Cellular and Molecular Gastroenterology and Hepatology

Gastrointestinal dysfunction is a prominent feature of neurodevelopmental disorders and contributes significantly to visceral discomfort, but the specific etiology is unclear. In particular, it is unknown which cell types within central, autonomic or enteric nervous systems, gut muscle, epithelium or immune system are the principal drivers of gut dysfunction. We report that the Mecp2 mouse model of Rett syndrome, a neurodevelopmental disorder with profound gut dysmotility, exhibits impaired whole gut transit times and constipation in vivo as well as striking alterations to propagating neurogenic contractions in ex vivo colons. Selective elimination of Mecp2 in neurons, but not glia or smooth muscle, recapitulates these dysmotility phenotypes. Bulk RNA sequencing of colonic external muscle, containing the myenteric compartment of the enteric nervous system, showed significant expression changes in specific neurochemical signaling pathways. Pharmacological modulation of one, the M1 acetylcholine receptor, modified the aberrant neurogenic contractile phenotype in mutant mice and reproduced it in wild-type mice. These results identify disruptions to neuronal muscarinic signaling in the enteric nervous system in a mouse model of Rett syndrome and provide an experimental strategy to identify translationally relevant cellular and molecular pathways underlying gastrointestinal dysmotility in neurodevelopmental disorders.

The role of aberrant glycosylation in pancreatitis remains an under-investigated area of research. Here, we determined that the ST6GAL1 sialyltransferase, which adds 2-6-linked sialic acids to N-glycoproteins, was upregulated in pancreatic tissues from patients with acute (AP) and chronic (CP) pancreatitis, and in mice with experimental AP. Within these tissues, ST6GAL1 was selectively expressed in acinar cells undergoing acinar to ductal metaplasia (ADM), a process whereby injured acinar cells de-differentiate and re-enter the cell cycle to enable tissue repair. To study the functional role of ST6GAL1 in pancreatitis, we used HPNE pancreatic epithelial cells with modulated ST6GAL1 expression, along with pancreatic organoids from mice with transgenic expression of ST6GAL1 (SC mice). In these models, ST6GAL1 activity promoted the activation of EGFR (a well-known ADM-driver), ERK, and AKT. ST6GAL1-expressing cells also displayed an ERK-dependent upregulation of the anti-apoptotic proteins, Mcl-1 and Bcl-xL, and impaired apoptotic signaling by the TNFR1 and Fas death receptors. Unbiased kinomics profiling revealed that ST6GAL1 induced the activation of many receptor tyrosine kinases associated with cell survival and proliferation (e.g., EGFR, PDGFR, MET). Furthermore, ST6GAL1 enhanced the survival and proliferation of cells exposed to stress-inducing conditions such as serum withdrawal. Based on these findings, we hypothesized that the pro-survival phenotype imparted by ST6GAL1 would facilitate tissue healing following a bout of pancreatitis. Accordingly, we induced AP in wild type and SC mice via L-arginine injection and found that SC mice had more rapid and efficient tissue repair, evidenced by accelerated restoration of the acini, diminished acinar apoptosis, and reduced immune cell infiltration. Together, these findings highlight a novel glycosylation-dependent mechanism that drives cell survival, positioning ST6GAL1 as a key adaptive mediator of pancreatic regeneration.

Background: The gut-liver axis is involved in Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD), but the host molecular actors involved in such dialogue have not yet been fully characterized. Objective: We investigated the sex-dependent role of the Constitutive Androstane Receptor (CAR) as a hepatic sensor of microbial signals in MASLD pathophysiology. Design: Male and female Car+/+ and Car-/- mice were fed a western diet (WD) for 4, 14 or 35 weeks. We performed plasma biochemistry, liver histology, bile acid and cholesterol quantification, untargeted multi-omics approach combining liver RNA-sequencing and 16S rRNA sequencing, together with co-housing, fecal microbiota transplantation and analysis of publicly available transcriptomics data in human cohorts. Results: Short-term WD revealed CAR-dependent protection against steatosis, together with perturbations in the transcriptional signatures of multiple transcription factors involved in hepatocyte identity in males. After 14 weeks, CAR deletion increased WD-induced liver damage, inflammation and fibrosis, and disrupted bile acid, cholesterol and gut microbiota homeostasis, in males only. Co-housing and fecal microbiota transplantations revealed that a Car+/+ gut microbiota protected against WD-induced liver damage, while a Car-/- microbiota was reshaped by a Car+/+ host. No CAR-dependent phenotype was observed in females. Finally, in human cohorts, hepatic CAR expression decreased in advanced clinical stages of MASLD. Conclusion: CAR is a sex-dependent protective nuclear receptor against MASLD progression, acting both as a transcriptional regulator of hepatocyte identity and as a mediator of the host-microbiota crosstalk.

