Cellular and Molecular Gastroenterology and Hepatology

Ulcerative colitis (UC) is characterized by chronic mucosal inflammation, recurrent epithelial injury, and impaired colonic mucosal wound healing. While WNT/{beta}-catenin dysregulation has been reported in UC, the mechanisms of such abnormalities remain unclear. To investigate epithelial intrinsic alterations associated with UC, we performed single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) multiomics on human primary colonic epithelial cells (colonoids) from healthy donors and patients with inactive or active UC. Colonoids were cultured in a 3D matrix recapitulating crypt base cells or grown as 2D monolayers in differentiation medium to recapitulate luminal epithelial cells. Colonoids from active UC had a unique cell population with elevated CTNNB1 and reduced APC expression. Chromatin profiling identified enrichment of RUNX2 motifs in this UC-associated cell population. Active UC colonoids exhibited reduced OLFM4 expression in 3D and the differentiation marker VIL1 in 2D, suggesting impaired self-renewal and maturation. RUNX2 inhibition using CADD522 reduced {beta}-catenin levels in 3D colonoids and restored VIL1 expression and junctional {beta}-catenin localization in 2D cultures. These findings reveal an intrinsic defect in epithelial renewal in UC, driven in part by RUNX2-dependent WNT dysregulation. Our study identifies RUNX2 as a transcriptional regulator of epithelial stem cell function and WNT signaling in the inflamed human colon. Graphical Abstract summarySingle-nucleus RNA and ATAC sequencing of UC patient-derived colonoids reveals a RUNX2-associated WNT signature in active inflammation. Elevated {beta}-catenin and reduced OLFM4 and VIL1 expression indicate impaired self-renewal and differentiation. Pharmacologic inhibition of RUNX2 restores epithelial maturation, identifying RUNX2 as a key regulator of epithelial dysfunction in UC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/688458v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@14c0093org.highwire.dtl.DTLVardef@b9b6a3org.highwire.dtl.DTLVardef@858ee2org.highwire.dtl.DTLVardef@67b98c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Circadian rhythms, 24-hour repeating oscillations in daily physiology, are implicated in maintaining intestinal homeostasis. These rhythms are driven by the circadian clock, a molecular timekeeper found throughout cells of the body, including those of the intestinal epithelium. Loss of clock function has been found to worsen colitis; however, it is not clear how the clock impacts regeneration which enables a tissue to return to its homeostatic set point following an injury. To investigate these questions, we used a conditional knockout of the core clock gene, Bmal1, in mouse colon epithelial cells. Our data show that prior to injury Bmal1 promotes colon mucus production, which increases in thickness and within goblet cells when mice are active and begin feeding. Bmal1 loss lowers mucus production but does not drive an apparent tissue phenotype until the system is injured and regenerates itself. In this context, Bmal1 epithelial loss drives a male-specific colitis phenotype and a delay in the ability of colon epithelial cells of both male and female mice to resolve injury to return to their homeostatic set point. Our data suggest that epithelial sex-specific clock rhythms are needed for optimal colon barrier homeostasis.

The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barretts esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 {micro}g/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-{kappa}B/P53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Clostridium perfringens, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1{beta}, Lbp, Lcn2, Myd88, Nfkb1, Tlr2 and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.

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.

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.

Microbial bile salt hydrolase (BSH) plays a central role in shaping bile acid composition and gut-liver metabolic signaling, yet its therapeutic potential in metabolic dysfunction-associated steatohepatitis (MASH) remains incompletely defined. Here, we evaluated the efficacy of the non-absorbable BSH inhibitor GR-7 in a diet induced mouse model of steatohepatitis using early and late intervention strategies with different dosing regimens. GR-7 reduced food intake and exerted stage- and dose-dependent therapeutic effects, with early intervention robustly suppressing hepatic fibrosis even at low dose, whereas late-stage administration of high-dose GR-7 markedly reduced hepatic steatosis and inflammation, as evidenced by decreased liver weight, hepatic triglyceride and cholesterol levels, and plasma ALT. Although late intervention did not result in statistically significant histological reversal of fibrosis, a trend toward improvement was observed, together with suppression of fibrogenic gene expression, suggesting that prolonged treatment may further enhance antifibrotic efficacy. Mechanistically, GR-7 effectively inhibited microbial BSH activity in vivo, leading to reduced cecal unconjugated primary and secondary bile acids--including deoxycholic acid and lithocholic acid, which was associated with improved gut barrier integrity and reduced hepatic inflammation. In parallel, BSH inhibition reprogrammed hepatic bile acid metabolism toward activation of the alternative CYP27A1-mediated synthesis pathway, accompanied by reduced food intake, thereby contributing to improved hepatic lipid accumulation. Furthermore, late-stage high-dose treatment selectively remodeled the hepatic immune landscape rather than fully restoring homeostasis, highlighting immune recalibration as a key component of therapeutic response. Together, these findings identify microbial BSH inhibition as a promising microbiome-targeted therapeutic strategy for MASH. HighlightsO_LIThe non-absorbable BSH inhibitor GR-7 improves steatosis, inflammation, and fibrosis in of Western diet-induced steatohepatitis model in mice in a dose-dependent manner. C_LIO_LIGR-7 reduces food intake and body weight gain. C_LIO_LIGR-7 reduces cytotoxic secondary bile acids, including DCA and LCA. C_LIO_LIGR-7 reprograms hepatic bile acid metabolism and immune responses. C_LI

