Pub Date : 2026-01-01DOI: 10.1016/j.jcmgh.2025.101681
Rainelli B. Koumangoye, Mohammed Z. Ferdaus, Xenia Davis, Julia K. Bohannon, Eric Delpire
Background & Aims
Potassium and chloride efflux have been reported to regulate nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Aberrant activation of NLRP3 inflammasome causes autoimmune and chronic inflammatory diseases. The Na+-K+-2Cl- cotransporter (NKCC1) maintains the intracellular concentrations of sodium, potassium, and chloride. Here we ask whether NKCC1 modulates NLRP3 inflammasome activation.
Methods
Mice with intestinal epithelial-specific deletion of NKCC1, CRISPR-Cas9 NKCC1-deleted Caco-2 and HT-29 cells, and human fibroblasts expressing mutant NKCC1 were used to evaluate NLRP3 inflammasome activation.
Results
We found that deletion of NKCC1 in established intestinal epithelial cells (IECs) in culture causes increased pyroptosis and interleukin (IL)-1β and IL-18 secretion upon NLRP3 inflammasome activation. Similarly, organoids derived from mice with conditional NKCC1 knockout in IECs also exhibit increased IL-1β secretion when stimulated with adenosine triphosphate (ATP). Moreover, fibroblasts from a patient with NKCC1 mutant also showed increased pyroptosis and IL-1β and IL-18 secretion. Loss of NKCC1 sensitizes IECs to changes in intracellular concentration of K+ and decreases the threshold required for NLRP3 inflammasome activation. Finally, we showed that NKCC1ΔIEC mice have increased infiltration of innate immune cells in the colon mucosa and peritoneal cavity.
Conclusions
NKCC1 functions as a negative regulator of NLRP3 inflammasome activation and this may explain why patients of loss-of-function mutations in NKCC1 are susceptible to inflammatory diseases.
背景：钾和氯的外排已被报道调节NLRP3炎性体的激活。NLRP3炎性小体的异常激活导致自身免疫性和慢性炎症性疾病。Na+- k +- 2cl -共转运体（NKCC1）维持细胞内钠、钾和氯化物的浓度。在这里，我们询问NKCC1是否调节NLRP3炎性体的激活。方法：采用肠上皮特异性缺失NKCC1的小鼠、CRISPR-Cas9缺失NKCC1的caco2和HT-29细胞以及表达突变型NKCC1的人成纤维细胞来评估NLRP3炎性体的激活。结果：我们发现在培养的小肠上皮细胞中缺失NKCC1导致NLRP3炎性体激活后的焦亡、IL-1β和IL-18分泌增加。同样，在肠上皮细胞（IECs）中NKCC1条件敲除小鼠的类器官在ATP刺激下也表现出IL-1β分泌增加。此外，NKCC1突变患者的成纤维细胞也显示出焦亡，IL-1β和IL-18分泌增加。NKCC1的缺失使IECs对细胞内K+浓度的变化敏感，并降低NLRP3炎性体激活所需的阈值。最后，我们发现NKCC1ΔIEC小鼠在结肠黏膜和腹膜腔中增加了先天免疫细胞的浸润。结论：NKCC1作为NLRP3炎性小体激活的负调节因子，这可能解释了为什么NKCC1功能缺失突变的患者易患炎性疾病。
{"title":"Loss of NKCC1 Activates the NLRP3 Inflammasome in Intestinal Epithelia","authors":"Rainelli B. Koumangoye, Mohammed Z. Ferdaus, Xenia Davis, Julia K. Bohannon, Eric Delpire","doi":"10.1016/j.jcmgh.2025.101681","DOIUrl":"10.1016/j.jcmgh.2025.101681","url":null,"abstract":"<div><h3>Background & Aims</h3><div>Potassium and chloride efflux have been reported to regulate nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Aberrant activation of NLRP3 inflammasome causes autoimmune and chronic inflammatory diseases. The Na<sup>+</sup>-K<sup>+</sup>-2Cl<sup>-</sup> cotransporter (NKCC1) maintains the intracellular concentrations of sodium, potassium, and chloride. Here we ask whether NKCC1 modulates NLRP3 inflammasome activation.</div></div><div><h3>Methods</h3><div>Mice with intestinal epithelial-specific deletion of NKCC1, CRISPR-Cas9 NKCC1-deleted Caco-2 and HT-29 cells, and human fibroblasts expressing mutant NKCC1 were used to evaluate NLRP3 inflammasome activation.</div></div><div><h3>Results</h3><div>We found that deletion of NKCC1 in established intestinal epithelial cells (IECs) in culture causes increased pyroptosis and interleukin (IL)-1β and IL-18 secretion upon NLRP3 inflammasome activation. Similarly, organoids derived from mice with conditional NKCC1 <em>knockout</em> in IECs also exhibit increased IL-1β secretion when stimulated with adenosine triphosphate (ATP). Moreover, fibroblasts from a patient with NKCC1 mutant also showed increased pyroptosis and IL-1β and IL-18 secretion. Loss of NKCC1 sensitizes IECs to changes in intracellular concentration of K<sup>+</sup> and decreases the threshold required for NLRP3 inflammasome activation. Finally, we showed that NKCC1<sup>ΔIEC</sup> mice have increased infiltration of innate immune cells in the colon mucosa and peritoneal cavity.</div></div><div><h3>Conclusions</h3><div>NKCC1 functions as a negative regulator of NLRP3 inflammasome activation and this may explain why patients of loss-of-function mutations in NKCC1 are susceptible to inflammatory diseases.</div></div>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":"20 3","pages":"Article 101681"},"PeriodicalIF":7.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145558291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1016/j.jcmgh.2025.101679
