Pathogenesis of necrotizing enterocolitis: mechanisms and mediators

Abstract

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of preterm neonatal infants. The hallmarks of the disease include severe intestinal necrosis and systemic inflammation leading to overwhelming sepsis. There remains significant morbidity and mortality attributed to NEC, raising the urgency to investigate potential causative mechanisms in order to treat and potentially prevent the disease. This review will summarize the established and emerging mechanisms leading to NEC, including toll-like receptor 4 activation by gram-negative bacteria, microbial dysbiosis, a maladapted immune response, impairment in the enteric nervous system and mitochondrial damage. Overriding each of these factors are potential genetic and epigenetic modulators, which may drive each of the above processes. Importantly, results of experiments from a variety of laboratories have suggested that an understanding of processes may lead to the development of novel preventative or treatment modalities, including small molecules and stem cells that can interfere with the deleterious pathways. By summarizing the latest mechanistic and therapeutic advances, this review hopes to provide insights into the ultimate goal of improving outcomes for patients with this devastating disease.

Introduction

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency among preterm neonates.^1–4 The disease is marked by intense inflammation and necrosis of the small bowel and colon which, if left untreated, may progress to intestinal perforation, overwhelming sepsis and death.^{2 5} NEC usually presents after the first week of life with non-specific gastrointestinal and systemic symptoms. Early signs and characteristics of NEC include feeding intolerance, abdominal distention and lethargy along with derangements in vital signs such as temperature instability, bradycardia and episodes of apnea. Gastrointestinal symptoms include emesis, bloody stool, abdominal tenderness, distention and abdominal wall discoloration.¹

NEC risk factors

The risk factors for NEC span prenatal, perinatal and postnatal domains. Major risk factors that are consistently implicated in NEC include extreme prematurity (<28 weeks’ gestation), extremely low birth weight (<1000 g), intrauterine growth restriction, formula feeding and intestinal dysbiosis.^{1–3 6} It is worth noting that formula feeding has traditionally been linked to NEC, but a 2-year surveillance study in the United Kingdom found that half of all NEC cases resulting in death or surgical intervention had only received human milk feeds prior to disease onset, thereby challenging this dogma.^7–10 The majority of studies strongly favor human milk to reduce NEC.^10–13 Other prenatal or perinatal risk factors associated with NEC development include intrauterine inflammation or infection, chorioamnionitis, maternal hypertension, preeclampsia, placental abruption and prolonged rupture of membranes.^{6 14–18} Postnatal concerns implicated as risk factors include patent ductus arteriosus, sepsis, empiric antibiotic use, severe anemia and blood transfusions.^{6 12 15 19–26} Although many risk factors have been identified, evidence for causality is actually quite sparse. Rather, these risk factors may be useful not only to predict who is more likely to develop NEC but also to develop appropriate animal model systems to determine mechanisms.

Mechanisms of NEC development: TLR4 signaling and beyond

The pathophysiology of NEC reflects the complex interplay between multiple pro-inflammatory factors and is initiated in part through activation of the lipopolysaccharide (LPS) receptor, namely toll-like receptor 4 (TLR4), in response to a dysbiotic microbiome within the intestinal lumen of the premature host as seen in figure 1.

Figure 1. (A) This image demonstrates the intestinal environment of a healthy premature infant. The infant’s mucosal barrier is intact and maintains a healthy homeostasis. Toll-like receptor 4 (TLR4) is present on the mucosal surface (1). There is a thick mucous barrier which contains antimicrobial peptides secreted by Paneth cells (2–3). A healthy microbiome populates the luminal surface (4). Components of the immune system such as lymphocytes and macrophages are present in the lamina propria (5). The mucosa is healthy and well perfused via intact microvascular structures (6). Key cell types and components are labeled. (B) This image demonstrates the intestinal environment of an infant with NEC, highlighting key mechanistic insults and potential intervention points. TLR4 binding to bacterial components leads to severe inflammation, leading to areas of necrosis and apoptosis which provide barrier defects to allow for translocation (1). Cell signaling activates the infant’s immune system, which further increases the inflammatory process by way of macrophage and lymphocyte activation (2 and 7). Overgrowth of harmful bacterial species outnumbers healthy gut flora (3). Additionally, the protective mucous barrier is more thin in the neonate with NEC (5). Stem cell therapies, such as human amniotic fluid stem cells (H. AFSC), can confer a benefit by facilitating healing and, possibly, regeneration (4). C34, a TLR4 inhibitor, can be seen binding TLR4 and preventing activation by bacterial components (6). Breast milk-derived IgA offers mucosal protection, with human milk oligosaccharides also playing a protective role (8). The mucosa is overall inflamed, with areas of microvascular damage leading to hypoxic injury (9). Key interactions are labeled. EEC, enteroendocrine cell; NEC, necrotizing enterocolitis; TLR4, toll-like receptor 4.

