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Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily rising in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel prevention or treatment approaches. We and others have shown that NEC arises in part from exaggerated signaling via the bacterial receptor, Toll-like receptor 4 (TLR4) on the intestinal epithelium, leading to widespread intestinal inflammation and intestinal ischemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC development in mouse and piglet models. We now seek to test the hypothesis that a secretome derived from amnion-derived cells can prevent or treat NEC in preclinical models of this disease via a process involving TLR4 inhibition. In support of this hypothesis, we show that the administration of this secretome, named ST266, to mice or piglets can prevent and treat experimental NEC. The protective effects of ST266 occurred in the presence of marked TLR4 inhibition in the intestinal epithelium of cultured epithelial cells, intestinal organoids, and human intestinal samples ex vivo, independent of epidermal growth factor. Strikingly, RNA-seq analysis of the intestinal epithelium in mice reveals that the ST266 upregulates critical genes associated with gut remodeling, intestinal immunity, gut differentiation. and energy metabolism. These findings show that the amnion-derived secretome ST266 can prevent and treat NEC, suggesting the possibility of novel therapeutic approaches for patients with this devastating disease.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in premature infants. One of the most devastating complications of NEC is the development of NEC-induced brain injury, which manifests as impaired cognition that persists beyond infancy, and which represents a pro-inflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyl lactose (2'-FL) or 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we now hypothesize that oral administration of these HMOs will reduce NEC-induced brain injury, and sought to determine the mechanisms involved. We now show that the administration of either 2'-FL or 6'-SL significantly attenuated NEC-induced brain injury, reversed myelin loss in the corpus callosum and midbrain of newborn mice, and prevented the impaired cognition observed in mice with NEC-induced brain injury. In seeking to define the mechanisms involved, 2'-FL or 6'-SL administration resulted in a restoration of the blood-brain barrier in newborn mice, and also had a direct anti-inflammatory effect on the brain as revealed through the study of brain organoids. Metabolites of 2'-FL were detected in the infant mouse brain by Nuclear Magnetic resonance (NMR), whereas intact 2'-FL was not. Strikingly, the beneficial effects of 2'-FL or 6'-SL against NEC-induced brain injury required the release of the neurotrophic factor BDNF, as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. These findings reveal that the HMOs 2'-FL or 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury.

Necrotising enterocolitis (NEC) is a devastating gastrointestinal disease of prematurity that typically develops after the administration of infant formula, suggesting a link between nutritional components and disease development. One of the most significant complications that develops in patients with NEC is severe lung injury. We have previously shown that the administration of a nutritional formula that is enriched in pre-digested Triacylglyceride that do not require lipase action can significantly reduce the severity of NEC in a mouse model. We now hypothesise that this ‘pre-digested fat (PDF) system’ may reduce NEC-associated lung injury. In support of this hypothesis, we now show that rearing newborn mice on a nutritional formula based on the ‘PDF system’ promotes lung development, as evidenced by increased tight junctions and surfactant protein expression. Mice that were administered this ‘PDF system’ were significantly less vulnerable to the development of NEC-induced lung inflammation, and the administration of the ‘PDF system’ conferred lung protection. In seeking to define the mechanisms involved, the administration of the ‘PDF system’ significantly enhanced lung maturation and reduced the production of reactive oxygen species (ROS). These findings suggest that the PDF system protects the development of NEC-induced lung injury through effects on lung maturation and reduced ROS in the lung and also increases lung maturation in non-NEC mice.

Necrotizing enterocolitis (NEC) is a disease of premature infants characterized by acute intestinal necrosis. Current dogma suggests that NEC develops in response to post-natal dietary and bacterial factors, and so a potential role for in utero factors in NEC remains unexplored. We now show that during pregnancy, administration of a diet rich in the aryl hydrocarbon receptor (AHR) ligand indole-3-carbinole (I3C), or of breast milk, activates AHR and prevents NEC in newborn mice by reducing Toll-like receptor 4 (TLR4) signaling in the newborn gut. Protection from NEC requires activation of AHR in the intestinal epithelium which is reduced in mouse and human NEC, and is independent of leukocyte activation. Finally, we identify an AHR ligand (“A18”) that limits TLR4 signaling in mouse and human intestine, and prevents NEC in mice when administered during pregnancy. In summary, AHR signaling is critical in NEC development, and maternally-delivered, AHR-based therapies may alleviate NEC., Necrotizing enterocolitis (NEC) is a disease of prematurity requiring Toll-like receptor 4 (TLR4) activation on the gut epithelium. Here the authors show that the aryl hydrocarbon receptor (AHR) mediates NEC pathogenesis via effects on TLR4, and that supplementing the diet with AHR ligands during pregnancy or postnatally prevents NEC.

