Inhibition of midkine alleviates experimental autoimmune encephalomyelitis through the expansion of regulatory T cell population Jinyan Wang*, Hideyuki Takeuchi*†, Yoshifumi Sonobe*, Shijie Jin*, Tetsuya Mizuno*, Shin Miyakawa‡, Masatoshi Fujiwara‡, Yoshikazu Nakamura§¶, Takuma Katoʈ, Hisako Muramatsu**, Takashi Muramatsu**††, and Akio Suzumura*† *Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; ‡RIBOMIC, Inc., 3-15-5-601 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan; §Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; ¶Core Research Evolutional Science and Technology, Japan Science and Technology Agency, Toyonaka, Osaka 560-8531, Japan; ʈDepartment of Bioregulation, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan; **Department of Biochemistry, Nagoya University Graduate School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; and ††Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, Nisshin, Aichi 470-0195, Japan Edited by Ethan Shevach, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board January 20, 2008 (received for review October 12, 2007) CD4؉CD25؉ regulatory T (Treg) cells are crucial mediators of nity, and abnormalities in Treg cell function may contribute to the autoimmune tolerance. The factors that regulate Treg cells, how- development of autoimmune diseases (15–17). Thus, expansion of ever, are largely unknown. Here, we show that deficiency in the Treg cell population could prevent autoimmune attacks, in- midkine (MK), a heparin-binding growth factor involved in onco- cluding EAE (12–14, 18, 19). genesis, inflammation, and tissue repair, attenuated experimental Here, we show that MK deficiency attenuates myelin oligoden- autoimmune encephalomyelitis (EAE) because of an expansion of drocyte glycoprotein (MOG)-induced EAE due to an expansion of the Treg cell population in peripheral lymph nodes and decreased the Treg cell population in the peripheral lymph nodes, followed by numbers of autoreactive T-helper type 1 (T 1) and T 17 cells. MK H H decreases in the numbers of autoreactive TH1 and TH17 cells. decreased the Treg cell population ex vivo in a dose-dependent Moreover, administration of anti-MK RNA aptamers that effec- manner by suppression of STAT5 phosphorylation that is essential tively neutralize MK induced expansion of the Treg cell population for Foxp3 expression. Moreover, administration of anti-MK RNA and reduced the symptoms of EAE. This demonstrates that MK is aptamers significantly expanded the Treg cell population and a critical suppressor of Treg cell expansion. Moreover, inhibition of alleviated EAE symptoms. These observations indicate that MK MK using RNA aptamers may be a potent therapeutic strategy serves as a critical suppressor of Treg cell expansion, and inhibition against autoimmune diseases, including MS. of MK using RNA aptamers may provide an effective therapeutic strategy against autoimmune diseases, including multiple sclerosis. Results MK Deficiency Attenuates MOG-Induced EAE. To examine the immu- aptamer ͉ multiple sclerosis nological roles of MK, we generated MOG35–55-induced EAE in C57BL/6J WT and MK-deficient (MdkϪ/Ϫ) mice. Most of the WT idkine (MK), a heparin-binding growth factor, exerts pleio- mice exhibited clinical signs of the disease Ϸ12 days after the initial Mtropic effects, including cell proliferation, cell migration, immunization and developed peak EAE Ϸ18 days after immuni- angiogenesis, and fibrinolysis, in a variety of tissues (1). MK also zation as described previously (20, 21). Almost all of the MdkϪ/Ϫ plays important roles in the induction of oncogenesis, inflamma- mice also developed EAE. However, the clinical scores of the Ϫ Ϫ tion, and tissue repair. The overexpression of MK has been ob- Mdk / mice were significantly lower than those of the WT mice MEDICAL SCIENCES served in a number of malignant tumors (1). However, MK- (Fig. 1a). deficient mice are reportedly resistant to ischemic renal injury (2) To confirm whether the suppression of EAE in the MdkϪ/Ϫ mice and neointima formation in atherosclerosis (3). A recent study was due to MK deficiency, we administrated MK to the MdkϪ/Ϫ proposed that MK deficiency suppresses the development of a mice with s.c.