Cellular and Molecular Gastroenterology and Hepatology

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.

Enteroendocrine cells (EECs) are specialized sensors of the gastrointestinal (GI) epithelium that regulate gut function and systemic metabolism through hormone secretion. The molecular pathways directing intestinal stem cell (ISC) differentiation into EECs are incompletely understood due, in part, to their rarity. We sought to identify novel regulators of human EEC differentiation using a high-throughput screen of FDA-approved drugs and human duodenal organoids. Two epidermal growth factor receptor inhibitors (EGFRi) commonly used in cancer therapy and known to cause GI side effects, erlotinib and lapatinib, emerged as strong inducers of EEC differentiation, dramatically increasing chromogranin A (CHGA) expression compared to controls, while maintaining ISC function and organoid growth. EGFRi-treated organoids revealed robust and broad upregulation of EEC hormones, including serotonin (5HT), motilin (MLN), and somatostatin (SST), among others. In agreement with these findings, analysis of a patient cohort with lung cancer revealed an association with erlotinib use and increased circulating levels of the above EEC hormones compared to matched controls. Supporting a direct effect of EGFRi on EEC differentiation, mice treated with erlotinib demonstrated increased EEC numbers and hormones and showed EGFRi-associated diarrhea (EAD), a limiting side effect of these medications. Mechanistically, EGFRi induced upregulation of interferon (IFN) signaling targets during early ISC-to-EEC differentiation. Consistent with this, inhibition of Signal Transducer and Activator of Transcription 1 (STAT1) attenuated EGFRi-induced EEC differentiation. These findings provide important insight into EEC differentiation that could inform treatment strategies for EAD, metabolic diseases, and GI diseases. Brief SummaryInhibition of EGFR signaling promotes human ISC-to-EEC differentiation through activation of STAT1 signaling.

Lethal sterile inflammatory diseases are linked to amino acid metabolism, but the role of serine remains unclear. Here, we show that dysregulated serine metabolism and reduced plasma serine levels correlate with disease severity of acute pancreatitis (AP) in patients and mouse models. Elevating serine levels via exogenous serine supplementation or pancreatic phosphoglycerate dehydrogenase (PHGDH) overexpression mitigates pancreatic injury, whereas a serine deprivation diet or pancreatic PHGDH knockdown exacerbates AP. Serine prevents cell death and oxidative stress in pancreatic acinar cells, human induced pluripotent stem cells-derived pancreatic organoids and mouse pancreatic tissue. Serine enhances cysteine and glutathione biosynthesis primarily by promoting solute carrier family 7 member 11 (SLC7A11)-dependent cystine uptake rather than by serving as a direct substrate. Mechanistically, the E3 ubiquitin ligase NEDD4 mediates ubiquitination and degradation of SLC7A11, whereas serine binds to NEDD4 and thereby inhibits SLC7A11 degradation. Similarly to serine, pharmacological inhibition of NEDD4 alleviates lipid peroxidation and pancreatic injury. These findings identify serine as a critical signaling regulator of SLC7A11 stability and oxidative stress, and provides a new therapeutic strategy for AP and associated sterile inflammatory disorders. HighlightsAcute pancreatitis (AP) is linked to abnormal serine metabolism and serine depletion. Serine prevents cell death in AP acinar cells, human pancreatic organoids and mice. Serine promotes SLC7A11-dependent cystine uptake and glutathione levels in acinar cells. Serine reduces NEDD4-mediated ubiquitination of SLC7A11. In briefSerine protects against cell death and pancreatic injury in acute pancreatitis by stabilizing SLC7A11 through disruption of NEDD4-mediated ubiquitination in acinar cells.

Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury, hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics (ST), immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early APAP-induced liver injury. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.

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.

