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The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis., Competing Interests: The authors have declared that no competing interests exist.

Although Indoleamine 2,3-dioxygenase (IDO), tryptophan-2,3-dioxygenase (TDO), and aryl hydrocarbon receptor (AHR) are involved in cancer immune escape, their prognostic impact on diffuse large B-cell lymphoma (DLBCL) is unknown.To examine the prognostic impact of IDO, TDO, and AHR on patients with DLBCL.This was a retrospective study on treatment-naïve patients with newly diagnosed DLBCL at the Henan Province People's Hospital between 01/2012 and 06/2015. Patients with inflammatory reactive lymph nodes were included as controls. All cases were reviewed by 2 pathologists. IDO, TDO, and AHR positivity was determined through immunochemistry. Survival was examined using the Kaplan-Meier method and multivariable Cox analyses.The positive expression of TDO (50.0% vs 16.7%, P = .005) and AHR (60.0% vs 8.3%, P < .001) were higher in DLBCL than in inflammatory control. The overall survival of IDO, TDO, and AHR positive expression in DLBCL patients was 34.6, 26.7, and 32.2 months, respectively, which is significantly shorter than that of the corresponding negative patients (49.0 months, P = .04; 58.2 months, P < .001; 58.0 months, P < .001; respectively). The multivariable analysis showed that TDO expression and Ann-Arbor stage were independently associated with PFS (TDO: HR = 8.347, 95%CI: 2.992-23.289, P < .001; stage: HR = 2.729, 95%CI: 1.571-4.739, P < .001) and OS (TDO: HR = 9.953, 95%CI: 3.228-30.686, P < .001; stage: HR = 2.681, 95%CI: 1.524-4.719, P = .001) in DLBCL patients.Overexpression of IDO, TDO, and AHR is associated with poor survival of patients with DLBCL and could be involved in the immune escape of cancer cells. Further studies are necessary to determine whether these proteins can be targeted by treatment regimens.

Tryptophan, an amino acid involved in routine energy metabolism, is a key modulator of sickness behaviors associated with inflammatory states and also plays roles in some psychiatric disorders. Tissue concentrations of tryptophan are regulated primarily by the enzymes indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO, encoded by TDO2). Altered IDO1 and TDO activities have been linked to the perturbed serotonergic neurotransmission that may underlie certain psychopathologies. Here we assessed mice genetically modified to be deficient in IDO1, IDO2 or TDO2 for their behavior and cognitive function using an automated home cage system, the IntelliCage™. A well-established behavioural and cognitive test battery was applied during two periods (Runs 1 and 2, "R1" and "R2") separated by one month. Various tryptophan-related neurochemicals also were measured in brain extracts. IDO1(-/-) mice displayed remarkable reductions of early diurnal exploration in the IntelliCage and this persisted in R2. In contrast, early diurnal hyperactivity was observed in IDO2(-/-) mice in both R1 and R2. TDO2(-/-) mice displayed increased diurnal and nocturnal exploration, but only in R2. Cognitive assessment suggested enhanced reference memory in IDO2(-/-) mice in a complex patrolling task, while TDO deficiency was associated with enhanced performance in complex patrolling and discrimination reversal tasks. Neurochemical measures showed attenuated brain serotonin levels in IDO1(-/-) mice and augmented tryptophan and serotonin levels in TDO2(-/-) animals, respectively. No neurochemical alterations were detected in IDO2(-/-) mice. Taken together, these findings reveal complex and dissimilar patterns of behavioral and cognitive changes induced by knockout of three different tryptophan-metabolizing enzymes., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Cancer patients mount T-lymphocyte responses against antigens expressed selectively by their malignancy, but these responses often fail to control their disease, because tumors select mechanisms that allow them to resist immune destruction. Among the numerous resistance mechanisms that have been proposed, metabolic inhibition of T cells by tryptophan catabolism deserves particular attention, because of the frequent expression of tryptophan-degrading enzymes in human tumors, and because in vitro and in vivo studies have shown that their enzymatic activity can be readily blocked by pharmacologic inhibitors, thereby restoring T-cell-mediated tumor cell killing and paving the way to targeted therapeutic intervention. In view of recent observations, and taking into account the differences between human and mouse data that differ in several aspects, in this Cancer Immunology at the Crossroads article, we discuss the role of the three enzymes that have been proposed to control tryptophan catabolism in tumoral immune resistance: indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase (TDO), and indoleamine 2,3-dioxygenase 2 (IDO2)., (©2015 American Association for Cancer Research.)

