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.

BackgroundSerrated neoplasia accounts for [~]25% of colorectal cancer. These cancers arise from serrated precursor lesions. Hyperplastic polyps initiated by either BRAF or KRAS mutation activating MAPK signalling are common, but premalignant sessile serrated lesions with KRAS mutation are rare. Here, we model Kras and Braf mutant neoplasia in vivo to compare histological, gene expression and DNA methylation manifestations associated with activation of these oncogenes. MethodsWe employ cre-recombinase dependent BrafV637 and KrasG12D murine models, and cross animals with those bearing the Villin-CreERT2 transgene to direct temporospatial activation of these oncogenes to the murine intestine. We examine histology, and genome-scale DNA methylation and gene expression via reduced representation bisulphite sequencing and RNA-Seq, respectively. We performed differential gene expression, methylation and pathways analysis to identify oncogene specific alterations. ResultsProlonged exposure to oncogenic Braf is associated with a time-dependent accumulation of murine serrated precursors (P=3x10-10) and advanced murine serrated lesions and invasive cancer (8x10- 8). Kras mutant animals acquire fewer precursor lesions (P=0.06) and have a significantly lower probability of developing advanced serrated lesions (P=0.004). Braf and Kras mutant animals develop pronounced hyperplasia, however the severity is significantly less in Kras mutant animals. Kras mutant advanced serrated lesions rarely develop aberrant WNT signaling activation (1/23). Gene expression profiling showed divergent transcriptomic profiles between Braf and Kras mutant intestines, with the former overexpressing genes associated with immune and inflammatory signaling. Deconvolution analysis revealed a comparably higher macrophage infiltrate (P=0.025) and upregulation of M1 macrophage gene sets in the Braf mutant intestine (P=0.0008), contributing to chronic inflammatory signalling. Both Kras and Braf mutations lead to accumulation of substantial temporal DNA methylation alterations, however a subset of CpG sites (1,306) show an attenuated rate of DNA methylation accumulation in the Kras mutant intestine compared with Braf mutant animals. ConclusionsIn this study, we show that Kras mutation can induce serrated intestinal neoplasia, however the latency period and penetrance is significantly lower when compared with Braf mutation. Aberrant WNT signalling is common in lesions arising in the context of Braf mutation, but rare in Kras mutant neoplasms. We show marked transcriptomic disparities between these models, with a tendency for the Braf mutant intestine to upregulate immunological processes. Our DNA methylation analysis reveals an attenuated CIMP-like phenotype that is specific to the Kras mutant intestine, consistent with our previous works in humans. These data have significant implications for our understanding of how MAPK-induced neoplasia develops within the intestine. SynopsisBRAF and KRAS mutant hyperplastic polyps have disparate malignant potential and the reason for this is unclear given both oncogenes activate MAPK signalling. We show that the DNA methylation alterations that follow Kras mutation are attenuated and that hyperactivation of WNT signaling is rare, providing a molecular mechanism that restrains malignant transformation.

Background and AimsMetabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Though MASH is closely tied to metabolic risk factors, the underlying pathogenic mechanisms remain scarcely understood. Recent research underscores the importance of the gut-liver axis in its pathogenesis, an aspect less explored in human studies. Here, we investigated whether the duodenal epithelium of MASH patients, could exhibit intrinsic dysfunctions. MethodsDuodenal epithelial organoids were generated from 16 MASH patients and 14 healthy controls. Biopsies and patient-derived organoid transcriptomes were then analyzed to evaluate if specific intestinal pathways were differentially modulated in MASH subjects. Functional assays were performed to assess the duodenal epithelial digestive potential and barrier functionality. ResultsOrganoid formation efficiency was similar between control-derived epithelial organoids (CDEOs) and MASH-derived epithelial organoids (MDEOs) (71% and 69%, respectively). Despite global heterogeneity in growth patterns, MDEOs frequently exhibited cystic spheroid morphology. MDEOs displayed altered digestive homeostasis associated with reduced mature absorptive cell fate, but they retained their lipid metabolic capacity, possibly mediated by lipid oxidation in stem/progenitor cells. Additionally, MDEOs misexpressed components of tight and adherens junctions and desmosomes compared to controls. However, MDEOs maintained pore and leak pathway integrity, indicating that the duodenal epithelial barrier remained functionally preserved under tested conditions. ConclusionsThis study provides evidence that the duodenal epithelium of MASH patients exhibits significant alterations in its digestive and barrier functions. This study sheds light on the intricate dynamics of duodenal epithelial alterations in MASH, highlighting potential therapeutic avenues for restoring intestinal homeostasis.

