Cellular and Molecular Gastroenterology and Hepatology

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.

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.

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent pediatric disorder with limited treatment options, primarily due to an incomplete understanding of its molecular drivers. Recent research underscores the role of microbial guilds in metabolic health, but the mechanisms by which dysbiosis driven by core species and co-abundant symbionts disrupt metabolic homeostasis in pediatric MASLD remain unclear. ResultsHere, we conducted integrated metagenomic and metabolomic analyses on 285 pediatric subjects including MASLD patients, obese and healthy controls. The gut dysbiosis in MASLD was characterized by a depletion of Phocaeicola vulgatus, Bacteroides uniformis, Parabacteroides distasonis, and Bacteroides thetaiotaomicron. Co-abundance network analysis, integrating our cohort with four public datasets, identified these species as core guild members associated with MASLD. Microbial enrichment analysis showed significant disruptions in carbohydrate metabolism, particularly the downregulation of the tricarboxylic acid (TCA) cycle, fructose and sucrose metabolism, and pentose and glucuronate interconversions. P. vulgatus and B. uniformis were identified as dominant species linked to the downregulation of KEGG orthologs (KOs) in these disrupted pathways that were inversely correlated with hepatic injury biomarkers. CAZyme database analysis further emphasized P. vulgatus as the primary contributor to glycoside hydrolases involved in monosaccharide utilization. Finally, both untargeted and targeted metabolomics analysis validated a disrupted metabolic network centered on the TCA cycle and monosaccharide metabolism in pediatric MASLD. ConclusionOur findings suggest the core guild species P. vulgatus and B. uniformis may serve as critical regulators of carbohydrate metabolism in pediatric MASLD, offering potential mechanistic targets for gut microbiome-based interventions.

Pain in pancreatic ductal adenocarcinoma (PDAC) is associated with poor survival, but whether altered pain processing carries prognostic significance is unknown. We analyzed a prospective cohort of 143 patients with PDAC who underwent pancreatic quantitative sensory testing (PQST) after diagnosis. Patients were classified as having normal pain processing (n=84), segmental hyperalgesia (n=30), or widespread hyperalgesia (n=29). Survival was measured from the date of P-QST assessment. During follow-up, 70 deaths occurred. Widespread hyperalgesia was associated with increased mortality in unadjusted Cox analysis (HR 1.96, 95% CI 1.14,3.35) and after adjustment for age, sex, tumor stage, comorbidity, opioid treatment, and body mass index (adjusted HR 2.33, 95% CI 1.30,4.15). Segmental hyperalgesia was not associated with mortality. Kaplan Meier analysis demonstrated lower survival probability in the widespread hyperalgesia group (log rank p=0.025). These findings suggest that widespread hyperalgesia, reflecting altered central pain processing, identifies a subgroup of PDAC patients at increased risk of mortality independent of conventional clinical factors.