This overview presents a hypothesis to bridge the gap between psychoneuroimmunological findings and recent results from pharmacological, neurochemical and genetic studies in schizophrenia. In schizophrenia, a glutamatergic hypofunction is discussed to be crucially involved in dopaminergic dysfunction. This view is supported by findings of the neuregulin- and dysbindin genes, which have functional impact on the glutamatergic system. Glutamatergic hypofunction is mediated by NMDA (N-methyl-D-aspartate) receptor antagonism. The only endogenous NMDA receptor antagonist identified up to now is kynurenic acid (KYN-A). KYN-A also blocks the nicotinergic acetycholine receptor, i.e. increased KYN-A levels can explain psychotic symptoms and cognitive deterioration. KYN-A levels are described to be higher in the CSF and in critical CNS regions of schizophrenics. Another line of evidence suggests that of the immune system in schizophrenic patients is characterized by an imbalance between the type-1 and the type-2 immune responses with a partial inhibition of the type-1 response, while the type-2 response is relatively over-activated. This immune constellation is associated with the inhibition of the enzyme indoleamine 2,3-dioxygenase (IDO), because type-2 cytokines are potent inhibitors of IDO. Due to the inhibition of IDO, tryptophan is predominantly metabolized by tryptophan 2,3-dioxygenase (TDO), which is located in astrocytes, but not in microglia cells. As indicated by increased levels of S100B, astrocytes are activated in schizophrenia. On the other hand, the kynurenine metabolism in astrocytes is restricted to the dead-end arm of KYN-A production. Accordingly, an increased TDO activity and an accumulation of KYN-A in the CNS of schizophrenics have been described. Thus, the immune-mediated glutamatergic-dopaminergic dysregulation may lead to the clinical symptoms of schizophrenia. Therapeutic consequences, e.g. the use of antiinflammatory cyclooxygenase-2 inhibitors, which also are able to directly decrease KYN-A, are discussed.

This investigation used cytosol fraction of rat liver to examine the effects of insulin (INS) on functional properties of glucocorticoid receptor (GR). Male Wistar rats (220-250 g b.wt.) were injected with INS (50 microg/200 g b.wt, i.p.) and 18 h after INS administration used for experiments. INS-stimulated dissociation of G-R complexes was significantly increased by 133% compared to control level. However, INS treatment significantly stimulated stability of GR protein by 138% above control value. Furthermore, results show that INS stimulated activation of formed cytosol [3H] TA-R complexes by 143% in respect to control. [3H]TA-R complexes from INS treated animals could be activated and accumulated at higher rate in cell nuclei of control animals. The physiological relevance of the data was confirmed by INS-related stimulation of Tryptophan oxigenase (TO) activity. It was observed that INS stimulated TO activity while INS injected to adrenalectomized rats, exhibited less effects compared to control. The results indicate that a glucocorticoid hormone (CORT) enhances INS induced stimulation of TO activity, as evidenced by enhanced enzyme activity. Presented data suggest: that INS treatment leads to modifications of the GR protein and the nuclear components and that INS activates the rat liver CORT signaling pathway which mediates, in part, the activity of TO.

By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-gamma- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-gamma may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.

Indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor, and it has since been isolated from several archaegastropods. The amino acid sequences and genomic structures of IDO-like Mbs show significant homology with those of mammalian IDOs, suggesting that they have evolved from a common ancestral gene. However, details of the evolutionary relationships between them remain unknown. Here, we isolated a novel multicopy gene from Sulculus named molluscan IDO-like protein (MIP). The amino acid sequences of MIPs show the highest homology (about 60% identity) with Sulculus IDO-like Mb, and their exon/intron structures are also highly homologous. However, MIPs are mainly expressed in the gut whereas IDO-like Mb was found only in the buccal mass, suggesting that MIPs are not simply isoforms of IDO-like Mb. A bacterial expression study showed that MIP is a heme-binding protein, and that His335 is the proximal ligand of heme. Although we could not detect IDO activity using a recombinant glutathione S-transferase (GST)-MIP fusion protein in the present study, MIP should have some function other than that of an oxygen carrier like myoglobin, and it might in fact be molluscan IDO.

