Gastroenterology
Abstract
Authors
Isin Y. Comba, Tijs Louwies, Ruben A. Mars, Yang Xiao, Prabhjot Kaur Sekhon, Brian S. Edwards, Adam Willits, Robin R. Shields-Cutler, Shreya Bellampalli, Arnaldo Mercado-Perez, Dennis R. Tienter, Lisa M. Till, David R. Linden, Gianrico Farrugia, Arthur Beyder, Kristen M. Smith-Edwards, Purna C. Kashyap
Abstract
Hypoxia in the tumor microenvironment promotes lymphatic metastasis, yet the role of cancer-associated fibroblasts (CAFs) in this process remains insufficiently elucidated in colorectal cancer (CRC). In this study, we developed a large language model–based cellular hypoxia–predicting classifier to identify hypoxic CAFs (HCAFs) at single-cell resolution. Our findings revealed that HCAFs enhance CRC lymphatic metastasis by secreting CLEC11A, a protein that binds to the LGR5 receptor on tumor cells, subsequently activating the WNT/β-catenin signaling pathway. This promotes epithelial-mesenchymal transition and lymphangiogenesis, facilitating the spread of tumor cells via the lymphatic system. Furthermore, we demonstrate that the hypoxia-induced transcription factor HIF1A regulates the conversion of normoxic CAFs to HCAFs, driving CLEC11A expression and promoting metastasis. In vivo and vitro experiments confirmed the pro-metastatic role of CLEC11A in CRC, with its inhibition reducing lymphatic metastasis. This effect was markedly reversed by targeting the LGR5 receptor on tumor cells or inhibiting the WNT/β-catenin pathway, further elucidating the underlying mechanisms of CLEC11A-driven metastasis. These findings underscore the potential of targeting the CLEC11A-LGR5 axis to prevent lymphatic dissemination in CRC. Our study highlights the role of HCAFs in CRC progression and reveals mechanisms of lymphatic metastasis for intervention.
Authors
Chuhan Zhang, Teng Pan, Yuyuan Zhang, Yushuai Wu, Anning Zuo, Shutong Liu, Yuhao Ba, Benyu Liu, Shuaixi Yang, Yukang Chen, Hui Xu, Peng Luo, Quan Cheng, Siyuan Weng, Long Liu, Xing Zhou, Jingyuan Ning, Xinwei Han, Jinhai Deng, Zaoqu Liu
Abstract
Single-cell studies have revealed that intestinal macrophages maintain gut homeostasis through the balanced actions of reactive (inflammatory) and tolerant (non-inflammatory) subpopulations. How such balance is impaired in inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), remains unresolved. Here, we define colon-specific macrophage states and reveal the critical role of non-inflammatory colon-associated macrophages (niColAMs) in IBD recovery. Through trans-scale analyses—integrating computational transcriptomics, proteomics, and in vivo interventional studies—we identified GIV (CCDC88A) as a key regulator of niColAMs. GIV emerged as the top-ranked gene in niColAMs that physically and functionally interacts with NOD2, an innate immune sensor implicated in CD and UC. Myeloid-specific GIV depletion exacerbates infectious colitis, prolongs disease, and abolishes the protective effects of the NOD2 ligand, muramyl dipeptide, in colitis and sepsis models. Mechanistically, GIV’s C-terminus binds the terminal leucine-rich repeat (LRR#10) of NOD2 and is required for NOD2 to dampen inflammation and clear microbes. The CD-associated 1007fs NOD2-variant, which lacks LRR#10, cannot bind GIV—providing critical insights into how this clinically relevant variant impairs microbial sensing and clearance. These findings illuminate a critical GIV-NOD2 axis essential for gut homeostasis and highlight its disruption as a driver of dysbiosis and inflammation in IBD.