Background: Heterogeneity in symptom presentation and treatment response in irritable bowel syndrome (IBS) remains poorly understood. The gut microbiota may contribute to this variability, but its role in shaping symptom trajectories and responses to self-management interventions is unclear. Objective: To identify symptom trajectory phenotypes and determine whether gut microbiota composition and function distinguish these phenotypes and predict multidimensional responses to pain self-management interventions in young adults with IBS. Design: Ancillary data analysis from a randomized control trial (NCT03332537). Methods: Participants with longitudinal data (n = 62) were analyzed using longitudinal k-means clustering (KML) based on trajectories of measures in IBS quality of life (QOL), Brief Pain Inventory (BPI), and psychoneurological outcomes (anxiety, applied cognition, depression, fatigue, global health, positive affect, and sleep disturbance) over 12 weeks. Baseline differences between clusters were assessed with Wilcoxon rank-sum tests, and longitudinal changes were evaluated with linear mixed models. Gut microbiota composition and predicted functional pathways were compared between phenotypes. Bayesian Additive Regression Trees (BART) models were used to identify baseline microbial taxa and pathways predictive of longitudinal changes in QOL, BPI pain interference, and severity. Results: Two distinct trajectory-defined response phenotypes were identified: a Constrained Response Phenotype (Phenotype A, n = 35) and an Adaptive Multidomain Response Phenotype (Phenotype B, n = 27). At baseline, Phenotype B showed lower pain severity and interference, but higher levels of anxiety, depression, and fatigue compared to Phenotype A. Over 12 weeks, both phenotypes showed improvements in pain outcomes (all p < 0.05), but only Phenotype B demonstrated broad improvements across psychoneurological domains and QOL (all p < 0.05). Phenotype A exhibited more limited improvements and worsening in several psychoneurological domains. Gut microbiota functional pathways differed between phenotypes, including pathways related to xenobiotic degradation, amino acid metabolism, bile secretion, and immune-related processes (all raw p < 0.05), although these did not remain significant after multiple testing correction. Machine learning models identified distinct, phenotype-specific microbial predictors of intervention response. In Phenotype A, genera such as Alistipes and Sutterella were consistently identified across models, whereas in Phenotype B, predictors included Phascolarctobacterium, Collinsella, and Parabacteroides. Functional pathways also differed between phenotypes, suggesting distinct microbiome-linked mechanisms underlying symptom trajectories and responses to pain interventions. Conclusions: Young adults with IBS exhibit distinct multidimensional response phenotypes that are associated with differential clinical and microbiome profiles. Baseline gut microbiota composition and functional capacity demonstrate phenotype-specific predictive signatures of treatment response, supporting a microbiome-informed framework for stratifying patients and advancing personalized self-management strategies in IBS.

Background Recent mouse model data demonstrate that chronic colonization with toxigenic Clostridioides difficile promotes colonic tumorigenesis via intraluminal toxin B (TcdB), its main virulence factor. In a prior multisite hospital cohort, we found that history of positive tcdB stool testing was associated with increased CRC risk in a dose-dependent manner, though limited by small sample size. We aimed to validate this association in a larger cohort with extended follow-up and greater geographic distribution using the Veterans Health Administration (VHA) Corporate Data Warehouse (CDW). Methods We conducted a retrospective cohort study among adults receiving care through the VA from 2000-2025 who underwent C. difficile testing. Data collected from the VHA CDW and National Death Index (NDI) included demographics, comorbidities, medications, CRC risk factors, and cancer incidence and death. The first C. difficile test date defined cohort entry; individuals with prior CRC were excluded. Ever C. difficile positivity was defined by a positive PCR or EIA results. The number of positive tests (episodes) was also determined to define recurrent positivity. Follow-up time ended at the first occurrence of CRC incidence or mortality, death from other causes, or censor date. Follow-up time was split for individuals who converted from negative to positive, with follow-up time updated accordingly. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HRs) for C. difficile exposure and CRC incidence and mortality after adjustment for confounders. Tests for linear trend and tests for interaction were conducted to assess effect modification by sex and IBD status, while time-lag intervals were evaluated for 1, 3, 5, and 10 years before the outcome. Results Among 806,844 veterans with C. difficile testing, those with positive tests were more likely to be older, male, to have diabetes, to use aspirin, and to have a lower BMI than those with negative tests. Race and IBD prevalence were similar between the groups. There was no overall association between ever C. difficile positivity and CRC incidence (HR = 0.99, 95% CI 0.93-1.05). However, recurrent C. difficile positivity was associated with increased risk in a dose-response manner [2-3 episodes HR = 1.30 (95% CI 1.16-1.47), and >3 episodes HR = 1.58 (95% CI 1.17-2.14) compared to negative tests; ptrend< 0.001]. Further, ever C. difficile positivity was associated with increased CRC mortality risk (HR = 1.21, 95% CI 1.13-1.30; p < 0.001). Recurrent C. difficile positivity was associated with increased mortality risk but was particularly strong for those with >3 episodes among individuals with IBD (HR=3.84, 95% CI 1.98-7.45). In sensitivity analyses, the increased risk of CRC incidence and mortality attenuated beyond 10 years. Conclusion Prior positive C. difficile testing was associated with increased CRC incidence and mortality in a dose-dependent manner, particularly among patients with IBD. These findings extend animal model evidence, epidemiologically establishing C. difficile presence as an independent risk factor for subsequent colorectal tumorigenesis and supporting investigation into recurrent CDI, especially among patients with IBD, as a potential modifiable CRC risk factor.

