Cellular and Molecular Gastroenterology and Hepatology

Background & AimsIntrahepatic biliary epithelial cell (BEC) heterogeneity remains challenging to define. Here, we sought to identify BEC subpopulations and biomarkers in mouse liver. MethodsWe performed scRNA-seq on Sox9EGFP+ liver epithelium from mice subjected to bile duct ligation (BDL) and sham controls. A machine learning algorithm, NSForest, identified minimal, multi-gene signatures for BEC subpopulations. These "metagenes" were validated using hybridization chain reaction (HCR) FISH in tissue sections from wild-type mice and on primary BECs expanded in vitro. Metagenes were used to match BDL subpopulations to their corresponding sham subpopulations for differential gene expression (DGE) analysis. ResultsWe identified 4 BEC subpopulations in sham controls, each associated with 1-2 gene metagenes. Spatial localization of metagene-defined BEC subpopulations by HCR FISH revealed heterogeneous cellular composition of intrahepatic bile ducts. BECs belonging to a given subpopulation were most likely to have neighbors of the same identity, forming homogenous cellular compartments within ducts. BDL downregulated subpopulation-specific genes and upregulated a damage-associated gene set. BDL samples also included a proliferative subpopulation not found in sham controls, which contained populations enriched for three of the four metagenes. All BEC subpopulations were also found in monolayers in vitro, where they clustered spatially with BECs of the same subtype. ConclusionsNovel metagene biomarkers of BEC subpopulations facilitated spatial localization of BECs in situ, identified subpopulation specific injury responses, and confirmed that BEC heterogeneity is preserved in vitro. The presence of locally homogenous BEC "neighborhoods" in vitro suggests some degree of BEC organization may be epithelial-autonomous.

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.

Background & AimsClinically relevant postoperative pancreatic fistula (CR-POPF) remains a major cause of morbidity following pancreatic surgery, potentially due to fatty acid release by lipase activity. This study investigated how the biochemical composition of CR-POPF effluents drives cellular injury and transcriptional stress responses. MethodsDrain effluents from 14 patients undergoing pancreatoduodenectomy (7 with CR-POPF and 7 controls) were analyzed using gas chromatography-mass spectrometry. Candidate lipids were tested on human foreskin fibroblasts, mesothelial cells, and pancreatic epithelial cells using viability and cytotoxicity assays. Effluents were applied directly to cultures, and RNA sequencing was performed on cells exposed to the two most cytotoxic CR-POPF samples. ResultsMetabolomic profiling revealed lipolytic traits characterized by long-chain saturated fatty acids, including palmitic and stearic acid, and the palmitic acid monoacylglycerol monopalmitin, in drain effluents. These fatty acids accounted for over 70% of the variance in multivariate metabolomic analyses between CR-POPF and control groups. Dose-response assays confirmed concentration-dependent cytotoxicity (p < 0.0001), with a subtoxic threshold of 0.2 mM. Two effluents (AES1448 and GR1479) consistently reduced cell viability across models (F > 19, p < 0.0001). Transcriptomic profiling showed enrichment of inflammatory, unfolded-protein, and stress-response pathways, along with suppression of proliferation modules. GR1479 induced metabolic adaptation, whereas AES1448 and monopalmitin triggered overt lipotoxic stress. ConclusionsLipolysis-derived lipids may mediate stromal and mesothelial injury in CR-POPF. Integrating metabolomic, functional, and transcriptomic data uncovers a spectrum of cellular responses, spanning from adaptive remodeling to lipolysis-driven proteotoxic stress. These findings support lipid toxicity as a biochemical property of CR-POPF and a potential target for prevention. SynopsisThis study identifies long-chain saturated fatty acids in postoperative pancreatic effluents as key mediators of cytotoxic and inflammatory stress. Integrating metabolomic and transcriptomic analyses link effluent composition directly to cellular injury and impaired healing after pancreatic surgery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/705517v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@2ba175org.highwire.dtl.DTLVardef@752522org.highwire.dtl.DTLVardef@d8f823org.highwire.dtl.DTLVardef@8acb7f_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Regeneration depends on tightly coordinated transcriptional programs governed by a dynamic epigenetic landscape to regulate cell identity, proliferation, and tissue remodelling following injury. The livers highly regenerative due to the ability to rapidly upregulate genes that drive the cell cycle and other genes important for regeneration. Trimethylation of histone 3 lysine 27 (H3K27me3) is deposited by the polycomb repressive complex 2 (PRC2) and many genes occupied by H3K27me3 in their promoters in uninjured livers become induced following PH. Here we test the hypothesis that depleting H3K27me3 by hepatocyte-specific deletion of Embryonic Ectoderm Development (EedHepKO), a key component of PRC2, changes the regenerative response in the liver. We show that Eed eliminates H3K27me3 in hepatocytes, resulting in reduced liver size, increased hepatocyte death, proliferation and fibrosis associated with upregulation of cell cycle and fibrogenic genes. Though these mice are less likely to survive two-thirds partial hepatectomy than wildtype controls, those that do survive increase liver mass faster than WTs. Importantly the genes that are occupied by H3K27me3 in control uninjured livers are upregulated in EEDHepKO and become further induced following PH. These data show that modulation of PRC2 activity disrupts epigenetic patterning, induces liver injury, and alters regenerative outcomes, suggesting that precise control of PRC2 function could be harnessed to enhance regenerative capacity.

