l-Tryptophan–Kynurenine Pathway Metabolites Regulate Type I IFNs of Acute Viral Myocarditis in Mice

Masato Hoshi, Keishi Matsumoto, Hiroyasu Ito, Hirofumi Ohtaki, Yuko Arioka, Yosuke Osawa, Yasuko Yamamoto, Hidetoshi Matsunami, Akira Hara, Mitsuru Seishima and Kuniaki Saito
J Immunol April 15, 2012, 188 (8) 3980-3987; DOI: https://doi.org/10.4049/jimmunol.1100997

Abstract

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 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 serum levels of Kyn increased, whereas those of Trp decreased, and IDO activity increased in the spleen and heart. The survival rate of IDO−/− or 1-MT–treated mice was significantly greater than that of IDO+/+ mice. Indeed, the viral load was suppressed in the IDO−/− or 1-MT–treated mice. Furthermore, the levels of type I IFNs in IDO−/− mice and IDO−/− bone marrow-transplanted IDO+/+ mice were significantly higher than those in IDO+/+ mice, and treatment of IDO−/− mice with Kyn metabolites eliminated the effects of IDO−/− 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 and enhance viral replication. We concluded that inhibition of the Trp–Kyn pathway ameliorates acute viral myocarditis.

L-Tryptophan (Trp) is an essential amino acid that is required for the biosynthesis of proteins and several other biologically important compounds such as kynurenine (Kyn), which is produced by l-TRP 2,3-dioxygenase, and IDO. These enzymes catabolize Trp via the Kyn pathway to form nicotinic acid, niacin, and NAD. Unlike l-TRP 2,3-dioxygenase, which is mainly localized in the liver and is upregulated by corticosteroids, IDO is expressed in diverse tissues in humans (1), including lymphoid organs and tumor cells (2), and is mostly expressed in the cells of the innate immune system, such as macrophages and dendritic cells (DC) (3), following microbial (4, 5) or viral infections (6, 7). Depending on the cell type, IDO expression can be constitutive or inducible by proinflammatory cytokines, TLR ligands, and costimulatory ligands such as CTLA4 (3, 8, 9) or IFN-γ (10). Previously, we showed that IDO induction has a significant synergistic effect with TNF-α, IL-6, or IL-1β; however, these proteins alone do not strongly induce IDO in THP-1 cells (11). Both animal and human studies demonstrate that cells that express IDO have an immunosuppressive function by increasing T lymphocyte tolerance (12). In addition, several other studies suggest that IDO-expressing cells deplete Trp from the extracellular milieu and secrete Trp metabolites (including Kyn, 3-hydroxy-kynurenine, 3-hydroxyanthranilic acid, and quinolonic acid), which induce T cell apoptosis and suppress immune responses in vitro (13–15). Recently, we demonstrated that type I IFN production is increased in the absence of IDO, resulting in the suppression of viral replication in chronic retrovirus-infected mice (16). However, the mechanism for this upregulation and the role of Trp catabolism in vivo after acute viral infection is not fully understood.

Encephalomyocarditis virus (EMCV), which is a member of the 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, dilated cardiomyopathy, and congestive heart failure (17). In this study, we examined the roles of IDO on immune regulation in EMCV infection by using IDO−/− mice or the IDO inhibitor 1-methyl-d,l-Trp (1-MT). We demonstrated that type I IFNs are upregulated, resulting in suppressed EMCV replication by IDO knockdown or inhibition; these results were reversed by Kyns administration. Thus, the inhibition of Trp–Kyn pathway ameliorates acute viral myocarditis.

Materials and Methods

Mice

Six-week-old male mice were used in this study. IDO1 gene-deficient (IDO−/−) mice of a C57BL/6J background were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice that were wild-type (+/+) or homozygous null (−/−) for targeted disruption of the IDO gene were selected from the offspring of heterozygous/homozygous matings by using the PCR tail DNA. C57BL/6J mice obtained from Japan SLC (Shizuoka, Japan) were used as WT (IDO+/+) controls. Furthermore, the IDO+/+ mice were divided into four groups: EMCV-infected mice with or without 1-MT treatment and uninfected mice with or without 1-MT treatment. These mice were administered 1-MT in their drinking water (5 mg⋅ml−1) after viral infection, and their average daily consumption was 3.5 ml. All experiments were performed in accordance with the Guidelines for Animal Care of Kyoto University.

