Gastroenterology

BackgroundIn the COVID-19 pandemic, a number of phase II and III randomized trials were launched to evaluate the effectiveness of camostat, an orally administered TMPRSS2 inhibitor previously approved for other indications, for treating SARS-CoV-2 infections. Owing to the rapidly changing landscape during the pandemic, many of these trials were unable to reach completion. Further, methods for synthesizing data for trials that were launched and not completed were critical. MethodsThis study aimed to consolidate global evidence by identifying placebo-controlled, randomized trials of camostat and analyzing their collective clinical and virologic impact on SARS-CoV-2 through an individual participant data meta-analysis. We harmonized data from the included studies and utilized Bayesian statistical models to assess virologic outcomes (measured by the rate of change in viral shedding) and clinical outcomes (based on the time to the first of two consecutive symptom-free days), adjusting for age and sex. FindingsThe meta-analysis incorporated data from six countries, totaling 431 patients across the studies; 118 patients contributed data for the primary virologic outcome and 240 for the clinical symptom outcome. Camostat did not improve the rate of change in viral load (difference in rate of change = 0.11 Ct value/day higher, 95% credible interval 2.04 lower to 2.23 higher) or time to symptom resolution (hazard ratio = 0.87, 95% credible interval 0.51, 1.55) when compared to placebo. InterpretationIn a meta-analysis prompted by a fast-changing landscape during the pandemic, we jointly synthesized evidence across multiple trials that did not meet their original recruitment goals. Despite its theoretically promising mode of action, camostat did not demonstrate a statistically significant virologic or clinical benefit in treating COVID-19, highlighting the complexity of drug repurposing in emergency health situations. FundingThis work was partially supported by The Lundbeck Foundation, LifeArc, Assistance Publique Hopitaux de Paris, anonymous donors, and awards from the National Institutes of Health. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCamostat mesilate, a therapy widely used in Japan for over two decades to treat pancreatitis and reflux esophagitis, showed promise against SARS-CoV-2 in early laboratory and animal studies. Numerous studies evaluating camostat as a treatment for COVID-19 were launched by autumn of 2020, but later stalled due to emerging treatments that altered the equipoise for placebo-controlled trials. Among the trials that reached publication, findings were mixed. Added value of this studyOur research brings a fresh perspective by comprehensively analyzing both published and previously unseen data from randomized clinical trials on camostat. By pooling data across studies, our analysis provides a more robust assessment of the effectiveness of camostat against viral and clinical outcomes than any single study could offer. Novel analytic approaches, data sharing efforts, and international collaboration during the global health emergency are additionally described. Implications of all the available evidenceAfter thorough analysis, our study concludes that, when considering all available data, camostat does not confer a virologic or clinical advantage in the treatment of COVID-19. This conclusion underscores the importance of pooling global research efforts to build a clearer understanding of potential treatments during health emergencies.

BackgroundA major unmet need in gastric cancer prevention is the lack of biomarkers to predict precancerous lesion progression, and the potential of chromosomal instability (CIN) for this role, particularly regarding early cancer and recurrence, remains unclear. ObjectiveTo assess the predictive role of CIN in lesion progression and postoperative recurrence following endoscopic submucosal dissection (ESD). DesignWe enrolled 106 patients from Taizhou Hospital Affiliated to Wenzhou Medical University (2011-2025) and collected 1,045 temporally continuous pathological samples. Copy number variations were profiled using low-coverage whole-genome sequencing (LC-WGS), and CIN scores were calculated. Associations between CIN and clinical outcomes were analyzed using survival analysis and ROC curves. ResultsCIN was detectable up to two years before gastric cancer diagnosis, with positivity rates increasing alongside lesion severity: 25.5% in intestinal metaplasia, 39.7% in low-grade dysplasia, and 83.0% in gastric cancer. CIN positivity independently predicted lesion progression (hazard ratio [HR] = 2.55, 95% CI: 1.19-5.47, p = 0.016), with the greatest risk for progression to carcinoma (HR = 16.61, p < 0.001). The predictive AUC was 0.81, improving to 0.88 when combined with age. Among 84 patients who underwent ESD, CIN positivity significantly increased recurrence risk (HR = 19.57, 95% CI: 2.59 - 147.61, p = 0.004; AUC = 0.80). Chromosomal arms 7p/q, 8p/q, and 20p/q showed high CIN frequencies, with MYC being the most frequently mutated oncogene. ConclusionCIN represents a reliable biomarker for early prediction of lesion progression and postoperative recurrence, enabling proactive surveillance and precision management of gastric cancer. Single Sentence SummaryChromosomal instability (CIN) acts as a reliable biomarker for predicting the progression of gastric precancerous lesions and postoperative recurrence.

