Deficiency in intestinal epithelial O‐GlcNAcylation predisposes to gut inflammation
2018; Springer Nature; Volume: 10; Issue: 8 Linguagem: Inglês
10.15252/emmm.201708736
ISSN1757-4684
AutoresMing Zhao, Xiwen Xiong, Kaiqun Ren, Bing Xu, Meng Cheng, Chinmayi Sahu, Kaichun Wu, Yongzhan Nie, Zan Huang, Richard S. Blumberg, Xiaonan Han, Hai‐Bin Ruan,
Tópico(s)Immune cells in cancer
ResumoResearch Article25 June 2018Open Access Source DataTransparent process Deficiency in intestinal epithelial O-GlcNAcylation predisposes to gut inflammation Ming Zhao Ming Zhao School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Xiwen Xiong Xiwen Xiong School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China Search for more papers by this author Kaiqun Ren Kaiqun Ren Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA College of Medicine, Hunan Normal University, Changsha, Hunan, China Search for more papers by this author Bing Xu Bing Xu State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China Search for more papers by this author Meng Cheng Meng Cheng Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Chinmayi Sahu Chinmayi Sahu Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Kaichun Wu Kaichun Wu State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China Search for more papers by this author Yongzhan Nie Yongzhan Nie State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, ChinaCorrection added on 7 August 2018 after first online publication: Yongzan Nie was corrected to Yongzhan Nie. Search for more papers by this author Zan Huang Zan Huang Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, College of Animal Science and Technology, Nanjing Agriculture University, Nanjing, Jiangsu, China National Center for International Research on Animal Gut Nutrition, Nanjing Agriculture University, Nanjing, Jiangsu, China Search for more papers by this author Richard S Blumberg Richard S Blumberg Division of Gastroenterology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xiaonan Han Xiaonan Han Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA MOH Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Science, Chinese Academy of Medical Science (CAMS) and Peking Union Medical College (PUMC), Beijing, China Search for more papers by this author Hai-Bin Ruan Corresponding Author Hai-Bin Ruan [email protected] orcid.org/0000-0002-3858-1272 School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Ming Zhao Ming Zhao School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Xiwen Xiong Xiwen Xiong School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China Search for more papers by this author Kaiqun Ren Kaiqun Ren Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA College of Medicine, Hunan Normal University, Changsha, Hunan, China Search for more papers by this author Bing Xu Bing Xu State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China Search for more papers by this author Meng Cheng Meng Cheng Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Chinmayi Sahu Chinmayi Sahu Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Kaichun Wu Kaichun Wu State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China Search for more papers by this author Yongzhan Nie Yongzhan Nie State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, ChinaCorrection added on 7 August 2018 after first online publication: Yongzan Nie was corrected to Yongzhan Nie. Search for more papers by this author Zan Huang Zan Huang Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, College of Animal Science and Technology, Nanjing Agriculture University, Nanjing, Jiangsu, China National Center for International Research on Animal Gut Nutrition, Nanjing Agriculture University, Nanjing, Jiangsu, China Search for more papers by this author Richard S Blumberg Richard S Blumberg Division of Gastroenterology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xiaonan Han Xiaonan Han Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA MOH Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Science, Chinese Academy of Medical Science (CAMS) and Peking Union Medical College (PUMC), Beijing, China Search for more papers by this author Hai-Bin Ruan Corresponding Author Hai-Bin Ruan [email protected] orcid.org/0000-0002-3858-1272 School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Author Information Ming Zhao1,2,‡, Xiwen Xiong1,‡, Kaiqun Ren2,3, Bing