American Journal of Physiology-Gastrointestinal and Liver Physiology

Ischemic damage to the intestinal epithelial barrier, such as in necrotizing enterocolitis or small intestinal volvulus, is associated with higher mortality rates in younger patients. We have recently reported a powerful pig model to investigate these age-dependent outcomes in which mucosal barrier restitution is strikingly absent in neonates but can be rescued by direct application of homogenized mucosa from older, juvenile pigs by a yet-undefined mechanism. Within the mucosa, a postnatally developing network of enteric glial cells (EGC) is gaining recognition as a key regulator of the mucosal barrier. Therefore, we hypothesized that the developing EGC network may play an important role in coordinating intestinal barrier repair in neonates. Neonatal and juvenile jejunal mucosa recovering from surgically induced intestinal ischemia was visualized by scanning electron microscopy and the transcriptomic phenotypes were assessed by bulk RNA sequencing. EGC network density and gliosis were examined by gene set enrichment analysis, three-dimensional volume imaging and western blot and its function in regulating epithelial restitution assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate, and in vivo by co-culture assay. Here we refine and elaborate our translational model, confirming a neonatal phenotype characterized by a complete lack of coordinated reparative signaling in the mucosal microenvironment. Further, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that EGC function in close proximity to wounded intestinal epithelium is critical to intestinal barrier restitution following ischemic injury. NEW & NOTEWORTHYThis study refines a powerful translational pig model, defining an age-dependent relationship between enteric glia and the intestinal epithelium during intestinal ischemic injury and confirming an important role of the enteric glial cell activity in driving mucosal barrier restitution. This study suggests that targeting the enteric glial network could lead to novel interventions to improve recovery from intestinal injury in neonatal patients.

BackgroundIn the United States over 10% of all neonates are born premature (less than 37 weeks gestational age), and many face complications related to prematurity, including necrotizing enterocolitis (NEC). NEC is the most deadly gastrointestinal disease and the leading cause of death in preterm neonates, with up to 50% mortality. Since there is no cure for NEC, prevention is the best strategy. Enhancing our understanding of intestinal epithelial cell (IEC) responses to NEC damage will provide novel therapeutic targets to prevent NEC. Published evidence suggests that the RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) plays roles in intestinal development, barrier function, and intestinal repair. Notably, however, roles for IMP1 in NEC are not defined. Goblet cells produce protective mucus in the intestine, and their mature function is dependent on the transcription factor Spdef. Emerging evidence suggests that goblet cell mucus complexity impacts barrier function and inflammation susceptibility. This study aimed to define the role of IMP1 in NEC pathogenesis using neonatal human enteroids and a model of NEC-like intestinal injury in mice with IEC-specific Imp1 overexpression and loss. HypothesisIMP1 expression is protective in NEC. MethodsThis study used mice with intestinal epithelial Imp1 overexpression or loss and corresponding wild-type controls. At post-natal day 3, mice of both sexes were randomly assigned to control or NEC groups. NEC was induced with the well-established experimental NEC-like intestinal injury model that includes stress, formula feeding, and hypoxia. Imp1 effects on experimental NEC were assessed using RNA sequencing, western blotting, and immunostaining. ResultsInflammatory bacteria induced IMP1 expression in neonatal human enteroids. Mice with Imp1 overexpression incur worse intestinal damage during NEC. Pathway analysis of RNA sequencing data revealed a significant enrichment of the Spdef transcriptional network in Imp1IEC-OE during NEC. This included significant upregulation of Spdef target genes such as Agr2 (in NEC WT: 617.8 {+/-} 33.56 vs Imp1IEC-OE: 812.9 {+/-} 111.3, p=0.02) and Fut2 (in NEC WT: 219.4 {+/-} 34 vs Imp1IEC-OE: 396.6 {+/-} 62.9, p=0.05). In silico analysis predicted Imp1 binding to Spdef and mucus glycosylation mediator mRNAs. Although genotype did not affect goblet cell number, Imp1IEC-OE mice with NEC exhibited significant increases (p0.05) in Spdef protein, genes responsible for goblet cell function (Spink4, Klk1, Tspg1) and mucus glycosylation (Gcnt3, B3gnt7, Qsox1). Ultimately, Imp1 overexpression led to increased mucus fucosylation during NEC. ConclusionOur data indicate that during NEC, upregulation of Imp1 promotes goblet cell function via Spdef, including enhanced goblet cell maturation and mucus fucosylation. NEW AND NOTEWORTHYExpression of the RNA-binding protein Imp1 is enhanced in neonatal human enteroids in response to inflammatory bacteria. Imp1 regulates goblet cell function in response to early intestinal inflammation in a mouse model of necrotizing enterocolitis. Specifically, Imp1 promotes a Spdef transcriptional program by upregulating Spdef, resulting in elevated gene expression of Spdef target genes. Additionally, Imp1 increases the gene expression of (1,2)fucosyltransferase, Fut2, and subsequently, production of fucosylated mucus marked by UEA1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/702645v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@189f615org.highwire.dtl.DTLVardef@7aeeedorg.highwire.dtl.DTLVardef@dc258corg.highwire.dtl.DTLVardef@1252b98_HPS_FORMAT_FIGEXP M_FIG C_FIG

