Neonatal Necrotizing Enterocolitis –Inflammation and Intestinal Immaturity

Abstract

Neonatal necrotizing enterocolitis is a devastating inflammatory bowel disease of premature infants. The pathogenesis remains incompletely understood and there is no specific treatment. Efforts are ongoing to understand aspects of intestinal immaturity which contribute to susceptibility to this disease. This review focuses on bacterial colonization patterns, intestinal barrier function, and inflammatory responses of immature enterocytes leading to a unique vulnerability of the preterm gut. In addition the possible therapeutic potential of factors in human milk and probiotic bacteria is discussed.

INTRODUCTION

Premature infants, defined as infants born at less than 37 weeks gestational age, account for over 12% of deliveries in the United States [1]. These infants have many acute problems, including fluid and electrolyte imbalance, impaired oxygenation associated with lung immaturity, and increased risk of sepsis all due to the premature assumption of roles expected to be performed by maternal circulation and the protective intra-uterine environment of fetal life. However, there is evidence that many of the long-term problems that account for much of the morbidity of prematurity are inflammatory in nature. The cytokine balance for preterm infants appears skewed toward inflammation [2]. Exposure to increased inflammatory cytokines comes from both maternal and infant sources, and risk for inflammatory conditions such as chronic lung disease, periventricular leukomalacia, cerebral palsy and necrotizing enterocolitis may be increased by maternal chorioamnionitis, postnatal sepsis, and intensive care unit interventions which increase the production of pro-inflammatory cytokines in preterm infants known to have lower levels of anti-inflammatory cytokines such as IL-10 [3–5]. This review will focus on the disease necrotizing enterocolitis (NEC) as an inflammatory disorder of the preterm infant.

NECROTIZING ENTEROCOLITIS

NEC is an inflammatory bowel necrosis that primarily afflicts premature infants after the initiation of enteral feeds. NEC affects about 10% of premature infants <1500grams, or 1–5% of all neonatal intensive care unit admissions. It characteristically strikes between 7 and 14 days of life, although, increasingly NEC has been documented several weeks after birth, particularly in very low birth weight infants [6–8]. Susceptibility to NEC is inversely related to gestational age [9]. Thus, more immature infants are more likely to get this disease and are at risk for a longer window of time suggesting that degree of immaturity plays an important role. While full term infants can develop a similar clinical picture, these infants generally have specific underlying risk factors for intestinal perfusion compromise such as birth asphyxia, polycythemia, exchange transfusion, intrauterine growth restriction, or cyanotic congenital heart disease. In these patients NEC often presents within the first days of life. Thus the pathophysiology in full term infants may be quite different from that in premature infants and will not be the focus of this review. There does not appear to be a sex or race predilection.

NEC presents with variable symptoms which are often non-specific signs of gastrointestinal dysfunction. These signs include abdominal distension, feeding intolerance, gastric aspirates, bilious vomiting and hematochezia; with sudden progression to pneumoperitoneum and/or systemic shock and rapid death in severe cases. The pathognomonic feature is pneumatosis intestinalis on abdominal radiograph which represents air tracking within the bowel wall as a result of bacterial fermentation of intraluminal substrates.

NEC classically affects the terminal ileum. The histopathologic landmark is coagulation necrosis indicating prior ischemia, with a combination of ischemic necrosis, acute and chronic inflammation, bacterial overgrowth, and tissue repair suggesting that NEC is an evolving process rather than an acute event [10, 11]. This disease can have focal, segmental, and non-continuous lesions. Inflammation can be limited to the mucosa and submucosa of the intestine, or progress to transmural involvement in the most severe cases. In addition, lesions may include submucosal or subserosal collections of gas which is what is seen as pneumatosis on abdominal radiograph.

Although numerous studies have been done on animals and humans and many theories have been proposed, the cellular basis of this disease remains poorly understood. Treatment is currently merely supportive and includes full bowel rest with immediate cessation of enteral feeds, institution of nasogastric suction, fluid resuscitation, close monitoring of acid/base and electrolyte balance, broad spectrum parenteral antibiotics, parenteral nutrition and frequent abdominal radiographs to document progression to pneumoperitoneum. Surgery is indicated for pneumoperitoneum, peritonitis, and intestinal obstruction. However, current treatment modalities are often inadequate because of the rapid progression of NEC from its initial diagnosis. In particular, the outcomes for these infants are very concerning. There is a high mortality rate recently documented at 16–42% depending on birth-weight, along with risk of intestinal stricture, short-gut syndrome, and the complications of difficulty providing adequate nutrition and parenteral nutrition-induced cholestasis [9]. Furthermore, studies have shown that NEC, particularly NEC requiring surgery, is an independent risk factor for poor neurodevelopmental outcome [12].