Studies evaluating the stool microbiome of premature infants with NEC have revealed that NEC develops in part due to intestinal dysbiosis marked by reduced microbial diversity. Notably, there is a preponderance of pathogenic gram-negative bacteria, such as Proteobacteria, and absence of beneficial commensals such as Bifidobacterium, Firmicutes and other strict anaerobes.^27–29 NEC tends to develop after bacterial colonization of the gastrointestinal tract has occurred and the central role of bacteria in NEC development is also underscored by the observation that NEC patients have enteric organisms and LPS present in circulation.

Several lines of evidence support increased intestinal TLR4 signaling and TLR4 gene expression as a driver of NEC. Our lab has previously established that Tlr4-mutant mice are protected from NEC development when compared with wild-type controls. Additionally, mice lacking Tlr4 in intestinal epithelial cells that are then subjected to experimental NEC show decreased severity of disease owing to reduced TLR4-activated autophagy and therefore improved mucosal healing.^{30 31} TLR4 activation in the gut can be inhibited by the nucleotide-binding oligomerization domain-containing 2 (NOD2) receptor, which is an intracellular pattern recognition receptor that recognizes muramyl dipeptide.³² NOD2 activation inhibits TLR4 activity in enterocytes and reverses the deleterious effects of TLR4 on mucosal injury. Moreover, mucosal injury allows for translocation of bacteria into circulation. Downstream interactions between bacteria and TLR4 on the endothelium of mesenteric tissue then leads to a reduction of endothelial nitric oxide synthase (eNOS). This drives vasoconstriction and mesenteric ischemia in animal models.^{33 34}

Human evidence for TLR4 in the pathogenesis of NEC

Shaw et al characterized the fecal microbiota of infants prior to NEC onset using shotgun metagenomic sequencing and quantitative PCR. They showed that infants who develop NEC have an increase in organisms expressing the TLR4 ligand LPS.³⁵ Further support evaluating TLR4 as a key mediator of NEC in human studies comes from studies demonstrating that breast milk reduces LPS binding to TLR4 via constituent oligosaccharides, thereby ameliorating intestinal inflammation. In mice, breast milk was found to inhibit TLR4 signaling by decreasing TLR4-induced NF-κB via epidermal growth factor (EGF) activation of the PI3K-AKT pathway.^{36 37} Finally, mutations in genes related to the single immunoglobulin and toll-interleukin 1 receptor (SIGIRR) signaling pathway, which is a negative regulator of TLR4 signaling, have been observed at higher frequencies in infants with NEC.^38–40

The cross-switching hypothesis in the pathogenesis of NEC

TLR4 expression is significantly higher in the premature intestine, an observation which led us to advance a unifying model for NEC development which we have termed the ‘cross-switching hypothesis’. In brief, the elevated expression of TLR4 in the gut reflects its non-immune role in the regulation of normal intestinal development, through TLR4-dependent effects on intestinal stem cells via the Notch signaling pathway.⁴¹ We note that the Drosophila homolog of TLR4 was originally discovered through its role in the regulation of thorax formation.⁴² The bacteria-free environment in which the fetus develops poses no threat to the elevated TLR4 expression. By contrast, in the setting of a premature birth, the persistently elevated TLR4 becomes activated by the dysbiotic gram-negative bacteria that colonize the intestinal lumen and the switch from a developmental to a proinflammatory role for TLR4 is the basis for the ‘cross-switching hypothesis’.⁴³ This approach to understanding NEC might explain why NEC does not develop immediately after birth but requires a period of time for gut colonization to occur. Testing this concept may also explain why the more premature the infant is, the longer it takes for NEC to develop, as the peak expression of TLR4 occurs at a fixed point relative to conception, and thus takes longer to be reached in more premature infants. The greatest value of the ‘cross-switching hypothesis’ may be the development of platforms for the design of TLR4 inhibitors as novel preventive or therapeutic approaches for NEC.

