Background & Aims: Necrotizing enterocolitis (NEC) is a life-threatening intestinal disease of preterm infants that causes epithelial injury and necrosis. Prematurity and postnatal formula administration are known contributors, but maternal nutrition may also influence offspring gut susceptibility. The biological relationship between maternal diet and neonatal intestinal vulnerability remains unclear. Methods: A maternal high-fat diet (HFD) mouse model with a neonatal NEC model were used to investigate how maternal HFD affects offspring outcomes. Bulk RNA sequencing of ileal tissue before NEC induction was used to assess baseline transcription profiles. Following NEC, survival rate was assessed with ileal histological injury, epithelial reactive oxygen species, and mitochondrial membrane depolarization, as well as ultrastructural characteristics on transmission electron microscopy. Results: Compared with standard diet, offspring born from HFD mothers showed altered mitochondria-related gene expression, shorter villi, and reduced MUC2+ goblet cells. Transmission electron microscopy showed region-specific mitochondria abnormalities, with crypt enterocytes showing reduced mitochondrial volume density and size, and increased mitochondria-endoplasmic reticulum contact site after NEC induction. Strikingly, HFD increased mortality and elevated epithelial reactive oxygen species after the induction of NEC, linking maternal dietary risk factors with NEC development. Conclusions: Maternal HFD disrupts mitochondrial architecture and transcriptional programming in the neonatal intestine, promoting a pre-injured state that increases NEC susceptibility, suggesting that maternal diet may be a modifiable target for early NEC prevention.

BACKGROUND:Necrotizing enterocolitis (NEC) is mediated by toll-like receptor 4 (TLR4)-induced inflammation and is preceded by reduced intestinal motility. Human milk oligosaccharides (HMOs) are non-digestible components of breast milk that prevent NEC in preclinical models. We now hypothesize that HMOs can reduce the risk of NEC through restoration of intestinal motility and reduced TLR4-mediated inflammation. METHODS:NEC was induced in C57-BL/6 mice through the combination of formula gavage, hypoxia, and oral administration of NEC stool. Mice were administered either 2'-FL (5 g/L), 6'-SL (5 g/L), or a blend of 5 specific HMOs (5 g/L) containing 2'-FL (2.606 g/L), 3'-FL (0.652 g/L), LNT (1.304 g/L), 3'-SL (0.174 g/L), and 6'-SL (0.260 g/L). Gastrointestinal motility was assessed by 70 Kd FITC-dextran transit time. Enteric glia were quantified by immunohistochemistry and qRT-PCR expression. RESULTS:Administration of either 2'-FL, 6'-SL, or HMO blend significantly attenuated NEC severity and reversed intestinal hypomotility. HMOs prevented enteric glia loss and regulated key genes critical for enteric glia maintenance, attenuated pro-apoptotic genes, and increased anti-apoptotic genes in vitro, resulting in a reduction in apoptosis. Strikingly, HMOs reduced LPS-TLR4-induced NFκB signaling and ROS generation in enteric glia. CONCLUSIONS:HMOs protect against NEC at least in part through protective effects on inflammation and the enteric nervous system. IMPACT:This study sheds light on the role of certain human milk oligosaccharides in a clinically relevant mouse model of NEC and adds additional insights into their underlying mechanism of action by revealing a protective effect on the enteric nervous system. These results reveal that HMOs prevent the loss of enteric glia in NEC and influence the expression of genes that regulate enteric glia maintenance. HMOs also limit TLR4-NFkB signaling, providing an additional mechanism of enteric glia maintenance.

BACKGROUND AND AIMS:Necrotizing enterocolitis (NEC) is a life-threatening condition in premature infants, marked by acute intestinal necrosis. NEC develops in part after activation of the lipopolysaccharide receptor toll-like receptor 4 (TLR4) by intestinal microbes in the intestinal epithelium. Previous authors have shown an increased risk of NEC in human infants after cytomegalovirus (CMV) infection, which can affect mitochondrial function. We now seek to explore the impact and the mechanisms of CMV infection on NEC severity and its relationship with TLR4 signaling and mitochondria function. METHODS:NEC was induced in newborn mice with and without CMV infection. RNA sequencing and gene set enrichment analysis were performed to identify effects on inflammatory and metabolic pathways. The role of TLR4 signaling and mitochondrial function were investigated in wild-type and Tlr4-deficient mice. The adenosine receptor agonist 5'-N-ethylcarboxamido adenosine was tested for its ability to reduce CMV-induced effects on NEC severity. RESULTS:CMV infection significantly increased NEC severity in wild-type mice. Mechanistically, CMV infection triggered proinflammatory pathways, disrupted cellular metabolism, and upregulated Tlr4 expression, leading to mitochondrial dysfunction and nuclear factor-kB translocation. These effects were notably absent in Tlr4-deficient mice. 5'-N-ethylcarboxamido adenosine treatment reversed CMV-induced NEC severity by reducing mitochondrial dysfunction and TLR4-driven nuclear factor-kB activation. CONCLUSIONS:CMV infection worsens NEC severity in mice by amplifying TLR4 signaling, inflammation, and mitochondrial dysfunction. Targeting CMV and its influence on TLR4 may offer novel therapeutic approaches for NEC.