Ulcerative Colitis (UC) is a chronic gastrointestinal condition with high morbidity. While modern medical therapies have revolutionized the care of UC, 10-25% of patients fail medications and still progress to surgery. Thus, developing new treatments is a core problem in UC. T-cells, especially Th17 cells, are strongly linked with UC and are major targets of medications in UC. Tissue-resident memory T-cells (TRM) are a distinct class of T-cells that are highly enriched in the intestine, closely aligned with the microbiota, and are implicated in the pathogenesis of UC. Unlike circulating T-cells, TRM are difficult to target because they do not recirculate. Thus, we focused on cytokines like IL-15 which act as a tissue danger signal and regulate T-cells in situ. We found that the IL15 axis is upregulated in UC and predicts treatment response. IL-15 was redundant for Th17 differentiation but could activate terminally differentiated Th17 cells to promote intestinal inflammation. Finally, in CD4+ TRM from patients with UC, IL-15 upregulated RORC, the master transcription factor for Th17 cells, via a Janus Kinase (JAK)1 pathway. Thus, IL-15 promotes terminally differentiated inflammatory Th17 cells in the intestine raising the possibility that IL-15 may be a target for UC treatments.

Background and AimsEpithelial barrier integrity in the gut must be tightly regulated to allow essential paracellular transport while limiting mucosal exposure to inflammatory microbes and toxins. Chronic inflammation in inflammatory bowel diseases is a major health burden and a key risk factor for colorectal cancer. Here, we identify the Rho-regulated kinase PKN2 as a non-redundant safeguard of epithelial barrier integrity and a candidate tumour suppressor in colorectal cancer. MethodsConditional PKN2 knockout mouse models were used to examine the impact of PKN2 loss on colitis severity and induction of colorectal adenomas. Intestinal organoid models, epithelial barrier assays and bioinformatic approaches explored PKN2 regulation of epithelial function. Human inflammatory bowel disease datasets are analysed for associations with PKN2 expression and signatures associated with compromised barrier integrity. ResultsPKN2 deletion sensitises mice to inflammatory bowel injury and promotes colorectal adenoma formation in a colitis-associated colorectal cancer model. Tumour burden tightly correlates with colitis severity suggesting that enhanced inflammation-driven tissue injury underlies tumour promotion. Mechanistically, PKN2 localises to epithelial tight junctions and is required to stabilise barrier integrity during epithelial injury in mouse and organoid models. Transcriptomic changes associated with epithelial PKN2 loss mimic inflammatory bowel diseases and correlate with diseases severity and therapy response in human datasets. ConclusionsPKN2 is a regulator of gut barrier integrity encoded at genomic loci previously associated with sensitivity to bowel inflammation and tumour suppression in both humans and mice. PKN2 loss recapitulates key features of human colitis and implicates PKN2 as a regulator tight junction integrity that shapes disease severity, treatment response and cancer risk.

BackgroundDysbiosis, an imbalance of the intestinal microbiota in critically ill patients, reportedly contributes to poor outcomes. Controlling the disruption of intestinal homeostasis could be promising tactics for infectious complications in acute pancreatitis. To improve the mortality rate of severe acute pancreatitis with high frequency of infectious complications, it is warranted to elucidate the pathophysiology for development of new treatment strategies. We hypothesized that patients with severe acute pancreatitis would demonstrate gut dysbiosis, leading to an onset of infectious complications and poor outcomes. MethodsWe will conduct a prospective study to compare the sequential changes in intestinal microbiota using 16s RNA metagenomics and metabolomics between patients with severe acute pancreatitis and mild acute pancreatitis. We will enroll adult patients (18 years of age or older) diagnosed with acute pancreatitis and newly admitted to the hospitals for 48 hours or longer. We will exclude patients with inflammatory bowel disease, patients with diarrhea prior to admission, patients who have received antimicrobial agents for more than 1 week in the 2 months prior to admission. We will collect stool and blood samples on day 1 and 6. The primary outcome is changes in various parameters of the intestinal microbiota, protein concentration in stool, and metabolite concentration. The secondary outcomes include relationship between each parameter and short- and long-term prognosis, correlation of each parameter with treatment details and clinical course during ICU stay, and associations among the amount of diarrhea and alpha-diversity parameters, protein concentration in each stool, and metabolite concentration.