Our understanding why a woman's immune system does not reject her histoincompatible fetus is still very limited. Distinct insights into the mechanisms involved in pregnancy maintenance may help us to prevent pregnancy complications, e.g., miscarriages or pre-eclampsia. Immune integration and tolerance at the feto-maternal interface appear to be indispensable for successful pregnancy maintenance. Little is known about the cross talk between ICAM-1, expressed on epithelium, endothelium, and APC, and its ligand, LFA-1, at the feto-maternal interface. However, based on the role of ICAM-1/LFA-1 in allograft acceptance or rejection upon transplantation, adhesion molecules are likely to interfere with successful pregnancy outcome. In this study, we tested the hypothesis that ICAM-1/LFA-1 pathways may be involved in pregnancy rejection in murine models. By blocking ICAM-1/LFA-1-mediated intercellular adhesion events, we show that fetal immune acceptance is restored in challenged pregnancies (e.g., upon exposure to sound stress), and adoptive transfer of LFA-1 cells into pregnant mice induces rejection only in abortion-prone mouse models. ICAM-1/LFA-1 cross talk leads to increased recruitment of proinflammatory cells to the implantation site, promotes dendritic cell maturation in the decidua, and subsequently induces additional local Th1 polarization via mature dendritic cells. Furthermore, our observations clearly point out that mechanisms of fetal tolerance, e.g., indoleamine 2,3-dioxygenase expression, presence of CD4+CD25bright regulatory T cells, and synthesis of asymmetric Abs, are ICAM-1/LFA-1 dependent. Hence, our data shed light on a hierarchical network of immune integration at the feto-maternal interface, in which ICAM-1/LFA-1 cross talk is clearly a proximate mediator capable of disrupting successful pregnancy maintenance.

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-gamma showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-gamma-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-gamma-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-gamma alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-gamma. Coculture of eosinophils with an IFN-gamma-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

Tumor escape is one major obstacle that has to be addressed prior to designing and delivering successful immunotherapy. There is compelling evidence to support the notion that immunogenic tumors, in murine models and cancer patients, can be rejected by the immune system under optimum conditions for activating adaptive and nonadaptive antitumor immune responses. Despite this capability, a large number of tumors continue to grow and evade recognition and/or destruction by the immune system. The limited success in current immunotherapeutic strategies may be due to a variety of reasons: failure of effector cells to compete with the growing tumor burden, production of humoral factors by tumors that locally block cytotoxicity, antigen/MHC loss, T-cell dysfunction, production of suppressor T cells-to name but a few causes for therapeutic ineffectiveness for the particular malignancy being treated. To optimize immunotherapy strategies, correction of immune-activating signals, eradication of inhibitory factors, and the evasion from newly developed immunoresistant tumor phenotypes need to be simultaneously considered.

Human monocyte-derived dendritic cells (DCs) are capable of expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which allows them to suppress Ag-driven proliferation of T cells in vitro. In DCs that express IDO, the activity of the enzyme is tightly regulated, with the protein being constitutively expressed, but functional activity requiring an additional set of triggering signals supplied during Ag presentation. We now show that triggering of functional IDO obligately requires ligation of B7-1/B7-2 molecules on the DCs by CTLA4/CD28 expressed on T cells. When this interaction was disrupted, IDO remained in the inactive state, and the DCs were unable to inhibit T cell proliferation. Inhibition could be fully restored by direct Ab-mediated cross-linking of B7-1/B7-2. Although both CD4(+) and CD8(+) T cells were susceptible to inhibition once IDO was induced, the ability to trigger functionally active IDO was strictly confined to the CD4(+) subset. Thus, the ability of CD4(+) T cells to induce IDO activity in DCs allowed the CD4(+) population to dominantly inhibit proliferation of the CD8(+) population via the bridge of a conditioned DC. We hypothesize that IDO activation via engagement of B7-1/B7-2 molecules on DCs, specifically, engagement by CTLA4 expressed on regulatory CD4(+) T cells, may function as a physiologic regulator of T cell responses in vivo.

