Viral Myocarditis in Mice Metabolites Regulate Type I Ifns of Acute

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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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. 8000; Tosoh, Tokyo, Japan) or fluorescence spectrometric detector (Hita- chi, Tokyo, Japan) or ECD-300 electrochemical detector (Eicom, Kyoto, Statistical analyses Japan) as described previously (1, 18). Results are expressed as mean (SD). The survival rates of mice were analyzed Downloaded from Enzymatic assay for IDO activity by the Kaplan–Meier method. Statistically significant differences between two groups were determined by using Student t test, and those among three groups IDO activity was assayed as described previously (19). Briefly, spleen, liver, were determined by using one-way ANOVA. We used StatView 4.5 for these lung, small intestine, and heart were homogenized with a Polytron ho- statistical analyses. The criterion for statistical significance was p , 0.05. mogenizer (Kinematica, Lucerne, Switzerland) in 1.5 vol of ice-cold 0.14 M KCl-20 mM potassium phosphate buffer (pH 7). Subsequently, the homogenate was centrifuged at 7000 3 g for 10 min at 4˚C. An aliquot of Results http://www.jimmunol.org/ the supernatant was used to measure the IDO activity. The reaction mixture Changes in serum L-Trp–Kyn pathway metabolism in EMCV- contained 50 ml enzyme preparation and 50 ml substrate solution (100 mM infected mice potassium phosphate buffer [pH 6.5], 50 mM methyleneblue, 20 mg cat- +/+ alase, 50 mM ascorbate, and 0.4 mM L- or D-Trp). After the reaction In IDO mice, the serum ALT, CK, and LD activities increased mixture was incubated at 37˚C, it was acidified with 3% perchloric acid significantly within 4 d after EMCV inoculation, suggesting that 3 and centrifuged at 7000 g for 10 min at 4˚C. Then, the concentrations of severe myocardial damage was induced by EMCV (Fig. 1A). The the enzymatic products were measured by using HPLC. The enzyme activity was expressed as the product content per hour per gram of tissue. serum Kyn levels increased significantly after 48 h of inoculation and peaked 4 d after inoculation (Fig. 1B), with the ratio of Kyn/ Histopathology Trp levels being significantly high as well as after 4 d, indicating