Activation of bile acid (BA) receptor, farnesoid X receptor (FXR) has been shown to inhibit inflammatory responses and improve tissue ischemia-reperfusion injury (IRI). This study investigated the effect of FXR deficiency on liver IRI, using a liver warm IRI mouse model. We demonstrate that liver IRI resulted in decreased FXR expression in the liver of WT mice. FXR-/-mice displayed greater liver damage and inflammatory responses than WT mice, characterized by significant increases in liver weight, serum AST and ALT, hepatocyte apoptosis and liver inflammatory cytokines. Liver IRI increased expression of X box binding protein 1 (XBP1) and FGF21 in WT liver, but not in FXR-/- liver, which conversely increased CHOP expression, suggesting a loss of ER stress protection in the absence of FXR. FXR deficiency increased circulating total BAs and altered BA composition with reduced TUDCA and hepatic BA synthesis markers. FXR deficiency also reshaped gut microbiota composition with increased Bacteroidetes and Proteobacteria and decreased Firmicutes. Curiously, Bacteroidetes were positively and Firmicutes were negatively correlated with serum ALT levels. Administration of FXR agonist CDCA inhibited NF-{kappa}B activity and TNF expression in vitro and improved liver IRI in vivo. Our findings demonstrate that FXR signaling plays an important role in the modulation of liver IRI.

Background & AimsAutophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. MethodsWe generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. ResultsGenetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA-Seq suggested increased autophagy in Cyto- IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-IDLow and Cyto- IDHigh cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. ConclusionsHigh AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo.

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 and AimsPrimary sclerosing cholangitis (PSC) associated inflammatory bowel diseases (IBD) increase colorectal dysplasia and malignancy risk. Current mouse models do not adequately replicate human PSC-IBD, limiting mechanistic understanding and therapeutic development. This study uses Mdr2/Il10 double knockout (DKO) mice to examine microbiota roles in mediating colitis, colitis-associated colorectal dysplasia and hepatobiliary inflammation/fibrosis. GoalDevelop and phenotype a chronic spontaneous PSC-IBD mouse model, emphasizing colitis, colonic dysplasia, hepatobiliary inflammation/ fibrosis and the functional roles of resident microbiota. MethodsWe utilized germ-free (GF) and specific-pathogen-free Mdr2/Il10 DKO, Il10-/- and Mdr2-/- mice to model PSC-IBD. We monitored colonic dysplasia progression, colitis kinetics and severity by lipocalin-2, cytokine measurement, and tissue evaluations of colon and liver. We manipulated the microbiome to assess its functional effects. ResultsDKO mice exhibited age- and region-specific accelerated colitis and spontaneous colonic dysplasia progressing to high-grade invasive adenocarcinomas. Despite aggressive colonic inflammation, DKO mice showed reduced hepatic fibrosis, increased hepatic reparative macrophages, and matrix metalloproteinase activity compared to Mdr2-/- mice. GF DKO had heightened liver inflammation and mortality with absent colitis and colonic dysplasia, reversed with microbial reconstitution from DKO mice. Changes in DKO primary/secondary bile acid profiles mirrored those in PSC-IBD. ConclusionThe Mdr2/Il10 DKO model mirrors key factors in PSC-IBD patients in terms of inflammation and carcinogenesis. We found an important role for the dysbiotic microbiota in DKO mice for disease onset and progression. Targeting microbiota and bile acid metabolism may provide promising strategies for developing effective PSC-IBD therapies.

Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is perturbed in esophageal pathologies both benign and malignant. Esophageal cancer is among the deadliest forms of human malignancy with 5-year survival rates of <20%. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are the two most common subtypes of esophageal cancer. Gastroesophageal reflux disease (GERD) is a primary risk factor for EAC. Although GERD and the food allergy-mediated condition eosinophilic esophagitis (EoE) are both associated with chronic esophageal inflammation and epithelial remodeling, including basal cell hyperplasia, epidemiological evidence suggests that EoE patients do not develop esophageal malignancy. Here, we perform single cell RNA-sequencing in murine models of EoE and ESCC to delineate the effects that these two conditions have specifically upon the cellular landscape of esophageal epithelium. In mice with EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect expansion of 4 suprabasal populations coupled with depletion of 4 basal cell populations. By contrast, mice with ESCC display expansion of 4 basal populations as well as depletion of 3 superficial populations. We further evaluated modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways were associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism were identified in ESCC-enriched clusters. Finally, by pairing murine models of EoE and ESCC, we demonstrate that exposure to EoE inflammation limits esophageal carcinogenesis. Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit act to esophageal carcinogenesis which may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

Background and AimsWNT2B mutations result in Diarrhea-9 (DIAR9), a congenital diarrhea syndrome with an extreme phenotype and unique histological defects. Attempts to model DIAR9 in rodents and study patient epithelial tissue have not been able to fully reproduce the human phenotype, making understanding this condition challenging. Here, we aimed to interrogate the mechanisms and the specific cellular compartment contributing to DIAR9 using a human intestinal organoid model. MethodsHuman intestinal organoids (HIOs) generated from both a patient-derived WNT2BR69*iPSC line and a control line were transplanted into immunocompromised mice for 10 weeks. Grafts were harvested and histologically compared. Bulk RNA sequencing was performed on both organoid groups and on patient-biopsy derived enteroids. In vitro recombination experiments were performed to describe the causative cellular compartment. ResultsLive and histological imaging revealed partial epithelial delamination in WNT2BR69*HIOs, which was absent in controls. A significant number of crypts in WNT2BR69* HIOs lacked OLFM4, a surrogate marker of stem cell activity. Key transcriptomic pathways altered between groups included trafficking of apical digestion proteins, which was confirmed via immunofluorescence. Patient derived enteroid proteomic analysis revealed similar results. Recombination experiments in HIOs revealed that while both epithelial and mesenchymal WNT2B are important for stem cell function, lack of mesenchymal WNT2B was sufficient to elicit the phenotype. ConclusionWe demonstrated that mesenchymal WNT2B is critical for supporting human intestinal epithelial development and function.

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

Background and AimsChronic pancreatitis (CP) is characterized by inflammation, fibrosis, and acinar-to-ductal metaplasia (ADM). PIN1, known to drive oncogenic signaling and cellular plasticity in cancer, has an unexplored role in CP. This study investigates PIN1s expression and function in CP pathogenesis using human tissues and mouse models. MethodsPIN1 expression was assessed in human CP tissue microarrays (TMAs) via immunohistochemistry (IHC) and cyclic immunofluorescence (CyCIF). Acute and chronic pancreatitis were induced in wild-type (WT) and PIN1 knockout (PIN1KO) mice using caerulein. Disease progression was monitored histologically, and immune profiling was conducted using flow cytometry. Pharmacological inhibition was performed using a small molecule PIN1 inhibitor-Sulfopin, and effects were evaluated by histology, qPCR, and cytokine analysis. Single-cell RNA sequencing (scRNA-seq) was performed on pancreatic tissues to perform pathway analysis and intercellular communication. ResultsPIN1 expression was elevated in human CP tissues, correlating with disease severity and ADM. In mice, both acute and chronic pancreatitis increased PIN1 expression, but only in our chronic PIN1KO mice displayed reduced pancreatic injury, fibrosis, ADM, and modulated immune infiltration. Pharmacological PIN1 inhibition mimicked the protective effects of genetic knockout, dampening inflammatory pathways. scRNA-seq revealed that PIN1 inhibition altered the intercellular communication networks between epithelial, immune, and stromal cells. ConclusionPIN1 drives cellular plasticity, immune modulation, and disease progression in CP. Targeting PIN1 may offer a therapeutic strategy to mitigate CP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/653850v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@67f9fcorg.highwire.dtl.DTLVardef@4d4c1forg.highwire.dtl.DTLVardef@c099d7org.highwire.dtl.DTLVardef@b41aa5_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Shah, V. (2025) https://BioRender.com/undefined