The mechanisms by which administration of interferon-alpha induces neuropsychiatric side effects, such as depressive symptoms and changes in cognitive function, are not clear as yet. Direct influence on serotonergic neurotransmission may contribute to these side effects. In addition, the enzyme indoleamine 2,3-dioxygenase (IDO), which converts tryptophan into kynurenine, may play an important role, first, because IDO activation leads to reduced levels of tryptophan, the precursor of serotonin (5-HT), and thus to reduced central 5-HT synthesis. Second, kynurenine metabolites such as 3-hydroxy-kynurenine (3-OH-KYN) and quinolinic acid (QUIN) have toxic effects on brain function. 3-OH-KYN is able to produce oxidative stress by increasing the production of reactive oxygen species (ROS), and QUIN may produce overstimulation of hippocampal N-methyl-D-aspartate (NMDA) receptors, which leads to apoptosis and hippocampal atrophy. Both ROS overproduction and hippocampal atrophy caused by NMDA overstimulation have been associated with depression.

Background & Aims: Indoleamine 2,3-dioxygenase (IDO), an interferon gamma-induced intracellular enzyme, inhibits lymphocyte proliferation through tryptophan degradation. IDO is highly expressed in the mammalian intestine. We sought to determine whether IDO played a regulatory role in the T-cell helper 1 (Th1)-mediated trinitrobenzene sulfonic acid (TNBS) model of colitis., Methods: Intrarectal TNBS was given to SJL/J mice along with either placebo or a specific IDO inhibitor. IDO protein and mRNA expression were assessed by Western blotting and real-time PCR. Colonic lamina propria mononuclear cells (LPMNCs) were isolated, fractionated, and cultured, in the presence and absence of IFN-gamma, to determine the cell type(s) expressing IDO., Results: IDO is expressed by professional antigen-presenting cells in the lamina propria. Induction of TNBS colitis resulted in a significant increase in IDO mRNA (P = 0.005) and protein expression. IDO inhibition during TNBS colitis resulted in an 80% mortality compared with 10% for placebo-treated animals (P = 0.0089). IDO inhibition resulted in a more severe colitis both histologically and morphologically (P < 0.05) and significantly increased colonic proinflammatory cytokine expression compared with placebo-treated animals., Conclusions: IDO is expressed in the normal colon and is up-regulated in the setting of TNBS colitis. Inhibition of IDO during TNBS colitis resulted in increased mortality and an augmentation of the normal inflammatory response. These findings suggest that IDO plays an important role in the down-regulation of Th1 responses within the gastrointestinal tract.

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8alpha, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.

The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-gamma. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-gamma fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

Respiratory viral infections increase inflammatory responses to concurrent or secondary bacterial challenges, thereby worsening disease outcome. This potentiation of inflammation is explained at least in part by IFN-gamma promoting increased sensitivity to TNF-alpha and LPS. We sought to determine whether and, if so, how IFN-gamma can modulate proinflammatory responses to TNF-alpha and LPS by epithelial cells, which are key effector cells in the airways. Preincubation of airway epithelial-like NCI-H292 cells with IFN-gamma resulted in a hyperresponsive IL-6 and IL-8 production to TNF-alpha and LPS. The underlying mechanism involved the induction of indoleamine 2,3-dioxygenase, which catabolized the essential amino acid, tryptophan. Depletion of tryptophan led to stabilization of IL-6 and IL-8 mRNA and increased IL-6 and IL-8 responses, whereas supplementing tryptophan largely restored these changes. This novel mechanism may be implicated in enhanced inflammatory responses to bacterial challenges following viral infection.