The adaptive immune system and its role in disease progression

T lymphocytes

Lymphocyte dysregulation has been shown to contribute to intestinal inflammation and injury related to NEC. Lymphocytes are abundant in intestinal samples from patients with NEC, and our lab has shown that Rag1-deficient mice (which lack mature T and B cells) are protected from experimental NEC.⁴⁴ Strikingly, transfer of intestinal lymphocytes from NEC mice into Rag1-deficient mice was sufficient to induce NEC, thereby supporting a pathogenic role of adaptive immunity.⁴⁴ This response is thought to be mediated by activation of TLR4 on the intestinal epithelium as activation drives lymphocyte recruitment through CCR9/CCL25 signaling and promotes a STAT3-dependent polarization toward proinflammatory responses. Similarly, there is a predominance of proinflammatory T helper 17 (Th17) cells, which release cytokines such as IL-17 and IL-22, and a reduction in regulatory T cells (Tregs). This Th17 polarization is critical for the development of NEC as IL-17 impairs enterocyte tight junctions, reduces proliferation and increases apoptosis.⁴⁴ Human studies confirm this imbalance of T cell populations as NEC patients show decreased Treg/CD4 and Treg/CD8 ratios relative to controls.⁴⁵

B lymphocytes

B cells appear to contribute in some capacity to the pathogenesis of NEC though they are not thought to be primary drivers of the inflammatory cascade. Single-cell RNA sequencing of human intestinal tissue from NEC patients identified B cells within the inflammatory milieu.⁴⁶ RNA sequencing from a separate group uncovered decreased expression of genes related to B cell receptor signaling in NEC patients such as CD19, BLK and IGHA1.⁴⁷ How this decrease in expression may portend a pathologic phenotype in NEC remains a matter of debate. Importantly, maternal immunoglobulin A (IgA) in breast milk provides critical protection from NEC development. Preterm infants who go on to develop NEC have a relative decrease in IgA-bound bacteria in stool samples with an increasing proportion of Enterobacteriaceae in the IgA-unbound fraction of the microbiota. Additionally, pups reared to IgA deficient mothers are susceptible to NEC development despite exposure to maternal milk.⁴⁸

The role of the enteric nervous system in the pathogenesis of NEC

Enteric nervous system (ENS) damage occurs in both human and experimental NEC. Evidence for ENS dysfunction includes neuronal apoptosis, disrupted myenteric plexus architecture and reduced expression of neuronal nitric oxide synthase (nNOS) in myenteric ganglia. Human intestinal samples show disrupted myenteric plexus architecture, reduced glial fibrillar acidic protein expression and loss of submucosal or mucosal innervation.^{49 50} These aberrancies in intestinal structure persist long term with ENS damage present at the time of stoma reversal for infants with NEC.⁴⁹ Importantly, ENS dysfunction was shown to precede the development of NEC and reflect a TLR4-dependent loss of enteric glia, resulting in a hyperinflammatory state within the intestinal mucosa that leads to NEC.⁵¹ Importantly, strategies that enhance motility,⁵¹ or which protect the ENS including heparin-binding EGF-like growth factor or stem cell transplantation, represent novel strategies for NEC prevention and treatment.⁵²