Necrotizing enterocolitis (NEC), a life-threatening intestinal disease, is becoming a larger proportionate cause of morbidity and mortality in premature infants. To date, therapeutic options remain elusive. Based on recent cell therapy studies, we investigated the effect of a human placental-derived stem cell (hPSC) therapy on intestinal damage in an experimental NEC rat pup model. NEC was induced in newborn Sprague-Dawley rat pups for 4 days via formula feeding, hypoxia, and LPS. NEC pups received intraperitoneal (ip) injections of either saline or hPSC (NEC-hPSC) at 32 and 56 hours into NEC induction. At 4 days, intestinal macroscopic and histological damage, epithelial cell composition, and inflammatory marker expression of the ileum was assessed. Breastfed (BF) littermates were used as controls. NEC pups developed significant bowel dilation and fragility in the ileum. Further, NEC induced loss of normal villi-crypt morphology, disruption of epithelial proliferation and apoptosis, and loss of Paneth cells and LGR5+ stem cells in the crypt. hPSC treatment improved macroscopic intestinal health with reduced ileal dilation and fragility. Histologically, hPSC administration had a significant reparative effect on the villi-crypt morphology and epithelium. In addition to a trend of decreased inflammatory marker expression, hPSC-NEC pups had increased epithelial proliferation and decreased apoptosis when compared to NEC littermates. Further, the intestinal stem cell niche of Paneth cells and LGR5+ stem cells was increased with hPSC therapy. Together, these data demonstrate hPSC can promote epithelial healing of NEC intestinal damage in part through support of the intestinal stem cell niche. New and NoteworthyThese studies demonstrate a human placental-derived stem cell (hPSC) therapeutic strategy for necrotizing enterocolitis (NEC). In an experimental model of NEC, hPSC administration improved macroscopic intestinal health, ameliorated epithelial morphology, and supported the intestinal stem cell niche. Our data suggest that hPSC are a potential therapeutic approach to attenuate established intestinal NEC damage. Further, we show hPSC are a novel research tool that can now be utilized to elucidate critical neonatal repair mechanisms to overcome NEC disease.

