The intricate interplay between the gut microbiome and bile acid metabolism via the gut-liver axis is fundamental to hepatic homeostasis. Perturbations in this axis are increasingly implicated in the pathogenesis of diverse liver diseases, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, cholestatic liver diseases, and hepatocellular carcinoma. This review integrates current understanding of hepatic bile acid synthesis, enterohepatic circulation, and gut microbial bile acid transformations, detailing how bile acids function as signaling molecules through nuclear receptors including farnesoid X receptor, pregnane X receptor, vitamin D receptor, constitutive androstane receptor, and G-protein-coupled receptors; G protein-coupled bile acid receptor 1 (also known as Takeda G protein-coupled receptor 5), and sphingosine-1-phosphate receptor 2. We explore disease-specific alterations in gut microbiota composition and bile acid profiles in metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, cholestatic liver diseases, and liver cancers, focusing on mechanisms linking gut dysbiosis, impaired intestinal barrier function, altered bile acid signaling, inflammation, and immune modulation to liver injury and progression. Furthermore, we discuss the clinical implications, highlighting the potential of microbiome signatures and bile acid profiles as diagnostic and prognostic biomarkers. Therapeutic strategies targeting the gut-liver axis, including probiotics, fecal microbiota transplantation, farnesoid X receptor agonists, and fibroblast growth factor 19 analogs, are reviewed. Finally, we address current challenges and future directions, emphasizing the need for multiomics integration, functional studies, and personalized medicine approaches to leverage the gut-liver axis for improved liver disease management. SIGNIFICANCE STATEMENT: Disruption of the gut microbiome-bile acid-liver axis is now recognized as a unifying mechanism driving multiple liver diseases, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, cholestatic liver diseases, and hepatocellular carcinoma. Unraveling the molecular and microbial interactions within this axis offers fundamental insights into disease pathogenesis and reveals novel therapeutic opportunities. Integrating multiomics technologies with artificial intelligence-based analytics will accelerate the discovery of predictive biomarkers and personalized interventions, advancing the field toward precision-based liver disease treatment protocols.

Abstract Introduction Inflammatory bowel diseases (IBD) exhibit a global disease burden, with pathogenesis driven by the dynamic interplay of genetic susceptibility, gut microbial dysbiosis, and immune dysregulation. We studied the tissue-specific regulatory role of Nlrp12 in gut microbial dysbiosis and colonic inflammation and revealed a complex regulatory link of myeloid-specific NLRP12 and the promotion of taurine to induce colitis-mitigating bacteria while restraining bacterial species that exacerbate gut inflammation. Methods We generated Nlrp12flox/flox (Nlrp12fl/fl) mice and created both myeloids-specific cre and epithelial-specific cre mouse. We used both conventional mouse and germ-free mouse model. For acute colitis induction, we used DSS for 5 days and then regular drinking water. DSS-AOM was used for colorectal cancer model. ELISA and western blot were used to study cytokine production and signaling pathways. Fecal microbiome profiling was done by 16s rRNA sequencing. We also have human fecal sample and qPCR was used to quantify specific strains. Fecal microbiome transfer was used to study the causality between genetics, microbial species and colitis. Results Myeloid-specific , but not epithelial-specifc expression of NLRP12 is protective against colitis. Deficiency of Nlrp12 in myeloid cells has minimal effects on colorectal cancer. Myeloid-specific NLRP12 does not result in inflammasome activation in the colitis model. Deficiency of Nlrp12 in myeloid cells promotes microbial dysbiosis. Fecal microbiota transplantation from WT mice attenuates colonic inflammation in Nlrp12ΔMye mice. Allobaculum exacerbate DSS-induced colitis in GF mice. Human IBD patient samples demonstrate similar microbial signatures. Fecal metabolomic analysis reveals alterations in taurine and bile acid metabolism in Nlrp12ΔMye mice. Conclusion This work reveals the profound effect of a host innate immune checkpoint that functions in a cell-specific fashion to protect against metabolomics changes and dysbiosis. that leads to colitis. Funding Source NIH Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Introduction:Biliary 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-β (TGFβ) 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. Methods:Human 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. Results:RUNX1 expression was significantly increased in cholangiocytes from PSC and PBC patients, and Mdr2 -/- mice. RUNX1 knockdown or inhibition reduced IL6, TNFα, and other proinflammatory cytokines in PSC-Cs and attenuated TGFβ-, LPS-, and TNFα-induced Il6 and Ccl2 expression in MLEs. ChIP-qPCR and ChIP-seq revealed TGFβ-induced RUNX1 binding to the Il6 promoter and 727 additional genomic sites enriched for fibrosis and inflammatory pathways; predicted upstream regulators included TGFβ, TNF, and NFκB signaling. Proteomic analysis identified TGFβ-induced RUNX1 interactions with SMAD2 and NFκB2. 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. Conclusion:RUNX1 is a central transcriptional hub integrating TGFβ 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.

Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis characterized by pulmonary microvascular dilation, hypoxemia, and increased mortality. Patients often exhibit unexplained restrictive ventilatory defects that correlate with circulating bile acids, suggesting superimposed alveolar dysfunction. To investigate this, we evaluated alveolar function, cell types, and the potential role of altered bile acids in the experimental HPS. Common bile duct ligation (CBDL) mice were assessed for pulmonary and surfactant function. AT2 cell-specific RiboTag RNA sequencing, single-cell RNA sequencing (scRNA-seq), and mass spectrometry-based bile acid profiling were performed. MLE12 cells were treated with bile acids in vitro, and an FXR agonist was administered in vivo to test effects on AT2 cell. CBDL mice developed HPS with restrictive defects due to reduced AT2 cell-derived surfactant-protein-C (SP-C), increased alveolar surface tension, and elevated plasma and bronchoalveolar bile acid levels. ScRNA-seq demonstrated a decrease in AT2 cells and an increase in AT2-to-AT1 transitional cells. AT2-specific RNA-seq revealed upregulated bile acid and cholesterol metabolism and downregulated proliferative pathways. In vitro, bile acids mimicking FXR antagonists reduced SP-C in MLE12 cells, while in vivo FXR agonist decreased circulating bile acids and restored SP-C-producing AT2 cells in CBDL mice. Our data demonstrates alterations in AT2 cell biology, including reduced surfactant expression, in the setting of elevated bile acids. These finding indicate an association between bile acid levels and AT2 cell alterations in cirrhosis and identify bile acid signaling and AT2 cell integrity as areas for future mechanistic investigation.

Background:Primary Sclerosing Cholangitis (PSC) is a chronic obstructive biliary disease and remains a high-burden cholestatic liver disease with no approved therapies and a substantial recurrence rate following liver transplantation. The long non-coding RNA H19 (H19) has emerged as a potential driver of PSC progression, yet its cell-type-specific and spatially resolved mechanisms remain poorly defined. Results:Age- and sex-matched wild type (WT), H19 knockout (H19KO), Mdr2 knockout (Mdr2KO), and double-knockout (DKO; Mdr2KO/H19KO) mice were used. The liver tissues were analyzed using single nucleus RNA sequencing (snRNAseq) and NanoString GeoMx spatial transcriptomics to elucidate H19-dependent cellular and spatial alternations in cholestatic liver injury. Machine learning models (logistic regression, XGBoost, neural network, and random forest) were developed to generate cell-type specific disease prediction signatures and validated using the publicly available human dataset GSE243981. Both spatial transcriptomics and snRNAseq identified a disease-associated cholangiocyte subcluster that was significantly expanded in Mdr2KO mice, but markedly diminished in DKO mice, demonstrating a requirement for H19 in sustaining pathogenic cholangiocyte state. SPP1 signaling was significantly dysregulated in cholestatic liver injury and ameliorated with H19 deletion. Novel murine markers were identified, including Gm13775 (healthy hepatocytes) and Clu and Spp1 (healthy cholangiocytes), all of which were markedly downregulaed in disease. Machine learning-based, cell type-specific disease prediction models achieved AUC values > 0.87 when validated in the GSE243981 human dataset. Noteably,Spp1 expression decreased in cholangiocytes but was ectopically upregulated in hepatocytes in diseased liver, highlighting disrupted intercellular signaling network. Spatial analyses showed that H19 deletion restored the disease-associated gene expression changes specifically within the bile duct region. Conclusion:H19 deletion mitigates cholestatic injury by suppressing pathogenic cholangiocyte states, normalizing SPP1-mediated signaling, and restoring bile-duct-localized transcriptional programs. These findings position H19 as a critical regulator of cholangiocyte-driven pathology and a potential therapeutic target in PSC.