Developmental programs establish tissue identity and organization, but how their reactivation contributes to adult tissue responses remains poorly understood. Here, we identify a functional contribution of apolipoprotein A-I (APOA1), which marks a late fetal colonic epithelial state that reappears after injury. Spatial and temporal profiling localized Apoa1 transcription predominantly to epithelial cells at the tips of developing proximal colonic folds. Apoa1 loss delayed fold maturation and altered the abundance and composition of developing enteroendocrine populations, linking this apolipoprotein to mucosal architecture and epithelial differentiation. APOA1 protein closely interacted with WNT5A during development and injury, while Apoa1 deficiency increased Wnt5a expression and altered its epithelial protein distribution. Following colonic injury, single-cell and spatial profiling identified genotype-associated transcriptional changes involving stress responses, barrier and immune functions, and metabolism, with a shared component across males and females. These changes accompanied reduced enteroendocrine representation and increased representation of inflammation-associated macrophages. Finally, regional and compartmental expression analyses, together with hindgut explant perturbation, identified HOXB7 transcription factor as a regulator of developmental Apoa1 expression. Together, these findings establish functional relevance for a component of a reactivated late fetal epithelial program, connecting APOA1 to colonic maturation and the epithelial and inflammatory organization of injured tissue.

The gut microbiome can influence cancer development, but its contribution to cancer predisposition in Li-Fraumeni Syndrome (LFS) remains poorly understood. Here, we investigated whether germline mutant p53 alters the intestinal environment using Trp53R172H/+ mice. Tumour-naive mutant mice exhibited increased intestinal permeability and a pro-inflammatory colonic phenotype, including elevated inflammatory cytokines and enhanced NF-{kappa}B signalling. Untargeted metabolomic profiling revealed a striking alteration in bile acid metabolism, with targeted analysis identifying increases in multiple primary and microbiota-derived secondary bile acids. Notably, these changes occurred despite no detectable differences in overall microbial community composition, suggesting altered host-microbial metabolic output in the absence of broad compositional change. Several faecal bile acids were positively associated with distal tumour burden, with the microbiota-derived secondary bile acid deoxycholic acid (DCA) showing the strongest relationship. Microbiota depletion reduced distal tumour growth in mutant mice and attenuated intestinal barrier dysfunction, colonic inflammation and the altered faecal bile acid profile, including DCA. Conversely, transfer of cell-free faecal filtrate from mutant donors to WT recipients induced colonic inflammation and enhanced distal tumour growth. Together, these findings identify a mutant p53-gut axis in which germline mutant p53 establishes a microbiota-shaped metabolic and inflammatory intestinal environment capable of promoting tumour growth beyond the gut.

The basal-like subtype of pancreatic ductal adenocarcinoma (PDAC) is associated with poor outcomes and increased chemoresistance. Expression of high mobility group AT-hook 2 (HMGA2), a chromatin architectural protein, marks basal PDAC cells and associates with specific features of the tumor microenvironment (TME), including more immunosuppressive cancer associated fibroblasts (CAFs) and fewer cytotoxic CD8+ T cells. Expression of HMGA2 by tumor cells leads to upregulation of the immunosuppressive markers interleukin 6 (IL6) and leucine-rich repeat containing protein 15 (LRRC15) in associated fibroblasts. Mechanistically, HMGA2 in the tumor cell binds the interleukin 1 alpha (IL1) promoter and upregulates transcription, increasing IL1 in the TME, in turn inducing immunosuppressive fibroblast polarization, increasing regulatory T cells, and decreasing CD8+ T cells. Blockade of the IL1 signaling axis disrupts this tumor-driven microenvironment phenotype and combines with inhibitors of oncogenic KRAS to improve tumor control.