The intestinal mucus network, primarily composed of O-glycosylated MUC2 mucin polymers, is essential for protecting the gastrointestinal tract from microbial threats. Sialic acid (Sia), a terminal monosaccharide on complex O-glycans, plays a key role in maintaining mucus integrity and is frequently modified by Casd1-dependent O-acetylation (OAc). Despite its prevalence, the biological significance of sialic acid OAc (OAc-Sia) modifications in human and murine mucus remains unclear. We hypothesized that OAc-Sia variants on mucus interact with the microbiota and are required for optimal mucus barrier function and host-microbe homeostasis in the colon. To test this, we profiled OAc-Sia on human and mouse MUC2 in situ using viral-derived probes with bacterial FISH and confocal microscopy; generated intestinal epithelial cell (IEC)-specific Casd1 null mice (IEC Casd1-/-); performed sialylomic and O-glycomic HPLC-MS analyses; assessed microbial communities by 16S rRNA sequencing with quantitative microbial profiling (QMP); and evaluated disease susceptibility using DSS colitis and Citrobacter rodentium infection models. Results revealed that both human and murine mucins are extensively O-acetylated and interact with the microbiota, suggesting biological relevance. IEC Casd1-/- mice were viable and displayed a complete loss of mucin OAc-Sia, indicating Casd1 is the sole contributor to OAc-status. Unexpectedly, mucus function was intact in IEC Casd1-/- mice, with no difference in structure or quality vs. WT co-housed littermates.16S rRNA analysis showed a modest but significant sex-specific reduction of microbial loads in male IEC Casd1-/-mice, and a clear trend toward reduced Turicibacter spp. vs. WT mice in both male and females, without impacting overall short-chain fatty acid (SCFA) production. DSS treatment led to more severe and extensive tissue damage in IEC Casd1-/- mice. C. rodentium infection led to increased damage in the cecum and distal colon of IEC Casd1-/- mice without affecting pathogen load, suggesting that OAc-Sia status has a role in tolerance defense. These findings establish intestinal epithelial Sia O-acetylation as a component dispensable for mucus and host-microbe homeostasis at baseline, but important in limiting damage to mucosal inflammatory insults.