The primary risk factors for NEC are prematurity, bacterial colonization, hypoxia/altered intestinal blood flow, and enteral feeding [13]. Prematurity with its associated immature GI host and blood flow regulation results in mucosal injury. Combined with enteral feeds and bacterial colonization, inflammatory mediators are released leading to a propagated inflammatory response with both pro and anti inflammatory influences. If counter-regulatory responses to these inflammatory events are insufficient, pathologic changes to gut mucosa occur. In the preterm infant the balance appears to favor pro inflammatory influences resulting in NEC.

SUSCEPTIBILITY OF THE IMMATURE INTESTINE

It has been hypothesized that the injury in NEC begins with a breach in the intestinal mucosal barrier leading to bacterial translocation across the epithelium, and exacerbation of the inflammatory cascade, resulting in the clinical signs of NEC. Thus to understand the intestinal inflammation progression, this review will focus on bacterial colonization patterns in preterm infants, means by which bacteria breach the intestinal barrier, and the inflammatory response of the immature enterocyte to these bacteria.

Bacterial Colonization and the Preterm Gut

As opposed to the adult intestinal microbiota which is comprised of more than 10¹³ microorganisms, the newborn gut is sterile at birth. Furthermore, colonization of the blank slate of the preterm intestine is influenced by iatrogenic manipulations in the neonatal intensive care unit (NICU). This includes a hospital environment; frequent use of broad spectrum antibiotics, opioids, and H2 blockers; and instrumentation with endotracheal tubes, feeding tubes, and suctioning tubes. Resulting altered microbial flora may have significant implications for development of the immature preterm gut and susceptibility to NEC.

Bacteria are believed to be important in the pathogenesis of NEC; however no specific pathogen has been identified. Previous studies have been limited by the inability of conventional microbiological cultivation techniques to thoroughly characterize the human gastrointestinal microbiota. It has been reported that 80% of the human colonic microbiota are not detected by conventional culture methods [14]. Molecular profiling of microbiomes are now possible by sequencing the highly conserved 16S small subunit bacterial ribosomal RNA gene (rRNA), allowing identification of previously undetectable microbes [15]. This approach therefore provides a more complete picture of the composition of human intestinal microbiota.

Sequencing of the intestinal microbiome of infants with and without NEC has demonstrated that sequences from NEC patients cluster separately from sequences from control patients, even between genetically identical twin pairs [16]. A specific pathogen has still not been identified, rather, the microbial community structure in NEC patients is distinct based on a significant decrease in diversity of microbial species with an increase in Proteobacteria dominance compared to other preterm infants, and a specific bloom of a single genus of Proteobacteria to >50% of the overall bacterial composition [16]. This data suggests that while bacteria are important in the pathogenesis of NEC, NEC does not appear to be an infection in classic sense. A specific organism is not causal, but rather a trigger for a rapid inflammatory cascade in the preterm gut, leading to signs and symptoms of disease. There are 2 sides to this interaction – the microbe and the host.

Host Defense in the Preterm Infant

The preterm gut is essentially a fetal gut, expecting conditions of the intrauterine environment. This includes absence of bacteria. Preterm infants may not yet be prepared for bacterial interaction when initially colonized and fed, potentially placing them at higher risk for NEC.

The mature intestine has many physical barriers to bacteria including peristalsis, gastric acid, proteolytic enzymes, intestinal mucus, cell surface glycoconjugates, and tight junctions between intestinal epithelial cells. These are designed to limit bacteria to the gut lumen and prevent attachment and translocation across the intestinal epithelium. Animal studies have shown that pathogenic organisms adhere to and translocate across the intestine to a greater extent in immature vs. mature animals Fig. (1). Abnormal peristaltic activity in these infants may increase bacterial adherence, allowing for bacterial overgrowth that could increase endotoxin exposure and predispose the infant to NEC [17–19]. Cell surface glycoconjugates which serve as adhesion sites for a variety of microbes, have a different pattern of carbohydrate residues in the immature compared to the adult intestine, which may result in increased pathogenic colonization in preterm infants [20–22]. Furthermore, it is known that intestinal mucus, which protects against bacterial and toxin invasion, is different in developing animals and perhaps in premature infants in terms of carbohydrate composition, density and possibly in inclusion of secretory immunoglobulin [23].

