Immature Intestine Research Articles

A CLINICAL REVIEW OF DIAGNOSTIC BIOMARKERS AND THE ROLE OF THE GUT MICROBIOTA IN PRETERM INFANTS WITH NECROTIZING ENTEROCOLITIS

The purpose of this work is to clarify the latest opinions of clinicians about the pathogenesis, diagnosis and treatment of necrotic enterocolitis, as well as the role of microflora in the pathogenesis of this pathology. Material and methods. Search Strategy: A literature search was conducted in the following electronic databases: PubMed, Web of Science (Thomson Reuters), Scopus, Cochrane Library, SpringerLink, and EBSCOhost ScienceDirect. The search spanned 20 years, from 2000 to 2020. In this review, we included 39 articles on this topic, which were available in full text and analyzed through critical evaluation. Results.According to the literature, necrotizing enterocolitis is primarily attributed to prematurity, compounded by the disruption of intestinal microflora composition due to artificial feeding, leading to an inflammatory reaction in the immature intestine. Delayed detection of the initial signs of necrotizing enterocolitis exacerbates treatment outcomes. In clinical practice, two main dilemmas in necrotizing enterocolitis diagnosis arise. Firstly, distinguishing early necrotizing enterocolitis from sepsis can be challenging, resulting in delayed diagnosis and treatment. Secondly, the optimal timing for surgical intervention remains unclear. Conclusion. There is a need to elucidate the latest opinions of clinicians regarding the pathogenesis, diagnosis, and treatment of necrotizing enterocolitis, including the role of microflora in its pathogenesis. Treatment strategies should integrate various models of clinical prognosis and biomarkers to accurately diagnose and tailor therapy for necrotizing enterocolitis.

259 Effect of very low calcium diet supplemented with microbial phytase in post-weaning piglets

Abstract Young piglets have an immature intestine that provides limited gastric acidity to digest nutrients compared with later stages of growth. Food has the intrinsic capacity to modify gastric acidity, particularly in relation to their concentrations of limestone, the ingredient with the greatest capacity to bind to acid. An experiment was conducted to evaluate the impacts of reducing dietary calcium (Ca) by reducing calcium supplementation immediately after weaning; compared with the recommendations of the NRC (2012), in association with phytase supplementation. The treatments were distributed during 3 phases [Phase 1 (P1, d 0-9), 2 (P2, d 10-16) and 3 (P3, d 17-35)]. Treatments were: 1) a diet low Ca that did not contained limestone during phase 1 (LowCa), P1 to P3 respectively, 0.51-0.60-0.65 % Ca and 0.48-0.43-0.38 % standardized ileal digestible P (STTD P), 2), diet 1 with microbial phytase (LowCa+PhytM); P1 to P3 respectively, 0.51-0.60-0.65 % Ca and 0.48-0.43-0.38% STTD P with 750 FTU phytase, and 3) a diet fulfilling requirement (NormalCa+PhytM) P1 to P3 respectively, 0.85-0.80-0.70 % Ca and 0.48-0.43-0.38 % STTD P with 750 FTU phytase. Piglets (n = 120) were allocated to 6 pens of 5 piglets per treatment in function of initial body weight (BW; 6.12 ± 0.028 kg). On d 0, 15 and 36, dual X-ray (DXA) bone densitometry was used to measure body fat, lean and mineral content (BMC) compositions using 8 piglets per treatment. At the end of phase 1 (d 9) a blood sample was collected. Data were analyzed using the SAS MIXED procedure with Tukey’s test to compare means. No differences were observed in growth performance or DXA measurements during the first phase. On d 9, LowCa piglets showed signs of hypocalcaemia with reduced plasma Ca (P = 0.03), increased plasma P (P = 0.02) and a lower Ca:P ratio in plasma (P = 0.04). From d 9-15, piglets that had received LowCa+PhytM showed greater average daily gain (ADG; P = 0.002) while other criteria did not differ between treatments. On d 15, the body composition evaluated by DXA showed no difference among dietary treatments. During phase 3, final BW and average daily feed intake were not modified by dietary treatments, while average daily gain and feed efficiency were greater in LowCa than in NormalCa+PhytM (+7.3 and 6.7%, respectively), and LowCa+PhytM was intermediate. Body composition on d 35 showed no difference among treatments while a tendency (P = 0.095) for greater BMC deposition (g/d) in LowCa in comparison with NormalCa+PhytM (+1.3 %). The implemented LowCa strategy resulted in momentary hypocalcemia which probably activated homeostatic mechanisms allowing piglets to be more efficient in using minerals and possibly other nutrients leading to better growth performance, without impact on bone mineralization.