Serotonin (5-HT), one of the evolutionary oldest central neurotransmitters, regulates the most extensive modulatory behavioral system in the brain of vertebrates. 5-HT projections are influenced by extrinsic and intrinsic impulses from different cortical brain areas, which reach Raphe nuclei over feedback loops, containing external and internal body information about planning, evaluation, motivation or excitation. Serotonergic neurotransmission adjusts neuromodulation with consecutive adequate stimulation of the neuronal network. This depends on appropriate equilibration of presynaptic 5-HT storage and release but also on 5-HT reuptake from synaptic cleft by 5-HT transporters. The associated pre and postsynaptic 5-HT receptor cooperation, postsynaptic second messenger response and phosphoinositide signaling mediated by postsynaptic 5-HT(2) receptor subpopulation alter signal transduction in which myristolated alanine rich C kinase substrate is prominently involved in regulation of further central 5-HT areas in the brain and corresponding functional neuronal changes. Even though the central function of 5-HT neurotransmission is dominating in the multifold behavioral regulation, peripheral concentration of tryptophan (TRP) adjusted by hepatic and non-hepatic TRP pyrrolase, TRP liberation from albumin especially by adrenergic stimulation of free fatty acids, TRP passage across the blood-brain barrier and TRP hydroxylase activity are also important for appropriate 5-HT neurotransmission as they affect central 5-HT synthesis. The high adaptability of 5-HT neurotransmission is able to compensate neuromodular dysfunctions in the brain by mechanisms which mediate 5-HT biosynthesis, release, reuptake, pre and postsynaptic receptor stimulation with the respective second messenger response and signal transduction to various areas of the brain which are involved in regulation of behavior, mood, memory, learning and attenuation of obsession, depending on the different vigilance states of the subject. Adequate 5-HT system function supports regulation of intercommunicative neuronal transmission in the brain, which optimizes behavioral neuromodulation during and after transient disturbances of neuromodular behavior caused by stress-induced exertions, but also in permanent disorder such as major depression. Serotonergic neurotransmission improves the clinical course due to compensatory 5-HT impulse correction. This hypothetical interpretation of the serotonergic central neuromodular regulation and interaction with the neuronal network is supported by findings both in functional disturbances and persistent impairments in mental disorders. A comparison of the symptomatology in permanent and transient disturbance of brain neuromodulation enhances our basic knowledge on the regulative factors e. g. in endogenous depression and depressive behavioral changes after exhaustive exercise. This consideration exhibits that the interaction between altered central neuromodulation and peripheral metabolic and hormonal dysfunctions is able to differentiate the etiology of the symptoms. It is suggested that the central neuromodular disturbance of stress-induced causes might initiate the manifestation of the impairment. The theoretical background of this hypothesis is discussed in the present review.

The murine conceptus is protected from maternal immunity by cells expressing indoleamine dioxygenase (IDO), which catabolizes tryptophan. Induction of lethal maternal anti-fetal immunity requires effective pharmacologic inhibition of IDO enzyme activity and the presence of maternal T cells, but not B cells and also depends on the degree of maternal-fetal tissue incompatibility. Based on these findings, we propose a model to explain the role of IDO in suppressing maternal immunity and the mechanism of fetal allograft rejection, when IDO activity is inhibited during gestation. This model incorporates observations that fetal allograft rejection is T cell dependent, antibody-independent and is accompanied by a novel type of inflammation involving extensive complement deposition at the maternal-fetal interface, when IDO activity is blocked during murine pregnancy.

