microfluidic-model-necrotizing-enterocolitis-incorporating-human

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Washington University School of Medicine

Sargramostim, granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulfate sodium (DSS)-induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs).

Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis.

Preterm infants are susceptible to unique pathology due to their immaturity. Mouse models are commonly used to study immature intestinal disease, including necrotizing enterocolitis (NEC). Current NEC models are performed at a variety of ages, but data directly comparing intestinal developmental stage equivalency between mice and humans are lacking.

A direct comparison of mouse and human intestinal development using epithelial gene expression patterns.

Necrotizing enterocolitis (NEC) in preterm infants is an often-fatal gastrointestinal tract emergency. A robust NEC biomarker that is not confounded by sepsis could improve bedside management, lead to lower morbidity and mortality, and permit patient selection in randomized clinical trials of possible therapeutic approaches.

Association of Intestinal Alkaline Phosphatase With Necrotizing Enterocolitis Among Premature Infants.

Necrotizing enterocolitis (NEC) remains one of the overall leading causes of death in premature infants, and the pathogenesis is unpredictable and not well characterized. The aim of our study was to determine the molecular phenotype of NEC via transcriptomic and epithelial cell-specific epigenomic analysis, with a specific focus on DNA methylation.

Global hypermethylation of intestinal epithelial cells is a hallmark feature of neonatal surgical necrotizing enterocolitis.

 Neonatal necrotizing enterocolitis (NEC) is a deadly and unpredictable gastrointestinal disease, for which no biomarker exists. We aimed to describe the methylation patterns in stool and colon from infants with NEC.  We performed a high-resolution genome-wide epigenomic analysis using solution-phase hybridization and next-generation sequencing of bisulfite-converted DNA.  Our data reveal significant genomic hypermethylation in NEC tissues compared with non-NEC controls. These changes were more pronounced in regions outside CpG islands and gene regulatory elements, suggesting that NEC-specific hypermethylation is not a nonspecific global phenomenon.  This study provides evidence of a methylomic signature associated with NEC that is detectable noninvasively and provides a new opportunity for the development of a novel diagnostic method for NEC.

Neonatal necrotizing enterocolitis-associated DNA methylation signatures in the colon are evident in stool samples of affected individuals.

Necrotizing enterocolitis (NEC) causes significant morbidity and mortality in premature infants; therefore, the identification of therapeutic and preventative strategies against NEC remains a high priority. The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) is well known to contribute to the regulation of intestinal microbial communities and amelioration of intestinal inflammation. However, the role of AhR signaling in NEC is unclear. Experimental NEC was induced in 4-d-old wild-type mice or mice lacking AhR expression in the intestinal epithelial cells or AhR expression in CD11c cells (AhR) by subjecting animals to twice daily hypoxic stress and gavage feeding with formula supplemented with LPS and enteric bacteria. During NEC, compared with wild-type mice treated with vehicle, littermates treated with an AhR proligand, indole-3-carbinol, had reduced expression of  and  a scavenger receptor that mediates dendritic cell activation and the recognition and clearance of bacterial pathogens by macrophages. Furthermore, indole-3-carbinol treatment led to the downregulation of genes involved in cytokine and chemokine, as revealed by pathway enrichment analysis. AhR expression in the intestinal epithelial cells and their cre-negative mouse littermates were similarly susceptible to experimental NEC, whereas AhR mice with NEC exhibited heightened inflammatory responses compared with their cre-negative mouse littermates. In seeking to determine the mechanisms involved in this increased inflammatory response, we identified the Tim-4 monocyte-dependent subset of macrophages as increased in AhR mice compared with their cre-negative littermates. Taken together, these findings demonstrate the potential for AhR ligands as a novel immunotherapeutic approach to the management of this devastating disease.

Indole-3-Carbinol-Dependent Aryl Hydrocarbon Receptor Signaling Attenuates the Inflammatory Response in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is an acute inflammatory disease that unforeseeably develops in very low birth weight premature infants. NEC is characterized by impairment of the intestinal barrier resulting in intestinal necrosis and multisystem organ failure. Animal models of NEC have contributed significantly to a better understanding of the underlying molecular mechanisms of the disease and facilitated the exploration of potential new therapeutic strategies. Here, we provide a detailed protocol that recapitulates some of the main histological and transcriptional features of human NEC in newborn mice.