Background Liver fibrosis (LF) represents a pivotal pathological phase in the advancement of chronic liver disorders toward cirrhosis. Amino acid metabolism reprogramming plays a pivotal role in its pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Methods Integrating three public datasets (GSE14323, GSE84044, and GSE136103) with amino acid metabolism-related gene sets, we performed consensus clustering, machine learning algorithms, functional enrichment analysis, immune microenvironment composition, regulatory network construction, and drug prediction. Results Fibrotic samples were classified into two amino acid metabolism-related subtypes with distinct immune landscapes and functional phenotypes. Through integrated analysis of differentially expressed genes (DEGs) common to both subtypes, fibrotic versus control comparisons, and amino acid metabolism-related gene sets, four biomarkers, GSTP1, LDHB, OXCT1, and PTGDS, were identified. These biomarkers were enriched in pathways related to epithelial-mesenchymal transition, interferon responses, and TNF/NF-{kappa}B signaling. Notably, GSTP1 and LDHB positively correlated with M1 macrophage infiltration and negatively with regulatory T cell abundance. Single-cell transcriptomic analysis revealed that cholangiocytes expressed all four biomarkers with elevated levels in fibrosis and interacted with macrophages/mesenchymal cells via MIF-CD74/CXCR4. Regulatory network analysis highlighted key modulators, including MALAT1, hsa-miR-3163, OXCT1, SMAD4, and RELA. Furthermore, 5-fluorouracil was predicted as a multi-target compound, with the strongest predicted binding affinity for OXCT1. In vitro validation confirmed the upregulation of GSTP1 and LDHB, aligning with the bioinformatics findings. Conclusion This study identified four amino acid metabolism-related biomarkers, revealing immune heterogeneity and cholangiocyte-centered intercellular communication in LF. These findings establish a foundation for biomarker-based diagnosis, subtype-guided patient stratification, and the development of cell-type-specific therapeutic strategies in LF.

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.

BackgroundTryptophan (Trp) metabolism is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. While individual Trp metabolites have been associated with disease severity and treatment response, systems-level frameworks to define metabolic subtypes in IBD are lacking. ObjectiveTo identify reproducible Trp-related metabolic subtypes ("metabotypes") in IBD and assess their association with disease activity, clinical outcomes, and early disease development. DesignWe applied unsupervised clustering to serum concentrations of 16 Trp-related metabolites in a discovery cohort of patients with IBD undergoing biologic induction therapy (n=134). Metabotypes were validated in three independent IBD cohorts (total n>2,800), a healthy reference population, and a prospective cohort of first-degree relatives at risk for Crohns disease. Associations with disease activity, longitudinal outcomes, and metabolic pathways were assessed using multivariable regression and survival analysis. ResultsFour reproducible metabotypes with distinct metabolite profiles were identified across cohorts: Low Kyna, High Kyna, High Quin, and Balanced. Low Kyna and High Quin metabotypes were consistently associated with increased inflammatory activity and adverse clinical outcomes, including increased risk of treatment escalation and disease progression. Pathway-level analyses revealed alterations in NAD-related, lipid, and amino acid pathways between inflammatory metabotypes. A metabotype resembling inflammatory disease states was enriched in individuals who later developed Crohns disease in a prospective pre-disease cohort. ConclusionTrp-linked metabotypes define reproducible immunometabolic states in IBD that associate with disease activity and clinical outcomes and may precede disease onset. These findings provide a framework for metabolic stratification and biomarker-guided clinical trials targeting immunometabolic pathways. What is already known on this topicTryptophan metabolism through the kynurenine pathway is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. Individual tryptophan metabolites have been associated with disease severity and treatment response, but their clinical utility for patient stratification remains limited. Systems-level approaches to define clinically meaningful metabolic subtypes in IBD are lacking. What this study addsWe identify four reproducible tryptophan-related metabolic subtypes ("metabotypes") that are consistently associated with disease activity across multiple independent IBD cohorts. Inflammation-associated metabotypes show distinct pathway-level alterations, including differences in NAD-related metabolism and broader metabolic programs. A metabotype resembling inflammatory disease states is detectable before clinical diagnosis in individuals who later develop Crohns disease. How this study might affect research, practice or policyMetabotype-based classification provides a framework for molecular stratification of patients in mechanistic studies and clinical trials targeting immunometabolic pathways. This approach may support biomarker-guided monitoring of disease activity and disease progression in IBD. Identification of preclinical metabolic states highlights the potential of metabolomics for early disease detection and prevention-oriented research strategies.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is characterized and initiated by the excessive accumulation of triacylglycerols (TG) and cholesteryl esters (CE) in the liver. Hepatic TG and CE synthesis, lipolysis and transport are tightly regulated by nutritional status, and disruption of this homeostasis contributes to MASLD pathogenesis. We have found that an endoplasmic reticulum-localized arylacetamide deacetylase (AADAC) catalyzes hepatic TG/CE turnover, and suppresses SREBP- and LXR-regulated lipogenesis and fatty acid esterification. Consequently, AADAC deficiency in mice leads to increased hepatic lipid synthesis, exacerbated steatosis, and impaired whole-body metabolism during Western-type diet feeding. These findings implicate AADAC as an important regulator of hepatic neutral lipid metabolism, linking endoplasmic reticulum cholesteryl ester hydrolysis as a modulator of lipid synthesis, and suggest its potential role in limiting MASLD pathogenesis under conditions of chronic overnutrition.