Tissues were fixed in 10% formalin in PBS overnight and then embedded in that IDO activity had increased. In contrast, serum Trp levels by guest on September 30, 2021 paraffin. Sections (thickness, 4 mm) were used for H&E staining and IDO decreased significantly after 7 d. Indeed, IDO activity in the spleen immunohistochemical staining as described in a previous report (16). Two and liver of the IDO+/+ had significantly increased within 24 h of pathologists who did not have any knowledge of the study design graded the extent of cellular infiltration and myocardial necrosis in tissue samples inoculation, and its activity in the lung and heart significantly according to the following scale: 0, no lesions; 1+, lesions involving increased after 48 h and 4 d, respectively. No significant difference ,25%; 2+, lesions involving 25–50%; 3+, lesions involving 50–75%; and was observed in the small intestine (Fig. 2A). Furthermore, 4+, lesions involving 75–100% (20). EMCV-infected IDO+/+ mice showed apparent cellular infiltration RNA extraction and real-time PCR analysis in the heart on day 7 postinfection (Fig. 2B). Notably, the infil- trated cells in the heart and splenocytes of the EMCV-infected Total RNAwas extracted from heart tissue with Isogen (Nippon Gene, Tokyo, +/+ Japan), and the RNA concentration was determined spectrophotometrically IDO mice strongly expressed IDO. at 260 nm. RT-PCR was performed by using High-Capacity cDNA Reverse Effect of IDO gene deficiency or IDO inhibition on survival Transcription kits (Applied Biosystems, Foster City, CA). The following PCR primers were synthesized: EMCV, sense, 59-GTCGTGAAGGAAG- rates and EMCV viral RNA in the heart CAGTTCC-39, and antisense, 59-CACGTGGCTTTTGGCCGCAGAGGC- To investigate the roles of IDO in EMCV-induced myocardial 39. EMCVand 18s were quantified by using real-time PCR in a LightCycler damage, the effects of IDO deficiency and of the IDO inhibitor 1- (Roche, Meylan, France). The EMCV and 18s reactions were performed 2/2 with a SYBR green PCR mix (Qiagen, Hilden, Germany) and a TaqMan MT were examined. On day 25, the survival rate of IDO mice probe (Applied Biosystems), respectively, as described previously (19). The after inoculation with EMCV was 62%, whereas the survival rate data were analyzed with LightCycler software (version 5.32; Roche). of IDO+/+ mice was 18% (Fig. 3A). Similarly, 1-MT improved the Flow cytometry analyses survival rate from 20 to 43% (Fig. 3B). On day 4, the levels of EMCV genomic RNA in the hearts of Splenocytes were purified by using Ficoll (IBL, Gunma, Japan) and washed 2/2 twice in PBS. Subsequently, these cells were incubated with anti-mouse IDO mice and 1-MT–treated mice were significantly less than +/+ CD11c Ab, anti-mouse CD11b Ab, and anti-mouse B220 Ab for 20 min those in IDO mice (Fig. 3C). Furthermore, the serum levels of at 4˚C (BD Biosciences, Franklin Lakes, NJ). Afterward, their expression LD and CK in IDO+/+ mice on day 4 were significantly worse than of membrane Ags was determined by using a FACScan flow cytometer those in IDO2/2 mice or 1-MT–treated mice (Table I). Indeed, (BD Biosciences). Viable cells were gated on forward and side scatter, and +/+ a minimum of 10,000 events was acquired for each sample. IDO mice exhibited more myocardial necrosis on day 7 than the IDO2/2 mice and 1-MT–treated mice did. Measurements of serum creatine kinase, lactate dehydrogenase, alanine aminotransferase, and blood urea Changes in the serum levels of type I IFNs and Kyn metabolites nitrogen in EMCV-infected mice The activities of creatine kinase (CK), lactate dehydrogenase (LD), alanine To determine the mechanisms by which IDO promotes myocardial aminotransferase (ALT), and blood urea nitrogen (BUN) concentrations in damage, changes in the serum levels of type I IFNs, which have 3982 ROLE OF Trp CATABOLISM IN ACUTE VIRAL MYOCARDITIS

FIGURE 1. Changes in the serum levels of Trp catabolism and biochem- ical makers in response to EMCV inoculation. (A) Serum levels of ALT, CK, and LDH activities and BUN concentrations in IDO+/+ mice at indicated times after EMCV inoculation. (B) Serum levels of L-Trp and L- Kyn, and the ratio of Trp/Kyn levels in IDO+/+ mice at indicated times after EMCV inoculation. The data are representative of three independent experiments and are expressed as the mean (SD) of six mice per time point per group. Statistically signiﬁ- cant differences between the groups were determined by using ANOVA; *p , 0.05. Downloaded from

