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 antibacterial 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 response 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, including 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 element (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 promoter reporter and ISRE re- poly(I:C) (5 mg/ml) or poly(dA:dT) (2 mg/ml) transfection, HEK293T or porter are promiscuous and may reflect the activation of IRF3 due THP-1 cells were treated with recombinant human TNF-a (10 ng/ml), IL- to other downstream factors, such as AP-1 and NF-kB (20). It is 1b (10 ng/ml), or IFN-g (10 ng/ml) for 12 h, or treated with CHX (20 rather difficult and time-consuming to measure IRF3 phosphory- mg/ml), TPCA-1 (1 mM), or GLPG0634 (2 mM) for 1 h. lation, dimerization, and nuclear translocation by Western blot, in IRF3-BiLC dimerization reporter and Dual-Luciferase particular for large-scale and extensive kinetic studies. Moreover, reporter assay none of these methods can be used directly and in a real-time manner to monitor IRF3 activation in vitro or in vivo. IRF3-BiLC reporters (20 ng each vector) or the indicated overexpression construct were transfected into HEK293T cells by Lipofectamine 2000. In this study, we created an IRF3 dimerization luciferase reporter Twenty-four hours after transfection, these cells were activated with the based on bimolecular luminescence complementation (BiLC) tech- indicated stimuli for another 12 h. Using the Renilla luciferase assay system nology, which provides a highly specific, sensitive, and direct readout (Promega, Madison, WI), the cells were lysed by Renilla lysis buffer, and Downloaded from of IRF3 activation. Using this novel IRF3 dimerization reporter the Gluc activity of the IRF3-BiLC reporter was measured according to the manufacturer’s instructions. HEK293T (IRF3-BiLC) or THP-1 (IRF3- system, we have observed distinct temporal patterns of IRF3 dimer- BiLC) cells were activated by the indicated amount of stimuli for the in- ization and activation in different cell types during their response to dicated times. The cells were lysed and the Gluc activity of IRF3-BiLC multiple PAMPs. We also identified previously unrecognized positive reporter was measured as described above. The ISRE-luciferase (Luc) re- regulation of IFN-a/b receptor (IFNAR)1 downstream signaling and porter construct and overexpression vectors were cotransfected with Renilla crosstalk with the NF-kB pathway, which occurred at the point luciferase reporter into HEK293T cells by Lipofectamine 2000. Twenty-four http://www.jimmunol.org/ hours after transfection, the cells were lysed using passive lysis buffer, and of IRF3 dimerization. the firefly luciferase activity of the ISRE-Luc reporter was measured and normalized by Renilla luciferase activity according to the Dual-Luciferase reporter assay system (Promega) protocol and a previous study (21). Material and Methods Western blot analysis of SDS-PAGE and native PAGE Cell culture and reagents For immunoblot analysis, cells were collected in Renilla or Triton X-100 HEK293T and THP-1 cell lines were obtained from American Type Culture lysis buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Collection (Manassas, VA). HEK293T cells were cultured in DMEM and Triton X-100, and 5% glycerol) containing complete protease inhibitors THP-1 cells were maintained in RPMI 1640 medium. Both media were

and phosphatase inhibitors (Roche). The extracts were loaded and sub- by guest on October 1, 2021 supplemented with 10% FCS (Thermo Fisher Scientific), 100 U/ml penicillin/ jected to SDS-PAGE, transferred onto a polyvinylidene difluoride mem- streptomycin, and 2 mM L-glutamine. Poly(deoxyadenylic-deoxythymidylic) brane (Millipore), probed with various Abs as indicated, and developed acid [poly(dA:dT)] was purchased from InvivoGen (San Diego, CA). LPS, using an ECL reagent (Millipore) and imaged using a ChemiDoc instru- poly(I:C), and cycloheximide (CHX) were from Sigma-Aldrich (St. Louis, ment (Bio-Rad). Native PAGE was performed as described previously MO). Recombinant human IFN-a,IFN-b,IFN-g,TNF-a, and IL-1b were (22). Cell extracts were subjected to native PAGE and transferred to a from PeproTech (Rocky Hill, NJ). Vesicular stomatitis virus (VSV) and polyvinylidene difluoride membrane, then probed and imaged using ECL. Sendai virus (SeV) were from Dr. Genhong Cheng’s laboratory (University of California Los Angeles). TPCA-1 and GLPG0634 were from Medchem Clustered regularly interspaced short palindromic repeat/ Express (Monmouth Junction, NJ). Anti–Gaussia luciferase (Gluc; E8023S) Cas9–mediated knockout of TBK1 and IFNAR1 in was from New England BioLabs; anti-TBK1 (3504S) and anti–p-Stat1 (9167S) were from Cell Signaling Technology (Danvers, MA); anti-Flag HEK293T cells (M2) (A8592) and anti–b-actin (A1978) were from Sigma-Aldrich; anti- To generate TBK12/2 and IFNAR12/2 cells, HEK293T cells were seeded IRF3 (sc-9082) and anti-STAT1 (sc-346) Abs were from Santa Cruz Bio- into 24-well plates one day prior to transfection at a density of 150,000 technology (Dallas, TX). Donkey anti-rabbit IgG(H+L) secondary Ab was cells per well. For each well of a 24-well plate, the cells were transfected from Jackson ImmunoResearch Laboratories (West Grove, PA). with clustered regularly interspaced short palindromic repeat (CRISPR)/ IRF3-BiLC reporter constructs Cas9 (300 ng) and single guide RNA (300 ng) expression plasmids by Lipofectamine 2000. The