Xu4, Meng Cheng2, Chinmayi Sahu2, Kaichun Wu4, Yongzhan Nie4, Zan Huang2,5,6, Richard S Blumberg7, Xiaonan Han8,9 and Hai-Bin Ruan *,1,2 1School of Forensic Medicine, Xinxiang Medical University, Xinxiang, Henan, China 2Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA 3College of Medicine, Hunan Normal University, Changsha, Hunan, China 4State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi, China 5Laboratory of Gastrointestinal Microbiology, Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, College of Animal Science and Technology, Nanjing Agriculture University, Nanjing, Jiangsu, China 6National Center for International Research on Animal Gut Nutrition, Nanjing Agriculture University, Nanjing, Jiangsu, China 7Division of Gastroenterology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 8Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA 9MOH Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Science, Chinese Academy of Medical Science (CAMS) and Peking Union Medical College (PUMC), Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +1 612 301 7686; Fax: +1 612 301 1229; E-mail: [email protected] EMBO Mol Med (2018)10:e8736https://doi.org/10.15252/emmm.201708736 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Post-translational modifications in intestinal epithelial cells (IECs) allow for precise control in intestinal homeostasis, the breakdown of which may precipitate the pathological damage and inflammation in inflammatory bowel disease. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification on intracellular proteins controls diverse biological processes; however, its roles in intestinal homeostasis are still largely unexplored. Here, we found that levels of protein O-GlcNAcylation and the expression of O-GlcNAc transferase (OGT), the enzyme adding the O-GlcNAc moiety, were reduced in IECs in human IBD patients. Deletion of OGT specifically in IECs resulted in disrupted epithelial barrier, microbial dysbiosis, Paneth cell dysfunction, and intestinal inflammation in mice. Using fecal microbiota transplantation in mice, we demonstrated that microbial dysbiosis although was insufficient to induce spontaneous inflammation but exacerbated chemical-induced colitis. Paneth cell-specific deletion of OGT led to Paneth cell dysfunction, which might predispose mice to chemical-induced colitis. On the other hand, the augmentation of O-GlcNAc signaling by inhibiting O-GlcNAcase, the enzyme removing O-GlcNAcylation, alleviated chemical-induced colitis. Our data reveal that protein O-GlcNAcylation in IECs controls key regulatory mechanisms to maintain mucosal homeostasis. Synopsis Intestinal epithelial cells (IECs) control multiple layers of intestinal homeostasis. IEC-specific O-GlcNAcylation-deficient mouse is a multi-hit model for inflammatory bowel disease (IBD). Restoring O-GlcNAcylation levels protected mice from chemical induction of inflammation. Levels of protein O-GlcNAcylation were reduced in IECs in human IBD patients. IEC-specific deficiency in O-GlcNAcylation resulted in permeable epithelial barrier, Paneth cell dysfunction, microbial dysbiosis, and ultimately intestinal inflammation in mice. Elevating O-GlcNAcylation levels increased barrier function and protected mice from chemically induced inflammation. Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a group of conditions characterized by chronic or recurring inflammation of the gastrointestinal tract. The induction and perpetuation of intestinal inflammation require the convergence of several abnormalities that affect overlapping layers of homeostatic modules including genetic predisposition, barrier dysfunction, microbial dysbiosis, and immune over-activation (Khor et al, 2011; Maloy & Powrie, 2011; Kayama & Takeda, 2012; Kamada et al, 2013; Knights et al, 2013; Sonnenberg & Artis, 2015). Intestinal epithelial cells (IECs) establish a barrier between luminal environment and the internal milieu, placing IECs at the center of interactions between the mucosal immune system and luminal antigens and metabolites. A healthy and robust layer of IECs maintains multiple layers of intestinal homeostasis. Dysfunction in IEC biology, such as epithelial barrier malfunction, uncontrolled cell death, and defective autophagy in Paneth cells, drives intestinal inflammation (Khor et al, 2011; Gilbert et al, 2012; Peterson & Artis, 2014). The epithelial barrier is primarily