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

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

Background and AimsThe tumor microenvironment in colorectal cancer (CRC) is richly innervated, yet the contribution of the enteric nervous system (ENS) to CRC biology remains poorly defined. ENS neurons express proenkephalin (PENK), which can be processed by proprotein convertase 1/3 (PCSK1) to generate Methionine-enkephalin (M-ENK), a bioactive peptide with growth-regulatory potential. We hypothesized that an ENS-derived PCSK1-M-ENK axis restrains CRC proliferation through opioid growth factor receptor (OGFr) signaling and is modulated by stress-associated glucocorticoid receptor (GR) signaling and GLP1 receptor (GLP1R) activity. MethodsPublicly available human CRC single-cell RNA-sequencing datasets were analyzed for OGFr expression. PCSK1 and M-ENK expression in murine ENS and tumor-associated tissue was assessed by immunofluorescence. Functional studies were performed using murine CRC organoids, and primary murine ENS neurons in mono- and co-culture. CRC proliferation was quantified by EdU incorporation following treatment with recombinant M-ENK, recombinant PCSK1, OGFr synthetic ligand naloxone, or PCSK1 inhibitors. Effects of dexamethasone and liraglutide on PCSK1 expression in ENS-containing murine tissue were evaluated. ResultsOGFr was enriched in CRC cells and positively associated with KRAS gene expression. A subset of adult murine colonic myenteric neurons expressed PCSK1 and M-ENK. M-ENK dose-dependently suppressed proliferation of CRC organoid cells. ENS neurons also suppressed CRC proliferation in a PCSK1-dependent manner. Dexamethasone reduced, whereas liraglutide increased, PCSK1 expression. ConclusionsThese findings define a previously unrecognized ENS-derived neuro-oncologic pathway that is associated with reduced CRC cell proliferation and identify the GR/GLP1R-PCSK1-M-ENK axis as a potentially actionable therapeutic node. SummaryThis study identifies a neuronal PCSK1 - M-ENK pathway in the ENS that directly suppresses colorectal cancer growth through local OGFr activation, revealing a previously unrecognized neuropeptidergic mechanism of tumor control within the intestinal microenvironment.

Fibrostenotic complications represent a major cause of morbidity in Crohns disease (CD), yet the cellular mechanisms that drive intestinal fibrosis independent of active inflammation remain poorly understood. Here, we identify impaired fatty acid oxidation (FAO) as a defining metabolic feature of fibroblasts in fibrostenotic CD. Untargeted lipidomics of non-inflamed colonic tissue from CD patients demonstrated enrichment of triacylglycerols and long-chain acylcarnitines, suggesting altered lipid utilization. Across three independent RNA-sequencing cohorts, including treatment-naive pediatric ileal biopsies, FAO genes (CPT1A, CPT2, SLC25A20) were selectively downregulated in patients with or destined to develop fibrostenotic disease. Single-cell RNA-sequencing localized these transcriptional alterations specifically to fibroblasts within strictured ileum. Primary fibroblasts derived from fibrostenotic CD exhibited increased neutral lipid accumulation, impaired mitochondrial fatty acid trafficking, and diminished responsiveness to PPAR{gamma}-mediated suppression of TGF{beta}-induced myofibroblast activation. Together, these findings demonstrate that FAO impairment is a conserved, fibroblast-specific metabolic program associated with intestinal fibrosis in CD and suggest that metabolic modulation of stromal cells represents a potential therapeutic strategy for fibrostenotic disease.

Patients with gastric intestinal metaplasia (GIM), a precancerous lesion, are at high risk for progressing to gastric cancer. Identifying these patients is critical to enable gastric cancer interception. Current approaches rely primarily on histologic evaluation of GIM severity and extent, which may be improved by incorporating molecular features that distinguish high-risk lesions. Our prior single-cell and spatial transcriptomics study identified differentially expressed genes associated with the highest-risk category of GIM. They included ANPEP expressed in enterocytes and CPS1 and OLFM4 expressed in intestinal stem-like or progenitor cells. We evaluated the protein expression and localization of these three markers to understand the cellular features associated with GIM risk and their spatial distribution within metaplastic tissues. Using multiplex immunofluorescence, whole slide image analysis and confocal microscopy, we examined protein expression from 100 tissue biopsies annotated for metaplasia severity using the Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM) system. Tissue samples included control gastric tissue, GIM, dysplasia and adenocarcinoma. Quantitative whole slide image analysis demonstrated that CPS1 expression had a modest association with disease severity. Although ANPEP was strongly associated with GIM severity, it was also frequently expressed in stromal regions outside epithelial glands. In contrast, OLFM4 expression was largely restricted to epithelial glands and showed a strong association with increased OLGIM severity. These OLFM4-positive epithelial cells were present in discrete glandular foci that expanded with increasing severity of metaplasia. Within individual metaplastic glands, OLFM4 expression was highest at the gland base with decreased expression toward the gland surface. Overall, these findings identified OLFM4 as a protein marker associated with high-risk GIM. The spatial organization of OLFM4-expressing cells at the base of metaplastic glands and their focal expansion within tissues suggest the presence of a stem cell-like epithelial compartment that may contribute to the progression of GIM towards gastric cancer.