Hypoxia and oxidative stress in the pathogenesis of NEC

Emerging evidence highlights the role of hypoxia in aggravating the inflammatory response in NEC and that delayed microvasculature development is part of the pathogenesis.⁵³ Nutrient absorption in the premature intestine requires a high oxygen demand after feeding. In neonates, inadequate intestinal blood flow and low arterial oxygen content contribute to a hypoxic environment, often attributed to premature cardiac impairment or respiratory distress. Moreover, studies indicate that intestinal microcirculation in premature infants and subsequent pressure flow autoregulation is impaired, which compromises sufficient oxygen supply.⁵⁴ In combination, these factors exacerbate the hypoxic environment and increase susceptibility to NEC. Hypoxic mucosal damage triggers oxidative stress by releasing reactive oxidative species and superoxide radicals, potentially leading to NEC, through multiple pathways. These include a disrupted antioxidant glutathione system with high amounts of lipid peroxidation products, which induces increased apoptosis,⁵⁵ intestinal iron levels and iron-dependent lipid peroxidation.⁵⁶ TNFα downstream signaling induces mitochondrial dysfunction and can alter the mitochondrial membrane potential resulting in mitochondrial apoptosis, which decreases ATP levels and shifts the cell towards anaerobic metabolism. A decline of mitochondrial function contributes to intestinal epithelial cell death and plays a critical role in disease progression.⁵⁷ A single-cell analysis identified a mitochondrial enzyme responsible for beta oxidation being a highly upregulated gene in NEC patients, namely acyl-CoA synthetase long chain family member 1 (ACSL1). ACSL1 expression is linked to immune cell infiltration and immune dysregulation in NEC.⁵⁸ Oxidative stress stimulates the release of HIF-1α. Interestingly, animal studies showed less intestinal injury in NEC models when HIF-1α stability was improved by dimethyloxalylglycine as well as by vascular endothelial growth factor (VEGF) plasmid injection,^{59 60} while mice with a knockdown of HIF-1α in intestinal epithelial cells revealed severe mucosal barrier defects.⁶¹

Structural immaturity of the gastrointestinal tract

Structural immaturity of the gastrointestinal tract contributes to NEC development. The intestinal epithelial cells act as a physical barrier to protect the rest of the body from the luminal environment, mainly via intraepithelial tight junctions, which are present as early as 10 weeks gestation.^62–64 However, in a premature infant, these tight junctions, which are primarily made via the interactions of occludins and claudins, may have increased permeability.⁶⁴ The physical barrier of the small intestine is also composed of the mucus covering that overlays the luminal surface. The thick outer layer serves as a direct barrier, protecting the mucosal surface, while the thinner underlayer plays a role in cell signaling and provides scaffolding for antimicrobial peptides (AMPs) and secretory IgA.^{62 65} Removal of the key mucin glycoprotein Muc2 gene in mouse models leads to increased susceptibility to irritative colitis as well as increased adhesion of bacteria to the mucosal surface.⁶⁶ In the case of the preterm infant, the quantity and quality of mucus produced changes with gestational age⁶⁴ (figure 1). While goblet cells may be present as early as 9 weeks gestation, the mucin glycoproteins that generate the protective layer do not reach adult levels until 27 weeks.^63,65 Mature mucins are also more viscous, more acidic and have a greater resistance to breakdown by deleterious enzymes, which all serve to better protect intestinal tract.^{63–65 67} This means that preterm infants have not only a lack of quantity of mucus but also a lack of mature mucins to confer appropriate protection. Importantly, exaggerated TLR4 signaling in the intestinal epithelium leads to a loss of goblet cells and reduced mucus production, thus increasing the likelihood for NEC development.⁴³

Microbial dysbiosis

Microbial dysbiosis has long been implicated in the pathogenesis of NEC. An increased risk of NEC has been associated with proliferation of bacteria of the Gammaproteobacteria class, which include Vibrionaceae, Enterobacteriaceae and Pseudomonadaceae.⁶⁸ Gram-negative bacteria are frequently found in peritoneal fluid cultures in patients with NEC, even if blood cultures remain sterile. Notably, premature babies have been found to have low bacterial diversity in fecal cultures, with up to 61% Proteobacteria 2 weeks before the development of NEC. A review analyzing the microbiomes of more than 300 preterm infants demonstrated not only decreased relative abundance of Streptococcus and Bifidobacterium but also reduced alpha diversity in fecal samples.⁶⁹ Although a subsequent meta-analysis by Pammi et al. identified inconsistencies among studies regarding alpha diversity in NEC, a consistent finding throughout analyses was an increased relative abundance of Proteobacteria accompanied by decreased levels of Bacteroidetes and Firmicutes—an inverse pattern compared with control infants.²⁸ Further impairment of intestinal defenses in premature babies, such as low mucin, decreased Paneth cells, and lower levels of secretory IgA, leave enterocytes more susceptible to interaction with these Gammaproteobacteria.⁶⁸ Gammaproteobacteria such as Enterobacteriaceae play a role in cultivating the newborn microbiome by creating the low-oxygen environment required for the proliferation of anaerobes such as Bifidobacteria and Clostridium. These bacteria play a critical role in the fermentation of the oligosaccharides and polysaccharides needed by the infant to digest human milk and, later, solid foods. However, in the premature infant gut, with its heightened sensitivity to gram-negative bacteria and overexpression of TLR4, the presence of Gammaproteobacteria can become detrimental. As gram-negative bacteria proliferate, exposure to LPS increases, resulting in overstimulation of TLR4. Furthermore, as these bacteria also express low levels of CpG in DNA, low activation of TLR 9 results, leading to insufficient downregulation of the TLR4 signaling pathway.⁷⁰