mediated by the formation of junction complexes between IECs, which connect adjacent IECs to form a continuous physical barrier that restricts luminal pathogens from invading the intestine. The turnover of IECs, such as apoptosis, provides an additional challenge to the maintenance of epithelial continuity. In IBD, dysregulation of junction complexes and cell death both contribute to the "leaky gut" and intestinal inflammation (Turner, 2009). The Paneth cell is a type of secretory IECs found at the base of the small intestine crypt. It contains large granules high in anti-microbial peptides (AMPs), which can alter the composition of gut microbiota and counteract enteric pathogens (Bevins & Salzman, 2011). A recent study has demonstrated Paneth cells as a site of origin for intestinal inflammation (Adolph et al, 2013). Several genetic susceptibility alleles for human IBD, such as ATG16L1, NOD2, and XBP1, all lead to Paneth cell dysfunction (Bevins & Salzman, 2011; Khor et al, 2011). Taken together, IECs control multi-layers of intestinal homeostasis, the disruption of which make a major contribution to the IBD pathogenesis. O-GlcNAcylation is the post-translational modification of serine and threonine residues with β-N-acetylglucosamine (O-GlcNAc) on intracellular proteins (Torres & Hart, 1984; Hart et al, 2007). This dynamic modification is attached by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Protein O-GlcNAcylation acts as a hormone and nutrient sensor to control many biological processes including cell signaling, metabolism, development, and aging (Hanover et al, 2012; Ruan et al, 2012, 2013b, 2014; Yang & Qian, 2017). Nevertheless, the role of intestinal epithelial O-GlcNAcylation in barrier function and inflammation is still largely unexplored. Herein, we found that levels of OGT and protein O-GlcNAcylation were downregulated in IECs of IBD patients. IEC-specific knockout of OGT in mice resulted in permeable epithelial barrier, Paneth cell dysfunction, microbial dysbiosis, and ultimately intestinal inflammation. Elevating intestinal O-GlcNAcylation levels increased barrier function and protected mice from chemical-induced inflammation. Our data demonstrate that protein O-GlcNAcylation in IECs is important for the intestinal homeostasis. Results Defective O-GlcNAc modification in IECs in IBD patients To explore the potential involvement of O-GlcNAc modification in intestinal inflammation, we performed the immunohistochemistry staining of OGT and protein O-GlcNAcylation on colon tissues of IBD patients (Appendix Table S1). In both UC and CD, levels of OGT and O-GlcNAc modification were robustly downregulated in IECs, compared to those in controls (Fig 1A and B). We also observed a similar reduction in levels of OGT and O-GlcNAcylation in a separate cohort of Chinese UC patients (Fig EV1A and B). Although not statistically significant, the expression of OGT gene in UC patients tended to decrease when compared to healthy subjects (Fig EV1C). To further evaluate whether there was any correlation between disease severity and levels of OGT and O-GlcNAcylation, we performed the immunohistochemistry staining on another set of intestine biopsies (Appendix Table S2) and confirmed the reduction in OGT and O-GlcNAcylation levels in IECs of both UC and CD (Fig 1C). Interestingly, intensities of both OGT and O-GlcNAcylation were negatively correlated with histological disease activities in UC and CD that were determined by Geboes and global histological activity (GHA) scores, respectively (Fig 1D and E, and Dataset EV1; van Loosdregt & Coffer, 2014). These data demonstrate that O-GlcNAc dysfunction in IECs is associated with IBD. Figure 1. Defective O-GlcNAc signaling in intestinal epithelial cells in IBD patients A, B. Representative images of OGT (A) and O-GlcNAc (B) immunohistochemistry on paraffin-embedded colon sections from control, UC, and CD patients (n = 4). Intensities of staining were scored on the right. Scale bars = 50 μm. (One-way ANOVA, OGT: Healthy versus UC P = 0.0002, Healthy versus CD P = 0.003; O-GlcNAc: Healthy versus UC P = 0.0365, Healthy versus CD P = 0.0125). C. Levels of intestinal epithelial OGT and O-GlcNAcylation in a second cohort of control, UC, and CD patients. (One-way ANOVA, OGT: Control n = 10, UC n = 9, CD n = 6; Control versus UC P = 0.035, Control versus CD P = 0.003; O-GlcNAc: Control n = 10, UC n = 8, CD