Virus inoculation

A myocarditic variant of EMCV was generously provided by Dr. Y. Seto (Keio University, Tokyo, Japan). The virus stock was stored at −80°C in HBSS with 0.1% BSA until use. The mice were injected i.p. with 500 PFU EMCV in 0.1 ml saline. Six-week-old male mice were inoculated and then housed in an isolated room. The day of virus inoculation was defined as day 0 in the following experiments. All experiments were performed in accordance with the institutional guidelines of Kyoto University.

Measurements of l-Trp and l-Kyn pathway metabolites

l-Trp, l-Kyn, 3-hydroxykynurenine, and 3-hydroxy-anthranilic acid were measured by using HPLC with a spectrophotometric detector (Ultraviolet-8000; Tosoh, Tokyo, Japan) or fluorescence spectrometric detector (Hitachi, Tokyo, Japan) or ECD-300 electrochemical detector (Eicom, Kyoto, Japan) as described previously (1, 18).

Enzymatic assay for IDO activity

IDO activity was assayed as described previously (19). Briefly, spleen, liver, lung, small intestine, and heart were homogenized with a Polytron homogenizer (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 × g for 10 min at 4°C. An aliquot of the supernatant was used to measure the IDO activity. The reaction mixture contained 50 μl enzyme preparation and 50 μl substrate solution (100 mM potassium phosphate buffer [pH 6.5], 50 μM methyleneblue, 20 μg catalase, 50 mM ascorbate, and 0.4 mM L- or d-Trp). After the reaction mixture was incubated at 37°C, it was acidified with 3% perchloric acid and centrifuged at 7000 × g for 10 min at 4°C. Then, the concentrations of the enzymatic products were measured by using HPLC. The enzyme activity was expressed as the product content per hour per gram of tissue.

Histopathology

Tissues were fixed in 10% formalin in PBS overnight and then embedded in paraffin. Sections (thickness, 4 μm) were used for H&E staining and IDO immunohistochemical staining as described in a previous report (16). Two pathologists who did not have any knowledge of the study design graded the extent of cellular infiltration and myocardial necrosis in tissue samples according to the following scale: 0, no lesions; 1+, lesions involving <25%; 2+, lesions involving 25–50%; 3+, lesions involving 50–75%; and 4+, lesions involving 75–100% (20).

RNA extraction and real-time PCR analysis

Total RNA was extracted from heart tissue with Isogen (Nippon Gene, Tokyo, Japan), and the RNA concentration was determined spectrophotometrically at 260 nm. RT-PCR was performed by using High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA). The following PCR primers were synthesized: EMCV, sense, 5′-GTCGTGAAGGAAGCAGTTCC-3′, and antisense, 5′-CACGTGGCTTTTGGCCGCAGAGGC-3′. EMCV and 18s were quantified by using real-time PCR in a LightCycler (Roche, Meylan, France). The EMCV and 18s reactions were performed with a SYBR green PCR mix (Qiagen, Hilden, Germany) and a TaqMan probe (Applied Biosystems), respectively, as described previously (19). The data were analyzed with LightCycler software (version 5.32; Roche).

Flow cytometry analyses

Splenocytes were purified by using Ficoll (IBL, Gunma, Japan) and washed twice in PBS. Subsequently, these cells were incubated with anti-mouse CD11c Ab, anti-mouse CD11b Ab, and anti-mouse B220 Ab for 20 min at 4°C (BD Biosciences, Franklin Lakes, NJ). Afterward, their expression of membrane Ags was determined by using a FACScan flow cytometer (BD Biosciences). Viable cells were gated on forward and side scatter, and a minimum of 10,000 events was acquired for each sample.