Fig. 1.

Aspects of immature intestinal host defense in the premature infant that may contribute to susceptibility to necrotizing enterocolitis. Reproduced with permission [146].

In addition, there is immaturity of the functional barrier that limits growth of bacteria that breach the physical barrier. This functional barrier is comprised of the immunologic host defense and various biochemical factors. It is known that numbers of intestinal B and T lymphocytes are decreased in neonates and do not approach adult levels until 3–4 weeks of life. Newborns also have reduced levels of secretory IgA in salivary samples, presumably reflecting decreased activity in the intestine [24–26]. Additionally, the premature infant has lower gastric acid production compared to older children, and immature proteolytic enzyme activity may lead to incomplete breakdown of toxins [24]. Finally, key proteins secreted from the intestinal epithelium such as intestinal trefoil factor are developmentally regulated and deficient in premature infants [27–29] while human defensins (or cryptidins) synthesized and secreted from paneth cells to protect against bacterial translocation are also altered in premature infants and those with NEC [30, 31]. All of these issues increase the likelihood that bacteria will interact with the immature enterocyte. A premature infant has a potentially more pathogenic bacterial balance and an immature gut defense to shield against bacteria, which collectively increases the risk for NEC.

Intestinal Barrier Disruption

A unique characteristic of the gastrointestinal system is its lining by a single epithelial layer in continuous contact with intestinal bacterial flora. This layer is a key host defense mechanism critical for confining pathogenic bacteria to the intestinal lumen, but must also allow passage of nutrients. Preterm infants have increased intestinal permeability, perhaps to allow expected passage of important macromolecules from amniotic fluid or breast milk [32]. However, this same increased permeability could lead to increased bacterial translocation. The inflammatory response of immature intestinal epithelial cells can be triggered by either commensal or pathogenic bacteria [33]. Disruption of the intestinal epithelial barrier increases this interaction and is thought to be an early event in the pathogenic cascade of NEC.

A breach in the mucosal barrier could be caused by disruption of the tight junctions between epithelial cells. The intestinal barrier normally consists of intestinal epithelial cells (IEC) connected by a system of both intracellular (zonula occludin proteins ZO-1,ZO-2, and ZO-3) and membrane-spanning (occludin, junction adhesion molecule (JAM) and the claudin family) proteins [34]. The epithelial tight junction (TJ) forms a selectively permeable barrier which allows fluids and solutes to cross while maintaining a protective barrier against other contents of the intestinal lumen. Occludin and claudin-3 have specifically shown to be altered in animal models of NEC [35].

Another means of barrier disruption is destruction of the cells themselves. One means of cell destruction is apoptosis, or programmed cell death. Apoptosis is a process of removing damaged cells characterized by cell shrinkage, chromatin condensation, and DNA fragmentation [36]. Caspase proteases are executors of apoptotic cell death. Caspase activation regulates endonucleases, resulting in cleavage of internucleosomal DNA and cell death [37]). In addition, there are caspase independent pathways in which proteases other than caspases initiate chromatin cleavage [38]. While apoptosis is a normal aspect of enterocyte turnover, accelerated apoptosis may lead to a breach in the critical intestinal mucosal barrier. Studies in animal models have demonstrated that apoptosis precedes necrosis in NEC, and that inhibition of apoptosis can decrease incidence of disease [39].

Apoptosis can be triggered by both intrinsic and extrinsic pathways [40]. In the intrinsic pathway, cell stress leads to binding of cytoplasmic proapoptotic proteins such as Bcl-2 family members BAX and BID to the mitochondria. This results in collapse of the mitochondrial potential. Cytochrome C is then released from mitochondria and binds to apoptotic protease activating factor 1 resulting in formation of the apoptosome. Exposure of the caspase recruitment domain recruits caspase -9 resulting in ultimate activation of caspase 3 and DNA fragmentation resulting in cell death. In the extrinsic pathway, binding of Fas ligand or tumor necrosis factor (TNF) to its receptor results in the recruitment of Fas associated death domain [41] proteins. Subsequent recruitment of caspase 8 results in the formation of the death inducing signal complex (DISC). This complex can directly activate caspase 3 in a mitochondria independent fashion, or act through BID to induce mitochondrial release of cytochrome C. Apoptosis can also result from DNA fragmentation induced by sustained calcium increases which act through activation of a Ca^2+/Mg²⁺ endonuclease or intracellular acidosis which can enhance activation of caspases and activate of pH sensitive endonucleases [42, 43].