Organoids from intestinal regions have proven to be useful tools to study intestinal epithelial responses to different conditions. Roux-en-Y gastric bypass (RYGB) has been associated with important intestinal adaptations, but the mechanisms underlying these changes are still poorly understood. Organoids could therefore be used to better decipher the intestinal adaptations associated with this surgery. Rat is a common model to assess RYGB responses in vivo, but surprisingly, very few studies managed to develop organoids from rat small intestine. The primary objective of this study was to establish a protocol for cultivating organoids derived from the small intestine of healthy rats. The second objective of this study focuses on the development of organoids from the small intestine of rats subjected to RYGB to evaluate whether phenotypic or gene expression differences emerge. We successfully devised a functional protocol for developing organoids from fresh or frozen rat small intestine tissues. The obtained organoids exhibit significant variability, making interpretation challenging. Variability is observed in size, shape, and the number of organoids developed from the same sample, but also gene expression, depending on samples prepared on different days or from fresh or frozen tissues. This protocol was then applied to the small intestine of RYGB or sham-operated rats. However, we did not detect any major difference in size between intestinal organoids derived from Sham rats and those from RYGB rats. The expression of several genes (peptide transporters, amino acid transporters, genes specific to certain types of intestinal cells, etc.) was also assessed, and inter-experiment variability was higher than any effect due to the operation on the rat the intestinal tissue was originating. In conclusion, this study has established a functional protocol to grow small intestine organoids in rats. Initial results suggest that in our experimental conditions, organoids obtained from rats subjected to RYGB do not differ from those obtained from Sham rats. However, increasing the sample size and improving reproducibility between experiments will be essential to confirm these findings.

Hirschsprungs disease (HSCR) is a congenital defect in which the enteric nervous system (ENS) does not develop in the distal bowel, requiring surgical removal of the portions of bowel without ENS ganglia ( aganglionic) and reattachment of the normal proximal bowel with ENS ganglia. Unfortunately, many HSCR patients have persistent dysmotility (e.g., constipation, incontinence) and enterocolitis after surgery, suggesting that the remaining bowel is not normal despite having ENS ganglia. Anatomical and neurochemical alterations have been observed in the ENS-innervated proximal bowel from HSCR patients and mice, but no studies have recorded ENS activity to define the circuit mechanisms underlying post-surgical HSCR dysfunction. Here, we generated a HSCR mouse model with a genetically-encoded calcium indicator to map the ENS connectome in the proximal colon. We identified abnormal spontaneous and synaptic ENS activity in proximal colons from GCaMP-Ednrb-/- mice with HSCR that corresponded to motor dysfunction. Many HSCR-associated defects were also observed in GCaMP-Ednrb+/- mice, despite complete ENS innervation. Results suggest that functional abnormalities in the ENS-innervated bowel contribute to post-surgical bowel complications in HSCR patients, and HSCR-related mutations that do not cause aganglionosis may cause chronic colon dysfunction in patients without a HSCR diagnosis.

Background & AimsIntestinal tuft cells have recently been the interest of studies in several human gastrointestinal diseases. However, the impact of tuft cell deletion on intestinal physiological functions are not fully understood. This study investigated the effects of acute tuft cell loss on nutrient absorption and cell lineage differentiation. MethodsTuft cell deletion was induced in DCLK1-IRES-GFP-CreERT2/+;Rosa-DTA (DCLK1-DTA) mice by a single tamoxifen injection concomitant with littermate controls. Intestinal tissues were analyzed two-, four-, or seven-days post tamoxifen injection. ResultsDCLK1-DTA mice showed significantly shortened small intestinal length and body weight loss on day 4. Impaired activities of Na+-dependent glucose transporter 1 (SGLT1) and cystic fibrosis transmembrane regulator (CFTR) were observed in Ussing chamber experiments. Tissue immunostaining revealed a transient deletion of intestinal and biliary tuft cells, which was maximal on day 4 and recovered by day 7. On day 4 post tamoxifen, cholecystokinin (CCK)+ enteroendocrine cell numbers were increased particularly in the ileum. Correlated with the tuft cell reduction, the frequency of mislocalized Paneth cells, which were co-labeled by Paneth and goblet cell markers, was increased in the villus regions. In the lamina propria, fewer mast cells and leukocytes were found in the day 4 DCLK1-DTA mice than in controls. ConclusionAblation of intestinal tuft cells may induce nutrient malabsorption through alterations in epithelial cell proliferation and differentiation along with changes in mucosal defense response. These observations elucidate a new role for tuft cells in regulating intestinal absorption and mucosal regeneration.