Kim H.H. Liss , Samantha Goldman , Mai He , Trevor M. Shew , Daniel Ferguson , Brian N. Finck
Background & Aims
Hepatic ischemia reperfusion injury (IRI) is unavoidable in most liver operations and is associated with poor patient and graft outcomes in the setting of liver transplantation. However, there are no pharmacological interventions available for treatment of IRI. Prior work has demonstrated that liver IRI leads to hepatic lipid accumulation, suggesting that increased adipocyte lipolytic rates may contribute to hepatic steatosis. Inhibition of adipose triglyceride lipase (ATGL), the rate-limiting enzyme involved in triglyceride hydrolysis, may be beneficial in cardiac injury and alcoholic liver disease, but its role in liver IRI has not been investigated. Our objective was to assess the effects of inhibition of adipose tissue lipolysis in the setting of liver IRI.
Methods
Wild-type mice were treated with Atglistatin, a small molecule inhibitor of ATGL, prior to IRI. Mice with hepatocyte- or adipocyte-specific deletion of Pnpla2, the gene encoding ATGL, were generated and subjected to a mouse model of IRI. Mouse hepatocytes were cultured with fatty acids in an in vitro model of IRI.
Results
We demonstrated that experimental IRI was associated with increased adipocyte lipolysis. Pharmacological and genetic inhibition of adipocyte lipolysis reduced plasma and hepatic free fatty acids and decreased circulating transaminases and liver inflammation following hepatic IRI. Furthermore, exogenous fatty acids were sufficient to increase cell death and the expression of inflammatory cytokines in in vitro IRI.
Conclusions
These data suggest that targeting adipocyte lipolysis may represent a novel therapeutic approach in the prevention of hepatic IRI, which could improve patient and graft outcomes following liver transplantation.
{"title":"Inhibition of Adipocyte Lipolysis Reduces Liver Injury in a Mouse Model of Ischemia Reperfusion Injury","authors":"Kim H.H. Liss , Samantha Goldman , Mai He , Trevor M. Shew , Daniel Ferguson , Brian N. Finck","doi":"10.1016/j.jcmgh.2025.101679","DOIUrl":"10.1016/j.jcmgh.2025.101679","url":null,"abstract":"<div><h3>Background & Aims</h3><div>Hepatic ischemia reperfusion injury (IRI) is unavoidable in most liver operations and is associated with poor patient and graft outcomes in the setting of liver transplantation. However, there are no pharmacological interventions available for treatment of IRI. Prior work has demonstrated that liver IRI leads to hepatic lipid accumulation, suggesting that increased adipocyte lipolytic rates may contribute to hepatic steatosis. Inhibition of adipose triglyceride lipase (ATGL), the rate-limiting enzyme involved in triglyceride hydrolysis, may be beneficial in cardiac injury and alcoholic liver disease, but its role in liver IRI has not been investigated. Our objective was to assess the effects of inhibition of adipose tissue lipolysis in the setting of liver IRI.</div></div><div><h3>Methods</h3><div>Wild-type mice were treated with Atglistatin, a small molecule inhibitor of ATGL, prior to IRI. Mice with hepatocyte- or adipocyte-specific deletion of <em>Pnpla2</em>, the gene encoding ATGL, were generated and subjected to a mouse model of IRI. Mouse hepatocytes were cultured with fatty acids in an in vitro model of IRI.