Gastroenterology

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.

Transient depletion of the transcription elongation factor SPT6 in the keratinocyte has been recently shown to inhibit epidermal differentiation and stratification; instead, they transdifferentiate into a gut-like lineage. We show here that this phenomenon of transdifferentiation recapitulates Barretts metaplasia, the only human pathophysiologic condition in which a stratified squamous epithelium that is injured due to chronic acid reflux is trans-committed into an intestinal fate. The evidence we present here not only lend support to the notion that the keratinocytes are the cell of origin of Barretts metaplasia, but also provide mechanistic insights linking transient acid exposure, downregulation of SPT6, stalled transcription of the master regulator of epidermal fate TP63, loss of epidermal fate and metaplastic progression. Because Barretts metaplasia in the esophagus (BE) is a pre-neoplastic condition with no preclinical human models, these findings have a profound impact on the modeling Barretts metaplasia-in-a-dish. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/442930v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@8ce08aorg.highwire.dtl.DTLVardef@e98c83org.highwire.dtl.DTLVardef@107f521org.highwire.dtl.DTLVardef@16e7523_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIKeratinocytes transdifferentiate into the gut lineage upon depletion of SPT6 C_LIO_LISuch transdifferentiation recapitulates Barretts metaplasia, not the healthy gut C_LIO_LIAcid downregulates SPT6, which derails the expression and functions of TP63 C_LIO_LISuch downregulation precedes the metaplasia-dysplasia-neoplasia cascade C_LI

Background and AimsIn inflammatory bowel disease, protein misfolding in the endoplasmic reticulum (ER) potentiates epithelial barrier dysfunction and impairs mucosal healing. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, acts as a chemical chaperone to reduce protein aggregation and colitis severity in preclinical models. We conducted an open label trial evaluating oral TUDCA as therapy in patients with active ulcerative colitis (UC). MethodsPatients with moderate-to-severely active UC (Mayo score [&ge;]6, endoscopic subscore [&ge;]1) received oral TUDCA at 1.75 or 2 g/day for 6 weeks. Exclusion criteria included known hepatic disorders or change in UC therapy within 60 days. Clinical disease activity questionnaires, endoscopy with biopsy, blood, and stool were collected at enrollment and after 6 weeks. The primary outcome measure was change in ER stress markers while safety, tolerability and change in UC disease activity were secondary outcomes. ResultsThirteen participants completed the study with eleven evaluable for clinical response. TUDCA was well-tolerated with transient dyspepsia being the most common side effect. Mucosal biopsies revealed significant reductions in ER stress and inflammation as well as an increase in markers of epithelial restitution. Clinical, endoscopic, and histologic disease activity were significantly improved at week 6 (mean total Mayo Score: 9 to 4.5, p<0.001). ConclusionsSix weeks of oral TUDCA treatment was well-tolerated in patients with active ulcerative colitis and promoted mucosal healing, lessened ER stress, and reduced clinical disease activity. A randomized controlled trial of adjunctive TUDCA therapy in patients with UC is warranted. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/25322684v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@bdccccorg.highwire.dtl.DTLVardef@d0843borg.highwire.dtl.DTLVardef@19966f0org.highwire.dtl.DTLVardef@352be6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Primary sclerosing cholangitis (PSC) is a chronic progressing cholestatic disease that often co-occurs with inflammatory bowel disease (PSC-IBD). PSC-IBD affecting the colon (PSC-UC) is likened clinically to ulcerative colitis (UC), however differences include a right colon dominance, less severe inflammatory presentation and a greater lifetime risk of colorectal cancer. To understand the basis of clinical differences, we combine single-cell mRNA and antigen receptor sequencing, 16S ribosomal DNA analysis and spatial transcriptomics on biopsies from multiple colon regions of both PSC-UC and UC patients in remission or at the time of relapse. We discover disease-specific cell and microbial profiles between these cohorts, highlighting a distinct landscape in the right colon of PSC-UC patients and an epithelial-endothelial cell state that may contribute to intestinal permeability in UC. We show the expansion of an activated mast cell state in both diseases during flare, and demonstrate the requirement of TMEM176B in sustaining this activated state. Together this work demonstrates that PSC-UC and UC are distinct diseases with common cell mechanisms during inflammation, providing cellular and microbial insights to improve treatment of both patient cohorts.