Platelet activating factor (PAF) is a phospholipid inter- and intra-cellular mediator which has been implicated in the pathology of inflammatory bowel disease and shown to induce intestinal epithelial cell apoptosis. However, the mechanism of PAF induced intestinal injury is incompletely understood. It is known that tissue and/or serum PAF levels are elevated in patients with Crohn’s disease, ulcerative colitis, and NEC and levels appear to correlate with disease severity [44–47]. In animal models of NEC, PAF receptor blockade or administration of the PAF-degrading enzyme PAF acetylhydrolase reduced the incidence of experimental NEC [48]. Moreover, in human studies of NEC, PAF has been shown to rise several days before the onset of clinical symptoms in some patients suggesting that this is a critical, and possibly initiating, factor in the development of the disease [49]. PAF levels have been shown to be increased by both hypoxia and enteral feeds, some of the specific risk factors implicated in NEC [50, 51].

Regulation begins at the receptor level where PAF acts through binding to a seven transmembrane domain G protein coupled receptor. Both high and low affinity PAF receptors have been identified [52]. Multiple signaling cascades are then linked to the PAF receptor. Depending on the route of administration and the animal model used, PAF can induce a variety of effects including platelet aggregation, hypotension, increased vascular permeability, vasoconstriction, intestinal ischemia, neutrophil recruitment, and production of reactive oxygen species [53]. In the intestine it is thought that PAF, once activated, initiates production of other inflammatory mediators such as TNFα, prostaglandins, thromboxane, and complement that then lead to the clinical signs and symptoms of inflammatory bowel disease [54–56]. However, several lines of evidence suggest that the pathologic consequences of elevated PAF levels in the intestine are not only the result of the initiation of an inflammatory cascade, but that PAF itself has direct effects on intestinal epithelial cells.

In isolated IEC lines and an animal model of NEC, PAF has been shown to induce apoptosis via three mechanisms [39, 57]. PAF induces translocation of BAX to mitochondria resulting in loss of mitochondrial membrane potential and caspase activation, an effect blocked by the anti-apoptotic protein Bcl2 [57]. PAF also inhibits the phosphatidylinositol 3-kinase/protein kinase B Akt signaling pathway [58]. Lastly, PAF has been shown to induce intracellular acidosis via activation of the Ca²⁺ dependent Cl⁻ channel ClC-3 resulting in apoptosis of IEC [59].

Studies of intestinal tissue of preterm human infants with NEC have noted additional markers to give clues about the mechanism of altered barrier function. These studies have demonstrated upregulation of inducible nitric oxide which correlated with the degree of intestinal injury and number of apoptotic nuclei, Local intestinal production of nitric oxide (NO) induced by cytokines leads to intestinal apoptosis, potentially exacerbating intestinal injury [60]. In addition increased levels of high mobility group box 1 (HMGB1) protein have been found, which is released by macrophages and necrotic cells and is associated with increased barrier dysfunction and bacterial translocation [61].

Intestinal Inflammation and NEC

The necrosis which follows apoptosis results from progression of the triggered inflammatory cascade. IL-8 is a chemokine that stimulates migration of neutrophils from intravascular to interstitial sites and can directly activate neutrophils and regulate the expression of neutrophil adhesion molecules [62–64]. Thus by recruiting and activating immune cells, IL-8 may play an important role in inflammation. Previous studies have shown that concentrations of serum IL-8 were significantly elevated in severe cases of NEC from its onset through the first 24 hours [65]. Surgical specimens of intestine from infants with acute NEC show upregulation of IL-8 mRNA throughout the serosa, muscularis, and intestinal epithelium compared to those with other inflammatory conditions or those without disease [66]. Studies also demonstrate increased serum levels of the pro-inflammatory cytokines IL-6 and TNFα in infants with NEC [51, 67, 68].

Preterm infants not only have altered bacterial colonization and immature host defense, but also an immature response to bacteria. It is potentially an important developmental step for enterocytes to decrease inflammatory responsiveness in order to prevent immune defense mechanisms against normal flora. Preterm infants may not have completed this maturation when initially fed and colonized by bacteria. Evidence suggests that the premature neonate may be predisposed to intestinal inflammation. Studies have demonstrated that compared to adult IEC, human fetal IEC have an exaggerated production of IL-8 in response to both pathogenic and commensal bacteria as well as to endogenous inflammatory mediators such as TNFα and IL-1γ [69, 70].