Vertical sleeve gastrectomy (VSG) is one of several bariatric procedures that substantially improves glycemia and energy homeostasis. Increased secretion of multiple gut peptides has been hypothesized to be a critical contributor to VSGs potent effects to reduce body weight and improve glucose regulation. VSG results in an increase in the number of hormone-secreting enteroendocrine cells (EECs) in the intestinal epithelium, but whether this increase is via proliferation or differentiation of EECs and their subtypes remains unclear. Notably, the beneficial effects of VSG are lost in a mouse model lacking the bile acid nuclear receptor, farnesoid X receptor (FXR). FXR is a nuclear transcription factor that has been shown to regulate intestinal stem cell (ISC) function in cancer models, but whether it plays a role specifically in normal intestinal differentiation remains unknown. Therefore, we hypothesized that the VSG-induced increase in EECs is due to changes in intestinal differentiation driven by an increase in bile acid signaling through FXR. To test this, we performed VSG in mice that express eGFP in ISC/progenitor cells and performed RNAseq on GFP-positive cells sorted from the intestinal epithelia. We also assessed changes in EEC number (marked by GLP-1) in mouse intestinal organoids following treatment with bile acids and/or an FXR antagonist. RNA-seq revealed that FXR is expressed in ISCs and that VSG explicitly alters ISC expression of several genes that regulate intestinal secretory cell development, including EEC differentiation. Mouse intestinal organoids treated with bile acids increased GLP-1-positive cell numbers, whereas a potent FXR antagonist blocked this effect. Taken together, these data indicate that VSG drives ISC fate towards EEC differentiation through FXR signaling.

BackgroundHepatocellular carcinoma (HCC) incidence is increasing worldwide due to the obesity epidemic, which drives metabolic dysfunction-associated steatohepatitis (MASH) that can lead to HCC. However, the molecular pathways that lead to MASH-HCC are poorly understood. We have previously reported that male mice with global haploinsufficiency of hypoxia-associated factor, HAF (SART1+/-) spontaneously develop MASH/HCC. However, the cell type(s) responsible for HCC associated with HAF loss are unclear. ResultsSART1-floxed mice were crossed with mice expressing Cre-recombinase within hepatocytes (Alb-Cre; hepS-/-) or macrophages (LysM-Cre, macS-/-). Only hepS-/- mice (both male and female) developed HCC suggesting that HAF protects against HCC primarily within hepatocytes. HAF-deficient macrophages showed decreased P-p65 and P-p50 and in many major components of the NF-{kappa}B pathway, which was recapitulated using HAF siRNA in vitro. HAF depletion increased apoptosis both in vitro and in vivo, suggesting that HAF mediates a tumor suppressor role by suppressing hepatocyte apoptosis. We show that HAF regulates NF-{kappa}B activity by controlling transcription of TRADD and RIPK1. Mice fed a high-fat diet (HFD) showed marked suppression of HAF, P-p65 and TRADD within their livers after 26 weeks, but manifest profound upregulation of HAF, P-65 and TRADD within their livers after 40 weeks of HFD, implicating deregulation of the HAF-NF-{kappa}B axis in the progression to MASH. In humans, HAF was significantly decreased in livers with simple steatosis but significantly increased in HCC compared to normal liver. ConclusionsHAF is novel transcriptional regulator of the NF-{kappa}B pathway that protects against hepatocyte apoptosis and is a key determinant of cell fate during progression to MASH and MASH-HCC.