n = 8; Control versus UC P = 0.0027, Control versus CD P = 0.008). D, E. Regression plots of average immune-staining scores of OGT and O-GlcNAcylation against histological scores in UC (D) and CD (E) (D: OGT n = 19, O-GlcNAc n = 18; E: OGT n = 15, O-GlcNAc n = 16). Data information: Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA with the Dunnett post hoc test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Defective O-GlcNAc signaling in epithelial cells of Chinese UC patients A, B. Representative images of OGT (A) and O-GlcNAc (B) immunohistochemistry in colon tissues from Chinese normal controls and UC subjects. Scale bars = 50 μm. C. mRNA levels of OGT in the colon from Chinese healthy and UC subjects (n = 12). Data information: Data are represented as scatter dot plots with lines at mean and SEM. Download figure Download PowerPoint Epithelial deficiency in OGT causes intestinal damages in mice To directly examine the role of protein O-GlcNAcylation in the intestinal epithelia, we generated IEC-specific Ogt gene knockout mice (Vil-Ogt KO) by crossing the Villin-Cre and Ogt-floxed mouse lines. Immunohistochemistry demonstrated that OGT and O-GlcNAcylation were specifically and efficiently depleted in both ileum and colon in Vil-Ogt KO mice (Fig EV2A and B). Male Vil-Ogt KO mice were viable, but substantially lighter in weight (Fig 2A) and gradually developed rectal prolapse, rectal bleeding, and diarrhea (Fig 2B and C). In females, heterozygous KO mice appeared normal and healthy, while homozygous KO females showed similar phenotypes as KO males including the body weight loss and the progressive rectal prolapse (Fig 2D and E). Later, we used male mice for most experiments. Histological analyses showed that intestinal architecture was disrupted in Vil-Ogt KO mice, such as the irregularity of the size and shape of crypts and increased crypt branching (Fig 2F). Click here to expand this figure. Figure EV2. Knockout specificity and efficiency in Vil-OGT KO mice Immunostaining of OGT and O-GlcNAc in the ileum and colon sections from male wild-type and Vil-Ogt KO mice. Scale bars = 50 μm. Immunoblotting of O-GlcNAc and OGT in the colon of female wild-type and Vil-Ogt KO mice. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. Loss of OGT in IECs causes intestinal damages in mice Body weight of male wild-type and Vil-Ogt KO mice at 6 weeks of age (n = 6, P = 0.000015). Incidence of rectal prolapse in male wild-type and Vil-Ogt KO mice (WT n = 17, KO n = 9, P < 0.0001). Representative colonoscopy images of male wild-type and Vil-Ogt KO mice. Body weight of female wild-type and Vil-Ogt KO mice at 9 weeks of age (WT n = 11, heterozygous KO n = 4, homozygous KO n = 5, P < 0.0001). Incidence of rectal prolapse in female wild-type and Vil-Ogt KO mice (WT n = 9, heterozygous KO n = 8, homozygous KO n = 6, P = 0.0439). H&E staining of duodenum, jejunum, ileum, colon, and rectum of 10-week-old male wild-type and Vil-Ogt KO mice. Scale bars = 100 μm. Data information: Data are represented as mean ± SEM. ***P < 0.001 by two-tailed t-test (A), Mantel–Cox test (B and E), or one-way ANOVA with the Dunnett post hoc test (D). Download figure Download PowerPoint We then utilized semi-quantitative pathology to qualify the pathological alterations in ileal and colonic mucosa (Erben et al, 2014; Gilbert et al, 2015). Significantly increased intestinal epithelial hypertrophy, epithelial hyperplasia, and mucosal thickness were observed in the Vil-Ogt KO mice (Fig 3A and B). Immunohistochemistry revealed a modest increase in infiltrating neutrophils, macrophages, and CD4 T cells at the crypt base of the ileum in Vil-Ogt KO mice (Fig 3C). Quantitative reverse transcription PCR (RT–qPCR) showed that the expression of inflammatory genes including Tnfa, Il6, Il1b, and Ifng was largely upregulated in the jejunum, ileum, and colon of Vil-Ogt KO mice when compared to control mice (Fig 3D). Immunofluorescence of Ki-67 showed significantly greater amounts of proliferating epithelial cells in the ileum of Vil-Ogt KO (Fig 3E). In addition, TUNEL assay and immunostaining of Cleaved-CASPASE3 showed increased apoptotic cells in the crypt region of ileum in Vil-Ogt KO mice (Fig 3F and G). Consistently, cultured ileal organoids from KO mice had a profound decrease in viability, when compared to control organoids (Fig 3H). Collectively, our data illustrate that loss of protein