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Effector Gene Expression Potential to Th17 Cells by Promoting Microrna

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Effector Gene Expression Potential to Th17 Cells by Promoting Microrna MicroRNA-155 Confers Encephalogenic Potential to Th17 Cells by Promoting Effector Gene Expression This information is current as Ruozhen Hu, Thomas B. Huffaker, Dominique A. Kagele, of October 1, 2021. Marah C. Runtsch, Erin Bake, Aadel A. Chaudhuri, June L. Round and Ryan M. O'Connell J Immunol 2013; 190:5972-5980; Prepublished online 17 May 2013; doi: 10.4049/jimmunol.1300351 http://www.jimmunol.org/content/190/12/5972 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/05/17/jimmunol.130035 Material 1.DC1 http://www.jimmunol.org/ References This article cites 45 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/190/12/5972.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 1, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology MicroRNA-155 Confers Encephalogenic Potential to Th17 Cells by Promoting Effector Gene Expression Ruozhen Hu,* Thomas B. Huffaker,* Dominique A. Kagele,* Marah C. Runtsch,* Erin Bake,* Aadel A. Chaudhuri,† June L. Round,* and Ryan M. O’Connell* Th17 cells are central to the pathogenesis of autoimmune disease, and recently specific noncoding microRNAs have been shown to regulate their development. However, it remains unclear whether microRNAs are also involved in modulating Th17 cell effector functions. Consequently, we examined the role of miR-155 in differentiated Th17 cells during their induction of experimental au- toimmune encephalomyelitis. Using adoptive transfer experiments, we found that highly purified, myelin oligodendrocyte glyco- protein Ag-specific Th17 cells lacking miR-155 were defective in their capacity to cause experimental autoimmune encephalomyelitis. Gene expression profiling of purified miR-1552/2IL-17F+ Th17 cells identified a subset of effector genes that are dependent on miR-155 for their proper expression through a mechanism involving repression of the transcription factor Ets1. Among the genes Downloaded from reduced in the absence of miR-155 was IL-23R, resulting in miR-1552/2 Th17 cells being hyporesponsive to IL-23. Taken together, our study demonstrates a critical role for miR-155 in Th17 cells as they unleash autoimmune inflammation and finds that this occurs through a signaling network involving miR-155, Ets1, and the clinically relevant IL-23–IL-23R pathway. The Journal of Immunology, 2013, 190: 5972–5980. utoimmunity occurs when dysregulated, autoreactive im- how miRNAs fit into the known regulatory circuits underlying mune cells inappropriately respond to self-Ags and cause Th17 cell biology remains an important area of investigation. http://www.jimmunol.org/ unwarranted inflammation that is destructive to sophis- miRNAs are small, ssRNA molecules that negatively regulate A + ticated tissue systems (1). Recently, Th17 cells, a subset of CD4 target gene expression posttranscriptionally. Specific miRNAs T cells defined by their expression of IL-17 cytokines, have emerged have been shown to support proper development of immune cells as key drivers of tissue inflammation. Th17 cells promote both the in mammals and have just recently been implicated in autoimmu- onset and persistence of inflammatory responses during autoimmune nity (20, 21). Among the miRNAs expressed in immune cells is disorders including multiple sclerosis, arthritis, psoriasis, lupus, and miR-155, which modulates the development of various inflam- inflammatory bowel disease (2, 3). matory T cell subsets, including Th1, Th17, and regulatory T cells Because of their central roles in driving disease, significant effort (18, 22–26). Demonstrating its importance during inflammation by guest on October 1, 2021 has gone into understanding the genes and pathways that regulate in vivo, we and others have recently shown that miR-1552/2 mice + Th17 cell development. Skewing of naive CD4 T cells toward the are highly resistant to distinct mouse models of autoimmunity, Th17 lineage is driven by the cytokines IL-6 and TGF-b,which including experimental autoimmune encephalomyelitis (EAE)— induce Th17 cell signature genes through such factors as Stat3, a model of human multiple sclerosis, T cell–dependent colitis, and RORgt, Ahr, Batf, and Irf4 (4-12). Furthermore, differentiated collagen-induced arthritis (18, 24, 27). Furthermore, dysregulated Th17 cells must receive additional signals from cytokines such as expression of miR-155 is also observed in mice and people with IL-23 to expand and achieve full inflammatory potential in vivo various types of autoimmune disorders (24, 28–30). Although (13–17). Recently, noncoding microRNAs (miRNAs) have also these reports reveal a