Background & AimsUnchecked inflammation in Eosinophilic esophagitis (EoE) leads to esophageal fibrosis and eventual stricture. Differentiated fibroblasts, termed myofibroblasts, are the main effector cells in fibrosis, responsible for secreting extracellular matrix proteins leading to tissue stiffness. Regulating myofibroblasts has not been explored as a therapeutic possibility in the fibrostenotic esophagus. Herein, we aim to investigate the efficacy of Prostaglandin E2 (PGE2) in dedifferentiation of the EoE myofibroblast. MethodsWe evaluated the efficacy and mechanism of myofibroblast dedifferentiation using fetal esophageal fibroblasts (FEF3), patient-derived fibroblasts, and a murine model of EoE. ResultsFibrosis markers (SMA, FN1, and COL1A1) and contractility of myofibroblasts were significantly decreased by PGE2 via the cAMP pathway. PGE2 treatment decreased nuclear accumulation of phospho-Smad2/3-YAP complex and induced phospho-YAP proteasomal degradation. Transcriptome analyses of FEF3 treated with TGF{beta} or PGE2 revealed that the Integrin1 pathway, and specifically thrombospondin 1 (THBS-1), was significantly upregulated by TGF{beta} and downregulated by PGE2, as supported by pseudo-bulk single-cell RNA-seq of EoE biopsies. THBS-1 was shown to be regulated by PGE2 via the cAMP/YAP pathway, and its knockdown induced myofibroblasts dedifferentiation. In a murine model of EoE, Butaprost, agonist of the E-prostanoid G protein-coupled receptor 2, treatment significantly reduced the expression of THBS-1, SMA, and FN1 along with a decrease in YAP nuclear translocation. Additionally, collagen fiber organization in the lamina propria was markedly reduced. ConclusionPGE2 promotes dedifferentiation of myofibroblasts in EoE via the cAMP/YAP/ THBS-1 pathway. Our data suggest that PGE2 is a promising treatment strategy for EoE with stenosis. What You Need to KnowO_ST_ABSBackground and ContextC_ST_ABSIn Eosinophilic esophagitis, unchecked inflammation and tissue stiffness drives fibroblast differentiation and fibrostenosis of the esophagus, yet targeting myofibroblasts as regulators of extracellular matrix deposition in fibrostenotic disease remains clinically unexplored. New FindingsProstaglandin E2 promotes dedifferentiation of myofibroblasts in eosinophilic esophagitis via the cAMP/YAP pathway, with Thrombospondin-1 identified as a critical YAP regulated target driving fibrostenosis. LimitationsThis study focused on fibroblast-specific mechanisms. The effects of PGE2 on esophageal epithelial differentiation, barrier function, and immune cell recruitment in EoE remain to be determined. Clinical Research RelevanceThis study demonstrates proof-of-concept that pharmacological reversal of established fibrosis is achievable in EoE. PGE2 and its EP2-selective agonists represent translatable therapeutic targets for fibrostenotic EoE--a patient population that remains treatment-refractory to current immunosuppressive approaches. Basic Research RelevanceThe cAMP/YAP/THBS-1 signaling in fibroblasts emerges as a critical therapeutic target for esophageal fibrosis. Importantly, this work demonstrates that terminally differentiated myofibroblasts retain remarkable plasticity and can dedifferentiate--challenging the paradigm that fibrosis is irreversible.

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

ObjectivesFree fatty acid receptors 2 and 3 (FFA2 and FFA3) are activated by nutrient-derived metabolites such as short-chain fatty acids (SCFAs) and ketone bodies, produced by the gut microbiota and host, respectively. This study aimed to investigate the intracellular signaling pathways recruited in glucagon-like peptide-1 (GLP-1) releasing enteroendocrine cells following activation of FFA2 and FFA3 to resolve the impact of nutrient status on enteroendocrine cell function. MethodsExperiments were performed using primary mouse colonic cultures and the mouse enteroendocrine cell line, GLUTag cells. Expression analysis by bulk RNA sequencing was used to determine expression of FFA2 and FFA3 in GLP-1 releasing cells. Measurement of GLP-1 secretion by sandwich ELISA was used to assess enteroendocrine cell function. Live-cell measurements of intracellular calcium and cAMP levels were performed to assess canonical second messenger signaling pathways. ResultsA SCFA mixture stimulated GLP-1 secretion from both primary mouse colonic cultures and GLUTag cells. In GLUTag cells, the FFA2 ligand 4-CMTB inhibited GLP-1 release independent of Gaq- and Gai-signaling as neither YM-254890 (Gaq inhibitor) nor pertussis toxin (Gai- uncoupler) altered its effect. However, 4-CMTB did elevate cAMP levels, suggesting an indirect mechanism for the increase in cAMP production. Stimulation of FFA2 with the Gai-biased ligand AZ1729 or the ketone body acetoacetate inhibited GLP-1 release and cAMP accumulation. AZ1729 was insensitive to pertussis toxin and OZITX, supporting atypical FFA2 signaling. Stimulation of FFA3 with AR420626 or the ketone body {beta}-hydroxybutyrate increased GLP-1 secretion from GLUTag cells, an effect that was not mediated by cAMP production. AR420626, but not {beta}-hydroxybutyrate increased intracellular calcium levels. ConclusionsOverall, activation of FFA2 inhibited secretory function in GLP-1-releasing enteroendocrine cells, whereas activation of FFA3 stimulated GLP-1 secretion via distinct intracellular signaling mechanisms. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/708924v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@175dbaforg.highwire.dtl.DTLVardef@a9cc3eorg.highwire.dtl.DTLVardef@1a026e5org.highwire.dtl.DTLVardef@15997af_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIExposure to physiological levels of SCFAs stimulates GLP-1 secretion from colonic EECs C_LIO_LIFFA2 and FFA3 regulate GLP-1 release via non-canonical signaling pathways C_LIO_LIKetone bodies activate SCFA receptors to differentially modulate GLP-1 levels C_LIO_LILigand bias enables nutrient-dependent tuning of EEC gut hormone secretion C_LI

