Green Tea Epigallocatechin-3-Gallate Mediates T Cellular NF-B Inhibition and Exerts Neuroprotection in Autoimmune Encephalomyelitis 1 Orhan Aktas, 2 * Timour Prozorovski, 2 * Alina Smorodchenko, 2 * Nicolai E. Savaskan, † Roland Lauster, ‡ Peter-Michael Kloetzel, § Carmen Infante-Duarte,* Stefan Brocke, ¶ and Frauke Zipp 3 * Recent studies in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), point to the fact that even in the early phase of inflammation, neuronal pathology plays a pivotal role in the sustained disability of affected individuals. We show that the major green tea constituent, ()-epigallocatechin-3-gallate (EGCG), dramatically suppresses EAE induced by proteolipid protein 139 –151. EGCG reduced clinical severity when given at initiation or after the onset of EAE by both limiting brain inflammation and reducing neuronal damage. In orally treated mice, we found abrogated proliferation and TNF- pro- duction of encephalitogenic T cells. In human myelin-specific CD4  T cells, cell cycle arrest was induced, down-regulating the cyclin-dependent kinase 4. Interference with both T cell growth and effector function was mediated by blockade of the catalytic activities of the 20S/26S proteasome complex, resulting in intracellular accumulation of IB- and subsequent inhibition of NF-B activation. Because its structure implicates additional antioxidative properties, EGCG was capable of protecting against neuronal injury in living brain tissue induced by N-methyl-D-aspartate or TRAIL and of directly blocking the formation of neurotoxic reactive oxygen species in neurons. Thus, a natural green tea constituent may open up a new therapeutic avenue for young disabled adults with inflammatory brain disease by combining, on one hand, anti-inflammatory and, on the other hand, neuroprotective capacities. The Journal of Immunology, 2004, 173: 5794 –5800. T ea (Camellia sinensis) and its constituent polyphenols have been reported to possess anticarcinogenic properties in a wide variety of experimental systems in vitro and in vivo. Drinking tea, especially green tea, is associated with a lower incidence of human cancer (1, 2). Subsequent studies demon- strated that certain fractions of green tea, among them (-)-epigal- locatechin-3-gallate (EGCG), 34 promote apoptosis and cell cycle arrest of transformed cells (3–5). Moreover, immunomodulatory properties of green tea extracts such as inhibiting endotoxin-in- duced TNF- production (6) and neutrophil-mediated effects have been recently described (7). In a mouse model of stroke, a neuro- protective role was observed (8) and linked in a Parkinson’s dis- ease model to regulation of superoxide dismutase and catalase ac- tivity in the brain (9). Multiple sclerosis (MS) is a multiphasic autoimmune disease of the CNS (10) in which myelin-specific CD4 + Th1 cells are thought to orchestrate the effector processes resulting in the destruction of the myelin sheath (11). Recent studies in MS and its animal model, experimental autoimmune encephalomyelitis (EAE) (12), suggest that already during the early phase of inflammation, neuronal pa- thology involving axonal transection and loss of parental cell bod- ies plays a pivotal role in disease severity (13, 14). It has become evident that long-term disability in MS correlates better with ax- onal damage than with the degree of demyelination (13, 14). We have very recently unraveled a process linking demyelination and neuronal damage (15). As a consequence of demyelination, the myelin breakdown product 7-ketocholesterol, which can be de- tected in the cerebrospinal fluid of MS patients, induces microglia- mediated apoptotic cell death of neurons in the brain stem motor region (15). However, currently approved therapies, which have to be administered parenterally in young disabled adults with neu- roinflammatory disorder, primarily focus on the inflammatory as- pect of the CNS disease (16), are only partly effective, and are often limited by side effects. In contrast, neuroprotective agents have not yet been established in this autoimmune disorder. In light of the pharmacological profile of EGCG, and its reported properties in immunity and neurodegeneration, we hypothesized a potential bene- ficial role of EGCG in the treatment of neuroinflammation and sub- sequently discovered the underlying mechanisms of action. Materials and Methods Induction, treatment, and evaluation of EAE Female SJL/J mice (6 – 8 wk; 20 g body weight; Charles River Labora- tories, Sulzfeld, Germany) were immunized s.c. with 75 g of proteolipid *Institute of Neuroimmunology, Neuroscience Research Center, Charite ´, Berlin, Ger- many; † Division of Cellular Biochemistry, The Netherlands Cancer Institute, Am- sterdam, The Netherlands; ‡ German Arthritis Research Center, Berlin, Germany; § In- stitute of Biochemistry, Charite ´, Berlin, Germany; ¶ Experimental Pathology, Department of Pathology, Hebrew University, Hadassah Medical School, Jerusalem, Israel Received for publication March 29, 2004. Accepted for publication August 26, 2004. 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 by grants from the Bundesministerium fu ¨r Bildung und Forschung, the Gemeinnu ¨tzige Hertie-Stiftung, and the Deutsche Forschungsgemein- schaft (SA 1041/3-1). 2 O.A., T.P., and A.S. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Frauke Zipp, Institute of Neu- roimmunology, Neuroscience Research Center, NWFZ 2680, Charite ´, 10098 Berlin, Germany. E-mail address: frauke.zipp@charite.de 4 Abbreviations used in this paper: EGCG, (-)-epigallocatechin-3-gallate; NMDA, N-methyl-D-aspartate; BSO, buthionine sulfoximine; MS, multiple sclerosis; TCL, T cell line; ROS, reactive oxygen species; EAE, experimental autoimmune encephalo- myelitis; CDK, cyclin-dependent kinase; PLP, proteolipid protein; MBP, myelin basic protein; APP, amyloid precursor protein. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00