l-Tryptophan−Kynurenine Pathway Metabolites Regulate Type I IFNs of Acute Viral Myocarditis in Mice This information is current as Masato Hoshi, Keishi Matsumoto, Hiroyasu Ito, Hirofumi of September 30, 2021. Ohtaki, Yuko Arioka, Yosuke Osawa, Yasuko Yamamoto, Hidetoshi Matsunami, Akira Hara, Mitsuru Seishima and Kuniaki Saito J Immunol 2012; 188:3980-3987; Prepublished online 14 March 2012; Downloaded from doi: 10.4049/jimmunol.1100997 http://www.jimmunol.org/content/188/8/3980 References This article cites 37 articles, 20 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/188/8/3980.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology L-Tryptophan–Kynurenine Pathway Metabolites Regulate Type I IFNs of Acute Viral Myocarditis in Mice Masato Hoshi,*,1 Keishi Matsumoto,†,1 Hiroyasu Ito,* Hirofumi Ohtaki,* Yuko Arioka,† Yosuke Osawa,* Yasuko Yamamoto,† Hidetoshi Matsunami,‡ Akira Hara,x Mitsuru Seishima,* and Kuniaki Saito† The activity of IDO that catalyzes the degradation of tryptophan (Trp) into kynurenine (Kyn) increases after diseases caused by different infectious agents. Previously, we demonstrated that IDO has an important immunomodulatory function in immune-related diseases. However, the pathophysiological role of IDO following acute viral infection is not fully understood. To investigate the role of IDO in the L-Trp–Kyn pathway during acute viral myocarditis, mice were infected with encephalomyocarditis virus, which 2/2 induces acute myocarditis. We used IDO-deficient (IDO ) mice and mice treated with 1-methyl-D,L-Trp (1-MT), an inhibitor of IDO, to study the importance of Trp–Kyn pathway metabolites. Postinfection with encephalomyocarditis virus infection, the Downloaded from serum levels of Kyn increased, whereas those of Trp decreased, and IDO activity increased in the spleen and heart. The survival rate of IDO2/2 or 1-MT–treated mice was significantly greater than that of IDO+/+ mice. Indeed, the viral load was suppressed in the IDO2/2 or 1-MT–treated mice. Furthermore, the levels of type I IFNs in IDO2/2 mice and IDO2/2 bone marrow-transplanted IDO+/+ mice were significantly higher than those in IDO+/+ mice, and treatment of IDO2/2 mice with Kyn metabolites eliminated the effects of IDO2/2 on the improved survival rates. These results suggest that IDO has an important role in acute viral myocarditis. Specifically, IDO increases the accumulation of Kyn pathway metabolites, which suppress type I IFNs production http://www.jimmunol.org/ and enhance viral replication. We concluded that inhibition of the Trp–Kyn pathway ameliorates acute viral myocarditis. The Journal of Immunology, 2012, 188: 3980–3987. -Tryptophan (Trp) is an essential amino acid that is required IL-6, or IL-1b; however, these proteins alone do not strongly induce for the biosynthesis of proteins and several other biolog- IDO in THP-1 cells (11). Both animal and human studies demon- L ically important compounds such as kynurenine (Kyn), strate that cells that express IDO have an immunosuppressive func- which is produced by L-TRP 2,3-dioxygenase, and IDO. These tion by increasing T lymphocyte tolerance (12). In addition, several enzymes catabolize Trp via the Kyn pathway to form nicotinic acid, other studies suggest that IDO-expressing cells deplete Trp from by guest on September 30, 2021 niacin, and NAD. Unlike L-TRP 2,3-dioxygenase, which is mainly the extracellular milieu and secrete Trp metabolites (including localized in the liver and is upregulated by corticosteroids, IDO is Kyn, 3-hydroxy-kynurenine, 3-hydroxyanthranilic acid, and quino- expressed in diverse tissues in humans (1), including lymphoid or- lonic acid), which induce T cell apoptosis and suppress immune re- gans and tumor cells (2), and is mostly expressed in the cells of the sponses in vitro (13–15). Recently, we demonstrated that type I IFN innate immune system, such as macrophages and dendritic cells production is increased in the absence of IDO, resulting in the sup- (DC) (3), following microbial (4, 5) or viral infections (6, 7). Depend- pression of viral replication in chronic retrovirus-infected mice (16). ing on the cell type, IDO expression can be constitutive or inducible However, the mechanism for this upregulation and the role of Trp by proinflammatory cytokines, TLR ligands, and costimulatory li- catabolism in vivo after acute viral infection is not fully understood. gandssuchasCTLA4(3,8,9)orIFN-g (10). Previously, we showed Encephalomyocarditis virus (EMCV), which is a member of the that IDO induction has a significant synergistic effect with TNF-a, Picornaviridae family, which