Gastroenterology
Abstract
Eosinophilic esophagitis (EoE) is an esophageal immune-mediated disease characterized by eosinophilic inflammation and epithelial remodeling, including basal cell hyperplasia (BCH). Although BCH is known to correlate with disease severity and with persistent symptoms in patients in histological remission, the molecular processes driving BCH remain poorly defined. Here, we demonstrated that BCH is predominantly characterized by an expansion of non-proliferative suprabasal cells that are still committed to early differentiation. Furthermore, we discovered that suprabasal and superficial esophageal epithelial cells retain progenitor identity programs in EoE, which was evidenced by increased quiescent cell identity scoring and the enrichment of signaling pathways regulating stem cell pluripotency. Enrichment and trajectory analyses identified SOX2 and KLF5 as potential drivers of the increased quiescent identity and epithelial remodeling observed in EoE. Notably, these alterations were not observed in GERD. These findings provide additional insights into the differentiation process in EoE and highlight the distinct characteristics of suprabasal and superficial esophageal epithelial cells in the disease.
Authors
Margarette H. Clevenger, Adam L. Karami, Dustin A. Carlson, Peter J. Kahrilas, Nirmala Gonsalves, John E. Pandolfino, Deborah R. Winter, Kelly A. Whelan, Marie-Pier Tétreault
Abstract
Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen–driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography–tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell–related proteins were among the most highly upregulated DEPs in EoE compared to controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all six subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of the epithelial cell through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.
Authors
Mark Rochman, Yrina Rochman, Julie M. Caldwell, Lydia Mack, John A Besse, Nathan P Manes, Sung Hwan Yoon, Tetsuo Shoda, Aleksandra Nita-Lazar, Marc Rothenberg
Abstract
Intestinal mucins play an essential role in the defense against bacterial invasion and the maintenance of gut microbiota, which is instrumental in the regulation of host immune systems; hence, its dysregulation is a hallmark of metabolic disease and intestinal inflammation. However, the mechanism by which intestinal mucins control the gut microbiota as well as disease phenotypes remains nebulous. Herein, we report that N-acetylglucosamine (GlcNAc)-6-O-sulfation of O-glycans on intestinal mucins performs a protective role against obesity and intestinal inflammation. Chst4-/- mice, lacking GlcNAc-6-O-sulfation of the mucin O-glycans, showed significant weight gain and increased susceptibility to dextran sodium sulfate-induced colitis as well as colitis-associated cancer accompanied by significantly reduced immunoglobulin A (IgA) production caused by impaired T follicular helper cell-mediated IgA response. Interestingly, the protective effects of GlcNAc-6-O-sulfation against obesity and intestinal inflammation depend on the gut microbiota, evidenced by the modulation of the gut microbiota by co-housing or microbiota transplantation reversing disease phenotypes and IgA production. Collectively, our findings provide novel insight into the significance of host glycosylation, more specifically GlcNAc-6-O-sulfation on intestinal mucins, in protecting against obesity and intestinal inflammation via regulation of the gut microbiota.
Authors
Hirohito Abo, Aoi Muraki, Akihito Harusato, Tetsuya Imura, Maki Suzuki, Kohta Takahashi, Timothy L. Denning, Hiroto Kawashima
Abstract
Thymic stromal lymphopoietin (TSLP) overexpression is widely associated with atopy. However, TSLP is expressed in normal barrier organs, suggesting a homeostatic function. To determine the function of TSLP in barrier sites, we investigated the impact of endogenous TSLP signaling on the homeostatic expansion of CD4+ T cells in adult mice. Surprisingly, incoming CD4+ T cells induced lethal colitis in adult Rag1-knockout animals that lacked the TSLP receptor (Rag1KOTslprKO). Endogenous TSLP signaling was required for reduced CD4+ T cell proliferation, Treg differentiation, and homeostatic cytokine production. CD4+ T cell expansion in Rag1KOTslprKO mice was dependent on the gut microbiome. The lethal colitis was rescued by parabiosis between Rag1KOTslprKO and Rag1KO animals and wild-type dendritic cells (DCs) suppressed CD4+ T cell–induced colitis in Rag1KOTslprKO mice. A compromised T cell tolerance was noted in TslprKO adult colon, which was exacerbated by anti–PD-1 and anti–CTLA-4 therapy. These results reveal a critical peripheral tolerance axis between TSLP and DCs in the colon that blocks CD4+ T cell activation against the commensal gut microbiome.
Authors
Jonathan L. Messerschmidt, Marjan Azin, Kaitlin E. Dempsey, Shadmehr Demehri
Abstract
The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unravel a novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2 and LoVo) with either knockdown or overexpression of LIN28B, we identified Claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and post-transcriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA-sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B-CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by post-transcriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.
Authors
Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Alice E. Shin, Nozomu Sakai, Hisahiro Matsubara, Masayuki Ohtsuka, Peter A. Sims, Christopher J. Lengner, Anil K. Rustgi
Abstract
Forkhead box M1 (FOXM1) plays a critical role in development physiologically and tumorigenesis pathologically. However, insufficient efforts have been dedicated to exploring the regulation, in particular the degradation of FOXM1. Here, the ON-TARGETplus siRNA library targeting E3 ligases was used to screen potential candidates to repress FOXM1. Of note, mechanism study revealed that RNF112 directly ubiquitinates FOXM1 in gastric cancer, resulting in a decreased FOXM1 transcriptional network and suppressing the proliferation and invasion of gastric cancer. Interestingly, the well-established small-molecule compound RCM-1 significantly enhanced the interaction between RNF112 and FOXM1, which further promoted FOXM1 ubiquitination and subsequently exerted promising anticancer effects in vitro and in vivo. Altogether, we demonstrate that RNF112 suppresses gastric cancer progression by ubiquitinating FOXM1 and highlight the RNF112/FOXM1 axis serves as both prognosis biomarker and therapeutic target in gastric cancer.