Cellular plasticity and transitional cellular states are crucial for tissue regeneration across multiple organs. In the pancreas, oncogenic Kras hijacks this program, acting on tissue-specific enhancers to prevent the resolution of acinar-to-ductal metaplasia (ADM) and lock regeneration into a pro-inflammatory state that progresses to cancer. Enhancer transcription, an early event during cellular state transitions, can generate stable enhancer-associated long noncoding RNAs (lncRNAs) positioned near key transcription factors and chromatin contact boundaries, often enriched for disease-associated variants. While enhancer-associated lncRNAs have been implicated in transcriptional regulation and genome organization, their role in pancreas regeneration and cancer initiation has remained unexplored. In this study, we investigated the expression of epithelial long noncoding RNAs (lncRNAs) and their target genes in PDAC precursor lesion formation. We focus on lncRNAs transcribed from enhancer elements near cell identity transcription factors. We demonstrate that LINC00673, expressed from a Sox9-associated super-enhancer during pancreatic development, is reactivated in PDAC. Conditional deletion of LINC00673 in the murine pancreatic epithelium accelerates resolution of ADM and significantly impairs PDAC initiation. Notably, LINC00673 harbors a variant associated with risk of developing PDAC. Our study identifies a critical function of LINC00673 in regulating both cell-autonomous and non-cell-autonomous processes during pancreas regeneration and Kras-driven cancer initiation. Furthermore, we highlight a previously unrecognized role of transcribed super-enhancers in facilitating long-range gene regulation during pancreatic cancer initiation. These findings reveal a novel regulatory layer linking developmental enhancer activity, cellular plasticity and pancreatic disease progression. TeaserLong noncoding RNAs from developmental enhancers play a role in long-range gene regulation with crucial biological impacts in development and cancer. Grant supportThis project has been supported by the Danish Cancer Society (R302-A17481). LA is supported by core funding of the Biotech Research and Innovation Center, the Danish Cancer Society (R302-A17481, R322-A17.350), The Novo Nordisk Foundation (NNF21OC0070884) and The Innovation Fund (Eurostars 2807). The Novo Nordisk Foundation Center for Stem Cell Biology was supported by Novo Nordisk Foundation grants NNF17CC0027852.

Background and AimsArtificial intelligence (AI) is increasingly applied to histological assessment in inflammatory bowel disease (IBD), but most approaches quantify features in isolation and ignore their anatomical location within the mucosa. We developed and validated PAIR-IBD (Perspectum AI Reading in IBD), an AI system that quantifies inflammatory cell populations, crypt injury, and epithelial damage within defined mucosal compartments to distinguish active disease, remission, and equivocal cases in ulcerative colitis (UC). MethodsA deep learning ensemble was trained on three IBD biopsy datasets to identify lymphocytes, neutrophils, eosinophils, and plasma cells, and to segment crypts, lamina propria (LP), and muscularis mucosae. Inflammatory cell densities and crypt injury metrics (mucin depletion, solidity, roughness, branching, and abscess formation) were quantified. PAIR-IBD outputs were compared between histologically active and remissive UC, evaluated in inconclusive cases, and correlated with manual pathology grading. ResultsNeutrophil density increased 3.5-fold in the LP and 15-fold within crypts in active UC (p<0.0001). Eosinophil density doubled and LP lymphocytes increased 1.4-fold. Active UC showed increased mucin depletion, crypt branching, and crypt abscesses, with reduced crypt solidity (p<0.0001 for all). PAIR-IBD metrics correlated with manual inflammatory and crypt injury scores (rs=0.23-0.72) and global indices (rs=0.27-0.65). Up to 89% of inconclusive cases aligned with remission-like profiles based on multiple independent AI metrics. ConclusionPAIR-IBD provides spatially resolved, quantitative assessment of inflammation and epithelial injury in UC, improving disease stratification and resolution of equivocal histology, with potential to support scoring consensus and improve accuracy of histological endpoints in clinical trials.

