Neutrophil extracellular trap inhibition increases inflammation, bacteraemia and mortality in murine necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC-like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case-matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.

Hyaluronan 35 kDa enhances epithelial barrier function and protects against the development of murine necrotizing enterocolitis.

BACKGROUND: Disruption of tight junctions (TJs) predisposes to bacterial translocation, intestinal inflammation, and necrotizing enterocolitis (NEC). Previously, studies showed that hyaluronan (HA), a glycosaminoglycan in human milk, maintains intestinal permeability, enhances intestinal immunity, and reduces intestinal infections. In this study, we investigated the effects of HA 35 kDa on a NEC-like murine model. METHODS: Pups were divided into Sham, NEC, NEC+HA 35, and HA 35. Severity of intestinal injury was compared using a modified macroscopic gut scoring and histologic injury grading. The effect of HA 35 on intestinal permeability was determined by measuring FITC dextran and bacterial translocation. RNA and protein expression of TJ proteins (claudin-2, -3, -4, occludin, and ZO-1) were compared between the groups. RESULTS: Pups in the NEC+HA 35 group had increased survival and lower intestinal injury compared to untreated NEC. In addition, HA 35 reduced intestinal permeability, bacterial translocation, and proinflammatory cytokine release. Ileal expression of claudin-2, -3, -4, occludin, and ZO-1 was upregulated in NEC+HA 35 and HA 35 compared to untreated NEC and shams. CONCLUSION: These findings suggest that HA 35 protects against NEC partly by upregulating intestinal TJs and enhancing intestinal barrier function.

Curcumin and Intestinal Inflammatory Diseases: Molecular Mechanisms of Protection.

Intestinal inflammatory diseases, such as Crohn's disease, ulcerative colitis, and necrotizing enterocolitis, are becoming increasingly prevalent. While knowledge of the pathogenesis of these related diseases is currently incomplete, each of these conditions is thought to involve a dysfunctional, or overstated, host immunological response to both bacteria and dietary antigens, resulting in unchecked intestinal inflammation and, often, alterations in the intestinal microbiome. This inflammation can result in an impaired intestinal barrier allowing for bacterial translocation, potentially resulting in systemic inflammation and, in severe cases, sepsis. Chronic inflammation of this nature, in the case of inflammatory bowel disease, can even spur cancer growth in the longer-term. Recent research has indicated certain natural products with anti-inflammatory properties, such as curcumin, can help tame the inflammation involved in intestinal inflammatory diseases, thus improving intestinal barrier function, and potentially, clinical outcomes. In this review, we explore the potential therapeutic properties of curcumin on intestinal inflammatory diseases, including its antimicrobial and immunomodulatory properties, as well as its potential to alter the intestinal microbiome. Curcumin may play a significant role in intestinal inflammatory disease treatment in the future, particularly as an adjuvant therapy.

Evolutionary bridges: how factors present in amniotic fluid and human milk help mature the gut.

Necrotizing enterocolitis (NEC) continues to be a leading cause of morbidity and mortality in preterm infants. As modern medicine significantly improves the survival of extremely premature infants, the persistence of NEC underscores our limited understanding of its pathogenesis. Due to early delivery, a preterm infant's exposure to amniotic fluid (AF) is abruptly truncated. Replete with bioactive molecules, AF plays an important role in fetal intestinal maturation and preparation for contact with the environment, thus its absence during development of the intestine may contribute to increased susceptibility to NEC. Human milk (HM), particularly during the initial phases of lactation, is a cornerstone of neonatal intestinal defense. The concentrations and activities of several bioactive factors in HM parallel those of AF, suggesting continuity of protection. In this review, we discuss the predominant overlapping bioactive components of HM and AF, with an emphasis on those associated with intestinal growth or reduction of NEC.

Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry.

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

Selenium Deficiency Exacerbates Hyperoxia-Induced Lung Injury in Newborn C3H/HeN Mice.