Background Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive liver cancer with a poor prognosis and rapid metastatic potential. Although circular RNAs (circRNAs) have emerged as important regulators in cancer biology, their translational potential and mechanistic contributions to ICC metastasis remain largely unexplored. Methods CircRNA-seq was performed on paired primary and recurrent ICC tissues to identify the differentially expressed circRNAs. Mass spectrometry and functional assays were used to characterize the novel protein encoded by circPICALM.The molecular mechanisms and biological functions of circPICALM and its encoded proteins were evaluated using in vitro and in vivo models, respectively. Results CircPICALM is significantly upregulated in recurrent ICC tumors and is associated with poor patient prognosis. Its biogenesis and expression are regulated by N6-methyladenosine (m6A) modifications within the introns flanking the circulating exons, facilitated by the m6A reader protein YTHDC1. Additionally, the RNA-binding protein, DEAD-box helicase 3 (DDX3), promotes circPICALM accumulation. Importantly, circPICALM encodes a novel protein, circPICALM-219aa, that drives ICC metastasis. Mechanistically, circPICALM-219aa disrupted the inhibitory interaction between SOCS3 and STAT3 by directly binding to both proteins. This interference alleviates SOCS3-mediated suppression of JAK activity and enhances IL-6/JAK/STAT3 signaling. Silencing circPICALM-219aa expression significantly suppressed the activation of this signaling pathway and metastatic potential. Conclusions This study identified circPICALM-219aa as a novel oncoprotein translated from an m6A-modified circRNA, and a key driver of ICC metastasis. Our findings uncover a previously unrecognized mechanism of m6A-mediated circRNA translation in ICC and highlight circPICALM-219aa as a promising therapeutic target for improving patient outcomes.

Hepatocellular carcinoma (HCC) commonly arises in metabolic dysfunction-associated steatohepatitis (MASH), alcohol-related liver disease (ALD), and metabolic dysfunction-associated ALD (MetALD), yet how zonal metabolic programs govern tumor lineage and immune responses remains unclear. Here, using complementary murine models of steatohepatitis-associated hepatocarcinogenesis, we show that CTNNB1-mutant MASH-HCC originates from periportal and midlobular hepatocytes through perivenous reprogramming. This transition is characterized by β-catenin activation, loss of periportal metabolic functions, and induction of the immunosuppressive IDO1-kynurenine-AhR axis. In contrast, ethanol exposure suppresses perivenous xenobiotic programs, destabilizes the β-catenin/AhR/CAR axis, and increases tumor heterogeneity by generating both progenitor/biliary- and hepatocyte-derived MetALD-HCC that remain sensitive to anti-programmed death-1 (aPD1) therapy. Pharmacologic AhR inhibition or hepatocyte-specific β-catenin deletion reduces MASH-HCC burden and restores sensitivity to aPD1 treatment. Together, these findings identify AhR as a central mediator of β-catenin-driven tumor immunosuppression and a potential therapeutic target in CTNNB1-mutant HCC, highlighting context-dependent mechanisms of immune escape in alcohol-associated HCC. Alcohol consumption and high fat diet can drive liver cancer through distinct pathways. Here, the authors characterize three murine models of steatohepatitis-associated hepatocarcinogenesis that recapitulate metabolic dysfunction-associated steatohepatitis (MASH), alcohol-related liver disease (ALD), and their overlapped condition, MetALD, showing that alcohol reshapes liver zonal plasticity and β-catenin-AhR signaling to alter tumor origin and increase immunotherapy sensitivity.

Abstract Background Despite recent advances, Primary Sclerosing Cholangitis (PSC)-a chronic obstructive biliary disease-still lacks effective therapies to prevent disease progression or the need for liver transplantation. Moreover, up to 30% of transplant recipients experience recurrence. Long non-coding RNA H19 (H19) has been implicated in promoting PSC progression, yet the cellular and molecular mechanisms underlying its pathogenic role remain incompletely understood. Results Liver tissues from age- and sex-matched wild type (WT), H19 knockout (H19KO), Mdr2 knockout (Mdr2KO), and double-knockout (DKO; Mdr2KO/H19KO) mice were analyzed using single-nucleus RNA sequencing (snRNAseq) and GeoMx spatial transcriptomics to define the cell type and spatially specific effects of H19 deletion in cholestatic liver injury. Machine learning models were built to develop cell-type-specific gene prediction signatures and cross-validated using the human dataset GSE243981. A disease-associated cholangiocyte subcluster that increased in Mdr2KO but was markedly reduced in DKO mice was identified. SPP1 signaling was significantly dysregulated in cholestatic liver injury and mitigated following H19 deletion. Translationally conserved healthy (Clu, Spp1) and diseased (Csmd1, Slco3a1, Cftr) cholangiocyte markers were identified. When validated in a human patient dataset (GSE243981), our machine-learning prediction models achieved AUC values > 0.869. Finally, spatial analyses demonstrated that the mitigation of disease-associated gene expression following H19 deletion was specifically restricted to hepatocytes within the bile duct region. Conclusions H19 deletion mitigates cholestatic injury by suppressing pathogenic cholangiocyte states, normalizing Spp1-mediated signaling, and shifting transcriptional programs specifically within the periductal niche. Furthermore, our machine-learning signatures demonstrate robust cross-species translation and may benefit future post-transplant analytics and disease recurrence predictions.

