Experimental autoimmune encephalomyelitis repressed by microglial paralysis Frank L Heppner 1 , Melanie Greter 1,2 , Denis Marino 1 , Jeppe Falsig 1 , Gennadij Raivich 3 , Nadine Hövelmeyer 4 , Ari Waisman 4 , Thomas Rülicke 5 , Marco Prinz 1,7 , Josef Priller 6 , Burkhard Becher 2 & Adriano Aguzzi 1 Although microglial activation occurs in inflammatory, degenerative and neoplastic central nervous system (CNS) disorders, its role in pathogenesis is unclear. We studied this question by generating CD11b-HSVTK transgenic mice, which express herpes simplex thymidine kinase in macrophages and microglia. Ganciclovir treatment of organotypic brain slice cultures derived from CD11b-HSVTK mice abolished microglial release of nitrite, proinflammatory cytokines and chemokines. Systemic ganciclovir administration to CD11b-HSVTK mice elicited hematopoietic toxicity, which was prevented by transfer of wild-type bone marrow. In bone marrow chimeras, ganciclovir blocked microglial activation in the facial nucleus upon axotomy and repressed the development of experimental autoimmune encephalomyelitis. We conclude that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions. The microglial compartment thus provides a potential therapeutic target in inflammatory CNS disorders. These results validate CD11b-HSVTK mice as a tool to study the impact of microglial activation on CNS diseases in vivo. Microglial cells are of hematopoietic origin and populate the CNS early during development to form a regularly spaced network of ram- ified cells 1 . Microglia become rapidly activated in most pathologi- cal conditions of the CNS. Although microglial activation has been described extensively in many CNS diseases 1 , its impact on disease pathogenesis remains ill defined 1,2 . In autoimmune diseases such as multiple sclerosis, most data point to a detrimental role of microglia, for example by producing neurotoxic molecules, proinflammatory cytokines, chemokines or by presenting self-antigens 3–6 . But there have been claims that microglial activation in other diseases may counteract the pathogenic changes by providing neurotrophic or immunosup- pressive factors 7,8 . We wished to investigate the role of activated microglia using a pharmacogenetically inducible in vivo model of microglial paralysis. We have therefore generated transgenic mice in which the thymidine kinase of herpes simplex virus (encoded by HSVTK) is driven by the CD11b promoter 9 . CD11b, encoded by Itgam, is the alpha chain of the Mac-1 integrin and is expressed in cells of myeloid origin, including macro- phages and microglia. We used a 1.7-kilobase CD11b promoter fragment which drives sustained expression in macrophages of transgenic mice at levels similar to those of the endogenous CD11b gene 9 . HSVTK is a suicide gene that converts antiviral nucleotide analog prodrugs such as ganciclovir (GCV) to a monophosphorylated form, which is then transformed into a toxic triphosphate by endogenous cellular kinases 10 . Expression of HSVTK renders preferentially proliferating cells sensi- tive to GCV, as the active metabolite competes with thymine for DNA synthesis. This strategy has been used to selectively ablate defined cell lineages, for example in transgenic mice 11,12 . Nondividing HSVTK + cells also show susceptibility to GCV, albeit at reduced levels: in this case, toxicity has been ascribed to interference with mitochondrial DNA synthesis 13 . Here we report that microglial cells of CD11b-HSVTK transgenic mice, although mostly resting in the adult CNS, are efficiently paralyzed by GCV administration. We took advantage of this phenomenon to study the contribution of micro- glial activation to two disease models: facial nerve transection and experimental autoimmune encephalitis. RESULTS Characterization of transgenic mice Before generating transgenic mice, the CD11b-HSVTK transgene was stably transfected into the BV-2 microglial cell line, and GCV was added to the culture medium. We detected efficient, dose-dependent killing of microglial cells with a significant difference between BV2- TK and controls at 2, 10 and 20 μm GCV (P < 0.001, one-way ANOVA test), confirming the functionality of the suicide approach (Fig. 1a). We then established two independent transgenic mouse lines denoted B6,D2-Tg(CD11b-HSVTK)618Zbz (tg618) and B6,D2-Tg (CD11b- HSVTK)620Zbz (tg620), and confirmed integration of the transgene by 1 Institute of Neuropathology, University Hospital Zurich, CH-8091 Zurich, Switzerland. 2 Department Neurology, Neuroimmunology Unit, University Hospital Zurich, CH-8091 Zurich, Switzerland. 3 Perinatal Brain Repair Group, Department of Obstetrics and Gynaecology and Department of Anatomy, University College London, WC1E 6HX London, UK. 4 Laboratory for Molecular Immunology, Institute for Genetics, University of Cologne, D-50931 Cologne, Germany. 5 Institute of Laboratory Animal Science, University of Zurich, CH-8091 Zurich, Switzerland. 6 Departments of Psychiatry and Experimental Neurology, Charité, Humboldt-University Berlin, 10117 Berlin, Germany. 7 Present address: Institute of Neuropathology, Georg-August-University Göttingen, D-37075 Göttingen, Germany. Correspondence should be addressed to A.A. (adriano@pathol.unizh.ch). Published online 23 January 2005; doi:10.1038/nm1177 146 VOLUME 11 | NUMBER 2 | FEBRUARY 2005 NATURE MEDICINE ARTICLES © 2005 Nature Publishing Group http://www.nature.com/naturemedicine