IFN Regulatory Factor 3-Dependent Induction of Type I IFNs by Intracellular Bacteria Is Mediated by a TLR- and Nod2-Independent Mechanism This information is current as of September 27, 2021. Silvia Stockinger, Benjamin Reutterer, Barbara Schaljo, Carola Schellack, Sylvia Brunner, Tilo Materna, Masahiro Yamamoto, Shizuo Akira, Tadatsugu Taniguchi, Peter J. Murray, Mathias Müller and Thomas Decker J Immunol 2004; 173:7416-7425; ; Downloaded from doi: 10.4049/jimmunol.173.12.7416 http://www.jimmunol.org/content/173/12/7416 http://www.jimmunol.org/ References This article cites 75 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/173/12/7416.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology IFN Regulatory Factor 3-Dependent Induction of Type I IFNs by Intracellular Bacteria Is Mediated by a TLR- and Nod2-Independent Mechanism1 Silvia Stockinger,2* Benjamin Reutterer,2* Barbara Schaljo,* Carola Schellack,† Sylvia Brunner,† Tilo Materna,* Masahiro Yamamoto,§ Shizuo Akira,§ Tadatsugu Taniguchi,¶ Peter J. Murray,ʈ Mathias Mu¨ller,‡ and Thomas Decker3* Like viruses, intracellular bacteria stimulate their host cells to produce type I IFNs (IFN-␣ and IFN-␤). In our study, we inves- tigated the signals and molecules relevant for the synthesis of and response to IFN by mouse macrophages infected with Listeria monocytogenes. We report that IFN-␤ is the critical immediate-early IFN made during infection, because the synthesis of all other type I IFN, expression of a subset of infection-induced genes, and the biological response to type I IFN was lost upon IFN-␤ Downloaded from deficiency. The induction of IFN-␤ mRNA and the IFN-␤-dependent sensitization of macrophages to bacteria-induced death, in turn, was absolutely dependent upon the presence of the transcription factor IFN regulatory factor 3 (IRF3). IFN-␤ synthesis and signal transduction occurred in macrophages deficient for TLR or their adaptors MyD88, TRIF, or TRAM. Expression of Nod2, a candidate receptor for intracellular bacteria, increased during infection, but the protein was not required for Listeria-induced signal transduction to the Ifn-␤ gene. Based on our data, we propose that IRF3 is a convergence point for signals derived from structurally unrelated intracellular pathogens, and that L. monocytogenes stimulates a novel TLR- and Nod2-independent path- http://www.jimmunol.org/ way to target IRF3 and the type I IFN genes. The Journal of Immunology, 2004, 173: 7416–7425. large number of pathogenic microbes enter cells of the through the type I IFNR and causes the formation of the transcrip- host organism to evade extracellular effector mecha- tion factor IFN-stimulated gene factor-3, a trimeric complex with A nisms of the immune system and to use the host cell Stat1, Stat2, and IRF9 subunits. IFN-stimulated gene factor-3 ini- machinery for their own propagation. To ensure clearance of such tiates transcription of the irf7 gene. IRF7 subsequently stimulates microbes, intracellular effector mechanisms must be set in action. transcription of many IFN-␣ subtypes, thus a second and delayed A prominent pair of intracellular pathogen and antimicrobial ef- wave of type I IFN synthesis (3). Much effort has been devoted by guest on September 27, 2021 fector mechanism are viruses and the antiviral state caused by type recently to the identification of sensor proteins of viral infection. It I IFNs (IFN-␣␤). In virus-infected cells, IFN regulatory factor 3 has become clear that viral sensor proteins exist in the host cell (IRF3)4 is rapidly activated, and is a transcription factor critically cytoplasm (4, 5). However, viruses and virus-associated molecular involved in the formation of an enhanceosome on the promoter of patterns can also stimulate signaling from the plasma membrane or the Ifn-␤ gene (1, 2). The synthesis of IFN-␤ stimulates signaling from within endocytic compartments. Four members of the TLR family, TLR3, -7, -8, and -9, have been shown to signal in response to different viral molecules, and these signals target the IFN-␤ *Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, De- gene (6–11). † partment of Microbiology and Genetics, University of Vienna, Intercell AG, and In this study, we examine