targeting sequences of TBK1 and IFNAR1 by IRF3-BiLC reporter constructs were cloned into the lentiviral vector backbone single guide RNAs were 59-GGAAATATCATGCGTGTTAT-39 and 59- FG11F plasmid (U.S. patent 20120201794 A1). GlucN (17–93 aa) and GlucC GACCCTAGTGCTCGTCGCCG-39, respectively. At 48 h after transfec- (94–185 aa) fragments (GenBank no. AY015993) were inserted between tion, cells were seeded into 96-well plates at a density of 0.5 cell per well 2/2 AscI and RsrII restriction sites of the FG11F vector. IRF3 (wild-type [WT]) with 100 ml DMEM. After amplification for 2–3 wk, the TBK1 and 2/2 and IRF3 (S386A) open reading frames were cloned into IRF3-BiLC re- IFNAR1 knockout cells were verified by Western blot, Dual-Luciferase porter system via the Gateway recombination cloning system (Invitrogen). assay, and genomic DNA PCR sequencing. Stable cell lines harboring IRF3-BiLC reporter HEK293T cells were seeded in 24-well plates the day before transfection. Results The lentiviral FG-EH-IRF3-GlucN and FG-EH-IRF3-GlucC plasmid were Design and verification of the IRF3 dimerization reporter cotransfected into HEK293T cells (50–60% confluency) together with lentiviral packaging plasmids psPAX2 and pMD2.G (Addgene nos. 12260 To create a reliable and sensitive system for quantifying the ac- and 12259, respectively) by calcium phosphate–mediated transfection tivation of the TBK1–IRF3 axis of IFN signaling, we designed an method. Ten hours after transfection, the media were replaced with 1 ml IRF3 dimerization reporter by taking advantage of BiLC tech- fresh prewarmed DMEM supplemented with 10% FCS. Forty-eight hours nology (23, 24). In this IRF3-BiLC reporter system, IRF3 was later, the IRF3-BiLC lentiviral particles were harvested for gene transduction. HEK293T or THP-1 cells (5000 cells/well) were seeded in 96-well fused with the N-terminal or C-terminal of split Gluc tags and plates for 18 h. These cells were transduced with the lentiviral IRF3-GlucN cloned into a construct with a lentiviral backbone (25), which can and lentiviral IRF3-GlucC viruses (100 mlplus100ml). At 72 h after be used to deliver the reporter in vitro and in vivo (Fig. 1A). Ideally, The Journal of Immunology 3 once viral or bacterial infection triggers PRRs, TBK1 is activated confirmed that the luciferase activity of the IRF3-BiLC reporter and subsequently phosphorylates IRF3, which leads to the dimer- highly correlates with IRF3 dimerization. Next, we overexpressed ization of IRF3. Dimerization will also lead to the reconstitution PRRs or their adaptor genes to activate IRF3 or NF-kB in HEK293T of the two halves of the Gluc. Therefore, IRF3 activation can be (IRF3-BiLC) cells and found that overexpression of IRF3 upstream measured using a simple standard bioluminescence assay (Fig. 1B). signaling molecules TIR domain-containing adapter protein induc- To test the validity of our IRF3-BiLC reporter system, we genet- ing IFN-b, RIG-I (2CARD), melanoma differentiation-associated ically engineered two cell lines, HEK293T (IRF3-BiLC) and THP-1 protein 5, mitochondrial antiviral-signaling protein, and cGAS/ (IRF3-BiLC), which stably expressed the paired IRF3-BiLC ex- STING induced IRF3-BiLC luciferase activity, but not MyD88 pression vectors via the lentiviral transduction system. Both cell lines (Fig.1E),whichisanNF-kB–stimulating factor. Collectively, these were then challenged with different cytokines, synthetic mimics of results validate the ability of the IRF3-BiLC reporter system to pathogen nucleic acids, and live viruses. These treatments are known monitor the activation of IRF3. to cause the activation of TBK1–IRF3 and/or NF-kB. We found that TBK1 is required for the activation of the IRF3-BiLC reporter treatment with poly(I:C), poly(dA:dT), or SeV robustly triggered IRF3-BiLC luciferase activity in HEK293T (IRF3-BiLC) cells TBK1 works as an essential kinase for its downstream IRF3 ac- (Fig. 1C). Similarly, significant elevation of IRF3-BiLC luciferase tivation and subsequent type I IFN production (7). To further activity was detected in THP-1 (IRF3-BiLC) cells after challenge by test the stringency of the IRF3-BiLC reporter, we generated a 2 2 LPS,poly(I:C),poly(dA:dT),orVSV(Fig.1D).However,TNF-a TBK1 / HEK293T cell line using the CRISPR/Cas9 gene and IL-1b treatment, which only activates NF-kB signaling, trig- knockout technique (26, 27). Knockout of TBK1 abolished the gered no IRF3-BiLC luciferase activity (Fig. 1C, 1D). These results ISRE luciferase activity induced by SeV infection (Fig. 2A), Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Design and verification of the IRF3-BiLC reporter system. (A) IRF3 with its N-terminal or C-terminal Gluc tags were cloned into FG11F lentiviral backbone vector to create an IRF3-BiLC reporter system, which can be used for transient and stable transfection for most cell types. (B) Principle of the IRF3-BiLC reporter system. Activation of PRRs by the bacterial and viral component triggers TBK1 phosphorylation and subsequently phosphorylates the downstream transcription factor IRF3. Phosphorylated IRF3 forms a homodimer complex, which makes the two split Gluc tags restore the luciferase enzyme activity and catalyze the oxidative decarboxylation of the substrate coelenterazine (CTZ), resulting in emission of light. (C) HEK293T (IRF3-BiLC) cells were treated with TNF-a, IL-1b, or infected with SeV, or transfected with poly(I:C) or poly(dA:dT) for indicated time points. IRF3- BiLC luciferase activity was measured. (D) THP-1 (IRF3-BiLC) cells were treated with TNF-a, IL-1b, or LPS, infected with VSV, or transfected with poly(I:C) or poly(dA:dT) for indicated time points. IRF3-BiLC luciferase activity was measured. (E) HEK293T (IRF3-BiLC) cells were transfected with expression plasmids encoding 50 ng empty vector (EV), MyD88, TIR domain-containing adapter protein inducing IFN-b (TRIF), RIG-I (2CARD), melanoma differentiation-associated protein (MDA-5), mitochondrial antiviral-signaling protein (MAVS), or cGAS plus STING (20 ng plus 40 ng) for 24 h, after which IRF3-BiLC luciferase activity was measured. Gluc tag expression of IRF3-transfected cells described in (C)–(E) was measured by Western blot, and the b-actin levels are shown as a loading control. Luciferase activity data of (C)–(E) are from three independent experiments (mean 6 SEM). ***p , 0.001 (Student t test). 4 IRF3 ACTIVATION REVEALED BY A DIMERIZATION REPORTER confirming the establishment of TBK12/2 cells. As expected, the whereas no luciferase activity was detected from the IRF3-BiLC 2 2 induction of IRF3-BiLC luciferase activity was also diminished in reporter (Fig. 2G). Therefore, using TBK1 / cells and side-by-side TBK12/2 cells when challenged by SeV (Fig. 2B), which was comparisons of the ISRE luciferase reporter and IRF3-BiLC reporter consistent with the absence of dimer formation of endogenous IRF3 activity, we confirmed that the IRF3-BiLC reporter is exclusively in SeV-infected TBK12/2 cells (Fig. 2C). Additionally, IRF3-BiLC dependent on the bona fide upstream signaling and can faithfully luciferase activity induced by poly(dA:dT) stimulation was also distinguish the stimuli with significant sensitivity and specificity. 2/2 obliterated in TBK1 cells (Fig. 2D). Nucleic acid sensors such as The S386 phosphorylation site is required for the activation of RIG-I and cGAS/STING could potently activate TBK1–IRF3 sig- the IRF3-BiLC reporter naling and induce downstream IFN-I production (28–30). However, To further demonstrate the fidelity and sensitivity of the IRF3-BiLC neither ISRE nor IRF3-BiLC luciferase activities were detected in reporter in monitoring the activation of the TBK1–IRF3 signaling 2/2 RIG-I (2CARD)– or cGAS/STING-overexpressed TBK1 cells axis, we created an IRF3 (S386A)–BiLC reporter, in which a (Fig. 2E, 2F). To further confirm the specificity of the IRF3-BiLC critical phosphorylation site of the IRF3 was mutated. Compared reporter for monitoring IRF3 activation, we showed that the treat- to the reporter using WT IRF3, the enforced expression of the ment of IFN-I (IFN-a and IFN-b) or type II IFN (IFN-g)could IRF3 (S386A) mutant could not activate the ISRE luciferase induce high levels of luciferase activity from the ISRE reporter, (Fig. 3A), verifying the importance of this phosphorylation site for Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. TBK1 is required for the activation of IRF3-BiLC reporter. (A) WT and TBK12/2 HEK293T cells were transfected with ISRE-Luc (50 ng) for 24 h, and then these cells were infected with SeV for 12 h, after which ISRE-Luc firefly luciferase activity was measured and normalized by Renilla luciferase activity. Relative ISRE-Luc activity was shown. (B) WT and TBK12/2 HEK293T cells were transfected with IRF3-BiLC reporter for 24 h, and then these cells were infected with SeV for 12 h, after which IRF3-BiLC luciferase activity was measured. Gluc tag expression of IRF3-transfected cells was measured by Western blot, and the b-actin levels were shown as a loading control. (C) WT and TBK12/2 HEK293T cells were transfected and infected as described in (B). IRF3 dimer and monomer in the cells were measured by Western blot (native PAGE). TBK1 and IRF3 were measured by Western blot (SDS-PAGE), and the b-actin levels are shown as a loading control. (D)WTandTBK12/2 HEK293T cells were transfected with IRF3-BiLC reporter for 24 h, and then these cells were activated by transfection of poly(dA:dT) for another 12 h, after which IRF3-BiLC luciferase activity was measured. (E) WT and TBK12/2 HEK293T cells were transfected with ISRE-Luc (50 ng) and 50 ng empty vector, RIG-I (2CARD), or cGAS plus STING (20 ng plus 40 ng) for 24 h. ISRE-Luc firefly luciferase activity was measured and normalized by Renilla luciferase activity. Relative ISRE-Luc activity is shown. (F) WT and TBK12/2 HEK293T cells were transfected with IRF3-BiLC reporter and other overexpression vectors as described in (E) for 24 h. IRF3-BiLC luciferase activity was measured. (G) HEK293T cells were transfected with ISRE-Luc (50 ng) or IRF3-BiLC reporter for 24 h, and then these cells were treated with 10 ng/ml IFN-a, IFN-b, or IFN-g for 24 h, after which ISRE-Luc (left) and IRF3-BiLC (right) luciferase activity was measured. Gluc tag expression of IRF3-transfected cells was measured by Western blot, and the b-actin levels were shown as a loading control. Luciferase activity data of (A), (B), and (D)– (G) are from three independent experiments (mean 6 SEM). Western blot data of (B), (C), and (G) are representative of three independent experiments. ***p , 0.001 (Student t test). The Journal of Immunology 5