Measurements of serum creatine kinase, lactate dehydrogenase, alanine aminotransferase, and blood urea nitrogen

The activities of creatine kinase (CK), lactate dehydrogenase (LD), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) concentrations in the serum were measured with commercially available kits and an automatic analyzer BM 2250.

Administration of Kyns

Kyns were administered as described previously (21, 22). Briefly, mice were i.p. injected with a mixture of Kyns (20 mg⋅kg−1⋅day−1 L-kynurenine, 3-hydroxykynurenine, and 3-hydroxy-anthranilic acid; Sigma-Aldrich, Tokyo, Japan) once per day.

Bone marrow transplantation

Bone marrow transplantation (BMT) was performed on mice 5 wk of age as described previously (20). The recipient mice (IDO+/+ and IDO−/− mice) were irradiated in fractionated dose (5 Gy twice with a 4-h interval) and reconstituted with the whole bone marrow cells injection (5 × 106 bone marrow cells/250 μl from young IDO+/+ and IDO−/− donor mice) via tail vein. These BMT mice were maintained under special pathogen-free conditions, given 500 U/ml gentamicin sulfate (Invitrogen, Grand Island, NY) and 100 μg/ml polymixicin B sulfate (Kayaku, Tokyo, Japan) in drinking water for 4 wk after cell transfer.

Statistical analyses

Results are expressed as mean (SD). The survival rates of mice were analyzed by the Kaplan–Meier method. Statistically significant differences between two groups were determined by using Student t test, and those among three groups were determined by using one-way ANOVA. We used StatView 4.5 for these statistical analyses. The criterion for statistical significance was p < 0.05.

Results

Changes in serum l-Trp–Kyn pathway metabolism in EMCV-infected mice

In IDO+/+ mice, the serum ALT, CK, and LD activities increased significantly within 4 d after EMCV inoculation, suggesting that severe myocardial damage was induced by EMCV (Fig. 1A). The serum Kyn levels increased significantly after 48 h of inoculation and peaked 4 d after inoculation (Fig. 1B), with the ratio of Kyn/Trp levels being significantly high as well as after 4 d, indicating that IDO activity had increased. In contrast, serum Trp levels decreased significantly after 7 d. Indeed, IDO activity in the spleen and liver of the IDO+/+ had significantly increased within 24 h of inoculation, and its activity in the lung and heart significantly increased after 48 h and 4 d, respectively. No significant difference was observed in the small intestine (Fig. 2A). Furthermore, EMCV-infected IDO+/+ mice showed apparent cellular infiltration in the heart on day 7 postinfection (Fig. 2B). Notably, the infiltrated cells in the heart and splenocytes of the EMCV-infected IDO+/+ mice strongly expressed IDO.

FIGURE 1.
FIGURE 1.

Changes in the serum levels of Trp catabolism and biochemical 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 significant differences between the groups were determined by using ANOVA; *p < 0.05.

FIGURE 2.
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 ×40) 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.

Effect of IDO gene deficiency or IDO inhibition on survival rates and EMCV viral RNA in the heart

To investigate the roles of IDO in EMCV-induced myocardial damage, the effects of IDO deficiency and of the IDO inhibitor 1-MT were examined. On day 25, the survival rate of IDO−/− mice after inoculation with EMCV was 62%, whereas the survival rate of IDO+/+ mice was 18% (Fig. 3A). Similarly, 1-MT improved the survival rate from 20 to 43% (Fig. 3B).

FIGURE 3.
FIGURE 3.

Survival rates and EMCV RNA levels in IDO−/− mice or 1-MT–treated mice after EMCV inoculation. (A) The survival rate of IDO−/− 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) also was higher than that of IDO+/+ mice (n = 15). The data are representative of three independent experiments. Statistically significant differences between the groups were determined using the log-rank test. (C) IDO+/+ mice, IDO−/− mice, and 1-MT–treated mice were inoculated with EMCV, 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 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 ANOVA; *p < 0.05, **p < 0.01.