Background: Gastric cancer surveillance in CDH1 pathogenic variant carriers is challenging, as predictors of localized (stage T1a) and advanced (stage >T1a) signet ring cell carcinoma (SRCC) are not well defined. We established the Group of investigAtors STriving toward Research In CDH1 (GASTRIC) consortium to identify clinicopathological factors associated with localized and advanced SRCC. Methods: A retrospective observational study (1998-2025) of CDH1 carriers across twelve academic centers was performed. Clinical, endoscopic, and pathological data were compared between carriers with and without SRCC on endoscopy, and between those with advanced versus localized or no cancer on gastrectomy specimens. Results: Overall, 390 CDH1 carriers from 235 families were included. Presence of SRCCs on endoscopy was significantly associated with thickened folds, nodularity, masses, and intestinal metaplasia, while gastritis was negatively associated. Of 196 carriers (52.4%) undergoing gastrectomy, 11 (5.6%) had advanced cancers, 10(90.9%) of which showed endoscopic abnormalities. Identification of SRCC on baseline endoscopy was the most sensitive feature for advanced disease (0.81) but had moderate specificity (0.74), whereas masses and thickened folds were highly specific (0.99 and 0.96, respectively) but less sensitive. Negative predictive values were high (0.94-1.0), while positive predictive values were modest (0.13-0.66). On multivariate analysis, masses and SRCC foci on baseline endoscopy were independent predictors of advanced disease. Conclusion: Among CDH1 carriers, absence of endoscopic findings was reassuring, whereas significance of detected endoscopic and pathological abnormalities was less certain. Advanced cancer occurred in a small number of carriers, with endoscopic abnormalities in nearly all cases. Endoscopic surveillance might be an alternative to surgery in carriers without worrisome mucosal findings.

Stem cells are critical for the development and maintenance of tissue integrity. An important example is intestinal stem cells (ISCs) that generate all epithelial cell types necessary for formation of the intestinal lining. HAT1, a histone acetyltransferase that acetylates newly synthesized histone H4 molecules on lysine residues 5 and 12 during replication-coupled chromatin assembly, is specifically expressed in intestinal stem and progenitor cells located in intestinal crypts. To determine if HAT1 is important for intestinal stem and progenitor cell function, we generated an inducible deletion of the HAT1 gene in intestinal epithelial cells. Loss of HAT1 resulted in morphological defects in the proximal end of the small intestine. Following loss of HAT1, intestinal crypts became elongated, with an increase in stem and progenitor cell proliferation and an increase in the population of OLFM+ cells. Loss of HAT1 also resulted in alterations in intestinal stem cell differentiation, including an increase in the number of Goblet cells and the mislocalization of Paneth cells into villi. HAT1 is specifically responsible for the acetylation of histone H4 lysine 5 (H4K5ac) in intestinal stem cells. Genome-wide characterization of HAT1-dependent H4K5ac in intestinal crypt cells indicates that the most significant loss of H4K5ac occurs in lamina-associated domains (LADs). Loss of H4K5ac in LADs is accompanied by an increase in histone H3 K9 tri-methylation indicating that HAT1 regulates LAD chromatin structure in intestinal crypt cells. A direct role for HAT1 in intestinal stem cell function was demonstrated using organoids in culture. HAT1 is required for differentiation in organoids and for the maintenance of Lgr5+ stem cells. These results indicate that HAT1 is required for the proper regulation of intestinal stem cell renewal and differentiation.

