Neonatal Intestine Research Articles

Neonatal Enteric Glia Enhance Intestinal Epithelial Restitution in vitro Following Exposure to Sterile Colonic Luminal Content of Mature but not Neonatal Pigs

Neonates exhibit significantly poorer outcomes from damage to the intestinal epithelium following ischemia for unknown reasons. In our pig model, we recently discovered an age-dependent epithelial restitution defect in ischemia-injured neonatal intestine that is rescued by application of homogenized juvenile mucosa, but the mechanism remains unknown. Enteric glial cells (EGC) have been shown to promote epithelial restitution following injury via paracrine signaling and are abundant in the intestinal mucosa of juvenile but not neonatal pigs. Co-culture studies have shown that pig EGC enhance restitution in epithelial monolayers following scratch wounding. In mice, postnatal maturation and maintenance of the EGC network is purportedly driven by colonization of gut microbiota. Furthermore, weaning of neonates leads to drastic shifts in the dynamics and populations of these gut microbiota. Therefore, we believe that changes to pig intestinal microbiota during weaning may play a key role in EGC maturation required for proper epithelial restitution. We hypothesized that treatment with mature (>6 weeks of age), but not neonatal (2 weeks of age), luminal content would improve restitution of neonatal epithelial monolayers co-cultured with neonatal EGC. We compared the effect of sterile-filtered neonatal or mature luminal content addition to the apical chamber on scratch wound restitution in neonatal porcine IPEC-J2 monolayers in monoculture or co-culture with primary porcine neonatal submucosal EGC using a transwell system (n = 9). Our results support a significant effect of luminal content treatment in the presence of EGC (P=0.0085). Specifically, mature luminal content treatment enhanced IPEC-J2 wound closure in co-culture with EGC but not in IPEC-J2 monoculture (P=0.0156). EGC co-culture alone or treated with neonatal lumen content did not affect restitution. Both neonatal and mature luminal content treatment decreased restitution in monoculture, but this effect was not significant. These data suggest that EGC provide secretory signals to the intestinal epithelium that promote repair in response to a component of mature luminal content, which we believe to be microbial products. Future work aims to determine what is driving these differences in restitution by analyzing neonatal and mature luminal secretomes. NIH 5 T35 OD 11070-12; NIH K01 OD 028207; NIH P30 DK 034987; NIH-NICHD R01 HD095876; USDA-NIFA VMCG-0065. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Different probiotic strains alter human cord blood monocyte responses

BackgroundProbiotics have a protective effect on various diseases. In neonatology, they are predominantly used to prevent necrotising enterocolitis (NEC), a severe inflammatory disease of the neonatal intestine. The mechanisms by which probiotics act are diverse; little is known about their direct effect on neonatal immune cells.MethodsIn this study, we investigated the effect of probiotics on the functions of neonatal monocytes in an in vitro model using three different strains (Lactobacillus rhamnosus (LR), Lactobacillus acidophilus (LA) and Bifidobacterium bifidum (BB)) and mononuclear cells isolated from cord blood.ResultsWe show that stimulation with LR induces proinflammatory effects in neonatal monocytes, such as increased expression of surface molecules involved in monocyte activation, increased production of pro-inflammatory and regulatory cytokines and increased production of reactive oxygen species (ROS). Similar effects were observed when monocytes were stimulated simultaneously with LPS. Stimulation with LA and BB alone or in combination also induced cytokine production in monocytes, with BB showing the least effects.ConclusionsOur results suggest that probiotics increase the defence functions of neonatal monocytes and thus possibly favourably influence the newborn’s ability to fight infections.ImpactProbiotics induce a proinflammatory response in neonatal monocytes in vitro.This is a previously unknown mechanism of how probiotics modulate the immune response of newborns.Probiotic application to neonates may increase their ability to fight off infections.

Maf family transcription factors are required for nutrient uptake in the mouse neonatal gut.