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, including all 10 Frizzled receptors (Fzds), has not been characterized in adult liver. To address this, we quantitatively mapped the spatial expression of Wnt/Fzd pathway components in adult mouse liver via multiplex fluorescent in situ hybridization. While all 10 Fzds are expressed within a metabolic unit, Fzds 1, 4, and 6 are the highest expressed. Though the majority of Wnt signaling occurs in zone 3, expression of most Fzds is not zonated. In contrast, Fzd6 is preferentially expressed in zone 1. We also discovered that Wnt2 and Wnt9b expression is highly zonated and primarily found in zone 3. Therefore, our results suggest that zonated Wnt expression is critical for zonation maintenance in healthy adult liver. Finally, we showed that Fzds and Wnts are not uniformly expressed by all hepatic cell types. Rather, there is broad distribution among both hepatocytes and non-parenchymal cells, including endothelial cells. Overall, our establishment of a definitive mRNA expression atlas of Wnt/Fzd pathway components opens the door to future functional characterization in healthy and disease states.

Metaplasia often involves a change from one cell type to another that was present during organogenesis. The embryonic esophagus is initially lined by columnar cells that are replaced by squamous cells, and metaplasia in Barretts esophagus (BE) involves a change from squamous to columnar cells in the setting of gastroesophageal reflux. Here, we explored the effect of ectopic expression of the essential developmental transcription factor GATA4 on squamous epithelial cell gene expression using human esophageal squamous epithelial cells. We found that GATA4 protein, although absent in mature human esophageal squamous epithelium, was present in BE and esophageal adenocarcinoma (EAC). Moreover, acid and bile induced GATA4 mRNA in esophageal squamous epithelial cells. Ectopic GATA4 expression in esophageal squamous epithelial cells generally compromised squamous cell marker gene expression, although the extent varied between cell lines studied. We observed GATA4 occupancy in the p63, KRT5, and KRT15 gene promoters, suggesting that GATA4 can directly repress expression of typical squamous epithelial cell marker genes. Overall, our data suggest a mechanism whereby GATA4 expression in abnormal esophageal cells, possibly induced by reflux, supports a columnar metaplastic cell identity by repressing expression of key genes required to program stratified squamous epithelial cell identity.

Epithelial cancers disrupt tissue architecture and are often driven by mutations in genes that play important roles in normal epithelial morphogenesis. The intrahepatic biliary system is an epithelial tubular network that forms within the developing liver via the de novo initiation and expansion of apical lumens. Intrahepatic biliary tumors (intrahepatic cholangiocarcinoma) commonly harbor activating genomic alterations in the FGFR2 receptor tyrosine kinase, which plays important roles in epithelial morphogenesis in other developmental settings. Using a physiologic and quantitative 3D model we demonstrate that FGFR signaling is important for biliary morphogenesis and that oncogenic FGFR2 fusions and in-frame deletions disrupt biliary architecture. Importantly, we show that the trafficking of and signaling from the FGFR2 mutants, as well as their phenotypic impacts, are governed by the epithelial state of the cell. Unexpectedly, we also found that distinct tumor-driving FGFR2 mutants disrupt biliary morphogenesis in completely different and clinically relevant ways, informing our understanding of morphogenesis and tumorigenesis and highlighting the importance of convergent studies of both. Summary statementUsing a physiologic 3D model of biliary tubulogenesis, Chiasson-MacKenzie et al. show that FGFR2 signaling is important for biliary morphogenesis and that different intrahepatic cholangiocarcinoma-causing FGFR2 alterations have unexpectedly distinct and epithelial state-specific impacts on biliary architecture.

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.