IRF3 dimerization and activation of downstream genes (31). As THP-1 is distantly related to a monocytic lineage of WBCs. These expected, no luciferase activity from the IRF3 (S386A)–BiLC two cell types together cover host responses to a broad range of reporter could be detected in the HEK293T cells infected by SeV, common and distinctive PAMPs and live viruses. Interestingly, whereas cells delivered the WT IRF3-BiLC reporter showed sig- poly(I:C), poly(dA:dT), and SeV trigger prolonged and continu- niﬁcant luciferase activity upon infection (Fig. 3B), which is ous IRF3 dimerization in HEK293T cells (Fig. 4A–C). However, consistent with the lack of dimer formation in S386A mutant– THP-1 cells only showed transient IRF3 dimer formation, peaking transfected cells (Fig. 3C). Additionally, neither transfection with at ∼12 h in response to both poly(I:C) and poly(dA:dT) stimula- poly(dA:dT) nor overexpression of RIG-I (2CARD) or cGAS/ tion (Fig. 4D, 4E). Interestingly, LPS stimulation led to an even STING could induce the luciferase activity of the IRF3 shorter period of IRF3-BiLC activity and IRF3 dimerization, (S386A)–BiLC reporter (Fig. 3D, 3E). Thus, these data verify that which peaked at 2 h (Fig. 4F). In contrast, continuous elevation of the S386 phosphorylation site is required for the activation of IRF3-BiLC luciferase activity was observed in THP-1 cells in- IRF3, and they indicate that IRF3-BiLC reporter luciferase ac- fected with SeV and VSV (Fig. 4G, 4H). Thus, these results tivity is completely determined by S386 phosphorylation- revealed that different cell types exhibit a characteristic temporal dependent IRF3 activation. pattern of IRF3 activation in response to different pathogens. Distinct temporal patterns of IRF3 dimerization in different IFNAR1 downstream signaling forms a positive feedback loop cell types that directly modulates IRF3 activation Activation of the TBK1–IRF3 signaling axis and production of Numerous genes, particularly the IFN-stimulated genes (ISGs), are IFN-I are sequential and temporally linked events during viral and induced during host antiviral immune responses (8, 32). We sought bacterial infection, and they determine the outcome of host anti- to determine whether these inducible genes are involved in the Downloaded from microbial immune responses (3, 4). Thus far, the temporal pattern feedback regulation of IRF3 activation. We found that treatment of IRF3 activation during the host response to various pathogens with CHX, which blocks new protein synthesis, signiﬁcantly at- has not been well characterized. Thinking that the IRF3-BiLC tenuated IRF3-BiLC reporter activity in poly(I:C)-activated reporter system could potentially be used for this purpose, we HEK293T and THP-1 cells (Fig. 5A, 5B). As the IFNAR- created HEK293T and THP-1 IRF3-BiLC reporter cell lines. dependent signaling pathway is known to be essential for signal

HEK293T is a cell type of epithelial/ﬁbroblast origin, whereas ampliﬁcation and induction of downstream ISGs, we set out to test http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. S386 phosphorylation site is required for the activation of IRF3-BiLC reporter. (A) HEK293T cells were transfected with ISRE-Luc reporter (50 ng) together with 100 ng empty vector, IRF3 (WT), or IRF3 (S386A) for 24 h. ISRE-Luc ﬁreﬂy luciferase activity was measured and normalized by Renilla luciferase activity. Relative ISRE-Luc activity is shown. Flag tag expression of WT and mutant IRF3-transfected cells was measured by Western blot, and the b-actin levels are shown as a loading control. (B) HEK293T cells were transfected with IRF3 (WT) or IRF3 (S386A) BiLC reporter or control plasmids, and 24 h after transfection, the cells were infected with SeV for 12 h, after which IRF3-BiLC luciferase activity was measured. Gluc tag expression of WT and mutant IRF3-transfected cells was measured by Western blot, and the b-actin levels are shown as a loading control. (C) HEK293T cells were transfected and infected as described in (B), IRF3 dimer and monomer in the cells were measured by Western blot (native-PAGE), and Flag tag expression was measured by Western blot (SDS-PAGE). The b-actin levels are shown as a loading control. (D) HEK293T cells were transfected as described in (B), and then the cells were activated by transfection of poly(dA:dT) for 12 h, after which IRF3-BiLC luciferase activity was measured. (E) HEK293T cells were transfected with IRF3 (WT) or IRF3 (S386A) BiLC reporter and 50 ng empty vector, RIG-I (2CARD), or cGAS plus STING (20 ng plus 40 ng) for 24 h. IRF3-BiLC luciferase activity was measured. Gluc tag expression of WT and mutant IRF3-transfected cells was measured by Western blot, and the b-actin levels are shown as a loading control. Luciferase activity data of (A), (B), (D), and (E) are from three independent experiments (mean 6 SEM). Western blot data of (A), (B), (C), and (E) are representative of three independent experiments. ***p , 0.001 (Student t test). 6 IRF3 ACTIVATION REVEALED BY A DIMERIZATION REPORTER

FIGURE 4. Distinct temporal patterns of IRF3 dimerization were observed in different cell types. (A–C) HEK293T (IRF3- BiLC) cells were transfected with poly(I:C) (A), poly(dA:dT) (B), or infected with SeV (C) for indicated time points. IRF3-BiLC luciferase activity was measured. Gluc expression levels in the cells were measured by Western blot, and the b-actin levels are shown as a loading control. (D–H) THP-1 (IRF3-BiLC) cells were transfected with poly(I:C) (D), poly(dA:dT) (E), or treated with LPS (F), or infected with SeV (G)or

VSV (H) for indicated time points. IRF3- Downloaded from BiLC luciferase activity was measured. Gluc expression levels in the cells were measured by Western blot, and the b-actin levels are shown as a loading control. Lu- ciferase activity data are from three independent experiments (mean 6 SEM).