Toll-like receptors (TLR) are a highly conserved family of pathogen associated molecular pattern (PAMP) receptors which recognize bacterial components. TLR4 specifically recognizes the bacterial cell wall component lipopolysaccharide (LPS). Interestingly, TLR4 expression decreases after birth in the intestines of healthy mother fed rat pups, but increases in the intestinal epithelium when pups are exposed to stresses common to preterm infants such as formula feeding and asphyxia [71]. Furthermore, animal models of NEC have demonstrated that TLR4 mutant mice were protected from NEC [71].

TLR activation, as well as TNFα and IL-1β can activate nuclear factor kappaB (NF-κB) proteins which lead to transcription of a wide variety of genes important in inflammatory and immune responses Fig. (2). In its resting state NF-κB dimers are bound in the cytoplasm to inhibitory κB (IκB) proteins [72]. Cell stimulation can trigger signaling pathways leading to phosphorylation, ubiquitination, and ultimately degradation of IκB by the 26S proteasome [73, 74]. NF-κB, thus liberated, moves to the nucleus where it activates gene transcription. Interestingly, NF-κB binding sites are located on the inhibitory IκBα promoter. Thus an elegant autoregulatory feedback loop exists in which NF-κB activation leads to IκBα synthesis to terminate the NF-κB response [75, 76]. However, in vitro and in vivo studies have shown that immature enterocytes have increased NF-κB activity associated with decreased baseline expression of IκB isoforms [33]. In addition, inflammatory stimuli trigger increased phosphorylation, ubiquitination, leading to increased degradation of IκBα and prolonged NF-κB activation in immature enterocytes compared to adult enterocytes [33, 77]. Differences in IκB levels appear to be key as increasing expression of IκBα by transfection in immature intestinal epithelial cells attenuated transcriptional up-regulation of inflammatory cytokines, and suggest that developmentally-regulated differences in IκB could contribute to the differential responsiveness of immature and mature enterocytes to inflammatory stimuli [33]. A critical balance between NF-κB activation and inhibition is important to allow appropriate defense against pathogens yet avoid host damaging inflammatory responses to commensal flora.

Fig. 2.

Nuclear factor kappa B signaling cascade. Asterisks denote steps with differential response to inflammatory stimuli in immature and mature intestinal epithelial cells.

POTENTIAL THERAPEUTIC OPTIONS FOR LIMITING INFLAMMATION IN NEC

NEC is a multi-factorial disease, leading to intestinal inflammation resulting in the signs and symptoms of disease. Many studies have investigated potential therapeutic agents to specifically limit the inflammatory cascade. Single studies in animal models of NEC have demonstrated the ability of various compounds to inhibit intestinal inflammatory injury when given prior to intestinal insult. These have included compounds to decrease NF-κB activation such as the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IκB kinase [78] within the NF-κB pathway and the peroxisome proliferation receptor γ (PPAR-γ) agonist 15d-PGJ₂ [79, 80]. PPARγ has been shown to directly associate with the nuclear NF-κB p65 protein, promoting its export from the nucleus, thus limiting its duration of action [81, 82]. The macrophage deactivator semapimod [61], decreased incidence of NEC potentially via limitation of production of pro-inflammatory mediators such as HMGB-1. Pentoxyphylline which inhibits the effects of the pro-inflammatory cytokine TNFα has also been shown to decrease the incidence of NEC [83]. While these studies add insight to mechanism of intestinal injury, therapeutic potential is unclear. These compounds, which inhibit fundamental pathways important in many tissues besides the intestine, raise concerns for unwanted side effects or collateral damage in the developing gut. Furthermore, studies have noted efficacy with administration prior to intestinal insult, which may be difficult to determine in the clinical setting of a disease without a discrete point of injury.

As previously described, factors contributing to the development of intestinal inflammation include a pathogenic bacterial colonization balance, disruption of the intestinal barrier, and immature regulation of NF-κB signaling. Targeting these deficiencies may allow prevention of disease or limitation of injury prior to onset of clinical symptoms. Two areas of promise are human milk and probiotics.

Human milk has been shown to be protective against NEC. A clinical study by Lucas and Cole demonstrated a 10–20 fold reduced incidence of NEC in human milk fed preterm infants compared to formula fed infants [84]. Recent studies suggest a dose-related association between human milk feeding and decreased incidence of NEC [85]. Many of the same factors present in amniotic fluid are present in human breast milk, and vary in amount with changing gestational age of the infant. Human milk may provide necessary factors for the preterm infant in the extra-uterine environment in the same way that amniotic fluid provides for the fetus in the intra-uterine environment [86].