Background and AimsPrimary sclerosing cholangitis (PSC) is a liver disease without medical therapy that significantly increases the risk of malignancies, acute cholangitis and cirrhosis. Approximately 2-7% of patients with inflammatory bowel disease (IBD) develop PSC. No drug has been shown to prevent de novo PSC in patients with IBD. Statin use, however, has been associated with increased liver transplant-free survival. Yet, the impact of statins on development of new PSC among those with IBD is unknown. MethodsWe conducted a retrospective cohort study of patients diagnosed with IBD with and without statin exposure using a large, representative national database. Patients were followed from the date of diagnosis of IBD. Patient demographics, co-morbidities, medications, and type of IBD were extracted. Unmatched and propensity score-matched Cox regression analyses were performed. ResultsOur analysis included 33,813 patients with IBD of whom 8,813 were exposed to statins. PSC developed in 181 patients over a median follow-up of 3.7 years. Only nine patients (0.1%) who were exposed to statins developed PSC compared to 173 (0.69%) in the non-exposed population. In propensity score-matched analysis, statin therapy was associated with an 86% lower risk of developing PSC (HR 0.14; 95% CI 0.06-0.33, p<0.001). The findings were consistent when accounting for unmeasured confounders (E-value) in sensitivity analyses, including stratification by age group, duration of statin use, and type of statin. ConclusionIn a propensity score-matched analysis, statin use was associated with an 86% risk reduction in new PSC diagnosis among patients with known IBD, suggesting a potential benefit as a prophylactic agent. These findings warrant prospective validation.

Neuropod cells are a newly discovered type of enteroendocrine cell (EEC) that connect the gut and brain functionally into one circuit. In the mouse colon, neuropod cells express various peptide hormones, such as Pyy and Glp1, presynaptic proteins, and make synaptic contacts with sensory neurons. While their function is not fully elucidated, they play a significant role in relaying signals to the brainstem upon sensing nutrients and microbial factors in the gut lumen. Their occurrence in the human gastrointestinal tract is currently not established. In this study, we showed that PYY-expressing cells (L-cells) in the human colon exhibit characteristics of neuropod cells. Utilizing advanced histological methods and confocal microscopy we found that L-cells of the healthy human colon possess distinctive morphology, express synaptic proteins, and exist proximal to sensory neurons. This agrees with our meta-analysis of single-cell RNA sequencing (scRNA-Seq) data that showed that human colonic L-cells express pre- and post-synaptic genes. As inflammatory conditions could affect colonic neuropod cells, we aimed to profile the phenotypic and transcriptional changes of neuropod cells both in human and murine colon in Inflammatory Bowel Disease (IBD) and experimental colitis, respectively. In human IBD, the abundance of neuropod cells and spatial proximity to sensory neurons were decreased in the colon of ulcerative colitis (UC) and Crohns disease (CD) patients. L-cells in IBD patients display genes related to innate and adaptive immunity, including antigen presentation genes suggesting a role in immune regulation. We further confirmed the effects of intestinal inflammation in neuropod cells by utilizing the DSS mouse model of colitis, where we showed that acute DSS colitis induced spatially distinct effects on the abundance of neuropod cells and impaired the synaptic connection with sensory neurons. Overall, these findings extend early murine characterizations to the human system and highlight the complex interactions between colonic neuropod cells and the enteric nervous and immune systems during inflammatory diseases.