Western blot data are representative of three http://www.jimmunol.org/ independent experiments. by guest on October 1, 2021 whether IFNAR signaling can modulate IRF3 activation during that IFNAR signaling can positively regulate IRF3 dimerization host response to viral infection and PAMP stimulation. To this during host antiviral immune responses. end, we generated an IFNAR12/2 HEK293T cell line using the NF-kB signaling positively regulates IRF3 activation CRISPR/Cas9 system (Supplemental Fig. 1A). Induction of ISRE luciferase activity (Supplemental Fig. 1B) and STAT1 phosphor- NF-kB–dependent genes are also induced during host antiviral ylation by IFN-b (Supplemental Fig. 1C) were absent in immune responses (2–4). We thus went on to determine the po- IFNAR12/2 cells, indicating that IFNAR1 downstream signaling tential crosstalk between the NF-kB and TBK1–IRF3 pathways. was indeed abolished in this knockout cell line. Interestingly, HEK293T cells expressing IRF3-BiLC were pretreated with the when we examined IRF3-BiLC reporter activation induced by NF-kB inhibitor TPCA-1 (33, 34), then stimulated with poly(I:C) poly(I:C) and poly(dA:dT) in IFNAR12/2 HEK293T cells, we and poly(dA:dT). Surprisingly, inhibition of NF-kB signaling observed that IRF3 activation is markedly attenuated in the ab- markedly attenuated IRF3-BiLC luciferase activity (Fig. 6A, 6B). sence of IFNAR1 (Fig. 5C, 5D), suggesting that IFNAR-dependent Moreover, treatment with TNF-a, which is known to activate NF- signaling is critical for the optimal IRF3 dimerization in kB signaling, potentiated poly(I:C)- and poly(dA:dT)-induced response to viral infections. As extended from this experiment, IRF3 dimerization in a dose-dependent manner (Fig. 6C, 6D, we found that addition of GLPG0634, a JAK1 inhibitor that can Supplemental Fig. 2C, 2D). Additionally, we found that the block IFNAR downstream signaling, also attenuated poly(I:C)- TPCA-1 pretreatment completely abolished the TNF-a–mediated triggered IRF3 dimerization in both HEK293T and THP-1 cells positive regulation of IRF3 dimerization in HEK293T stimulated (Fig. 5E, 5F). Consistent with these results, IFN-b pretreatment by poly(I:C) and poly(dA:dT) (Fig. 6E, 6F).Furthermore, similar potentiated poly(I:C)- and poly(dA:dT)-induced IRF3-BiLC lu- phenomena were also observed in THP-1 cells: TPCA-1 pre- ciferase activity in WT but not IFNAR12/2 cells (Fig. 5G–J, treatment attenuated poly(I:C)-induced IRF3 activation, whereas Supplemental Fig. 2A, 2B). Further experiments showed that IFN- TNF-a pretreatment enhanced it (Fig. 6G, 6H). Thus, these results b–induced boosting of IRF3 activation was mainly mediated by collectively point to the fact that NF-kB signaling positively JAK/TYK downstream of IFNAR, as GLPG0634 can largely regulates IRF3 dimerization. dampen such potentiation (Fig. 5K–N). However, JAK/TYK may not be the only pathway downstream of IFNAR acting in the Discussion positive feedback regulation of IRF3 activation. As in the case of Proper activation of IRF3 is essential for host antiviral immunity, poly(dA:dT) stimulation (Fig. 5L, 5N), IFN-b–induced elevated whereas overactivation of IRF3 can lead to autoimmune inﬂam- IRF3-BiLC reporter activity was not completely blocked by matory diseases and tumorigenesis (1, 14, 17–19). Therefore, GLPG0634. Taken together, we have provided unequivocal evidence quantiﬁcation of IRF3 dimerization in various physiological and The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. IFNAR1 downstream signaling positively regulates IRF3 dimerization. (A and B) HEK293T (IRF3-BiLC) (A) and THP-1 (IRF3-BiLC) (B) cells were pretreated with CHX for 1 h, and these cells were transfected with poly(I:C) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (C and D) WT and IFNAR12/2 HEK293T (IRF3-BiLC) cells were transfected with poly(I:C) (C) or poly(dA:dT) (D) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (E and F) HEK293T (IRF3-BiLC) (E) or THP-1 (IRF3-BiLC) (F) cells were pretreated with GLPG0634 for 1 h and then transfected with poly(I:C) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (G and H) HEK293T (IRF3-BiLC) cells were pretreated with IFN-b and then transfected with poly(I:C) (G) or poly(dA:dT) (H) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (I and J) WT and IFNAR12/2 HEK293T (IRF3-BiLC) cells were pretreated with IFN-b for 12 h and then transfected with poly(I:C) (I) or poly(dA:dT) (J) for 12 h, after which IRF3-BiLC luciferase activity was measured. (K–N) HEK293T (IRF3-BiLC) and THP-1 (IRF3-BiLC) cells were pretreated with GLPG0634 for 1 h, treated with IFN-b for 12 h, and then transfected with poly(I:C) or poly(dA:dT) for 10 h, after which IRF3-BiLC luciferase activity was measured. Data are from three independent experiments (mean 6 SEM). *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test). pathophysiological conditions is very important. However, there cell lines. Importantly, although activation of the NF-kB pathway have only been indirect or relatively complicated approaches for often coincides with IFN signaling, it will not induce luciferase measuring IRF3 activation thus far. Currently available ap- activity in this reporter system. As expected, knockout of the IRF3 proaches such as measuring the luciferase activity of the IFN-b upstream kinase TBK1, or mutation of the critical serine phos- promoter reporter, ISRE reporter, or detecting phosphorylated phorylation site of the