Probiotics are living microorganisms in food and dietary supplements which have beneficial health effects beyond their inherent nutritive value. Probiotics are most commonly Lactobacillus or Bifidobacterium strains, which are the dominant species in the intestinal microbiota of breast fed infants. Since bacteria are necessary for maturation of the intestine and appropriate containment of inflammatory responses. It may be important to influence bacterial colonization. Health may not hinge on the presence of bad vs. good bacteria but the creation of optimal bacterial communities versus pathogenic bacterial communities. Thus for a premature infant it has been suggested that optimizing the bacterial flora can enhance intestinal maturation and decrease the incidence of NEC [87]. Probiotics have been shown to be beneficial in treating inflammatory bowel diseases, rotavirus and antibiotic associated diarrhea, and C. difficile colitis [88–91].

Four recent clinical studies have suggested a beneficial effect of probiotics on the incidence of necrotizing enterocolitis. Hoyos et al. administered Infloran Berna7 containing a mixture of Lactobacillus acidophilus and Bifidobacterium infantis to all newborns admitted to the neonatal intensive care unit at Hospital Simon Bolivar in Colombia and compared incidence of NEC in this cohort to the incidence of NEC in historical controls from the previous year [92]. This study found a significantly lower incidence of NEC – 2.9% in the probiotic treated infants vs. 6.6 % in historical controls, and no adverse events. Lin et al. also evaluated the efficacy of the probiotic product Infloran in a prospective, masked randomized control trial of 367 infants <1500gm at China Medical University Hospital in Taiwan and documented decreased incidence of NEC (≥ Bell stage 2) from 5.3 to 1.1% (P = 0.04) with no reported adverse effects [93]. This trial was recently repeated as a prospective, blinded, randomized, multi-center controlled trial at 7 neonatal intensive care units in Taiwan and enrolled 434 infants <1500gm [94]. Patients received Infloran with a new formulation containing a combination of L. Acidophilus 10⁹ cfu and Bifidobacterium bifidum 10⁹ cfu and incidence of NEC (≥ Bell stage 2) was again decreased from 6.5% to 1.8% in the probiotic treatment group with no reported adverse events. Last, Binnun et al. conducted a trial at the Shaare Zedek Medical Center in Israel. 145 infants <1500gm were enrolled in a prospective, randomized, double blind study of ABC Dophilus containing B. infantis, Streptococcus thermophilus, and Bifidobacterium bifidus [95]. This study demonstrated a significant decrease in incidence of NEC (≥ Bell stage 2) from 14% to 1% (P=0.013) with no adverse effects.

Both human milk and probiotics have potential to influence many of the features of NEC pathogenesis including bacterial colonization and translocation, intestinal barrier dysfunction, and the intestinal inflammatory response.

Optimizing Bacterial Colonization

Many factors contribute to the acquisition of intestinal flora including mode of delivery, gestational age, exposure to antibiotics in either the mother or the infants, and importantly, feeding. In breast fed infants, Bifidobacterium is a primary organism with Lactobacillus and Streptococcus as minor components. In formula-fed infants, similar amounts of Bacteroides and Bifidobacterium are found with minor components of the more pathogenic species Staphylococcus, Escherichia coli, and Clostridia [96–100]. As previously noted, oligosaccharides on the surface of intestinal epithelial cells function as binding sites for bacteria, and have different carbohydrate residue patterns in immature vs. mature intestine, potentially contributing to a different adherent bacterial profile in preterm infants. Human milk also contains free and conjugated oligosaccharides known as glycans, which protect breast fed infants against infection by functioning as soluble receptors to inhibit pathogens from adhering to the gastrointestinal mucosa [101]. Human milk contains many other factors which may influence bacterial colonization including secretory IgA which can compensate for the decreased intestinal IgA in immature gut and the antimicrobial factors lysozyme and lactoferrin. [102–104]. Human milk has even been described as “Bifidogenic” - a reference to a complex interaction of proteins and growth factors which appear to function as prebiotics to promote the specific growth of beneficial Bifidobacterium species [105].

Probiotics may directly influence bacterial colonization patterns. Necessary conditions of care of the preterm infant such as frequent use of broad spectrum antibiotics, instrumentation, and formula feeding may predispose to altered bacterial balance. It has been suggested that probiotic treatment may improve this balance. Multiple studies have shown that different organisms have unique properties, and even different strains of the same organism may behave differently [106–108]. Probiotics may influence bacterial colonization patterns via the production of ammonia, hydrogen peroxide, and bacteriocins which directly inhibit the growth of pathogenic bacteria [109–114].