Background and AimsGlissons capsule is the interstitial connective tissue that surrounds the liver. As part of its normal physiology, it withstands significant daily changes in liver size. The pathophysiology of the capsule in disease is not well understood. The aim of this study was to characterize the changes in capsule matrix, cellular composition, and mechanical properties that occur in liver disease and to determine whether these correlate with disease severity or etiology. Methods10 control, 6 steatotic, 7 moderately fibrotic and 37 cirrhotic patient samples were collected from autopsies, intraoperative biopsies and liver explants. Matrix proteins and cell markers were assessed by staining and second harmonic generation imaging. Mechanical tensile testing was performed on a test frame. ResultsCapsule thickness was significantly increased in cirrhotic samples compared to normal controls irrespective of disease etiology (69.62 {+/-} 9.99 and 171.269 {+/-} 16.65 {micro}m respectively), whereas steatosis and moderate fibrosis had no effect on thickness (62.15 {+/-} 4.97 {micro}m). Changes in cirrhosis included an increase in cell number (fibroblasts, vascular cells, infiltrating immune cells and biliary epithelial cells). Key matrix components (collagens 1 and 3, hyaluronan, versican and elastin) were all deposited in the lower capsule although only the relative amounts per area of hyaluronan and versican were increased. Organizational features including crimping and alignment of collagen fibers were also altered in cirrhosis. Unexpectedly, capsules from cirrhotic livers had decreased resistance to loading in comparison to controls. ConclusionsThe liver capsule, like the parenchyma, is an active site of disease, demonstrating changes in matrix and cell composition as well as mechanical properties. Lay summaryWe assessed the changes in composition and response to stretching of the liver outer sheath, the capsule, in human liver disease. We find an increase in key structural components and numbers of cells as well as a change in matrix organization of the capsule in the later stages of disease. This allows the diseased capsule to stretch more under any given force, suggesting it is less stiff than healthy tissue. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/505570v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@10a9b60org.highwire.dtl.DTLVardef@15eea52org.highwire.dtl.DTLVardef@69b874org.highwire.dtl.DTLVardef@cccd03_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe capsule is an active site of disease: thickness and cellularity increase markedly in cirrhosis C_LIO_LIExtracellular matrix composition and organization change in cirrhosis C_LIO_LIThe cirrhotic capsule stretches more and is less stiff C_LI

Intestinal organoids are promising tools in the context of animal experiment reduction. Yet, a thorough characterization of the impact of the origin of intestinal stem cells (ISC) on organoid phenotype is needed to routinely use this cellular model. Our objective was to evaluate the effect of ISC donor age on the growth, morphology and cellular composition of intestinal organoids derived from pig, a valuable model of Humans. Organoids were derived from jejunal and colonic ISC obtained from 1, 7, 28, 36 and 180-day old pigs and passaged three times. We first confirmed by qPCR that the expression of 18% of the >80 studied genes related to various intestinal functions differed between jejunal and colonic organoids after two passages (P<0.05). Growth and morphology of organoids depended on intestinal location (greater number and larger organoids derived from colonic than jejunal ISC, P<0.05) but also pig age. Indeed, when ISC were derived from young piglets, the ratio of organoids to spheroids was greater (P<0.05), spheroids were larger during the primary culture but smaller after two passages (P<0.05) and organoids smaller after one passage (P>0.05) compared to ISC from older pigs. Finally, no difference in cellular composition, evaluated by immunostaining of markers of the major intestinal cell types (absorptive, enteroendocrine and goblet cells) were observed between organoids originating from 7 or 180-day old pigs, while difference between intestinal site origin were noticed. In conclusion, while the age of the tissue donor affected organoid growth and morphology, it did not influence their phenotype.