IRF3, will completely abolish the IRF3- IRF3 and IRF3 dimers by Western blot are indirect and time- BiLC luciferase activity, which can be triggered by SeV and poly consuming. In this study, we report the development of a novel (dA:dT). In addition to its speciﬁcity and sensitivity, the IRF3- BiLC-based IRF3 dimerization reporter system, IRF3-BiLC, BiLC reporter system makes the detection of IRF3 activation which generates strong luciferase activity as soon as the IRF3 much easier and faster than previous methods. Thus, our novel dimer forms. The IRF3-BiLC reporter system is highly reliable, IRF3-BiLC reporter system can be used for high-throughput sensitive, provides clear quantiﬁcation of activity, and can even be screening of genes or drugs that can control or modulate IRF3 used for large-scale high-throughput studies. Activation of the activation. TBK1–IRF3 signaling axis by pathogen-derived or synthetic The IRF3-BiLC reporter system was cloned into a lentiviral mimics of pathogen nucleic acids, or through viral infection, in- backbone construct for easy transduction both in vitro and in vivo. duces robust IRF3-BiLC luciferase reporter activity in a variety of Using two cell lines of different origin, that is, HEK293T (IRF3- 8 IRF3 ACTIVATION REVEALED BY A DIMERIZATION REPORTER

FIGURE 6. NF-kB signaling positively regulates IRF3 dimerization. (A and B) HEK293T (IRF3-BiLC) cells were pretreated with TPCA-1 for 1 h and Downloaded from then transfected with poly(I:C) (A) or poly(dA:dT) (B) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (C and D) HEK293T (IRF3-BiLC) cells were pretreated with TNF-a and then transfected with poly(I:C) (C) or poly(dA:dT) (D) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (E and F) HEK293T (IRF3-BiLC) cells were pretreated with TPCA-1 for 1 h, stimulated with TNF-a for 12 h, and then transfected with poly(I:C) (E) or poly(dA:dT) (F) for 12 h, after which IRF3-BiLC luciferase activity was measured. (G) THP-1 (IRF3-BiLC) cells were pretreated with TPCA-1 for 1 h and then transfected with poly(I:C) for indicated time points, after which IRF3-BiLC luciferase activity was measured. (H) THP-1(IRF3-BiLC) cells were pretreated TNF-a and transfected with poly(I:C) for 10 h, after which IRF3-BiLC luciferase activity was measured. Data are from three independent experiments (mean 6 SEM). **p , 0.01, ***p , 0.001 (Student t test). http://www.jimmunol.org/

BiLC) and THP-1 (IRF3-BiLC), we found that PAMPs triggered aid in the identification of ISGs that contribute to the IFN-I pos- highly distinctive temporal patterns of IRF3 activation. Particu- itive feedback loop. larly, stimulation with poly(I:C) and poly(dA:dT) gradually in- Transcriptional activation of the IFN-b gene requires the as- duced IRF3 dimerization in HEK293T cells from 6 to 24 h, whereas sembly of an enhanceosome containing ATF-2/c-Jun, IRF3/IRF7, peak dimerization was observed at ∼12 h in THP-1 cells. These and NF-kB (20), which indicates that NF-kB binding is essential results suggest that negative feedback regulation pathways might for IFN-b production. Our results suggest that activation of NF- exist in THP1 cells, but not in the HEK293T cells. Thus, identi- kB is also required for the optimal dimerization of IRF3. As by guest on October 1, 2021 fication and verification of the negative regulators in innate im- shown using our IRF3-BiLC luciferase assay, the pharmacological munity is a potential application of our IRF3-BiLC reporter inhibition of NF-kB will also attenuate poly(I:C)- and poly(dA:dT)- system. During microbial infection in vivo, IRF3 will be activated triggered IRF3 dimerization. Likewise, the activation of NF-kB and IFN-I will be produced by multiple cell types, including both by pretreatment with TNF-a will enhance IRF3 dimerization immune and nonimmune cells. However, the intensity and dura- triggered by those PAMPs. These results suggest that NF-kB tion of IRF3 activation may differ greatly between cell types. signaling plays a role in the upstream regulation of IFN-I, in ad- Using a combination of our IRF3-BiLC reporter system with a live dition to its direct interaction with the IFN-b promoter. However, imaging system could be used for real-time monitoring of IRF3 further experiments are required to fully understand the detailed activation in vivo. mechanisms in NF-kB–mediated regulation of IRF3 activation The IRF3–IFN-b–IFNAR–IRF7–IFN-a/b signaling axis is a during microbial infection. well-established positive feedback loop for the induction of IFN-I In this study, we have demonstrated that the complex and dy- during viral infection (35–37). It is commonly accepted that namic regulation of IRF3 activation during the host immune re- downstream signaling of IFNAR positively regulates IFN-I pro- sponse can be quantitatively measured and monitored using a novel duction through the induction of a secondary transcription factor, IRF3-BiLC dimerization reporter system. As there are many other IRF7. To our surprise, our present work has shown that IFNAR- molecules used in immune signaling that undergo dimerization, JAK–dependent signaling can lead to another positive feed- such as TLRs, TBK1, IRF7, and STING, we anticipate that this forward pathway triggered by IRF3 dimerization. It has been reporter system could be significantly applied to many innate reported that induction of ISG15 by IFN-I during viral infection immune-related studies. This will in turn broaden our