antiviral effects, were compared between IDO+/+ and IDO2/2 IDO2/2 mice than in IDO+/+ mice. To conﬁrm whether Kyn mice after inoculation with EMCV (Fig. 4A). Serum levels of pathway regulates the production of type I IFNs, we compared type I IFNs after 48 h of inoculation were signiﬁcantly higher in the changes in the serum levels of the metabolites of Kyn http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. IDO activity after EMCV inoculation. (A) IDO activity in the heart, spleen, liver, intestine, and lungs of IDO+/+ mice at various times after EMCV inoculation. The data are representative of three independent experiments and are expressed as the mean (SD) of five mice per time point per group. Statistically significant differences between groups were determined by using ANOVA; *p , 0.05. (B) Histopahologic findings are demonstrated using H&E staining and IDO immunohistochemical staining (original magnification 340) of both heart and spleen from uninfected mice (day 0, upper panel) and EMCV-infected mice (day 7, bottom panel). The results of one of the five analyses with similar results are shown in this study. The Journal of Immunology 3983 0.6 0.5 0.7 6 6 6 0.50.5* 1.9 2.3 0.5* 2.4 Score on Day 7 Histopathological 6 6 6 360211* 2.3 1.3 322* 1.6 6 6 6 561411* 1620 992 350* 1030 6 6 6 Downloaded from 19687* 2788 1661 101* 1830 6 6 6 CK (IU/l) http://www.jimmunol.org/ 137266 1314 881 199 911 6 6 6 7588 496 437 110 468 6 6 6 by guest on September 30, 2021

2/2 201105* 230 210 FIGURE 3. Survival rates and EMCV RNA levels in IDO mice or 1- 211* 135 2 2 6 6 MT–treated mice after EMCV inoculation. (A) The survival rate of IDO / 6 mice (n = 15) was signiﬁcantly higher than that of IDO+/+ mice (n =15).(B)

Consistent with these results, the survival rate of 1-MT–treated mice (n =15) mice 2 also was higher than that of IDO+/+ mice (n = 15). The data are represen- / 2 tative of three independent experiments. Statistically significant differences 541623* 1055 860 between the groups were determined using the log-rank test. (C)IDO+/+ 399* 813 6 6 mice, IDO2/2 mice, and 1-MT–treated mice were inoculated with EMCV, 6 and then, the amount of EMCV RNA in the heart from each group of mice was determined using real-time quantitative PCR. The data are representative SD. of three independent experiments, and are expressed as the mean (SD) of five 6 mice per time point per group. Statistically significant differences between 658583* 2481 1623 the groups were determined using ANOVA; *p , 0.05, **p , 0.01. 409* 1799 6 6 6 LDH (IU/l) pathway in IDO2/2 mice and IDO+/+ mice after inoculation with = 7. Values are mean

EMCV. The serum Kyn and 3-hydroxykynurenine levels in n 250162 2248 1514 IDO+/+ mice 48 h after inoculation signiﬁcantly increased 101 1816 6 6 compared with those in IDO2/2 mice(Fig.4B).Moreover, 6 treatment of Kyns decreased the induction of type I IFNs in IDO2/2 mice (Fig. 4C). We assessed the myocardial damage in 2 2 Kyns-treated IDO / mice. The serum levels of ALT, LD, and . 8085 498 570 101 508 2/2 +/+ 6 6 CK in Kyns-treated IDO mice were signiﬁcantly elevated 6

2/2 Day 0 Day 2 Day 4 Day 7 Day 10 Day 0 Day 2 Day 4 Day 7 Day 10 Necrosis Inﬁltration than those in untreated IDO mice (Fig. 5A). Moreover, Kyns- , and 1-MT–treated mice; 210 250 2 2/2 / treated IDO mice exhibited pronounced myocardial necrosis 2 4 d after inoculation with EMCV compared with the untreated 2 / 2 2 ,IDO / 0.01 versus IDO +/+ 2

IDO mice (Fig. 5B). In addition, on day 25, the survival rate +/+ 2/2 , IDO of the untreated IDO mice was 62%, whereas all the Kyns- IDO 1-MT 315 IDO 2/2 *p