</div></div><div><h3>Results</h3><div>We demonstrated that experimental IRI was associated with increased adipocyte lipolysis. Pharmacological and genetic inhibition of adipocyte lipolysis reduced plasma and hepatic free fatty acids and decreased circulating transaminases and liver inflammation following hepatic IRI. Furthermore, exogenous fatty acids were sufficient to increase cell death and the expression of inflammatory cytokines in in vitro IRI.</div></div><div><h3>Conclusions</h3><div>These data suggest that targeting adipocyte lipolysis may represent a novel therapeutic approach in the prevention of hepatic IRI, which could improve patient and graft outcomes following liver transplantation.</div></div>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":"20 3","pages":"Article 101679"},"PeriodicalIF":7.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145566188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1016/j.jcmgh.2025.101673
Angela M. Halstead , Chinye Nwokolo , Stella Hoft , Jinsheng Yu , Lifei Zhu , Brendan Tuley , Nancy Vargas , RuiRui Liu , Francisco Ramirez Victorino , Simrin Phatak , Wandy Beatty , Chun-Kan Chen , Richard DiPaolo , Paul Cliften , Tarin M. Bigley , José B. Sáenz
Background & Aims
Recent evidence suggests that endogenously derived double-stranded RNA (dsRNA) impacts multiple cellular processes, although its role in epithelial injury remains understudied. We previously identified the response to dsRNA as the most upregulated pathway across 2 distinct murine models of spasmolytic polypeptide-expressing metaplasia (SPEM), a critical pre-neoplastic transition in the progression to gastric cancer. The aim of this study was to define how dysregulation of the dsRNA response within gastric epithelium impacts gastric pre-neoplasia.
Methods
We specifically deleted ADAR1, a central regulator of dsRNA signaling, from gastric parietal cells (Adar1ΔPC). Adar1ΔPC and age-matched controls stomachs were histologically, transcriptionally, and immunologically profiled. The source of dsRNA in Adar1ΔPC gastric epithelium was assessed by dsRNA immunoprecipitation and immuno-electron microscopy. Finally, to define the contributions of interferon (IFN) signaling, Adar1ΔPC;Ifnar1-/-and Adar1ΔPC;Ifnlr1-/- mice, defective in type I and type III IFN signaling, respectively, were characterized.
Results
Adar1ΔPC mice spontaneously developed SPEM and gastric dysplasia, in the absence of exogenous injury. Our phenotype depended on Mavs, a key dsRNA signaling hub, implying that our model of gastric pre-neoplasia was specific to dsRNA signaling. Further characterization of this pre-neoplastic environment by single-cell RNA sequencing and flow cytometry noted a chronic and sustained transcriptional upregulation of the dsRNA response throughout gastric epithelium that was independent of adaptive immunity and that depended on both type I and type III IFN signaling. Finally, we identified an enrichment of mitochondrial dsRNA within the gastric epithelium of Adar1ΔPC stomachs.
Conclusions
Our new genetic model implicates ADAR1-mediated dsRNA signaling in gastric pre-neoplasia.
{"title":"A New Model of Gastric Pre-neoplasia Induced by Aberrant ADAR1-mediated Double-stranded RNA Signaling","authors":"Angela M. Halstead , Chinye Nwokolo , Stella Hoft , Jinsheng Yu , Lifei Zhu , Brendan Tuley , Nancy Vargas , RuiRui Liu , Francisco Ramirez Victorino , Simrin Phatak , Wandy Beatty , Chun-Kan Chen , Richard DiPaolo , Paul Cliften , Tarin M. Bigley , José B. Sáenz","doi":"10.1016/j.jcmgh.2025.101673","DOIUrl":"10.1016/j.jcmgh.2025.101673","url":null,"abstract":"<div><h3>Background & Aims</h3><div>Recent evidence suggests that endogenously derived double-stranded RNA (dsRNA) impacts multiple cellular processes, although its role in epithelial injury remains understudied. We previously identified the response to dsRNA as the most upregulated pathway across 2 distinct murine models of spasmolytic polypeptide-expressing metaplasia (SPEM), a critical pre-neoplastic transition in the progression to gastric cancer. The aim of this study was to define how dysregulation of the dsRNA response within gastric epithelium impacts gastric pre-neoplasia.