Strengthening of the Intestinal Barrier

Disruption of the intestinal barrier is a known aspect of NEC thought to lead to bacterial translocation, thus increasing inflammatory stimuli and furthering intestinal injury. Both disruption of the tight junctions between IEC as well as destruction of the cells themselves via direct oxidant injury, apoptosis, or necrosis have been linked to disruption of this key host defense mechanism. Several factors in breast milk and probiotics bacteria have demonstrated specific ability to improve barrier function.

Epidermal growth factor (EGF) is a heat and acid stable protein found in both amniotic fluid and breast milk in amounts that vary with gestational age [115]. It has been shown to have many trophic effects on the intestine. EGF receptors are present throughout the gastrointestinal tract on the basolateral membrane, which may be more accessible in the preterm intestine with increased permeability [116, 117]. It promotes intestinal growth, stimulates intestinal repair, and protects against intestinal epithelial cell apoptosis [118, 119]. EGF levels are decreased in the saliva and serum of patients with NEC versus nondiseased age matched controls. Animal studies have shown a decreased incidence and severity of NEC in a rat model treated with EGF associated with normalization of occludin and claudin 3 tight junction proteins [120, 121]. There has also been a case report of an infant with enterocolitis improving after treatment with intravenous EGF along with reports of improved intestinal repair measured in rectal biopsy samples of preterm infants with NEC receiving recombinant EGF [122, 115]. Heparin-binding EGF is another growth factor in the EGF family also found in both breast milk and amniotic fluid and upregulated in response to tissue damage. It has been shown to protect intestinal barrier function, protect against proinflammatory cytokine induced apoptosis, and induce intestinal barrier restitution following injury. In addition it has specifically been shown to decrease the incidence of NEC in animal models [123, 124].

Erythropoietin (Epo) is a glycoprotein originally described as a hormone important for red blood cell production. However, it is also present in both breast milk and amniotic fluid. Epo receptors have been demonstrated on human fetal and postnatal intestine. Enterally administered Epo appears to have limited systemic absorption, but rather acts locally [125]. It has been shown to increase small bowel length and villus surface area and decrease TNFα induced IEC apoptosis [126]. Interestingly a retrospective study of preterm infants showed a decreased incidence of NEC in infants given Epo compared to infants who did not receive Epo [127].

Glutamine is found in increasing amounts throughout lactation in human milk. It has been shown to maintain intestinal mucosal integrity, and glutamine deprivation has been shown in in vitro models to increase bacterial translocation. Studies have further specifically found decreases in the tight junction proteins claudin-1, occludin, and ZO-1 under conditions of glutamine deprivation [128].

Probiotics can also improve intestinal barrier function. The immature gut is “leaky” compared to mature IEC as demonstrated by permeability studies. One means of evaluating barrier function is measuring transepithelial resistance (TER). Studies have demonstrated that when intestinal cells are treated with commensal bacteria such as E. coli, Streptococcus thermophilus, Lactobacillus, and Bifidobacterium, TER increases, while treatment with pathogenic Salmonella decreases TER [129]. The specific combination of live Streptococcus thermophilus and Lactobacillus acidophilus has been shown in cell culture models to improve TER, associated with maintenance of tight junction protein phosphorylation [130]. Utilizing a model of directly measuring intestinal permeability to dextran or mannitol in the presence of different bacteria, it was similarly found that colonization with E. coli, Klebsiella, or Streptococcus viridans increased permeability while colonization with Lactobacillus brevis decreased permeability [131]. This has clinical relevance as a clinical study in infants demonstrated that when intestinal flora was manipulated to increase levels of anaerobic bacteria including Bifidobacterium, there were lower levels of serum endotoxin, suggesting decreased translocation of endotoxin producing organisms [132]. Furthermore, Ussing chamber experiments of transport of intact proteins, have demonstrated that Lactobacillus GG can specifically protect against increased intestinal permeability in suckling rats [133]. L. plantarum has also been shown to protect against E. coli induced intestinal permeability [134].

Modulation of the Inflammatory Response

Lastly, human milk and probiotics contain factors that can be viewed as anti inflammatory. Human milk specifically contains soluble TNF receptor, IL-1 receptor antagonist, and the PAF degrading enzyme acetylhydrolase [135]. In addition transforming growth factor beta and Epo in human milk have been shown to dampen the exaggerated IL-8 production of immature enterocytes exposed to TNFα or IL-1β [69].