Background and ObjectivesThe etiology of biliary obstruction has undergone notable shifts over recent decades, yet long-term epidemiological studies addressing these changes remain scarce. With the widespread clinical adoption of endoscopic ultrasound (EUS), its role in altering diagnostic patterns warrants investigation. This study aimed to characterize the evolving disease patterns of biliary obstruction and specifically evaluate the impact of EUS adoption on driving these perceived etiological shifts over a 15-year period. MethodsThis retrospective, single-center study analyzed data from patients with biliary obstruction over a 15-year period. Time-series analysis was employed to characterize evolving disease patterns. To investigate the drivers underlying the observed trends, we applied a difference-in-differences (DID) analytical framework, uniquely treating the widespread clinical adoption of EUS as a natural experiment. Furthermore, multivariable logistic regression was utilized to identify independent predictors for malignant biliary obstruction of pancreatic origin. ResultsAmong 5,672 patients with pathological diagnoses, the disease spectrum shifted significantly toward malignant etiologies, particularly pancreatic and ampullary cancers, over the study period. The DID analysis confirmed that the broad adoption of EUS was associated with a significant relative increase in the precise diagnosis of malignancies detectable by this modality. Multivariable analysis further identified the EUS promotion era and calendar year as independent predictors for the pancreatic origin of malignancy. ConclusionsThe observed increase in pancreatic and ampullary cancers among patients with biliary obstruction is significantly associated with the enhanced diagnostic capabilities brought by EUS. This suggests that the diagnostic evolution driven by the widespread adoption of EUS, alongside potential epidemiological changes, is a major contributing factor to the perceived etiological shifts in biliary obstruction.

