American Journal of Physiology-Gastrointestinal and Liver Physiology

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.

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.

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.

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.

BACKGROUNDPatients with inflammatory bowel disease (IBD) are at increased risk of cardiovascular disease, yet the mechanisms linking chronic intestinal inflammation to cardiac dysfunction remain poorly understood. IBD is characterized by profound gut microbiota dysbiosis, which we hypothesize drives systemic immune dysregulation and contributes to cardiac dysfunction. METHODSA chronic colitis mouse model was used to assess gut microbiota dysbiosis, systemic immune cell metabolism, and cardiac remodeling. Cardiac outcomes were evaluated by echocardiography, histology, and molecular analyses. Mechanisms were examined using fecal microbiota transplantation, immune cell depletion, exosome transfer, bone marrow chimeras, RNA-seq, co-immunoprecipitation, confocal microscopy, and siRNA-mediated gene silencing. RESULTSChronic DSS colitis induced cardiac dysfunction, hypertrophy, and fibrosis in mice. These changes were accompanied by sustained gut microbiota dysbiosis, metabolic reprogramming, and mitochondrial dysfunction in circulating immune cells. Fecal microbiota transfer experiments demonstrated that colitis-associated microbiota were sufficient to reprogram systemic immune cells and promote cardiac dysfunction. Immune cell depletion studies identified macrophages as key mediators of colitis-associated cardiac injury. Colitis increased systemic lipopolysaccharide (LPS) translocation, bone marrow chimera experiments demonstrated that hematopoietic TLR4 signaling was required for immune cell metabolic remodeling and cardiac dysfunction during chronic colitis. Transcriptomic analysis identified guanylate-binding protein 2b (GBP2b/GBP1, hereafter referred to as GBP1) as a key downstream effector of LPS-TLR4 signaling. Upon LPS stimulation, GBP1 localized to mitochondria, where it interacted with DRP1 and FIS1 to promote mitochondrial fission, oxidative stress, and enhanced immune cell migration into the heart. In addition, GBP1 was secreted via exosomes, which were taken up by cardiomyocytes and contributed to hypertrophic remodeling, and cardiac dysfunction. CONCLUSIONSThese findings establish the LPS-TLR4-GBP1 axis as a key driver of colitis-associated cardiovascular dysfunction and highlight this pathway as a promising therapeutic target for reducing cardiovascular risk in patients with IBD. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIPatients with inflammatory bowel disease have an increased risk of cardiovascular dysfunction that cannot be fully explained by traditional cardiovascular risk factors. C_LIO_LIGut microbiota dysbiosis and chronic innate immune activation are hallmarks of inflammatory bowel disease, but their direct contribution to cardiac remodeling remains unclear. C_LI What New Information Does This Article Contribute?O_LIChronic colitis-associated gut microbiota dysbiosis induces systemic immune cell metabolic and mitochondrial reprogramming that is sufficient to drive cardiomyocyte hypertrophy and cardiac dysfunction. C_LIO_LIHematopoietic Toll-like receptor 4 signaling links colitis associated gut microbiota to immune metabolic dysfunction and cardiac impairment, establishing a causal gut-immune-heart axis. C_LIO_LIGuanylate-binding protein 2b (GBP2b/GBP1) is identified as a critical downstream effector that promotes mitochondrial fission, oxidative stress, immune cell cardiac infiltration, and exosome-mediated cardiac remodeling. C_LI