Background and AimsEpithelial permeability barrier dysfunction is a central pathogenic driver of Crohns disease (CD), fueling microbial translocation, chronic inflammation, and progressive tissue injury. While current therapies suppress inflammation, none directly restore epithelial barrier function. Importantly, in CD patients, permeability barrier healing (BH) rather than mucosal healing is associated with long-term remission and a reduced risk of disease complications. Yet BH remains an unaddressed therapeutic target in CD. Here, we investigated whether pharmacologic inhibition of the integrated stress response (ISR) and RIPK3-mediated necroptosis, two convergent pathways of epithelial injury, can promote epithelial viability, regeneration, and barrier integrity in CD. MethodsWe employed villin-1/gelsolin double knockout (DKO) mice with epithelial-intrinsic ISR activation, Tnf{Delta}ARE/+ mice with chronic inflammation, and CD patient-derived enteroids (PDEs). Animals and PDE were treated with ISR inhibitor ISRIB, RIPK3 inhibitor Necrostatin-1 (Nec-1), or FDA-approved cancer drugs pazopanib and ponatinib, repurposed as potent RIPK3 inhibitors. Epithelial survival, regenerative growth (enteroid formation, budding), and barrier function (transepithelial electrical resistance, TEER) were assessed. ResultsChronic ISR activation and necroptosis were prominent in both murine models and CD PDEs, causing epithelial death, Paneth cell expansion, impaired enteroid survival, and regenerative failure. Pharmacologic inhibition with ISRIB, Nec-1, pazopanib, or ponatinib restored villus architecture, reduced inflammation, enhanced epithelial survival and regeneration, and significantly improved TEER. ConclusionsISR activation and RIPK3-mediated necroptosis converge to drive epithelial injury and barrier dysfunction in CD. Repurposing pazopanib and ponatinib offers a potentially translatable approach, to restore barrier integrity in CD. SynopsisISR activation and RIPK3-mediated necroptosis drive epithelial injury in Crohns disease. Repurposed RIPK3 inhibitors, pazopanib and ponatinib, restore epithelial homeostasis and permeability barrier function, providing a translational strategy to achieve sustained remission in CD.

BackgroundThe incidence of Barrett esophagus (BE) and Gastroesophageal Adenocarcinoma (GEAC) correlates with obesity and a diet rich in fat. Bile acids (BA) support fat digestion and undergo microbial metabolization in the gut. The farnesoid X receptor (FXR) is an important modulator of the BA homeostasis. The capacity of inhibiting cancer-related processes when activated, make FXR an appealing therapeutic target. In this work, we assess the role of diet on the microbiota-BA axis and evaluate the role of FXR in disease progression. ResultsHere we show that high fat diet (HFD) accelerated tumorigenesis in L2-IL1B mice (BE- and GEAC- mouse model) while increasing BA levels and enriching gut microbiota that convert primary to secondary BA. While upregulated in BE, expression of FXR was downregulated in GEAC in mice and humans. In L2-IL1B mice, FXR knockout enhanced the dysplastic phenotype and increased Lgr5 progenitor cell numbers. Treatment of murine organoids and L2-IL1B mice with the FXR agonist obeticholic acid (OCA) deacelerated GEAC progression. ConclusionWe provide a novel concept of GEAC carcinogenesis being accelerated via the diet-microbiome-metabolome axis and FXR inhibition on progenitor cells. Further, FXR activation protected with OCA ameliorated the phenotype in vitro and in vivo, suggesting that FXR agonists have potential as differentiation therapy in GEAC prevention. Statement of significanceIf its inhibition is linked to disease progression and its activation to cancer prevention, exploring the potential of FXR as a therapeutic target has great clinical relevance in GEAC context.