under- might stabilize the IRF3 protein and sustain IRF3 activation (38, standing of multiple branches of immune signaling and enable 39). In response to RNA virus infection, induction of TRIM25 is high-throughput screening of new drugs or genes that modulate essential for RIG-I ubiquitination and affects downstream sig- inflammatory disorders and infectious diseases. naling events, including IRF3 activation and IFN-b production (40, 41). Moreover, recent studies have shown that DNA sensors Acknowledgments cGAS and STING also are ISGs, and that induction of these DNA We thank Drs. Jun Cui, Wenbin Ma, and Yong Zhao for providing reagents sensors can enhance IFN-I production via the cGAS/STING– and helpful discussions. We thank Saba Aliyari and Connie Au for critical IRF3–IFN-b feedback loop (42, 43). These studies have expanded reading of this manuscript. the horizon of IFN regulation, suggesting that ISGs might positively modulate IFN-I production at multiple levels during viral Disclosures infection. The IRF3-BiLC reporter system thus will undoubtedly The authors have no financial conflicts of interest. The Journal of Immunology 9

References 24. Cassonnet, P., C. Rolloy, G. Neveu, P. O. Vidalain, T. Chantier, J. Pellet, L. Jones, M. Muller, C. Demeret, G. Gaud, et al. 2011. Benchmarking a lucif- 1. McNab, F., K. Mayer-Barber, A. Sher, A. Wack, and A. O’Garra. 2015. Type I erase complementation assay for detecting protein complexes. Nat. Methods 8: interferons in infectious disease. Nat. Rev. Immunol. 15: 87–103. 990–992. 2. Goubau, D., S. Deddouche, and C. Reis e Sousa. 2013. Cytosolic sensing of 25. Qin, X. F., D. S. An, I. S. Chen, and D. Baltimore. 2003. Inhibiting HIV-1 in- viruses. Immunity 38: 855–869. fection in human T cells by lentiviral-mediated delivery of small interfering 3. Newton, K., and V. M. Dixit. 2012. Signaling in innate immunity and inflammation. RNA against CCR5. Proc. Natl. Acad. Sci. USA 100: 183–188. Cold Spring Harb. Perspect. Biol. 4: 4 doi:10.1101/cshperspect.a006049. 26. Cong, L., F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, 4. Thaiss, C. A., M. Levy, S. Itav, and E. Elinav. 2016. Integration of innate im- W. Jiang, L. A. Marraffini, and F. Zhang. 2013. Multiplex genome engineering mune signaling. Trends Immunol. 37: 84–101. using CRISPR/Cas systems. Science 339: 819–823. 5. Honda, K., and T. Taniguchi. 2006. IRFs: master regulators of signalling by Toll- 27. Ran, F. A., P. D. Hsu, J. Wright, V. Agarwala, D. A. Scott, and F. Zhang. 2013. like receptors and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6: Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8: 2281– 644–658. 2308. 6. Ikushima, H., H. Negishi, and T. Taniguchi. 2013. The IRF family transcription 28. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, factors at the interface of innate and adaptive immune responses. Cold Spring M. Miyagishi, K. Taira, S. Akira, and T. Fujita. 2004. The RNA helicase RIG-I Harb. Symp. Quant. Biol. 78: 105–116. has an essential function in double-stranded RNA-induced innate antiviral re- 7. Fitzgerald, K. A., S. M. McWhirter,K.L.Faia,D.C.Rowe,E.Latz, sponses. Nat. Immunol. 5: 730–737. ε D. T. Golenbock, A. J. Coyle, S. M. Liao, and T. Maniatis. 2003. IKK and TBK1 29. Ablasser, A., M. Goldeck, T. Cavlar, T. Deimling, G. Witte, I. Ro¨hl, are essential components of the IRF3 signaling pathway. Nat. Immunol. 4: 491–496. K. P. Hopfner, J. Ludwig, and V. Hornung. 2013. cGAS produces a 29-59-linked 8. Sadler, A. J., and B. R. Williams. 2008. Interferon-inducible antiviral effectors. cyclic dinucleotide second messenger that activates STING. Nature 498: 380– Nat. Rev. Immunol. 8: 559–568. 384. € 9. Decker, T., M. Muller, and S. Stockinger. 2005. The Yin and Yang of type I 30. Cai, X., Y. H. Chiu, and Z. J. Chen. 2014. The cGAS-cGAMP-STING pathway interferon activity in bacterial infection. Nat. Rev. Immunol. 5: 675–687. of cytosolic DNA sensing and signaling. Mol. Cell 54: 289–296. 10. Wilson, E. B., D. H. Yamada, H. Elsaesser, J. Herskovitz, J. Deng, G. Cheng, 31. Takahasi, K., M. Horiuchi, K. Fujii, S. Nakamura, N. N. Noda, M. Yoneyama, B. J. Aronow, C. L. Karp, and D. G. Brooks. 2013. Blockade of chronic type I T. Fujita, and F. Inagaki. 2010. Ser386 phosphorylation of transcription factor interferon signaling to control persistent LCMV infection. Science 340: 202– IRF-3 induces dimerization and association with CBP/p300 without overall Downloaded from 207. conformational change. Genes Cells 15: 901–910. 11. Teles, R. M., T. G. Graeber, S. R. Krutzik, D. Montoya, M. Schenk, D. J. Lee, 32. Schneider, W. M., M. D. Chevillotte, and C. M. Rice. 2014. Interferon- E. Komisopoulou, K. Kelly-Scumpia, R. Chun, S. S. Iyer, et al. 2013. Type I stimulated genes: a complex web of host defenses. Annu. Rev. Immunol. 32: interferon suppresses type II interferon-triggered human anti-mycobacterial re- 513–545. sponses. Science 339: 1448–1453. 33. Birrell, M. A., E. Hardaker, S. Wong, K. McCluskie, M. Catley, J. De Alba, 12.Teijaro,J.R.,C.Ng,A.M.Lee,B.M.Sullivan, K. C. Sheehan, M. Welch, R. Newton, S. Haj-Yahia, K. T. Pun, C. J. Watts, et al. 2005. Ik-B kinase-2 in- R. D. Schreiber, J. C. de la Torre, and M. B. Oldstone. 2013. Persistent LCMV inhibitor blocks inflammation in human airway smooth muscle and a rat model of fection is controlled by blockade of type I interferon signaling. Science 340: 207–211. asthma. Am. J. Respir. Crit. Care Med. 172: 962–971.

