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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 proinflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL) and 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we hypothesized that oral administration of these HMOs would 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 brain-derived neurotrophic factor (BDNF), as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. Taken in aggregate, these findings reveal that the HMOs 2'-FL and 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury. NEW & NOTEWORTHY This study reveals that the administration of human milk oligosaccharides, which are present in human breast milk, can interfere with the proinflammatory gut-brain axis and prevent neuroinflammation in the setting of necrotizing enterocolitis, a major intestinal disorder seen in premature infants.

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 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. NEW & NOTEWORTHY This work provides hope for children who develop NEC, a devastating disease of premature infants that is often fatal, by revealing that the secreted product of amniotic progenitor cells (called ST266) can prevent or treat NEC in mice, piglet, and "NEC-in-a-dish" models of this disease. Mechanistically, ST266 prevented bacterial signaling, and a detailed transcriptomic analysis revealed effects on gut differentiation, immunity, and metabolism. Thus, an amniotic secretome may offer novel approaches for NEC.

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 that 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 revealed enteric glia depletion, and glia-deficient mice ( Plp1 ΔDTR , Sox10 ΔDTR , and Bdnf ΔDTR ) showed increased NEC severity compared with wild-type mice. 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. Last, 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 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) 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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: 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., Methods: 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., 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., Conclusions: 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., Impact: 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.

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)