Impaired intestinal microcirculation

Preterm neonates are characterized by an impaired intestinal microvascular development that makes them susceptible to NEC.⁵³ More precisely, real-time blood flow of rat ileum showed smaller arterioles with a ‘stop and go’ flow pattern in a rat NEC model.⁷¹ An example of maladaptive blood vessel response was observed in piglets exposed to severe hypoxemia, where vasoconstriction occurred, leading to intestinal ischemia.⁷² Additionally, nitric oxide (NO)-mediated vasodilatation was defective in intestinal samples of human NEC patients.⁷³ Mechanistically, endothelial TLR4 signaling suppresses the expression of eNOS resulting in reduced NO production.³³ Assessment of endothelin-1 (ET-1) in human preterm intestine samples, which acts as a potent vasoconstrictor, revealed higher ET-1 levels in NEC specimens.⁷⁴ In animal models mimicking NEC, a significantly reduced vascular density and enlarged vessel-free areas have been observed in the first 48 hours of NEC induction as illustrated in figure 1. Notably, these changes occur before intestinal mucosal destruction can be seen in ileal cross sections.⁷⁵ A pivotal signaling molecule in angiogenesis of intestinal microvasculature is VEGF-A/VEGF receptor 2 (VEGFR2), which is upregulated in the last days of gestation. VEGF-A/VEGFR2 is a downstream target of hypoxia-induced factor 1α (HIF-1α), which drives angiogenic sprouting in the hypoxemic environment in utero. A therapeutic approach to improve microcirculation is remote ischemic conditioning (RIC), which entails exposing mice to intermittent hind limb ischemia. Applied RIC leads to preserved vasodilatation and ameliorated intestinal morphology in NEC models via reduced intestinal inflammation, partially mediated by NO release.⁷⁶

Paneth cell impairment

Paneth cells are located throughout the small intestine, residing at the base of crypts of Lieberkühn, they contain large secretory granules.⁷⁷ These cells play an important role in cell signaling and mediation of innate immunity in the human gut. They are the key secretors of AMPs into the intestinal lumen that serve to disable or kill destructive microorganisms.^{62 65 77} Exposing murine crypts, rich in Paneth cells, to Escherichia coli or Staphylococcus aureus results in secretion of α-defensin and lysozyme.^{65 78} This results in a significant decrease in bacterial populations.⁷⁸ In the developing fetus, Paneth cells are initially noted at the end of the first trimester.⁶⁵ However, all infants have fewer Paneth cells when compared with the adult population, leading to a reduced ability to secrete AMPs, α-defensin and lysozyme.^{65 79} It has been disputed whether there is a difference in quantity of Paneth cells in human tissue affected by NEC, with potentially fewer lysozyme-positive cells in NEC samples being discussed as a key role in NEC susceptibility.^80–83 It has been proposed that TNFα modulation of S-adenosyl-l-methionine-pointed domain-containing E-twenty-six family transcription factor leads to changes in Paneth cell differentiation in an inflammatory environment.⁸² Hyperplasia and metaplasia of Paneth cells after recovery have also been noted, indicating a role for Paneth cells in intestinal recovery as well.⁸¹ These cells have also been interrogated in animal models.