</div></div><div><h3>Methods</h3><div>We specifically deleted ADAR1, a central regulator of dsRNA signaling, from gastric parietal cells (<em>Adar1</em><sup><em>ΔPC</em></sup>). <em>Adar1</em><sup><em>ΔPC</em></sup> and age-matched controls stomachs were histologically, transcriptionally, and immunologically profiled. The source of dsRNA in <em>Adar1</em><sup><em>ΔPC</em></sup> gastric epithelium was assessed by dsRNA immunoprecipitation and immuno-electron microscopy. Finally, to define the contributions of interferon (IFN) signaling, <em>Adar1</em><sup><em>ΔPC</em></sup><em>;Ifnar1</em><sup><em>-/-</em></sup> <em>and Adar1</em><sup><em>ΔPC</em></sup><em>;Ifnlr1</em><sup><em>-/-</em></sup> mice, defective in type I and type III IFN signaling, respectively, were characterized.</div></div><div><h3>Results</h3><div><em>Adar1</em><sup><em>ΔPC</em></sup> mice spontaneously developed SPEM and gastric dysplasia, in the absence of exogenous injury. Our phenotype depended on <em>Mavs</em>, a key dsRNA signaling hub, implying that our model of gastric pre-neoplasia was specific to dsRNA signaling. Further characterization of this pre-neoplastic environment by single-cell RNA sequencing and flow cytometry noted a chronic and sustained transcriptional upregulation of the dsRNA response throughout gastric epithelium that was independent of adaptive immunity and that depended on both type I and type III IFN signaling. Finally, we identified an enrichment of mitochondrial dsRNA within the gastric epithelium of <em>Adar1</em><sup><em>ΔPC</em></sup> stomachs.</div></div><div><h3>Conclusions</h3><div>Our new genetic model implicates ADAR1-mediated dsRNA signaling in gastric pre-neoplasia.</div></div>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":"20 3","pages":"Article 101673"},"PeriodicalIF":7.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145460738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1016/j.jcmgh.2025.101683
Xu Han , Yutong Hou , Taoying Chen , Zhenyu Jia , Fuqiang Yuan , Mingxin Wang , Yinling Su , Ru Yan , Xiaolei Wang , Weilai Jin , Yahui Zhou , Yun Li , Le Zhang
Background & Aims
Breast milk contains abundant glutamine and glutamate, yet their roles in neonatal gut health remain controversial. We aimed to investigate how these amino acids influence neonatal enteritis and the underlying mechanisms.
Methods
We used a neonatal rat necrotizing enterocolitis (NEC) model to test the effects of glutamine and glutamate given either before disease onset or during NEC progression. Previously published human neonatal NEC single-cell RNA psequencing (scRNA-seq) data were reanalyzed to assess immune cell composition and pathway activity. Bone marrow-derived macrophages (BMDMs) were used to examine inflammatory responses in vitro. Flow cytometry and transcriptomics were applied to analyze metabolic reprogramming of ileal macrophages.
Results
Glutamine and glutamate prevented NEC when administered prophylactically but worsened disease when given during NEC progression. scRNA-seq revealed enrichment of macrophages with activated inflammatory pathways in NEC ileum. In vitro, pretreatment with glutamine or glutamate reduced lipopolysaccharide-induced cytokine expression in BMDMs, whereas administration after stimulation had no benefit. In vivo, glutamine pretreatment decreased CD45+F4/80+CD11b/c+TNFα+ macrophages, whereas treatment during NEC increased this subset. Integrated analyses showed NEC upregulated glutaminase (GLS) and downregulated glutamate dehydrogenase (GLUD1) in ileal macrophages. Pretreatment with glutamine or glutamate restored GLUD1 expression, favoring α-ketoglutarate (α-KG) rather than succinate metabolism. Supplementation with α-KG reversed glutamine-induced macrophage activation during NEC, whereas succinate abolished the protective effect of glutamine pretreatment.
Conclusions
Glutamine and glutamate exert dual, context-dependent effects in neonatal enteritis by modulating macrophage glutamine metabolism. These findings provide mechanistic insight and suggest a basis for personalized glutamine supplementation strategies in neonatal gut disorders.