BackgroundKeratins, a major subgroup of intermediate filament proteins, play a critical role in maintaining epithelial barrier and intracellular epithelial integrity. Studies have demonstrated possible links between inflammatory signaling and colonic keratins type II K8, and type I K18, K19 and K20, in animal models of colitis, and in patients with Inflammatory Bowel Disease (IBD). K7 is de novo expressed in patients with the IBD subtypes Ulcerative Colitis (UC) and Crohns Disease (CD). However, the histopathological roles of colonocyte keratins across IBD, microscopic colitis (MC), and Irritable Bowel Syndrome (IBS) remain poorly understood. Given the established utility as biomarkers in cancer diagnostics, we investigated whether keratin expression patterns could be used to distinguish inflammatory and functional colonic disorders. MethodsBiobank samples from patients with IBD (n=27), MC (n=18), IBS (n=32) and healthy controls (n=31), were collected and immunohistochemically stained for K7, K8, K18, K19, and K20. Digital image analysis quantified staining intensities, which were correlated with histopathological severity scores and clinical parameters. ResultsColonic keratin expression was significantly elevated in IBD, particularly in UC, while they were decreased in MC, and unaltered in IBS. Notably, K8 and K19 expression were strongly associated with areas of severe epithelial damage in IBD. Keratin expression was most pronounced in patients who had undergone colectomy due to treatment-resistant IBD. DiscussionKeratin changes in IBD and MC but not in IBS highlight their importance in maintaining barrier homeostasis. Whether these changes are causes or consequences for these diseases will warrant further research.

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.

Time-restricted feeding (TRF) is widely considered metabolically beneficial, yet its impact on chronic liver disease progression remains poorly defined. This study investigates the effects of TRF on liver fibrogenesis. Using carbon tetrachloride (CCl4)-induced, bile duct ligation (BDL)-induced, and choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced murine models of liver fibrosis, we demonstrate that TRF consistently exacerbates fibrotic injury. Mechanistically, TRF induces the systemic elevation of the ketone body {beta}-hydroxybutyrate (BHB). We identify the ketolytic enzyme 3-hydroxybutyrate dehydrogenase 1 (BDH1) as a critical mediator of this process within hepatic stellate cells (HSCs). BDH1 expression is markedly upregulated in activated HSCs, enabling these cells to metabolize BHB. This BDH1-dependent ketolysis redirects BHB-derived carbons into the tricarboxylic acid cycle, supplying acetyl-CoA and citrate to drive de novo lipogenesis and support a profibrogenic metabolic state. Both the genetic ablation of Bdh1 specifically in HSCs and the inhibition of hepatic ketogenesis successfully abolished the pro-fibrotic effects of TRF and exogenous BHB administration. Conversely, exogenous BHB alone was sufficient to recapitulate the exacerbated fibrotic phenotype observed with TRF. These findings reveal a context-dependent, detrimental role for TRF during chronic liver injury, driven by BDH1-mediated metabolic reprogramming in HSCs. Consequently, dietary interventions that elevate systemic ketone bodies should be approached with caution in the setting of active liver fibrosis.