BackgroundShort bowel syndrome (SBS) resulting from extensive small bowel resection is characterized by severe malabsorption and represents the leading cause of intestinal failure. Although spontaneous intestinal adaptation can partially restore nutrient absorption, the temporal coordination and hierarchy of the adaptive mechanisms involved--particularly those linking the gut microbiota, enteroendocrine function, hyperphagia, and intestinal remodeling-- remain incompletely understood. MethodsWe investigated the kinetics of spontaneous intestinal adaptation in a rat model mimicking type 2 SBS over a 28-day postoperative period. Body weight, food intake, gastrointestinal transit, fecal losses, intestinal morphology, enteroendocrine hormone secretion, hypothalamic neuropeptide expression, and gut microbiota composition were assessed longitudinally in SBS and SHAM-operated rats. ResultsExtensive small bowel resection induced marked early weight loss, accelerated intestinal transit, diarrhea, and increased fecal energy losses that persisted throughout the follow-up. Profound gut microbiota remodeling occurred as early as day 7, remained largely stable thereafter, and was characterized by reduced diversity and enrichment in Lactobacillaceae and Enterobacteriaceae. Early elongation of remaining colon and epithelial remodeling were observed, preceding the jejunal hyperplasia, which became evident from day 14 onward. Enteroendocrine adaptation was marked by an early increase in plasma peptide YY levels, whereas glucagon-like peptide-1 showed a modest response. Food intake was increased in SBS rats from day 7 onward, and hyperphagia developed gradually and reached a plateau by the end of the third postoperative week, in parallel with increased hypothalamic AgRP levels and reduced POMC levels. No significant improvement of intestinal transit and fecal energy losses was observed during the study period. ConclusionIntestinal adaptation to extensive resection follows a time-dependent sequence in which early gut microbiota remodeling and colonic adaptation precede hyperphagia and small intestinal remodeling. These findings highlight the gut microbiota and the colon as central components of the early post-resection adaptation and potential therapeutic targets in SBS.

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B{Delta}IEC) and Vil1-CreERT2;Myo5bflox/G519R(MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B{Delta}IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B{Delta}IEC tissues and enteroids. The present study demonstrates that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that may improve the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B. NEW & NOTEWOTHYThis study demonstrates the importance of MYO5B for cellular lipid metabolism and mitochondria in intestinal epithelial cells, a previously unexplored function of MYO5B. Alterations in cellular metabolism may underlie the progenitor cell malfunction observed in microvillus inclusion disease (MVID). To examine the therapeutic potential of progenitor-targeted treatments, the effects of LPAR5-preferred agonist, Compound-1, was investigated utilizing several MVID model mice and enteroids. Our observations suggests that Compound-1 may provide a therapeutic approach for treating MVID. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/610579v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@54b5borg.highwire.dtl.DTLVardef@1966a5aorg.highwire.dtl.DTLVardef@2073f1org.highwire.dtl.DTLVardef@9c12e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Diarrhea occurs in 2-50% of cases of COVID-19 ([~]8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for [~]24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by [~]8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of [~]10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