prominent, clinically relevant role for miR- been found to regulate Th17 cell development (18, 19). However, 155 during autoimmunity, they have not determined whether this is a consequence of reduced inflammatory T cell numbers or compromised effector cell function. Furthermore, it remains un- *Division of Microbiology and Immunology, Department of Pathology, University of clear how miR-155 functions at the molecular level to instruct † Utah, Salt Lake City, UT, 84112; and Department of Radiation Oncology, Stanford Th17 cell biology. University School of Medicine, Stanford, CA, 94305 In the current study, we have investigated the role of miR-155 Received for publication February 6, 2013. Accepted for publication April 8, 2013. in differentiated Th17 cells. This was accomplished by generat- This work was supported by National Institutes of Health Grant 5R00HL102228-04. ing a novel mouse strain that allows for the isolation and analy- The microarray data presented in this article have been deposited in the National sis of viable, miR-1552/2 IL-17F–expressing CD4+ T cells that Center for Biotechnology Information Gene Expression Omnibus database (http:// www.ncbi.nlm.nih.gov/geo/) under accession number GSE45122. specifically recognize the myelin oligodendrocyte glycoprotein 2/2 Address correspondence and reprint requests to Dr. Ryan M. O’Connell, Division (MOG)35–55 Ag. Our studies find that purified miR-155 Th17 of Microbiology and Immunology, Department of Pathology, University of Utah, 15 cells are extremely defective in causing EAE following adoptive North Medical Drive East, JMRB, Salt Lake City, UT, 84112. E-mail address: ryan. [email protected] transfer when compared with wild-type (Wt) controls. Further- The online version of this article contains supplemental material. more, we demonstrate that miR-155 directly targets the tran- scription factor Ets1 to regulate a subset of Th17 cell–effector Abbreviations used in this article: EAE, experimental autoimmune encephalomyeli- 2/2 tis; miRNA, microRNA; MOG, myelin oligodendrocyte glycoprotein; qPCR, quan- genes, which includes the IL-23R. Consequently, miR-155 titative real-time PCR; RFP, red fluorescent protein; shRNA, short hairpin RNA; 39- Th17 cells are hyporesponsive to IL-23, revealing a new link UTR, 39-untranslated region; Wt, wild-type. between miR-155 and the highly relevant IL-23–IL-23R pathway Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 (31–36). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300351 The Journal of Immunology 5973 Materials and Methods bus database under accession number GSE45122 (http://www.ncbi.nlm. Mice nih.gov/geo/). All experiments were approved by the University of Utah Institutional ELISAs Animal Care and Use Committee. miR-155+/+, miR-1552/2, Rag12/2, IL- 2 ELISAs to detect expression of IL-17A and IFN-g were performed with 17F red fluorescent protein (RFP)+/ , 2D2 TCR Tg, and combinations of 2 2 cytokine-specific kits (eBioscience), according to the manufacturer’s these mice are all on a C57BL/6 genetic background. miR-155 / mice 2 2 instructions. were crossed with IL-17F RFP+/+ to create miR-155+/ IL-17F RFP+/ +/+ mice, which were crossed further to create both miR-155 and miR- Immunoblotting 1552/2IL-17F RFP+/2 mice. miR-155+/+IL-17F RFP+/+ and miR-1552/2 IL-17F RFP+/+ mice were crossed with Wt 2D2+ to create miR-155+/+IL- Cell pellets were lysed in 8 M urea buffer. Protein extracts were subjected 17F RFP+/22D2+ and miR-1552/2IL-17F RFP+/22D2+ mice, respectively. to gel electrophoresis and transferred onto a nitrocellulose membrane, Genotyping was performed as described previously (22, 37, 38). followed by Ab staining (Ets1 and b-actin; Santa Cruz Biotechnology) and detection as described previously (40). Expression levels were quantified Mouse models of EAE using National Institutes of Health ImageJ software. EAE was induced in mice as described previously (18). Briefly, MOG35–55 Luciferase reporter assays peptide (BD Biosciences) was emulsified in CFA (100 mg/ml) and injected s.c. into the base of the mouse’s tail. Pertussis toxin was injected i.p. into A region of the 39-untranslated region (39-UTR) of mouse Ets1 containing mice on days 0 and 2. Clinical symptoms of EAE were scored according to the conserved miR-155 binding site was cloned downstream from lucif- the following criteria: 0, no symptoms; 0.5, partially limp tail; 1, com- erase in the pmiReport plasmid. Site-directed mutagenesis was used to pletely limp tail; 1.5, impaired righting reflex; 2, hind-limb paresis; 2.5, disrupt the seed sequence. The forward and reverse primer sequences for hind-limb paralysis; 3, forelimb weakness; 4, complete paralysis; and 5, cloning Ets1 are 59-gtaactagtTACCCGAAACATGGAAGACTC-39 and death.
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