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Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System This information is current as Zining Wang, Jingyun Ji, Di Peng, Feng Ma, Genhong of October 1, 2021. Cheng and F. Xiao-Feng Qin J Immunol published online 4 April 2016 http://www.jimmunol.org/content/early/2016/04/02/jimmun ol.1502458 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/04/02/jimmunol.150245 Material 8.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 *average by guest on October 1, 2021 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 4, 2016, doi:10.4049/jimmunol.1502458 The Journal of Immunology Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System Zining Wang,* Jingyun Ji,* Di Peng,* Feng Ma,†,‡,x Genhong Cheng,†,‡,x and F. Xiao-Feng Qin*,†,‡ Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in an- tibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in re- sponse to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream Downloaded from kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type–specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-a/b receptor–mediated feed- forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, http://www.jimmunol.org/ including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation. The Journal of Immunology, 2016, 196: 000–000. he rapid and robust induction of type I IFN (IFN-I) is a key (cGAS) and stimulator of IFN genes (STING), recruit downstream step in the activation of host innate immunity by invading adaptor proteins to activate the TANK-binding kinase 1 (TBK1)– T viruses and bacteria (1, 2). During microbial infection, IFN regulatory factor 3 (IRF3) signaling axis, which triggers the pathogen-associated molecular patterns (PAMPs) such as LPS and production of large amounts of IFN-I (3, 4). IRF3 is the master and pathogenic nucleic acids are detected by the pattern recognition primary transcription activator of IFN-b and IFN-a4, the main receptors (PRRs) of the host cells (3, 4). These PRRs, which in- components of the first-wave IFN-I production (5, 6). Phosphory- by guest on October 1, 2021 clude TLRs, retinoic acid–inducible gene I (RIG-I)–like receptors, lation by the serine/threonine kinase TBK1 and homodimerization and cytosolic DNA sensors such as cyclic GMP–AMP synthase are both essential for the transcription activity of IRF3 (5–7). Overall, the induction of IFN-I involves a cascade of events, in- cluding IRF3 phosphorylation, dimerization, nuclear translocation, *Key Laboratory of Gene Engineering of the Ministry of Education and State Key and binding to the promoter and enhancer regions of IFN-I genes. Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China; Although induction of IFN-I is considered the first line of de- †Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; fense against many viral infections (1, 8), it plays detrimental roles ‡Suzhou Institute of Systems Medicine, Suzhou, Jiangsu 215123, China; and xDepart- during bacterial and persistent viral infections (9–12). For exam- ment of Microbiology, Immunology and Molecular Genetics, University of California ple, elevated expression of IFN-I in human lepromatous (L-lep)– Los Angeles, Los Angeles, CA 90095 type lesions suppresses type II IFN–triggered antimycobacterial Received for publication November 20, 2015. Accepted for publication March 5, 2016. responses (11). Chronic IFN-I signaling is associated with hy- This work was supported by National Natural Science Foundation of China Grant perimmune activation and disease progression in persistent lym- 31170832, Guangdong Innovative Research Team Program Grant 201001Y0104687244, phocytic choriomeningitis virus infections (10, 12). Additionally, Ministry of Science and Technology Project Preparation Grant 2014CB745203, and by it is well known that constitutive activation of the TBK1–IRF3 Ministry of Health Grant 201302018. signaling axis leads to excess IFN-I production, which contributes Address correspondence and reprint requests to Prof. F. Xiao-Feng Qin, Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Med- to numerous human autoimmune diseases and autoinflammatory ical Sciences and Peking Union Medical College, Beijing 100005, China and Suzhou syndromes such as rheumatoid arthritis, systemic lupus eryth- Institute of Systems Medicine, Suzhou, Jiangsu 215123, China. E-mail address: ematosus, systemic sclerosis, inflammatory bowel disease, chronic [email protected] obstructive pulmonary disease, and type II diabetes (13–16). The online version of this article contains supplemental material. Several recent studies have also shown that the activity of the Abbreviations used in this article: BiLC, bimolecular luminescence complementation; TBK1–IRF3 signaling axis is elevated in cancer cells and poten- cGAS, cyclic GMP–AMP synthase; CHX, cycloheximide; CRISPR, clustered regularly interspaced short palindromic repeat; Gluc, Gaussia luciferase; IFNAR, IFN-a/b re- tially promotes tumorigenesis (17–19). Therefore, the proper ceptor; IFN-I, type I IFN; IRF3, IFN regulatory factor 3; ISG, IFN-stimulated gene; control of TBK1–IRF3 activation and IFN-I production is criti- ISRE, IFN-sensitive response element; Luc, luciferase; PAMP, pathogen-associated molecular pattern; poly(dA:dT), poly(deoxyadenylic-deoxythymidylic) acid; PRR, pat- cally important. Developing a sensitive and reliable method to tern recognition receptor; RIG-I, retinoic acid–inducible gene I; SeV, Sendai virus; monitor IRF3 activation is crucial for investigating therapeutic in- STING, stimulator of IFN genes; TBK1, TANK-binding kinase 1; VSV, vesicular terventions and taking advantage of the beneficial effect of IFN-I stomatitis virus; WT, wild-type. in host antiviral immunity. Additionally, it will allow us to avoid Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 the detrimental effect of excess IRF3 activation and IFN-I pro- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502458 2 IRF3 ACTIVATION REVEALED BY A DIMERIZATION REPORTER duction in antibacterial immunity, persistent viral infections, and infection, the cells were transferred to 24-well plates, followed by autoimmune diseases. blasticidin (10 mg/ml) and puromycin (2 mg/ml) selection for another Currently, several direct and indirect assays are used to measure 72 h. Cells surviving this double selection were considered to have the IRF3-BiLC reporter stably expressed and were labeled as recombinant IRF3 activation and subsequent IFN-I production, including the HEK293T (IRF3-BiLC) and THP-1 (IRF3-BiLC) cells. IFN-b promoter luciferase reporter, IFN-stimulated response ele- ment (ISRE) luciferase reporter, native PAGE Western blot for Activation and inhibition of signaling pathway IRF3 dimerization, SDS-PAGE Western blot and immunohisto- To activate recombinant HEK293T or THP-1 cells, they were stimulated chemistry for phosphorylated IRF3, and Western blot to detect with LPS (1 mg/ml), VSV (MOI of 1), or SeV (50 hemagglutinating units/ IRF3 nuclear translocation. However, these assays also have im- ml) or transfected with poly(I:C) or poly(dA:dT) using Lipofectamine 2000 (Life Technologies) as per the manufacturer’s instructions. Prior to portant limitations. The IFN-b
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