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease, and liver fibrosis is the principal determinant of disease progression and clinical outcome. We recently showed that non-apoptotic caspase-8 in hepatocytes is upregulated in MASH and leads to the induction and secretion of meteorin, which promotes hepatic stellate cell (HSC) activation and liver fibrosis. We address here the major mechanistic question arising from this study, namely how non-apoptotic caspase-8 induces pro-fibrotic meteorin in hepatocytes in MASH. Methods: Mechanistic studies were performed using AML12 hepatocytes; primary mouse and human hepatocytes; and diet-induced mouse models of MASH with hepatocyte-specific caspase-8 deletion. YY1 post-translational modifications were identified by LC-MS/MS and investigated using phosphorylation-defective and phosphomimetic YY1 mutants. Genetic and biochemical approaches were used to interrogate the caspase-8 DED-SRC-GSK3b; pathway, and its relevance was assessed in human MASH liver specimens. Results: In vitro mechanistic studies showed that the amino-terminal death effector domain (DED) of caspase-8 activated a SRC-GSK3b signaling cascade that promoted phosphorylation of YY1 at Ser247, resulting in YY1 nuclear localization and Metrn induction. Disruption of the conserved DED RXDLL motif impaired SRC activation, YY1 phosphorylation, and Metrn expression. Hepatocyte YY1 overexpression increased Metrn expression and fibrosis in wild-type MASH mice but not in hepatocyte-caspase-8-deficient mice. In contrast, phosphomimetic YY1-S247D restored these endpoints even when hepatocyte caspase-8 was lacking. Studies in primary mouse and human hepatocytes and human MASH liver specimens supported conservation of this signaling pathway. Conclusions: These findings identify YY1 Ser247 phosphorylation as a critical molecular event linking non-apoptotic hepatocyte caspase-8 signaling to METRN induction and MASH fibrosis and define a previously unrecognized DED-SRC-GSK3b-YY1 signaling axis with potential therapeutic relevance.

Defining molecular patterns underlying pediatric end-stage liver disease and its treatment with liver transplantation requires rich datasets spanning a multitude of diagnoses and transplant outcomes. Here, we describe the spatial molecular landscape of approximately five million liver cell profiles from 98 liver tissue samples. The samples span donor liver, used here as a reference tissue, and end-stage diseases including biliary atresia, progressive familial intrahepatic cholestasis, hepatoblastoma, acute liver failure, and gestational alloimmune liver disease. Post-transplant liver allograft biopsies include those without and with T-cell-mediated and antibody-mediated rejection, mixed pathology, and outcome groups from immunosuppression withdrawal studies. Samples were profiled using the CosMx Human Universal Cell Characterization RNA Panel (1K) and the CosMx Human 6K Discovery Panel (6K). Preliminary exploratory analyses include cellular composition and gene abundance and variability across clinical cohorts. A twelve-gene signature comprising the genes with the highest variance implicates acute-phase response genes (e.g., SAA1) and profibrotic genes (e.g., COL1A1) as a shared link across these otherwise distinct conditions. This injury-repair-remodeling gene network warrants further investigation through higher-resolution, zone-specific spatial mapping to help clarify disease pathogenesis.