Authors
Gajanan D. Katkar, Mahitha Shree Anandachar, Stella-Rita C. Ibeawuchi, Ella G. McLaren, Megan L. Estanol, Kennith Carpio-Perkins, Shu-Ting Hsu, Celia R. Espinoza, Jane E. Coates, Yashaswat S. Malhotra, Madhubanti Mullick, Vanessa Castillo, Daniella Vo, Saptarshi Sinha, Pradipta Ghosh
Abstract
LRRK2 contains a kinase domain where the N2081D Crohn’s disease (CD) risk and the G2019S Parkinson’s disease (PD) pathogenic variants are located. It is not clear how the N2081D variant increases CD risk or how these adjacent mutations give rise to distinct disorders. To investigate the pathophysiology of the CD-linked LRRK2 N2081D variant, we generated a knock-in (KI) mouse model and compared its effects with those of the LRRK2-G2019S mutation. Lrrk2N2081D KI mice demonstrated heightened sensitivity to induced colitis, resulting in more severe intestinal damage than in Lrrk2G2019S KI and WT mice. Analysis of colon tissue revealed distinct mutation-dependent LRRK2 RAB substrate phosphorylation, with significantly elevated phosphorylated RAB10 levels in Lrrk2N2081D mice. In cells, we demonstrated that the N2081D mutation activates LRRK2 through a mechanism distinct from that of LRRK2-G2019S. We also found that proinflammatory stimulation enhances LRRK2 kinase activity, leading to mutation-dependent differences in RAB phosphorylation and inflammatory responses in dendritic cells (DCs). Finally, we show that knockout of Rab12, but not pharmacological LRRK2 kinase inhibition, significantly reduced colitis severity in Lrrk2N2081D mice. Our study characterizes the pathogenic mechanisms of LRRK2-linked CD, highlights structural and functional differences between disease-associated LRRK2 variants, and suggests RAB proteins as promising therapeutic targets for modulating LRRK2 activity in CD treatment.
Authors
George R. Heaton, Xingjian Li, Xianting Li, Xiaoting Zhou, Yuanxi Zhang, Duc Tung Vu, Marc Oeller, Ozge Karayel, Quyen Q. Hoang, Meltem Ece Kars, Nitika Kamath, Minghui Wang, Leonid Tarassishin, Matthias Mann, Inga Peter, Zhenyu Yue
Abstract
A20, encoded by the TNFAIP3 gene, is a protein linked to Crohn’s disease and celiac disease in humans. We now find that mice expressing point mutations in A20’s M1-ubiquitin–binding zinc finger 7 (ZF7) motif spontaneously develop proximal enteritis that requires both luminal microbes and T cells. Cellular and transcriptomic profiling reveals expansion of Th17 cells and exuberant expression of IL-17A and IL-22 in intestinal lamina propria of A20ZF7 mice. While deletion of IL-17A from A20ZF7/ZF7 mice exacerbates enteritis, deletion of IL-22 abrogates intestinal epithelial cell hyperproliferation, barrier dysfunction, and alarmin expression. Colonization of adult germ-free mice with microbiota from adult WT specific pathogen–free mice drives duodenal IL-22 expression and duodenitis. A20ZF7/ZF7 Th17 cells autonomously express more RORγt and IL-22 after differentiation in vitro. ATAC sequencing identified an enhancer region upstream of the Il22 gene, and this enhancer demonstrated increased activating histone acetylation coupled with exaggerated Il22 transcription in A20ZF7/ZF7 T cells. Acute inhibition of RORγt normalized histone acetylation at this enhancer. Finally, CRISPR/Cas9–mediated ablation of A20ZF7 in human T cells increases RORγt expression and IL22 transcription. These studies link A20’s M1-ubiquitin binding function with RORγt expression, expansion of Th17 cells, and epigenetic activation of IL-22–driven enteritis.