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

Liver sinusoidal endothelial cells (LSECs) separate the blood from the hepatic parenchyma and thus are at the frontline as scavengers of blood-borne waste, pathogens and metabolic stimuli. LSECs are also critical for sensing systemic iron availability by controlling the synthesis of bone morphogenetic protein (BMP) 6, which is essential for hepcidin expression in hepatocytes. Hepcidin maintains systemic iron homeostasis by inhibiting dietary iron uptake and iron release from iron recycling macrophages. Hepcidin is also an acute-phase protein and its activation by inflammation requires active BMP signaling. It is incompletely understood how signals derived from inflammation, cellular damage and iron are integrated by the liver to assure adequate hepcidin expression. Here, we show that Bmp6 expression is activated in primary LSEC cultures upon their exposure to danger-associated molecular patterns (DAMPs), such as heme and myoglobin, pathogen-associated molecular pattern (PAMPs), such as lipopolysaccharide (LPS) and Fibroblast-Stimulating Lipopeptide-1 (FSL1), or oxidative stress inducers (H2O2). Interestingly, all regulatory cues converge at the MAPK signaling pathway, although the specific signaling branches involved are stimulus-specific. Of note, Bmp6 upregulation in LSECs in response to all signals tested is strongly enhanced by the hepatocyte secretome. As hepatocytes critically depend on active BMP/SMAD signaling to control hepcidin activation, our results reveal that multiple sources of signaling input activating Bmp6 in LSECs and hepcidin in hepatocytes serve to determine BMP/SMAD signaling strength. Furthermore, our findings identify hypoferremia (low plasma iron levels), the result of high hepcidin levels due to elevated Bmp6, as a convergent response in conditions of inflammation, oxidative stress and cellular damage. HighlightsO_LIDAMPs (heme and myoglobin), PAMPs (LPS) and oxidative stress activate Bmp6 mRNA expression via the MAPK signaling pathway C_LIO_LIThe TLR/MAPK/BMP6 regulatory axis integrates inflammatory and iron signals C_LIO_LIOur work uncovers a novel connection between innate immune sensing, oxidative stress and hepatic iron homeostasis C_LI

Inflammatory bowel disease (IBD), comprising Ulcerative colitis (UC) and Crohns Disease, is a chronic relapsing immune-mediated inflammatory disorder of the gut. The intestinal mucus layer is a protective barrier that safeguards direct exposure of epithelium to luminal microbes and antigens. A prolonged disruption of the mucus layer may contribute to the development of IBD. Loss of mucin-producing goblet cells is a hallmark of UC. The underlying molecular mechanism controlling goblet regulation remains poorly understood. In the current work, we show a key role for NCoR1 (Nuclear corepressor 1) in goblet cell regulation. A specific downregulation of NCoR1 in intestinal crypts and goblet cells was observed in human UC and mice models. While NCoR1 was upregulated during goblet cell differentiation, inflammatory cues downregulated its expression. Experimental loss of NCoR1 resulted in exacerbated disease in a murine model of colitis, whereas its upregulation via Vitamin D led to a rescue. ChIP-seq led to the identification of KLF-16, a transcription factor, as a target of NCoR1. NCoR1 -KLF16 regulatory axis regulated key goblet cell proteins, including MUC2. Mechanistically, the regulation of MUC2 is modulated by the NCoR1-KLF16 axis, via mTOR signalling. In conclusion, this work shows a critical involvement of NCoR1-KLF16 in governing goblet cell function and intestinal homeostasis.

ABSTRACTUlcerative colitis is a chronic inflammatory bowel disease that can progress from dysplasia to cancer. Inflammatory responses are critical drivers in this process, typically triggered by epithelial lesions and the ensuing infiltration of microbiota into the interstitial layer. Here, we focus on the pro-inflammatory state of the interstitial fibroblasts, which promotes immune infiltration and augments disease progression. The study aims to provide a mechanistic link how fibroblasts of the colitis-associated microenvironment integrate inflammatory signals, microbial infiltration and cellular memory. To this end, we investigated a large number of primary colon fibroblasts obtained from normal, colitis and colon cancer samples using a range of in vitro approaches and an in vivo co-inoculation cancer model. mRNA sequencing analysis identified that the disease-associated fibroblasts are exhibit a cellular inflammatory status, which involves the injury-induced senescence pathway. Using CXCL8, a potent chemokine upregulated in colitis and cancer colon fibroblasts, as a paradigm, this inflammatory status is triggered by the activation of the NF{kappa}B signaling via immune-derived cytokines (TNF, IL-1{beta}), bacterial signals (LPS) and the microbiome itself using mycoplasma as a paradigm. Finally, iPSC reprogramming studies indicate that fibroblasts from ulcerative colitis retain an epigenetic memory that sustains elevated CXCL8 expression. Together, our findings demonstrate that the senescence associated secretory phenotype of colon fibroblasts is a robust indicator for inflammation-driven colon tumorigenesis.

