Hidetsugu Fujigaki

Nitration and Inactivation of IDO by Peroxynitrite

IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

Programmable multivalent display of receptor ligands using peptide nucleic acid nanoscaffolds

Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models.

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.

Indoleamine 2,3-dioxygenase is highly expressed in human adult T-cell leukemia/lymphoma and chemotherapy changes tryptophan catabolism in serum and reduced activity

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell lymphotropic virus type 1 (HTLV-1). Indoleamine 2,3-dioxygenase (IDO), the l-tryptophan (l-TRP)-degrading enzyme, plays a key role in the powerful immunomodulatory effects of several different types of immune cells. In this study, we investigated the IDO expression in ATLL cells and the effect of chemotherapy on IDO-initiating l-TRP catabolism in patients with ATLL. Serum l-kynurenine (l-KYN) concentrations, l-KYN/l-TRP ratio, and the level of IDO mRNA expression in ATLL cells were significantly increased in ATLL patients compared to those in healthy and HTLV-positive carrier subjects. On the other hand, l-TRP level was significantly decreased in ATLL patients compared to that in healthy subjects. In the immunohistochemical staining, IDO was strongly expressed in cytoplasm of ATLL cells. Interestingly, serum l-KYN as well as soluble IL-2 receptor concentrations was significantly reduced, and l-TRP concentrations were significantly increased after chemotherapy. These data provide evidence that IDO is highly expressed in ATLL cells, and that IDO-initiating l-TRP catabolism changes with chemotherapy.

L-Tryptophan-kynurenine pathway enzymes are therapeutic target for neuropsychiatric diseases: Focus on cell type differences

ABSTRACT The kynurenine pathway (KP) is the major route for tryptophan (TRP) metabolism in most mammalian tissues. The KP metabolizes TRP into a number of neuroactive metabolites, such as kynurenine (KYN), kynurenic acid (KYNA), 3‐hydroxykynurenine (3‐HK), and quinolinic acid (QUIN). Elevated metabolite concentrations in the central nervous system are associated with the pathophysiology of several inflammation‐related neuropsychiatric diseases. During an inflammatory response, the initial KP metabolic step is primarily regulated by indoleamine 2,3‐dioxygenase 1 (IDO1), which produces KYN from TRP. Following this initial step, the KP has 2 distinct branches; one branch is regulated by kynurenine 3‐monooxygenase (KMO) and is primarily responsible for the 3‐HK and QUIN production, and the other branch is regulated by kynurenine aminotransferase (KAT), which produces KYNA, an N‐methyl‐d‐aspartate receptor and alpha‐7‐nicotinic acetylcholine receptor antagonist. Unbalanced KP metabolism has been demonstrated in distinct neuropsychiatric diseases; thus, understanding the mechanisms that regulate KP enzyme expression and activity is important. These enzymes are expressed by specific cell types, and the induction of enzyme expression by inflammatory stimuli also shows cell type specificity. This review provides an overview and discusses the current understanding of the influence of KP enzyme expression and activity in different cell types on the pathophysiological mechanisms of specific neuropsychiatric diseases. Moreover, the potential use of KP enzyme inhibition as a therapeutic strategy for treating neurological diseases is briefly discussed. This article is part of the Special Issue entitled ‘The Kynurenine Pathway in Health and Disease’. HIGHLIGHTSThe kynurenine pathway (KP) is associated with neurological diseases because several KP metabolites are neuroactive.The KP enzymes show cell‐type specific expression.KP manipulation by KP enzyme inhibition is a potential therapeutic strategy for neurological diseases.

Marked increases in hippocampal neuron indoleamine 2, 3-dioxygenase via IFN-γ-independent pathway following transient global ischemia in mouse

Indoleamine 2, 3-dioxygenase (IDO), which catabolizes L-tryptophan (L-TRP) to L-kynurenine (L-KYN), is an immunoregulatory factor that is up-regulated via an interferon-gamma (IFN-gamma)-dependent and/or -independent mechanism. In this study, we investigated the localization of IDO and whether induction of IDO expression is an IFN-gamma-dependent and/or -independent mechanism in the CNS after cerebral ischemia. The expressions of IDO protein and mRNA were investigated at different time points following cerebral ischemia using immunohistochemistry, immunofluorescence and RT-PCR. Hippocampal neuron IDO mRNA and immunohistochemical staining were significantly up-regulated 72h after transient global ischemia. Although IFN-gamma is a dominant inducer of IDO, hippocampal neuron IDO was clearly up-regulated in IFN-gamma KO mice. In summary, this is the first finding that up-regulation of IDO in hippocampal neurons after transient global ischemia occurs via INF-gamma-independent mechanisms.

Posttranslational modification of indoleamine 2,3-dioxygenase

Protein posttranslational modifications (PTMs) perform essential roles in the biological regulation of a cell. PTMs are extremely important because they can change a protein’s physical or chemical properties, conformation, activity, cellular location, or stability. In fact, most proteins are altered by the addition or removal of a chemical moiety on either an amino acid or the protein’s N- or C-terminus. Some PTMs can be added and removed dynamically as a mechanism for reversibly controlling protein function. Thus, identifying the PTM sites is critical to fully understand the biological roles of any given protein. Mass spectrometry (MS) is a widely used analytical strategy to identify PTMs. We have used an automated two-dimensional liquid chromatography (LC) system coupled with electrospray ionization quadrupole ion-trap MS to identify PTMs for indoleamine 2,3-dioxygenase 1 (IDO1), one of the tryptophan catabolic enzymes. IDO1 promotes immune tolerance by suppressing local T-cell responses under various physiological and pathophysiological conditions, such as pregnancy in mammals, tumor resistance, autoimmunity, and chronic inflammation. Although many studies have demonstrated the biological importance of IDO activity, the PTMs of IDO enzymes remain largely unknown. Only a few important PTMs of IDO1 have been found, such as nitration, N-terminal acetylation, and phosphorylation. In this review, we analyze the PTMs of IDO1 using our two-dimensional LC-MS/MS system, and provide an overview of our current understanding.