On day 4, the levels of EMCV genomic RNA in the hearts of IDO−/− mice and 1-MT–treated mice were significantly less than those in IDO+/+ mice (Fig. 3C). Furthermore, the serum levels of LD and CK in IDO+/+ mice on day 4 were significantly worse than those in IDO−/− mice or 1-MT–treated mice (Table I). Indeed, IDO+/+ mice exhibited more myocardial necrosis on day 7 than the IDO−/− mice and 1-MT–treated mice did.

View this table:
Table I. Changes in myocardial marker and histopathologic findings in IDO−/− mice

Changes in the serum levels of type I IFNs and Kyn metabolites in EMCV-infected mice

To determine the mechanisms by which IDO promotes myocardial damage, changes in the serum levels of type I IFNs, which have antiviral effects, were compared between IDO+/+ and IDO−/− mice after inoculation with EMCV (Fig. 4A). Serum levels of type I IFNs after 48 h of inoculation were significantly higher in IDO−/− mice than in IDO+/+ mice. To confirm whether Kyn pathway regulates the production of type I IFNs, we compared the changes in the serum levels of the metabolites of Kyn pathway in IDO−/− mice and IDO+/+ mice after inoculation with EMCV. The serum Kyn and 3-hydroxykynurenine levels in IDO+/+ mice 48 h after inoculation significantly increased compared with those in IDO−/− mice (Fig. 4B). Moreover, treatment of Kyns decreased the induction of type I IFNs in IDO−/− mice (Fig. 4C). We assessed the myocardial damage in Kyns-treated IDO−/− mice. The serum levels of ALT, LD, and CK in Kyns-treated IDO−/− mice were significantly elevated than those in untreated IDO−/− mice (Fig. 5A). Moreover, Kyns-treated IDO−/− mice exhibited pronounced myocardial necrosis 4 d after inoculation with EMCV compared with the untreated IDO−/− mice (Fig. 5B). In addition, on day 25, the survival rate of the untreated IDO−/− mice was 62%, whereas all the Kyns-treated IDO−/− mice had died on day 25 (Fig. 5C). These results suggest that increased levels Kyns as a result of IDO activity might increase viral load, thereby promoting myocardial damage by inhibition of induction of type I IFNs.

FIGURE 4.
FIGURE 4.

Changes in serum levels of type I IFN in response to EMCV inoculation. (A) IDO+/+ and IDO−/− 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 IDO−/− 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 IDO−/− mice (**p < 0.05). (C) IDO−/− 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.

FIGURE 5.
FIGURE 5.

Effect of Kyns treatment on acute viral myocarditis. (A) ALT, CK, and LDH activities and BUN concentrations in the serum of IDO−/− or Kyns-treated 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 significant differences between the groups were determined using Student t test; *p < 0.05. (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-treated IDO−/− mice showed more cellular infiltration and myocardial necrosis than IDO−/− mice did. Representative images from five independent analyses are shown. Scale bars, 100 μm. (C) The survival rates of IDO−/− mice (n = 15) were significantly higher than those of Kyns-treated IDO−/− 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.

Increased type I IFN levels in IDO gene-deficient bone marrow cells after EMCV infection

Bone marrow-derived cells are considered a source of type I IFNs. To explore the involvement of IDO in the bone marrow-derived cells, we generated IDO-chimeric mice by using a combination of irradiation and bone marrow transplants. IFN-β levels in the IDO−/− bone marrow-transplanted IDO+/+ mice was significantly higher than those in the IDO+/+ bone marrow-transplanted IDO−/− mice 48 h after inoculation with EMCV (Fig. 6A). In addition, on day 25, the survival rate of the IDO−/− bone marrow- transplanted IDO+/+ was 64%, whereas that of IDO+/+ bone marrow-transplanted IDO−/− was 23% (Fig. 6B). These results suggest that increased levels of type I IFNs in IDO−/− mice can be attributed to bone marrow-derived cells from IDO+/+ mice and not non-bone marrow-derived cells.