Pediatric endoscopy is performed only when clinically indicated, limiting access to healthy duodenal tissue. Biopsies with duodenal no pathologic abnormality (NPA) are often used as controls despite the presence of symptoms or inflammatory disease found elsewhere in the gastrointestinal (GI) tract. We characterized pediatric duodenal NPA tissue across clinical, histologic, cellular, and transcriptomic domains. Methods Archival duodenal NPA biopsies were obtained with clinical metadata and hematoxylin-and-eosin whole-slide images (WSIs). Duodenal mRNA-seq data were analyzed from a subset of patients with duodenal NPA. Clinical metadata and WSIs underwent machine-learning analysis, cell populations were quantified from WSIs, and RNA-seq data underwent differential expression and pathway-enrichment analyses. Results The primary cohort included 195 patients with duodenal NPA. Comparisons between patients with non-duodenal GI disease and those with no GI disease showed differences in inflammatory biomarkers and follow-up utilization. Unsupervised clinical clustering identified three clusters with partial enrichment for IBD with colonic inflammation and Eosinophilic Esophagitis (EoE) with esophageal inflammation. Supervised clinical classification showed modest discrimination. WSI clustering showed limited disease-status discrimination, and cell quantification showed no significant group differences. In the separate RNA-seq cohort of 43 patients, differential-expression and pathway-enrichment analyses identified transcriptional and pathway-level differences between disease-status groups. Conclusions This multi-level characterization indicates that pediatric duodenal NPA tissue should not be treated as a uniform control category. Clinical metadata and transcriptomics revealed clinical and molecular heterogeneity, while histologic and cell analyses showed limited disease-status separation, supporting a refined definition of control tissue.

Intestinal stem and progenitor cells balance proliferation and lineage commitment to maintain epithelial homeostasis and adapt to environmental challenges. Tuft cells are a rare chemosensory lineage that rapidly expands in response to the cytokine IL-13 during helminth infection, yet how epithelial progenitor states influence this response remains incompletely understood. Using genetic mouse models, intestinal organoids, and single-cell approaches, we identify the Ras guanine nucleotide exchange factor RasGRP1 as an epithelial-intrinsic promoter of efficient tuft cell differentiation. Rasgrp1 is expressed in rare crypt epithelial cells distinct from Lgr5-positive stem cells. Once cells entered the tuft cell differentiation trajectory, Rasgrp1 deficiency has little impact on tuft cell maturation, including expression of the Pou2f3 tuft cell transcription factor. Instead, RasGRP1 acts at the progenitor stage, where it restrains EGFR-driven proliferation and supports a transcriptionally heterogeneous transit-amplifying state, including a subset of cells expressing Pou2f3, that is largely lost upon Rasgrp1 deficiency. Consistent with this model, EGFR inhibition restores tuft cell numbers in Rasgrp1-deficient organoids and cooperates with IL-13 to enhance tuft cell differentiation. Rasgrp1-deficient mice exhibit delayed tuft cell hyperplasia and inefficient helminth clearance. Conversely, constitutive epithelial RasGRP1 overexpression, uncoupled from its endogenous regulation, drives spontaneous tuft cell hyperplasia and accelerates parasite clearance, but alters the balance of intestinal epithelial lineages. Together, our findings identify RasGRP1 as a cell-intrinsic regulator that restrains progenitor proliferation and promotes responsiveness to IL-13, thereby coupling growth factor signaling, cell cycle control, and immune-driven epithelial lineage specification.