Intestinal Stem Cells Remodel in Response to Acute Notch Inhibition and Paneth Cell Loss

INTRODUCTION: Intestinal epithelial cells are rapidly renewed throughout life by intestinal stem cells (ISCs). ISC function is regulated by niche factors that orchestrate cellular proliferation and differentiation. Of these, Notch signaling is essential for ISC self-renewal and regulates the cell fate choice between absorptive cells (enterocyte) vs secretory (Paneth, goblet, enteroendocrine, and tuft) cells. Yet, the Notch niche is not fully defined. In the mature small intestine, Paneth cells neighbor each ISC and act as Notch niche cells, providing the Notch signal by expressing the Notch ligands DLL1 and DLL4 on their surface. However, studies in mice have shown that ISCs can function in the immature intestine before Paneth cells are formed, and when Paneth cells are depleted in the adult intestine. We previously observed that acute Notch inhibition via the inhibitor dibenzazepine (DBZ) resulted in Paneth cell apoptosis with intact ISCs displaying transient dysfunction. Here, we aimed to define the Notch niche after Paneth cell apoptosis in adult mice and in the developing intestine. We hypothesized that during acute Notch inhibition, ISCs remodel to compensate for Paneth cell loss by activating Notch ligand expression, and that this remodeling mimics the state of immature ISCs prior to Paneth cell emergence. METHODS: We isolated Lgr5+ ISCs by FACS from Lgr5-GFP-CreERT2 mice 24 hours after DBZ or vehicle treatment and performed bulk RNAseq analysis. To define the Notch niche during development, we analyzed postnatal intestines from Notch ligand reporter mice (Dll1-mCherry and Dll4-mCherry) crossed with the ISC reporter mice (Lgr5-GFP-CreERT2). RESULTS: ISCs isolated from Paneth cell-depleted intestines upregulated expression of Notch ligands Dll4 and Dll4 and various secretory cell markers, with expected downregulation of Notch target genes, but no alteration of other stem cell genes. This suggests that after acute Notch inhibition, Lgr5+ ISCs maintain partial stem cell identity while acquiring a signature resembling that of secretory progenitors. In the immature intestine, we showed by immunohistology that sporadic cells expressing secretory cell markers (LYZ, MUC2, and CHGA) localized to the intervillus region and co-stained with Dll1- or Dll4-mCherry+. This suggests that multiple differentiated cell types could provide the Notch signal to immature ISCs. We also observed occasional Lgr5-GFP+ ISCs that were co-positive for Dll1-mCherry, suggesting that immature ISCs can express Notch ligands at homeostasis. Further experimentation will determine whether these Dll1+ ISCs are similar to the remodeled adult ISCs after Paneth cell depletion. CONCLUSIONS: Acute Notch inhibition directly remodels ISCs to express the Notch ligands, Dll1 and Dll4 in the adult intestine. In the immature intestine, multiple secretory cell types appear to comprise the immature Notch niche. This project was funded by NIH R01 DK118023 awarded to Dr. Linda Samuelson. This is the full abstract presented at the American Physiology Summit 2024 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.

Paneth cell ontogeny in term and preterm ovine models.

Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent. Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry. Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon. Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

Modulation of pectin on intestinal barrier function via changes in microbial functional potential and bile acid metabolism

Weaning is one of the major factors that cause stress and intestinal infection in infants and in young animals due to an immature intestine and not fully developed immune functions. Pectin (PEC), a prebiotic polysaccharide, has attracted considerable attention in intestinal epithelial signaling and function via modulation of the microbial community. A total of 16 weaned piglets (21-d-old) were randomly assigned into two groups: control group and PEC group. Supplementation of 5% pectin improved intestinal mucosal barrier function by modulating the composition of the bile acid pool in piglets. Specifically, piglets in PEC group had less serum D-lactate content and alkaline phosphatase activity. In the ileum, dietary pectin increased the number of crypt PAS/AB-positive goblet cells and the mRNA expressions of MUC2, ZO-1, and Occludin. Piglets in PEC group displayed a decreased abundance of Enterococcus (2.71 vs. 65.92%), but the abundances of Lactobacillus (30.80 vs. 7.93%), Streptococcus (21.41 vs. 14.81%), and Clostridium_sensu_stricto_1 (28.34 vs. 0.01%) were increased. Elevated concentrations of bile acids especially hyocholic acid species (HCAs) including HCA, HDCA, and THDCA were also observed. Besides, correlation analysis revealed that dietary pectin supplementation may have beneficial effects through stimulation of the crosstalk between gut microbes and bile acid synthesis within the enterohepatic circulation. Thus, dietary pectin supplementation exhibited a further positive effect on the healthy growth and development of weaned piglets. These findings suggest pectin supplementation as the prebiotic is beneficial for gut health and improvement of weaned stress via regulating microbiota and bile acid metabolism.

Congenitally underdeveloped intestine drives autism-related gut microbiota and behavior

Autism spectrum disorder (ASD) is a neurological and developmental disorder accompanied by gut dysbiosis and gastrointestinal symptoms in most cases. However, the development of the autism-related gut microbiota and its relationship with intestinal dysfunction in ASD remain unclear. Using a valproic acid (VPA)-induced ASD mouse model, we showed a congenitally immature intestine of VPA-exposed mice accompanied by prominent oxidative stress and inflammation. Of note, the gut microbiota composition of VPA-exposed mice resembled that of control mice within 24 h after birth; however, their gut microbiota compositions differed on postnatal days 7 and 21. Oral administration of superoxide dismutase (SOD) to attenuate intestinal oxidative stress either before weaning or during juvenile restored the autism-associated gut microbiota, leading to the amelioration of autism-related behaviors. These findings collectively suggest the congenitally underdeveloped intestine as an early driving force shaping the autism-associated gut microbiota and host neurodevelopment through enhancing oxidative stress.

Ex Vivo and In Vitro Studies Revealed Underlying Mechanisms of Immature Intestinal Inflammatory Responses Caused by Aflatoxin M1 Together with Ochratoxin A.

Aflatoxin M1 (AFM1) and ochratoxin A (OTA), which are occasionally detected in milk and commercial baby foods, could easily enter and reach the gastrointestinal tract, posing impairment to the first line of defense and causing dysfunction of the tissue. The objective of this study was to investigate the immunostimulatory roles of individual and combined AFM1 and OTA on the immature intestine. Thus, we used ELISA assays to evaluate the generation of cytokines from ex vivo CD-1 fetal mouse jejunum induced by AFM1 and OTA and explored the related regulatory pathways and pivot genes using RNA-seq analysis. It was found that OTA exhibited much stronger ability in stimulating pro-inflammatory cytokine IL-6 from jejunum tissues than AFM1 (OTA of 4 μM versus AFM1 of 50 μM), whereas the combination of the two toxins seemed to exert antagonistic actions. In addition, transcriptomics also showed that most gene members in the enriched pathway ‘cytokine–cytokine receptor interaction’ were more highly expressed in OTA than the AFM1 group. By means of PPI network analysis, NFKB1 and RelB were regarded as hub genes in response to OTA but not AFM1. In the human FHs 74 Int cell line, both AFM1 and OTA enhanced the content of reactive oxygen species, and the oxidative response was more apparent in OTA-treated cells in comparison with AFM1. Furthermore, OTA and AFM1 + OTA raised the protein abundance of p50/RelB, and triggered the translocation of the dimer from cytosol to nucleus. Therefore, the experimental data ex vivo and in vitro showed that OTA-induced inflammation was thought to be bound up with the up-regulation and translocation of NF-κB, though AFM1 seemed to have no obvious impact. Since it was the first attempt to uncover the appearances and inner mechanisms regarding inflammation provoked by AFM1 and OTA on immature intestinal models, further efforts are needed to understand the detailed metabolic steps of the toxin in cells and to clarify their causal relationship with the signals proposed from current research.