Ablation of Paneth cells in neonatal rats with dithizone followed by exposure to Escherichia coli leads to increased mortality and significant intestinal necrosis, presenting similarly to NEC.^{65 79 84} There is also a well-established mouse model of NEC that relies on Paneth cell ablation. Mice treated with both dithizone followed by Klebsiella show significant small intestinal damage, most notably in the ileum, with colon sparing consistent with classic murine NEC models induced by hypoxia and/or LPS exposure. Inflammatory cytokines are also elevated with Paneth cell ablation in mice.⁸³ Moreover, P14–P16 mice treated with dithizone, and bacteria show an increase in cecal Enterobacteriaceae species that is phenotypically similar to human NEC.⁸⁵ Without Paneth cells, and by extension AMPs and lysozyme, the natural defense of the small intestine is significantly weakened leading to bacterial overgrowth and invasion. With the data in both human samples and animal models together, it can be inferred that the underlying deficiency may compound with inflammatory dysfunction of Paneth cells in the neonate, which plays a role in the development of NEC.

Genetic and epigenetic links

Given the multifactorial nature of NEC pathogenesis, the contribution of genetic susceptibility has become an area of increasing investigation. SIGIRR, a negative regulator of TLR signaling, has emerged as a candidate gene of interest. In exome sequencing performed on an infant with NEC totalis, Sampath et al identified both a stop mutation (p.Y168X) and a missense variant (p.S80Y) in the SIGIRR gene.⁸⁶ Further studies conducted in embryonic cell lines demonstrated that SIGIRR modulates LPS-induced IL-6 production. However, this effect was lost following transfection with the identified mutations, suggesting loss-of-function effects by these variants.³⁹ Further sequencing analyses also demonstrated the presence of SIGIRR mutations in infants with NEC when compared with controls.^{39 86} Given that increase in NFKβ is found in in-vivo models of NEC, the NFKB signaling pathway has also been investigated as potential genetic contributor. In further study by Sampath et al of 271 infants, two variants—NFKB1 and NFKBIA—were identified as potentially relevant. NFKB1 (g.-24519delATTG) was found in all 15 infants who developed NEC, compared with 65% of those who did not develop NEC. Conversely, the NFKBIA variant was found to have a protective effect and was found in 13.3% of NEC infants but in 49% of unaffected patients.⁸⁶ NOD2, a pattern recognition receptor associated with inflammatory bowel diseases, also may have a role in NEC. Although some studies have found no association between NOD2 variants and NEC, a multicenter study of 9082 very low birth weight infants in Germany found that those with two or more variants of NOD2 were at an increased risk of developing NEC.^{86 87}

Epigenetic regulation and DNA methylation have also emerged as areas of interest. Recent research has demonstrated increased DNA methylation patterns in both colonic and ileal tissue from infants with NEC.

Using whole genome bisulfite sequencing of isolated colonic and ileal epithelium, Good et al. demonstrated 7087 differentially methylated regions that resulted in altered gene transcription between infants with surgical NEC versus controls without NEC.^{88 89} In the ileum, 652 CpG sites were hypermethylated in NEC samples, whereas 38 809 CpG sites were hypermethylated in the colon. Genes demonstrating significant hypermethylation included hepatocyte nuclear factor 4 alpha and G protein-coupled receptor 35. In addition, stool samples from patients with NEC have demonstrated increased methylation of the TLR4 gene.⁸⁸

Viral triggers

Cytomegalovirus infection, while typically asymptomatic in immunocompetent adults, may also contribute to NEC pathogenesis during the perinatal period. Findings from Scheese et al. suggest this may occur through upregulation of genes involved in TLR4-mediated inflammation such as 2 H2k1, H2d1m H2t23, and Stat1. In a murine model, CMV infection in mice decreased methylation of the Tlr4 promoter, resulting in increased Tlr4 expression.⁹⁰ In addition to upregulating Tlr4 expression, CMV infection was shown to disrupt genes involved in cellular metabolism. CMV induced mitochondrial dysfunction through activation of the mitochondrial fission gene Drp1, mitophagy pathway Pink1, and through fragmentation of mitochondrial DNA. Furthermore, these disturbances occur in tandem with the CMV-mediated reduction of ATP and the genes governing its metabolism.⁹⁰ Specifically, CMV upregulated ATP catabolic enzymes Entpd1/Cd39 and Nt5e/Cd73, while also downregulating enzymes responsible for ATP synthesis.