Disorders of gut-brain interaction (DGBIs), formerly known as functional gastrointestinal disorders, are extremely common and historically difficult to manage. This is largely because their cellular and molecular mechanisms have remained poorly understood and understudied. One approach to unravel the molecular underpinnings of complex disorders such as DGBIs is performing genome wide association studies (GWASs). However, due to the heterogenous and non-specific nature of GI symptoms, it has been difficult to accurately classify cases and controls. Thus, to perform reliable studies, we need to access large patient populations which has been difficult to date. Here, we leveraged the UK Biobank (UKBB) database, containing genetic and medical record data of over half a million individuals, to perform GWAS for five DGBI categories: functional chest pain, functional diarrhea, functional dyspepsia, functional dysphagia, and functional fecal incontinence. By applying strict inclusion and exclusion criteria, we resolved patient populations and identified genes significantly associated with each condition. Leveraging multiple human single-cell RNA-sequencing datasets, we found that the disease associated genes were highly expressed in enteric neurons, which innervate and control GI functions. Further expression and association testing-based analyses revealed specific enteric neuron subtypes consistently linked with each DGBI. Furthermore, protein-protein interaction analysis of each of the disease associated genes revealed protein networks specific to each DGBI, including hedgehog signaling for functional chest pain and neuronal function and neurotransmission for functional diarrhea and functional dyspepsia. Finally, through retrospective medical record analysis we found that drugs that inhibit these networks are associated with an increased disease risk, including serine/threonine kinase 32B drugs for functional chest pain, solute carrier organic anion transporter family member 4C1, mitogen-activated protein kinase 6, and dual serine/threonine and tyrosine protein kinase drugs for functional dyspepsia, and serotonin transporter drugs for functional diarrhea. This study presents a robust strategy for uncovering the tissues, cell types, and genes involved in DGBIs, presenting novel predictions of the mechanisms underlying these historically intractable and poorly understood diseases.

Pediatric Inflammatory Bowel Disease (IBD) remains challenging to treat and difficult to prognosticate. Although multiple immune cell types coordinate pathology in both Crohns disease (CD) and ulcerative colitis (UC), specifying which cell types and cell states portend better or worse response to major IBD treatment strategies, including anti-TNF therapies (the only FDA-approved therapy for pediatric IBD), remains challenging. Here, we present the results of the PREDICT study, which enrolled 79 treatment-naive pediatric patients at the time of diagnostic endoscopy, enabling a comprehensive transcriptomic, histologic, and serologic analysis of 40 CD patients and 16 UC patients, as well as 23 patients with functional gastrointestinal disorders (FGID) who served as pediatric non-inflamed controls. Leveraging these data, we performed a comprehensive analysis of colonic immunology in each of these clinical conditions. Our results indicate that, within the complex landscape of immune pathology in pediatric CD and UC, there is a coordinated shift across the TH1-to-TH17 immune activation continuum among T cells that is pertinent to anti-TNF response. For CD, this shift defines partial response to anti-TNF treatment. For UC, the landscape is more complex, with both TH17 and TFH biology defining disease, and pre-treatment TH17 biology contributing to anti-TNF treatment resistance. Related to this, polyreactive TCR phenotypes within UC TFH cells are correlated with both germinal center activity and the frequency of IgG1 plasma cells, yet opposed to the TH17 signatures associated with anti-TNF nonresponse. The elucidation of these distinct mechanisms of T cell-dependent disease pathology, treatment response, and TCR polyreactivity suggests a model in which sustained TH17 signaling in CD and baseline TH17 signaling in UC are associated with disease pathogenesis, forming a basis for a generalized understanding of IBD pathogenesis and underscoring the need for endotype-specific approaches to IBD therapy.