Mast cells (MCs) are myeloid cells of the innate immune system. As a first line of defence they fulfill effector functions and immune modulatory properties. Upon activation they release pro-inflammatory mediators such as cytokines and proteases. It has been suggested that MCs may contribute to the development of liver fibrosis. However, investigating hepatic MC biology in mice is challenging due to low MC numbers and a lack of suitable detection techniques relying on MC proteins and their modifications. Here, we evaluated whether the expression strength of MC markers correlates with the degree of liver fibrosis in mice and aimed to determine the frequency and localization of hepatic MCs. We applied both a toxic (DEN/CCl4 treatment) and a genetic (Mdr2-/- mice) liver fibrosis model in C57BL/6 mice and found a significant correlation between fibrosis grade and the expression of several established mast cell markers. This correlation was further supported in patients with fibrosis and hepatocellular carcinoma (HCC) using publicly available transcriptomics datasets. We used FACS to purify and isolate MCs from fibrotic mouse livers and verified MC signatures by qPCR analysis of MC-specific gene expression. Hepatic MCs were predominantly negative for Mast-Cell-Protease 5 (Mcpt5) and occurred at a low frequency (approximately 1-2% of leukocytes). Using Molecular CartographyTM of fibrotic liver sections, we determined the spatial localization, expression signature, abundance (approximately 2 cells/mm2) and cellular environment of murine hepatic MCs. In summary, we demonstrated the existence of MCs in murine fibrotic livers and defined an MC expression signature that correlates with the strength of liver fibrosis. These findings will help to study MC biology in murine models of liver disease more effectively in the future.

Identifying biological roles for glycosyltransferases is a continuing challenge and important for defining morbidities associated with congenital disorders of glycosylation. Here we investigate the consequences to intestinal development of conditionally deleting Lfng alone or Lfng, Mfng and Rfng together in a mixed or Eogt-null genetic background. Each Fringe transfers N-acetylglucosamine (GlcNAc) to fucose (Fuc) attached to Ser or Thr by POFUT1 in a consensus sequence found in certain epithelial growth factor-like (EGF) repeats. EOGT transfers GlcNAc directly to Ser/Thr in a separate consensus sequence of the EGF repeat. Notch receptors and Notch ligands contain the largest number of EGF repeats with consensus sites for these O-glycans. Conditional deletion of Pofut1 in mouse intestine causes similar developmental defects to deletion of Notch1 and Notch2 or Dll1 and Dll4. LFNG also contributes to optimal Notch signaling in mouse intestine. In this work, we generated Lfng[F/F]:Villin-Cre and Lfng[F/F]Mfng[-/-]Rfng[-/-]:Villin-Cre mice in which extension of O-Fuc on EGF repeats was inhibited or prevented in intestinal epithelium. Conditional deletion of either Lfng alone or all three Fringe activities together led to defective intestinal development with a marked increase in goblet and Paneth cells, increased crypt width and reduced villus length. Unexpectedly, in mice globally lacking EOGT, conditional inactivation of the three Fringe genes did not lead to defective intestinal development. Thus, the absence of EOGT prevented disruption of development in Fringe-null intestine, identifying a novel role for EOGT in regulating intestinal development.

Cirrhosis, the end stage of chronic liver disease marked by fibrosis and impaired liver function, is associated with cirrhosis-associated immune dysfunction, a condition in which systemic inflammation coexists with impaired host defense and increased susceptibility to infections. However, intestinal intraepithelial lymphocytes (IELs), key mediators of epithelial immune defense, remain poorly characterized in this context. Using high-dimensional profiling of paired duodenal biopsies and peripheral blood across disease stages, we define IEL alterations in cirrhosis. Contrary to prior reports of immune exhaustion, lymphocyte effector function was preserved, while disease progression was marked by systemic inflammatory remodeling and increased tumor necrosis factor (TNF) production by circulating T cells. The IEL compartment was markedly altered, with loss of CD8{beta} IELs, expansion of natural killer (NK) IELs, and reduced CCR9CD8{beta} IELs, suggesting altered gut homing. These findings refine cirrhosis-associated immune dysfunction as inflammatory immune reprogramming coupled to impaired epithelial immune surveillance. HighlightsPeripheral lymphocytes from cirrhosis patients retain effector capacity with enhanced inflammatory activity Cirrhosis reshapes the duodenal intraepithelial lymphocyte landscape Reduced frequency of CCR9+CD8{beta} IELs indicates altered gut-homing in cirrhosis