While this lab has a focus on CMV, other viruses have also been linked to NEC. Several single-center retrospective case–control studies have linked outbreaks of norovirus and rotavirus in the NICU to NEC, with both a spatial and temporal relationship indicating a potential ‘transmission of NEC’ between patients without significant clinical differences.^91–94 Additionally, potential caregiver transmission of these viral gastrointestinal diseases has been documented, highlighting the importance of good hand hygiene.^{92 93} Postoperatively, adenovirus infection in patients who experienced surgical resection for NEC has been linked with longer hospital stays, most notably in neonates with ostomies.⁹⁵ Mani et al published a comprehensive review of viral infections associated with NEC, looking at over 50 either review or meta-analysis studies. Rotavirus, norovirus and CMV were among the most studied associated viral infections, with odds ratios (ORs) of 3.96 (95% confidence interval (CI) 1.12 to 13.95), 11.95 (95% CI 2.05 to 69.84), and 3.50 (95% CI 1.60 to 7.65), respectively.⁹⁶ Many of these studies use postdiagnosis stool tests to confirm viral infection, making it difficult to definitively state whether the viral infection predated NEC. Besides the viruses previously discussed, coronavirus, torovirus, coxsackievirus, astrovirus and HIV have also been linked to NEC in neonates, though mechanisms remain poorly explored.⁹⁷ Potential theories involve components of bacterial dysbiosis leading to increased susceptibility to viral infection, immature immune response to viral pathogens and the relatively novel idea of the viral microbiome.

Much like the healthy bacterial population in the human gut, it is now proposed there is a normally present healthy viral population as well. Infants, including premature neonates, are born with sterile gastrointestinal tracts that subsequently become colonized with healthy gut flora through interaction with parents/caregivers, the environment and feeding. Analysis of an infant’s meconium compared with stool at 1 week of life showed no viral particles in meconium but 10⁸ viral particles per gram wet weight of stool by the end of the first week, which is still far less than adult stool samples.⁹⁸ One study looking specifically at infants with NEC found that the so-called ‘virome’ in neonates with NEC converged approximately 10 days prior to diagnosis. Members of the Myoviridae, Podoviridae and Siphoviridae families were represented during this time, with additional unknown species that were hypothesized to be lytic viruses.⁹⁹ This indicates that, much like bacterial dysbiosis, there may be an opportunity for viral dysbiosis in the preterm neonate.

Translational potential based on mechanistic insights

To date, there is no specific treatment for NEC and most therapies are started after the insult has occurred. Investigating the potential factors that lead to NEC is imperative to finding novel strategies for both prevention and treatment to be used in the clinical setting. Without understanding the mechanism of injury, it is difficult to derive a solution. Advancements in technology allow for comprehensive mechanistic analysis. Based on the current understanding of NEC, novel therapeutic strategies include a combination of prebiotics, probiotics, biologics, small molecule medications and stem cell-based therapies. It has long been known that breast fed infants suffer from NEC at lower rates compared with formula fed infants. Components of breast milk were analyzed and it was found that the HMOs 2’-fucosyllactose and 6’-sialyllactose reduced NEC severity via inhibition of TLR4 signaling, increased mucin production and improved vasodilation.^{36 100 101} This prebiotic benefit is preserved in the absence of normal gut bacteria and facilitates healthy bacterial growth, meaning the strategy shows promise in the underdeveloped gut.^102–104 Human studies have proposed that infants receiving breast milk with higher quantities of disialyllacto-N-tetraose have a lower risk of developing NEC, which provides another potential supplement to investigate.¹⁰⁵

Probiotic supplementation, such as Bifidobacterium plus Lactobacillus, has been shown to improve outcomes and severity in NEC compared with placebo in several clinical trials.^106–108 Different formulations of probiotics have recently been proposed, with a promising biofilm formulation showing protection of both the brain and intestines in piglet and rat models of NEC.^{109 110} However, there have been concerns raised about the potential for infection, leading to sepsis when the preterm gut is exposed to probiotics. A meta-analysis completed in 2025 looking at 63 studies showed less than 0.04% of infants given probiotics were diagnosed with probiotics sepsis.¹¹¹ This indicates that there is a small risk that should be considered when utilizing this otherwise largely beneficial strategy.