-implanted microosmotic pumps for Ϸ16 days after rheumatoid arthritis model by preventing inflammatory leukocyte MOG immunization as described previously (3, 4). As shown in Fig. migration and osteoclast differentiation (4). Furthermore, MK Ϫ Ϫ 1, MK administration to Mdk / mice abolished the suppression of expression is up-regulated in the spinal cord during the induction EAE (MdkϪ/Ϫ ϩ MK) (Fig. 1a). However, the cessation of MK and progression phases of experimental autoimmune encephalo- administration resulted in regaining the attenuation of EAE in myelitis (EAE) (5). However, the precise immunological functions Ϫ/Ϫ ϩ of MK have yet to be elucidated. Mdk MK mice (starting 22 days after MOG immunization). Multiple sclerosis (MS) and its animal model, EAE, are auto- Histological analysis of the lumbar spinal cord revealed that immune diseases characterized by inflammatory demyelination in the CNS (6). Although MS and EAE have been described as Author contributions: J.W., H.T., and Y.S. contributed equally to this work; H.T. and A.S. T-helper type 1 (TH1) cell-mediated autoimmune diseases, TH17 designed research; J.W., H.T., Y.S., S.J., T.M., and T.K. performed research; S.M., M.F., Y.N., ϩ cells, a newly discovered lineage of CD4 T cells that secrete IL-17, H.M., and T.M. contributed new reagents/analytic tools; J.W., H.T., and Y.S. analyzed data; have recently been found to be critical for the development of and H.T. and A.S. wrote the paper. autoimmune diseases, including EAE (7–10). Various types of The authors declare no conflict of interest. immune cells and soluble mediators contribute to the complex This article is a PNAS Direct Submission. E.S. is a guest editor invited by the Editorial Board. mechanism underlying the onset and progression of MS, which is Freely available online through the PNAS open access option. characterized by autoreactive T cell infiltration and the activation †To whom correspondence may be addressed. E-mail: [email protected] or of microglia, the resident antigen-presenting cells in the CNS. In [email protected]. ϩ ϩ particular, CD4 CD25 regulatory T (Treg) cells have received a This article contains supporting information online at www.pnas.org/cgi/content/full/ great deal of attention as negative regulators of MS pathogenesis 0709592105/DC1. (11–14). Treg cells regulate peripheral tolerance and autoimmu- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709592105 PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3915–3920 Downloaded by guest on September 27, 2021 MdkϪ/Ϫ-EAE mice had fewer infiltrating inflammatory cells than WT-EAE mice (WT mice) (Fig. 1 b and e)(MdkϪ/Ϫ mice) (Fig. 1 c and f). MK administration also reversed this reduced level of CNS inflammation in the MdkϪ/Ϫ mice (MdkϪ/Ϫ ϩ MK mice) (Fig. 1 d and g). These histological findings agreed with the clinical data. Taken together, these data indicate that MK plays an important role in the induction and development of EAE and that these immunological effects of MK are transient and reversible. MK Deficiency Induces the Expansion of the CD4؉CD25؉Foxp3؉ Treg Cell Population in Peripheral Lymph Nodes. We then assessed the immunological differences between WT and MdkϪ/Ϫ mice after MOG immunization. First, we compared the expression profiles of antigen-presenting molecules [MHC class II, B7–1, B7–2, intercel- lular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1], cytokines (TNF-␣, IL-1␤, IL-6, TGF-␤, and IL-12), chemokines [IFN-␥-inducible 10-kDa protein (IP-10) reg- ulated upon activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein-1␣ (MIP-1␣), and monocyte chemoattractant protein-1 (MCP-1)], and iNOS in mi- croglia or macrophages (as the antigen-presenting cells in the CNS or periphery, respectively). Before stimulation with LPS and IFN-␥, Ϫ/Ϫ ␤ Fig. 1. Attenuation of MOG-induced EAE in MK-deficient mice. (a) EAE Mdk microglia had higher B7–2, MCP-1, and IL-1 expression clinical scores for WT (filled squares; n ϭ 16), MdkϪ/Ϫ (open circles; n ϭ 13), and levels and lower ICAM-1 and MIP-1␣ expression levels than WT Ϫ Ϫ MK-administered MdkϪ/Ϫ mice (MdkϪ/Ϫ ϩ MK) (filled circles; n ϭ 13). MdkϪ/Ϫ microglia. In contrast, Mdk / macrophages had higher B7–1 and mice exhibited significantly lower clinical scores than WT mice (*, P Ͻ 0.01 vs. B7–2 expression levels than WT macrophages. After stimulation WT). MK administration to MdkϪ/Ϫ mice abrogated the attenuation of EAE. with LPS and IFN-␥, however, these differences were no longer After MK administration ceased (starting 22 days after MOG immunization), observed, except for decreased expression of VCAM-1 and MIP-1␣ Ϫ/Ϫ ϩ Mdk MK mice showed the partial suppression of EAE symptoms again (†, in MdkϪ/Ϫ microglia [supporting information (SI) Fig. 6a]. The total P Ͻ 0.05 vs. WT). (b–g) H&E staining of lumbar spinal cords from peak EAE mice. e, f, and g are higher-magnification images of the boxed areas in b, c, and d, number of mononuclear cells from the peripheral lymph nodes was Ϫ/Ϫ respectively. (Original magnification: b–d, ϫ100; e–g, ϫ400.) (b and e)WT similar between WT and Mdk mice (data not shown). Thus, we mice (score, 5) showed inflammatory cell infiltration in the spinal cord. (c and assessed subpopulations of T cells in the peripheral lymph nodes.