{"title":"The Glutamine Metabolic Switch Influences the Response of Neonatal Intestinal Macrophages to Breast Milk","authors":"Xu Han , Yutong Hou , Taoying Chen , Zhenyu Jia , Fuqiang Yuan , Mingxin Wang , Yinling Su , Ru Yan , Xiaolei Wang , Weilai Jin , Yahui Zhou , Yun Li , Le Zhang","doi":"10.1016/j.jcmgh.2025.101683","DOIUrl":"10.1016/j.jcmgh.2025.101683","url":null,"abstract":"<div><h3>Background & Aims</h3><div>Breast milk contains abundant glutamine and glutamate, yet their roles in neonatal gut health remain controversial. We aimed to investigate how these amino acids influence neonatal enteritis and the underlying mechanisms.</div></div><div><h3>Methods</h3><div>We used a neonatal rat necrotizing enterocolitis (NEC) model to test the effects of glutamine and glutamate given either before disease onset or during NEC progression. Previously published human neonatal NEC single-cell RNA psequencing (scRNA-seq) data were reanalyzed to assess immune cell composition and pathway activity. Bone marrow-derived macrophages (BMDMs) were used to examine inflammatory responses in vitro. Flow cytometry and transcriptomics were applied to analyze metabolic reprogramming of ileal macrophages.</div></div><div><h3>Results</h3><div>Glutamine and glutamate prevented NEC when administered prophylactically but worsened disease when given during NEC progression. scRNA-seq revealed enrichment of macrophages with activated inflammatory pathways in NEC ileum. In vitro, pretreatment with glutamine or glutamate reduced lipopolysaccharide-induced cytokine expression in BMDMs, whereas administration after stimulation had no benefit. In vivo, glutamine pretreatment decreased CD45<sup>+</sup>F4/80<sup>+</sup>CD11b/c<sup>+</sup>TNFα<sup>+</sup> macrophages, whereas treatment during NEC increased this subset. Integrated analyses showed NEC upregulated glutaminase (GLS) and downregulated glutamate dehydrogenase (GLUD1) in ileal macrophages. Pretreatment with glutamine or glutamate restored GLUD1 expression, favoring α-ketoglutarate (α-KG) rather than succinate metabolism. Supplementation with α-KG reversed glutamine-induced macrophage activation during NEC, whereas succinate abolished the protective effect of glutamine pretreatment.</div></div><div><h3>Conclusions</h3><div>Glutamine and glutamate exert dual, context-dependent effects in neonatal enteritis by modulating macrophage glutamine metabolism. These findings provide mechanistic insight and suggest a basis for personalized glutamine supplementation strategies in neonatal gut disorders.</div></div>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":"20 3","pages":"Article 101683"},"PeriodicalIF":7.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145650222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1016/j.jcmgh.2025.101650
Hanchao Lin, Qiongzhu Dong
{"title":"Anesthetic Agents as Modulators of Antitumor Immunity: Repurposing Etomidate for Hepatocellular Carcinoma Therapy","authors":"Hanchao Lin, Qiongzhu Dong","doi":"10.1016/j.jcmgh.2025.101650","DOIUrl":"10.1016/j.jcmgh.2025.101650","url":null,"abstract":"","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":"20 2","pages":"Article 101650"},"PeriodicalIF":7.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145208129","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1016/j.jcmgh.2025.101684
Rodrigo Velázquez-Moctezuma , Rani Burm , Lieven Verhoye , Laura Collignon , Kenn Holmbeck , Erick Giang , Mansun Law , Jens Bukh , Philip Meuleman , Jannick Prentoe
Background & Aims
Mechanisms of hepatitis C virus (HCV) adaptation and escape from broadly neutralizing antibodies (bNAbs) have been primarily studied in vitro. Here, we used a previously developed in vivo adapted J6/JFH1A876P virus and the highly bNAb sensitive hypervariable region 1 (HVR1) deleted variant, J6/JFH1A876P,ΔHVR1, to study adaptation and bNAb AR5A escape in the HCV-permissive human-liver chimeric mouse model.
Methods
In vitro identified AR5A escape substitution, L665S, was introduced into J6/JFH1A876P with or without HVR1. The infection of human liver chimeric mice with these recombinants revealed adaptive mutations, and the potential mechanism of adaptation was extensively characterized in vitro. Finally, we tested the barrier to resistance of AR5A in vivo by challenging passively immunized animals with HVR1-deleted viruses, either with or without the AR5A escape substitution, L665S.
Results
L665S was found to be an escape substitution in vivo. Furthermore, sequence analysis showed that the escape substitution L665S arose as early as 2 weeks post infection. At week 8, we also identified antibody escape substitutions as well as several potential in vivo adaptive substitutions in E2. For J6/JFH1A876P, S449P and M702L increased cell-free particle infection and broadly affected antibody sensitivity for virus with HVR1. For J6/JFH1A876P,ΔHVR1, N430D and M702L substitutions increased both cell-free particle mediated infection and cell-to-cell spread, whereas N430D also increased thermal stability at 37°C.
Conclusions
We show that L665S is an AR5A escape mutation in vivo, supporting the use of cost-effective vaccine escape studies in vitro. We also identify novel in vivo adaptive mutations and characterize their mechanism of action, thus facilitating interpretation of future HCV in vivo studies.