O-GlcNAcylation in IECs results in dysregulated proliferative/apoptotic homeostasis and susceptibility to inflammation in mice. Figure 3. IEC-specific loss of OGT disrupts intestinal proliferative/apoptotic homeostasis A. Representative images of the H&E staining of ileum and colon of 10-week-old male wild-type and Vil-Ogt KO mice. Scale bars = 100 μm. B. Combined scores of the mucosal injury in 10-week-old male wild-type and Vil-Ogt KO mice (n = 11, ileum: P < 0.0001, colon: P = 0.0012). C. Representative images of MPO, F4/80, and CD4 immunohistochemistry in the ileum tissue of male wild-type and Vil-Ogt KO mice. Scale bars = 50 μm. D. RT–qPCR of inflammatory markers in jejunum, ileum, and colon (n = 5, jejunum: Il6 P = 0.029, Ifng P = 0.0009; ileum: Tnfa P = 0.0094, Ifng P = 0.0066; colon: Tnfa P = 0.028). E. Immunofluorescent staining of Ki-67 in ileal tissues (n = 5). Scale bars = 50 μm. F, G. TUNEL staining (F) and immunostaining of Cleaved-CASPASE3 (G) in the ileum. Quantification of the numbers of positive cells is shown at the right. (TUNEL WT n = 9, KO n = 6, P = 0.0003; Cleaved Cas3 WT = 8, KO = 7, P < 0.0001). H. Numbers of survived organoids per 1000 isolated ileal crypts (WT n = 4, KO n = 23, P < 0.0001). Data information: Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed t-test. Download figure Download PowerPoint Defective intestinal barrier in Vil-Ogt KO mice Intestinal barrier dysfunction potentiates and sustains intestinal inflammation (Turner, 2009), and we then sought to test whether the deficiency in O-GlcNAcylation in IECs alters epithelial barrier function. Vil-Ogt KO mice exhibited increased plasma levels of fluorescein isothiocyanate (FITC) after oral gavage of FITC-dextran (Fig 4A) and increased fecal albumin (Fig 4B), indicating that Vil-Ogt KO mice had increased intestinal permeability. A major component of the mucosal barrier is the junction complex between IECs (Turner, 2009). Electron microscopy of the wild-type intestine clearly showed the tight junctions (arrows), the adherens junctions (arrowheads), and desmosomes (stars) (Fig 4C). In Vil-Ogt KO mice, however, the density of perijunctional ring was profoundly decreased (Fig 4C). Desmosomes were reduced in number, which sometime caused open paracellular space (Fig 4C). We also observed splenomegaly (Fig EV3A) and increased levels of inflammatory gene expression in the liver (Fig EV3B), indicating that the leaky gut in Vil-Ogt KO mice caused systemic inflammation. Figure 4. Intestinal barrier dysfunction in Vil-Ogt KO mice A, B. Intestinal barrier functional assays measuring serum FITC-dextran (A) and albumin from fecal sample (B) of male wild-type and Vil-Ogt KO mice. (A: WT n = 5, KO n = 6, P = 0.0015; B: WT n = 9, KO n = 6, P = 0.0053). C. Representative EM pictures of ileum epithelial cells. Arrows, arrowheads, and stars indicate tight junctions, adherens junctions, and desmosomes, respectively. Scale bars = 1 μm. D. Immunoblotting of protein markers of tight junction (ZO-1), adherens junction (β-CATENIN, α-E-CATENIN, and PLAKOGLOBIN), and desmosome (PLAKOGLOBIN and DESMOPLAKIN) in colon. E. Densitometric analysis of PLAKOGLOBIN protein levels in (D). (WT n = 6, KO n = 5, P = 0.021). F. Representative images of ZO-1, ZO-2, PLAKOGLOBIN, and β-CATENIN immunofluorescent staining in the ileum tissue of male wild-type and Vil-Ogt KO mice. Arrow and arrowhead indicate ZO-1 and PLAKOGLOBIN, respectively. Scale bars = 10 μm. Data information: Data are represented as mean ± SEM. *P < 0.05; **P < 0.01 by two-tailed t-test. Source data are available online for this figure. Source Data for Figure 4D [emmm201708736-sup-0004-SDataFig4.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Systemic inflammation in Vil-Ogt KO mice A, B. Gross morphology of spleen (A) and RT–qPCR of inflammatory markers in liver (B) from male wild-type and Vil-Ogt KO mice (WT n = 4, KO n = 6, Il6 P = 0.0271). C. Immunoblotting of protein markers of tight junction including OCCLUDIN, CLAUDIN 3 and 5 in colon. The blots were from the same experiment as shown in Fig 7A. Data information: Data are represented as mean ± SEM. *P < 0.05 by two-tailed t-test. Source data are available online for this figure. Download figure Download PowerPoint We then performed Western blotting and immunofluorescence using antibodies against markers of junction complexes and found that PLAKOGLOBIN, a critical component