There are fundamental differences in how neonatal and adult intestines absorb nutrients. In adults, macromolecules are broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely on internalization of whole macromolecules and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors MAFB and c-MAF as markers of terminally differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α and HNF4γ, master regulators of enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of macromolecular nutrient uptake. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number, in response to loss of Maf proteins. Finally, we show that loss of BLIMP1, a repressor of adult enterocyte genes, shows highly overlapping changes in gene expression and similar defects in macromolecular uptake. This work defines transcriptional regulators that are necessary for nutrient uptake in neonatal enterocytes.

Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction.

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

Effect of Breastmilk Microbiota and Sialylated Oligosaccharides on the Colonization of Infant Gut Microbial Community and Fecal Metabolome.

The complex microbiota and sialylated oligosaccharides in breastmilk are important bioactive components that affect the gut microbiota. However, the effect of breastmilk microbiota and sialylated oligosaccharides on the gut microbiota during the neonatal period has been largely overlooked. Here, 16S rRNA gene sequencing and metabolomics analysis were applied to the breastmilk and feces of 69 newborns to clarify the link between breastmilk components and the newborn gut. Results showed that Staphylococcus, Enterococcus, and Bacteroides were commonly shared and positively correlated between breastmilk and the neonatal intestine and they were the main bacteria of breastmilk that interacted with the newborn fecal metabolome. Breastmilk Staphylococcus mainly interacted with amino acids, whereas Bacteroides was involved in the tryptophan, nucleotide, and vitamin metabolism. Breastmilk sialylated oligosaccharides were related to Bacteroides and amino acids of the newborn fecal metabolites. Moreover, Bacteroides was related to the interaction between breastmilk 3'-sialyllactose and newborn fecal metabolites in the mediation effect models. Finally, we pointed out that breastmilk Bacteroides was important in the milk-gut interaction, and it was negatively associated with waist circumference in infants aged 1 year. Our study provides a scientific basis for understanding the role of breastmilk in the development of newborn gut microbiota and metabolome.

Circumumbilical incision for neonatal abdominal surgery: additional skin incision when there is difficulty in manipulating the intestine.

This study aimed to evaluate the safety and effectiveness of circumumbilical incision (CUI) for neonates requiring intestinal anastomosis. Seventy neonates requiring intestinal anastomosis at our institution between 2003 and 2020 were included in this retrospective case-control study. Patients were classified into the CUI (25 patients: 36%) and transverse incision (TI) groups (45 patients: 64%). Postoperative complications and surgical outcomes were compared between the two groups. Intestinal perforation at the non-anastomotic site occurred significantly more often in the CUI group than in the TI group (3 patients: 12%, and 0 patients: 0%, respectively (p = 0.042)). There were no between-group differences regarding anastomotic leakages, anastomotic strictures, time to enteral feeding, operative time, and blood loss. Neonatal intestinal surgery employing CUI might be associated with increased intestinal perforation at the non-anastomotic site. Hesitating to enlarge the skin incision to maintain favorable cosmetic outcomes might cause severe injury to the delicate neonatal intestine during the surgical procedure owing to the restricted surgical field. When performing CUI, we suggest that the skin incision should be extended without hesitation whenever there is difficulty in manipulating the intestine.

Pharmacodynamic characterization of rytvela, a novel allosteric anti-inflammatory therapeutic, to prevent preterm birth and improve fetal and neonatal outcomes