Objective: Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2’-Fucosyllactose (2’-FL) and 6’-Sialyllactose (6’-SL), can reduce NEC through inhibition of TLR4 signaling. Design: NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2’-FL, 6’-SL or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, human tissue explants, and via in silico modeling. Results: Supplementation of infant formula with either 2’-FL and/or 6’-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2’-FL and 6’-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2’-FL and 6’-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling. Conclusion: 2’-FL and 6’-SL, but not lactose, prevent NEC in mice and piglet models, and attenuate NEC-inflammation in human ileum, in part through TLR4 inhibition.

Background & Aims In short-bowel syndrome (SBS), inadequate intestinal adaptation is responsible for the majority of complications, including sepsis, liver failure, and death. In this study, we sought to further delineate the adaptive response to identify potential therapeutic targets. Methods We performed a 75% small-bowel resection (SBR) or sham operation on C57Bl/6J wild-type (WT), lipocalin-2 (LCN2)-/-, and interleukin 22 (IL22)-/- mice. Exogenous IL22 was administered to SBR WT mice. Cecal fecal matter from SBR WT and SBR LCN2-/- mice were transplanted into germ-free mice. Intestinal permeability, inflammation, proliferation, and the microbiome were evaluated 1 week after surgery. CD4+IL22+ laminal propria lymphocytes were sorted by flow cytometry. Naïve T cells were polarized to T-helper cells with or without LCN2. Results A 75% SBR in a mouse re-creates the increased intestinal permeability, enterocyte proliferation, and intestinal dysbiosis seen in SBS. LCN2 expression increases after 75% SBR, and this increase can be abrogated with broad-spectrum antibiotic treatment. LCN2-/- mice have less intestinal inflammation, increased IL22 expression, and greater adaptation as evidenced by less intestinal permeability, increased carbohydrate enzyme expression, less weight loss, and less dysbiosis after 75% SBR than WT mice. The proinflammatory and anti-adaptive effects of LCN2 can be transferred to germ-free mice via a fecal transplant. Administration of exogenous IL22 improves adaptation and restores the normal microbiome after 75% SBR in WT mice. Conclusions LCN2 promotes inflammation and slows intestinal adaptation through changes in the microbiome and IL22 inhibition in a mouse SBS model. Strategies to reduce LCN2 may offer novel therapeutic approaches to enhance adaptation in SBS., Graphical abstract