Background: Inflammatory bowel disease (IBD) has been linked to a two- to eightfold increase in colorectal cancer (CRC) risk. However, particular genes that bridge intestinal dysbiosis and oncogenic transcriptional reprogramming remain unclear. The Hippo/YAP pathway is a major regulator of intestinal epithelial homeostasis whose effector YAP is hyperactivated in CRC, but its transcriptional engagement in IBD mucosa and its link to specific cancer-risk genes has not been thoroughly mapped. Methods: Bulk RNA-seq data were integrated from two independent cohorts: GSE235236 (IBD colon mucosa; UC/CD vs. healthy controls; n = 56) and TCGA-COAD/READ (CRC tumors stratified by YAP activity; n = 592). Differential expression analysis was conducted separately within each cohort using Welch's t-test with Benjamini-Hochberg FDR correction. YAP transcriptional activity was estimated per sample as the mean row-wise Z-score of seven canonical YAP target genes, and TCGA tumors were stratified into Hippo-High (Q4) and Hippo-Low (Q1) groups. Genes showing consistent dysregulation across both cohorts were identified and ranked to define a Dysbiosis-Hippo Response (DHR) module, which was further organized into functional gene archetypes. Results: IBD mucosa showed 2,848 significantly dysregulated genes (FDR < 0.05). Comparison with the CRC cohort identified 120 genes showing concordant dysregulation, forming the DHR module. These genes were significantly enriched in TNF-alpha signaling via NF-kB (adjusted p = 0.006), inflammatory response (adjusted p = 0.013), and Myogenesis (adjusted p = 0.013). DHR gene classification revealed three functional archetypes: (i) innate immune activation (SPI1, OSM, IL11, LILRA2, SLC11A1) reflecting convergent myeloid reprogramming; (ii) epithelial-mesenchymal transition (TWIST1, SNAI1, PRRX2, MMP9) priming invasive potential before malignancy; and (iii) tumor microenvironment immune regulation (TN-FRSF4, TNFRSF18, PDCD1, VASN, SPON2) shaping immune tolerance. Interestingly, the canonical YAP target genes (CTGF, CYR61, ANKRD1, AMOTL2, and FSTL1), sharply upregulated in Hippo-High CRC, were essentially unchanged in IBD mucosa. This signifies that the shared DHR programme operates through non-canonical YAP effectors. And yet, YAP target scores are positively correlated with DHR module expression across IBD samples (Spearman rho = 0.726, p = 2.5 x 10-10). Conclusions: Our findings define a three-archetype DHR module that links gut dysbiosis to YAP-driven oncogenesis at single-gene resolution. The programme shared between IBD and CRC is driven by innate immune activation, EMT priming, and remodeling of the immune microenvironment, not by the canonical CTGF/CYR61 YAP target axis, which appears only at the CRC stage. This gene-level framework provides tractable mechanistic targets for chemoprevention and biomarker development at the inflammation-to-cancer transition.

Treatments for pancreatic ductal adenocarcinoma (PDAC) often fail because of the cancer's ability to adapt its signaling and metabolic pathways to circumvent treatment mechanisms. PDAC hallmarks that promote treatment resistance (increased fatty acid metabolism, survival signaling, desmoplasia, and macropinocytosis) are extensively regulated by heparan sulfate proteoglycans (HSPGs) and their binding partners. The pro-tumor functions of HSPGs are controlled by site-specific cleavage of their heparan sulfate chains by mammalian heparanase; however, cleavage by bacterial heparinase III (HepIII) represses these functions and prevents downstream, pro-tumor signaling. Using tumor-targeting, attenuated Salmonella typhimurium (strain YS1646), we have limited HepIII transgene expression to tumor tissue. Degradation of tumor cell heparan sulfates by HepIII resulted in decreased macropinocytosis, reduced survival signaling, lipid storage depletion, and decreased expression of translational machinery. This tumor-targeting treatment has the potential to improve the efficacy of several approved pancreatic cancer therapies, which is predicted to increase patient survival.

Introduction: Sporadic colorectal cancers (CRCs) form following initiating APC mutations, inducing constitutive {beta}-catenin/TCF signaling. Similarly, the guanylyl cyclase C (GUCY2C) tumor suppressor axis is universally silenced in CRC. While the GUCY2C receptor is retained, loss of its paracrine hormones guanylin (GUCA2A) and uroguanylin (GUCA2B) is among the earliest events in transformation. Mutant APC/{beta}-catenin/TCF signaling driving CRC transcriptionally silences GUCY2C hormone expression while suppressing that oncogenic pathway reconstitutes hormone production opposing tumorigenesis. p38 opposes tumor initiation, and its loss promotes transformation in chemical and genetic mouse models of CRC. In healthy intestine, p38 supports a gradient of epithelial differentiation along the crypt-surface axis. Here, we reveal that p38 links regulation of oncogenic APC/{beta}-catenin/TCF signaling and GUCY2C hormone expression. Methods: Regulation of p38 by APC/{beta}-catenin/TCF signaling was defined in four genetically engineered human CRC cell lines with conditional oncogenic APC/{beta}-catenin/TCF signaling and healthy human colon organoids. Expression of p38 was eliminated from conditional human CRC cell lines by pharmacological inhibition, RNA knockdown, and CRISPR/Cas9 gene editing. Transcriptomic analysis of these cells by RNA sequencing defined the set of genes regulated by APC/{beta}-catenin/TCF signaling controlled through p38. Results: In CRC cell lines, APC/{beta}-catenin/TCF signaling suppressed p38 phosphorylation and activity, whereas inhibition of APC/{beta}-catenin/TCF restored p38 signaling. In the absence of APC/{beta}-catenin/TCF signaling, inhibiting p38 repressed GUCY2C hormone expression. Inhibiting APC/{beta}-catenin/TCF signaling enriched phosphorylated p38 at the GUCA2A promoter. In that context, analysis of the CPTAC proteomic database revealed that phosphorylation of p38 and its upstream kinases MKK3 and MKK4 was reduced in human CRC. Finally, transcriptomic analysis revealed that p38 is a critical signaling node controlling the expression of a substantial subset of APC/{beta}-catenin/TCF-regulated genes in human CRC cells and patient tumors. Conclusions: APC/{beta}-catenin/TCF signaling inhibits p38 phosphorylation and transcriptional activity, suppressing GUCA2A and GUCA2B expression and functionally silencing the retained GUCY2C receptor. These findings identify p38 as a signaling intermediary integrating oncogenic APC/{beta}-catenin/TCF activity to extinction of the GUCY2C tumor-suppressor axis and reveal a broader p38-dependent component of the APC/{beta}-catenin/TCF-regulated transcriptome.