FIGURE 6.
FIGURE 6.

Effect of type I IFN production and survival rates after BMT. (A) IDO+/+ bone marrow-transplanted IDO−/− mice and IDO−/− bone marrow-transplanted IDO+/+ mice were inoculated with EMCV. At 48 h postinfection, the serum levels of IFN-β 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 IDO−/− bone marrow-transplanted IDO+/+ mice (n = 12) was significantly higher than that of IDO+/+ bone marrow-transplanted IDO−/− mice (n = 12). The data are representative of three independent experiments. Statistically significant differences between the groups were determined using the log-rank test.

Increased macrophage levels in the spleen of IDO−/− mice after EMCV infection

To explore the cell subsets producing type I IFN levels in bone marrow-derived cells, we next used flow cytometry to quantify the number of conventional DCs (CD11c+B220−), plasmacytoid DCs (pDCs; CD11clowB220+), and macrophages (CD11b+CD11c−) in the spleen of IDO+/+ mice, IDO−/− mice, and Kyns-treated IDO−/− mice after inoculation with EMCV (Fig. 7A). The gated percentages of macrophages in the spleen of IDO−/− mice were significantly higher than those in the IDO+/+ mice or Kyns-treated IDO−/− mice 48 h after inoculation. In addition, the number of splenocytes in IDO−/− mice was significantly higher than that in IDO+/+ mice or Kyns-treated IDO−/− mice (Fig. 7B). The expression of IDO mRNA at 48 h, and the level of IFN-β in the medium supernatant at 72 h, was significantly enhanced in the spleen CD11c+ cells and CD11b+ cells (Fig. 7C, 7D). These results suggest that the source of type I IFNs is the macrophage, which is regulated by Kyns.

FIGURE 7.
FIGURE 7.

Cell populations in the spleen of IDO−/− mice producing type I IFN. (A) Gated percentages of CD11b+CD11c− cells, CD11b+CD11c+ cells, CD11+B220+ cells, and CD11c+B220− cells in the spleen of IDO+/+ mice, IDO−/− mice, or Kyns-treated IDO−/− mice at 0 and 48 h after EMCV inoculation. Statistically significant differences between the groups were determined using ANOVA; *p < 0.05. (B) The number of splenocytes in IDO+/+ mice, IDO−/− mice, or Kyns-treated IDO−/− 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 IDO−/− 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. The expression of IDO mRNA in these cells was analyzed using real-time quantitative PCR. (D) The levels of IFN-β in the medium supernatant at 72 h of incubation of these indicated cells (1 × 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.

