Hirofumi Ohtaki

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

Indoleamine 2,3-dioxygenase, the l-tryptophan–degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO−/−) and 1-methyl-d-l-tryptophan–treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO−/− mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO−/− mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO−/− mice or 1-methyl-d-l-tryptophan–treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO−/− mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

l-Tryptophan-mediated Enhancement of Susceptibility to Nonalcoholic Fatty Liver Disease Is Dependent on the Mammalian Target of Rapamycin

Nonalcoholic fatty liver disease is one of the most common liver diseases. l-Tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. However, it is unclear whether l-tryptophan promotes hepatic steatosis. To explore this issue, we examined the role of l-tryptophan in mouse hepatic steatosis by using a high fat and high fructose diet (HFHFD) model. l-Tryptophan treatment in combination with an HFHFD exacerbated hepatic steatosis, expression of HNE-modified proteins, hydroxyproline content, and serum alanine aminotransaminase levels, whereas l-tryptophan alone did not result in these effects. We also found that l-tryptophan treatment increases serum serotonin levels. The introduction of adenoviral aromatic amino acid decarboxylase, which stimulates the serotonin synthesis from l-tryptophan, aggravated hepatic steatosis induced by the HFHFD. The fatty acid-induced accumulation of lipid was further increased by serotonin treatment in cultured hepatocytes. These results suggest that l-tryptophan increases the sensitivity to hepatic steatosis through serotonin production. Furthermore, l-tryptophan treatment, adenoviral AADC introduction, and serotonin treatment induced phosphorylation of the mammalian target of rapamycin (mTOR), and a potent mTOR inhibitor rapamycin attenuated hepatocyte lipid accumulation induced by fatty acid with serotonin. These results suggest the importance of mTOR activation for the exacerbation of hepatic steatosis. In conclusion, l-tryptophan exacerbates hepatic steatosis induced by HFHFD through serotonin-mediated activation of mTOR.

Ability of IDO To Attenuate Liver Injury in α-Galactosylceramide–Induced Hepatitis Model

IDO converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, we examined the effect of IDO in α-galactosylceramide (α-GalCer)–induced hepatitis. The increase in IDO expression in the liver of wild-type (WT) mice administered α-GalCer was confirmed by real-time PCR, Western blotting, and IDO immunohistochemical analysis. The serum alanine aminotransferase levels in IDO-knockout (KO) mice after α-GalCer injection significantly increased compared with those in WT mice. 1-Methyl-d-tryptophan also exacerbated liver injury in this murine hepatitis model. In α-GalCer–induced hepatitis models, TNF-α is critical in the development of liver injury. The mRNA expression and protein level of TNF-α in the liver from IDO-KO mice were more enhanced compared with those in WT mice. The phenotypes of intrahepatic lymphocytes from WT mice and IDO-KO mice treated with α-GalCer were analyzed by flow cytometry, and the numbers of CD49b+ and CD11b+ cells were found to have increased in IDO-KO mice. Moreover, as a result of the increase in the number of NK cells and macrophages in the liver of IDO-KO mice injected with α-GalCer, TNF-α secretion in these mice was greater than that in WT mice. Deficiency of IDO exacerbated liver injury in α-GalCer–induced hepatitis. IDO induced by proinflammatory cytokines may decrease the number of TNF-α–producing immune cells in the liver. Thus, IDO may suppress overactive immune response in the α-GalCer–induced hepatitis model.

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

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.

IDO1 plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate-induced colitis in mice.

IDO, an enzyme that degrades the essential amino acid l-tryptophan to N-formylkynurenine, is known to exert immunomodulatory effects in a number of diseases and disorders. IDO expression is increased in tumors, where it is thought to be involved in tumor evasion by suppressing the immune response. A competitive inhibitor of IDO is currently being tested in clinical trials for relapsed or refractory solid tumors; however, there remains a concern that attenuation of the immunosuppressive function of IDO might exacerbate inflammatory responses. In this study, we investigated the role of IDO in 2,4,6-trinitrobenzene sulfate (TNBS)–induced colitis in mice by gene deletion and pharmacological inhibition. TNBS treatment induced significantly more severe colitis in Ido1 gene–deficient (Ido1−/−) mice than in Ido1 wild-type (Ido1+/+) mice, indicating a role for IDO1 in suppression of acute colitis. Consistent with this, the expression of Ido1 was increased in the colonic interstitial tissues of TNBS-treated Ido1+/+ mice. Furthermore, transplantation of Ido1+/+ bone marrow cells into Ido1−/− mice reduced the pathological damage associated with colitis, altered the expression of cytokines, including IFN-γ, TNF-α, and IL-10, and increased the number of CD4+ Foxp3+ regulatory T cells in the colon. Pharmacological inhibition of IDO enzymatic activity by oral administration of 1-methyltryptophan (1-methyl-l-tryptophan or 1-methyl-d-tryptophan) significantly increased the severity of TNBS-induced colitis in mice, demonstrating that both stereoisomers can promote colitis. Collectively, our data indicate that IDO1 plays an important immunoregulatory role in the colon.