Angiotensin-converting enzyme 2 (ACE2) is a potent negative regulator capable of restraining overactivation of the renin–angiotensin system, which contributes to exuberant inflammation after bacterial infection. However, the mechanism through which ACE2 modulates this inflammatory response is not well understood. Accumulating evidence indicates that infectious insults perturb ACE2 activity, allowing for uncontrolled inflammation. In the current study, we demonstrate that pulmonary ACE2 levels are dynamically varied during bacterial lung infection, and the fluctuation is critical in determining the severity of bacterial pneumonia. Specifically, we found that a pre-existing and persistent deficiency of active ACE2 led to excessive neutrophil accumulation in mouse lungs subjected to bacterial infection, resulting in a hyperinflammatory response and lung damage. In contrast, pre-existing and persistent increased ACE2 activity reduces neutrophil infiltration and compromises host defense, leading to overwhelming bacterial infection. Further, we found that the interruption of pulmonary ACE2 restitution in the model of bacterial lung infection delays the recovery process from neutrophilic lung inflammation. We observed the beneficial effects of recombinant ACE2 when administered to bacterially infected mouse lungs following an initial inflammatory response. In seeking to elucidate the mechanisms involved, we discovered that ACE2 inhibits neutrophil infiltration and lung inflammation by limiting IL-17 signaling by reducing the activity of the STAT3 pathway. The results suggest that the alteration of active ACE2 is not only a consequence of bacterial lung infection but also a critical component of host defense through modulation of the innate immune response to bacterial lung infection by regulating neutrophil influx.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants whose pathogenesis remains incompletely understood, although activation of the gram-negative bacterial receptor Toll-like receptor-4 (TLR4) on the intestinal epithelium plays a critical role. Patients with NEC typically display gastrointestinal dysmotility before systemic disease is manifest, suggesting dysmotility could drive NEC development. Both intestinal motility and inflammation are governed by the enteric nervous system, a network of enteric neurons and glia. We hypothesized here that enteric glia loss in the premature intestine could lead to dysmotility, exaggerated TLR4 signaling and NEC development. We found that intestinal motility is reduced early in NEC in mice, preceding the onset of intestinal inflammation, whereas pharmacologic restoration of intestinal motility reduced NEC severity. Ileal samples from mouse, piglet and human NEC presented enteric glia depletion, and three strains of glia-deficient mice (Plp1(ΔDTR), Sox10(ΔDTR), Bdnf(ΔDTR)) showed increased NEC severity compared with wildtype control animals. Mice lacking TLR4 on enteric glia (Sox10-Tlr4(ko)) did not show NEC-induced enteric glia depletion and were protected from NEC. Mechanistically, brain-derived neurotrophic factor (BDNF) from enteric glia restrained TLR4 signaling on the intestine to prevent NEC. BDNF was reduced in mouse and human NEC, and BDNF administration reduced both TLR4 signaling and NEC severity in enteric glia-deficient mice. Finally, we identified an agent (J11) that enhanced enteric glial BDNF release, inhibited intestinal TLR4, restored motility and prevented NEC in mice. Thus, enteric glia loss might contribute to NEC through intestinal dysmotility and increased TLR4 activation, suggesting enteric glia therapies for this disorder.

Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal disease of the premature infant. We have recently shown that NEC development occurs after an increase in proinflammatory CD4+Th17 (Th17) cells and reduced anti-inflammatory Foxp3+ regulatory T cells (Tregs) to the premature small intestine of mice and humans, which can be experimentally reversed in mice by administration of all-trans retinoic acid (ATRA). We have also shown that NEC is characterized by apoptosis of Lgr5-positive intestinal stem cells (ISCs–Lgr5+cells) within the crypts of Lieberkühn, which are subsequently essential for intestinal homeostasis. We now hypothesize that the normal lymphocyte balance within the lamina propria of the intestine can be achieved via administration of ATRA which restores mucosal integrity by preventing the loss of intestinal stem cells. Utilizing both in vivo and in vitro strategies we now demonstrate that Th17 recruitment and Treg depletion lead to increased apoptosis within ISC niches, significantly impairing proliferative capacity and mucosal healing. ATRA exerted its protective effects by preventing T cell imbalance, ultimately leading to the protection of the ISC pool preventing the development of NEC in mice. These findings raise the exciting possibility that dietary manipulations could prevent and treat NEC by modulating lymphocyte balance and the ISC pool within the newborn small intestine.

Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can reduce NEC through inhibition of TLR4 signaling.NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2'-FL, 6'-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling.Supplementation of infant formula with either 2'-FL and/or 6'-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2'-FL and 6'-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2'-FL and 6'-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling.2'-FL and 6'-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition.Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2'-FL and 6'-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2'-FL and 6'-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2'-FL and 6'-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants, and is associated with the development of severe lung inflammation. The pathogenesis of NEC-induced lung injury remains unknown, yet infiltrating immune cells may play a role. In support of this possibility, we now show that NEC in mice and humans was associated with the development of profound lung injury that was characterized by an influx of Th17 cells and a reduction in T regulatory lymphocytes (Tregs). Importantly, the adoptive transfer of CD4 T cells isolated from lungs of mice with NEC into the lungs of immune incompetent mice (Rag1 mice) induced profound inflammation in the lung, while the depletion of Tregs exacerbated NEC induced lung injury, demonstrating that imbalance of Th17/Treg in the lung is required for the induction of injury. In seeking to define the mechanisms involved, the selective deletion of toll-like receptor 4 (TLR4) from the Sftpc1 pulmonary epithelial cells reversed lung injury, while TLR4 activation induced the Th17 recruiting chemokine (C-C motif) ligand 25 (CCL25) in the lungs of mice with NEC. Strikingly, the aerosolized inhibition of both CCL25 and TLR4 and the administration of all trans retinoic acid restored Tregs attenuated NEC-induced lung injury. In summary, we show that TLR4 activation in Surfactant protein C-1 (Sftpc1) cells disrupts the Treg/Th17 balance in the lung via CCL25 leading to lung injury after NEC and reveal that inhibition of TLR4 and stabilization of Th17/Treg balance in the neonatal lung may prevent this devastating complication of NEC.