Abstract Pancreatic cancer (PC) remains one of the most lethal malignancies worldwide, characterized by dense desmoplastic stroma, profound immunosuppression, and poor responsiveness to conventional therapies. Accumulating evidence positions the intratumoral microbiome, encompassing bacteria as well as understudied fungal and viral components, as a critical regulator of PC pathogenesis, tumor microenvironment (TME) remodeling, and therapeutic efficacy. In this review, we synthesize the current understanding of the pancreatic intratumoral microbiome, including its distinct composition relative to normal pancreatic tissue, gut microbiota, and oral microbiota. The PC intratumoral microbiome is characterized by enrichment of validated pro‐tumorigenic drivers (e.g., Pseudomonas, Bacteroides species) alongside protective taxa (e.g., Lactobacillus species). These taxonomic profiles vary significantly based on disease stage, patient demographics, and treatment history. We dissected the mechanisms by which microbes and their derivatives drive PC initiation and progression, including immune suppression through modulation of myeloid‐derived suppressor cells and regulatory T cells, metabolic reprogramming involving short‐chain fatty acids and the tryptophan metabolite 3‐indoleacetic acid (3‐IAA), and stromal remodeling via activation of cancer‐associated fibroblasts. The influence of the microbiome on therapeutic response is examined, with particular emphasis on microbial‐mediated mechanisms that undermine treatment efficacy. We further summarize diagnostic and prognostic biomarkers, including salivary microbial signatures with moderate sensitivity and specificity, circulating microbial DNA, and composite microbial risk scores that integrate multiple bacterial species to predict survival. Therapeutic strategies targeting the microbiome are discussed, distinguishing clinically translatable approaches from experimental concepts requiring further validation. Finally, we outline challenges in standardizing profiling methods, validating microbial signatures across diverse populations, and translating preclinical insights to clinical practice, emphasizing the need for multi‐omics integration and interdisciplinary collaboration to advance microbiome‐driven PC management.

Metabolic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), alcohol-associated liver disease (ALD), and the metabolic dysfunction and alcohol-associated liver disease (MetALD) phenotype, are major causes of fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Circular RNAs (circRNAs) are covalently closed RNA molecules generated mainly by back-splicing or, in selected cases, by read-through transcription followed by circularization. Their resistance to exonuclease degradation, context-specific expression, and enrichment in extracellular vesicles (EVs) make them attractive candidates for mechanistic discovery, liquid-biopsy biomarker development, and RNA-targeted therapy. Here, we review current evidence on circRNA biogenesis and mechanisms of action in metabolic liver diseases, with emphasis on MASLD/MASH, ALD/MetALD-related fibrosis, cirrhosis, and HCC. In MASLD/MASH, circRNAs have been implicated in nutrient-response signaling, lipid handling, mitochondrial stress, autophagy, inflammatory amplification, macrophage polarization, and hepatic stellate cell (HSC) activation. In ALD and MetALD, direct MetALD-specific circRNA evidence remains limited; available data mainly support ethanol-induced hepatic circRNA remodeling, macrophage inflammatory responses, and apoptosis. Fibrosis-associated circRNAs connect inflammatory and transforming growth factor β (TGF-β)-centered signaling to extracellular matrix remodeling, whereas HCC currently provides the strongest near-term biomarker opportunity, particularly through circulating and exosomal circRNAs that may complement alpha-fetoprotein (AFP). However, most candidate mechanisms and biomarker signatures remain discovery-stage findings that require circRNA-specific validation, etiology-specific cohorts, clinically relevant comparator groups, and standardized assays. Overall, circRNAs should be viewed not only as microRNA (miRNA) sponges but also as multifaceted regulators of RNA-binding protein (RBP) interactions, organelle function, translation, EV-mediated signaling, disease endotyping, and therapeutic response. Clinical translation will require standardized detection methods, multicenter longitudinal validation, pharmacokinetic and pharmacodynamic evaluation, and liver-cell-specific delivery strategies.