BackgroundBarretts esophagus (BE), characterized by specialized intestinal metaplasia (SIM), is the precursor to esophageal adenocarcinoma (EAC). Despite published BE screening guidelines for at-risk individuals, uptake of endoscopic screening remains low. We present 18 months of real-world data on non-endoscopic BE screening using EsoGuard(R) (EG), the first commercially available U.S. molecular biomarker test for this purpose, performed on esophageal cell samples collected with the swallowable EsoCheck(R) (EC) balloon-capsule device. MethodsWe retrospectively analyzed EC performance and EG results in patients tested commercially from January 2023 to June 2024. A subset enrolled in a registry underwent follow-up endoscopy. Multivariable logistic regression was used to evaluate risk factors associated with (1) positive EG results, and (2) confirmed BE (SIM [&ge;]1 cm). ResultsAmong 11,991 tested patients, 11,355 (94.7%) had successful EC cell collection, averaging under 2 minutes with no serious adverse events. EG was positive in 16.6% of patients, with positivity increasing by age; age > 50 years was the strongest individual risk factor for predicting a positive EG result. Among 177 EG-positive registry patients who underwent endoscopy, 59 (33.3%) had SIM, of which 33 met American College of Gastroenterology criteria for BE and 26 had ultra-short SIM (<1 cm). Dysplasia was found in 3 patients: 1 HGD, 1 LGD, and 1 indefinite for dysplasia (IND). ConclusionsWe report here the largest real-world experience of EG and EC to date, demonstrating excellent safety, tolerability, and scalability. EG detects both guideline-recognized and ultra-short SIM, supporting its utility as a non-invasive BE screening tool.

The microbiota plays a pivotal role in gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17a (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. While it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown. Furthermore, it was not clear whether 3-oxoLCA and other immunomodulatory bile acids are associated with gut inflammatory pathologies in humans. Using a high-throughput screen, we identified human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Like 3-oxoLCA, isoLCA suppressed TH17 differentiation by inhibiting ROR{gamma}t (retinoic acid receptor-related orphan nuclear receptor {gamma}t), a key TH17 cell-promoting transcription factor. Levels of both 3-oxoLCA and isoLCA and the 3-hydroxysteroid dehydrogenase (3-HSDH) genes required for their biosynthesis were significantly reduced in patients with inflammatory bowel diseases (IBD). Moreover, levels of these bile acids were inversely correlated with expression of TH17 cell-associated genes. Overall, our data suggest that bacterially produced TH17 cell-inhibitory bile acids may reduce the risk of autoimmune and inflammatory disorders such as IBD.

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.

European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines.

Paneth cells regulate host-microbial homeostasis and defects in autophagy and host defense pathways have been associated with inflammatory bowel diseases (IBD). Genetic variants in TL1A (TNFSF15) and its receptor DR3 (TNFRSF25) have been associated with IBD. TL1A expression is increased in IBD patients, particularly in TL1A risk allele carriers. However, effects of TL1A on Paneth cells, resident microbiota, and development of ileitis remain unknown. TL1A overexpression in mice induces Paneth cell hyperplasia and morphological abnormalities preceding the development of ileitis. In Crohns disease (CD) patients, ileal TL1A expression was associated with abnormal Paneth cell phenotypes. We confirmed direct effects of TL1A on Paneth cells in human iPSC-derived human intestinal organoids and mouse Paneth cell-enriched organoids. Resident microbiota was required for TL1A-mediated Paneth cell dysfunction, and ileitis. Tl1a-tg mice were enriched in short chain fatty acid-producing bacteria and the metabolite acetate. Acetate supplementation in WT or Tl1a-tg mice caused ileal inflammation, suggesting that acetate is sufficient to cause ileitis. DR3-deficiency in Paneth cells resulted in Paneth cell abnormalities and microbiome composition changes. Our findings provide a mechanistic link between overexpression of TL1A in CD patients, Paneth cell dysfunction, and enrichment of acetate-producing bacteria and acetate that promotes ileal inflammation. Brief SummaryOverexpression of TL1A drives Paneth cell dysfunction in Crohns Disease and mice leading to microbial and metabolomic changes that promote small bowel inflammation.