The expression and secretion of sulfated colonic-type mucins is a feature of high-risk metaplasias of the gastrointestinal foregut (Barretts esophagus, type III intestinal metaplasia of the stomach, and pancreatic intraepithelial neoplasia). Galectin-3 is a lectin that preferentially associates with galactose modified by a 3-O-sulfate relative to its unmodified counterparts and is upregulated as the tissue transitions to high-risk metaplasia, dysplasia, and cancer. Since both galectin-3 and sulfated glycotopes are aberrantly and concurrently overexpressed in high-risk premalignant and malignant tissue transformations, we sought to investigate the role of galectin-3 in the metaplastic reaction. We found that injury induces the expression of Lgals3 at the RNA and protein levels. Unlike cancer cell lines, we show that in vivo galectin-3 colocalized with sulfomucins in zymogenic granules of the gastric chief cell. Utilizing a synchronous, chemically-induced murine model that produces spasmolytic polypeptide expressing metaplasia, we found that galectin-3 facilitates cathartocytosis of the vesicles it resides in, but not organelles lacking LGALS3. Inhibition of cellular downscaling resulted in delayed expression of the metaplastic transcription factor Sox9 as well as proliferation. Here, we present a new role for galectin-3 in promoting the transition from normal, homeostatic tissue to metaplasia and our data suggest that cathartocytosis represents an unconventional secretory pathway for galectin-3, which has been a matter of controversy as galectins are not secreted via canonical pathways.

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.

Liver injury induces rapid transcriptional responses in hepatocytes, yet the chromatin features that distinguish injured hepatocytes from healthy hepatocytes remain poorly understood. Using an integrated functional genomics approach combining bulk RNA-seq, ATAC-seq, and CUT&Tag profiling of H3K27ac and H3K27me3, we define the transcriptional and chromatin landscape of Sox9-expressing hepatocytes, which exhibit gene expression consistent with both hepatocyte and biliary identity. Under homeostatic conditions, Sox9+ hybrid hepatocytes (HybHeps) are rare and confined to the periportal space, while chronic injury induces an expansion of Sox9+ metaplastic hepatocytes (MetHeps). We identify three classes of differentially expressed genes associated with injury-responsive, state-associated, or shared regulatory programs and demonstrate that these classes are governed by distinct chromatin mechanisms. Injury-responsive transcription is driven primarily by dynamic chromatin accessibility remodeling at NF-{kappa}B- and AP-1-enriched regulatory elements, while state-associated and shared programs are reinforced through selective H3K27ac and H3K27me3 modification with comparatively stable accessibility. Relative to conventional hepatocytes, HybHeps encode a permissive chromatin landscape at injury-responsive loci under homeostatic conditions, consistent with epigenetic priming that facilitates rapid inflammatory activation. Projection of mouse-derived gene programs onto a human liver single-cell atlas encompassing both healthy and diseased hepatocytes confirms that SOX9-expressing hepatocytes preferentially engage injury-associated inflammatory modules while attenuating hepatocyte metabolic identity programs. Together, these findings define a chromatin-based regulatory dichotomy between inflammatory responsiveness and hybrid hepatocyte cell state stability, providing mechanistic insight into how differentiated epithelial cells integrate inflammatory signals while preserving cell state.

Patients with long-standing ulcerative colitis (UC) remain at risk for colorectal neoplasia even after overt inflammatory activity improves, suggesting that repaired mucosa may retain residual tissue-level abnormalities. Whether extracellular matrix remodeling leaves a persistent mechanical cue that contributes to early neoplastic remodeling is unknown. Here we identify post-inflammatory matrix stiffening as a premalignant mechanical niche in UC-associated neoplasia. In human colonic biopsies, collagen remodeling and atomic-force-microscopy-based mucosal stiffness increased from non-UC controls to UC and UC-associated dysplasia. A stiffness-associated transcriptional program was also enriched in histologically non-dysplastic mucosa from patients with UC-associated neoplasia. In mouse models, colonic stiffness increased along the colitis-to-tumorigenesis axis and remained elevated during apparent recovery, when epithelial permeability, junctional protein loss, crypt proliferation and nuclear {beta}-catenin accumulation persisted despite reduced disease activity. Stiffness-controlled intestinal epithelial cultures showed that stiff substrates were sufficient to disrupt ZO-1 organization and enhance {beta}-catenin redistribution, particularly under TNF- stimulation. Pharmacological matrix normalization with {beta}-aminopropionitrile partially restored barrier organization and attenuated early epithelial remodeling in vivo. Single-cell profiling identified a stiffness-associated MMP7-positive/YAP-active epithelial state that was enriched in collagen-rich regions and reduced after matrix softening. Inhibition of YAP or MMP7 attenuated stiffness-associated junctional disruption and {beta}-catenin redistribution. These findings suggest that inflammatory recovery and mechanical recovery can be uncoupled, and that persistent mucosal stiffening provides a tissue-level mechanism by which chronically injured UC mucosa may remain biologically vulnerable to premalignant epithelial remodeling.