In clinical trials for ulcerative colitis (UC), pathologists assess disease severity through standardized histological indices, including the Geboes Score, Robarts Histopathology Index (RHI), and Nancy Histologic Index (NHI). Despite strong associations with clinical outcomes, histologic scoring suffers from inter- and intra-reader variability, and consensus criteria for histologic remission remain uncertain. Through a consortium approach, we developed an artificial intelligence-based measurement (AIM) tool for scoring histology in UC mucosal biopsies (AIM-HI UC). This model, trained on a large dataset of UC biopsies (N=10,230), utilizes additive multiple instance learning models leveraging PLUTO, a pathology foundation model, that predict each of the Geboes subgrades, from which the Geboes grade-level score, RHI, and NHI can be calculated. Evaluation of this model on a standalone verification set including clinical trial specimens established algorithm non-inferiority and/or superiority relative to standard qualified pathologists through comparison of algorithm-consensus and pathologist-consensus agreement metrics (non-inferior if difference >-0.1, superior if difference >0, inclusive of confidence intervals). AIM-HI UC was determined to be non-inferior to pathologists (N=3) for the prediction of all seven Geboes subgrades, grade-level Geboes, RHI, NHI, histologic improvement (GS<3.1), 2A histologic remission (GS<2A.0), and 2B histologic remission (GS<2B.0). AIM-HI UC was superior to pathologists for several Geboes subgrades (GS 0, GS 1, GS 2B, and GS 5), as well as grade-level Geboes, RHI, and positive percent agreement of 2A histologic remission. The model was shown to be greater than 99% repeatable for all histologic scoring metrics examined. Model-derived scores were shown to strongly correlate with canonical histologic features of inflammation, including the proportion of total epithelium that is inflamed (Spearman r=0.83; p<0.01), the proportion of neutrophils localized within crypt epithelium (Spearman r=0.83, p<0.01), and the amount of mucosal area classified as erosion or ulceration (Spearman r=0.80, p<0.01). Overall, these results suggest that AIM-HI UC has the potential to improve consistency of UC histology interpretation, providing a path toward standardization of UC histology scoring in clinical trials.

BACKGROUNDMetabolic dysfunction-associated steatotic liver disease (MASLD) is a systemic immunometabolic disorder rapidly increasing worldwide, affecting nearly 38% of adults. Gut dysbiosis and host genetic factors, such as PNPLA3 I148M variant, modulate disease development and progression. Through the gut-liver axis, increased intestinal permeability enables microbial translocation to the liver, promoting inflammation and metabolic disruption. However, the composition and functional potential of the hepatic microbiome remain poorly characterized. Understanding its relationship with histological injury and genetic susceptibility may provide novel mechanistic insights. We hypothesized that the hepatic microbiome composition and function are associated with histological severity and PNPLA3 genotype in this disease. AIMTo characterize the hepatic microbiome and assess its association with histological severity and PNPLA3 genotype. METHODSThis cross-sectional observational study included 30 patients with MASLD from a tertiary care hospital. Liver tissue underwent shotgun metagenomic sequencing. Histological severity was assessed using the NAFLD Activity Score (NAS). PNPLA3 genotype was determined by PCR. Differential abundance and functional enrichment analyses were performed using MaAsLin2. Somatic variants were identified using Mutect2. Correlation networks were constructed using Spearmans correlation coefficients. RESULTSPatients with advanced histological injury (NAS [&ge;]5) and PNPLA3 I148M carriers showed a trend toward higher somatic mutational load and a markedly reduced microbial abundance. Analyses revealed broad compositional shifts across bacterial, fungal, viral, and eukaryotic taxa, affecting both commensal and context-dependent pathobiont lineages. Pseudomonas species were enriched, whereas Siphoviridae phages were depleted in advanced disease and PNPLA3 I148M carriers. Functional analysis revealed enrichment of pathways related to nutrient transport and metabolic stress adaptation, while TonB-associated functions were enriched in advanced liver injury but depleted in PNPLA3 I148M carriers. Network analysis identified Sphingomonas leidyi as a keystone node associated with hexosamine metabolism. Salmonella enterica abundance positively correlated with somatic variant burden, suggesting a link between microbial signatures and genomic instability. Histological progression and the risk PNPLA3 genotype were accompanied by marked topological simplification, reflecting less resilient community structures. CONCLUSIONSThe hepatic microbiome in MASLD is a low-biomass, polymicrobial ecosystem shaped by the host genetic background. Its functional activity, taxonomic composition and system architecture bidirectionally relate to liver DNA instability and the severity of histological damage. Core tipThis study characterizes the multi-kingdom hepatic microbiome in MASLD using FFPE-derived metagenomics. We demonstrate that microbial abundance-including bacteria, fungi, protozoa, and viruses- significantly decreases with increased histological severity and the PNPLA3 risk genotype. Rather than global diversity shifts, results showed that disease progression could be linked to specific functional adaptations and simplified microbial network connectivity. In addition, we described associations between specific taxa and somatic mutational burden, suggesting a link between microbial signals and genomic instability. These findings indicate that changes in the liver microbiome as a whole, rather than specific taxonomic modifications, influence MASLD pathophysiology.