Authors
Christopher J. Bowman, Dorothea M. Stibor, Xiaofei Sun, Nika Lenci, Hiromichi Shimizu, Emily F. Yamashita, Rommel Advincula, Min Cheol Kim, Jessie A. Turnbaugh, Yang Sun, Bahram Razani, Peter J. Turnbaugh, Chun Jimmie Ye, Barbara A. Malynn, Averil Ma
Abstract
Crypt hyperplasia is a key feature of celiac disease and several other small intestinal inflammatory conditions. Analysis of the gut epithelial crypt zone by mass spectrometry-based tissue proteomics revealed a strong interferon-γ (IFN-γ) signal in active celiac disease. This signal, hallmarked by increased expression of MHC molecules, was paralleled by diminished expression of proteins associated with fatty acid metabolism. Crypt hyperplasia and the same proteomic changes were observed in wild type mice administered IFN-γ. In mice with conditional knockout of the IFN-γ receptor in gut epithelial cells these signature morphological and proteomic changes were not induced on IFN-γ administration. IFN-γ is thus a driver of crypt hyperplasia in celiac disease by acting directly on crypt epithelial cells. The results are relevant to other enteropathies with involvement of IFN-γ.
Authors
Jorunn Stamnaes, Daniel Stray, M. Fleur du Pré, Louise F. Risnes, Alisa E. Dewan, Jakeer Shaik, Maria Stensland, Knut E.A. Lundin, Ludvig M. Sollid
Abstract
Hypertonic and hyperosmolar stimuli frequently pose challenges to the intestinal tract. Therefore, a resilient epithelial barrier is essential for maintaining gut homeostasis in the presence of osmotic perturbations. NFAT5, an osmosensitive transcription factor, primarily maintains cellular homeostasis under hypertonic conditions. However, the osmoprotective role of NFAT5 in enterocyte homeostasis is poorly understood. Here, we demonstrate that NFAT5 is critical for the survival and proliferation of intestinal epithelial cells (IECs) and that its deficiency accelerates chemically induced or spontaneous colitis in mice. Mechanistically, NFAT5 promotes the survival of IECs and the renewal of intestinal stem cells, thereby regulating the production of mucus and antimicrobial compounds, including RegIII and lysozyme, which consequently shape the gut microbial composition to prevent colitis. Transcriptome analysis identifies HSP70 as a key downstream target of NFAT5 in epithelial regeneration. Loss- and gain-of-function experiments of HSP70 revealed that NFAT5 mitigates experimental colitis through IEC Hsp70, which protected stem cells from inflammation-induced injury and maintained barrier function. In conclusion, our study demonstrates a previously unknown role for NFAT5 in dictating the crosstalk between intestinal stem cells and the microbiota, underscoring the importance of the NFAT5–HSP70 axis in maintaining epithelial regeneration related to gut barrier function, balancing microbial composition, and subsequently preventing colitis progression.
Authors
Se Hyeon Park, Dae Hee Cheon, Yu-Mi Kim, Yeji Choi, Yong-Joon Cho, Bong-Ki Hong, Sang-Hyun Cho, Mi‑Na Kweon, Hyug Moo Kwon, Eugene B. Chang, Donghyun Kim, Wan-Uk Kim
Abstract
Somatic mutations that increase clone fitness or resist disease are positively selected, but the impact of these mutations on organismal health remains unclear. We previously showed that Tbx3 deletion increases hepatocyte fitness within fatty livers. Here, we detected TBX3 somatic mutations in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). In mice, Tbx3 deletion protected against, whereas Tbx3 overexpression exacerbated MASLD. Tbx3 deletion reduced lipid overload by accelerating VLDL secretion. Choline deficient diets, which block VLDL secretion, abrogated this protective effect. TBX3 transcriptionally suppressed the conventional secretory pathway and cholesterol biosynthesis. Hdlbp is a direct target of TBX3 that is responsible for the altered VLDL secretion. In contrast to wild-type TBX3, the TBX3 I155S and A280S mutations found in patients failed to suppress VLDL secretion. In conclusion, TBX3 mutant clones expand during MASLD through increased lipid disposal, demonstrating that clonal fitness can benefit the liver at the cost of hyperlipidemia.