BackgroundIntraductal papillary mucinous neoplasms (IPMNs) are clinically detectable precursors of pancreatic adenocarcinoma, yet the mechanisms initiating their development remain poorly defined. Although KRAS mutations are highly frequent in human IPMNs, KRAS activation in pancreatic ductal cells alone fails to recapitulate IPMN development in murine models, indicating that additional tumor-suppressive mechanisms must be overcome. ObjectiveThe objective was to determine whether loss of the transcription factor HNF1B predisposes to initiation of IPMN. DesignWe assessed HNF1B nuclear expression and promoter methylation in resected human IPMN specimens. To model IPMN initiation, we generated mice with ductal-specific inactivation of Hnf1b, alone or combined with KRASG12D. Ductal organoids and RNA-sequencing were used to investigate molecular mechanisms. Transcriptomic analyses were also performed on human IPMN surgical specimens. MRI from germline HNF1B mutation/deletion carriers was re-evaluated for IPMN prevalence. ResultsHuman IPMNs showed loss of HNF1B by immunochemistry, with enrichment to promoter methylation that increased with dysplasia grade. The KHC model recapitulated the key features of IPMN development including ductal dilation, high proliferation, papillary architecture and mucin production. Loss of Hnf1b together with Kras activation induced loss of primary cilia, cellular reprogramming and engaged oncogenic YAP and Wnt/{beta}-catenin signaling, similar to human IPMNs. Moreover, germline HNF1B carriers exhibited a markedly increased prevalence of branch-duct IPMN. ConclusionHNF1B functions as a tumor-suppressive gatekeeper of pancreatic ductal cells. These findings highlight HNF1B inactivation as a potential biomarker and therapeutic entry point for early interception of IPMN-driven pancreatic cancer. They also have implications for the surveillance of HNF1B-syndrome.

Background/AimsIntrahepatic cholangiocarcinoma (iCCA) represents an unmet clinical need due to its increasing incidence, aggressive biology, and limited treatment options. The extremely low-response rates to current systemic regimens and the emergence of adaptive resistance to targeted therapies underscore the urgent need for alternative therapeutic strategies. Given that the lineage-defining transcription factors SOX9 and YAP1 are central regulators of cholangiocyte and iCCA identity, we investigated their functional roles as potential therapeutic vulnerabilities across multiple preclinical models. MethodsPatient tissue-microarray (TMA) analysis, Sleeping-Beauty hydrodynamic tail vein injection-based iCCA models, and Cre-mediated inducible gene deletion systems were used to investigate the roles of Sox9 and Yap1. Deep-learning-based prediction, RNA-seq, ChIP-seq and immunohistochemistry analyses were performed to delineate transcriptional networks and downstream effectors associated with SOX9/YAP1 signaling. ResultsDual deletion of Sox9 and Yap1 effectively eradicated advanced iCCA while preserving intrahepatic bile ducts, regardless of oncogenic drivers. Mechanistically, SOX9 and YAP1 transcriptionally compensated for each other when one was absent, and ILF2, MGAT5, and WWTR1 were identified as key downstream effectors mediating this compensatory mechanism. Loss of Ilf2, Mgat5, or Taz suppressed iCCA, whereas overexpression of Ilf2 or Taz following Sox9/Yap1 co-deletion restored tumor development, indicating that ILF2 or TAZ can functionally substitute for YAP1 and SOX9 in sustaining iCCA. ConclusionsCo-targeting SOX9 and YAP1 offers a promising and safe broad-spectrum preventive/therapeutic approach for iCCA, potentially overcoming resistance to YAP1 inhibition. The adaptive resistance mechanism identified may extend to other malignancies, providing insights for addressing the advanced resistant to YAP1-TEAD-directed therapies.