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, in part due to a dense and highly plastic tumor microenvironment dominated by cancer-associated fibroblasts (CAFs). In previous work, we identified a CAF population expressing a neural-associated NES+SOX4+NOTCH1+ program that increased with disease progression. The signals driving this state and the mechanisms regulating it remained unclear. Here, we investigated how tumor-derived signals activate this program and whether it represents a stable fibroblast identity or a reversible state. Using spatial transcriptomic data spanning normal pancreas to invasive carcinoma, we identified EGF, FGF2 and BDNF as candidate tumor-to-fibroblast signals associated with disease progression. Functional experiments in patient-derived CAFs and human fibroblast cell lines showed that EGF and FGF2 are sufficient to induce the NES+SOX4+NOTCH1+ program. Growth factor withdrawal reversed the NES+SOX4+NOTCH1+ program at both the protein and transcriptional levels, indicating that this state is dynamic and environment dependent. Single-cell transcriptomic analysis further revealed a multi-phase response involving early loss of fibroblast-associated features, transient activation of neural-associated genes, and a later stress-associated state. Regulatory analyses identified ETS-family transcription factors as key regulators of the program, while phosphoproteomic profiling and inhibitor studies showed that MAPK/ERK signaling plays a central role in its transcriptional activation. Finally, pan-cancer single-cell analysis showed that this state is enriched in CAFs and increases from normal to tumor tissue across multiple cancer types. Together, these findings define a reversible, growth factor-driven CAF program and identify signaling mechanisms that may provide opportunities to selectively modulate tumor-supportive fibroblast states.

Introduction: Adipose tissue fibrosis has been associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and suboptimal weight loss after bariatric surgery. The objective was to characterize changes in adipose tissue fibrosis and extracellular matrix (ECM) components in adipose tissues from two abdominal fat compartments, subcutaneous and omental, in response to bariatric surgery; and determine the effect of weight loss on the association between adipose tissue fibrosis and MASLD feature changes with weight loss. Methods: Liver and adipose tissue biopsy samples were obtained before and after weight loss from patients who underwent two-stage bariatric surgery. Adipose tissue pericellular fibrosis was evaluated histologically with picrosirius red staining for collagen quantification and normalized to adipocyte count per microscope field. Transcript levels of ECM remodeling and inflammatory genes were measured by RNA sequencing in subcutaneous adipose tissue pre- and post-surgery. Results: At baseline, adipose tissue pericellular fibrosis did not associate with MASLD features, metabolic dysfunction-associated steatohepatitis or hepatic fibrosis in patients with severe obesity. Changes in liver histology after surgery were unrelated to changes in adipose tissue fibrosis. Transcripts for most genes involved in ECM remodeling and inflammation were downregulated in subcutaneous fat one year after bariatric surgery. Omental, but not subcutaneous, adipose tissue fibrosis decreased after surgery when adjusted for cell number (mean {+/-} SD: 0.41 {+/-} 0.12 vs 0.32 {+/-} 0.08 {micro}m2/cell, p<0.005). Higher subcutaneous adipose tissue fibrosis correlated with reduced weight loss (estimate {+/-} SE: -2.04 {+/-} 0.99, p<0.05) and BMI loss (-0.73 {+/-} 0.35, p<0.05), while increased omental adipose tissue fibrosis after weight loss was associated with worsening hepatic fibrosis (0.31 {+/-} 0.14, p<0.05). Conclusion: In individuals with severe obesity undergoing bariatric surgery, adipose tissue fibrosis is poorly correlated with MASLD features. Notably, pericellular fibrosis in subcutaneous abdominal, but not omental, adipose tissue was associated with a reduced weight-loss response to surgery.

