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Double-Stranded RNA Induces an Antiviral Defense Status in Epidermal Keratinocytes through TLR3-, PKR-, and MDA5/RIG-I-Mediated Differential Signaling¹

Behnam Naderi Kalali^*,, Gabriele Köllisch^*,, Jörg Mages, Thomas Müller, Stefan Bauer^2,, Hermann Wagner, Johannes Ring^*,, Roland Lang, Martin Mempel^3,*, and Markus Ollert^3,4,*,

^* Department of Dermatology and Allergy Biederstein and Clinical Research Division of Molecular and Clinical Allergotoxicology, Technische Universität München (TUM), Munich, Germany; Division of Environmental Dermatology and Allergy HMGU/TUM, Helmholtz Zentrum München, German National Research Center for Environment Health, Munich-Neuherberg, Germany; and Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München (TUM), Munich, Germany

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 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 development grant from the Deutsche Forschungsgemeinschaft (DFG) (Me 1708/1-2). The Microarray and Bioinformatics Core Unit at the Institute of Medical Microbiology, Immunology and Hygiene is supported by the Bundesministerium für 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 Research Division of Molecular and Clinical Allergotoxicology, Department of Dermatology and Allergy, Technische Universität München (TUM), Biedersteiner Strasse 29, D-80802 Munich, Germany. E-mail address: ollert{at}lrz.tum.de

⁵ Abbreviations used in this paper: PRR, pathogen recognition receptor; BFA, bafilomycine 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-β; 2-AP, 2-aminopurine.

⁶ The online version of this article contains supplemental material.

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