Authors
Shengwei Zhang, Jing Wang, Weichao Hu, Lijiao He, Qingyun Tang, Jie Li, Mengmeng Jie, Xinzhe Li, Cheng Liu, Qin Ouyang, Shiming Yang, Changjiang Hu
Abstract
Ulcerative colitis (UC), Crohn’s disease (CD), and celiac disease are prevalent intestinal inflammatory disorders with non-satisfactory therapeutic interventions. Analyzing patient data-driven cohorts can highlight disease pathways and new targets for interventions. Long non-coding RNAs (lncRNAs) are attractive candidates as they are readily targetable by RNA therapeutics, show relative cell-specific expression, and play key cellular functions. Uniformly analyzing gut mucosal transcriptomics from 696 subjects, we highlight lncRNA expression along the gastrointestinal (GI) tract, demonstrating that in control samples, lncRNAs have a more location-specific expression in comparison to protein-coding genes. We defined dysregulation of lncRNAs in treatment-naïve UC, CD, and celiac diseases using independent test and validation cohorts. Using the PROTECT inception UC cohort, we define and prioritize lncRNA linked with UC severity and prospective outcomes, and highlight lncRNAs linked with gut microbes previously implicated in mucosal homeostasis. HNF1A-AS1 lncRNA was reduced in all 3 conditions and was further reduced in more severe UC form. Similarly, the reduction of HNF1A-AS1 ortholog in mice gut epithelial showed higher sensitivity to dextran sodium sulfate-induced colitis which was coupled with alteration in the gut microbial community. These analyses highlight prioritized dysregulated lncRNAs that can guide future preclinical studies for testing them as novel potential targets.
Authors
Tzipi Braun, Katya E. Sosnovski, Amnon Amir, Marina BenShoshan, Kelli L. VanDussen, Rebekah Karns, Nina Levhar, Haya Abbas-Egbariya, Rotem Hadar, Gilat Efroni, David Castel, Camila Avivi, Michael J. Rosen, Anne M. Grifiths, Thomas D. Walters, David R. Mack, Brendan M. Boyle, Syed Asad Ali, Sean R. Moore, Melanie Schirmer, Ramnik J. Xavier, Subra Kugathasan, Anil G. Jegga, Batya Weiss, Chen Mayer, Iris Barshack, Shomron Ben-Horin, Igor Ulitsky, Anthony Beucher, Jorge Ferrer, Jeffrey S. Hyams, Lee A. Denson, Yael Haberman
Abstract
Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism, which may become overactivated in pathological states resulting intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP, and inhibition of Orai1 could prevent the development of end-stage CP.
Authors
Viktória Szabó, Noémi Csákány-Papp, Marietta Görög, Tamara Madacsy, Árpád Varga, Aletta Kiss, Balint Tel, Boldizsár Jójárt, Tim Crul, Krisztina Dudás, Mária Bagyánszki, Nikolett Bódi, Ferhan Ayaydin, Shyam Jee, Laszlo Tiszlavicz, Kenneth A. Stauderman, Sudarshan Hebbar, Petra Pallagi, József Maléth
Abstract
The incidence of early onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 alpha (HNF1AA98V, Rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9 and the mice were placed on either a high fat (HFD) or high sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however,19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes and Wnt/β-catenin signaling components in the HNF1A mutant relative to the wildtype mice. Mouse polyps and colon cancers from subjects carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated β-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared to wildtype HNF1A. Collectively, our study shows that the HNF1AA98V variant plus HFD promotes the formation of colonic polyps by activating β-catenin via decreasing Cdx2 expression.
Authors
Heyu Song, Ricky A. Sontz, Matthew J. Vance, Julia M. Morris, Sulaiman Sheriff, Songli Zhu, Suzann Duan, Jiping Zeng, Erika Koeppe, Ritu Pandey, Curtis A. Thorne, Elena M. Stoffel, Juanita L. Merchant
Abstract
Altered mitochondrial function without a well-defined cause has been documented in the patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homologue, CLUH, only in the active UC tissues compared to the unaffected areas from the same patient and healthy controls. Stimulation with bacterial toll like receptor (TLR) ligands similarly reduced CLUH expression in the human primary macrophages. Further, CLUH negatively regulated secretion of pro-inflammatory cytokines IL6, TNF-α and rendered a pro-inflammatory niche in the TLR stimulated macrophage. CLUH was further found to bind to mitochondrial fission protein DRP-1 and also regulated DRP-1 transcription in the human macrophages. In the TLR ligand stimulated macrophages, absence of CLUH led to enhanced DRP-1 availability for mitochondrial fission and smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production, reduced mitophagy and lysosomal function in the CLUH knockout macrophages. Remarkably, our studies in the mice model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report signifying the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.
Authors
Shaziya Khan, Desh Raj, Shikha Sahu, Anam Naseer, Nishakumari C. Singh, Sunaina Kumari, Sharmeen Ishteyaque, Jyotsna Sharma, Promila Lakra, Madhav Nilakanth Mugale, Arun Kumar Trivedi, Mrigank Srivastava, Tulika Chandra, Vivek Bhosale, Manoj Kumar Barthwal, Shashi Kumar Gupta, Kalyan Mitra, Aamir Nazir, Uday C. Ghoshal, Amit Lahiri
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