OBJECTIVEH. pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbor a nearly two-fold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking. DESIGNWe infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterized and evaluated in germ-free mice and via stool transfer experiments. RESULTSH. pylori infection accelerated tumor development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterized by a reduction in regulatory T-cells and proinflammatory T-cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signaling and a loss of goblet cells, changes that have been shown to contribute - in combination with pro-inflammatory and mucus degrading microbial signatures - to tumor development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalized the tumor incidence to the level of uninfected controls. CONCLUSIONSOur studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.

Metastatic pancreatic ductal adenocarcinoma (PDAC) remains deadly, with minimal improvement in prognosis over the past 20 years despite expansion of our chemotherapeutic arsenal. The complex tumor microenvironment (TME) of PDAC in the advanced stage, which often accompany clinical diagnosis, likely contributes to the limited efficacy of current standard of care chemotherapy. Informed by mechanistic preclinical studies, we evaluated the impact of inhibition of Hedgehog (Hh) signaling to prime PDAC TME and leverage anti-tumor efficacy of Gemcitabine plus nab-Paclitaxel (GnP) together with anti-CTLA-4 immune check point inhibitor (ICI, zalifrelimab). Hh inhibition using NLM-001 (an oral small molecule inhibitor of Smo) aimed to polarize the PDAC TME, including cancer associated fibroblasts (CAFs) and intratumoral immune profile, and to foster an immunosuppressive milieu that engages ICI. In this Phase 1b/2, open label, single arm study, patients with metastatic PDAC received standard GnP every 28-day cycles. In addition, NLM-001 was given at 800 mg daily on days -4 to -1 and days 10 to 13 of the GnP cycles 1 to 3, 6 to 8, 11 to 13 onwards (3 cycles on, followed by 2 rest cycles). Anti CTLA-4 inhibitor, zalifrelimab, was administered at 1mg/kg on day 15 of cycle 1 and every 6 weeks thereafter. The primary end point was to assess efficacy by objective response rate (ORR) as per RECIST v1.1. Treatment was overall well tolerated in the 28 patients enrolled. Most frequent grade 3-4 adverse events (AEs) were neutropenia (46.4%), asthenia (21.4%), and neurotoxicity (14.3%). No patient discontinued treatment due to toxicity. ORR was 50% [95% CI, 29.1-70.9] and disease control rate was 95.5% [95% CI, 86.8 - 100.0]. Median progression-free survival (PFS) was 7.3 months 95.5% [95% CI, 5.564 - 9.041] and median overall survival (OS) was 11.5 months [95% CI, 10.23 -12.73]; 1-year PFS was 18.2% [95% CI, 2.1 - 34.3] and 1-year OS was 50% [95% CI, 29.0 - 710]. Patients who achieved ctDNA clearance at cycle 4 had a significant better PFS (10.7 vs 6.0 months; p<0.0001). Paired biopsies immunolabeling and spatial transcriptomic analyses showed polarization of the TME, with increased CD4+ and CD8+ T cells infiltration, down trending Tregs, and decreased SMA/FAP ratio. Hedgehog inhibitor NLM-001 in combination with gemcitabine/nab-paclitaxel and zalifrelimab was safe and well tolerated and showed encouraging objective responses in the first line treatment of advanced PDAC. Clinical Trial RegistrationEudraCT: 2020-004932-52; NCT04827953.