BackgroundUbiquitin-dependent signaling is essential for maintaining intestinal homeostasis and its dysregulation contributes to chronic intestinal disorders, such as Inflammatory Bowel Disease (IBD). Ube4A is a U-box E3/E4 ubiquitin ligase involved in lipid metabolism and insulin signaling in metabolic tissues. Autoantibodies against Ube4A have been identified in patients with IBD and are associated with disease long-term complications. Despite these clinical associations, the physiological role of Ube4A in the gastrointestinal tract remains unknown. This study aimed to define the function of Ube4A in the colon and determine how its loss influences susceptibility to experimental colitis. MethodsUBE4A expression in human colonic tissue from healthy individuals and patients with IBD was analyzed using publicly available single-cell RNA sequencing datasets. The role of Ube4A in colonic homeostasis and colitis pathogenesis was examined using global Ube4A knockout (UKO) mice subjected to dextran sulfate sodium (DSS)-induced colitis. UKO colon phenotypes were characterized using transcriptomic analyses, immunofluorescence, and flow cytometry. ResultsUBE4A is highly expressed in human colonic epithelial cells, and its expression is reduced from healthy to IBD inflamed tissues. In mice, Ube4A deficiency significantly exacerbated DSS-induced colitis, as evidenced by increased weight loss, disease activity scores, shortened colon length, and more severe histological injury. Transcriptomic profiling revealed enhanced inflammatory signaling, alongside dysregulation of lipid transport and storage, as well as antimicrobial defense pathways. DSS-treated UKO mice also exhibited increased mast cell activation and elevated expression of matrix metalloproteinases. Importantly, colons from UKO mice displayed baseline transcriptional alterations indicative of epithelial stress and disrupted lipid metabolic programs, even in the absence of injury. ConclusionsUbe4A is a previously unrecognized regulator of colon homeostasis. Its loss induces existing epithelial stress and metabolic reprogramming that sensitize the colon to exaggerated inflammatory responses during injury such as experimental colitis.

The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically modified mouse models that allow temporal and spatial activation of oncogenic KRAS (KRASG12D). The most commonly used model involves targeted insertion of a cre recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRASG12D). The goal of this study was to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRASG12D before and after induction of pancreatic injury. We performed morphological and molecular analysis of three mouse lines that express a tamoxifen-inducible cre recombinase to activate KRASG12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a and Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Up to two months after tamoxifen induction of KRASG12D, morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in stark differences in tissue morphology between lines within seven days, which were maintained for at least five weeks after injury. Ptf1acreERT pancreata showed widespread PanIN lesions and fibrosis, while the Mist1creERT and Ela-creERT models showed reduced amounts of pre-neoplastic lesions. RNA-seq analysis prior to inducing injury suggested Ptf1acreERT and Mist1creERT lines have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the lines. These findings suggest understanding the mechanisms underlying the differential response to KRASG12D will help in further defining the intrinsic KRAS-driven mechanisms of neoplasia initiation.

Hepatic stellate cells (HSC) are non-parenchymal liver cells that produce extracellular matrix comprising fibrotic lesions in chronic liver diseases. Prior work demonstrated that mitochondrial pyruvate carrier (MPC) inhibitors suppress HSC activation and fibrosis in a mouse model of metabolic dysfunction-associated steatohepatitis (MASH). In the present study, pharmacologic or genetic inhibition of the MPC in HSC decreased expression of markers of activation in vitro. MPC knockdown also reduced the abundance of several intermediates of the TCA cycle, and diminished -ketoglutarate played a key role in attenuating HSC activation by suppressing hypoxia inducible factor-1 signaling. On high fat diets, mice with HSC-specific MPC deletion exhibited reduced circulating transaminases, numbers of HSC, and hepatic expression of markers of HSC activation and inflammation compared to wild-type mice. These data suggest that MPC inhibition modulates HSC metabolism to attenuate activation and illuminate mechanisms by which MPC inhibitors could prove therapeutically beneficial for treating MASH.