Background & AimsBiliary atresia (BA), the leading cause of liver transplantation in children, presents in neonates with jaundice and progressive extrahepatic bile duct obstruction, yet its etiology and pathogenesis remain unknown. Here, we aimed to investigate the molecular mechanisms underlying BA and the susceptibility of cholangiocytes in the extrahepatic biliary tree using patient-derived extrahepatic cholangiocyte organoids (EHCOs). MethodsEHCOs were derived from common bile ducts remnants of BA patients undergoing Kasai portoenterostomy and from non-BA controls at the time of liver transplantation. Transcriptomic profiling was performed via bulk RNA sequencing, and analyzed in two ways: differentially expressed pathways and perturbation analysis to predict aberrant functions. Key findings were validated through mechanistic assays, immunofluorescence staining, qPCR and transmission electron microscopy (TEM). ResultsTranscriptomic analysis predicted significant alteration in endoplasmic reticulum (ER) stress, dysregulations of drug metabolism, alongside pronounced alterations in cellular adhesion and polarity-related genes in BA-derived EHCOs. Cell-to-cell alterations were observed with various proteins including E-cadherin, RhoU, Sox17 and CFTR. BA EHCOs had an increased endoplasmic reticulum (ER) stress response, exemplified by elevated PERK, BiP, and ATF4 along with abnormal ER on TEM. Furthermore CHOP, ERO1A, WFS1, and SOD3 were decreased suggestive of abnormal ER stress response. BA EHCOs displayed increased toxicity to biliatresone-induced injury and inhibition of cytochrome P450 resulted in attenuation of the ER stress markers PERK, BiP and ATF4. Finally, liver hilum biopsies from BA patients undergoing Kasai portoenterostomy confirmed elevated PERK and PGR78(BiP) consistent with the EHCOs analysis. ConclusionsBA EHCOs exhibit disrupted polarity, ER stress, and increased susceptibility to drug toxicity. These findings highlight key pathogenic mechanisms in BA and suggest that targeting these pathways may help mitigate cholangiocyte injury in BA. Impact and implicationsThis study provides the first transcriptomic and functional analysis of human extrahepatic cholangiocyte organoids (EHCOs) derived from biliary atresia (BA) patients. By focusing on the extra-hepatic biliary tree, we identified key mechanisms of cholangiocyte injury, including persistent ER stress, impaired stress response pathways, altered drug metabolism and disrupted epithelial polarity. These findings highlight ER stress and metabolic vulnerability as potential therapeutic targets and establish EHCOs as a tractable model for investigating BA pathogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/649927v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@150ab7org.highwire.dtl.DTLVardef@172953forg.highwire.dtl.DTLVardef@1a46e4eorg.highwire.dtl.DTLVardef@45c5c6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst transcriptomic profiling of extrahepatic cholangiocyte organoids (EHCOs) from BA patients, C_LIO_LIrevealing distinct molecular alterations compared to controls. C_LIO_LIBA EHCOs exhibit disrupted epithelial polarity, with downregulation of E-cadherin and Sox17 and upregulation of CFTR. C_LIO_LIER stress is a hallmark of BA cholangiocytes, with elevated PERK, BiP, and ATF4, and C_LIO_LIdysregulation of downstream effectors including CHOP, ERO1A, and SOD3. C_LIO_LIBA EHCOs are more susceptible to biliatresone-induced injury, with enhanced ER stress and structural damage. C_LIO_LIInhibition of cytochrome P450 activity (CYP4A) reduces ER stress markers. C_LI

IntroductionBiliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause; however, the response of the fetal EHBD to injury remains unknown. The objective of this study was to define the fetal response to EHBD injury and to determine whether it follows a fetal wound healing paradigm. MethodsMouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. ResultsA wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland (PBG) hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. ConclusionThe fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with high levels of HA typical of the regenerative, scarless program termed fetal wound healing. Although generally beneficial, the expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD.