Mucosal healing following inflammatory injury is poorly understood and often neglected, despite being the best indicator of long-term outcomes in inflammatory bowel diseases. We report here that the enigmatic small molecular weight heat shock protein, Hsp25 (the human form is Hsp27), plays a vital role in converging microbial and host factors to promote pSTAT3-mediated mucosal healing. In wild type mice, the proximal-to-distal gradient of intestinal epithelial cell (IEC) Hsp25 expression is dependent on microbial cues. Patients with left-sided ulcerative colitis, however, show reduced levels of Hsp27 expression in both uninvolved and involved areas compared to normal colons of non-IBD patients. In mice with global or IEC-specific Hsp25 gene-targeted deletion, impaired mucosal healing with development of hallmarks of chronic disease are observed following DSS-induced or TNBS-induced colitis, whereas mucosal restitution is accelerated in IEC-specific overexpressing Hsp25 transgenic mice. In colonic IECs derived from these murine lines, Hsp25 binds and stabilizes a phospho-STAT3/YAP nuclear complex stimulated by IL-22 to sustain its wound healing gene programming. Thus, our findings provide insight into the mechanism of action of IEC Hsp25/27 in integrating host and microbial drivers of mucosal restitution, which can be leveraged to develop novel approaches for achieving and maintaining remission in complex immune disorders like IBD.

Enhancer of Zeste Homologue 2 (EZH2) is part of the Polycomb Repressor Complex 2, which induces trimethylation of lysine 27 on histone 3 (H3K27me3) and promotes genes repression. EZH2 is overexpressed in many cancers including pancreatic ductal adenocarcinoma (PDAC). Previous studies in mice attributed both pro-oncogenic and tumor suppressive functions to EZH2. Deletion of the EZH2 enhances de novo KRAS-driven neoplasia following pancreatic injury by preventing acinar cell regeneration, while increased EZH2 expression in PDAC is correlated to poor prognosis, suggesting a context-dependant effect for EZH2 in PDAC progression. In this study, we examined EZH2 function in pre-and early neoplastic stages of PDAC. Using an inducible model to generate deletion of EZH2 only in adult acinar cells (EZH2{Delta}SET), we showed loss of EZH2 activity did not prevent acinar cell regeneration in the absence of oncogenic KRAS (KRASG12D), nor lead to increased PanIN formation in the presence of KRASG12D in adult mice. However, loss of EZH2 did reduce recruitment of inflammatory cells and, when combined with a PDAC model, promoted widespread PDAC progression. Loss of EZH2 function also correlated to remodeling of the tumor microenvironment, which favors cancer cell progression. This study suggests expression of EZH2 in adult acinar cells restricts PDAC initiation and progression by affecting both the tumour microenvironment and acinar cell differentiation.

Doxorubicin (DXR) is a widely used chemotherapy drug that can induce severe intestinal mucositis. While the influence of gut bacteria on DXR-induced damage has been documented, the role of eukaryotic commensals remains unexplored. We discovered Tritrichomonas muris (Tmu) in one of our mouse colonies exhibiting abnormal tuft cell hyperplasia, prompting an investigation into its impact on DXR-induced intestinal injury. Mice from Tmu-colonized and Tmu-excluded facilities were injected with DXR, and tissue morphology and gene expression were evaluated at acute injury (6 h) and peak regeneration (120 h) phases. Contrary to previous reports, DXR did not significantly alter villus height, crypt depth, or crypt density in any mice. However, we did observe apoptosis, measured by cleaved caspase 3 (CC3) staining, in intestinal crypts at 6 h post-DXR that was significantly higher in mice colonized by Tmu. Interestingly, while DXR did not alter the expression of active and facultative intestinal stem cell (ISC) marker genes in control mice, it significantly reduced their expression in Tmu+ mice. Tmu, but not DXR, is also associated with increased inflammation and expression of the type 2 cytokines IL-5 and IL-13. However, pre-treatment of intestinal organoids with these cytokines is not sufficient to drive elevated DXR-induced apoptosis. These findings highlight the significant influence of commensal microbiota, particularly eukaryotic organisms like Tmu, on intestinal biology and response to chemotherapy, underscoring the complexity of gut microbiota interactions in drug-induced mucositis. NEW & NOTEWORTHYOur study found that the eukaryotic commensal Tritrichomonas muris (Tmu) significantly increases DXR-induced intestinal apoptosis in mice, despite no changes in tissue morphology. Tmu also reduces intestinal stem cell gene expression post-DXR injury, and elevates inflammation and type 2 cytokine expression in the absence of injury. In vitro organoid assays suggest that type 2 cytokines alone are insufficient to promote increased DXR-associated apoptosis. These findings emphasize the complex role of gut microbiota in drug-induced intestinal damage.