Extremely preterm infants are often treated with supraphysiological oxygen, which contributes to the development of bronchopulmonary dysplasia (BPD). These same infants exhibit compromised antioxidant capacities due in part to selenium (Se) deficiency. Se is essential for basal and inducible antioxidant responses. The present study utilized a perinatal Se deficiency (SeD) mouse model to identify the combined effects of newborn hyperoxia exposure and SeD on alveolarization and antioxidant responses, including the identification of affected developmental pathways. Se-sufficient (SeS) and SeD C3H/HeN breeding pairs were generated, and pups were exposed to room air or 85% O2 from birth to 14 d. Survival, antioxidant protein expression, and RNA seq analyses were performed. Greater than 40% mortality was observed in hyperoxia-exposed SeD pups. Surviving SeD pups had greater lung growth deficits than hyperoxia-exposed SeS pups. Gpx2 and 4 protein and Gpx activity were significantly decreased in SeD pups. Nrf2-regulated proteins, Nqo1 and Gclc were increased in SeD pups exposed to hyperoxia. RNA seq revealed significant decreases in the Wnt/ß-catenin and Notch pathways. Se is a biologically relevant modulator of perinatal lung development and antioxidant responses, especially in the context of hyperoxia exposure. The RNA seq analyses suggest pathways essential for normal lung development are dysregulated by Se deficiency.

The Nonbacterial Microbiome: Fungal and Viral Contributions to the Preterm Infant Gut in Health and Disease.

The intestinal microbiome is frequently implicated in necrotizing enterocolitis (NEC) pathogenesis. While no particular organism has been associated with NEC development, a general reduction in bacterial diversity and increase in pathobiont abundance has been noted preceding disease onset. However, nearly all evaluations of the preterm infant microbiome focus exclusively on the bacterial constituents, completely ignoring any fungi, protozoa, archaea, and viruses present. The abundance, diversity, and function of these nonbacterial microbes within the preterm intestinal ecosystem are largely unknown. Here, we review findings on the role of fungi and viruses, including bacteriophages, in preterm intestinal development and neonatal intestinal inflammation, with potential roles in NEC pathogenesis yet to be determined. In addition, we highlight the importance of host and environmental influences, interkingdom interactions, and the role of human milk in shaping fungal and viral abundance, diversity, and function within the preterm intestinal ecosystem.

Biomarkers of necrotizing enterocolitis in the era of machine learning and omics.

Necrotizing enterocolitis (NEC) continues to be a major cause of morbidity and mortality in preterm infants. Despite decades of research in NEC, no reliable biomarkers can accurately diagnose NEC or predict patient prognosis. The recent emergence of multi-omics could potentially shift NEC biomarker discovery, particularly when evaluated using systems biology techniques. Furthermore, the use of machine learning and artificial intelligence in analyzing this 'big data' could enable novel interpretations of NEC subtypes, disease progression, and potential therapeutic targets, allowing for integration with personalized medicine approaches. In this review, we evaluate studies using omics technologies and machine learning in the diagnosis of NEC. Future implications and challenges inherent to the field are also discussed.

Impact of neonatal nutrition on necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in preterm infants. NEC is multifactorial and the result of a complex interaction of feeding, dysbiosis, and exaggerated inflammatory response. Feeding practices in the neonatal intensive care units (NICUs) can vary among institutions and have significant impact on the vulnerable gastointestinal tract of preterm infants. . These practices encompass factors such as the type of feeding and fortification, duration of feeding, and rate of advancement, among others. The purpose of this article is to review the data on some of the most common feeding practices in the NICU and their impact on the development of NEC in preterm infants. Data on the human milk bioactive component glycosaminoglycans, specifically hyaluronan, will also be discussed in the context of postnatal intestinal development and NEC prevention.

Neonatal Microbiome, Intestinal Inflammation, and Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC), the most common gastrointestinal emergency in the neonatal intensive care unit (NICU), is a leading cause of preterm infant morbidity and mortality [...].

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-α for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-α, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing using Enteroid-derived Monolayers.

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 µg/mL, 100 µg/mL, 200 µg/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ. The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

Clinical Characteristics and Potential Pathogenesis of Cardiac Necrotizing Enterocolitis in Neonates with Congenital Heart Disease: A Narrative Review.