Background & AimsHepatocellular carcinoma (HCC) frequently exhibits resistance to immune checkpoint inhibitors (ICIs), particularly in {beta} -catenin-driven tumors characterized by immune exclusion. While the Unfolded Protein Response (UPR) and the Integrated Stress Responses (ISR) enable tumor adaptation to metabolic stress their role in shaping tumor immunogenicity remains incompletely understood. We investigated whether ATF4, a central effector of the integrated stress response, couples metabolic reprogramming to suppression of anti-tumor immunity in HCC. MethodsWe combined transcriptomic analyses across three independent human HCC cohorts with mechanistic studies using an immunotherapy-resistant MYC/{beta}-catenin-driven murine HCC model. We integrated CRISPR/Cas9-mediated deletion of Atf4 with RNA-sequencing and targeted metabolomics. The impact of tumor-derived metabolites on macrophage differentiation and polarization was evaluated using primary bone marrow-derived cells. Therapeutic responses were evaluated in orthotopic and subcutaneous models treated with anti-PD-1 and anti-VEGFA. ResultsATF4 and XBP1 transcriptional signatures are selectively enriched in human HCC and associate with poor prognosis, vascular invasion, and an immunosuppressive myeloid-enriched tumor microenvironment. Genetic ablation of Atf4 markedly suppressed tumor growth in immunocompetent but not immunodeficient hosts, establishing a requirement for immune-mediated tumor control. Mechanistically, Atf4 loss downregulated Aldh18a1 and disrupted proline biosynthesis, resulting in extracellular proline depletion. This proline-deficient environment abrogated monocyte-to-macrophage differentiation and decreased M2 polarization, thereby reshaping the tumor microenvironment toward enhanced T cell infiltration and activation. Functionally, Atf4-deficient tumors exhibited restored sensitivity to anti-PD-1 monotherapy and showed pronounced responses to combined anti-PD-1/anti-VEGFA treatment in aggressive orthotopic models. ConclusionATF4 programs a proline-dependent metabolic axis that sustains macrophage-mediated immunosuppression and immune evasion in {beta}-catenin-driven HCC. Disruption of this pathway converts immune-excluded tumors into T cell-inflamed states and restores responsiveness to immunotherapy. By governing proline homeostasis and macrophage-mediated immunosuppression, ATF4 is a key metabolic checkpoint for immune evasion, linking stress adaptation to immune escape and a candidate therapeutic target in HCC. Impact and implicationsWe identify ATF4 as a crucial metabolic-immune orchestrator that sustains myeloid-driven immune evasion in {beta}-catenin-dependent HCC through proline-dependent circuitry. Disrupting the ATF4-proline axis converts immune-desert tumors into T cell-inflamed lesions by blocking macrophage differentiation, thereby sensitizing tumors to immune checkpoint therapy. This work positions ATF4 as a tractable therapeutic target to overcome immunotherapy resistance in HCC. Graphical abstract Highlights- ATF4 orchestrates an immunosuppressive tumor microenvironment in HCC by coupling metabolic stress adaptation to immune evasion. - Ablation of ATF4 disrupts proline biosynthesis, leading to a marked depletion of extracellular proline. - Cancer cell-derived proline availability contributes to macrophage differentiation and M2 polarization; its loss restores T cell-mediated anti-tumor surveillance and sensitizes beta-catenin-driven HCC to immune checkpoint blockade.

The mitochondrial membrane protein phosphoglycerate mutase 5 (PGAM5) is a protein of interest in the complex transition from hepatic steatosis to hepatocellular carcinoma. PGAM5 is a serine/threonine/histidine phosphatase that plays a role in mitochondrial biogenesis, mitophagy, and multiple cell death pathways. Increased expression of PGAM5 in hepatocellular carcinoma is correlated with reduced patient survival. In this study, we demonstrate that loss of PGAM5 alters the bioenergetic landscape of liver cancer by promoting mitochondrial oxidant injury and suppressing the glycerophospholipid and lysophospholipid pathways, leading to accumulation of the bioactive phospholipid lysophosphatidylcholine. Additionally, PGAM5 deletion downregulates fatty acid biosynthesis, resulting in reduced cellular diacylglycerol concentrations through two probable mechanisms: attenuated long chain fatty acid uptake and suppressed de novo synthesis. These findings underscore the broad impact of a single phosphatase on mitochondrial function and provide a rationale for therapeutically targeting PGAM5 to disrupt lipid metabolism in hepatocellular carcinoma.

