IFIT2 Is an Effector Protein of Type I IFN− Mediated Amplification of Lipopolysaccharide (LPS)-Induced TNF- α Secretion and LPS-Induced Endotoxin Shock This information is current as of September 27, 2021. Alexandra Siegfried, Susanne Berchtold, Birgit Manncke, Eva Deuschle, Julia Reber, Thomas Ott, Michaela Weber, Ulrich Kalinke, Markus J. Hofer, Bastian Hatesuer, Klaus Schughart, Valérie Gailus-Durner, Helmut Fuchs, Martin Hrabe de Angelis, Friedemann Weber, Mathias W. Hornef, Ingo B. Autenrieth and Erwin Bohn Downloaded from J Immunol published online 6 September 2013 http://www.jimmunol.org/content/early/2013/09/06/jimmun ol.1203305 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/09/06/jimmunol.120330 Material 5.DC1 Why The JI? Submit online. by guest on September 27, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published September 6, 2013, doi:10.4049/jimmunol.1203305 The Journal of Immunology IFIT2 Is an Effector Protein of Type I IFN–Mediated Amplification of Lipopolysaccharide (LPS)-Induced TNF-a Secretion and LPS-Induced Endotoxin Shock Alexandra Siegfried,*,1 Susanne Berchtold,*,1 Birgit Manncke,* Eva Deuschle,* Julia Reber,* Thomas Ott,† Michaela Weber,‡ Ulrich Kalinke,x Markus J. Hofer,{ Bastian Hatesuer,‖,#,** Klaus Schughart,‖,#,** Vale´rie Gailus-Durner,†† Helmut Fuchs,†† Martin Hrabe de Angelis,††,‡‡ Friedemann Weber,‡ Mathias W. Hornef,xx Ingo B. Autenrieth,* and Erwin Bohn* a Type I IFN signaling amplifies the secretion of LPS-induced proinflammatory cytokines such as TNF- or IL-6 and might thus Downloaded from contribute to the high mortality associated with Gram-negative septic shock in humans. The underlying molecular mechanism, however, is ill defined. In this study, we report the generation of mice deficient in IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and demonstrate that Ifit2 is a critical signaling intermediate for LPS-induced septic shock. Ifit2 expression was significantly upregulated in response to LPS challenge in an IFN-a receptor– and IFN regulatory factor (Irf)9–dependent manner. Also, LPS induced secretion of IL-6 and TNF-a by bone marrow–derived macrophages (BMDMs) was significantly enhanced in the presence of Ifit2. In accordance, Ifit2-deficient mice exhibited significantly reduced serum levels of IL-6 and TNF-a and http://www.jimmunol.org/ reduced mortality in an endotoxin shock model. Investigation of the underlying signal transduction events revealed that Ifit2 upregulates Irf3 phosphorylation. In the absence of Irf3, reduced Ifn-b mRNA expression and Ifit2 protein expression after LPS stimulation was found. Also, Tnf-a and Il-6 secretion but not Tnf-a and Il-6 mRNA expression levels were reduced. Thus, IFN- stimulated Ifit2 via enhanced Irf3 phosphorylation upregulates the secretion of proinflammatory cytokines. It thereby amplifies LPS-induced cytokine production and critically influences the outcome of endotoxin shock. The Journal of Immunology, 2013, 191: 000–000. ipopolysaccharide derived from Gram-negative bacteria is recognition (1, 2). TRIF-mediated signaling induces expression by guest on September 27, 2021 recognized by TLR4 and initiates downstream signaling and secretion of type I IFNs that in turn stimulate the IFN-a/b L via MyD88, TIRAP/Mal, IL-1R–associated kinases 1, 2, receptor (IFNAR) consisting of an IFNAR1 and IFNAR2 chain. and 4, and TNFR-associated factor (TRAF)6 (1, 2). Activation of Receptor dimerization induces autophosphorylation of the receptor- the IkB kinase and MAPK signal transduction cascades results in associated kinases TYK2 and JAK1. This leads to phosphorylation activation and nuclear translocation of the transcription factors of STAT1 and STAT2 and the assembly of the IFN-stimulated gene NF-kB, IFN regulatory factor (IRF)3, and AP-1. Additionally, factor (ISGF)3, a transcription factor complex consisting of STAT1, Toll/IL-1R domain–containing adapter inducing IFN-b (TRIF)– STAT2, and IRF9. This complex translocates into the nucleus and dependent signaling is activated downstream of TLR4 upon LPS activates a large number of IFN-stimulated genes (ISGs) (3–5). *Interfakulta¨res Institut fu¨r Mikrobiologie und Infektionsmedizin, Eberhard Karl Uni- the European Union (Grant LSHG-2006-037188 to the European Mouse Disease versita¨t Tuebingen, 72076 Tuebingen, Germany; †Interdisciplinary Center for Clinical Clinic and Infrafrontier Contract 211404 to the German Mouse Clinic). F.W. is Research Transgenic Facility, University Hospital Tuebingen, 72076 Tuebingen, Ger- supported by Research Funding Grant 47/2012MR in cooperative agreement with x many; ‡Institute for Virology, 35043 Marburg, Germany; Institute for Experimental the Universita¨tsklinikum Giessen und Marburg and the Leibniz Graduate School for Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Re- Emerging Infectious Diseases. search, 30625 Hannover, Germany; {Department of Neuropathology, University Clinic A.S. and S.B. performed research and wrote the paper; S.B. and T.O. designed of Marburg and Giessen, 35043 Marburg, Germany; ||Department of Infection Genetics, transgenic mice; B.M., E.D., M.W., and J.R. performed research; M.J.H., B.H., Helmholtz Center for Infection Research, 38124 Braunschweig, Germany; #University K.S., and U.K. provided mice and discussed data; V.G.