Background and AimsBetter biomarkers for prediction of ulcerative colitis (UC) development and prognostication are needed. Anti-integrin v{beta}6 autoantibodies (anti-v{beta}6) have been described in UC patients. Here, we tested for the presence of anti-v{beta}6 antibodies in the pre-clinical phase of UC and studied their association with disease-related outcomes after diagnosis. MethodsAnti-v{beta}6 were measured in 4 longitudinal serum samples collected from 82 subjects who later developed UC and 82 matched controls from a Department of Defense pre-clinical cohort (PREDICTS). In a distinct, external validation cohort (GEM), we tested 12 pre-UC subjects and 49 matched controls. Further, anti-v{beta}6 were measured in 2 incident UC cohorts (COMPASS n=55 and OSCCAR n=104) and associations between anti-v{beta}6 and UC-related outcomes were defined using Cox proportional-hazards model. ResultsAnti-v{beta}6 were significantly higher among individuals who developed UC compared to controls up to 10 years before diagnosis in PREDICTS. The anti-v{beta}6 seropositivity was 12.2% 10 years before diagnosis and increased to 52.4% at the time of diagnosis in subjects who developed UC compared with 2.7% in controls across the 4 timepoints. Anti-v{beta}6 predicted UC development with an AUC of at least 0.8 up to 10 years before diagnosis. The presence of anti-v{beta}6 in pre-clinical UC samples was validated in the GEM cohort. Finally, high anti-v{beta}6 was associated with a composite of adverse UC-outcomes including hospitalization, disease extension, colectomy, systemic steroid use and/or escalation to biologic therapy in recently diagnosed UC. ConclusionAnti-integrin v{beta}6 auto-antibodies precede the clinical diagnosis of UC by up to 10 years and are associated with adverse UC-related outcomes.

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the United States. This is due in part to the limited availability of effective treatment options for patients, highlighting a significant need for new targets and approaches. Deregulated metabolism is a hallmark feature of PDA that has gained attention as a promising inroad for therapy. The aspartate transaminases (glutamate oxaloacetate transaminases, cytosolic GOT1 and mitochondrial GOT2) have several important metabolic functions, including maintaining energy and redox balance and generating aspartate, an essential building block in protein and nucleotide biosynthesis. Previous studies of GOT proteins in preclinical tumor transplant models have yielded conflicting results regarding the requirement of GOT1 and GOT2 for PDA tumor growth. To assess the role of GOT proteins in tumor development and tumor maintenance, we generated conditional knockout mice for Got1 and Got2 and crossed these into pancreas-specific models. Whereas loss of either Got does not impact pancreas development, double Got1 and Got2 knockout results in markedly reduced pancreas size and cellularity without overtly impacting endocrine or exocrine function. In genetically engineered cancer models, single Got loss does not impact lesion formation, tumor size, animal survival, or the composition of the tumor microenvironment. Identical results were also observed in orthotopic allograft mouse models. Together, these findings add to a growing body of work illustrating the adaptability of metabolism in cancer. They also emphasize the importance of model selection, the use of multiple independent models, and the in vivo context when studying the role of metabolic programs in cancer.

Barretts esophagus containing intestinal metaplasia predisposes to cancer, yet the majority of cases are undiagnosed. The length of a Barretts segment is a key indicator of cancer risk, but measuring it has so far relied on endoscopy, which is expensive and invasive. Cytosponge-TFF3 is a minimally-invasive test that identifies intestinal metaplasia for endoscopic confirmation. We report a machine learning technique to quantify the extent of intestinal metaplasia and predict Barretts segment length from whole-slide image tile counts automatically generated from Cytosponge-TFF3 histology slides. Utilizing data from 529 patients, our segment length prediction model achieves an average validation fold accuracy of 0.84. Applying this algorithm to an independent test set of 162 patients from a screening trial shows a precision of 0.90 for identifying short-segment disease. This advance will enable higher-risk patients to be prioritized for endoscopy while saving more than half of Cytosponge-TFF3-positive patients from endoscopy in the screening setting.