Background and aimsTransfer RNA (tRNA) modifications determine translation fidelity and efficiency. It occurs through the action of specific enzymes that modify the nucleotides within the tRNA molecule. Our previous study demonstrated tRNA modopathies and altered queuine-related metabolites in inflammatory bowel diseases. Queuine tRNA-ribosyltransferase catalytic subunit 1 (QTRT1) and QTRT 2 co-localize in mitochondria and form a heterodimeric TGT participating in tRNA Queuosine (tRNA-Q) modification. Human body acquires Queuine/Vitamin Q from intestinal microbiota or from diet. However, the roles of tRNA-Q modifications in the maintenance of intestinal mitochondrial homeostasis and microbiome are still unclear. MethodsWe used publicly available human IBD datasets, QTRT1 knockout (KO) mice, QTRT1 intestinal epithelial conditional KO (QTRT1{Delta}IEC) mice, cultured cell lines with QTRT1-specific siRNA, and organoids from patients with IBD to investigate the mechanism of tRNA-Q modifications in intestinal mitochondrial homeostasis and therapeutic potential in anti-inflammation. ResultsIn single cell RNA sequencing datasets of human IBD, we identified significant reduced intestinal epithelial QTRT1 expression in the patients with Crohns Disease. Using publicly available datasets, we identified significantly changes of Vitamin Q-associated bacteria in human IBD, compared to the healthy control. Qtrt1-/- mice had significant reduction of Q-associated bacteria, e.g., Bacteroides. Alcian Blue and Mucin-2 staining revealed mucosal barrier damage and disrupted homeostasis, with reduced colonic cell proliferation. Intestinal tight junction integrity was impaired in QTRT1-KO mice, as evidenced by reduced ZO-1 and increased Claudin-10 expression. QTRT1{Delta}IEC mice also showed dysbiosis and disrupted TJs. ATP synthesis was significantly decreased in the colon of QTRT1-KO mice, accompanied by severe mitochondrial dysfunction: reduced mitochondrial quality, Cytochrome-C release, and mitochondrial DNA (mtDNA) leakage. Mitochondrial dysfunction contributed to colonic cell death, as shown by elevated expressions of Cleaved Caspase-3 and Cleaved Caspase-1, increased BAX/Bcl-2 ratio, and positive TUNEL signals. Elevated CDC42, CD14, and CD4 levels in QTRT1-KO colon suggested mucosal immune activation and tissue repair responses. QTRT1-deficient CaCO2-BBE cells showed mitochondrial dysfunction. Cytochrome-C and mito-DNA release leading to cell death characterized by elevated expressions of Cleaved Caspase-3 and Caspase-1, increased BAX/Bcl-2 ratio, and higher apoptosis rate. Organoids isolated from patients with IBD showed reduced levels of QTRT1 and dysfunctional mitochondria. Restoring mitochondrial function leads to enhanced QTRT1. ConclusionsThese findings underscore the critical role of QTRT1/Q-tRNA modification in maintaining intestinal and microbial homeostasis. Mechanistically, QTRT1 loss impacts mitochondrial integrity and mucosal homeostasis. Our study highlights the novel roles of tRNA-Q modification in maintaining mucosal barriers and innate immunity in intestinal health. What is already known about this subject?O_LIEukaryotes acquire queuine (q), also known as Vitamin Q, as a micronutrient factor from intestinal microbiota or from diet. C_LIO_LIVitamin Q is needed for queuosine (Q) modification of tRNAs for the protein translation rate and fidelity. C_LIO_LIQueuine tRNA-ribosyltransferase catalytic subunit 1 (QTRT1) is reduced in human IBD. C_LIO_LIHowever, health consequences of disturbed availability of queuine and altered Q-tRNA modification in digestive diseases remain to be investigated. C_LI What are the new findings?O_LIQTRT1 deficiency leads to altered microbiome and reduced Vitamin Q-associated bacteria in human IBD and a QTRT1 KO animal model. C_LIO_LIQTRT1 protects the host against losing intestinal integrity during inflammation. C_LIO_LIQTRT1 localizes in mitochondria and plays novel functions by maintaining intestinal mitochondrial function. QTRT1 loss impacts tRNA modification in the intestine, linking to mitochondrial integrity and mucosal homeostasis. C_LIO_LIHuman IBD showed reduced levels of QTRT1 and dysfunctional mitochondria. Restoring mitochondrial function leads to enhanced QTRT1. C_LI How might it impact on clinical practice in the foreseeable future?Targeting tRNA-Q modification in enhancing mitochondrial function will be a novel method to maintain intestinal health.