Electronic cigarettes, also known as e-cigarettes or vapes, are battery-operated devices that heat and produce aerosols for inhalation. Maternal nicotine e-cigarette use during pregnancy can have detrimental effects on fetal growth and development, which may be in part due to deleterious effects of nicotine vapor exposure on innate immunity of the developing fetus tethered to the harmful chemicals and toxins present in the vaping device. Understanding these effects is crucial for identifying potential risks to fetal health and developing interventions that may mitigate the impact of maternal stress on the developing immune system. We hypothesized that murine maternal exposure to nicotine e-cigarette vapor would lead to neonatal innate immune activation. Our preliminary studies showed that pregnant mice chronically (five times per week, from gestational day 14 through 21, or birth) exposed to e-cigarette vapor in-utero exhibited alterations in inflammatory response, as demonstrated through the increased production of pro-inflammatory cytokines in the gut, including TLR4, IL-6, TNF, and IL-1b, by RT-PCR. We also show the activation of neonatal immune cells, including microglia in the brain and neutrophils in the lung via immunohistochemistry. Consistent with our hypothesis, these findings reveal maternal vaping induces immunological signaling and leads to innate immune activation in neonatal mouse lung, gut, and brain, suggesting a role in adverse immunological outcomes. Supported by NIH/NIGMS R35GM141956; Vivien Thomas Scholars Initiative. Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Short bowel syndrome (SBS) leads to severe morbidity and mortality. Intestinal adaptation is crucial in improving outcomes. To understand the human gene pathways associated with adaptation, we perform single-cell transcriptomic analysis of human small intestinal organoids explanted from mice with experimental SBS. We show that transmembrane ion pathways, specifically the transepithelial zinc transport pathway genes SLC39A4 and SLC39A5, are upregulated in SBS. This discovery is corroborated by an external dataset, bulk RT-qPCR, and Western blots. Oral zinc supplementation is shown to improve survival and weight gain of SBS mice and increase the proliferation of intestinal crypt cells in vitro. Finally, we identify the upregulation of SLC39A5 and associated transcription factor KLF5 in biopsied intestinal tissue specimens from patients with SBS. Thus, we identify zinc supplementation as a potential therapy for SBS and describe a xenotransplantation model that provides a platform for discovery in other intestinal diseases. SBS leads to severe morbidity and mortality. Here, using a xenotransplantation model, the authors uncover the role of zinc transport pathways in intestinal adaptation in SBS, highlighting the potential of zinc supplementation as a therapeutic approach.

BACKGROUND & AIMS: The abdominal discomfort experienced by patients with colitis may be attributable in part to the presence of small intestinal dysmotility, yet mechanisms linking colonic inflammation with small-bowel motility remain largely unexplored. We hypothesize that colitis results in small intestinal hypomotility owing to a loss of enteroendocrine cells (EECs) within the small intestine that can be rescued using serotonergic-modulating agents. METHODS: Male C57BL/6J mice, as well as mice that over- express (EECOVER) or lack (EECDEL) NeuroD1 & thorn; enteroendocrine cells, were exposed to dextran sulfate sodium (DSS) colitis (2.5% or 5% for 7 days) and small intestinal motility was assessed by 70-kilodalton fluorescein isothiocyanate-dextran fluorescence transit. EEC number and differentiation were evaluated by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, and quantitative reversetranscriptase polymerase chain reaction. Mice were treated with the 5-hydroxytryptamine receptor 4 agonist prucalopride (5 mg/kg orally, daily) to restore serotonin signaling. RESULTS: DSS-induced colitis was associated with a significant small-bowel hypomotility that developed in the absence of significant inflammation in the small intestine and was associated with a significant reduction in EEC density. EEC loss occurred in conjunction with alterations in the expression of key serotonin synthesis and transporter genes, including Tph1, Ddc, and Slc6a4. Importantly, mice overexpressing EECs revealed improved small intestinal motility, whereas mice lacking EECs had worse intestinal motility when exposed to DSS. Finally, treatment of DSSexposed mice with the 5-hydroxytryptamine receptor 4 agonist prucalopride restored small intestinal motility and attenuated colitis. CONCLUSIONS: Experimental DSS colitis induces significant small-bowel dysmotility in mice owing to enteroendocrine loss that can be reversed by genetic modulation of EEC or administering serotonin analogs, suggesting novel therapeutic approaches for patients with symptomatic colitis.

BackgroundProbiotic administration may decrease the incidence of necrotizing enterocolitis (NEC) through mechanisms that are largely unknown. We investigated the effects of probiotics on intestinal epigenetics and assessed their effects on intestinal inflammation and motility using both ileum-predominant and combined ileo-colitis mouse NEC models.MethodsC57BL/6 J mice were gavage-fed a multi-strain probiotic from postnatal days 3-11, consisting of B. infantis, B. lactis, and S. thermophilus. From p8, mice were exposed to ileo-colitis NEC involving formula containing NEC bacteria and 0.5% DSS. DNA methylation was measured using the Infinium Methylation Assay. Gastrointestinal motility was assessed by 70 Kd FITC-dextran transit time. Probiotic colonization was measured in probiotic-fed mice by qPCR.ResultsProbiotic administration caused significant changes in the small intestine's epigenetic signature, a reduction in NEC severity, and improved intestinal motility. The effects of probiotics were more pronounced in the ileo-colitis NEC model.ConclusionsThese findings shed light on the role of probiotics in two clinically relevant models of NEC, add additional insights into their underlying mechanism of action, and reveal unanticipated epigenetic modifications to the intestinal mucosa after their use.ImpactThese findings shed light on the role of multi-strain probiotics in two clinically relevant animal models of NEC, and add additional insights into their underlying mechanism of actionThis study provides a new, clinically relevant model for the study of NEC including administration of 0.5% DSS, to include ileal dominant and ileo-colonic dominant phenotypes of the disease.These results reveal that clinically relevant strains of probiotic bacteria can exert epigenetic effects on the small intestine in mice, and can attenuate the epigenetic changes induced by NEC.

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.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily ris-ing in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel preven-tion 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 is-chemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC develop-ment 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 epi-thelial 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 remodel-ing, 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.

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.

Brain injury in necrotizing enterocolitis requires an influx of gut-derived IFN-γ–releasing T lymphocytes into the developing mouse and human brain.

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.