Necrotizing enterocolitis (NEC) causes acute intestinal necrosis in premature infants and is associated with severe neurological impairment. In NEC, Toll-like receptor 4 is activated in the intestinal epithelium, and NEC-associated brain injury is characterized by microglial activation and white matter loss through mechanisms that remain unclear. We now show that the brains of mice and humans with NEC contained CD4(+) T lymphocytes that were required for the development of brain injury. Inhibition of T lymphocyte influx into the brains of neonatal mice with NEC reduced inflammation and prevented myelin loss. Adoptive intracerebroventricular delivery of gut T lymphocytes from mice with NEC into Rag1(−/−) recipient mice lacking CD4(+) T cells resulted in brain injury. Brain organoids derived from mice with or without NEC and from human neuronal progenitor cells revealed that IFN-γ release by CD4(+) T lymphocytes induced microglial activation and myelin loss in the organoids. IFN-γ knockdown in CD4(+) T cells derived from mice with NEC abrogated the induction of NEC-associated brain injury after adoptive transfer to naïve Rag1(−/−) recipient mice. T cell receptor sequencing revealed that NEC mouse brain-derived T lymphocytes shared homology with gut T lymphocytes from NEC mice. Intraperitoneal injection of NEC gut-derived CD4(+) T lymphocytes into naïve Rag1(−/−) recipient mice induced brain injury, suggesting that gut-derived T lymphocytes could mediate neuroinflammation in NEC. These findings indicate that NEC-associated brain injury may be induced by gut-derived IFN-γ–releasing CD4(+) T cells, suggesting that early management of intestinal inflammation in children with NEC could improve neurological outcomes.

Background & Aims Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by Toll-like receptor 4 (TLR4)-dependent intestinal inflammation and enterocyte death. Given that necroptosis is a proinflammatory cell death process that is linked to bacterial signaling, we investigated its potential role in NEC, and the mechanisms involved. Methods Human and mouse NEC intestine were analyzed for necroptosis gene expression (ie, RIPK1, RIPK3, and MLKL), and protein activation (phosphorylated RIPK3). To evaluate a potential role for necroptosis in NEC, the effects of genetic (ie, Ripk3 knockout or Mlkl knockout) or pharmacologic (ie, Nec1s) inhibition of intestinal inflammation were assessed in a mouse NEC model, and a possible upstream role of TLR4 was assessed in Tlr4-deficient mice. The NEC-protective effects of human breast milk and its constituent milk oligosaccharides on necroptosis were assessed in a NEC-in-a-dish model, in which mouse intestinal organoids were cultured as either undifferentiated or differentiated epithelium in the presence of NEC bacteria and hypoxia. Results Necroptosis was activated in the intestines of human and mouse NEC in a TLR4-dependent manner, and was up-regulated specifically in differentiated epithelium of the immature ileum. Inhibition of necroptosis genetically and pharmacologically reduced intestinal–epithelial cell death and mucosal inflammation in experimental NEC, and ex vivo in the NEC-in-a-dish system. Strikingly, the addition of human breast milk, or the human milk oligosaccharide 2 fucosyllactose in the ex vivo system, reduced necroptosis and inflammation. Conclusions Necroptosis is activated in the intestinal epithelium upon TLR4 signaling and is required for NEC development, and explains in part the protective effects of breast milk., Graphical abstract

