Double-Stranded RNA Induces an Antiviral Defense Status in Epidermal Keratinocytes through TLR3-, PKR-, and MDA5/RIG-I-Mediated Differential Signaling 1 Behnam Naderi Kalali,* † Gabriele Ko ¨llisch,* ‡ Jo ¨ rg Mages, § Thomas Mu ¨ ller, § Stefan Bauer, 2§ Hermann Wagner, § Johannes Ring,* ‡ Roland Lang, § Martin Mempel, 3 * † and Markus Ollert 3,4 * † Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-B, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-B but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin. The Journal of Immunology, 2008, 181: 2694 –2704. T he functions of lymphocytes, macrophages, neutrophils, and dendritic cells in innate immunity have been well characterized (1). In contrast, the role of epithelial cells such as keratinocytes in innate immunity is less well understood, although keratinocytes represent the major cellular component of human skin and are thus the first line of encounter for various pathogens. Human skin is known to be constantly exposed to var- ious pathogens of prokaryotic, eukaryotic, and viral origin. Emerg- ing evidence suggests, in addition to an essential contribution of keratinocytes in building a physical skin barrier, an important role for epidermal keratinocytes in innate immune mechanisms against skin infections (2). To fulfill the latter function, keratinocytes are equipped with a set of pathogen recognition receptors (PRRs) 5 and antimicrobial defensins enabling them to mount an efficient and sustained immune response (2). Among the identified and at least partially characterized PRRs in keratinocytes are also members of the TLR family. Like other epithelial cells, keratinocytes express a broad set of TLR, mainly of the extracellular type (3). We have previously shown that the expressed TLR in keratinocytes are functional and respond to their respective ligands by activating the central transcription factor NF-B and by up-regulating proinflam- matory mediators such as IL-8, inducible NO synthase, and cyclo- oxygenase (3, 4). Interestingly, keratinocytes respond best to poly(I:C), a synthetic analog of viral double-stranded RNA (dsRNA), which occurs as an important metabolite during viral infection. The corresponding receptor of the TLR family on human cells is TLR3, which is expressed at high levels in human kera- tinocytes (3, 5, 6). TLR3 is the only TLR that does not use the crucial adaptor molecule MyD88 for intracellular signal transmis- sion (7). In addition to direct NF-B activation, TLR3 can also use *Department of Dermatology and Allergy Biederstein and † Clinical Research Divi- sion of Molecular and Clinical Allergotoxicology, Technische Universita ¨t Mu ¨nchen (TUM), Munich, Germany; ‡ Division of Environmental Dermatology and Allergy HMGU/TUM, Helmholtz Zentrum Mu ¨nchen, German National Research Center for Environment Health, Munich-Neuherberg, Germany; and § Institute of Medical Mi- crobiology, Immunology and Hygiene, Technische Universita ¨t Mu ¨ nchen (TUM), Mu- nich, Germany Received for publication June 26, 2007. Accepted for publication June 10, 2008. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grant 01GC0104 from the German Federal Ministery of Education and Science (BMBF) to M.O., by Grant UW-S15T03 from the German National Genome research network (to M.O. and J.R.), and a grant from the GALDERMA foundation to M.M. M.M. was supported by a Heisenberg career de- velopment grant from the Deutsche Forschungsgemeinschaft (DFG) (Me 1708/1-2). The Microarray and Bioinformatics Core Unit at the Institute of Medical Microbiol- ogy, Immunology and Hygiene is supported by the Bundesministerium fu ¨r Bildung und Forschung (BMBF-NGFN Network Infection and Inflammation FKZ 01G0402, TP 37 to R.L., R. Hoffmann, and H.W.). 2 Current address: Institute of Immunology, University of Marburg, Marburg, Germany. 3 M.O. and M.M. contributed equally to this work. 4 Address correspondence and reprint requests to Dr. Markus Ollert, Clinical Re- search Division of Molecular and Clinical Allergotoxicology, Department of Derma- tology and Allergy, Technische Universita ¨t Mu ¨nchen (TUM), Biedersteiner Strasse 29, D-80802 Munich, Germany. E-mail address: ollert@lrz.tum.de 5 Abbreviations used in this paper: PRR, pathogen recognition receptor; BFA, bafilo- mycine A1; dsRNA, double-stranded RNA; eIF2-, eukaryotic initiation factor 2-; PKR, protein kinase R; RIG-I, retinoic acid-inducible gene I; siRNA, small interfering RNA; ssRNA, single-stranded RNA; TBK-1, TANK-binding kinase-1; TRIF, TIR domain-containing adaptor including IFN-; 2-AP, 2-aminopurine. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org