{"title":"In Vivo Determinants of Hepatitis C Virus Adaptation and Escape From Neutralizing Antibody AR5A","authors":"Rodrigo Velázquez-Moctezuma , Rani Burm , Lieven Verhoye , Laura Collignon , Kenn Holmbeck , Erick Giang , Mansun Law , Jens Bukh , Philip Meuleman , Jannick Prentoe","doi":"10.1016/j.jcmgh.2025.101684","DOIUrl":"10.1016/j.jcmgh.2025.101684","url":null,"abstract":"<div><h3>Background & Aims</h3><div>Mechanisms of hepatitis C virus (HCV) adaptation and escape from broadly neutralizing antibodies (bNAbs) have been primarily studied in vitro. Here, we used a previously developed in vivo adapted J6/JFH1<sub>A876P</sub> virus and the highly bNAb sensitive hypervariable region 1 (HVR1) deleted variant, J6/JFH1<sub>A876P,ΔHVR1</sub>, to study adaptation and bNAb AR5A escape in the HCV-permissive human-liver chimeric mouse model.</div></div><div><h3>Methods</h3><div>In vitro identified AR5A escape substitution, L665S, was introduced into J6/JFH1<sub>A876P</sub> with or without HVR1. The infection of human liver chimeric mice with these recombinants revealed adaptive mutations, and the potential mechanism of adaptation was extensively characterized in vitro. Finally, we tested the barrier to resistance of AR5A in vivo by challenging passively immunized animals with HVR1-deleted viruses, either with or without the AR5A escape substitution, L665S.</div></div><div><h3>Results</h3><div>L665S was found to be an escape substitution in vivo. Furthermore, sequence analysis showed that the escape substitution L665S arose as early as 2 weeks post infection. At week 8, we also identified antibody escape substitutions as well as several potential in vivo adaptive substitutions in E2. For J6/JFH1<sub>A876P</sub>, S449P and M702L increased cell-free particle infection and broadly affected antibody sensitivity for virus with HVR1. For J6/JFH1<sub>A876P,ΔHVR1</sub>, N430D and M702L substitutions increased both cell-free particle mediated infection and cell-to-cell spread, whereas N430D also increased thermal stability at 37°C.</div></div><div><h3>Conclusions</h3><div>We show that L665S is an AR5A escape mutation in vivo, supporting the use of cost-effective vaccine escape studies in vitro. We also identify novel in vivo adaptive mutations and characterize their mechanism of action, thus facilitating interpretation of future HCV in vivo studies.</div></div>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":"20 4","pages":"Article 101684"},"PeriodicalIF":7.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145558307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-30DOI: 10.1016/j.jcmgh.2025.101718
Beatriz Elias Ribeiro, Isadora Schmukler de Lima, Karen Cristina da Silva E Souza, Siane Lopes Bittencourt Rosas, Patrícia Teixeira Santana, Gilda Amaral, João Carlos Machado, Rodrigo Pereira de Oliveira, Camille Leal, Cristiane Thompson, Fabiano Thompson, Morgana Teixeira Lima Castelo-Branco, Robson Coutinho-Silva, Heitor Siffert Pereira de Souza
Background & aims: Considering the role of the P2X7 receptor in intestinal inflammation, we examined its potential involvement in fibrosis development.
Methods: Colonic biopsies from patients with inflammatory bowel disease (IBD) were analyzed via double immunofluorescence under confocal microscopy. Colon fibroblasts were used to analyze P2X7 receptor modulation and chemotaxis. Experimental chronic colitis was induced with 3 cycles of oral dextran sodium sulfate (DSS) treatment in P2X7+/+ and P2X7-/- mice. The mice were evaluated via follow-up video endoscopy with an endoluminal ultrasound biomicroscopic (eUBM) system, histological scoring, immunohistochemistry, cytokine measurement in colon explants, gene expression analysis of P2X7 signaling targets via quantitative real-time polymerase chain reaction (qRT-PCR), and microbiome composition analysis.
Results: Colocalization studies revealed a greater density of P2X7+/alpha smooth muscle actin (α-SMA)+ cells in colon sections from patients than in those from controls, especially in patients with Crohn's disease (P < .05). Activation of the adenosine triphosphate (ATP)-P2X7 pathway in human fibroblasts increased cell migration, calcium influx, and collagen production. Video colonoscopy with the eUBM system revealed significantly more inflammation, with greater wall thickness and stiffness, in P2X7+/+ mice than in P2X7-/- and P2X7+/+ mice treated with A740003 (a P2X7-selective inhibitor). P2X7+/+ mice exhibited increased caspase-1 and NLRP3 expression, as well as nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) activation, accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) expression. In the supernatants of colon explants, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, transforming growth factor (TGF)-β, IL-10, and collagen production were increased in P2X7+/+ mice. Various microbial changes were observed in P2X7-/- and P2X7+/+ mice.