Neonates with congenital heart disease (CHD) are at an increased risk of developing necrotizing enterocolitis (NEC), an acute inflammatory intestinal injury most commonly associated with preterm infants. The rarity of this complex disease, termed cardiac NEC, has resulted in a dearth of information on its pathophysiology. However, a higher incidence in term infants, effects on more distal regions of the intestine, and potentially a differential immune response may distinguish cardiac NEC as a distinct condition from the more common preterm, classical NEC. In this review, risk factors, differentiated from those of classical NEC, are discussed according to their potential contribution to the disease process, and a general pathogenesis is postulated for cardiac NEC. Additionally, biomarkers specific to cardiac NEC, clinical outcomes, and strategies for achieving enteral feeds are discussed. Working towards an understanding of the mechanisms underlying cardiac NEC may aid in future diagnosis of the condition and provide potential therapeutic targets.

Hyaluronic Acid 35 kDa Protects against a Hyperosmotic, Formula Feeding Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC), an inflammatory disease of the intestine, is a common gastrointestinal emergency among preterm infants. Intestinal barrier dysfunction, hyperactivation of the premature immune system, and dysbiosis are thought to play major roles in the disease. Human milk (HM) is protective, but the mechanisms underpinning formula feeding as a risk factor in the development of NEC are incompletely understood. Hyaluronic acid 35 kDa (HA35), a bioactive glycosaminoglycan of HM, accelerates intestinal development in murine pups during homeostasis. In addition, HA35 prevents inflammation-induced tissue damage in pups subjected to murine NEC, incorporating Paneth cell dysfunction and dysbiosis. We hypothesized HA35 treatment would reduce histological injury and mortality in a secondary mouse model of NEC incorporating formula feeding. NEC-like injury was induced in 14-day mice by dithizone-induced disruption of Paneth cells and oral gavage of rodent milk substitute. Mortality and histological injury, serum and tissue cytokine levels, stool bacterial sequencing, and bulk RNA-Seq comparisons were analyzed. HA35 significantly reduced the severity of illness in this model, with a trend toward reduced mortality, while RNA-Seq analysis demonstrated HA35 upregulated genes associated with goblet cell function and innate immunity. Activation of these critical protective and reparative mechanisms of the small intestine likely play a role in the reduced pathology and enhanced survival trends of HA-treated pups subjected to intestinal inflammation in this secondary model of NEC, providing potentially interesting translational targets for the human preterm disease.

Placental Neutrophil Infiltration Associated with Tobacco Exposure but Not Development of Bronchopulmonary Dysplasia.

Objective: In utero inflammation is associated with bronchopulmonary dysplasia (BPD) in preterm infants. We hypothesized that maternal tobacco exposure (TE) might induce placental neutrophil infiltration, increasing the risk for BPD. Study design: We compared the composite outcome of BPD and death in a prospective pilot study of TE and no-TE mothers and their infants born <32 weeks. Placental neutrophil infiltration was approximated by neutrophil gelatinase-associated lipocalin (NGAL) ELISA, and total RNA expression was analyzed via NanoString© (Seattle, WA, USA). Result: Of 39 enrolled patients, 44% were classified as tobacco exposure. No significant difference was noted in the infant's composite outcome of BPD or death based on maternal tobacco exposure. NGAL was higher in placentas of TE vs. non-TE mothers (p < 0.05). Placental RNA analysis identified the upregulation of key inflammatory genes associated with maternal tobacco exposure. Conclusion: Tobacco exposure during pregnancy was associated with increased placental neutrophil markers and upregulated inflammatory gene expression. These findings were not associated with BPD.

Early Antibiotic Exposure Alters Intestinal Development and Increases Susceptibility to Necrotizing Enterocolitis: A Mechanistic Study.