of both desmosomes and adherens junctions that anchors transmembrane cadherins to intermediate filaments, was downregulated in Vil-Ogt KO mice (Fig 4D–F). Even though the expression level was not changed, the tight junction protein zonula occludens-1 (ZO-1) remarkably lost its localization to the cell membrane in Vil-Ogt KO mice (Fig 4D and F). We did not observe any changes in other junction complex markers including ZO-2, β-CATENIN, α-E-CATENIN, DESMOPLAKIN, CLAUDIN 3 and 5, and OCCLUDIN (Figs 4D and F, and EV3C). Taken together, these findings demonstrate that the loss of OGT in IECs impairs epithelial barrier function, which may potentiate the development of intestinal inflammation. Microbial dysbiosis in Vil-Ogt KO mice Microbial dysbiosis plays an important role in inflammatory bowel disease (Bevins & Salzman, 2011), and we then sought to determine whether the deficiency in intestinal epithelial O-GlcNAcylation interferes with gut microbiota. 16S rRNA gene sequencing was used to interrogate differences in fecal microbiota between singly housed wild-type and Vil-Ogt KO mice. The phylogenetic diversity was comparable between control and KO mice (Fig EV4A). Principal coordinate analysis (PCoA) of the unweighted UniFrac showed that bacterial communities within the same genotype had similar bacterial composition no matter where they were housed (Fig 5A). Linear discriminant analysis (LDA) effect size (LEfSe) highlighted differentially abundant taxonomic clades (Fig EV4B). The phylum Firmicutes, the Coriobacteriia class within the Actinobacteria phylum, and Mycoplasmatales order within the Tenericutes phylum were decreased, while the Bacteriodetes family Bacteriodaceae and the Gammaproteobacteria class within the Proteobacteria phylum were enriched in Vil-Ogt KO mice (Figs 5B and EV4C and D). Interestingly, the most consistent observations of microbial dysbiosis in IBD patients are a reduction in Firmicutes and an increase in Proteobacteria (Matsuoka & Kanai, 2015). Click here to expand this figure. Figure EV4. Changes in microbial composition in Vil-Ogt KO mice A. Phylogenetic diversity of fecal bacteria from mice that were individually housed at UMN (WT n = 5, KO n = 4) or Yale (n = 5). B. Differentially abundant taxomonic clades analyzed by LEfSe. C, D. Abundance histogram plots of taxonomic groups detected by LEfSe that were downregulated (C) or upregulated (D) in Vil-Ogt KO mice (n = 4–5). E–H. Antibiotic-treated mice were transplanted with gut microbiota from control or Vil-Ogt KO mice (n = 5). (E) PCoA plot of unweighted UniFrac distance of bacterial communities. (F) H&E staining of colon. Scale bars = 100 μm. (G) Levels of serum FITC-dextran. (H) Albumin from fecal samples. Data information: Data are represented as mean ± SEM. Download figure Download PowerPoint Figure 5. Microbial dysbiosis in OGT-deficient mice A. PCoA plot of unweighted UniFrac distance of bacterial communities in wild-type and Vil-Ogt KO mice that were singly housed at Yale or UMN (UMN KO n = 4, rest n = 5). B. Cladogram showing the most discriminative bacterial clades between wild-type and Vil-Ogt KO mice that were identified by LEfSe. Regions in red indicate clades that were enriched in Vil-Ogt KO mice, while regions in green indicate clades that were enriched in wild-type mice. C–H. Antibiotic-treated C57BL/6 male mice were transplanted with fecal microbiota from wild-type or Vil-Ogt KO mice and then induced with DSS for colitis (WT n = 5, KO n = 7). (C) Daily changes in body weight. (D) Colitis scores. (E) Intestinal barrier functional assays measuring serum FITC-dextran on Day 10. (F) H&E staining of colon tissues. Ulcer areas are designated by red circles. Scale bars = 200 μm. (G) Pathological scores of the mucosal injury. (H) RT–qPCR of inflammatory markers in the colon. (D: P = 0.0409; E: P = 0.0457; G: P = 0.032). Data information: Data are represented as mean ± SEM. *P < 0.05 by two-way ANOVA followed by Bonferroni corrections (D) and two-tailed t-test (E and G). Download figure Download PowerPoint To determine whether microbial dysbiosis in Vil-Ogt KO mice contributes to the pathogenesis of intestinal inflammation, we first performed fecal microbiota transplantation (FMT) from control and Vil-Ogt KO mice into antibiotic-treated wild-type mice. Compositional differences between the microbiota from control and
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