13. Corr, M., D. L. Boyle, L. Ronacher, N. Flores, and G. S. Firestein. 2009. Syn- 34. Podolin, P. L., J. F. Callahan, B. J. Bolognese, Y. H. Li, K. Carlson, T. G. Davis, http://www.jimmunol.org/ ergistic benefit in inflammatory arthritis by targeting IkB kinase epsilon and G. W. Mellor, C. Evans, and A. K. Roshak. 2005. Attenuation of murine interferon b. Ann. Rheum. Dis. 68: 257–263. collagen-induced arthritis by a novel, potent, selective small molecule inhibitor 14. Baechler, E. C., F. M. Batliwalla, G. Karypis, P. M. Gaffney, W. A. Ortmann, of IkB kinase 2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3- K. J. Espe, K. B. Shark, W. J. Grande, K. M. Hughes, V. Kapur, et al. 2003. thiophenecarboxamide), occurs via reduction of proinflammatory cytokines Interferon-inducible gene expression signature in peripheral blood cells of pa- and antigen-induced T cell proliferation. J. Pharmacol. Exp. Ther. 312: 373–381. tients with severe lupus. Proc. Natl. Acad. Sci. USA 100: 2610–2615. 35. Marie´, I., J. E. Durbin, and D. E. Levy. 1998. Differential viral induction of 15. Wang, Q., D. R. Nagarkar, E. R. Bowman, D. Schneider, B. Gosangi, J. Lei, distinct interferon-a genes by positive feedback through interferon regulatory Y. Zhao, C. L. McHenry, R. V. Burgens, D. J. Miller, et al. 2009. Role of double- factor-7. EMBO J. 17: 6660–6669. stranded RNA pattern recognition receptors in rhinovirus-induced airway epi- 36. Sato, M., N. Hata, M. Asagiri, T. Nakaya, T. Taniguchi, and N. Tanaka. 1998. thelial cell responses. J. Immunol. 183: 6989–6997. Positive feedback regulation of type I IFN genes by the IFN-inducible tran- 16. Cario, E. 2010. Toll-like receptors in inflammatory bowel diseases: a decade scription factor IRF-7. FEBS Lett. 441: 106–110. later. Inflamm. Bowel Dis. 16: 1583–1597. 37. Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya, by guest on October 1, 2021 17. Korherr, C., H. Gille, R. Scha¨fer, K. Koenig-Hoffmann, J. Dixelius, M. Katsuki, S. Noguchi, N. Tanaka, and T. Taniguchi. 2000. Distinct and es- K. A. Egland, I. Pastan, and U. Brinkmann. 2006. Identification of proangiogenic sential roles of transcription factors IRF-3 and IRF-7 in response to viruses for genes and pathways by high-throughput functional genomics: TBK1 and the IFN-a/b gene induction. Immunity 13: 539–548. IRF3 pathway. Proc. Natl. Acad. Sci. USA 103: 4240–4245. 38. Lu, G., J. T. Reinert, I. Pitha-Rowe, A. Okumura, M. Kellum, K. P. Knobeloch, 18. Chien, Y., S. Kim, R. Bumeister, Y. M. Loo, S. W. Kwon, C. L. Johnson, B. Hassel, and P. M. Pitha. 2006. ISG15 enhances the innate antiviral response M. G. Balakireva, Y. Romeo, L. Kopelovich, M. Gale, Jr., et al. 2006. RalB by inhibition of IRF-3 degradation. Cell. Mol. Biol. (Noisy-le-grand) 52: 29–41. GTPase-mediated activation of the IkB family kinase TBK1 couples innate 39. Shi, H. X., K. Yang, X. Liu, X. Y. Liu, B. Wei, Y. F. Shan, L. H. Zhu, and immune signaling to tumor cell survival. Cell 127: 157–170. C. Wang. 2010. Positive regulation of interferon regulatory factor 3 activation by 19. Barbie, D. A., P. Tamayo, J. S. Boehm, S. Y. Kim, S. E. Moody, I. F. Dunn, Herc5 via ISG15 modification. Mol. Cell. Biol. 30: 2424–2436. A. C. Schinzel, P. Sandy, E. Meylan, C. Scholl, et al. 2009. Systematic RNA 40. Gack, M. U., Y. C. Shin, C. H. Joo, T. Urano, C. Liang, L. Sun, O. Takeuchi, interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature S. Akira, Z. Chen, S. Inoue, and J. U. Jung. 2007. TRIM25 RING-finger E3 462: 108–112. ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446: 20. Panne, D., T. Maniatis, and S. C. Harrison. 2007. An atomic model of the in- 916–920. terferon-b enhanceosome. Cell 129: 1111–1123. 41. Versteeg, G. A., R. Rajsbaum, M. T. Sańchez-Aparicio, A. M. Maestre, 21. Ma, F., S. Y. Liu, B. Razani, N. Arora, B. Li, H. Kagechika, P. Tontonoz, J. Valdiviezo, M. Shi, K. S. Inn, A. Fernandez-Sesma, J. Jung, and A. Garcıá- V. Nuń˜ez, M. Ricote, and G. Cheng. 2014. Retinoid X receptor a attenuates host Sastre. 2013. The E3-ligase TRIM family of proteins regulates signaling path- antiviral response by suppressing type I interferon. Nat. Commun. 5: 5494 doi: ways triggered by innate immune pattern-recognition receptors. Immunity 38: 10.1038/ncomms6494. 384–398. 22. Iwamura, T., M. Yoneyama, K. Yamaguchi, W. Suhara, W. Mori, K. Shiota, 42. Ma, F., B. Li, S. Y. Liu, S. S. Iyer, Y. Yu, A. Wu, and G. Cheng. 2015. Positive Y. Okabe, H. Namiki, and T. Fujita. 2001. Induction of IRF-3/-7 kinase and NF- feedback regulation of type I IFN production by the IFN-inducible DNA sensor kB in response to double-stranded RNA and virus infection: common and unique cGAS. J. Immunol. 194: 1545–1554. pathways. Genes Cells 6: 375–388. 43. Ma, F., B. Li, Y. Yu, S. S. Iyer, M. Sun, and G. Cheng. 2015. Positive feedback 23. Remy, I., and S. W. Michnick. 2006. A highly sensitive protein-protein inter- regulation of type I interferon by the interferon-stimulated gene STING. EMBO action assay based on Gaussia luciferase. Nat. Methods 3: 977–979. Rep. 16: 202–212.