Background & AimsBiliary atresia (BA) is an obstructive newborn jaundice disease that leads to liver failure in the majority of affected infants. Viral infection is an important environmental trigger of BA. The aim of the study is to establish how viral infection rewires the cellular and metabolic processes of the digestive systems in at-risk infants and leads to BA development. MethodsSingle cell RNA (scRNA) transcriptomes and V(D)J sequences were generated using small intestine and liver biopsies from BA and control infants. Candidate risk genes were identified by genome-wide association study. Patient specimens, mouse model of experimental biliary atresia, and a myeloid-specific Folr2 knockout mice (folr2Mko) were used to determine immune pathologies that lead to BA development. An open label clinical trial was conducted to determine the therapeutic effect of folic acid on post-Kasais outcomes of patients with BA. ResultsType I interferon (IFN-I) signaling is persistently activated in infants with BA. This promotes expression of hepcidin in hepatic TREM2+ macrophages and hepatocytes, which impairs SLC40A1-mediated iron excretion from the small intestine, leading to iron accumulation, lipid peroxidation, dysbiosis and folic acid deficiency. By genetic ablation of Folr2, we show that folinate supplementation halts persistent IFN-I activation and suppresses hepcidin expression by TREM2+ macrophages. In an open label clinical study, folic acid supplementation decreased post-Kasais cholangitis incidences and liver transplantation rates by 70%. ConclusionPersistent IFN-I signaling plays a critical role in virally induced pathological jaundice in infants, and that folic acid supplementation is an effective therapy for BA.

Background and AimsEpithelial permeability barrier dysfunction is a central pathogenic driver of Crohns disease (CD), fueling microbial translocation, chronic inflammation, and progressive tissue injury. While current therapies suppress inflammation, none directly restore epithelial barrier function. Importantly, in CD patients, permeability barrier healing (BH) rather than mucosal healing is associated with long-term remission and a reduced risk of disease complications. Yet BH remains an unaddressed therapeutic target in CD. Here, we investigated whether pharmacologic inhibition of the integrated stress response (ISR) and RIPK3-mediated necroptosis, two convergent pathways of epithelial injury, can promote epithelial viability, regeneration, and barrier integrity in CD. MethodsWe employed villin-1/gelsolin double knockout (DKO) mice with epithelial-intrinsic ISR activation, TNF{Delta}ARE/+ mice with chronic inflammation, and CD patient-derived enteroids (PDEs). Animals and PDE were treated with ISR inhibitor ISRIB, RIPK3 inhibitor Necrostatin-1 (Nec-1), or FDA-approved cancer drugs pazopanib and ponatinib, repurposed as potent RIPK3 inhibitors. Epithelial survival, regenerative growth (enteroid formation, budding), and barrier function (transepithelial electrical resistance, TEER) were assessed. ResultsChronic ISR activation and necroptosis were prominent in both murine models and CD PDEs, causing epithelial death, Paneth cell expansion, impaired enteroid survival, and regenerative failure. Pharmacologic inhibition with ISRIB, Nec-1, pazopanib, or ponatinib restored villus architecture, reduced inflammation, enhanced epithelial survival and regeneration, and significantly improved TEER. ConclusionsISR activation and RIPK3-mediated necroptosis converge to drive epithelial injury and barrier dysfunction in CD. Repurposing pazopanib and ponatinib restored epithelial regeneration and BH, offering an immediately translatable therapeutic strategy for sustained remission in CD. SynopsisISR activation and RIPK3-mediated necroptosis drive epithelial injury in Crohns disease. Repurposed RIPK3 inhibitors, pazopanib and ponatinib, restore epithelial homeostasis and permeability barrier function, providing a translational strategy to achieve sustained remission in CD.

Inflammatory bowel disease (IBD) is a multifactorial disease of the gastrointestinal tract without curative treatment. Previous studies highlighted that altered fecal bile acid levels correlate with intestinal microbiota composition changes and inflammation in IBD. Lithocholic acid (LCA) is a secondary bile acid (SBA) drastically reduced during active IBD but mediates beneficial effects at the mucosal intestinal barrier during intestinal homeostasis. In a dextran sodium sulfate (DSS)-induced colitis-on-chip model, it was investigated whether the administration of LCA has a protective impact on inflammation-mediated tissue damage. Physiological responses were successfully recapitulated in the human colitis model, enabling the dissection of individual cell responses. Treatment with LCA concentrations similar to healthy human intestinal levels efficiently ameliorated the colitis-like phenotype. LCA treatment stimulated epithelial cell proliferation, thereby maintaining villus morphology, intestinal barrier integrity, and reducing inflammation. The protective effects of LCA were mainly mediated by the activation of the farnesoid X receptor (FXR).