Preterm birth is the leading cause of neonatal morbidity and mortality. Studies have shown that interleukin 1 plays a major role in the pathophysiology of preterm birth by inducing the production of proinflammatory mediators and uterine activation proteins leading to labor. More importantly, uteroplacental inflammation, associated with preterm birth parturition pathways, is detrimental to fetal tissues and leads to long-term sequelae. Our group has developed an allosteric antagonist of the interleukin 1 receptor, rytvela, found to be potent and safe in preventing preterm birth by suppressing inflammation via the inhibition of the mitogen-activated protein kinase pathway while preserving the Nuclear factor kappa B pathway (important in immune vigilance). Rytvela has been shown to inhibit inflammatory up-regulation and uterine activation while preserving fetal development. This study aimed to further the preclinical development of rytvela by evaluating its optimal dose and minimal duration of treatment to inhibit the inflammatory cascade, prolong gestation, and promote neonatal outcomes. Pregnant CD-1 mice were administered with lipopolysaccharide (10 μg, intraperitoneal administration) or interleukin 1 (1 μg/kg, intrauterine administration) on gestational day 16 to induce preterm labor. Rytvela was administered at different doses (0.1, 0.5, 1.0, 2.0, 4.0 mg/kg/d subcutaneously) from gestational days 16 to 18.5. To evaluate the minimal duration of treatment, the mice were administered with rytvela (2 mg/kg/d subcutaneously) for 24, 36, or 48 hours. The rate of prematurity (gestational day <18.5) and neonate survival and weightwere evaluated. Gestational tissues were collected at gestationalday 17.5 to quantify cytokines, proinflammatory mediators, and uterine activating proteins by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The neonatal lungs and intestines were collected from postnatal days 5 to 7 and analyzed by histology. Rytvela exhibited a dose-response profile and achieved maximum efficacy at a dose of 2 mg/kg/d by reducing 70% of lipopolysaccharide-induced preterm births and 60% of interleukin 1β-induced preterm births. In addition, rytvela attained maximum efficacy at a dose of 1 mg/kg/d by increasing neonate survival by up to 65% in both models of preterm birth. Rytvela protected fetuses from inflammatory insult as of 24 hours, preserving lung and intestinal integrity, and prevented preterm birth and fetal mortality by 60% and 50%, respectively, as of 36 hours of treatment. The maximum efficacy of rytvela was achieved at 2 mg/kg/d with improved birth outcomes and prevented inflammatory up-regulation upon 36 hours (only) of treatment. Rytvela exhibited desirable properties for the safe prevention of preterm birth and fetal protection.

Early life dynamics of ARG and MGE associated with intestinal virome in neonatal piglets

The pre- and post-weaning stages for piglets are critical periods for the maturation of intestinal functions and contamination with antibiotic resistant bacterial pathogens will threaten their intestinal health. The presence of bacteriophage can also alter bacterial populations in the intestine but whether transmission of antibiotic resistance genes (ARG) is affected by phage during maturation of the neonatal piglet intestine is not known. We therefore identified the intestinal virome along with ARGs and mobile genetic elements (MGE) from piglet fecal samples collected from 3 to 28 days representing the different growth stages. We found wide fluctuations for the intestinal virome of weaning piglets and most virus - related antibiotic resistance was derived from temperate phage suggesting a reservoir of multidrug resistance was present in the neonatal porcine gut. Our results provide a comprehensive understanding of ARGs associated with the intestinal virome that therefore represents a potential risk for horizontal ARG transfer to pathogenic bacteria.

Temporal and spatial development of intestinal smooth muscle layers of human embryos and fetuses.

The sequential occurrence of three layers of smooth muscle layers (SML) in human embryos and fetus is not known. Here, we investigated the process of gut SML development in human embryos and fetuses and compared the morphology of SML in fetuses and neonates. The H&E, Masson trichrome staining, and Immunohistochemistry were conducted on 6-12 gestation week human embryos and fetuses and on normal neonatal intestine. We showed that no lumen was seen in 6-7th gestation week embryonic gut, neither gut wall nor SML was developed in this period. In 8-9th gestation week embryonic and fetal gut, primitive inner circular SML (IC-SML) was identified in a narrow and discontinuous gut lumen with some vacuoles. In 10th gestation week fetal gut, the outer longitudinal SML (OL-SML) in gut wall was clearly identifiable, both the inner and outer SML expressed α-SMA. In 11-12th gestation week fetal gut, in addition to the IC-SML and OL-SML, the muscularis mucosae started to develop as revealed by α-SMA immune-reactivity beneath the developing mucosal epithelial layer. Comparing with the gut of fetuses of 11-12th week of gestation, the muscularis mucosae, IC-SML, and OL-SML of neonatal intestine displayed different morphology, including branching into glands of lamina propria in mucosa and increased thickness. In conclusions, in the human developing gut between week-8 to week-12 of gestation, the IC-SML develops and forms at week-8, followed by the formation of OL-SML at week-10, and the muscularis mucosae develops and forms last at week-12.