Effects of Indoleamine 2,3-Dioxygenase Deficiency on High-Fat Diet-Induced Hepatic Inflammation

Hepatic immune regulation is associated with the progression from simple steatosis to non-alcoholic steatohepatitis, a severe condition of inflamed fatty liver. Indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that mediates the catabolism of L-tryptophan to L-kynurenine, plays an important role in hepatic immune regulation. In the present study, we examined the effects of IDO gene silencing on high-fat diet (HFD)-induced liver inflammation and fibrosis in mice. After being fed a HFD for 26 weeks, the IDO-knockout (KO) mice showed a marked infiltration of inflammatory cells, especially macrophages and T lymphocytes, in the liver. The expression levels of F4/80, IFNγ, IL-1β, and IL-6 mRNA in the liver and the expression levels of F4/80 and TNF-α mRNA in the white adipose tissue were significantly increased in IDO-KO mice, although hepatic steatosis, the accumulation of intrahepatic triglycerides, and the amount of oxidative stress were lower than those in IDO-wild-type mice. IDO-KO mice also developed marked pericellular fibrosis in the liver, accumulated hepatic hydroxyproline, and exhibited increased expression levels of hepatic TGF-β1 mRNA. These findings suggest that IDO-KO renders the mice more susceptible to HFD-induced hepatic inflammation and fibrosis. Therefore, IDO may have a protective effect against hepatic fibrosis, at least in this HFD-induced liver injury model.

Toll-like Receptor agonists and alpha-galactosylceramide synergistically enhance the production of interferon-gamma in murine splenocytes

Vα14 natural killer T (iNKT) cells activated by alpha-galactosylceramide (GalCer) secrete a large amount of cytokines. Toll-like receptors (TLRs) play a critical role in the innate immune responses via the recognition of pathological antigen. Previously we demonstrated that the iNKT cells activated by GalCer augmented LPS-induced NO production in peritoneal cells. In this study, we examined the effect of GalCer and TLR agonists by IFN-γ production from splenocytes. Splenocytes pretreated with GalCer induced TLR3, 4, 7/8, and 9 agonists in vitro, resulting in the enhancement of IFN-γ mRNA expression. In particular, IFN-γ stimulated by GalCer and LPS was increased in NK cells and CD8 T cells, and inhibited by a neutralizing anti-IL-12 antibody. Pretreatment with GalCer enhanced the phosphorylation of IκB-α induced by LPS stimulation. The present study showed that co-stimulation of GalCer and TLR agonists powerfully induced the production of IFN-γ from splenocytes.

Vα14 NKT cells activated by alpha-galactosylceramide augment lipopolysaccharide-induced nitric oxide production in mouse intra-hepatic lymphocytes

Valpha14 natural killer T (Valpha14 NKT) cells activated by alpha-galactosylceramide (alpha-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-gamma plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by alpha-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of alpha-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by alpha-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, alpha-GalCer did not augment LPS-induced NO production in IHLs from IFN-gamma knockout mice. In flow cytometry analysis of IHLs from alpha-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Valpha14 NKT cells were activated.

Inhibition of indoleamine 2,3‐dioxygenase 1 expression alters immune response in colon tumor microenvironment in mice

Indoleamine 2,3‐dioxygenase (IDO), an enzyme that degrades the essential amino acid l‐tryptophan along the kynurenine pathway, exerts immunomodulatory effects in a number of diseases. IDO expression is increased in tumor tissue and in draining lymph nodes; this increase is thought to play a role in tumor evasion by suppressing the immune response. A competitive inhibitor of IDO is currently being tested in clinical trials for the treatment of relapsed or refractory solid tumors, but the efficacy of IDO inhibition in colorectal tumors remains to be fully elucidated. In this study, we investigated the effect of IDO deficiency on colon tumorigenesis in mice by genetic deletion and pharmacological inhibition. Ido1‐deficient(−/−) mice were crossed with ApcMin/+ mice or were administered azoxymethane with or without dextran sodium sulfate. Ido1 deficiency did not lead to significant differences in the size and number of colon tumors. Similarly, the pharmacological inhibition of IDO using 1‐methyltryptophan (1‐mT) also resulted in no significant differences in tumor size and number in ApcMin/+ mice. However, Ido1 deficiency altered the immune response in the tumor microenvironment, showing a significant increase in mRNA expression of pro‐inflammatory cytokines and a significant decrease in the number of Foxp3‐positive regulatory T cells in the colon tumors of Ido1(−/−) mice. Importantly, 1‐mT treatment also significantly altered cytokine expression in the colon tumor tissues. These results suggest that IDO inhibition alone cannot sufficiently suppress colon cancer development in mice despite its immunomodulatory activity in the tumor microenvironment.

Blockade of Indoleamine 2,3-Dioxygenase Reduces Mortality from Peritonitis and Sepsis in Mice by Regulating Functions of CD11b+ Peritoneal Cells

ABSTRACT Indoleamine 2,3-dioxygenase-1 (Ido), which catalyzes the first and limiting step of tryptophan catabolism, has been implicated in immune tolerance. However, the roles of Ido in systemic bacterial infection are complicated and remain controversial. To explore this issue, we examined the roles of Ido in bacterial peritonitis and sepsis after cecal ligation and puncture (CLP) in mice by using the Ido inhibitor 1-methyl-d,l-tryptophan (1-MT), by comparing Ido+/+ and Ido−/− mice, or by using chimeric mice in which Ido in the bone marrow-derived cells was deficient. Ido expression in the peritoneal CD11b+ cells and its metabolite l-kynurenine in the serum were increased after CLP. 1-MT treatment or Ido deficiency, especially in bone marrow-derived cells, reduced mortality after CLP. Compared to Ido+/+ mice, Ido−/− mice showed increased recruitment of neutrophils and mononuclear cells into the peritoneal cavity and a decreased bacterial count in the blood accompanied by increased CXCL-2 and CXCL-1 mRNA in the peritoneal cells. Ido has an inhibitory effect on LPS-induced CXCL-2 and CXCL-1 production in cultured peritoneal cells. These findings indicate that inhibition of Ido reduces mortality from peritonitis and sepsis after CLP via recruitment of neutrophils and mononuclear cells by chemokine production in peritoneal CD11b+ cells. Thus, blockade of Ido plays a beneficial role in host protection during bacterial peritonitis and sepsis.