A Mouse Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal that  expression in neonatal murine intestine is negligible until weaning, and both human and murine neonates lack IL-22 production during NEC. Mice deficient in IL-22 or lacking the IL-22 receptor in the intestine display a similar susceptibility to NEC, consistent with the lack of endogenous IL-22 during development. Strikingly, treatment with recombinant IL-22 during NEC substantially reduces inflammation and enhances epithelial regeneration. These findings may provide a new therapeutic strategy to attenuate NEC.

Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration.

The application of metabolomics in neonatology offers an approach to investigate the complex relationship between nutrition and infant health. Characterization of the metabolome of human milk enables an investigation into nutrients that affect the neonatal metabolism and identification of dietary interventions for infants at risk of diseases such as necrotizing enterocolitis (NEC). In this study, we aimed to identify differences in the metabolome of breast milk of 48 mothers with preterm infants with NEC and non-NEC healthy controls. A minimum significant difference was observed in the human milk metabolome between the mothers of infants with NEC and mothers of healthy control infants. However, significant differences in the metabolome related to fatty acid metabolism, oligosaccharides, amino sugars, amino acids, vitamins and oxidative stress-related metabolites were observed when comparing milk from mothers with control infants of ≤1.0 kg birth weight and >1.5 kg birth weight. Understanding the functional biological features of mothers' milk that may modulate infant health is important in the future of tailored nutrition and care of the preterm newborn.

Untargeted Metabolomic Analysis of Human Milk from Mothers of Preterm Infants.

Recapitulating human NEC using animal models has been insightful in dissecting the signaling pathways, immune-mediated mechanisms, genetic signatures, and the intestinal architecture of NEC. This protocol describes an  murine NEC model, using hypoxia and formula containing lipopolysaccharide and enteric bacteria derived from an infant with NEC. With this mouse model, we aim to further dissect NEC pathogenesis and develop new therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Mihi et al. (2021).

A protocol for the induction of experimental necrotizing enterocolitis in neonatal mice.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Necrotizing enterocolitis (NEC) is the most common and lethal gastrointestinal disease affecting preterm infants. NEC develops suddenly and is characterized by gut barrier destruction, an inflammatory response, intestinal necrosis and multi-system organ failure. There is currently no method for early NEC detection, and the pathogenesis of NEC remains unclear.

Selective hypermethylation is evident in small intestine samples from infants with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease.

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip.

Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology.

Necrotizing enterocolitis (NEC) is a potentially fatal intestinal disease primarily affecting preterm infants. Early diagnosis of neonates with NEC is crucial to improving outcomes; however, traditional diagnostic tools remain inadequate. Biomarkers represent an opportunity to improve the speed and accuracy of diagnosis, but they are not routinely used in clinical practice.

Article	Year
Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis. Inflammatory bowel diseases\| PubMed ID: 17932977	2008
A direct comparison of mouse and human intestinal development using epithelial gene expression patterns. Pediatric research\| PubMed ID: 31242501	2019
Association of Intestinal Alkaline Phosphatase With Necrotizing Enterocolitis Among Premature Infants. JAMA network open\| PubMed ID: 31702803	2019
Global hypermethylation of intestinal epithelial cells is a hallmark feature of neonatal surgical necrotizing enterocolitis. Clinical epigenetics\| PubMed ID: 33308304	2020
Neonatal necrotizing enterocolitis-associated DNA methylation signatures in the colon are evident in stool samples of affected individuals. Epigenomics\| PubMed ID: 33905263	2021
Indole-3-Carbinol-Dependent Aryl Hydrocarbon Receptor Signaling Attenuates the Inflammatory Response in Experimental Necrotizing Enterocolitis. ImmunoHorizons\| PubMed ID: 33906960	2021
A Mouse Model of Necrotizing Enterocolitis. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34048010	2021
Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration. Cell reports. Medicine\| PubMed ID: 34195684	2021
Untargeted Metabolomic Analysis of Human Milk from Mothers of Preterm Infants. Nutrients\| PubMed ID: 34684605	2021
A protocol for the induction of experimental necrotizing enterocolitis in neonatal mice. STAR protocols\| PubMed ID: 34816129	2021
Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins. Communications biology\| PubMed ID: 35022507	2022
Selective hypermethylation is evident in small intestine samples from infants with necrotizing enterocolitis. Clinical epigenetics\| PubMed ID: 35410447	2022
Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip. JCI insight\| PubMed ID: 36881475	2023
Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology. Cancer immunology, immunotherapy : CII\| PubMed ID: 37179276	2023
Identification of serum biomarkers for necrotizing enterocolitis using aptamer-based proteomics. Frontiers in pediatrics\| PubMed ID: 37325361	2023

Qingqing Gong

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