Maternal gut microbiome-induced IgG regulates neonatal gut microbiome and immunity.

The gut microbiome elicits antigen-specific immunoglobulin G (IgG) at steady state that cross-reacts to pathogens to confer protection against systemic infection. The role of gut microbiome-specific IgG antibodies in the development of the gut microbiome and immunity against enteric pathogens in early life, however, remains largely undefined. In this study, we show that gut microbiome-induced maternal IgG is transferred to the neonatal intestine through maternal milk via the neonatal Fc receptor and directly inhibits Citrobacter rodentium colonization and attachment to the mucosa. Enhanced neonatal immunity against oral C. rodentium infection was observed after maternal immunization with a gut microbiome-derived IgG antigen, outer membrane protein A, or induction of IgG-inducing gut bacteria. Furthermore, by generating a gene-targeted mouse model with complete IgG deficiency, we demonstrate that IgG knockout neonates are more susceptible to C. rodentium infection and exhibit alterations of the gut microbiome that promote differentiation of interleukin-17A-producing γδ T cells in the intestine, which persist into adulthood and contribute to increased disease severity in a dextran sulfate sodium-induced mouse model of colitis. Together, our studies have defined a critical role for maternal gut microbiome-specific IgG antibodies in promoting immunity against enteric pathogens and shaping the development of the gut microbiome and immune cells in early life.

Gut microbiome regulates serotonin production in the neonatal intestine to promote immune tolerance in early life

Abstract The gut microbiome in neonates is vastly different from that in adults due to dietary changes and gut maturation. However, the neonatal gut metabolome, largely shaped by gut bacteria, remains largely undefined. Using high-throughput metabolomics, we compared &amp;gt;500 metabolites in the ileum of specific pathogen-free (SPF) wild-type neonatal and adult mice. We found that most metabolites at significantly higher levels in neonates were neurotransmitters, including serotonin (5-HT), a neurotransmitter critical in the regulation of gut motility and mood stabilization. SPF neonates had significantly higher 5-HT levels in the ileum compared to both SPF adults and germ-free (GF) neonates, suggesting the gut microbiome as a key driver of generation in the neonatal intestine. Gut bacteria in neonates modulate the enzymes TPH-1 and MAO-A involved in the synthesis and breakdown of 5-HT. In addition, unlike in the adult intestine where 5-HT is produced primarily by enterochromaffin cells, gut bacteria are major producers of 5-HT in the neonatal intestine. The immunomodulatory role of 5-HT in the neonatal intestine has not been explored. We demonstrate that 5-HT directly promotes the differentiation regulatory T cells while diminishing interferon gamma and interleukin-17 produced in CD4 T cells, ex vivo and in vivo, in the neonatal intestine. Oral gavage of 5-HT into neonatal mice enhanced immune tolerance toward both commensal bacteria and dietary antigens. Our study has elucidated unique mechanisms by gut microbiota to regulate 5-HT generation in the neonatal intestine, and a novel immunomodulatory role for intestinal 5-HT to promote immune tolerance towards both dietary antigens and commensal bacteria during the early developmental stage.