Discussion

IDO is expressed in human (1) and mouse tumor cells (2), DCs, and macrophages following microbial (4, 5) or viral infections (6, 7). In this study, we examined the roles of IDO in the Trp-Kyn metabolic pathway with respect to the immune regulation in EMCV infections by using IDO−/− mice and the IDO inhibitor 1-MT. We demonstrated that type I IFNs are upregulated, resulting in suppressed EMCV replication by IDO knockdown or inhibition, and that these observations are reversed by administration of Kyns. IDO has been thought to have beneficial actions. For example, recent studies have shown that TLR3 ligand poly (I:C) induces IDO activation in astrocytes and causes an antiviral response (23). In addition, IFN-γ–induced IDO has an antiviral effect in measles virus infection of epithelial and endothelial cells in vitro (24). In contrast, disadvantageous functions of IDO have been also reported in viral infections. CTLA-4 blockade has been reported to decrease the expression of TGF-β, IDO, and viral RNA in the tissues from SIVmac251-infected macaques (25), and the IDO inhibitor 1-MT may be a promising candidate for enhancing immunity, including anti-HIV immunity, in HIV-infected patients (26). In this study, IDO was induced by EMCV, which promoted viral replication and tissue damage, suggesting that IDO has a disadvantageous function in acute EMCV infection. IDO-deficient and inhibitor-treated mice had higher survival rates, resulting in greater suppression of EMCV replication (Fig. 3). When pathogens invade host cells, they activate the innate immune system and elicit the production of cytokines and chemokines, which recruit immune cells that mediate pathogen clearance. In particular, type I IFNs are important mediators of innate immunity that limit the adverse effects of many viruses (27). Postinfection, EMCV is initially recognized by the melanoma differentiation-associated gene 5 receptor, which induces antiviral responses including the production of type I IFNs and proinflammatory cytokines in DCs and macrophages (28, 29). IFN-α and IFN-β suppress the progression of EMCV infection (30). Indeed, the serum levels of type I IFNs was increased in IDO−/− mice 48 h after inoculation with EMCV and were significantly higher than those in IDO+/+ mice, resulting in reduced myocardial damage in the IDO−/− mice or 1-MT–treated mice. Currently, there are two hypotheses regarding the role of Trp catabolism in the induction of tolerance. One hypothesis proposes that the downstream metabolites of Trp suppress immune reactivity by directly interacting with effector T lymphocytes and other types of immune cells (13, 14, 31). An alternative hypothesis suggests that the breakdown of Trp suppresses T cell proliferation by reducing the availability of this essential amino acid under local tissue microenvironments (32). In this study, Kyns mixture reversed the effects of IDO knockdown, suggesting that the increase in Kyn metabolites via upregulation of IDO after EMCV inoculation regulated the production of type I IFNs. Our results are consistent with a previous study that showed that the cytotoxicity of Trp metabolites tends to affect macrophages as well as T, B, and NK cells in vitro but not DCs (13). In addition, it has been previously reported that Kyn metabolites inhibit NF-κB activation, which is a transcription factor that regulates type I IFN production, upon T cell Ag receptor engagement, by specifically targeting phosphoinositide-dependent protein kinase-1 (33). Moreover, IFN-β levels in the IDO−/− bone marrow-transplanted IDO+/+ mice were significantly higher than those in the IDO+/+ bone marrow-transplanted IDO−/− mice, suggesting that IDO regulates the production of type I IFNs in the bone marrow cells. The number of type I IFN-producing macrophages in IDO−/− mice was significantly increased compared with that in IDO+/+ mice (Fig. 7), suggesting that the increase in the levels of type I IFNs may be due to the regulation of macrophage number via inhibition of Kyns accumulation. This is consistent with our previous results demonstrating that the number of intrahepatic CD11b+ cells in IDO−/− mice was significantly greater than that in IDO+/+ mice in an α-galactosylceramide–induced hepatitis model (34). However, we do not exclude other possibilities (e.g., Kyns enhance regulatory T cell activity or suppress Th17 cells). In addition, the increase in the type I IFN levels in IDO−/− mice can be also explained by alternate mechanisms. Recent studies have demonstrated that production of type I IFN in pDCs is regulated by the mammalian target of rapamycin, which is known to inhibit via amino-acid starvation (35, 36), suggesting that depletion of local Trp by IDO induction might regulate type I IFN production via mammalian target of rapamycin. In addition, IDO is involved with TGF-β in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs (37). Therefore, IDO inhibits type I IFNs not only by suppressing the macrophage number but also by regulating the IFNs production in macrophages.

In conclusion, our findings show that Kyn metabolites regulate the production of type I IFNs by decreasing the number macrophages. Consequently, modulation of the Trp–Kyn pathway may be an effective strategy for treating acute viral myocarditis.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • 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.

  • Abbreviations used in this article:

    ALT
    alanine aminotransferase
    BMT
    bone marrow transplantation
    BUN
    blood urea nitrogen
    CK
    creatine kinase
    DC
    dendritic cell
    EMCV
    encephalomyocarditis virus
    Kyn
    kynurenine
    LD
    lactate dehydrogenase
    1-MT
    1-methyl-d,l-Trp
    pDC
    plasmacytoid dendritic cell
    Trp
    tryptophan.

  • Received April 6, 2011.
  • Accepted February 8, 2012.

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