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Negatively Regulated by ADAM15 TRIF-Mediated TLR3 and TLR4 TRIF-Mediated TLR3 and TLR4 Signaling Is Negatively Regulated by ADAM15 Suaad Ahmed, Ashwini Maratha, Aisha Qasim Butt, Enda Shevlin and Sinead M. Miggin This information is current as of September 27, 2021. J Immunol 2013; 190:2217-2228; Prepublished online 30 January 2013; doi: 10.4049/jimmunol.1201630 http://www.jimmunol.org/content/190/5/2217 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/01/30/jimmunol.120163 Material 0.DC1 References This article cites 57 articles, 20 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/190/5/2217.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • 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 TRIF-Mediated TLR3 and TLR4 Signaling Is Negatively Regulated by ADAM15 Suaad Ahmed, Ashwini Maratha, Aisha Qasim Butt, Enda Shevlin, and Sinead M. Miggin TLRs are a group of pattern-recognition receptors that play a crucial role in danger recognition and induction of the innate immune response against bacterial and viral infections. The TLR adaptor molecule, Toll/IL-1R domain-containing adaptor inducing IFN (TRIF), facilitates TLR3 and TLR4 signaling and concomitant activation of the transcription factors, NF-kB and IFN regulatory factor 3, leading to proinflammatory cytokine production. Whereas numerous studies have been undertaken toward understand- ing the role of TRIF in TLR signaling, little is known about the signaling components that regulate TRIF-dependent TLR signaling. To this end, TRIF-interacting partners were identified by immunoprecipitation of the TRIF signaling complex, followed by protein identification using liquid chromatography mass spectrometry. Following stimulation of cells with a TLR3 or TLR4 ligand, we identified a disintegrin and metalloprotease (ADAM)15 as a novel TRIF-interacting partner. Toward the functional Downloaded from characterization of the TRIF:ADAM15 interaction, we show that ADAM15 acts as a negative regulator of TRIF-mediated NF-kB and IFN-b reporter gene activity. Also, suppression of ADAM15 expression enhanced polyriboinosinic polyribocytidylic acid and LPS-mediated proinflammatory cytokine production via TRIF. In addition, suppression of ADAM15 expression enhanced rhino- virus 16 and vesicular stomatitis virus–mediated proinflammatory cytokine production. Interestingly, ADAM15 mediated the proteolytic cleavage of TRIF. Thus, ADAM15 serves to curtail TRIF-dependent TLR3 and TLR4 signaling and, in doing so, protects the host from excessive production of proinflammatory cytokines and matrix metalloproteinases. In conclusion, to our http://www.jimmunol.org/ knowledge, our study clearly shows for the first time that ADAM15 plays an unexpected role in TLR signaling, acting as an anti- inflammatory molecule through impairment of TRIF-mediated TLR signaling. The Journal of Immunology, 2013, 190: 2217–2228. he innate immune response is highly conserved and rep- of the MyD88-dependent and TRIF-dependent pathways. Whereas resents the first line of defense against microbial path- MyD88 is used by TLR2, TLR5, TLR7, TLR8, and TLR9, TRIF T ogens (1). The TLR family is a conserved component of is used by TLR3. Both the MyD88 and TRIF-dependent path- the innate immune system that responds to specific pathogen- ways are used by TLR4 toward the induction of proinflammatory associated molecular pattern from viruses, bacteria, fungi, and cytokines and IFN-stimulated genes (5). Additionally, activation parasites (2, 3). Signaling through TLRs can be broadly divided of the TLR pathway also leads to the induction of matrix metal- by guest on September 27, 2021 into two pathways that are linked to downstream activation of loproteinases (MMPs), including MMP-9 (gelatinase B) and NF-kB, MAPKs, and IFN regulatory factors (IRFs) (4). All TLRs MMP-10 (stromelysin 2) (6, 7). Activation of MMPs has been have a Toll/IL-1R domain that facilitates their interaction with associated with the maintenance of normal homeostasis, and the TLR adaptor proteins, five of which have been identified to date, upregulation of MMPs has been linked with inflammatory re- namely MyD88, MyD88 adaptor-like, Toll/IL-1R domain- sponses, cancer, and arthritis (7). containing adaptor inducing IFN (TRIF), TRIF-related adaptor During TLR3 and TLR4 signaling, TRIF, in association with molecule (TRAM), and Sterile-alpha