Cell plasticity governs tissue regeneration but can also drive metaplasia, the replacement of one cell type with another. This process increases the risk of cancer development in several tissues, including esophagus. Esophageal metaplasia development partly depends on keratinocyte plasticity, making regulation of esophageal progenitor fate critical. We previously identified Sox9 as instrumental in regulating esophageal cell plasticity following Hedgehog pathway activation. Our current study reveals that Hedgehog indirectly regulates Sox9 by modifying epithelial-stromal communication. This activates TGF-{beta} and BMP pathways in epithelial cells, which synergistically regulate Sox9 and stimulate a transcriptomic program resembling squamo-columnar junction progenitors, which are prone to initiate metaplasia. Importantly, we demonstrate pharmacological modulation of this plasticity in vivo. Indeed, Ibuprofen inhibits Hedgehog-induced Sox9 expression by directly targeting epithelial cells, providing proof of concept for pharmacological intervention in cell plasticity with implications for regenerative medicine and metaplasia treatment.

Reg4+ secretory cells are upregulated in colitis and promote epithelial repair, but their regulation is poorly understood. We show that TNF-TNFR1 signaling controls Reg4+ cell numbers in mouse and human colonoids and in vivo, with TNFR1 deficiency reducing these cells and TNF restoring them dose-dependently. In UC patients and DSS-colitis, REG4+ cells mark regenerating crypts, and persistent REG4, DUOX2, and TNFR1 expression identifies non-responders to anti-TNF therapy. These findings reveal a TNFR1-dependent mechanism regulating Reg4+ cells and suggest potential strategies to improve therapeutic responses in IBD.

BackgroundImmune checkpoint inhibitors (ICIs) are effective for many cancers but often cause immune-related adverse events, particularly gastrointestinal (GI) complications such as colitis. Identifying risk factors for ICI colitis (CIC) could improve patient selection and discover potential mechanisms relevant to idiopathic inflammatory bowel disease (IBD). Prior studies on ICI risk factors have methodological limitations. MethodsWe used electronic health record data from six University of California institutions with 10,260 adults on ICIs. Baseline and time-varying predictors were defined based on prior IBD epidemiology studies, including interactions. LASSO Cox regression was applied to data from UCSF Central Data Warehouse to select the most predictive model, with coefficients estimated using multi-center data. Results and conclusionFourteen significant predictors of CIC were identified. The strongest predictors were concurrent use of antibiotics (HR 2.72, 95% CI: 2.41-3.07) and non-steroid anti-inflammatory drugs (1.50, 1.16-1.94). Notable GI risk factors included gastroesophageal reflux disorder (1.31, 1.14-1.50), other GI disorders (1.32, 1.16-1.50), and disorders of the gallbladder, biliary tract, and pancreas (1.33, 1.15-1.57). High BMI before ICI administration increased risk (per unit 1.07, 1.04-1.09), while higher BMI after ICI administration decreased risk (per unit 0.93, 0.90-0.95). Melanoma, anxiety, and depression also increased risk. GI oncologists should consider ICI colitis in patients with comorbid GI disorders or those using antibiotics or NSAIDs. Baseline fecal calprotectin testing is recommended for these patients. Future studies are needed to explore the underlying mechanisms. Our findings suggest microbiome-and immune system-altering factors play roles in CIC and idiopathic GI disorders.

Colorectal cancer (CRC) remains the third leading cause of cancer death in the United States with an alarming rise among young (<50-years-old) patients.1 Epidemiologically, obesity appears to be a risk factor for CRC.1 Although bariatric surgery has been shown to be associated with decreased risk for most cancers, studies to date on bariatric surgery and CRC continue to yield conflicting results.2 One possible explanation for this seeming irreconcilability is the inherent heterogeneity of CRC with its varied mechanisms. This is likely compounded by the differing bariatric operations currently employed. Here, we sharpen our focus and investigate how the most performed bariatric operation, sleeve gastrectomy (SG), affects colitis-associated CRC. Using a murine model, we found that SG significantly exacerbates both colitis and colitis-associated CRC. Using a germ-free (GF) microbiota transplant model, we found that the post-SG microbiota, when transplanted into GF mice, is capable of independently recapitulating the tumor-promoting phenotype of SG. Our results suggest that the postsurgical microbiome plays a key causal role in the increased risk for CRC after SG. This finding represents the first step in our understanding of this complex relationship that is at the intersection of two rising public health threats.