CD206+MHCII− macrophages present in the neonatal but not adult intestine do not derive from LysM monocytes and are decreased in experimental Necrotizing Enterocolitis

Abstract Necrotizing enterocolitis (NEC) is an intestinal disease targeting premature infants. While not well understood, its pathogenesis involves an underdeveloped intestinal immune system. In the neonate, this system is primarily innate, with Cx3cr1+ intestinal macrophages (Mφ) being the predominant population. In the murine neonatal intestine, the Cx3cr1+ compartment contains embryonic self-renewing Mφ (Ly6C−CD64+MHCII+Tim4+) that are largely replaced by monocyte-derived-Mφ at weaning, while only a small population of Tim4+ cells persist in adults. However, how inflammation affects the neonatal intestinal Mφ compartment is not known. Using flow cytometry, we found the neonatal but not adult murine intestine to contain a population of Cx3cr1HiCD64HiMHCII− cells (30.62% ± 1.6 of Cx3cr1+ cells). 28.7% ± 1.8 of these cells express CD206, a known marker of embryonic origin in other tissues. While 34.4% ± 1.2 of total CD206+ cells expressed Tim4 in dam fed pups, it increased to 49.3% ± 0.51 p≤ 0.01 in pups exposed to experimental NEC for 24 hours. This CD206+Tim4+ subset was mainly MHCII+. When Cx3cr1-expressing cells were depleted (Cx3cr1-Cre CSFR1-DTR mice), all Cx3cr1-expressing cells were depleted, including CD206+MHCII−and+, but not Ly6C+ monocytes. In contrast, when LysM-Cre was used (LysM-Cre CSFR1-DTR mice), pups had decreased Ly6C+ monocytes but the CD206+MHCII−and+ populations were not depleted. These results were confirmed by immunofluorescent staining for CD206 and IBA-1. Thus, we conclude that neonatal intestinal CD206+MHCII−or+ cells do not derive from LysM+ monocytes. We speculate that CD206+MHCII− cells acquire MHCII during NEC thus decreasing the CD206+MHCII− pool. Supported by NIH DK116568 (IDP)

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

SUMMARYResident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Group B Streptococcus does not translocate the gut epithelium of neonatal mice.

Abstract Neonatal sepsis remains a leading cause of death in preterm and low-birthweight infants and can be separated into two groups: early-onset sepsis (EOS) and late-onset sepsis (LOS). Previous studies have shown that LOS can result from translocation of enteric bacteria across the intestinal epithelium, resulting in bloodstream infection. Through metagenomic sequencing we have observed, unsurprisingly, potential sepsis-causing organisms such as Escherichia coli, Staphylococcus aureus, Enterobacter cloacae, and Group B Streptococcus (GBS) in the stool of neonatal intensive care unit patients. Our previous work has shown E. coli can disseminate from the intestines via goblet cells in a model of LOS, but how other bacterial pathogens disseminate remains unclear. GBS is often associated with EOS following in utero infections and vertical transmission from the mother during pregnancy and delivery. However, the ability of gut-residing GBS to cause sepsis in a murine model of LOS is still unknown. Here we infected five-day-old mice by oral gavage with different strains of GBS, but we did not observe GBS translocating the intestinal epithelium in the pups, unlike E. coli. We next induced EOS in newborn mice by vaginally inoculating pregnant mice to assess GBS dissemination and compare the EOS model to our LOS model. After in utero infection, most pups were intestinally colonized with GBS and, interestingly, only pups with GBS colonies in the lungs had systemic GBS infections. This data suggests neonates are at risk of GBS infection in utero, but not following delivery, even if GBS colonizes the neonatal intestine. This work could elucidate the pathogenesis of EOS as compared to LOS, leading to a deeper understanding of modes of GBS transmission. Supported by NIH: DK122187

Macrophage-derived IGF-1 protects the neonatal intestine against necrotizing enterocolitis by promoting microvascular development

Necrotizing enterocolitis (NEC) is a deadly bowel necrotic disease of premature infants. Low levels of plasma IGF-1 predispose premature infants to NEC. While increasing evidence suggests that defective perinatal intestinal microvascular development plays a role in NEC, the involved mechanism remains incompletely understood. We report here that serum and intestinal IGF-1 are developmentally regulated during the perinatal period in mice and decrease during experimental NEC. Neonatal intestinal macrophages produce IGF-1 and promote endothelial cell sprouting in vitro via IGF-1 signaling. In vivo, in the neonatal intestine, macrophage-derived IGF-1 promotes VEGF expression and endothelial cell proliferation and protects against experimental NEC. Exogenous IGF-1 preserves intestinal microvascular density and protects against experimental NEC. In human NEC tissues, villous endothelial cell proliferation and IGF-1- producing macrophages are decreased compared to controls. Together, our results suggest that defective IGF-1-production by neonatal macrophages impairs neonatal intestinal microvascular development and predisposes the intestine to necrotizing enterocolitis.