Early-life microbial exposures can shape lifelong disease risk, yet how developmental timing influences the host-microbe interaction remains unclear. During neonatal colonization by enterotoxigenic Bacteroides fragilis (ETBF), the metalloprotease toxin BFT enables lamina propria niche entry and remodeling of colonic epithelial cell states. Early-life ETBF exposure expands murine Lgr5+ stem cells, enhances Atoh1+ secretory differentiation, and induces distal colon-specific Wnt hyperactivation. These epithelial alterations coincide with increased formation of precancerous lesions in ApcMin/+ mice. ETBF preferentially forms intracellular bacterial aggregates within these lesions, establishing a persistent reservoir that reinforces epithelial remodeling. Colonization with non-toxigenic B. fragilis during this critical developmental window prevents lesion formation, revealing that developmental context dictates whether microbial colonization imprints protection or predisposition toward colorectal cancer.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a common, clinically heterogeneous condition associated with liver-related and cardiometabolic morbidity. Sex differences in MASLD prevalence and progression are well recognized, but how they extend across the liver and visceral adipose tissue (VAT), a major source of lipid and endocrine signals to the liver, remains unclear. Here, we profiled paired liver and VAT biopsies from women and men with and without MASLD, generating single-nucleus datasets of 34 liver samples and 40 VAT samples, including 32 patient-matched pairs. In liver, women with MASLD showed a higher Kupffer-cell triglyceride-catabolism score (1.30-SD female-male difference; FDR=0.0022), accompanied by directionally consistent hepatocyte and endothelial programs. In VAT, women with MASLD showed higher adipocyte lipid and oxidative metabolism (0.87-1.15 SD; FDR<0.05 across seven pathways) together with macrophage immune activation, whereas men with MASLD showed stronger stromal communication routes. Across matched patients, gene modules linked VAT adipocyte and macrophage states to liver hepatocyte stress and lipid-receipt states. Bulk RNA-seq module scoring and paired liver-VAT analyses in 293 patients supported hepatic stress in both sexes, lower adipocyte signaling in women with increasing steatosis, and cross-tissue adipocyte-hepatocyte coupling (r=0.30; FDR=2.1e-6). Together, these findings identify distinct sex-associated transcriptional states in liver and VAT in MASLD and show that these states are coordinated across both tissues within the same individuals.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects nearly a third of adults worldwide, yet how diet shapes the microbiota's contribution remains unclear. In a diet-induced mouse model varying protein content independently of caloric intake, microbiota depletion ameliorated hepatic steatosis and liver injury under protein- sufficient but not protein-restricted conditions. Caecal and plasma metabolomics implicated microbial phenylalanine catabolism and its host conjugates phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly). PAGln supplementation exacerbated steatosis and liver injury without altering body weight, adiposity or glucose homeostasis. Individual-antibiotic perturbation with metagenomics linked bacterial phenylalanine catabolic capacity to disease severity, and in gnotobiotic mice, genetic disruption of bacterial phenylalanine-to-phenylacetic acid conversion lowered circulating conjugates and attenuated steatosis and fibrosis. In a human cohort, circulating PAGln was selectively elevated in cardiometabolic MASLD. These findings identify dietary protein availability as a modifier of the contribution of microbial phenylalanine metabolism to MASLD and highlight PAGln as a microbiota-derived metabolite that exacerbates hepatic disease.