ObjectiveTo establish a fetal lamb model of complex gastroschisis and characterize the impact on the intestines over time. Summary Background DataGastroschisis is a congenital abdominal wall defect and in its complex form is associated with serious morbidity. Robust large-animal models may help understanding are lacking. MethodsAt gestational day 75, gastroschisis was induced by creating a 1-cm abdominal wall defect reinforced by a silicone ring. Fetuses were assessed either at term or at mid-gestation (13-21 days post-induction). The primary outcome was complex gastroschisis occurrence, defined by bowel stenosis, atresia, volvulus, perforation or necrosis; otherwise classified as simple. At mid-gestation, occurrence was compared between early (13-16 days) and late (17-21 days) intervals. Secondary outcomes included prenatal ultrasound findings, in vivo bowel motility and morphology, ex-vivo bowel contractility, amniotic fluid composition, and histology across complex, simple, and normal groups. ResultsGastroschisis was induced in 32 fetuses. At term (n=14), all survivors (7/14; 50%) had complex gastroschisis, with impaired bowel motility, altered enteric neural contractile responses and smooth muscle remodeling. At mid-gestation (n=18), complex gastroschisis occurred more frequently in the late than in the early group (71% vs. 11%; p=0.035). Mid-gestation gastroschisis fetuses showed greater intra-abdominal bowel dilatation on ultrasound and higher amniotic fluid digestive enzyme levels compared with non-operated littermates, with the greatest dilation observed in complex gastroschisis. ConclusionsThis model consistently reproduces complex gastroschisis in term survivors. After induction, complex gastroschisis occurrence increases with disease duration and is accompanied by structural and functional bowel changes.

Background/AimsIntrahepatic cholangiocarcinoma (iCCA) represents an unmet clinical need due to its increasing incidence, aggressive biology, and limited treatment options. The extremely low-response rates to current systemic regimens and the emergence of adaptive resistance to targeted therapies underscore the urgent need for alternative therapeutic strategies. Given that the lineage-defining transcription factors SOX9 and YAP1 are central regulators of cholangiocyte and iCCA identity, we investigated their functional roles as potential therapeutic vulnerabilities across multiple preclinical models. MethodsPatient tissue-microarray (TMA) analysis, Sleeping-Beauty hydrodynamic tail vein injection-based iCCA models, and Cre-mediated inducible gene deletion systems were used to investigate the roles of Sox9 and Yap1. Deep-learning-based prediction, RNA-seq, ChIP-seq and immunohistochemistry analyses were performed to delineate transcriptional networks and downstream effectors associated with SOX9/YAP1 signaling. ResultsDual deletion of Sox9 and Yap1 effectively eradicated advanced iCCA while preserving intrahepatic bile ducts, regardless of oncogenic drivers. Mechanistically, SOX9 and YAP1 transcriptionally compensated for each other when one was absent, and ILF2, MGAT5, and WWTR1 were identified as key downstream effectors mediating this compensatory mechanism. Loss of Ilf2, Mgat5, or Taz suppressed iCCA, whereas overexpression of Ilf2 or Taz following Sox9/Yap1 co-deletion restored tumor development, indicating that ILF2 or TAZ can functionally substitute for YAP1 and SOX9 in sustaining iCCA. ConclusionsCo-targeting SOX9 and YAP1 offers a promising and safe broad-spectrum preventive/therapeutic approach for iCCA, potentially overcoming resistance to YAP1 inhibition. The adaptive resistance mechanism identified may extend to other malignancies, providing insights for addressing the advanced resistant to YAP1-TEAD-directed therapies.