Human colonoids derived from adult intestinal tissue provide powerful models for epithelial biology but lack several rare or regionally restricted cell types. To overcome this limitation, we engineered doxycycline-inducible lentiviral constructs to drive expression of transcription factors that control lineage specification, including KLF4, POU2F3, SPIB, NEUROG3, and NEUROG3 T2A NKX6-3. Transduction of human colonoids with these constructs revealed that transient KLF4 induction is sufficient to generate TFF3 and WFDC2 expressing goblet cells. Unexpectedly KLF4 also induced GUCA2A positive colonocytes. In contrast, SPIB and POU2F3 were not sufficient to specify M-cells or mature tuft cells respectively, suggesting the requirement for additional permissive cues. Notably, co-expression of NEUROG3 and NKX6-3 induced enteroendocrine subtypes characteristic of the duodenum. These findings establish a set of lentiviral tools for induction of specific epithelial lineages in human colonoids providing a platform for replenishing cell types that are lost or altered in intestinal diseases.

Gastric mucosal homeostasis is maintained by tissue-resident stem and progenitor cells residing in the isthmus region. Following mucosal injury, surviving cells contribute to regeneration, coinciding with characteristic pathological changes such as atrophic gastritis and metaplasia. To comprehensively understand the cellular dynamics involved in this process, we performed single-cell and spatial transcriptomics using newly generated transgenic mice. In human samples and mouse models, loss of gastric chief cells precedes, and even induces, loss of parietal cells during the progression of atrophy and metaplasia, validating the causal relationship underlying the decrease of these two lineages. Single-cell analysis confirmed robust stemness and metaplastic changes in the Muc6-expressing neck lineage following either chief or parietal cell ablation, and lineage-tracing experiments revealed that Muc6-expressing isthmus progenitors serve as a source of metaplasia and regeneration. Mechanistically, mucosal injury recruits IL-1-expressing myeloid cells, which stimulates NRG1 production in stromal fibroblasts, leading to mucosal proliferation and regeneration mediated by Myc activation in isthmus progenitors. These findings highlight the injury-responsible stem cell-like function of Muc6-expressing isthmal progenitors, which play a critical role in mucosal homeostasis and disease progression. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/648856v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@10fc2eaorg.highwire.dtl.DTLVardef@1c5b82corg.highwire.dtl.DTLVardef@1be747dorg.highwire.dtl.DTLVardef@d1d722_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fibrosing cholangiopathies, including biliary atresia and primary sclerosing cholangitis, involve immune-mediated bile duct epithelial injury and hepatic bile acid (BA) retention (cholestasis). Regulatory T-cells (Tregs) can prevent auto-reactive lymphocyte activation, yet the effects of BA on this CD4 lymphocyte subset are unknown. Gene regulatory networks for hepatic CD4 lymphocytes in a murine cholestasis model revealed Tregs are polarized to Th17 during cholestasis. Following bile duct ligation, Stat3 deletion in CD4 lymphocytes preserved hepatic Treg responses. While pharmacological reduction of hepatic BA in MDR2-/- mice prompted Treg expansion and diminished liver injury, this improvement subsided with Treg depletion. A cluster of patients diagnosed with biliary atresia showed both increased hepatic Treg responses and improved 2-year native liver survival, supporting that Tregs might protect against neonatal bile duct obstruction. Together, these findings suggest liver BA determine Treg function and should be considered as a therapeutic target to restore protective hepatic immune responses.