Conclusions: Regulatory mechanisms downstream of P2X7, combined with signals from a dysbiotic microbiota, activate intracellular signaling pathways and the inflammasome, leading to intestinal inflammation and promoting fibrogenesis.
{"title":"The P2X7 Receptor Promotes Intestinal Fibrosis by Modulating the Gut Microbiota and the Inflammasome.","authors":"Beatriz Elias Ribeiro, Isadora Schmukler de Lima, Karen Cristina da Silva E Souza, Siane Lopes Bittencourt Rosas, Patrícia Teixeira Santana, Gilda Amaral, João Carlos Machado, Rodrigo Pereira de Oliveira, Camille Leal, Cristiane Thompson, Fabiano Thompson, Morgana Teixeira Lima Castelo-Branco, Robson Coutinho-Silva, Heitor Siffert Pereira de Souza","doi":"10.1016/j.jcmgh.2025.101718","DOIUrl":"10.1016/j.jcmgh.2025.101718","url":null,"abstract":"<p><strong>Background & aims: </strong>Considering the role of the P2X7 receptor in intestinal inflammation, we examined its potential involvement in fibrosis development.</p><p><strong>Methods: </strong>Colonic biopsies from patients with inflammatory bowel disease (IBD) were analyzed via double immunofluorescence under confocal microscopy. Colon fibroblasts were used to analyze P2X7 receptor modulation and chemotaxis. Experimental chronic colitis was induced with 3 cycles of oral dextran sodium sulfate (DSS) treatment in P2X7<sup>+/+</sup> and P2X7<sup>-/-</sup> mice. The mice were evaluated via follow-up video endoscopy with an endoluminal ultrasound biomicroscopic (eUBM) system, histological scoring, immunohistochemistry, cytokine measurement in colon explants, gene expression analysis of P2X7 signaling targets via quantitative real-time polymerase chain reaction (qRT-PCR), and microbiome composition analysis.</p><p><strong>Results: </strong>Colocalization studies revealed a greater density of P2X7<sup>+</sup>/alpha smooth muscle actin (α-SMA)<sup>+</sup> cells in colon sections from patients than in those from controls, especially in patients with Crohn's disease (P < .05). Activation of the adenosine triphosphate (ATP)-P2X7 pathway in human fibroblasts increased cell migration, calcium influx, and collagen production. Video colonoscopy with the eUBM system revealed significantly more inflammation, with greater wall thickness and stiffness, in P2X7<sup>+/+</sup> mice than in P2X7<sup>-/-</sup> and P2X7<sup>+/+</sup> mice treated with A740003 (a P2X7-selective inhibitor). P2X7<sup>+/+</sup> mice exhibited increased caspase-1 and NLRP3 expression, as well as nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) activation, accompanied by decreased peroxisome proliferator-activated receptor gamma (PPARγ) expression. In the supernatants of colon explants, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, transforming growth factor (TGF)-β, IL-10, and collagen production were increased in P2X7<sup>+/+</sup> mice. Various microbial changes were observed in P2X7<sup>-/-</sup> and P2X7<sup>+/+</sup> mice.</p><p><strong>Conclusions: </strong>Regulatory mechanisms downstream of P2X7, combined with signals from a dysbiotic microbiota, activate intracellular signaling pathways and the inflammasome, leading to intestinal inflammation and promoting fibrogenesis.</p>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":" ","pages":"101718"},"PeriodicalIF":7.1,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145890609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-25DOI: 10.1016/j.jcmgh.2025.101715
Katarina Coolens, Melissa Van Der Vliet, Jente Van Campenhout, Natalia Del Pozo, Alejo Torres-Cano, Catharina Olson, Jianming Xu, Heiko Lickert, Meritxell Rovira, Isabelle Houbracken, Jonathan Baldan, Francisco X Real, Francesca M Spagnoli, Ilse Rooman
{"title":"ΔNP63 Defines an Exocrine-committed Subset of Murine Pancreatic Progenitor Cells.","authors":"Katarina Coolens, Melissa Van Der Vliet, Jente Van Campenhout, Natalia Del Pozo, Alejo Torres-Cano, Catharina Olson, Jianming Xu, Heiko Lickert, Meritxell Rovira, Isabelle Houbracken, Jonathan Baldan, Francisco X Real, Francesca M Spagnoli, Ilse Rooman","doi":"10.1016/j.jcmgh.2025.101715","DOIUrl":"10.1016/j.jcmgh.2025.101715","url":null,"abstract":"","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":" ","pages":"101715"},"PeriodicalIF":7.1,"publicationDate":"2025-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145844307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-25DOI: 10.1016/j.jcmgh.2025.101717
Katherine J Aney, Woo-Jeong Jeong, Pal Koak, Anders W Ohman, Canh Hiep Nguyen, Brian M Wolpin, Jonathan A Nowak, Sahar Nissim
Background & aims: In response to injury, pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), marked by loss of acinar identity and acquisition of ductal features. Although ADM can resolve to support tissue repair, it may also persist and serve as a precursor to pancreatic cancer. Whether diverse pancreatic stressors drive a shared or context-specific ADM program remains unclear. We sought to comprehensively define metaplastic responses to clinically relevant exocrine pancreas diseases known to increase cancer risk.
