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Uhrf1-Mediated Tnf-Α Gene Methylation Controls

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Uhrf1-Mediated Tnf-Α Gene Methylation Controls Uhrf1-Mediated Tnf-α Gene Methylation Controls Proinflammatory Macrophages in Experimental Colitis Resembling Inflammatory Bowel Disease This information is current as of September 30, 2021. Shanshan Qi, Yongkui Li, Zheng Dai, Mengxi Xiang, Guobin Wang, Lin Wang and Zheng Wang J Immunol published online 14 October 2019 http://www.jimmunol.org/content/early/2019/10/12/jimmun ol.1900467 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/10/12/jimmunol.190046 Material 7.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 30, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 14, 2019, doi:10.4049/jimmunol.1900467 The Journal of Immunology Uhrf1-Mediated Tnf-a Gene Methylation Controls Proinflammatory Macrophages in Experimental Colitis Resembling Inflammatory Bowel Disease Shanshan Qi,*,1 Yongkui Li,*,1 Zheng Dai,* Mengxi Xiang,* Guobin Wang,*,† Lin Wang,*,‡ and Zheng Wang*,† Macrophages drive the pathological process of inflammatory bowel diseases (IBD) mostly by secreting proinflammatory cytokines, such as Tnf-a. Recent studies have indicated the association between epigenetic modifications and macrophage functions. How- ever, epigenetic mechanisms regulating macrophages’ functional involvement in IBD remain unknown. In this study, we inves- tigated whether the epigenetic regulator Uhrf1 plays a role in innate immunity by functionally regulating macrophages in fl/fl intestines. We employed two transgenic strains of mice (one with Uhrf1 deficiency in macrophages [Uhrf1 Lyz2-Cre mice] Downloaded from and the other with the two mutations at Uhrf1’s DNA methylation regulatory site [Uhrf1YP187/188AA mice]) to assess their susceptibility to dextran sodium sulfate–induced colitis. We examined the cytokines derived from Uhrf1fl/flLyz2-Cre and Uhrf1YP187/188AA macrophages in response to LPS stimulation. We also analyzed the effects of proinflammatory cytokines on Uhrf1 expression in macrophages. The data demonstrated that Uhrf1 deficiency and Uhrf1YP187/188AA mutation resulted in severe colitis in the dextran sodium sulfate–treated mice. In vitro analysis revealed the hypomethylation of Tnf-a promoter and the fl/fl YP187/188AA increased Tnf-a expression in Uhrf1 Lyz2-Cre and Uhrf1 macrophages in response to LPS stimulation, and anti–Tnf- http://www.jimmunol.org/ a therapy implied the key role of Tnf-a to the aggravated colitis in Uhrf1-deficient mice. Exogenous Tnf-a destabilized Uhrf1 protein through ubiquitination-mediated protein degradation, triggering macrophage activation. In conclusion, we identified that Uhrf1-mediated DNA methylation controls Tnf-a expression of macrophages in the experimental colitis resembling IBD. The epigenetic mechanisms that activate macrophages may provide new therapeutic targets for IBD treatment. The Journal of Immunology, 2019, 203: 000–000. nflammatory bowel disease (IBD) is a gastrointestinal in- to dextran sodium sulfate (DSS) treatment (11). These observa- flammation disorder characterized by abdominal pain, tions reveal a promoting role of macrophages in IBD. I diarrhea, and bloody diarrhea (1, 2). Although the exact Macrophages acquire and maintain different phenotypes by guest on September 30, 2021 mechanisms of IBD are not fully understood, breakdown of in- depending on transcriptional and posttranscriptional regulations flammatory homeostasis is reportedly critical for IBD pathogen- (12, 13). Epigenetic modifications are thought to be involved in esis (3–5). Macrophages, a critical member of innate immunity, macrophage activation and differentiation. For instance, DNA are abundant in normal intestines (6, 7). In active IBD, the in- methyltransferase Dnmt1, an epigenetic regulator responsible creased number of macrophages infiltrate into lamia propria (8) for epigenetically silencing target genes via methylating their and produce cytokines, such as Tnf-a (9). Macrophage depletion promoter DNA (14, 15), can hypermethylate PPAR-g pro- reportedly represses the development of spontaneous colitis in moter, consequently inducing the production of proinflammatory Il102/2 mice (10). Furthermore, M-CSF–deficient mice that lack cytokines (Tnf-a and Il-1b) (16). Administration