A1 USING HUMAN NEONATAL ORGANOIDS TO EXPLORE GUT-IMMUNE SYSTEM INTERACTIONS OF THE NEONATAL INTESTINE

BackgroundNecrotizing Enterocolitis (NEC) affects around 10% of preterm babies and is one of the leading causes of death for newborns. NEC is characterized by exaggerated inflammation of the intestinal mucosa, possibly triggered by aberrant exposure to gut microbes, leading to hypoxic conditions and the death of intestinal tissues. It has been hypothesized that NEC develops when the immature intestine (epithelium and immune system) is unable to properly balance these new microbial interactions. To date, NEC is poorly understood and due to the difficulty of modeling the human neonatal intestine, few therapeutic options are available.Intestinal epithelial cells (IEC) are important players in promoting beneficial host-microbe interactions in the gut, being the primary barrier that separates the host’s mucosal immune system from luminal microbiota, as well as key players in mediating signaling between microbes and the host. Based on their location, IEC are also subject to injury associated with maladaptive immune responses against gut microbes. Many studies have shown that immune cells (such as T helper 17 cells) interact with IEC to promote gut health and function. These interactions include educating IEC on how to respond to, and fight pathogenic microbes, yet also remain tolerant to commensal microbes.AimsThis project seeks to develop an in vitro human neonatal intestinal organoid model to study developmental changes in IEC and their functional interactions with neonatal Th17 cells.Methods3D organoids were established from human neonatal intestinal biopsies and then co-cultured with the supernatant of differentiated Th17 cells or with recombinant cytokines, IL-17 and IL-22. Changes in barrier function, cell proliferation, production of mucins and anti-microbial peptides (AMP) were analyzed by qPCR and immunostaining.ResultsUsing 3D neonatal organoids we observed that the supernatants from neonatal Th17 cells (containing IL-17, IL-22 etc.) promoted the proliferation, differentiation and barrier function of the neonatal epithelium. By using specific recombinant cytokine (IL22, IL17) and neutralizing IL-22 antibodies in parallel, we demonstrated that the high levels of IL-22 produced by neonatal Th17 cells specifically induced proliferation of IEC, AMP and mucus production as compared to control media treated organoids, as shown by increases in Ki67, Reg3γ and Muc2 markers.ConclusionsThis experimental model mimicking the neonatal intestinal environment can be used to study interactions between neonatal IEC and immune cells. Our findings can provide clinically relevant information and clues to how developmental changes in the newborn intestine can influence susceptibility to NEC while demonstrating our development of a simple, yet accurate and clinically applicable model of the neonatal gut.Funding AgenciesCAG, CIHRBCCHRI, CCC, C.H.I.L.D. Fdn

DMBT1 expression and neutrophil-to-lymphocyte ratio during necrotizing enterocolitis are influenced by impaired perfusion due to cardiac anomalies