Multiple different biologic agents have been investigated to explore their possible therapeutic effects in NEC. Lactoferrin is a protein abundant in human colostrum with both antimicrobial and anti-inflammatory properties. Lactoferrin administration in murine NEC results in less inflammation and less severity via intestinal proliferation.¹¹² Supplementation of lactoferrin has been explored in human neonates in clinical trials with mixed results.^{113 114} Granulocyte-colony stimulating factor is a cytokine, which stimulates white blood cell production and enhances immune response that has been shown in a pilot study in preterm infants to aid in healing and recovery after NEC.¹¹⁵ EGF works to promote intestinal growth and repair at the cellular level. In preclinical animal models and small human-based clinical trials, it has been shown to reduce NEC severity and promote intestinal healing.^{116 117} Further studies are required to fully elucidate the benefits and discover any possible side effects of the aforementioned biologic agents.¹⁰⁵

Understanding NEC on a molecular level has given rise to potential small molecule-based therapeutic interventions. Our lab identified C34, a 2-acetamidopyranoside oligosaccharide, that acts to block TLR4, preventing NEC in mice and piglets when administered orally or intravenously¹¹⁸ (figure 1). The same molecule has also been proven to reduce NEC-associated lung injury by blocking TLR4.¹¹⁹ Pregnane X receptor (PXR), which is a microbial sensor and signaling molecule for host-bacterial metabolites, reduces TLR4 expression. When treated with a PXR agonist, mice had lower NEC severity.¹²⁰ Another small molecule that targets enteric glial cells to induce brain-derived neurotrophic factor (BDNF) secretion was proven to also inhibit TLR4 signaling as well as restore intestinal motility, thereby reducing NEC severity in mice.^{33 51} This strategy extends to prenatal interventions as well with the administration of aryl hydrocarbon receptor (AHR) ligand indole-3-carbinol (I3C) during pregnancy in preclinical studies showing a reduction in NEC.¹²¹ Mice in experimental NEC supplemented with AHR proligand I3C also showed reduced severity.^{121 122} Additional investigation is needed to assess the feasibility of both short-term and long-term safety of TLR4 inhibition in neonates.^{44 48 123}

Intraperitoneal injections of mesenchymal stem cells in mice show improved intestinal inflammation, less lung injury and less microglial damage in brains compared with controls.^124–126 Human placental stem cells have also shown protection in experimental NEC and were found to target damaged intestine in rat models.^{127 128} Attention has also been placed on IP administration of stem cell exosomes, which contain RNA, miRNA, DNA and protein, with possible therapeutic effects in NEC.¹²⁹ The mechanisms by which stem cells enact their effects are not completely understood at this time. Potential challenges with stem cell therapy include optimal dosage, timing, and delivery method, with added risks including possible rejection.

Conclusions and future directions

NEC remains a multifactorial disease, which appears to be triggered by dysregulated TLR4 signaling in response to microbial dysbiosis, leading to a variety of downstream effects including hypoxia/oxidative stress, microcirculatory failure, ENS injury and deficient mucosal defenses. While there are no specific NEC therapies at this time, several emerging areas of study suggest that mechanism-driven therapies may be on the horizon. For instance, treatment through targeted modulation of innate pathways with TLR4 inhibitors, NOD2 agonists and HMOs suggest efficacy in animal models, and several are in the process of development for clinical trials. Intestinal microcirculatory can be targeted with trials of NO precursors, remote ischemic conditioning, and HIF-1/VEGF-axis stabilizers with careful monitoring for off-target effects. Moreover, translational success will likely depend on early identification of at-risk infants, and stratification of infants into relevant subtypes based on molecular signatures. By integrating mechanistic insights with precision neonatal care, the field is promising to reduce NEC incidence, limit surgery and long-term sequelae and improve survival and quality of life for preterm infants.

Data availability statement

No data are available.