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

Anti-inflammatory actions of acetate, propionate, and butyrate in fetal mouse jejunum cultures ex vivo and immature small intestinal cells in vitro.

Necrotizing enterocolitis (NEC) is an intestinal disease that frequently occurs in premature infants. Presently, there is no effective therapy for NEC. Therefore, the key to reduce the incidence rate of NEC is to take effective intervention measures as early as possible. Short‐chain fatty acids (SCFAs) (acetate, propionate, and butyrate), the principal terminal products of enterobacteria fermentation, play anti‐inflammatory actions in mature intestinal cells. However, few studies focus on their roles in immature intestine. Here, we evaluated the anti‐inflammatory actions of SCFAs ex vivo with ICR fetal mouse jejunum cultures and explored the potential anti‐inflammatory regulators through RNA‐seq and then verified them in vitro with human fetal small intestinal epithelial FHs 74 Int cells. In this study, we found that acetate, propionate, and butyrate decreased IL‐1β‐induced production of CXCL2 ex vivo and IL‐8 and IL‐6 in vitro significantly (p < .05). Furthermore, the inhibitors of NF‐κB p65, JNK1/2, and ERK1/2 pathways, which were selected from RNA‐seq and depressed by SCFAs, also significantly decreased IL‐8 and IL‐6 productions induced by IL‐1β (p < .05). Therefore, our results showed that acetate, propionate, and butyrate ameliorated the fetal small intestine inflammatory response induced by IL‐1β through inhibiting ERK1/2 pathway; NF‐κB p65, JNK1/2, and ERK1/2 pathways; or NF‐κB p65 and ERK1/2 pathways, respectively. These findings suggested that SCFAs may be a new therapy agent for NEC.

The microbiome, guard or threat to infant health.

Despite improvements in survival for very low birthweight (VLBW) premature infants, there continues to be significant morbidity for these infants at remarkable cost to the healthcare system. Concurrent development of the preterm infant intestine alongside the gut microbiome in the clinical setting rather than in the protected in utero environment where it would usually occur creates significant vulnerabilities for the infant's immature intestine and immune system, resulting in devastating illness and neurological injury. However, the microbiome also has the capacity to promote healthy development. Studies of parallel gut microbiome and preterm infant development have given key insight into the impact of the microbiome on intestinal as well as neural development and may provide potential therapeutic targets to prevent preterm infant morbidities.

Epigenetics in Necrotizing Enterocolitis.

Epigenetic alterations in our genetic material can lead to heritable changes in the risk, clinical manifestations, course, and outcomes of many diseases. Understanding these epigenetic mechanisms can help in identifying potential therapeutic targets. This is especially important in necrotizing enterocolitis (NEC), where prenatal as well as postnatal factors impact susceptibility to this devastating condition, but our therapeutic options are limited. Developmental factors affecting intestinal structure and function, our immune system, gut microbiome, and postnatal enteral nutrition are all thought to play a prominent role in this disease. In this manuscript, we have reviewed the epigenetic mechanisms involved in NEC. These include key developmental changes in DNA methylation in the immature intestine, the role of long non-coding RNA (lncRNA) in maintaining intestinal barrier function, epigenetic influences of prenatal inflammation on immunological pathways in NEC pathogenesis such as Toll-Like Receptor 4 (TLR4) and epigenetic changes associated with enteral feeding causing upregulation of pro-inflammatory genes. We have assimilated research findings from our own laboratory with an extensive review of the literature utilizing key terms in multiple databases, including PubMed, EMBASE, and Science Direct.