Background and Aims: Diet plays a critical role in managing Crohns disease (CD) inflammation. We assessed whether dietary replacement of animal protein (AnimalP) by soy-pea protein (SoyP) decreases the pro-inflammatory potential of gut microbiota and intestinal inflammation in CD patients. Design: In an open-label, randomized controlled feeding trial at University Hospitals Cleveland Medical Center, CD participants and healthy controls were randomized (1:1) to a soy-pea or animal protein diet for 7-days. Primary outcomes were the absolute difference (d7-d0) in; Crohns Disease Activity Index (CDAI) score and fecal myeloperoxidase (MPO). Secondary outcomes included fecal calprotectin (FC) and high-sensitivity C-reactive protein (hsCRP). Murine fecal transplantation experiments were performed to determine the inflammatory potential of diet-altered gut microbiota. Results: The study randomized 66 participants and 60 were included in the final analysis (n=31 CD, n=29 HC). After 7 days, CD-SoyP participants were more likely than CD-AnimalP to show reductions in HBI (RR=4.68, 95% CI: 1.22-17.98, P=0.009) and fecal MPO (RR=2.30, 95% CI: 1.04-4.85, P=0.032), with a similar directional trend for CDAI (RR=1.52, 95% CI: 0.89-2.58, P=0.135). No participants experienced worsening of CDAI. The rank-based composite CDAI-MPO score was lower in the CD-SoyP vs CD-AnimalP group (median [IQR]: 5 [4-6] vs 8 [7-9]; P=0.012). Stratified analyses showed significant reductions in fecal MPO among CD participants with lower baseline disease activity (CDAI <150; P<0.0001), but not in those with higher activity (P=0.799) Conclusion: Short-term addition of plant-based soy-pea protein within a controlled diet exerted a beneficial, anti-inflammatory effect in CD, with evidence of greater effects among participants with lower baseline disease activity. ClinicalTrials.gov, Number NCT04065048.

BackgroundPostoperative pancreatic fistula (POPF) is a major cause of morbidity after pancreatoduodenectomy, particularly in patients with high-risk pancreatic remnants. Preventive strategies based solely on surgical technique have yielded inconsistent results, and thus there has been growing interest in strategies aiming to modify the biological behavior of the pancreatic remnant. This preclinical study evaluated the biological and histopathological effects of preoperative endoluminal radiofrequency ablation (ERFA) of the main pancreatic duct (MPD) performed 4 weeks before pancreatic transection in a porcine model. MethodsAnimals underwent laparoscopic MPD occlusion followed by pancreatic transection at 4 weeks and necropsy 15 days thereafter. Feasibility, safety, histological atrophy, and macroscopic findings associated with POPF risk were assessed. As a secondary objective, outcomes were compared with a that underwent MPD occlusion using cyanoacrylate glue. ResultsPreoperative ERFA was technically feasible and safe. At 4 weeks, ERFA induced marked and homogeneous acinar atrophy that was significantly greater than that observed after glue occlusion (p = 0.018), indicating effective biological conditioning of the pancreatic remnant. At necropsy, pseudocyst formation and intra-abdominal adhesions, known surrogate markers of pancreatic fistula in pigs, were significantly more frequent in the glue group and absent in ERFA-treated animals. Serum amylase levels, postoperative weight gain, complication rates, and preservation of endocrine architecture were comparable between groups. ConclusionsDuctal ablation of the MPD via ERFA induced stable, progressive exocrine pancreatic atrophy, effectively preconditioning the gland prior to pancreatic transection. Experimental evidence suggests that its biological effects stabilize approximately 4 weeks after treatment. Compared to cyanoacrylate occlusion, ERFA achieved more homogeneous early biological effects and fewer fistula-related macroscopic complications. These findings support the further investigation of preoperative pancreatic conditioning as a potential adjunct strategy for POPF risk reduction, although clinical studies are needed to clarify its role alongside established reconstructive approaches.