We seek to define the mechanisms leading to the development of lung disease in the setting of neonatal necrotizing enterocolitis (NEC), a life-threatening gastrointestinal disease of premature infants characterized by the sudden onset of intestinal necrosis. NEC development in mice requires activation of the LPS receptor TLR4 on the intestinal epithelium, through its effects on modulating epithelial injury and repair. Although NEC-associated lung injury is more severe than the lung injury that occurs in premature infants without NEC, the mechanisms leading to its development remain unknown. In this study, we now show that TLR4 expression in the lung gradually increases during postnatal development, and that mice and humans with NEC-associated lung inflammation express higher levels of pulmonary TLR4 than do age-matched controls. NEC in wild-type newborn mice resulted in significant pulmonary injury that was prevented by deletion of TLR4 from the pulmonary epithelium, indicating a role for pulmonary TLR4 in lung injury development. Mechanistically, intestinal epithelial TLR4 activation induced high-mobility group box 1 release from the intestine, which activated pulmonary epithelial TLR4, leading to the induction of the neutrophil recruiting CXCL5 and the influx of proinflammatory neutrophils to the lung. Strikingly, the aerosolized administration of a novel carbohydrate TLR4 inhibitor prevented CXCL5 upregulation and blocked NEC-induced lung injury in mice. These findings illustrate the critical role of pulmonary TLR4 in the development of NEC-associated lung injury, and they suggest that inhibition of this innate immune receptor in the neonatal lung may prevent this devastating complication of NEC.

Necrotising enterocolitis (NEC) is a devastating disease that typically affects formula-fed premature infants, suggesting that dietary components may influence disease pathogenesis. TAG are the major fat components of infant formula, and their digestion requires pancreatic lipases, which may be naturally deficient in premature neonates. We hypothesise that NEC develops partly from the accumulation of incompletely digested long-chain TAG-containing unsaturated fatty acids within the intestinal epithelial cells, leading to oxidative stress and enterocyte damage. We further hypothesise that the administration of a formula that contains reduced TAG (‘pre-digested fat’) that do not require lipase action may reduce NEC severity. To test these hypotheses, we induced NEC in neonatal mice using three different fat formulations, namely ‘standard fat’, ‘pre-digested fat’ or ‘very low fat’, and determined that mice fed ‘standard fat’ developed severe NEC, which was significantly reduced in mice fed ‘pre-digested fat’ or ‘very low fat’. The expression level of the critical fat-digesting enzyme carboxyl ester lipase was significantly lower in the newborn compared with older pups, leading to impaired fat digestion. The accumulation of mal-digested fat resulted in the significant accumulation of fat droplets within the intestinal epithelium of the distal ileum, resulting in the generation of reactive oxygen species and intestinal inflammation. Strikingly, these changes were prevented in pups fed ‘pre-digested fat’ or ‘very low fat’ formulas. These findings suggest that nutritional formula containing a pre-digested fat system may overcome the natural lipase deficiency of the premature gut, and serve as a novel approach to prevent NEC.

Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease that is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), whereas selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Overexpression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of glycogen synthase kinase-3β (GSK3β). Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis.

The low-molecular weight isopropyl 2-acetamido-α-glucoside 16 (C34) inhibits toll-like receptor 4 (TLR4) in enterocytes and macrophages in vitro, and reduces systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. We used a copper(II)-mediated solvolysis of anomeric oxazolines and an acid-mediated conversion of β-glucosamine and β-galactosamine pentaacetates to generate analogs of 16 at the anomeric carbon and at C-4 of the pyranose ring. These compounds were evaluated for their influence on TLR4-mediated inflammatory signaling in cultured enterocytes and monocytes. Their efficacy was confirmed using a NF-kB-luciferase reporter mouse, thus establishing the first structure-activity relationship (SAR) study in this series and identifying the more efficacious isopropyl 2-acetamido-α-galactoside 17.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.