Cell division cycle 42 (Cdc42) is a member of the Rho family of small GTPases, which plays crucial roles in regulating cytoskeletal remodeling, and membrane trafficking. While previous studies implicated Cdc42 in controlling intestinal epithelial homeostasis, the involvement of this small GTPase in the process of intestinal fibrogenesis remains unexplored. Our study was designed to determine whether Cdc42 regulates the fibrogenic activation of intestinal myofibroblasts in vitro. The study was conducted using a CCD-18Co normal human colonic fibroblast cell line, and primary human intestinal myofibroblasts (HIMF) isolated from Crohns disease (CD) patients. CCD-18Co and HIMF cells were stimulated by transforming growth factor-{beta}1 (TGF-{beta}1). Cdc42 was inhibited either genetically, using siRNA-mediated knockdown, or pharmacologically using specific Cdc42 inhibitors, ML141 and CASIN. Genetic and pharmacologic inhibition of Cdc42 markedly reduced TGF-{beta}1 induced expression of the major contractile cytoskeletal proteins, -smooth muscle actin, calponin 1 and L-caldesmon. Furthermore, Cdc42 inhibition significantly attenuated expression of key extracellular matrix (ECM) proteins, fibronectin and collagen I, in activated CCD-18Co cells and HIMF. Interestingly, decreased expression of contractile and ECM proteins in Cdc42-depleted myofibroblasts was not due to downregulation of the TGF-{beta}1 signaling, decreased mRNA transcription or increased lysosomal or proteasomal degradation of these proteins. Such suppressed pro-fibrotic activation of Cdc42-deficient CCD-18Co cells and HIMF involved a selective inhibition of protein translation due to inactivation of the AKT-mammalian target of rapamycin (mTOR) signaling module. These findings highlight Cdc42 as a key regulator of intestinal fibrosis that controls mTOR activation to enhance ECM production and contractile actomyosin cytoskeleton in intestinal myofibroblasts.

STRUCTURED ABSTRACTO_ST_ABSObjectiveC_ST_ABSTo characterize intestinal transcriptional profiles in gastroschisis, their temporal evolution, and response to fetal intervention. Summary Background DataGastroschisis causes significant intestinal dysfunction, with intra-abdominal bowel dilation clinically shown to correlate with worse outcomes. While inflammation and neurovascular impairment have been implicated, genome-wide transcriptional characterization of disease severity remains lacking. MethodsUsing a fetal ovine model of complex gastroschisis, in which all gastroschisis animals demonstrated significant intra-abdominal bowel dilation at term, bulk RNA sequencing was performed on proximal small intestinal tissue from mid-gestation and term fetuses across three groups: normal, gastroschisis, and prenatally repaired gastroschisis. Differential gene expression (FDR [&le;] .05, |log2 fold change| [&ge;] 1.5) and pathway enrichment analyses were performed, with targeted interrogation of extracellular matrix (ECM), enteric nervous system (ENS), angiogenic, and inflammatory pathways. ResultsAt mid-gestation, gastroschisis intestine showed minimal transcriptional differences (150 differentially expressed genes [DEGs]) and some bowel dilation. By term, dysregulation was substantial (2,423 DEGs) alongside significant dilation. Normal ontogenetic intestinal maturation patterns were altered, with fewer expected developmental gene changes and discordant pathway regulation. ECM pathway aberrations emerged early and persisted, while ENS, angiogenic, and inflammatory pathways were only dysregulated at term. Fetal repair was associated with normalization of gene expression at term (29 DEGs vs controls). ConclusionIntestinal transcriptional changes in experimental gastroschisis parallel progressive bowel dilation, consistent with a mechanical stress contribution to intestinal injury. Prenatal repair normalizes both dilation and gene expression, indicating a dynamic and potentially modifiable transcriptional program that supports the rationale for early fetal intervention. Mini AbstractIn a fetal ovine model, progressive bowel dilation in gastroschisis parallels transcriptomic dysregulation of ECM remodeling, neurovascular impairment, and inflammation which is normalized by prenatal repair.