Stem cell renewal and crypt survival are tightly controlled processes critical for gut repair. Defining key regulators of intestinal healing is critical for the development of new epithelial-targeted therapies. We previously showed that the nuclear receptor LRH-1 (NR5A2) maintains intestinal epithelial health and protects against inflammatory damage. Here, using lineage tracing and selective LRH-1 knockout in the Atoh1+ secretory lineage we show LRH-1 is vital for intestinal stem cell (ISC) regeneration in complementary in vivo and ex vivo injury-recovery models. Transcriptomic profiling and pathway analysis reveal downregulation of ER stress and unfolded protein response (UPR) programs. Using a new in vivo model to ascertain how LRH-1 directly impacts intestinal cell responses, we identify key ER stress response genes Ire1 and Xbp1 as potential LRH-1 targets. Together our results uncover a novel mechanism whereby LRH-1 sustains the IRE1-XBP1 arm of the UPR to support injury-induced dedifferentiation and ISC regeneration. Our findings highlight LRH-1 as a promising therapeutic target for restoring epithelial integrity in inflammatory intestinal disorders.

ABSTACTGalectin-8 is a lectin that binds N-acetyllactosamine moieties with preference towards those with acidic, terminal modifications (-O-sialyated, 3-O-sulfated). As CD44-variants (CD44v) are biomarkers of metaplasia and cancer, a hyaluronic acid receptor that modulates STAT3 signaling, and specifically expresses 3-Sialyl-LeA/X glycotopes, we asked whether galectin-8 might play a role in gastric metaplasia. Using a synchronous, chemically induced murine model that produces gastric spasmolytic polypeptide expressing metaplasia (SPEM), we compared Lgals8-/- mice to congenic wild-type C57BL/6J mice. We found that galectin-8 was necessary for membrane localization of CD44v on SPEM cells at the base of the glands, suggesting a physical interaction between galectin-8 and CD44v. Metaplastic glands from Lgals8-/- mice had an increase in nuclear pSTAT3 compared to C57BL/6J mice, suggesting that galectin-8 restrains CD44 -> STAT3 signaling. This effect was more prominent in the neck compared to the base, which has greater abundance of CD44v after injury in Lgals8-/- mice. Derepression of STAT3 signaling may explain why low galectin-8 levels is associated with a worse prognosis in gastric cancer.