Authors
Gregory Mannino, Gabriella Quinn, Min Zhu, Zixi Wang, Xun Wang, Boyuan Li, Meng-Hsiung Hsieh, Thomas Mathews, Lauren Zacharias, Wen Gu, Purva Gopal, Natalia Brzozowska, Peter Campbell, Matt Hoare, Glen Liszczak, Hao Zhu
Abstract
Perianal fistulizing Crohn’s disease (PCD) is a common and debilitating complication with elusive pathophysiology. To define actionable immunologic targets in PCD, we recruited patients with PCD (n = 24), CD without perianal disease (NPCD, n = 10), and idiopathic/cryptoglandular perianal fistulas (IPF, n = 29). Biopsies from fistula tracts, fistula opening, and rectal mucosa were analyzed using single-cell RNA-sequencing (scRNA-seq), mass cytometry (CyTOF), and spatial transcriptomics (ST). Global hyperactivation of IFN-g pathways distinguished PCD from idiopathic perianal fistulas and CD without perianal disease in the fistula tracts and/or intestinal mucosa. IFN-g and TNF-a signaling directly induced genes involved in epithelial-to-mesenchymal transition in PCD rectal epithelial cells. Enhanced IFN-g signaling in PCD was driven by pathogenic Th17 (pTh17) cells, which were recruited and activated by myeloid cells overexpressing LPS signature (LPS_myeloid). pTh17 and LPS_myeloid cells co-localized adjacent to PCD fistula tracts on ST and drove local IFN-g signaling. Anti-TNFs facilitated fistula healing by downregulating T and myeloid cell signatures, while promoting mucosal barrier repair and immunoregulatory processes. Key single-cell findings were validated by bulk RNA-seq data of an independent CD cohort. To summarize, we identified IFN-g-driven mechanisms contributing to pathogenesis and highlighted its blockade as a therapeutic strategy for PCD.
Authors
Siyan Cao, Khai M. Nguyen, Kaiming Ma, Tingyi Tan, Xin Yao, Ta-Chiang Liu, Malek Ayoub, Jalpa Devi, Sami Samaan, Yizhou Liu, Radhika Smith, Matthew L. Silviera, Steven R. Hunt, Paul E. Wise, Matthew G. Mutch, Sean C. Glasgow, William C. Chapman Jr, Michelle L. Cowan, Mathew A. Ciorba, Marco Colonna, Parakkal Deepak
Abstract
Esophageal adenocarcinoma (EA) is increasingly prevalent and is thought to arise from Barrett’s esophagus (BE), a metaplastic condition in which chronic acid and bile reflux transforms the esophageal squamous epithelium into a gastric-intestinal glandular mucosa. The molecular determinants driving this metaplasia are poorly understood. We developed a human BE organoid biobank that recapitulates BE’s molecular heterogeneity. Bulk and single-cell transcriptomics, supported by patient tissue analysis, revealed that BE differentiation reflects a balance between SOX2 (foregut/esophageal) and CDX2 (hindgut/intestinal) transcription factors. Using squamous-specific inducible Sox2 knockout (Krt5CreER/+; Sox2∆/∆; ROSA26tdTomato/+) mice, we observed increased basal proliferation, reduced squamous differentiation, and expanded metaplastic glands at the squamocolumnar junction, some tracing back to Krt5-expressing cells. CUT&RUN analysis showed SOX2 bound and promoted differentiation-associated (e.g., Krt13) and repressed proliferation-associated (e.g., Mki67) targets. Thus, SOX2 is critical for foregut squamous epithelial differentiation and its decreased expression is likely an initiating step in progression to BE and thence to EA.
Authors
Ramon U. Jin, Yuanwei Xu, Tung-Shing Lih, Yang-Zhe Huang, Toni M. Nittolo, Blake E. Sells, Olivia M. Dres, Jean S. Wang, Qing Kay Li, Hui Zhang, Jason C. Mills
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)