Little is known about the pathogenesis of metabolic syndrome, although Toll-like receptor 4 (TLR4) has been implicated. We investigated whether TLR4 in the intestinal epithelium regulates metabolic syndrome by coordinating interactions between the luminal microbiota and host genes that regulate metabolism. Mice lacking TLR4 in the intestinal epithelium (TLR4ΔIEC), but not mice lacking TLR4 in myeloid cells nor mice lacking TLR4 globally, developed metabolic syndrome; these features were not observed in TLR4ΔIEC mice given antibiotics. Metagenomic analysis of the fecal microbiota revealed differences between TLR4ΔIEC and wild type mice, while meta-transcriptome analysis of the microbiota showed that intestinal TLR4 affected the expression of microbial genes involved in the metabolism of lipids, amino acids, and nucleotides. Genes regulated by peroxisome proliferator-activated receptors (PPARs) and the antimicrobial peptide lysozyme were significantly down-regulated in TLR4ΔIEC mice, suggesting a mechanism by which intestinal TLR4 could exert its effects on the microbiota and metabolic syndrome. Supportingly, antibiotics prevented both downregulation of PPAR genes and the development of metabolic syndrome, while PPAR agonists prevented development of metabolic syndrome in TLR4ΔIEC mice. Thus, intestinal epithelial TLR4 regulates metabolic syndrome through altered host-bacterial signaling, suggesting that microbial or PPAR based strategies might have therapeutic potential for this disease.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease of the premature infant. One of the most important long-term complications observed in children who survive NEC early in life is the development of profound neurological impairments. However, the pathways leading to NEC-associated neurological impairments remain unknown, thus limiting the development of prevention strategies. We have recently shown that NEC development is dependent on the expression of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium, whose activation by bacteria in the newborn gut leads to mucosal inflammation. Here, we hypothesized that damage-induced production of TLR4 endogenous ligands in the intestine might lead to activation of microglial cells in the brain and promote cognitive impairments. We identified a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR4 signaling led to release of high-mobility group box 1 in the intestine that, in turn, promoted microglial activation in the brain and neurological dysfunction. We further demonstrated that an orally administered dendrimer-based nanotherapeutic approach to targeting activated microglia could prevent NEC-associated neurological dysfunction in neonatal mice. These findings shed light on the molecular pathways leading to the development of NEC-associated brain injury, provide a rationale for early removal of diseased intestine in NEC, and indicate the potential of targeted therapies that protect the developing brain in the treatment of NEC in early childhood.

Necrotising enterocolitis (NEC) is a common disease in premature infants characterised by intestinal ischaemia and necrosis. The only effective preventative strategy against NEC is the administration of breast milk, although the protective mechanisms remain unknown. We hypothesise that an abundant human milk oligosaccharide (HMO) in breast milk, 2′-fucosyllactose (2′FL), protects against NEC by enhancing intestinal mucosal blood flow, and we sought to determine the mechanisms underlying this protection. Administration of HMO-2′FL protected against NEC in neonatal wild-type mice, resulted in a decrease in pro-inflammatory markers and preserved the small intestinal mucosal architecture. These protective effects occurred via restoration of intestinal perfusion through up-regulation of the vasodilatory molecule endothelial nitric oxide synthase (eNOS), as administration of HMO-2′FL toeNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC, and by 16S analysis HMO-2′FL affected the microbiota of the neonatal mouse gut, although these changes do not seem to be the primary mechanism of protection. Induction of eNOS by HMO-2′FL was also observed in cultured endothelial cells, providing a link between eNOS and HMO in the endothelium. These data demonstrate that HMO-2′FL protects against NEC in part through maintaining mesenteric perfusion via increased eNOS expression, and suggest that the 2′FL found in human milk may be mediating some of the protective benefits of breast milk in the clinical setting against NEC.

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)—a devastating inflammatory disease of the premature intestine—upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR- or peroxisome proliferator-activated receptor γ–deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.

Background & Aims Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of β-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired β-catenin signaling. Methods Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4 −/− mice after induction of NEC or endotoxemia. β-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of β-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. Results TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. β-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3β, causing β-catenin degradation. NEC in both mouse and humans was associated with decreased β-catenin and increased mucosal GSK3β expression. Strikingly, the inhibition of enterocyte β-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. Conclusion We now report a novel pathway linking TLR4 with inhibition of β-catenin signaling via GSK3β activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.