ABSTRACTGATA6 and GATA4 play key roles in pancreatic development and are essential to maintain the classical transcriptional program in pancreatic ductal adenocarcinoma (PDAC). Using genetic mouse models we show that, in contrast to GATA6, GATA4 is dispensable for the maintenance of acinar homeostasis in the adult pancreas. Deletion of Gata4 in mice expressing mutant Kras in the embryonic pancreas (KG4C) leads to PDAC development in the absence of tissue remodeling, pancreatic intraepithelial neoplasia (PanIN), or other canonical precursor lesions present in Gata4-proficient (KC) mice. Similar observations were made when Gata4 was selectively inactivated in adult, Kras-mutant, acinar cells. We identify Pale Acinar Lesions (PALes) as a previously unrecognized pancreatic lesion, distinct from acino-ductal metaplasia (ADM) and PanINs, present in KC and KG4C mice but not in wild type mice. PALes display weak expression of acinar and ductal markers and lack mucins; they have lower proliferation rates than PanINs. RNA-seq and ChIP-seq reveal that GATA4 and GATA6 partially share genomic binding sites and transcriptomic effects, but they exert opposing influences on mutant Kras-induced, haematopoietic cell-dependent, transcriptional inflammatory programs. Adenoviral-mediated pancreatic expression of IL17 restored the formation of ductal lesions in KG4C mice but failed to rescue PanIN development. Our data indicate that GATA4 functions through the coordinated action of multiple inflammatory factors that are required for ADM/PanIN formation but are dispensable for PDAC development. Collectively, these findings challenge current paradigms of PDAC initiation and progression.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related deaths in the United States. Intraductal papillary mucinous neoplasms (IPMNs) are neoplastic lesions of ductal origin that seed 10-25% of PDAC. There are currently no markers that distinguish between IPMN that will remain benign and those that will progress to cancer. A heterogenous population of secretory cells, including chemosensory tuft cells and hormone-expressing enteroendocrine cells (EECs), form during metaplasia and neoplastic progression in the pancreas, but the relevance of these populations as it relates to IPMN progression is not well characterized. Here, we performed spatial transcriptomics as well as multiplex immunostaining and spatial statistics on surgically resected IPMN from 60 patients to characterize these populations in all subtypes (gastric foveolar, intestinal, pancreatobiliary) and grades (low-grade, high-grade, invasive). We found that POU2F3+ tuft-like cells, CHGA+ EECs, and a subset of pancreatic endocrine cells ([a] and {gamma} cells) were present in all types of IPMN. Further, serotonin-expressing enterochromaffin cells made up the bulk of EECs in low-grade disease. Enterochromaffin, tuft-like, and glucagon-expressing alpha cells were not evenly distributed and instead were significantly enriched in a spatial manner, which is overlooked using conventional whole tissue quantification approaches. Tuft-like cell clusters were enriched with monocytes and resident memory T cells and anti-correlated to activated fibroblasts (myCAFs, iCAFs). Overall, these secretory cell clusters may reflect clonal expansion resulting in formation of distinct stromal niches with unknown consequences for disease progression.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease for which the vast majority of the approved therapies target the immune system. We aimed to identify consistently dysregulated genes and pathways across independent UC transcriptomic cohorts, distinguishing constitutive from inflammation-dependent changes. We performed a random-effects meta-analysis of 14 microarray datasets from the Gene Expression Omnibus (972 mucosal biopsies, 9 platforms), comparing inflamed UC, uninflamed UC, and inflamed Crohns disease (CD) to controls, as well as UC to CD directly. The inflamed UC analysis revealed an upregulated inflammatory transcriptomic profile in UC, providing a rationale for the use of all approved anti-inflammatory therapies. In parallel, the predominant downregulated signal was metabolic, driven by PPARGC1A, PPARGC1B, and ES-RRA, indicating a coordinated, inflammation-dependent collapse. The uninflamed UC analysis revealed a separate set of potentially constitutive vulnerabilities -- fully suppressed glucuronidation, upregulated translation, complement priming, and altered iron export -- that are not downstream of the energy collapse. The metabolic deficit was more severe in UC than in CD, while immune pathways were shared. These findings suggest a two-layer model of UC pathology: a constitutive impairment of metabolic pathways that is further exacerbated by inflammation. The inflammation analysis reveals new targets for immune suppression while the constitutive analysis identifies targets for proactive intervention between flares directed at the metabolic deficiency.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease for which the mechanisms linking lipid dysregulation to fibrosis remain poorly defined. Hepatic phosphatidylcholine (PC) content is reduced in MASH, but how this alteration drives disease progression is unclear. Here, we identify a role for copper (Cu) homeostasis as a downstream effector of impaired PC biosynthesis. Using single-nucleus RNA sequencing in complementary genetic and dietary mouse models, we found that reduced hepatic PC is associated with marked depletion of hepatic Cu and a concomitant increase in circulating Cu, indicating disrupted Cu distribution. Mechanistically, PC depletion impaired plasma membrane localization of the high-affinity Cu transporter CTR1 (SLC31A1) in hepatocytes, limiting Cu uptake. In human hepatic stellate cells, Cu promoted fibrogenic activation, whereas suppression of Cu import or pharmacologic inhibition of MAPK signaling attenuated fibronectin deposition. In vivo, liver-directed Cu supplementation restored hepatic Cu levels and reduced steatosis but failed to improve fibrosis. In contrast, pharmacologic Cu chelation with bathocuproinedisulfonic acid (BCS) reduced fibrosis without affecting inflammation. Together, these findings identify Cu redistribution as a consequence of impaired PC biosynthesis and implicate Cu-dependent signaling in stellate cell activation, fibrogenesis and MASH pathogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/723926v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@144d748org.highwire.dtl.DTLVardef@91dd8corg.highwire.dtl.DTLVardef@683686org.highwire.dtl.DTLVardef@1d3a0da_HPS_FORMAT_FIGEXP M_FIG C_FIG