Understanding intestinal health in nursery pigs and the relevant nutritional strategies.

In the modern pig production, pigs are weaned at early age with immature intestine. Dietary and environmental factors challenge the intestine, specifically the jejunum, causing inflammation and oxidative stress followed by destruction of epithelial barrier and villus structures in the jejunum. Crypt cell proliferation increases to repair damages in the jejunum. Challenges to maintain the intestinal health have been shown to be related to changes in the profile of mucosa-associated microbiota in the jejunum of nursery pigs. All these processes can be quantified as biomarkers to determine status of intestinal health related to growth potential of nursery pigs. Nursery pigs with impaired intestinal health show reduced ability of nutrient digestion and thus reduced growth. A tremendous amount of research effort has been made to determine nutritional strategies to maintain or improve intestinal health and microbiota in nursery pigs. A large number of feed additives have been evaluated for their effectiveness on improving intestinal health and balancing intestinal microbiota in nursery pigs. Selected prebiotics, probiotics, postbiotics, and other bioactive compounds can be used in feeds to handle issues with intestinal health. Selection of these feed additives should aim modulating biomarkers indicating intestinal health. This review aims to define intestinal health and introduce examples of nutritional approaches to handle intestinal health in nursery pigs.

DRG1 Maintains Intestinal Epithelial Cell Junctions and Barrier Function by Regulating RAC1 Activity in Necrotizing Enterocolitis.

An immature intestine is a high-risk factor for necrotizing enterocolitis (NEC), which is a serious intestinal disease in newborns. The regulation of developmentally regulated GTP-binding protein 1 (DRG1) during organ development suggests a potential role of DRG1 in the maturation process of the intestine. To illustrate the function of DRG1 during the pathogenesis of NEC. DRG1 expression in the intestine was measured using immunohistochemistry and q-PCR. Immunoprecipitation coupled with mass spectrometry was used to identify the interacting proteins of DRG1. The biological functions of the potential interactors were annotated with the Database for Annotation, Visualization and Integrated Discovery. Caco2 and FHs74Int cells with stable DRG1 silencing or overexpression were used to investigate the influence of DRG1 on cell junctions and intestinal barrier permeability and to elucidate the downstream mechanism. DRG1 was constitutively expressed during the intestinal maturation process but significantly decreased in the ileum in the context of NEC. Protein interaction analysis revealed that DRG1 was closely correlated with cell junctions. DRG1 deficiency destabilized the E-cadherin and occludin proteins near the cell membrane and increased the permeability of the epithelial cell monolayer, while DRG1 overexpression prevented lipopolysaccharide-induced disruption of E-cadherin and occludin expression and cell monolayer integrity. Further investigation suggested that DRG1 maintained cell junctions, especially adherens junctions, by regulating RAC1 activity, and RAC1 inhibition with NSC23766 attenuated intestinal injury and led to improved barrier integrity in experimental NEC. Our findings illustrate the mechanism underlying the effect of DRG1 deficiency on epithelial cell permeability regulation and provide evidence supporting the application of RAC1 inhibitors for protection against NEC.

Rapid Gut Adaptation to Preterm Birth Involves Feeding-Related DNA Methylation Reprogramming of Intestinal Genes in Pigs

Following preterm birth, the immature gut function and immunology must rapidly adapt to cope with bacterial colonization and enteral milk feeding. We hypothesized that intestinal epigenetic changes are involved in the gut response to preterm birth and the first feeding. Using piglets as models for infants, preterm, and term pigs were fed total parenteral nutrition (TPN) or partial enteral feeding for 5 days, followed by exclusive enteral feeding with bovine milk until day 26 (weaning age). Intestinal structure, function, microbiome, DNA methylome, and gene expressions were compared between preterm and term pigs on days 0, 5, and 26 (n = 8 in each group). At birth, the intestine of preterm pigs showed villus atrophy and global hypermethylation, affecting genes related to the Wnt signaling pathway. Hypermethylation-associated lowered expression of lipopolysaccharide-binding protein and genes related to the Toll-like receptor 4 pathway were evident during the first 5 days of life, but most early methylation differences disappeared by day 26. Regardless, sucrase and maltase activities (adult-type brush border enzymes) remained reduced, and the gut microbiota altered (fewer Akkermansia, more Lachnoclostridia and Lactobacilli) until day 26 in preterm pigs. During the 0- to 5-day period, many new preterm–term methylation differences appeared, but mainly when no enteral feed was provided (TPN feeding). These methylation differences affected intestinal genes related to cell metabolism, including increased GCK (glucokinase) expression via promoter hypomethylation. In conclusion, the immature intestine has a remarkable capacity to adapt its gene methylation and expression after preterm birth, and only few preterm-related defects persisted until weaning. Early enteral feeding may be important to stimulate the methylation reprogramming of intestinal genes, allowing rapid intestinal adaptation to preterm birth.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine.

Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.

Effects of dietary fatty acids on gut health and function of pigs pre- and post-weaning.

Fatty acids (FA) play a major role in relation to mucosal immune responses, epithelial barrier functions, oxidative stress, and inflammatory reactions. The dietary FA composition and the molecular structures (chain length and number of double bonds) influence digestion, absorption and metabolism, and the bioactivity of the FA. Piglets post-weaning having an immature intestine and not fully formed immune functions are very vulnerable to invading microorganisms. Manipulation of the milk FA composition via sow nutrition, or inclusion of dietary fat sources in the feed for newly weaned pigs, may be used as a strategic tool to enhance pig performance and their gut health and function pre- and post-weaning. Medium-chain fatty acids (MCFA) are absorbed directly into the portal blood and may contribute to immediate energy for the enterocytes. In addition, the MCFA, similarly to the short-chain fatty acids (SCFA), possess antibacterial effects and may thereby prevent overgrowth of pathogenic bacteria in the gastrointestinal tract. The essential FA, linoleic (LA) and α-linolenic (ALA) FA, form the building blocks for the long-chain polyunsaturated n-3 and n-6 FA. The conversion of ALA and LA into n-3 and n-6 eicosanoids, respectively, influences the molecular structures of metabolites and inflammatory reactions and other immune responses upon bacterial challenges. Dietary manipulation of the lactating sow influences the transfer of the n-3 and n-6 polyunsaturated fatty acids (PUFA) from the sow milk to the piglet and the incorporation of the FA into piglet enteric tissues and cell membranes, which exerts bioactivity of importance for immune responses and the epithelial barrier function. Especially, the n-3 PUFA present in fish oil seem to influence the gut health and function of pigs, and this is of importance during the transition periods such as post-weaning in which piglets are prone to inflammation. The proportion of unsaturated FA in the cell membranes influences the susceptibility to oxidative stress. Oxidative stress accompanies infectious diseases, and the development of lipid peroxides and other reactive oxygen products may be harmful to the epithelial barrier function. Fatty acid peroxides from the feed may also be absorbed with other lipid-solubles and thereby harm the intestinal function. Hence, antioxidative protection is important for the enteric cells. In conclusion, manipulation of the dietary FA composition can influence the gut health and function in pigs and may support a normal immune system and modulate resistance to infectious diseases during especially stressful phases of a pig's life such as post-weaning.

Postnatal Gut Immunity and Microbiota Development Is Minimally Affected by Prenatal Inflammation in Preterm Pigs.

