particular TLR 4, whose ability to respond to bacteria associated with the intestinal epithelium may in part explain why some premature infants are susceptible to NEC. More than ten TLRs have so far been identified in humans . These receptors form part of the innate immune response and interact with different components of bacteria and viruses. TLR4, for example, is known to be the receptor for bacterial lipopolysaccharide. As NEC has often been shown to develop after gut colonisation with Gram-negative strains of bacteria, a putative role for TLR4 in the pathogenesis of this disease has been suggested. Mice with mutations in TLR4 or lacking TLR4 do not develop NEC [24,165]. Activation of enterocyte TLR4 leads to increased death of cells in the intestine through the mechanism of apoptosis . TLR4 activation results in stimulation of IL-1R kinase via adaptor molecules MyD88 and MD2 resulting in activation through NFκB and up-regulation of pro-inflammatory cytokines . Gribar et al have demonstrated that TLR4 expression is higher during gestation in the mouse and falls off shortly before birth . They have also postulated a link between TLR4 levels and TLR9 levels in the pathogenesis of NEC. TLR9 recognises bacterial DNA as opposed to LPS recognised by TLR4. TLR4 levels are increased in the bowel of infants with NEC compared to control bowel samples . In a mouse model of NEC, Leaphart et al demonstrated that physiological stressors such as hypoxia and LPS associated with the development of NEC sensitise the epithelium to LPS through the up-regulation of TLR4 and that if animals had a mutation in TLR4 the severity of NEC was reduced due to increased healing capacity of the epithelium . Thus the effect of TLR4 appears to be two-fold by promoting damage to the small intestine through up-regulation of inflammatory cytokines and in reducing mucosal repair. As reported in the last section, there is also a known relationship between TLR4 up-regulation and PAF, one of the molecules implicated in the pathogenesis of NEC.
The relationship between TLR4 and TLR 9 and the signalling from both these receptors has an important role to play in the development of NEC . In cases of NEC, in both humans and mice, increased TLR4 and decreased TLR9 expression was measured. TLR9 recognises CpG motifs of bacterial DNA. Bacterial DNA differs from human DNA in that is enriched with CpG motifs and is largely unmethylated . Using a murine model of NEC, Gibrar et al demonstrated that NEC occurs when there is increased TLR4 and decreased TLR9 expression in developing intestinal mucosa. Furthermore, they were able to show that activation of TLR9 with CpG-DNA inhibited TLR4 mediated signalling in enterocytes. The mechanism of inhibition was dependent upon the inhibitory signalling molecule IL-1R kinase. When CpG-DNA was administered to newborn mice the incidence of experimental NEC was significantly reduced. Other studies have also implicated TLR2 in the pathogenesis of NEC. TLR2 mRNA expression was increased along with TLR4 mRNA and activated NFκB in a neonatal rat model of NEC 48 hours prior to lesions being identified histologically [171,172]. TLR2 mediates host response to Gram-positive bacteria and yeasts through the NFκB pathway .
Signalling pathways of the innate immune system therefore play an important role in the development of NEC. Understanding of the interactions in this system may lead to the development of new therapeutic treatments for NEC e.g. probiotics with known effects on signalling pathways, anti-inflammatory molecules.
4.2. Cell damage
When pieces of bowel are removed during surgery for NEC and examined histologically, a large number of apoptotic nuclei signifying programmed cell death are observed in the tissues . Studies using an animal model of NEC have demonstrated that apoptosis occurs prior to gross histological damage. When apoptosis was prevented using caspase inhibitors, the development of NEC was significantly reduced . Also, the neonatal pathogen
5. Hypothetical model of NEC
NEC is a complex disease influenced by several risk factors that may act alone and together. The innate immune system appears to play a large role in the establishment of NEC. We propose that in the preterm infant susceptible to NEC, scanty mucus production allows interaction between commensal bacteria and TLR triggering the innate immune response leading to loosening of intercellular tight junctions, enterocyte apoptosis and necrosis, resulting in translocation of bacteria (Figure 2a). Additional factors may also increase translocation, including infection with enteric viruses, presence of microbial cytotoxins or other toxic substances, and a bloom of certain bacteria in the gut lumen causing increased phagocytic uptake by enterocytes. In comparison, the intestinal epithelium of the healthy term neonate secretes adequate mucus, trapping enteric bacteria in the upper layers. In addition, the presence of sIgA and other antimicrobial substance inhibit bacterial colonisation of enterocytes, and any commensal bacteria that do adhere are recognised as not harmful, dampening TLR-mediated inflammatory signals (Figure 2b). In essence, a pivotal event in the development of NEC may be whether the innate immune system of the preterm infant intestine views bacteria reaching the enterocyte surface as friend or foe.
Microbial succession ensures that the intestines of healthy neonates are readily colonised with probiotic bacteria such as
Trials of probiotics in preterm infants have had varying results. Those reporting a reduction in NEC have often employed
Cario et al demonstrated that TLR -2 was able to control mucosal inflammation in both in vivo and in vitro models by preserving TJ associated barrier assembly against stress-induced damage via MyD88. When colitis was induced in wild type mice using dextran sodium sulphate followed by treatment with a TLR-2 agonist, clinical signs of colitis were abrogated in all animals when compared to mice which didn’t undergo treatment . The probiotic
Whether probiotic therapy in preterm infants should include supplementation with prebiotics is open to question. Prebiotics promoting the growth of bifidobacteria and lactobacilli are naturally present as oligosaccharides in human milk . The few existing trials of prebiotics in preterm infant have indicated an increase in stool counts of bifidobacteria and lactobacilli occurs. However, until more data is available, routine feed supplementation with prebiotics or synbiotics (probiotic, prebiotic mixtures) is not recommended .
7. Future directions
An article rather pessimistically entitled ‘Necrotizing enterocolitis – 150 years of fruitless search for the cause’ was published in 2011 . We believe the search has been fruitful and a fuller picture of NEC is now beginning to emerge. NEC is a far more complex disease than early researchers anticipated, involving intrinsic gastrointestinal barriers, the innate immune response, signalling pathways and bacterial colonisation patterns. It seems likely that in individual cases of NEC there will be differences in both the aetiology and pathogenesis. Research into this disease will always be hampered by the fragility and vulnerability of the patients and ethical considerations, leading to a greater dependence on in vitro and animal model experimentation than is usual. Although necessary, antibiotic therapy is often a confounding factor in NEC research because it may eliminate the microbes that initiated the infection. Prospective studies represent a promising avenue of research especially when combined with DNA or RNA based methods for studying changes in the enteric microbiota, initiation of the inflammatory pathway and cell signalling. Through increasing our understanding of NEC, new targets for intervention will be identified. Further investigation of the mechanisms of action of probiotic bacteria will hopefully identify the ideal probiotic for NEC prevention. There is every reason not to be pessimistic about our ability to treat, and more importantly to prevent, this potentially devastating disease of preterm infants in the future.