BackgroundIntestinal barrier dysfunction is a hallmark of inflammatory bowel diseases (IBD). This condition causes intoxication and immune hyperactivation. Understanding the events underlying epithelial barrier disruption during chronic inflammation is key to developing barrier-restoring therapies. Filamentous actin (F-actin) is essential for maintaining polarity and junctional integrity. However, the contribution of F-actin dynamics to IBD-associated barrier dysfunction remains unclear. ObjectiveWe aimed to examine actin cytoskeleton integrity during chronic colitis across mouse models and human patients and identify potential regulators of cytoskeleton dynamics. DesignF-actin and junctional proteins were analyzed in three models of chronic colitis (Muc2 KO, DSS-induced colitis, adoptive transfer colitis) using confocal microscopy. Claudin-3 interactors were identified by immunoprecipitation and proteomics. Intestinal organoids were used to assess the effect of F-actin disruption on barrier integrity. Metabolomic and gene expression analyses identified candidate pathways, further validated by chemical inhibition. Biopsies from patients with ulcerative colitis (UC) were examined using transmission electron microscopy and confocal microscopy. ResultsDisrupted actin dynamics emerged as a critical driver of epithelial barrier dysfunction in chronic colitis. An imbalance between polymeric and monomeric actin impaired barrier integrity in vivo and in 3D organoids. Immunoprecipitation identified actin and associated factors as the primary interactors of claudin-3 with reduced interaction during inflammation. Ceramide metabolism was revealed as a potential regulator of F-actin and intestinal barrier. In UC patients, we confirmed concurrent disruption of junctions and F-actin. ConclusionsF-actin dysregulation contributes to barrier dysfunction in IBD and targeting its modulators, including ceramide biosynthesis, represents a novel therapeutic strategy. WHAT IS ALREADY KNOWN ON THIS TOPICO_LIEpithelial damage and increased paracellular permeability are key characteristics of inflammatory bowel diseases. C_LIO_LIParacellular permeability is partially attributed to the downregulation of junction proteins but this mechanism does not explain all clinical observations. C_LIO_LIIn the Muc2 KO mouse model of chronic colitis, F-actin organization and membrane localization of tight junction protein claudin-3 are disrupted, although protein expression levels remain unchanged. C_LI WHAT THIS STUDY ADDSO_LIF-actin dynamics is impaired in the intestinal epithelium across three different mouse models of chronic colitis and IBD patients. C_LIO_LIDisruption of F-actin dynamics leads to impaired membrane localization of tight and adherens junction proteins and increased intestinal epithelial permeability in vivo and in colonic organoids. C_LIO_LIInhibition of ceramide biosynthesis rescues F-actin polymerization and intestinal barrier integrity in mouse chronic colitis models. C_LI HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYO_LITargeting F-actin dynamics is a promising approach to improve gut epithelial integrity. C_LIO_LI"Ceramide-F-actin-junction" axis is proposed as one of the mechanisms behind epithelial barrier disruption in colitis. Therapeutic targeting of this axis represents a promising path for restoring gut integrity. C_LI

The circadian clock coordinates physiology and behavior through [~]24-h rhythms, and disruption of core clock genes has been implicated in tumorigenesis. However, the impact of Per1, a major clock gene, on colorectal tumor development remains unclear. Here, we investigated how Per1 deletion influences intestinal tumorigenesis using the ApcMin/+ mouse model and ApcMin/+Per1-/-mice generated by crossing ApcMin/+ and Per1-/- lines (C57BL/6J background). Mice were maintained under controlled light-dark conditions, and we assessed survival, intestinal polyp burden, histopathology using Swiss-roll sections, {beta}-catenin protein abundance (immunofluorescence and western blotting), Ctnnb1 mRNA expression (RT-qPCR), and crypt proliferation (5-bromo-2-deoxyuridine (BrdU) immunohistochemistry). Per1 deletion did not significantly alter overall survival in ApcMin/+ mice but increased inter-individual variability. In contrast, polyp number was markedly increased by Per1 deletion, affecting both small (<2 mm) and large ([&ge;]2 mm) polyps across intestinal segments. Histology confirmed aberrant crypt foci and polyps in both ApcMin/+ genotypes. {beta}-Catenin protein levels in the whole intestine were significantly increased by Per1 deficiency and Apc mutation (two-way ANOVA), whereas Ctnnb1 mRNA was largely unchanged across regions. BrdU-based crypt proliferation was increased by the Apc mutation but not by Per1 deletion. These results indicate that Per1 loss exacerbates intestinal polyp formation and elevates {beta}-catenin predominantly through non-transcriptional mechanisms, supporting a tumor-suppressive role of Per1 in colorectal tumorigenesis.