macrophages infected with Listeria ‡Institute of Animal Breeding and Genetics, Veterinary University of Vienna, Vienna, Austria; §Department of Host Defense, Research Institute for Microbial Diseases, monocytogenes to address the question whether activation of the Osaka University, Osaka, Japan; ¶Graduate School of Medicine and Faculty of Med- IRF3 pathway and subsequent waves of type I IFN production are icine, University of Tokyo, Tokyo, Japan; and ʈDepartment of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105 general responses to intracellular pathogens and whether all intra- Received for publication July 16, 2004. Accepted for publication October 1, 2004. cellular pathogens achieve this goal at least in part via TLRs. We also examine a possible involvement of nucleotide-binding oli- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance gomerization domain 2 (Nod2), a cytoplasmic protein shown to with 18 U.S.C. Section 1734 solely to indicate this fact. bind bacterial peptidoglycan and capable of initiating signal trans- 1 This study was supported by the Austrian Science Foundation (Grant P15272 to duction through its caspase recruitment domain (CARD) (12, 13). T.D.), the Viennese Foundation for Science, Research and Technology (Project L. monocytogenes LS133 to M.M. and T.D.), and the Austrian Ministry of Education and Science The Gram-positive bacterium is endowed with (Project BM:BWK OEZBT GZ200.074/I-IV/Ia/2002 to M.M.). a cluster of virulence genes that allow intracellular survival and 2 S.S. and B.R. contributed equally to this study. multiplication as well as spreading between cells (14, 15). Follow- 3 Address correspondence and reprint requests to Dr. Thomas Decker, Max F. Perutz ing internalization or phagocytic uptake, L. monocytogenes es- Laboratories, University Departments at the Vienna Biocenter, Department of Mi- capes from internal vacuolar compartments to the cytoplasm. Cy- crobiology and Genetics, University of Vienna, Dr. Bohr-Gasse 9/4, A1030 Vienna, toplasmic escape requires the lytic activity of listeriolysin O Austria. E-mail address: [email protected] (LLO), the major virulence factor encoded in the L. monocyto- 4 Abbreviations used in this paper: IRF, IFN regulatory factor; Nod, nucleotide-bind- ing oligomerization domain; CARD, caspase recruitment domain; LLO, listeriolysin genes virulence gene cluster. IFN-␥-activated macrophages effi- O; TIR, Toll/IL-1R; TRIF, TIR domain-containing adaptor inducing IFN-␤; TRAM, ciently kill ingested Listeria, whereas resting macrophages provide TRIF-related adaptor molecule; wt, wild type; iNOS, inducible NO synthase; GBP, guanylate-binding protein; f, forward; r, reverse; MOI, multiplicity of infection; a suitable cytoplasmic environment for bacterial multiplication MDP, muramyl dipeptide; ISG, IFN-stimulated gene. (16). Infection with L. monocytogenes ultimately kills resting-state Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 7417 macrophage hosts through signals and intracellular effector mech- anisms that are largely unknown (17, 18). In the early phase of infection, L. monocytogenes induces mac- rophages to synthesize IFN-␤ and to initiate Jak-Stat signaling through the type I IFNR. Genetic disruption of the type I IFNR subunits, or of Stat1, results in an increased resistance of macro- phages to Listeria-induced death, implicating type I IFN in bacte- ria-mediated killing of their cellular host (18). Strikingly, LLO- mutant bacteria, which are nonpathogenic and do not kill macrophages, do not stimulate IFN-␤ synthesis (18, 19). This find- ing can either be explained with a role of LLO in signal transduc- tion, as has recently been shown for the production of IL-12 and IFN-␥, the stimulation of Ca2ϩ signaling, lipid mediator release, or an apoptotic response (20–24), or it may reflect the role of LLO in phagosome escape, which would allow recognition by cytoplasmic receptors (18, 19). Like probably all bacterial pathogens, L. monocytogenes stim- ulates signal transduction from members of the TLR family (25– 28). Whether these receptors contribute to the onset of Ifn-␤ tran- Downloaded from scription, or whether the gene is targeted by signals from intracellular receptors like