Chorioamnionitis (CA), resulting from intra-amniotic inflammation, is a frequent cause of preterm birth and exposes the immature intestine to bacterial toxins and/or inflammatory mediators before birth via fetal swallowing. This may affect intestinal immune development, interacting with the effects of enteral feeding and gut microbiota colonization just after birth. Using preterm pigs as model for preterm infants, we hypothesized that prenatal exposure to gram-negative endotoxin influences postnatal bacterial colonization and gut immune development. Pig fetuses were given intra-amniotic lipopolysaccharide (LPS) 3 days before preterm delivery by cesarean section and were compared with littermate controls (CON) at birth and after 5 days of formula feeding and spontaneous bacterial colonization. Amniotic fluid was collected for analysis of leukocyte counts and cytokines, and the distal small intestine was analyzed for endotoxin level, morphology, and immune cell counts. Intestinal gene expression and microbiota were analyzed by transcriptomics and metagenomics, respectively. At birth, LPS-exposed pigs showed higher intestinal endotoxin, neutrophil/macrophage density, and shorter villi. About 1.0% of intestinal genes were affected at birth, and DMBT1, a regulator of mucosal immune defense, was identified as the hub gene in the co-expression network. Genes related to innate immune response (TLR2, LBP, CD14, C3, SFTPD), neutrophil chemotaxis (C5AR1, CSF3R, CCL5), and antigen processing (MHC II genes and CD4) were also affected, and expression levels correlated with intestinal neutrophil/macrophage density and amniotic fluid cytokine levels. On day 5, LPS and CON pigs showed similar sensitivity to necrotizing enterocolitis, endotoxin levels, morphology, immune cell counts, gene expressions, and microbiota composition (except for difference in some low-abundant species). Our results show that CA markedly affects intestinal genes at preterm birth, including genes related to immune cell infiltration. However, a few days later, following the physiological adaptations to preterm birth, CA had limited effects on intestinal structure, function, gene expression, bacterial colonization, and necrotizing enterocolitis sensitivity. We conclude that short-term, prenatal intra-amniotic inflammation is unlikely to exert marked effects on intestinal immune development in preterm neonates beyond the immediate neonatal period.

Short chain fatty acids produced by colonizing intestinal commensal bacterial interaction with expressed breast milk are anti-inflammatory in human immature enterocytes.

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother’s expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.

Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine.

Background:Necrotizing enterocolitis (NEC), a necrotic inflammation of the intestine, represents a major health problem in the very premature infant. Although prevention is difficult, the combination of ingestion of maternal-expressed breastmilk in conjunction with a probiotic provides the best protection. In this study, we establish a mechanism for breastmilk/probiotic protection.Methods:Ultra-high-performance liquid chromatography-tandem mass spectrometry of B. infantis secretions was used to identify an anti-inflammatory molecule. Indole-3-lactic acid (ILA) was then tested in an established human immature small intestinal cell line, necrotizing colitis enterocytes and other immature human enteroids for anti-inflammatory effects and to establish developmental function. ILA was also examined in immature and mature enterocytes.Results:We have identified indole-3-lactic acid, a metabolite of breastmilk tryptophan, as the anti-inflammatory molecule. This molecule is developmentally functional in immature but not mature intestinal enterocytes, ILA reduces the IL-8 response after IL-1β stimulus. It interacts with the transcription factor aryl hydrocarbon receptor (AHR) and prevents transcription of the inflammatory cytokine IL-8.Conclusions:This molecule produced by B. infantis (ATCC No. 15697) interaction with ingested breastmilk functions in a complementary manner and could become useful in the treatment of all at risk premature infants for necrotizing enterocolitis if safety and clinical studies are performed.

Breast Milk and Microbiota in the Premature Gut: A Method of Preventing Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating inflammatory condition of the intestine, which affects premature infants and causes untold damage. Its pathogenesis has to do with how colonizing bacteria interact with the immature newborn intestine. An immature innate immune response with increased TLR-4 on the cell surface and increased signaling molecules, such as NF-κB, can cause excessive inflammation. This is in conjunction with a decrease in the appearance of regulatory molecules which effect the control of innate responses. This condition is so devastating that it must be prevented and not treated. Fortunately, breast milk and probiotics can affect the condition leading to reduced inflammation. How does this effect work? We have shown that breast milk tryptophan and Bifidobacterium infantis result in a metabolite (indole-3-lactic acid) response, which is anti-inflammatory via inhibition of the aryl hydrocarbon receptor transcription factor which stimulates an IL-8 response. We have also shown that breast milk complex carbohydrates interacting with Bacteroides fragilis can cause short-chain fatty acids which exert anti-inflammatory effects on the newborn intestine. These breast milk metabolites could help prevent NEC if shown to be effective clinically.