Methods: We profiled ADM and the surrounding microenvironment across mouse models of exocrine disease-including acute, recurrent, and chronic pancreatitis, as well as in the setting of oncogenic Kras-capturing over 300,000 single cells. To enable high-quality transcriptomic profiling in enzyme-rich tissue, we leveraged FixNCut, a method that preserves RNA integrity in the exocrine pancreas. Findings were validated in human pancreas tissue using CosMx spatial transcriptomics.
Results: We identify a conserved acinar response across disease contexts that gives rise to previously unrecognized distinct metaplastic states, including a "gateway" ADM population that precedes more advanced metaplastic states marked by complete loss of acinar identity. In pancreatic intraepithelial neoplasia (PanIN) precancerous lesions, we detect classical-like and basal-like states, suggesting that pancreatic cancer subtypes are specified much earlier than previously appreciated. In Kras-mutant tissue, we identify a second wave of inflammation and the emergence of an immunosuppressive niche, coinciding with PanIN formation.
Conclusions: Our findings define a conserved program of acinar plasticity across exocrine pancreas diseases. We further link unresolved ADM to immune remodeling during precursor lesion formation and observe the emergence of pancreatic cancer subtypes in early PanIN lesions.
{"title":"Novel Acinar Metaplastic States Uncovered in Exocrine Pancreas Disease.","authors":"Katherine J Aney, Woo-Jeong Jeong, Pal Koak, Anders W Ohman, Canh Hiep Nguyen, Brian M Wolpin, Jonathan A Nowak, Sahar Nissim","doi":"10.1016/j.jcmgh.2025.101717","DOIUrl":"10.1016/j.jcmgh.2025.101717","url":null,"abstract":"<p><strong>Background & aims: </strong>In response to injury, pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), marked by loss of acinar identity and acquisition of ductal features. Although ADM can resolve to support tissue repair, it may also persist and serve as a precursor to pancreatic cancer. Whether diverse pancreatic stressors drive a shared or context-specific ADM program remains unclear. We sought to comprehensively define metaplastic responses to clinically relevant exocrine pancreas diseases known to increase cancer risk.</p><p><strong>Methods: </strong>We profiled ADM and the surrounding microenvironment across mouse models of exocrine disease-including acute, recurrent, and chronic pancreatitis, as well as in the setting of oncogenic Kras-capturing over 300,000 single cells. To enable high-quality transcriptomic profiling in enzyme-rich tissue, we leveraged FixNCut, a method that preserves RNA integrity in the exocrine pancreas. Findings were validated in human pancreas tissue using CosMx spatial transcriptomics.</p><p><strong>Results: </strong>We identify a conserved acinar response across disease contexts that gives rise to previously unrecognized distinct metaplastic states, including a \"gateway\" ADM population that precedes more advanced metaplastic states marked by complete loss of acinar identity. In pancreatic intraepithelial neoplasia (PanIN) precancerous lesions, we detect classical-like and basal-like states, suggesting that pancreatic cancer subtypes are specified much earlier than previously appreciated. In Kras-mutant tissue, we identify a second wave of inflammation and the emergence of an immunosuppressive niche, coinciding with PanIN formation.</p><p><strong>Conclusions: </strong>Our findings define a conserved program of acinar plasticity across exocrine pancreas diseases. We further link unresolved ADM to immune remodeling during precursor lesion formation and observe the emergence of pancreatic cancer subtypes in early PanIN lesions.</p>","PeriodicalId":55974,"journal":{"name":"Cellular and Molecular Gastroenterology and Hepatology","volume":" ","pages":"101717"},"PeriodicalIF":7.1,"publicationDate":"2025-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145844549","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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