BackgroundDeleted in malignant brain tumors 1 (DMBT1) is involved in innate immunity and epithelial differentiation. It has been proven to play a role in various states of inflammation or hypoxia of fetal gastrointestinal and pulmonary diseases. Discrimination of pathogenesis in necrotizing enterocolitis (NEC) based on cardiac status improves the understanding of NEC in different patient subgroups. We aimed at examining DMBT1 expressions regarding their association with cardiac status leading to impaired intestinal perfusion, intraoperative bacteria proof, and a fulminant course of NEC.MethodsTwenty-eight patients with NEC were treated surgically between 2010 and 2019 at our institution. DMBT1 expression was examined in intestinal sections using immunohistochemistry to detect DMBT1 protein. Associations of clinical parameters and DMBT1 expression were analyzed.ResultsWe examined DMBT1 levels in 10 patients without cardiac defects and 18 patients with persisting ductus arteriosus (PDA) and congenital heart defects (CHD). Compared to patients without cardiac malformations, DMBT1 levels tended to score higher in patients with PDA/CHD (p = 0.2113) and were negatively correlated with C-reactive protein in these infants (p = 0.0172; r = − 0.5533). The number of DMBT1-expressing macrophages was elevated in the PDA/CHD-subgroup (p = 0.0399). Ratios of neutrophils and monocytes to lymphocytes were significantly higher in infants with PDA/CHD (p = 0.0319 and 0.0493). DMBT1 expression was significantly associated with positive bacterial culture of intraoperative swabs (p = 0.0252) and DMBT1 expression of the serosa was associated with a fulminant course of NEC (p = 0.0239).ConclusionsThis study demonstrates that DMBT1 expression may be influenced by cardiac anomalies with an impaired intestinal perfusion in the neonatal intestine. NEC in PDA/CHD infants is associated with more DMBT1-positive macrophages and a significantly elevated neutrophil-to-lymphocyte ratio.

Insulin is expressed by enteroendocrine cells during human fetal development.

Generation of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here we reconstruct a single-cell atlas of the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and expose its molecular blueprint.

Priming with Retinoic Acid, an Active Metabolite of Vitamin A, Increases Vitamin A Uptake in the Small Intestine of Neonatal Rats.

Given that combined vitamin A (VA) and retinoic acid (RA) supplementation stimulated the intestinal uptake of plasma retinyl esters in neonatal rats, we administrated an RA dose as a pretreatment before VA supplementation to investigate the distinct effect of RA on intestinal VA kinetics. On postnatal days (P) 2 and 3, half of the pups received an oral dose of RA (RA group), while the remaining received canola oil as the control (CN). On P4, after receiving an oral dose of 3H-labeled VA, pups were euthanized at selected times (n = 4–6/treatment/time) and intestine was collected. In both CN and RA groups, intestinal VA mass increased dramatically after VA supplementation; however, RA-pretreated pups had relatively higher VA levels from 10 h and accumulated 30% more VA over the 30-h study. Labeled VA rapidly peaked in the intestine of CN pups and then declined from 13 h, while a continuous increase was observed in the RA group, with a second peak at 10 h and nearly twice the accumulation of 3H-labeled VA compared to CN. Our findings indicate that RA pretreatment may stimulate the influx of supplemental VA into the intestine, and the increased VA accumulation suggests a potential VA storage capacity in neonatal intestine.

Dystrophin in the Neonatal and Adult Rat Intestine.

Background: Gastrointestinal (GI) complaints are frequently noted in aging dystrophinopathy patients, yet their underlying molecular mechanisms are largely unknown. As dystrophin protein isoform 71 (Dp71) is particularly implicated in the development of smooth muscle cells, we evaluated its distribution in the neonatal and adult rat intestine in this study. Methods: Dp71 expression levels were assessed in the proximal (duodenum, jejunum and ileum) and distal (caecum, colon and rectum) intestine by Western blotting and qPCR. In addition, the cellular distribution of total Dp was evaluated in the duodenum and colon by immunohistochemical colocalization studies with alpha-smooth muscle actin (aSMA), Hu RNA binding proteins C and D (HuC/HuD) for neurons and vimentin (VIM) for interstitial cells. Results: In neonatal and adult rats, the distal intestine expressed 2.5 times more Dp71 protein than the proximal part (p < 0.01). This regional difference was not observed in Dp71 mRNA. During both stages, Dp-immunoreactivity was predominant in the muscularis propria, where it co-localized with aSMA and HuC/HuD. Conclusions: In neonatal and adult rats, Dp71 was expressed highest in the distal intestine. Together with the observation that Dp may be expressed by myenteric neurons, this warrants a paradigm shift in the treatment of GI comorbidities.