IntroductionBiliary fibrosis and inflammation are central to the pathogenesis of cholangiopathies such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Inflammatory and fibrogenic stimuli, such as transforming growth factor-{beta} (TGF{beta}) and lipopolysaccharide (LPS) signaling, drive these processes, but their underlying transcriptional mechanisms in cholangiocytes remain incompletely defined. We investigated the role of Runt-related transcription factor 1 (RUNX1) as a transcriptional co-regulator of fibroinflammatory signaling in cholangiocytes. MethodsHuman PSC-derived cholangiocytes (PSC-Cs) and mouse large biliary epithelial cells (MLEs) were subjected to RUNX1 knockdown or pharmacologic inhibition (Ro5-3335 or AI-10-104). Cytokine secretion was profiled by Luminex multiplexing; RUNX1 genomic binding and protein interactome were assessed by ChIP-qPCR, ChIP-seq, and LC-MS/MS. In vivo, Mdr2-/- mice received Ro5-3335, and cholangiocyte-selective Runx1 knockout mice (Krt19-CreERT) were challenged with a DDC diet, followed by evaluation of fibrosis and inflammation. ResultsRUNX1 expression was significantly increased in cholangiocytes from PSC and PBC patients, and Mdr2-/- mice. RUNX1 knockdown or inhibition reduced IL6, TNF, and other proinflammatory cytokines in PSC-Cs and attenuated TGF{beta}-, LPS-, and TNF-induced Il6 and Ccl2 expression in MLEs. ChIP-qPCR and ChIP-seq revealed TGF{beta}-induced RUNX1 binding to the Il6 promoter and 727 additional genomic sites enriched for fibrosis and inflammatory pathways; predicted upstream regulators included TGF{beta}, TNF, and NF{kappa}B signaling. Proteomic analysis identified TGF{beta}-induced RUNX1 interactions with SMAD2 and NF{kappa}B2. In vivo, Ro5-3335 treatment in Mdr2-/- mice reduced hepatic collagen, ECM gene expression, immune cell infiltration, and serum liver injury markers and bile acids. Similarly, cholangiocyte-specific Runx1 deletion mitigated fibrosis, inflammation, and liver injury in DDC-fed mice. ConclusionRUNX1 is a central transcriptional hub integrating TGF{beta} and inflammatory signals in cholangiocytes. Its inhibition attenuates biliary fibrosis and inflammation in cholestatic models, supporting RUNX1 as a potential therapeutic target in fibroinflammatory cholangiopathies.

The intestinal epithelium relies on rapid repair to maintain homeostasis after injury, and dysregulation of this process contributes to the pathogenesis of inflammatory bowel disease and colorectal cancer. Interleukin-11 (IL-11), a fibroblast-derived cytokine elevated in these diseases, has well-documented effects on stromal cells, but its direct action on intestinal epithelial cells remains poorly characterized. Here, we used mouse colon organoids as an isolated epithelial system to directly examine the effects of IL-11 on epithelial cells. IL-11 stimulation activated the canonical JAK/STAT3 pathway, as evidenced by increased STAT3 phosphorylation and Socs3 induction in a concentration-dependent manner. In a pipetting-based mechanical disruption model, IL-11 significantly increased the number of organoids recovered. Although mechanical disruption dominated the overall transcriptional landscape, RNA-seq analysis identified coordinated upregulation of STAT3 target genes and proliferation-related pathways specifically in response to IL-11. Pharmacological inhibition of STAT3 attenuated the IL-11-induced promotion of organoid recovery, indicating that STAT3 signaling mediates the epithelial response to IL-11 and maintains organoid size under basal conditions. Together, these findings demonstrate that IL-11 directly promotes intestinal epithelial repair after mechanical disruption through STAT3-dependent signaling, providing a mechanistic basis for its protective role in acute colonic injury.

Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

EGF-Containing Fibulin Extracellular Matrix Protein 1 (EFEMP1, also called fibulin 3) is an extracellular matrix protein linked in a genome-wide association study to biliary atresia, a fibro-inflammatory disease of the neonatal extrahepatic bile duct. EFEMP1 is expressed in most tissues and Efemp1 null mice have decreased elastic fibers in visceral fascia; however, in contrast to other short fibulins (fibulins 4 and 5), EFEMP1 does not have a role in the development of large elastic fibers, and its overall function remains unclear. We demonstrated that EFEMP1 is expressed in the submucosa of both neonatal and adult mouse and human extrahepatic bile ducts and that, in adult Efemp1+/- mice, elastin organization into fibers is decreased. We used pressure myography, a technique developed to study the mechanics of the vasculature, to show that Efemp1+/- extrahepatic bile ducts are more compliant to luminal pressure, leading to increased circumferential stretch. We conclude that EFEMP1 has an important role in the formation of elastic fibers and mechanical properties of the extrahepatic bile duct. These data suggest that altered expression of EFEMP1 in the extrahepatic bile duct leads to an abnormal response to mechanical stress such as obstruction, potentially explaining the role of EFEMP1 in biliary atresia.