treated IDO mice had died on day 25 (Fig. 5C). These results Table I. Changes in myocardial marker and histopathologic ﬁndings in IDO 3984 ROLE OF Trp CATABOLISM IN ACUTE VIRAL MYOCARDITIS Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. Changes in serum levels of type I IFN in response to EMCV inoculation. (A) IDO+/+ and IDO2/2 mice were inoculated with EMCV, and the serum levels of type I IFNs were subsequently quantified using an ELISA. The data are representative of three independent experiments and are expressed as the mean (SD) of five mice per time point per group. Statistically significant differences between the groups were determined using a Student t test; *p , 0.05. (B) Serum Trp metabolite levels in IDO+/+ mice and IDO2/2 mice with or without EMCV infection. The data are representative of three independent experiments and are expressed as the mean (SD) of five mice per group. Statistically significant differences between the uninfected IDO+/+ mice and EMCV-infected IDO+/+ mice (*p , 0.05) or between EMCV-infected IDO+/+ mice and EMCV-infected IDO2/2 mice (**p , 0.05). (C) IDO2/2 mice were inoculated with EMCV and then i.p. injected with a Kyns mixture once per day. The serum levels of type I IFNs were analyzed using ELISA. The data are representative of three independent experiments and are expressed as the mean (SD) of five mice per time point per group. Statistically significant differences between the groups were determined using Student t test; *p , 0.001. suggest that increased levels Kyns as a result of IDO activity tributed to bone marrow-derived cells from IDO+/+ mice and not might increase viral load, thereby promoting myocardial damage non-bone marrow-derived cells. by inhibition of induction of type I IFNs. Increased macrophage levels in the spleen of IDO2/2 mice Increased type I IFN levels in IDO gene-deficient bone marrow after EMCV infection cells after EMCV infection To explore the cell subsets producing type I IFN levels in bone Bone marrow-derived cells are considered a source of type I IFNs. marrow-derived cells, we next used flow cytometry to quantify the 2 To explore the involvement of IDO in the bone marrow-derived number of conventional DCs (CD11c+B220 ), plasmacytoid DCs 2 cells, we generated IDO-chimeric mice by using a combination (pDCs; CD11clowB220+), and macrophages (CD11b+CD11c )in 2 2 2 2 of irradiation and bone marrow transplants. IFN-b levels in the the spleen of IDO+/+ mice, IDO / mice, and Kyns-treated IDO / IDO2/2 bone marrow-transplanted IDO+/+ mice was significantly mice after inoculation with EMCV (Fig. 7A). The gated per- 2 2 higher than those in the IDO+/+ bone marrow-transplanted IDO2/2 centages of macrophages in the spleen of IDO / mice were mice 48 h after inoculation with EMCV (Fig. 6A). In addition, on significantly higher than those in the IDO+/+ mice or Kyns-treated 2 2 day 25, the survival rate of the IDO2/2 bone marrow- transplanted IDO / mice 48 h after inoculation. In addition, the number of 2 2 IDO+/+ was 64%, whereas that of IDO+/+ bone marrow- splenocytes in IDO / mice was significantly higher than that in 2 2 transplanted IDO2/2 was 23% (Fig. 6B). These results suggest IDO+/+ mice or Kyns-treated IDO / mice (Fig. 7B). The ex- that increased levels of type I IFNs in IDO2/2 mice can be at- pression of IDO mRNA at 48 h, and the level of IFN-b in the The Journal of Immunology 3985 Downloaded from

FIGURE 5. Effect of Kyns treatment on acute viral myocarditis. (A) ALT, CK, and LDH activities and BUN concentrations in the serum of IDO2/2 or Kyns-treated IDO2/2 mice at indicated times after EMCV inoculation. The data are representative of three independent experiments and are expressed as the mean (SD) of six mice per time point per group. Statistically significant differences between the groups were determined using Student t test; *p , 0.05. 2/2 2/2 (B) Histopathological analysis of the H&E-stained heart tissue sections of IDO mice or Kyns-treated IDO mice 4 d after EMCV inoculation. Kyns- http://www.jimmunol.org/ treated IDO2/2 mice showed more cellular infiltration and myocardial necrosis than IDO2/2 mice did. Representative images from five independent analyses are shown. Scale bars, 100 mm. (C) The survival rates of IDO2/2 mice (n = 15) were significantly higher than those of Kyns-treated IDO2/2 mice (n = 13). The data are representative of three independent experiments. Statistically significant differences between the groups were determined by using the log-rank test.