and Armadillo motif-con- TRAM in the case of TLR4, initiates a signaling pathway through taining protein (SARM) (4); these adaptors facilitate activation TRAF3, TANK-binding kinase 1 (TBK1), and inhibitor of nuclear factor-kB(IkB)–kinase complex [IKKε]), which mediates the direct phosphorylation of IRF3 and IRF7 and the formation of Immune Signaling Group, Institute of Immunology, Department of Biology, National IRF3 and IRF7 hetero- or homodimers. The IRF3/7 dimers then University of Ireland Maynooth, Maynooth, County Kildare, Ireland translocate to the nucleus, in association with transcriptional Received for publication June 13, 2012. Accepted for publication December 21, coactivators, such as CBP and p300, and bind to target sequences 2012. in DNA, such as IFN-stimulated response elements (8, 9). Also, This work was supported by the Science Foundation of Ireland. the IRFs, together with the transcription factors ATF-2/c-Jun and Address correspondence and reprint requests to Dr. Sinead M. Miggin, Immune Signaling Group, Institute of Immunology, Department of Biology, National Univer- NF-kB from the IFN-b enhanceosome, bind to a nucleosome-free sity of Ireland Maynooth, Maynooth, County Kildare, Ireland. E-mail address: enhancer region within the IFN-b promoter upstream of the [email protected] transcription start site, leading to transcriptional activation of the The online version of this article contains supplemental material. IFN-b gene (10). The enhancer itself is divided into four positive Abbreviations used in this article: ADAM, a disintegrin and metalloprotease; regulatory domains (PRD) whereby NF-kB binds to the PRDII esiRNA, endoribonuclease-prepared siRNA; HA, hemagglutinin; IRF, IFN regulatory element within the IFN-b enhancer region, ATF-2/c-Jun binds to factor; LC-MS, liquid chromatography mass spectrometry; MEF, murine embryonic fibroblast; MDA5, melanoma differentiation–associated protein 5; MMP, matrix met- the PRDIV element and is activated by JNK, and IRF3 and IRF7 alloproteinase; poly(I:C), polyriboinosinic polyribocytidylic acid; PRD, positive reg- bind to the PRDI-III element (8). Also, TRIF-dependent activation ulatory domain; RGD, arginine-glycine-aspartic acid; RLR, RIG-I–like receptor; RV, rhinovirus; siRNA, small interfering RNA; TBK1, TANK-binding kinase 1; of NF-kB occurs through binding of TRAF6 to TRIF and subse- TRAM, Toll/IL-1R domain-containing adaptor inducing IFN–related adaptor quent ubiquitination-dependent recruitment and activation of molecule; TRIF, Toll/IL-1R domain-containing adaptor inducing IFN; VSV, TAK1 (11). Despite the numerous studies that have been under- vesicular stomatitis virus; WT, wild-type. taken toward understanding the role of TRIF in TLR signaling, Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 little is known about the signaling components that regulate the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201630 2218 TRIF-MEDIATED TLR SIGNALING IS INHIBITED BY ADAM15 dependent TLR signaling pathway. In this study, we sought to medium was supplemented with blasticidin (100 mg/ml). The HEK293-Blue identify novel components of the TRIF signaling pathway. To this IFN-ab culture medium was supplemented with zeocin (100 mg/ml) and end, TRIF-interacting partners were identified by immunoprecip- blasticidin (10 mg/ml). HEK293-TLR9 was as described (18). U373-CD14 cells were cultured in RPMI 1640 with GlutaMAX (Life Technologies-BRL) itation of the TRIF signaling complex, followed by protein iden- supplemented with 10% FBS, 1% penicillin-streptomycin, 1% fungizone, tification using liquid chromatography mass spectrometry (LC- and 250 mg/ml gentamicin (G418; Sigma-Aldrich). Highly purified protein- MS). Following stimulation of cells with a TLR3 or TLR4 li- free LPS derived from Escherichia coli strain 011:B4 (Alexis) and naked gand, we identified a disintegrin and metalloprotease (ADAM)15 poly(I:C) (InvivoGen) were used for all treatments. ADAM15 small inter- fering RNA (siRNA) was purchased from Sigma-Aldrich (catalog as a novel TRIF-interacting partner. ADAM15, also known as EHU076821) and siRNA control from Ambion (catalog AM16106). RV16 metalloprotease–arginine-glycine-aspartic acid (RGD)–disintegrin was from S. Goodbourn (University of London). VSV was from American protein (Metargidin), is a type I transmembrane glycoprotein be-
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