Increasing evidence suggests that prolonged antibiotic therapy in preterm infants is associated with increased mortality and morbidities, such as necrotizing enterocolitis (NEC), a devastating gastrointestinal pathology characterized by intestinal inflammation and necrosis. While a clinical correlation exists between antibiotic use and the development of NEC, the potential causality of antibiotics in NEC development has not yet been demonstrated. Here, we tested the effects of systemic standard-of-care antibiotic therapy for ten days on intestinal development in neonatal mice. Systemic antibiotic treatment impaired the intestinal development by reducing intestinal cell proliferation, villi height, crypt depth, and goblet and Paneth cell numbers. Oral bacterial challenge in pups who received antibiotics resulted in NEC-like intestinal injury in more than half the pups, likely due to a reduction in mucous-producing cells affecting microbial-epithelial interactions. These data support a novel mechanism that could explain why preterm infants exposed to prolonged antibiotics after birth have a higher incidence of NEC and other gastrointestinal disorders.

Lipid Composition, Digestion, and Absorption Differences among Neonatal Feeding Strategies: Potential Implications for Intestinal Inflammation in Preterm Infants.

Necrotizing enterocolitis (NEC) is a significant cause of morbidity and mortality in the neonatal population. Formula feeding is among the many risk factors for developing the condition, a practice often required in the cohort most often afflicted with NEC, preterm infants. While the virtues of many bioactive components of breast milk have been extolled, the ability to digest and assimilate the nutritional components of breast milk is often overlooked. The structure of formula differs from that of breast milk, both in lipid composition and chemical configuration. In addition, formula lacks a critical digestive enzyme produced by the mammary gland, bile salt-stimulated lipase (BSSL). The gastrointestinal system of premature infants is often incapable of secreting sufficient pancreatic enzymes for fat digestion, and pasteurization of donor milk (DM) has been shown to inactivate BSSL, among other important compounds. Incompletely digested lipids may oxidize and accumulate in the distal gut. These lipid fragments are thought to induce intestinal inflammation in the neonate, potentially hastening the development of diseases such as NEC. In this review, differences in breast milk, pasteurized DM, and formula lipids are highlighted, with a focus on the ability of those lipids to be digested and subsequently absorbed by neonates, especially those born prematurely and at risk for NEC.

Intestinal and Hepatic Uptake of Dietary Peroxidized Lipids and Their Decomposition Products, and Their Subsequent Effects on Apolipoprotein A1 and Paraoxonase1.

Both pro- and antiatherosclerotic effects have been ascribed to dietary peroxidized lipids. Confusion on the role of peroxidized lipids in atherosclerotic cardiovascular disease is punctuated by a lack of understanding regarding the metabolic fate and potential physiological effects of dietary peroxidized lipids and their decomposition products. This study sought to determine the metabolic fate and physiological ramifications of 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-HODE (13-hydroxyoctadecadienoic acid) supplementation in intestinal and hepatic cell lines, as well as any effects resulting from 13-HPODE or 13-HODE degradation products. In the presence of Caco-2 cells, 13-HPODE was rapidly reduced to 13-HODE. Upon entering the cell, 13-HODE appears to undergo decomposition, followed by esterification. Moreover, 13-HPODE undergoes autodecomposition to produce aldehydes such as 9-oxononanoic acid (9-ONA). Results indicate that 9-ONA was oxidized to azelaic acid (AzA) rapidly in cell culture media, but AzA was poorly absorbed by intestinal cells and remained detectable in cell culture media for up to 18 h. An increased apolipoprotein A1 (ApoA1) secretion was observed in Caco-2 cells in the presence of 13-HPODE, 9-ONA, and AzA, whereas such induction was not observed in HepG2 cells. However, 13-HPODE treatments suppressed paraoxonase 1 (PON1) activity, suggesting the induction of ApoA1 secretion by 13-HPODE may not represent functional high-density lipoprotein (HDL) capable of reducing oxidative stress. Alternatively, AzA induced both ApoA1 secretion and PON1 activity while suppressing ApoB secretion in differentiated Caco-2 cells but not in HepG2. These results suggest oxidation of 9-ONA to AzA might be an important phenomenon, resulting in the accumulation of potentially beneficial dietary peroxidized lipid-derived aldehydes.

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice.

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.