medium supernatant at 72 h, was signiﬁcantly enhanced in the and that these observations are reversed by administration of spleen CD11c+ cells and CD11b+ cells (Fig. 7C, 7D). These Kyns. IDO has been thought to have beneﬁcial actions. For ex- results suggest that the source of type I IFNs is the macrophage, ample, recent studies have shown that TLR3 ligand poly (I:C) by guest on September 30, 2021 which is regulated by Kyns. induces IDO activation in astrocytes and causes an antiviral response (23). In addition, IFN-g–induced IDO has an antiviral Discussion effect in measles virus infection of epithelial and endothelial cells IDO is expressed in human (1) and mouse tumor cells (2), DCs, in vitro (24). In contrast, disadvantageous functions of IDO have and macrophages following microbial (4, 5) or viral infections (6, been also reported in viral infections. CTLA-4 blockade has been 7). In this study, we examined the roles of IDO in the Trp-Kyn reported to decrease the expression of TGF-b, IDO, and viral metabolic pathway with respect to the immune regulation in RNA in the tissues from SIVmac251-infected macaques (25), and EMCV infections by using IDO2/2 mice and the IDO inhibitor 1- the IDO inhibitor 1-MT may be a promising candidate for en- MT. We demonstrated that type I IFNs are upregulated, resulting hancing immunity, including anti-HIV immunity, in HIV-infected in suppressed EMCV replication by IDO knockdown or inhibition, patients (26). In this study, IDO was induced by EMCV, which

FIGURE 6. Effect of type I IFN production and survival rates after BMT. (A) IDO+/+ bone marrow-transplanted IDO2/2 mice and IDO2/2 bone marrow- transplanted IDO+/+ mice were inoculated with EMCV. At 48 h postinfection, the serum levels of IFN-b were quantified using ELISA. The data are representative of three independent experiments and are expressed as the mean (SD) of seven mice per time point per group. Statistically significant differences between the groups were determined using Student t test; *p , 0.05. (B) The survival rate of IDO2/2 bone marrow-transplanted IDO+/+ mice (n = 12) was significantly higher than that of IDO+/+ bone marrow-transplanted IDO2/2 mice (n = 12). The data are representative of three independent experiments. Statistically significant differences between the groups were determined using the log-rank test. 3986 ROLE OF Trp CATABOLISM IN ACUTE VIRAL MYOCARDITIS

FIGURE 7. Cell populations in the spleen of IDO2/2 mice producing type I IFN. (A) Gated percentages of CD11b+CD11c2 cells, CD11b+CD11c+ cells, CD11+ B220+ cells, and CD11c+B2202 cells in the spleen of IDO+/+ mice, IDO2/2 mice, or Kyns-treated IDO2/2 mice at 0 and 48 h after EMCV inoculation. Statisti- cally significant differences between the groups were determined using ANOVA; *p , 0.05. (B) The number of splenocytes in IDO+/+ mice, IDO2/2 mice, or Kyns-treated IDO2/2 mice at 0 and 48 h after EMCV inoculation. The data are representative of three independent experiments and are expressed as the mean (SD) of five mice per group. Statistically significant differences between EMCV-infected IDO+/+ mice or EMCV-infected Kyns-treated IDO2/2 mice; *p , 0.05. (C) These indicated cells (CD11c+, CD11b+, DX5+, CD8+, CD4+, and other) were isolated from the spleens of IDO+/+ mice 48 h after EMCV inoculation. Downloaded from The expression of IDO mRNA in these cells was analyzed using real-time quantitative PCR. (D) The levels of IFN-b in the medium supernatant at 72 h of incubation of these indicated cells (1 3 106/well) was quantified using ELISA. These data are representative of three independent experiments and are expressed as the mean (SD) of seven mice per group. http://www.jimmunol.org/