The delta-9 desaturase stearoyl-CoA desaturase-1 (SCD1) catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids (MUFA) and is highly expressed in liver and adipocytes. Previous studies have demonstrated that treating mice with dextran sulfate sodium (DSS), a chemical inducer of ulcerative colitis, results in severe downregulation of SCD1 in the liver. However, the specific role of hepatic SCD1 in modulating colitis severity, as well as the impact of DSS on SCD1 and other lipogenic factors in other tissues has not been investigated. Here we show that downregulation of hepatic SCD1 following DSS treatment is not accompanied by changes to other lipogenic genes in the liver. In contrast, adipose tissue demonstrates coordinated reductions in lipogenic genes, including SCD1 and SCD2, while the colon does not display any perturbation of these targets. Furthermore, we demonstrate that the downregulation of hepatic SCD1 occurs independently of sterol regulatory element binding protein-1c (SREBP-1c) and does not require an intact gut microbiome. Interestingly, a distinct model of colitis induced by IL-10 deficiency does not result in downregulation of hepatic SCD1. Concomitant with transcriptional changes, DSS treatment is associated with significant remodeling of the hepatic lipidome, including reductions in total phospholipids (PLs) and reduced MUFA-containing PLs and triacyglycerols (TAGs), consistent with the observed reduction in SCD1. Interestingly, hepatic cholesterol esters and plasma lipids including free cholesterol and glycerophospholipids were significantly elevated following DSS treatment. Given the significant reduction in hepatic SCD1 following DSS treatment, we tested a role for liver SCD1 in modulating colitis sensitivity. Mice with a targeted deletion of hepatic SCD1 were not more prone to colitis, indicating that the loss of hepatic SCD1, while a consequence of DSS-induced colitis, does not mediate colitis sensitivity in vivo. SynopsisHepatic SCD1 does not modulate colitis severity upon DSS exposure. However, DSS-induced colitis elicits significant lipid metabolism dysfunction, demonstrated by elevated plasma and liver lipids, particularly plasma cholesterol and hepatic cholesterol esters, highlighting a role for gutliver crosstalk following colonic inflammation.

Background & AimsPrimary sclerosing cholangitis (PSC) is a progressive cholangiopathy characterized by ductular remodeling, inflammation, and periportal fibrosis, for which effective medical therapies remain limited. The Hippo pathway effector TEAD1 has been implicated in liver regeneration and fibrogenesis; however, its role in cholestatic injury remains poorly defined. We investigated whether hepatocyte TEAD1 regulates injury-associated remodeling in a PSC-mimicking model and whether this mechanism is conserved in human PSC liver. MethodsHepatocyte-specific TEAD1 knockout mice (Alb-TEAD1-/-) and littermate controls were subjected to DDC-induced cholestatic injury. Ductular reaction, fibrosis, inflammation, and bile acid-related gene programs were assessed by histology, immunostaining, and gene expression analyses. Translational relevance was evaluated using bulk and single-cell transcriptomic datasets from human PSC liver. ResultsHepatocyte TEAD1 deletion attenuated DDC-induced fibrosis, ductular expansion, and inflammatory cell accumulation, while preserving hepatocyte proliferative responses. TEAD1-deficient livers exhibited reduced expression of profibrotic mediators, including Spp1, Ctgf, and Cyr61, with decreased extracellular matrix deposition. In contrast, canonical transcriptional adaptations to cholestatic stress, including suppression of bile acid uptake, induction of efflux pathways, and repression of bile acid synthesis genes, were preserved in the absence of TEAD1. Analysis of human PSC datasets demonstrated coordinated upregulation of TEAD1 and TEAD-associated target genes. Single-cell transcriptomic analysis further revealed hepatocyte-enriched TEAD1 expression and activation of a TEAD1 target gene program across all hepatic zones in PSC, with effect sizes exceeding those observed in non-parenchymal populations. TEAD1 activation was accompanied by co-expression of profibrotic mediators and downregulation of hepatocyte differentiation markers, consistent with a maladaptive hepatocyte state. ConclusionsHepatocyte TEAD1 drives ductular, inflammatory, and fibrogenic remodeling during cholestatic injury without disrupting bile acid metabolic adaptation. These findings identify TEAD1 as a hepatocyte-intrinsic regulator of epithelial-stromal crosstalk and establish conserved activation of this pathway in human PSC, supporting TEAD-directed signaling as a therapeutic target.