Depressive symptoms as a side effect of Interferon-α therapy induced by induction of indoleamine 2,3-dioxygenase 1.

Depression is known to occur frequently in chronic hepatitis C viral (HCV) patients receiving interferon (IFN)-α therapy. In this study, we investigated whether indoleamine 2,3-dioxygenase1 (IDO1)-mediated tryptophan (TRP) metabolism plays a critical role in depression occurring as a side effect of IFN-α therapy. Increases in serum kynurenine (KYN) and 3-hydroxykynurenine (3-HK) concentrations and in the ratios of KYN/TRP and 3-HK/kynurenic acid (KA) were much larger in depressive HCV patients than in non-depressed patients following therapy. Furthermore, transfection of a plasmid continuously expressing murine IFN-γ into normal mice significantly increased depression-like behavior. IFN-γ gene transfer also resulted in a decrease in serum TRP levels in the mice while KYN and 3-HK levels were significantly increased in both serum and frontal cortex. Genetic deletion of IDO1 in mice abrogated both the increase in depression-like behavior and the elevation in TRP metabolites’ levels, and the turnover of serotonin in the frontal cortex after IFN-γ gene transfer. These results indicate that the KYN pathway of IDO1-mediated TRP metabolism plays a critical role in depressive symptoms associated with IFN-α therapy.

Genotyping of hepatitis C virus by melting curve analysis with SYBR Green I

Background: Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. We needed to develop a convenient screening test for HCV genotypes 1 and 2. Method: We tested 55 patients with known chronic HCV infection. Viral RNA was extracted from serum samples using an automatic viral RNA purification system, and HCV genotypes were determined by reverse transcriptase-polymerase chain reaction using LightCycler melting curve analysis with SYBR Green I. Results: HCV RNA was detected in all samples and each genotype was determined. The mean (standard deviation) melting temperatures for subtypes 1b (n = 32), 2a (n = 15) and 2b (n = 8) were 93.14°C (0.51°C), 91.08°C (0.49°C) and 91.77°C (0.27°C), respectively. Genotypes 1 and 2 were differentiated within 3 h by this method. Conclusions: Our melting curve analysis is a rapid and convenient screening test for differential identification of HCV genotypes 1 and 2.

Changes in serum tryptophan catabolism as an indicator of disease activity in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm that develops in a small population of human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals and is characterized by mostly CD4þ and CD25þ mature T-cell phenotypes, and onset at middle age or later [1–4]. The exact mechanism of immune modulation in ATLL remains unknown. The essential amino acid Ltryptophan (L-TRP) is required for the biosynthesis of proteins and is precursor for several biologically important compounds: (a) 5-hydroxy-tryptamine (serotonin) which is formed by tryptophan 5-hydroxylase following decarboxylation; (b) kynurenine which is produced by L-tryptophan 2,3-dioxygenase (TDO); and indoleamine 2,3-dioxygenase (IDO). The latter two enzymes catabolize tryptophan via the so-called kynurenine-pathway synthesizing nicotinic acid, the vitamin niacin and nicotinamide adenine dinucleotides as end products. Although TDO is localized in the liver and is up-regulated by corticosteroids, IDO is expressed by a variety of cells and is inducible by various cytokines, especially interferon-g [5]. Recently, IDO has been identified as an enzyme endowed with powerful immunomodulatory effects, resulting from its enzymatic activity that leads to catabolism of the essential amino acid L-TRP. For example, certain IDO-generated L-TRP-derived metabolites, in particular L-kynurenine (L-KYN), have been reported to block Ag-driven specific T-cell proliferation and even to induce T-cell death [6]. On the other hand, IDO has been found in various tumors of different histotypes and increments of IDO activity correlate with tumor progression [7]. In addition, we have been reported that IDO is highly expressed in ATLL cells [8]. Therefore, the provided L-TRP catabolism as a result of IDO activity may play an important role in the immune regulation exerted by the antigen-presenting cells. In this study, we investigated whether the level of L-TRP metabolites in blood could be a useful parameter of chemotherapy efficacy for ATLL patients. The patients analysed are a 74-year-old woman in acute type ATLL without chemotherapy and a 67year-old woman in acute type ATLL with chemotherapy. The diagnosis of ATLL was based on clinical features, hematological characteristics, presence of serum antibodies to ATLL-associated antigens, and presence of HTLV-1 proviral genome in DNA from leukemic cells. Informed consent was provided according to the Declaration of Helsinki. Combination chemotherapy was used according to a multi-agent chemotherapy protocol LSG15. Chemotherapy consisted of two cycles of VCAP (vincristine 1 mg/m, cyclophosphamide 350 mg/m, doxorubicin 40 mg/m and prednisone 40 mg/m), AMP (doxorubicin 30 mg/m, ranimustine 60 mg/m and prednisone 40 mg/m), and VECP (vindesine 2.4 mg/m, etoposide 100 mg/m, carboplatin 250 mg/m and prednisone 40 mg/m). G-CSF was administered during the intervals of chemotherapy