The intestinal mesentery has been recently classified as a 'new' organ and contains various cell types including adipocytes, preadipocytes, endothelial cells, and immune cells. In addition, neuronal cell bodies are found in the small intestinal mesentery and are situated either individually or clustered together with glial cells in small ganglion structures close to the gut wall. However, little is known about the origin or function of these extra-intestinal mesenteric neurons. The aim of this study was to better these characterize mesenteric neurons and to examine their connectivity with the ENS using calcium imaging in adult mouse ileum with the mesentery attached. Here we show that neurons in the mesentery express typical ENS neurochemical markers, respond to 5-HT, ATP and the nicotinic agonist DMPP, and receive nicotinic synaptic inputs. Furthermore, using labeling with the neuronal tracer DiI, some mesenteric neurons were found to project into the gut wall and can provide functional excitatory inputs to myenteric neurons. By contrast, we did not find evidence for mesenteric neurons providing inputs to other extrinsic neuronal targets, suggesting that they preferentially interact with the ENS. We also demonstrate that mesenteric neurons can be activated by intestinal distension and that the mesentery provides a source of inhibition to the myenteric plexus. Taken together, we show that the ENS not only interacts with vagal and spinal afferents, and sympathetic and parasympathetic nerves, but also neurons situated in the mesentery. Finally, our data suggest that these neurons may provide a form of negative feedback to the myenteric plexus such as in the event of intestinal distension. These findings have important implications for the regulation of intestinal motility in physiological and pathophysiological conditions.

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.

Iron absorption in the small intestine has classically been described by the duodenal DMT1/FPN1 pathway for inorganic non-heme iron, yet emerging evidence suggests that chemically distinct iron forms may use region-specific routes. Nicotianamine (NA), a plant-derived metal chelator, can form NA-iron (NA-Fe) complexes and has been proposed to support intestinal iron absorption through amino acid transporter pathways. However, direct comparisons of transepithelial transfer of inorganic iron and NA-Fe across defined small intestinal regions under controlled epithelial conditions remain limited. Here, we established region-specific 2D epithelial monolayers derived from duodenal and proximal jejunal crypt organoids from male ICR mice cultured on Transwell inserts. Transcriptomic profiling indicated partial retention of regional identity, and barrier integrity was confirmed by junctional marker localization, transepithelial electrical resistance, and low paracellular permeability. We then examined expression and polarized localization of candidate transporters for inorganic iron (Dmt1/Fpn1) and NA-Fe (Pat1/Lat2). Finally, we quantified transepithelial transport using apical loading of isotope-labeled iron (55Fe) or NA-55Fe and measured radioactivity appearing in the basolateral compartment as the primary readout of transepithelial flux. Basolateral appearance of inorganic 55Fe was comparable between duodenum- and proximal jejunum-derived monolayers, whereas NA-55Fe exhibited significantly greater basolateral appearance in proximal jejunum-derived monolayers. These findings demonstrate that organoid derived, region-specific monolayers provide a tractable epithelial platform to evaluate iron form-dependent, region-specific transepithelial transfer and to enable further mechanistic dissection of NA-Fe transport. NEW & NOTEWORTHYNon-heme iron absorption may depend on iron chemical form and intestinal region, but direct epithelial comparisons are scarce. We established duodenum and proximal jejunum derived murine intestinal organoid monolayers on Transwells and quantified transepithelial flux using isotope-labeled iron. Inorganic 55Fe showed no clear regional difference, whereas NA-55Fe displayed greater basolateral appearance in proximal jejunum-derived monolayers. This platform enables mechanistic studies of NA-iron complex transport.

BackgroundLymphangiogenesis plays a critical role in various liver diseases, yet its function in liver fibrosis remains controversial. This study aimed to explore the role of lymphangiogenesis in liver fibrogenesis and its underlying regulatory mechanisms. MethodsLiver fibrotic mice were established by carbon tetrachloride (CCl4) or Thioacetamide (TAA)-induced injection or bile duct ligation. Lymphatic vessels were marked by podoplain (Pdpn) staining in mice and D2-40 staining in clinical samples. Lymphatic vessels area and density were measured to indicate lymphangiogenesis. Multiplexing immunohistochemistry was used to detect co-localization of proteins. ResultsIn the present study, we first verified increased lymphangiogenesis in human and murine fibrotic livers. Afterwards, we identified VEGFC rather than VEGFD as the primary driver of lymphangiogenesis in liver fibrosis. Furthermore, we demonstrated that M1 macrophages serve as the major source of VEGFC. Founctional studies revealed that VEGFC-mediated lymphangiogenesis exacerbates hepatic fibrosis, while its inhibition alleviated fibrosis. Bioinformatic analysis uncovered Midkine (MDK) as a key downstream of lymphangiogenesis. Both in vivo and in vitro studies confirmed that exogenous MDK promotes liver fibrosis via activating hepatic stellate cells (HSCs), whereas MDK inhibition counteracts the profibrotic effects of VEGFC-induced lymphangiogenesis. Importantly, we discovered that MDK activates HSCs through the Hippo/YAP signaling pathway. ConclusionsM1 macrophage-mediated lymphangiogenesis aggravates liver fibrosis via MDK secretion, which activates HSCs. These findings provide novel insights into coordinated crosstalk between macrophages, lymphatic endothelial cells and HSCs in liver fibrosis and suggest lymphangiogenesis and MDK as potential therapeutic targets for fibrotic liver diseases.