The low-molecular weight isopropyl 2-acetamido-α-glucoside 16 (C34) inhibits toll-like receptor 4 (TLR4) in enterocytes and macrophages in vitro, and reduces systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. We used a copper(II)-mediated solvolysis of anomeric oxazolines and an acid-mediated conversion of β-glucosamine and β-galactosamine pentaacetates to generate analogs of 16 at the anomeric carbon and at C-4 of the pyranose ring. These compounds were evaluated for their influence on TLR4-mediated inflammatory signaling in cultured enterocytes and monocytes. Their efficacy was confirmed using a NF-kB-luciferase reporter mouse, thus establishing the first structure-activity relationship (SAR) study in this series and identifying the more efficacious isopropyl 2-acetamido-α-galactoside 17.

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS −/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate—a precursor for enteral generation of nitrite and nitric oxide—and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate–nitrite–NO signaling.

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.

Background & Aims Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. Methods Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout ( TLR4 −/− ) mice were generated by breeding TLR4 loxp/loxp mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. Results TLR4 −/− mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. Conclusions TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.

Intestinal mucosal injury occurs after remote trauma although the mechanisms that sense remote injury and lead to intestinal epithelial disruption remain incompletely understood. We now hypothesize that Toll-like receptor 4 (TLR4) signaling on enterocytes after remote injury, potentially through the endogenous TLR4 ligand high-mobility group box-1 (HMGB1), could lead to intestinal dysfunction and bacterial translocation and that activation of TLR9 with DNA could reverse these effects. In support of this hypothesis, exposure of TLR4-expressing mice to bilateral femur fracture and systemic hypotension resulted in increased TLR4 expression and signaling and disruption of the ileal mucosa, leading to bacterial translocation, which was not observed in TLR4-mutant mice. TLR4 signaling in enterocytes, not immune cells, was required for this effect, as adenoviral-mediated inhibition of TLR4 in enterocytes prevented these findings. In seeking to identify the endogenous TLR4 ligands involved, the expression of HMGB1 was increased in the intestinal mucosa after injury in wild-type, but not TLR4-mutant, mice, and administration of anti-HMGB1 antibodies reduced both intestinal mucosal TLR4 signaling and bacterial translocation after remote trauma. Strikingly, mucosal injury was significantly increased in TLR9-mutant mice, whereas administration of exogenous DNA reduced the extent of TLR4-mediated enterocyte apoptosis, restored mucosal healing, and maintained the histological integrity of the intestinal barrier after remote injury. Taken together, these findings identify a novel link between remote injury and enterocyte TLR4 signaling leading to barrier injury, potentially through HMGB1 as a ligand, and demonstrate the reversal of these adverse effects through activation of TLR9.

Background & Aims Factors that regulate enterocyte apoptosis in necrotizing enterocolitis (NEC) remain incompletely understood, although Toll-like receptor-4 (TLR4) signaling in enterocytes plays a major role. Nucleotide-binding oligomerization domain-2 (NOD2) is an immune receptor that regulates other branches of the immune system, although its effects on TLR4 in enterocytes and its role in NEC remain unknown. We now hypothesize that activation of NOD2 in the newborn intestine inhibits TLR4, and that failure of NOD2 signaling leads to NEC through increased TLR4-mediated enterocyte apoptosis. Methods The effects of NOD2 on enterocyte TLR4 signaling and intestinal injury and repair were assessed in enterocytes lacking TLR4 or NOD2, in mice with intestinal-specific wild-type or dominant-negative TLR4 or NOD2, and in mice with NEC. A protein array was performed on NOD2-activated enterocytes to identify novel effector molecules involved. Results TLR4 activation caused apoptosis in newborn but not adult small intestine or colon, and its intestinal expression was influenced by NOD2. NOD2 activation inhibited TLR4 in enterocytes, but not macrophages, and reversed the effects of TLR4 on intestinal mucosal injury and repair. Protection from TLR4-induced enterocyte apoptosis by NOD2 required a novel pathway linking NOD2 with the apoptosis mediator second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (SMAC-DIABLO), both in vitro and in vivo. Strikingly, activation of NOD2 reduced SMAC-DIABLO expression, attenuated the extent of enterocyte apoptosis, and reduced the severity of NEC. Conclusions These findings reveal a novel inhibitory interaction between TLR4 and NOD2 signaling in enterocytes leading to the regulation of enterocyte apoptosis and suggest a therapeutic role for NOD2 in the protection of intestinal diseases such as NEC.