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.

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.

WNT2B is Wnt ligand which is able to support intestinal stem cells (ISC) in culture and support the intestinal epithelium in vivo. We have previously shown that WNT2B is critical for resistance to colitis, but not small intestinal injury, in the adult mouse. WNT2B is thought to coordinate with WNT3 in supporting ISC, and we have also shown that WNT3 expression is low in the early postnatal ileum in mice. Here, we hypothesized that WNT2B may be more critical in the small intestine during early development, and we challenged Wnt2b KO mice and controls with experimental necrotizing enterocolitis (NEC) on postnatal days 5-8. Wnt2b KO mice had similar ileum histology and injury scores to control mice. Molecular analyses showed that Wnt2b KO mice have differences in Lgr5 and Tlr4 expression compared to wild type controls in untreated conditions, but under experimental NEC expression of epithelial markers and inflammatory genes associated with NEC were similar to wild type. Periodic acid Schiff positive cells were lower in the villi of Wnt2b KO mice during NEC, however expression of goblet cell markers was not different compared to wild type mice. We also used an organoid-based NEC model to highlight the epithelium in isolation and also found no impact of WNT2B KO in the setting of NEC. These data further affirm that WNT2B is critical for inflammation responses in the mouse colon, but does not appear to play a major role in the small intestine, no matter the developmental period.

Microfold cells (M cells) are a specialized subset of epithelial intestinal cells responsible for immunosurveillance of the gastrointestinal tract. M cells are located in the Peyers patches and are crucial for monitoring and the transcytosis of antigens, microorganisms, and pathogens via their mature receptor GP2. A mature M cell with Gp2 receptor aids in the uptake of antigens, which are passed through the single layer of epithelium and presented to underlying antigen-presenting cells and processed further down-stream with B cells, T cells, and dendritic cells. Recent studies revealed several transcription factors and ligands responsible for the development and differentiation of mature M cells however, an exhaustive list of factors remains to be elucidated. Our recent work on the epigenetic regulation of M cell development found 12 critical transcription factors that were controlled by the polycomb recessive complex 2. Musculoaponeurotic fibrosarcoma transcription factor (Maf) was identified as a gene regulated by the polycomb repressive complex (PRC2) during the development of M cells. In this paper, we explore Mafs critical role in M cell differentiation and maturation. Maf falls under the purview of RANKL signaling, is localized in the Peyers patches of the intestine, and is expressed by M cells. Given that, complete knockout of the Maf gene leads to a lethal phenotype, organoids isolated from Maf knockout mice and treated with RANKL exhibited impaired M cell development and a significant decrease in Gp2 expression. These findings reveal that Maf is an important regulator for M cell development and differentiation.

The cellular census of the colonic crypt is tightly regulated, yet the molecular mechanisms that regulate this census are not fully understood. Lrig3, a transmembrane protein, is expressed in colonic crypt epithelial cells, including the stem, progenitor, and differentiated cell types. Mice missing Lrig3 have a disruption in their cellular census: using a novel Lrig3-/- mouse we demonstrate that Lrig3-/- mice have more cells per crypt, a greater mucosal area, and longer colons compared to wildtype mice, suggesting the expression of Lrig3 is required for both the total number of epithelial cells in the mouse colon, as well as colon length. In addition, we show Lrig3-/- mice have significantly more stem, progenitor, and deep crypt secretory cells, yet harbor a normal complement of enteroendocrine, Tuft, and absorptive cells. Lrig3-/- mice also have a concomitant decrease in phosphorylated Extracellular signal-related kinases, indicating the loss of Lrig3 leads to an expansion of the colonic stem cell compartment, in an Erk-dependent manner. Our study describes the expression of Lrig3 within the colon, defines perturbations in mice lacking Lrig3, and supports a role for Lrig3 in the establishment of both colonic crypt structure and cellular census, defined as the epithelial cell type and number in colon crypts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/483523v3_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@244c29org.highwire.dtl.DTLVardef@1045457org.highwire.dtl.DTLVardef@13d4134org.highwire.dtl.DTLVardef@83db4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.