Background & AimsHepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. Here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. MethodsAdult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. ResultsHBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, HBx expression increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative, less differentiated state. ConclusionsHBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. These findings support a model in which HBx promotes a survival-permissive less differentiated state that may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implicationsUnderstanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and disrupts hepatocyte identity, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.

Background & AimsSystemic metabolic dysfunction promotes degenerative diseases in many organs, including liver and kidney. The liver is a master regulator of systemic metal ion homeostasis. Hepatic copper deficiency is increasingly observed in metabolic dysfunction associated steatotic liver disease (MASLD) and is associated with greater disease severity and poor outcomes. However, mechanisms linking copper dysregulation to MASLD and its co-morbidities remain poorly defined. We investigated whether impaired mitochondrial copper homeostasis contributes to MASLD-related pathobiology and represents a modifiable therapeutic axis. Methods & ResultsUsing dietary mouse models of MASLD and in vitro systems, we found that dietary copper deficiency induces lipotoxicity and suppresses mitochondrial metabolic programs. MASLD livers exhibited marked depletion of copper, impaired cytochrome c oxidase integrity, and bioenergetic failure. Targeted restoration of mitochondrial copper with the copper ionophore elesclomol normalized copper-handling programs, improved mitochondrial function, and suppressed ferroptotic stress, hepatocyte senescence, and fibroinflammatory remodeling. Mechanistically, reduced expression of the mitochondrial copper transporter SLC25A3 and MT-CO1 disrupted the SLC25A3-SCO1-MT-CO1-CTR1 axis, limited copper uptake and destabilized copper-iron balance, promoting maladaptive cell fate changes. Across multiple human cohorts and mouse models, copper-iron imbalance tracks with MASLD progression, clinical outcomes, and multiple extrahepatic comorbidities; restoring copper homeostasis in mice with MASLD attenuates both liver and kidney inflammation and fibrosis. ConclusionsMitochondrial copper deficiency is a mechanistically actionable driver of MASLD that promotes bioenergetic failure, ferroptosis, senescence and fibroinflammatory damage in the liver and other organs. Targeting copper-centered mitochondrial regulation represents a novel biomarker and therapeutic strategy for MASLD and its systemic complications.

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: Gut injury after pediatric cardiac surgery remains an ongoing challenge, resulting in increased morbidity and mortality for children with congenital heart disease (CHD) and a significant burden on the healthcare system. It remains unclear what the driving forces are that result in this pro-inflammatory state following pediatric cardiac surgery with cardiopulmonary bypass. Understanding key components involved in the gut composition, gut barrier function, and systemic inflammation in children with CHD after cardiac surgery is critical to improve outcomes. Methods: A prospective study of patients aged 0-5 years with CHD undergoing cardiac surgery (CPB group) or non-CHD undergoing non-cardiac surgery (Comparison group). We collected pre-operative and post-operative stool and plasma to evaluate the microbiome, metabolites, markers of gut barrier function, and inflammatory cytokines. Clinical variables were collected to evaluate markers of inflammation. These variables were compared between the two groups to evaluate signatures and develop unique biomarker profiles. Results: We enrolled 62 patients (CPB, n=46; Comp, n=16). CPB patients had increased pro-inflammatory microbiota and reduced diversity metrics pre-operatively, which were exacerbated post-operatively. The CPB group also had increased pro-inflammatory eicosanoids and reduced gut and heart protective short-chain fatty acids versus the Comparison group. The CPB group had increased pro-inflammatory and reduced anti-inflammatory cytokines post-operatively. The CPB group also had increased markers of gut barrier dysfunction versus the Comparison group. Mediation analysis showed the microbial functional shift was associated with increased PGE2 and reduced butyric acid in the CPB group, associated with increased cytokines and clinical markers of inflammation post-operatively. Conclusion: We demonstrate unique gut microbial and metabolites profiles associated with gut permeability and systemic inflammation in children with CHD undergoing cardiac surgery highlighting a unique microbiome-inflammation axis in this patient population. Further studies to evaluate causal links with these profiles will identify potential targets to improve outcomes for these patients.