Some probiotics also possess anti-inflammatory properties and specifically inhibit activity of NF-κB [136–138]. The previously cited clinical studies of probiotics and NEC were not sufficiently powered to fully assess the safety of administration of live bacteria to preterm infants. Thus, despite potential biologic plausibility for the use of probiotics in the treatment of NEC, safety is a primary concern. Live probiotic bacteria have the potential to become pathogenic when host defenses are compromised [139–142]. Studies reporting increased infant mortality in probiotic-treated animal models have raised questions as to the prudence of using live bacteria in relatively immunocompromised premature infants [143].

While some protective effects of probiotics require direct bacterial-epithelial cell-to-cell contact with live bacteria, other studies have shown that beneficial effects can be conferred by synthesized bioactive factors. Thus research is progressing to investigate the possibility of using secreted bacterial products to confer protection without the risk of live organisms. Conditioned media from probiotic organisms is the broth used to grow the probiotic bacteria subsequently filtered to remove all bacteria to result in a complex formulation of natural bioactive components synthesized and secreted by the probiotics in a solution that is bacteria-free. Particularly relevant to the premature infant with increased activation of NF-κB dependent inflammatory cytokine production, the combination of probiotic organisms called VSL#3 containing a mixture of Streptococcus, Lactobacillus and Bifidobacterium can decrease NF-κB activation by inhibition of the proteasome, preventing degradation of the inhibitor IκB [136]. Recent studies have demonstrated that conditioned media from Lactobacillus plantarum alone is similarly able to directly inhibit the chymotrypsin-like activity of the 26S proteasome [144]. Preserving IκB may be specifically important as this is a point of developmental differentiation between immature and mature IEC [33]. Other studies have shown that bacterial CpG-DNA can activate TLR 9 signaling in intestinal epithelial cells and dampen the LPS-mediated TLR 4 inflammatory signaling which has been shown to be important in NEC. Thus activation of TLR9 by bacterial DNA may be another means by which probiotics can offer protection, and suggests another potential bacterial product that could be harnessed separate from live bacteria as a therapeutic option [71, 145]. However, further research is required to determine which probiotic organisms are optimal and means of effecting the greatest benefit while minimizing harm.

CONCLUSIONS

Much work has been done in cell culture and animal models regarding possible mechanisms of injury which must now be translated to clinical utility. Future research must be focused on identifying means to predict which preterm infants are at greatest risk for this disease, an early point in the inflammatory cascade at which intervention can be beneficial prior to clinical symptoms, as well as means to appropriately mature host defense and inflammatory responses of the preterm infant gut in order to prevent this disease. Unique characteristics of the preterm gut predispose to an exaggerated inflammatory response. These include limited diversity and Proteobacteria predominance of the intestinal microbiota, immature intestinal host defense, and immature NF-κB regulation leading to exaggerated production of inflammatory cytokines. Identifying means of predicting which infants are at highest risk for this disease by microbial profiling would allow early intervention, perhaps utilizing probiotic agents to improve the microbial profile.

NEC is a multifactorial disease and likely requires a multifactorial intervention. A combination of human milk and probiotic factors targeting areas of susceptibility of the immature gut may be necessary. Breast milk feeding should be encouraged for all preterm infants. In addition much work remains to understand the beneficial aspects of human milk, as well as beneficial probiotic organisms for this patient population. Human studies to translate bench research into therapeutic options are still required. However, factors in human milk as well as factors secreted by probiotic bacteria hold great promise as means to mature responses, strengthen intestinal barrier function, as well as modulate inflammation to improve the health of preterm infants.

ABBREVIATIONS

NEC

Necrotizing enterocolitis

NICU

Neonatal intensive care unit

IEC

Intestinal epithelial cell

JAM

Junction adhesion molecule

ZO

Zonula occludin

TJ

Tight junction

TNF

Tumor necrosis factor

DISC

Death inducing signal complex

PAF

Platelet activating factor

NO

Nitric oxide

HMGB1

High mobility group box 1

TLR

Toll-like receptor

PAMP

Pathogen associated molecular pattern

LPS

Lipopolysaccharide

NF-κB

Nuclear factor kappaB

IκB

Inhibitor of kappB

NBD

NEMO binding domain

PPARγ

Peroxisome proliferation receptor gamma

EGF

Epidermal growth factor

Epo

Erythropoietin

TER

Transepithelial resistance