with DNA mature macrophages develop attenuated acute colitis in response methylation inhibitor 5-Aza-2-deoxycytidine or Dnmt1 deletion *Research Center for Tissue Engineering and Regenerative Medicine, Union and the Academic Medical Doctor Supporting Program (2018) of Tongji Medical Hospital, Tongji Medical College, Huazhong University of Science and Technol- College, Huazhong University of Science and Technology. ogy, Wuhan 430022, China; †Department of Gastrointestinal Surgery, Union Hos- L.W., Z.W., and G.W. designed the experiments and wrote the manuscript. S.Q. and pital, Tongji Medical College, Huazhong University of Science and Technology, Y.L. performed the experiments and analyzed the data. Z.D. and M.X. generated the Wuhan 430022, China; and ‡Department of Clinical Laboratory, Union Hospital, Uhrf1fl/flLyz2-Cre mice. Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China The sequences presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136311) under 1S.Q. and Y.L. contributed equally to this work. accession number GSE136311. ORCID: 0000-0002-9330-0728 (Z.W.). Address correspondence and reprint requests to Dr. Guobin Wang, Dr. Lin Wang, and Received for publication April 25, 2019. Accepted for publication September 19, Dr. Zheng Wang, Research Center for Tissue Engineering and Regenerative Medicine, 2019. Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, China. E-mail addresses: [email protected] (G.W.), This work was supported by the Major State Basic Research Development Program [email protected] (L.W.), and [email protected] (Z.W.) of China (973 Program, 2015CB554007), National Natural Science Foundation of China programs (81272559, 81572866, 81502572, 81773263, and 81773104), the The online version of this article contains supplemental material. International Science and Technology Cooperation Program of Chinese Ministry Abbreviations used in this article: BMDM, bone marrow–derived macrophage; ChIP, of Science and Technology (2014DFA32920), the Science and Technology Program chromatin immunoprecipitation; CKO, conditional knockout; DSS, dextran sodium of Chinese Ministry of Education (113044A), the Frontier Exploration Program of sulfate; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; qPCR, quan- Huazhong University of Science and Technology (2015TS153), the Natural Science titative real-time PCR; RIPA, radioimmunoprecipitation assay; Uhrf1, ubiquitin-like Foundation Program of Hubei Province (2015CFA049), the Research Fund of Public with PHD and RING finger domains 1; WT, wild-type. Welfare in Health Industry (201402015) from the Health and Family Plan Committee of China, the Integrated Innovative Team for Major Human Diseases Program (2017), Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900467 2 Uhrf1 AND INFLAMMATORY BOWEL DISEASES promotes M2 macrophage phenotype characteristic of anti- (extensive damage and extension into deeper structure of the bowel wall). inflammation (17, 18). These observations support the idea that For Tnf-neutralizing therapy, the mice were injected i.p. with Tnf-a Ab epigenetic regulators play a key role in regulating the functions (no. 506332; 20 mg/mouse/d; BioLegend) on days 3–7 of the DSS-induced colitis experiment. of macrophages. However, epigenetic regulations on inflamma- tory macrophages during IBD are poorly understood. Bone marrow–derived macrophage Genome-wide association studies implicated the association of The protocol of bone marrow–derived macrophage (BMDM) production IBD with ubiquitin-like with PHD and RING finger domains 1 was according to the work (33). Briefly, the 6–8-wk-old mice were sac- (Uhrf1) (19, 20). Uhrf1 is a key epigenetic regulator (also known rificed. Femurs were cut off and flushed with 10 ml 10% FBS (Life as Np95 in mice and ICBP90 in humans), acting as a primary Technologies) DMEM medium (GE Healthcare Life Sciences). The mass was removed by 40-mm filters (no. REF352350; Corning), and RBCs were mediator of DNA methylation (21–23). Uhrf1 binds to H3K9me2/ lysed with ammonium/chloride/potassium buffer (no. C3720; Beyotime 3 (di- and trimethyl K9 histone H3) of the newly assembled nu- Biotechnology). Suspended cells were collected and cultured with 10% cleosomes, which is a prerequisite for Uhrf1 to recruit Dnmt1 onto FBS (Life Technologies)
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