The ketogenic diet (KD) promotes ketone body synthesis and has been used as an effective treatment for disorders such as epilepsy. Although elevated ketone bodies, including {beta}-hydroxybutyrate ({beta}HB) and acetoacetate, are thought to meditate the beneficial effects of the KD, the mechanisms underlying their metabolic actions remain incompletely understood. In this study, we focused on GPR109A, a receptor for {beta}HB with an unclear role in metabolic homeostasis. We employed KD and fasting models to examine metabolic changes under two distinct ketogenic conditions. Under KD conditions, Gpr109a-/- mice exhibited increased hepatic lipid accumulation, and subsequent hepatic inflammation and fibrosis. However, Gpr109a deletion did not exacerbate hepatic lipid accumulation or inflammation during short-term fasting, suggesting that GPR109A-mediated liver protection is specific to KD-induced metabolic stress rather than under fasting conditions. Mechanistic analysis revealed that GPR109A protects the liver from inflammation by maintaining intestinal barrier integrity. These findings highlight the novel protective mechanism of GPR109A, via the gut-liver axis, to sustain metabolic homeostasis during the KD. This study provides valuable insights into the physiological effects of ketone bodies.

Background and AimsStem cell-derived organoids are promising platforms for therapeutic screening in inflammatory bowel disease (IBD), but identifying functional organoid readouts with translational utility is challenging. Colon epithelial organoids from patients with ulcerative colitis (UC) overexpress chemokines CXCL1, CXCL11, CCL2, and CCL28, yet whether these inflammatory signatures correlate with disease activity and treatment response is unknown. This short report investigates whether organoid-retained chemokines correlate with disease activity and therapeutic outcomes. MethodsWe interrogated three bulk and two single-cell transcriptomic datasets from IBD clinical trials encompassing anti-TNF and anti-integrin therapies to determine whether epithelial chemokines retained in UC organoids track clinical response and distinguish treatment responders from non-responders to biologic therapy across multiple IBD patient cohorts. ResultsIn bulk transcriptomic data, CXCL1, CXCL11, and CCL2 were elevated in active UC and normalized only in patients achieving clinical remission, independent of therapy class, with persistent chemokine overexpression in non-responders. Single-cell analysis demonstrated widespread chemokine overexpression in UC epithelial clusters, with consistent normalization of CXCL1, CXCL11, and CCL28 in LGR5-positive stem compartment of patients who achieved clinical remission, but not in non-responders. In Crohns disease, the resolution of these epithelial chemokines was not associated with clinical response. ConclusionsEpithelial chemokines, particularly CXCL1, CXCL11, and CCL28, track clinical remission in UC and represent candidate biomarkers and functional endpoints for epithelial-directed therapeutic strategies using stem cell-derived UC organoid models.

The intestine plays a crucial role in physiology, nutrition, and immune function, but intestinal anatomy and cell types have yet to be fully characterized in many fish species, the most diverse group of vertebrates. To address this gap, we characterized the structure and composition of the intestine of threespine stickleback (Gasterosteus aculeatus), an emerging model teleost in biological research. Using histology, myeloperoxidase staining, single-cell RNA sequencing, and RNA in situ hybridization, we defined major intestinal epithelial, immune, stromal, and stem/progenitor populations. Goblet cells were abundant in proximal and hindgut, while myeloperoxidase-positive granulocytes were evenly distributed throughout the intestine. To facilitate future experimental studies of stickleback intestinal function, we also developed the first intestinal organoid culture from stickleback and show that these cultures recapitulate epithelial architecture and retain expression of canonical intestinal epithelial markers. This organoid platform enables future functional studies of mucosal immunity, host-microbe interactions, and intestinal physiology in stickleback and related teleosts. Together, our integrated approach provides a comprehensive cell atlas and a novel experimental model for studying digestive and immune functions in threespine stickleback.