Intestinal barrier dysfunction is widely observed in patients with irritable bowel syndrome (IBS) and significantly contributes to the persistence of IBS symptoms. The association between gut dysbiosis and the pathogenesis of IBS, as well as its connection to intestinal barrier dysfunction, has been established. However, the precise roles of gut bacteria in inducing intestinal barrier dysfunction and the underlying mechanisms of IBS remain unclear. In the present study, we showed that microbiota-derived lithocholic acid (LCA) is positively associated with intestinal barrier dysfunction biomarkers in patients with IBS-D. We found excessive LCA disrupts intestinal barrier function in normal mice and aggravates colonic inflammation in a mouse model of experimental colitis. Mechanistically, LCA modulates phospholipid metabolism and compromises the intestinal barrier by directly activating lysophosphatidylcholine acyltransferase 1 (LPCAT1). Our results demonstrated that LCA significantly upregulates LPCAT1 expression, and its overexpression leads to intestinal barrier dysfunction and promotes colonic inflammation via the activation of matrix metalloproteinase-1 (MMP1). Furthermore, we found inhibition of LPCAT1 ameliorates intestinal barrier dysfunction, diarrhea symptoms and colonic inflammation in LCA-treated mice and those with experimental colitis, highlighting LPCAT1 as a potential therapeutic target for gastrointestinal diseases characterized by intestinal barrier dysfunction and colonic inflammation. Additionally, our findings showed a positive correlation between LPCAT1 and biomarkers of intestinal barrier dysfunction and colonic inflammation in patients with ulcerative colitis. Collectively, these findings reveal the essential role of gut microbiota-derived LCA in the development of intestinal barrier dysfunction by activating LPCAT1, which worsens diarrhea symptoms in IBS and colonic inflammation in IBD. Inhibiting LPCAT1, therefore, presents a promising therapeutic strategy for both IBS-D and ulcerative colitis. This study provides insights into the molecular mechanisms involving LCA and an associated gut dysbiosis pattern in the pathogenesis of IBS-D, while also identifying new therapeutic targets aimed at maintaining intestinal homeostasis in gastrointestinal diseases, particularly in reducing IBD risks among the IBS-D population.

Backgrounds and aimsTransfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine (Q)-modification is a fundamental process for fidelity and efficiency of translation from RNA to proteins. In eukaryotes, tRNA-Q-modification relies on the intestinal microbial product queuine. However, the roles and potential mechanisms of Q-tRNA modifications in IBD are unknown. MethodsWe explored the Q-tRNA modifications and expression of Q tRNA ribosyltransferase catalytic subunit 1 (QTRT1) in patients with IBD by investigating human biopsies and reanalyzing datasets. We used colitis models, organoids, and cultured cells for loss- and gain-of-function studies to investigate the molecular mechanisms of Q-tRNA modifications in intestinal inflammation. ResultsQTRT1 expression was significantly downregulated in ulcerative colitis and Crohns disease patients. The four Q-tRNA-related tRNA synthetases (asparaginyl-aspartyl-, histidyl-, and tyrosyl-tRNA synthetase) were decreased in IBD patients. This reduction was further confirmed in DSS-induced colitis and IL10-deficient mice. Reduced QTRT1 was significantly correlated with cell proliferation and intestinal junctions, including downregulated {beta}-catenin and Claudin-5 and upregulated Claudin-2. These alterations were confirmed in vitro by deleting QTRT1 from cells. Queuine treatment significantly enhanced cell proliferation and junction functions in cell lines and human colonoids. Queuine treatment also reduced inflammation in epithelial cells. Moreover, altered QTRT1-related metabolites were found in human IBD. ConclusiontRNA modifications play an unexplored novel role in the pathogenesis of intestinal inflammation by altering epithelial proliferation and junctions. Investigations on tRNA modification will uncover novel molecular mechanisms for potential prevention and therapy for IBD.