includes the Enterovirus genus, can cause acute myocarditis in various animals including mice. EMCV infection in mice is an established model for viral myocarditis, di- *Department of Informative Clinical Medicine, Gifu University Graduate School lated cardiomyopathy, and congestive heart failure (17). In this study, of Medicine, Gifu 501-1194, Japan; †Human Health Sciences, Kyoto University Graduate School of Medicine and Faculty of Medicine, Kyoto 606-8507, Japan; we examined the roles of IDO on immune regulation in EMCV in- ‡ 2/2 Department of Medicine, Matsunami General Hospital, Kasamatsu Cho, Gifu 501- fection by using IDO mice or the IDO inhibitor 1-methyl-D,L-Trp 6062, Japan; and xDepartment of Tumor Pathology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan (1-MT). We demonstrated that type I IFNs are upregulated, resulting in suppressed EMCV replication by IDO knockdown or inhibition; 1M.H. and K.M. contributed equally to this study. these results were reversed by Kyns administration. Thus, the in- Received for publication April 6, 2011. Accepted for publication February 8, 2012. hibition of Trp–Kyn pathway ameliorates acute viral myocarditis. This work was supported by Grants-in-Aid for Scientific Research (20390167 and 23790787) from the Ministry for Education, Culture, Sports, Science and Technology of Japan. Materials and Methods Address correspondence and reprint requests to Dr. Kuniaki Saito, Human Health Mice Sciences, Kyoto University Graduate School of Medicine and Faculty of Medicine, Kyoto 606-8507, Japan. E-mail address: [email protected] Six-week-old male mice were used in this study. IDO1 gene-deficient (IDO2/2) mice of a C57BL/6J background were obtained from The Jack- Abbreviations used in this article: ALT, alanine aminotransferase; BMT, bone mar- son Laboratory (Bar Harbor, ME). Mice that were wild-type (+/+) or ho- row transplantation; BUN, blood urea nitrogen; CK, creatine kinase; DC, dendritic 2/2 cell; EMCV, encephalomyocarditis virus; Kyn, kynurenine; LD, lactate dehydroge- mozygous null ( ) for targeted disruption of the IDO gene were selected nase; 1-MT, 1-methyl-D,L-Trp; pDC, plasmacytoid dendritic cell; Trp, tryptophan. from the offspring of heterozygous/homozygous matings by using the PCR tail DNA. C57BL/6J mice obtained from Japan SLC (Shizuoka, Japan) Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 were used as WT (IDO+/+) controls. Furthermore, the IDO+/+ mice were www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100997 The Journal of Immunology 3981 divided into four groups: EMCV-infected mice with or without 1-MT the serum were measured with commercially available kits and an automatic treatment and uninfected mice with or without 1-MT treatment. These analyzer BM 2250. mice were administered 1-MT in their drinking water (5 mg×ml21) after viral infection, and their average daily consumption was 3.5 ml. All ex- Administration of Kyns periments were performed in accordance with the Guidelines for Animal Kyns were administered as described previously (21, 22). Briefly, mice Care of Kyoto University. were i.p. injected with a mixture of Kyns (20 mg×kg21×day21 L-kynurenine, Virus inoculation 3-hydroxykynurenine, and 3-hydroxy-anthranilic acid; Sigma-Aldrich, Tokyo, Japan) once per day. A myocarditic variant of EMCV was generously provided by Dr. Y. Seto (Keio University, Tokyo, Japan). The virus stock was stored at 280˚C in Bone marrow transplantation HBSS with 0.1% BSA until use. The mice were injected i.p. with 500 PFU Bone marrow transplantation (BMT) was performed on mice 5 wk of age as EMCV in 0.1 ml saline. Six-week-old male mice were inoculated and then described previously (20). The recipient mice (IDO+/+ and IDO2/2 mice) housed in an isolated room. The day of virus inoculation was defined as were irradiated in fractionated dose (5 Gy twice with a 4-h interval) and day 0 in the following experiments. All experiments were performed in reconstituted with the whole bone marrow cells injection (5 3 106 bone accordance with the institutional guidelines of Kyoto University. marrow cells/250 ml from young IDO+/+ and IDO2/2 donor mice) via tail Measurements of L-Trp and L-Kyn pathway metabolites vein. These BMT mice were maintained under special pathogen-free conditions, given 500 U/ml gentamicin sulfate (Invitrogen, Grand Island, L-Trp, L-Kyn, 3-hydroxykynurenine, and 3-hydroxy-anthranilic acid were NY) and 100 mg/ml polymixicin B sulfate (Kayaku, Tokyo, Japan) in measured by using HPLC with a spectrophotometric detector (Ultraviolet- drinking water for 4 wk after cell transfer.