PurposeHirschsprung-associated enterocolitis remains a major postoperative complication of Hirschsprungs disease (HD), and impaired epithelial barrier integrity has been proposed as a contributing factor. In this study, we investigated whether 12-hydroxyheptadecatrienoic acid (12-HHT), an endogenous leukotriene B4 receptor 2 (BLT-2) agonist, enhances the epithelial barrier and exerts anti-inflammatory effects in patient-derived colonic organoids. MethodsNormoganglionic specimens from rectal/rectosigmoid HD at pull-through (HD-N; n = 8) and transverse colon specimens from anorectal malformation (ARM) at colostomy closure (n = 10) were used to generate colonic organoids. Epithelia were isolated using ethylenediaminetetraacetic acid and subsequently embedded in Matrigel. Baseline expression of TJP1, TJP2, F11R (encoding junctional adhesion molecule-A), JAM2, CLDN1, CLDN3, CLDN4) and LTB4R2 (encoding BLT-2) was assessed by qPCR and immunoblotting. Organoids were then treated with 12-HHT (0.4, 2, or 10 M) for 7 days, followed by qPCR. Additional experiments assessed cytokine expression (IL1B, IL6) and TJPs after 24 h with tumor necrosis factor- (TNF-, 100 ng/mL) plus phosphate buffered saline or 12-HHT. Barrier function was evaluated using FITC-dextran influx assays. ResultsHD-N and ARM organoids exhibited similar growth efficiencies. Baseline expression for F11R, JAM2, CLDN1, CLDN3, CLDN4, and LTB4R2 was significantly lower in HD-N than in ARM. TJPs were upregulated by 12-HHT at 2 and 10 M in both groups, with stronger effects in ARM. In HD-N organoids, 10 M 12-HHT suppressed TNF--induced IL1B and IL6 elevation mitigated tight junction proteins (TJPs) downregulation more effectively than 2 M. 12-HHT attenuated TNF--induced FITC-dextran influx in HD-N organoids. Conclusion12-HHT may exert anti-inflammatory effects by integrating TJPs of HD-N.

Background and aimsWNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. MethodsWe investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3{chi} antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. ResultsMice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3{chi} antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. ConclusionWNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine. WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency StatementAll RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Enteric glial cells (EGC) are a prominent cell type of all layers of the gut wall, virtually controlling all gastrointestinal functions. While the development of transgenic mice has led to major advances in understanding EGC biology, in vitro models are still fairly limited and do not allow for the robust and reproducible establishment of primary cultures discriminating EGC from the inner versus outer layers of the gut wall. Here we report a novel method to separately grow EGC from the inner and outer layers of the intestinal wall from the same mouse with a high degree of purity and cell heterogeneity. Our results indicate that EGC from the inner layers of the gut wall exhibit higher calcium response to ATP when compared to EGC from the outer layers. We also show that inner EGC cultures express lower levels of the transcription factor Sox 10 as compared to outer EGC cultures, which mirrors in situ differential expression of Sox10 in submucosal (inner) versus myenteric (outer) plexus assessed using wholemounts. Confocal microscopy analyses of wholemounts further demonstrate that a majority of calretinin-expressing ganglionic cells of the submucosal plexus express the EGC marker S-100{beta}, while this population is marginally represented in ganglia of the myenteric plexus. Altogether this study describes a novel method of EGC primary cultures permitting for the first time to compare inner versus outer EGC and provides in vitro and ex vivo evidence that inner EGC and outer EGC are phenotypically and functionally distinct.