promoted viral replication and tissue damage, suggesting that IDO which is a transcription factor that regulates type I IFN production, has a disadvantageous function in acute EMCV infection. IDO- upon T cell Ag receptor engagement, by speciﬁcally targeting

deficient and inhibitor-treated mice had higher survival rates, phosphoinositide-dependent protein kinase-1 (33). Moreover, by guest on September 30, 2021 resulting in greater suppression of EMCV replication (Fig. 3). IFN-b levels in the IDO2/2 bone marrow-transplanted IDO+/+ When pathogens invade host cells, they activate the innate im- mice were significantly higher than those in the IDO+/+ bone mune system and elicit the production of cytokines and chemo- marrow-transplanted IDO2/2 mice, suggesting that IDO regulates kines, which recruit immune cells that mediate pathogen the production of type I IFNs in the bone marrow cells. The clearance. In particular, type I IFNs are important mediators of number of type I IFN-producing macrophages in IDO2/2 mice was innate immunity that limit the adverse effects of many viruses significantly increased compared with that in IDO+/+ mice (Fig. 7), (27). Postinfection, EMCV is initially recognized by the mela- suggesting that the increase in the levels of type I IFNs may be due noma differentiation-associated gene 5 receptor, which induces to the regulation of macrophage number via inhibition of Kyns antiviral responses including the production of type I IFNs and accumulation. This is consistent with our previous results demon- proinflammatory cytokines in DCs and macrophages (28, 29). strating that the number of intrahepatic CD11b+ cells in IDO2/2 IFN-a and IFN-b suppress the progression of EMCV infection mice was significantly greater than that in IDO+/+ mice in an (30). Indeed, the serum levels of type I IFNs was increased in a-galactosylceramide–induced hepatitis model (34). However, we IDO2/2 mice 48 h after inoculation with EMCV and were sig- do not exclude other possibilities (e.g., Kyns enhance regulatory nificantly higher than those in IDO+/+ mice, resulting in reduced T cell activity or suppress Th17 cells). In addition, the increase in myocardial damage in the IDO2/2 mice or 1-MT–treated mice. the type I IFN levels in IDO2/2 mice can be also explained by Currently, there are two hypotheses regarding the role of Trp ca- alternate mechanisms. Recent studies have demonstrated that pro- tabolism in the induction of tolerance. One hypothesis proposes duction of type I IFN in pDCs is regulated by the mammalian target that the downstream metabolites of Trp suppress immune reac- of rapamycin, which is known to inhibit via amino-acid starvation tivity by directly interacting with effector T lymphocytes and (35, 36), suggesting that depletion of local Trp by IDO induction other types of immune cells (13, 14, 31). An alternative hypothesis might regulate type I IFN production via mammalian target of suggests that the breakdown of Trp suppresses T cell proliferation rapamycin. In addition, IDO is involved with TGF-b in intracel- by reducing the availability of this essential amino acid under lular signaling events responsible for the self-amplification and local tissue microenvironments (32). In this study, Kyns mixture maintenance of a stably regulatory phenotype in pDCs (37). reversed the effects of IDO knockdown, suggesting that the in- Therefore, IDO inhibits type I IFNs not only by suppressing the crease in Kyn metabolites via upregulation of IDO after EMCV macrophage number but also by regulating the IFNs production in inoculation regulated the production of type I IFNs. Our results macrophages. are consistent with a previous study that showed that the cyto- In conclusion, our findings show that Kyn metabolites regulate toxicity of Trp metabolites tends to affect macrophages as well as the production of type I IFNs by decreasing the number macro- T, B, and NK cells in vitro but not DCs (13). In addition, it has been phages. Consequently, modulation of the Trp–Kyn pathway may be previously reported that Kyn metabolites inhibit NF-kB activation, an effective strategy for treating acute viral myocarditis. The Journal of Immunology 3987

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