-D., H.F., and M.H.d.A. de- of Veterinary Medicine, Hannover, Germany; **University of Tennessee Health Science signed and organized the phenotyping at the German Mouse Center and analyzed and Center, Memphis, TN 38163; ††German Mouse Clinic, Institute of Experimental Ge- discussed data; F.W., I.B.A., and M.W.H. analyzed and discussed data; and E.B. netics, Helmholtz Center Munich, German Research Center for Environmental Health, designed the study and wrote the paper. 85764 Neuherberg–Munich, Germany; ‡‡Chair of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technical University of Munich, 85350 Freis- Address correspondence and reprint requests to Dr. Erwin Bohn, Interfakulta¨res xx ing–Weihenstephan, Germany; and Institute for Medical Microbiology and Hospital Institut fu¨r Mikrobiologie und Infektionsmedizin, Eberhard Karl Universita¨t Tuebin- Epidemiology, Hannover Medical School, 30625 Hannover, Germany gen, Elfriede-Aulhorn-Strasse 6, 72076 Tuebingen, Germany. E-mail address: erwin. [email protected] 1A.S. and S.B. contributed equally to this work. The online version of this article contains supplemental material. Received for publication November 30, 2012. Accepted for publication July 27, 2013. Abbreviations used in this article: BMDM, bone marrow–derived macrophage; eIF3, eukaryotic initiation factor 3; ES, embryonic stem; IFIT, IFN-induced protein with This work was supported by Deutsche Forschungsgemeinschaft Grant Bo1527/3-2. tetratricopeptide repeats; IFNAR, IFN-a/b receptor; IRF, IFN regulatory factor; ISG, M.W.H. was supported by German Ministry for Science and Education Grant DLR IFN-stimulated gene; ISGF3, IFN-stimulated gene factor 3; TRAF, TNFR-associated 01KI1003D, German Research Foundation Grant Ho2236/8-1, and Collaborative factor; TRIF, Toll/IL-1R domain–containing adapter inducing IFN-b. Research Center Grants SFB621 and SFB900. This work was also partially supported by the German Federal Ministry of Education and Research (NGFN-Plus Grant Ó 01GS0850 and Infrafrontier Grant 01KX1012 to the German Mouse Clinic) and by Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203305 2 ROLE OF IFIT2 IN ENDOTOXEMIA LPS is the main immunostimulatory agent in the pathogenesis of GCC AGT CCT GCA CTG-39), P1 (59-CTG ATC TAG GAG CTT CTT endotoxin shock. The TLR4-induced release of cytokines such as GTA AAG-39), and P2 (59-CGG AGA TGA GGA AGA GGA GAA AAG- TNF-a, IL-6, IL-1, IL-12, and NO causes peripheral vascular di- 39), resulting in the 39 fragmentP3-P4(5kb)andthe59 fragment P1-P2 (3 kb). A selected ES cell clone was used for injection into blastocysts. latation, capillary leakage, and impaired organ perfusion, the clin- Chimeras were carried out by foster mothers. Deletion of the Neo ical hallmarks of septic shock (6). Studies using Escherichia coli or cassette was achieved by breeding the offspring with Pgk-Cre mice (26). LPS revealed that TLR4 stimulation additionally triggers type I IFN Breeding with Pgk-Cre mice led to exertion of the Neo cassette at its loxP secretion. Interestingly, IFN binding to IFNAR amplifies the secre- sites. Genotyping of these mice was performed using the oligonucleotides P5 (59-CAG TCT TGC AAG GAA ACC CAA TC-39) and P7 (59-CTC CAG tion of cytokines such as TNF-a and IL-6 (7, 8). Also in vivo, type I TGA CTC CTT ACT CGT TG-39)andP8(59-CAA GGA TGG AAC ATT IFN was shown to promote the development of septic shock using CAC CCA TG-39), resulting in the amplicons P5-P7 (299 bp, wild-type) and models of LPS challenge or bacterial infection (9–11). The molec- P5-P8 (231 bp, transgene). Mice were backcrossed 10 generations to a ular mechanisms and signaling intermediates of the crosstalk be- C57BL/6N Crl background (N10) with speed congenics. Wild-type lit- termates were kept in this C57BL/6N Crl background and were bred in tween IFN signaling and the induction of sepsis-inducing cytokines, 2 2 parallel to IFIT2 / mice. The mice have been phenotypically analyzed in however, have remained ill defined.