1. The physiological importance of human placental indoleamine 2,3-dioxygenase (EC 1.13.11.42), the first and rate-limiting enzyme in tryptophan metabolism, in regulating feto-maternal immunology has been studied. 2. Concentrations were measured in placental villous explant conditioned media of 14 amino acids that are known to be required for lymphocyte proliferation. In the absence of interferon-gamma only tryptophan and threonine were significantly lowered; in the presence of interferon-gamma (known to stimulate indoleamine 2,3-dioxygenase) tryptophan but not threonine depletion was much greater. 3. Peripheral blood mononuclear cell proliferation determined by measuring thymidine incorporation into DNA following culture in the medium previously conditioned by culture of villous explants was markedly reduced when placental indoleamine 2,3-dioxygenase was stimulated with interferon-gamma. Inhibition of placental indoleamine 2,3-dioxygenase by 1-methyl-tryptophan prevented inhibition of thymidine incorporation. Supplementation of the conditioned medium with tryptophan but no other amino acid completely reversed the inhibition of thymidine incorporation. 4. Flow cytometric analysis showed that CD4-positive T lymphocyte division was specifically suppressed by indoleamine 2,3-dioxygenase-mediated tryptophan depletion. This inhibition of T cell proliferation was due to arrest of cell cycle progression. 5. To study the mechanism of tryptophan sensing we examined the ability of 11 L-tryptophan analogues to support lymphocyte proliferation. Only L-tryptophan methyl and ethyl esters were able to stimulate proliferation in tryptophan-free media. Since both of these molecules are readily degraded to tryptophan by intracellular esterases this suggests that the tryptophan sensor is intracellular. 6. Our results show that mechanisms are present in the human placenta which are able to regulate cellular proliferation of the maternal immune system. This mechanism is dependent both on placental indoleamine 2,3-dioxygenase-mediated tryptophan degradation and on tryptophan sensing systems within lymphocytes.

Background: The tryptophan catabolizing enzyme, indoleamine 2,3, dioxygenase (IDO) is one of two mammalian enzymes, which can catabolize the rarest essential amino acid, tryptophan. IDO is inducible by cytokines such as interferon-gamma and plays a role in inflammation and maternal tolerance of fetal allografts, although its exact mode of action is unclear. Therefore, we investigated the circumstances under which IDO is expressed in vitro together with the effects of overexpression of IDO on the growth and morphology of cells., Results: Overexpression of IDO in the murine macrophage cell line RAW 264.7 and the murine fibrosarcoma cell line MC57, resulted in the growth of macroscopic cell foci, with altered cell adhesion properties. The expression of IDO was also detected during adhesion of wild type, nontransfected cells in tissue culture to standard cell growth substrates. Inhibition of this expression, likewise resulted in alterations in cell adhesion. Overexpression of IDO or inhibition of endogenous IDO expression was accompanied by changes in metalloproteinase expression and also in the expression and activity of the cyclooxygenase enzymes. In the case of RAW cells, IDO effects on cell growth could be reversed by adding back prostaglandins., Conclusions: These results suggest that catabolism of the rarest essential amino acid may regulate processes such as cell adhesion and prostaglandin synthesis.

The mechanism of killing of obligately intracellular Rickettsia conorii within human target cells, mainly endothelium and, to a lesser extent, macrophages and hepatocytes, has not been determined. It has been a controversial issue as to whether or not human cells produce nitric oxide. AKN-1 cells (human hepatocytes) stimulated by gamma interferon, tumor necrosis factor alpha, interleukin 1beta, and RANTES (regulated by activation, normal T-cell-expressed and -secreted chemokine) killed intracellular rickettsiae by a nitric oxide-dependent mechanism. Human umbilical vein endothelial cells (HUVECs), when stimulated with the same concentrations of cytokines and RANTES, differed in their capacity to kill rickettsiae by a nitric oxide-dependent mechanism and in the quantity of nitric oxide synthesized. Hydrogen peroxide-dependent intracellular killing of R. conorii was demonstrated in HUVECs, THP-1 cells (human macrophages), and human peripheral blood monocytes activated with the cytokines. Rickettsial killing in the human macrophage cell line was also mediated by a limitation of the availability of tryptophan in association with the expression of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase. The rates of survival of all of the cell types investigated under the conditions of activation and infection in these experiments indicated that death of the host cells was not the explanation for the control of rickettsial infection. This finding represents the first demonstration that activated human hepatocytes and, in some cases, endothelium can kill intracellular pathogens via nitric oxide and that RANTES plays a role in immunity to rickettsiae. Human cells are capable of controlling rickettsial infections intracellularly, the most relevant location in these infections, by one or a combination of three mechanisms involving nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation.

Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been implicated as a potential risk factor in atherosclerosis, possibly because the pathogen can exist in a persistent form similar to that described for Chlamydia trachomatis. The present study investigated whether gamma interferon (IFN-gamma) can induce indoleamine 2,3-dioxygenase (IDO) activity in aortic smooth muscle cells, leading to a marked inhibition of C. pneumoniae growth. Our data indicate a stimulation of IDO mRNA expression and dose-dependent enzymatic activity following IFN-gamma treatment. IDO-mediated increase in tryptophan catabolism resulted in a dose-dependent marked inhibition of C. pneumoniae replication.

We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-gamma (IFN-gamma)-activated macrophages of the murine BACl.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-gamma induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.

Indoleamine 2,3-dioxygenase (IDO) is expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity. We isolated a cDNA for tryptophan 2,3-dioxygenase (TDO), another key catabolizing enzyme of tryptophan, from a mouse uterus cDNA library enriched with pregnancy-induced genes. Northern blot and in situ hybridization analyses demonstrated that the TDO mRNA was induced in the decidualized stromal cells around the implanted embryo at the time of implantation. The expression was then upregulated and primarily localized at the mesometrial decidua. TDO mRNA was induced by deciduoma formation as well as embryo transfer but not by ovarian steroid hormones. These findings demonstrated that TDO is induced in the endometrial stromal cells concomitant with decidualization and suggested its involvement in the implantation process by regulating the tryptophan level at the implantation site., (Copyright 2000 Academic Press.)

Human T cell lymphotropic virus type I (HTLV-I) is associated with a chronic neurologic disease called HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The potential mechanisms of HAM/TSP pathogenesis were assessed by examination of 2 pathways initiated by interferon-gamma, a predominant cytokine in HAM/TSP. Jamaican HAM/TSP patients (n=17) were compared with patients with other neurologic diseases (ONDs; n=13) with respect to cerebrospinal fluid levels of the following: neopterin; nitrite plus nitrate, a stable indicator of nitric oxide; and tryptophan and kynurenine, metabolites of the indoleamine-2,3-dioxygenase (IDO) pathway. HAM/TSP patients had significantly elevated levels of neopterin (P=.003) and kynurenine (P=.05) and a significantly decreased level of tryptophan (P=.003), compared with patients with ONDs. These results support immune activation within the central nervous system and activation of the IDO pathway. Thus, activation of the IDO pathway may play a role in HAM/TSP.

In macrophages, interleukin-1 (IL-1) and lipopolysaccharide (LPS) enhance the antichlamydial effect of interferon-gamma (IFN-gamma) by increasing indoleamine 2,3-dioxygenase (IDO) activity in a dose-dependent manner. Our objectives were to characterize the antichlamydial effect of tumor necrosis factor-alpha (TNF-alpha) on IFN-induced IDO activity and to establish the relationship between LPS and TNF-alpha in IDO potentiation. TNF-alpha inhibited chlamydial growth in a dose-dependent manner only in IFN-treated macrophages. Furthermore, excess tryptophan reversed the effect of combined cytokine treatment, indicating that IDO alone was responsible for chlamydial inhibition. The promonocyte THP-1 cell line, previously used to model the effect of IL-1 on IDO mRNA expression, was treated with IFN-gamma and increasing concentrations of LPS or TNF-alpha. IDO mRNA was quantified by RT-PCR, and IDO activity was measured by HPLC at 24 and 48 h after treatment, respectively. Both LPS and TNF-alpha enhanced IDO activity and IDO mRNA expression, with maximal IDO induction at 100 ng/ml LPS or 5 ng/ml TNF-alpha. Anti-TNF-alpha failed to neutralize the effects of LPS treatment, and insufficient TNF-alpha or IL-1 was produced by LPS-treated THP-1 cells to account for the enhancing effect of LPS, indicating that the effect of LPS on IDO was independent of TNF-alpha and IL-1.

Dendritic cells (DCs) play a key role in the activation and regulation of B and T lymphocytes. Production of indoleamine 2, 3-dioxygenase (IDO) by macrophages has recently been described to result in inhibition of T cell proliferation through tryptophan degradation. Since DCs can be derived from monocytes, we sought to determine whether DCs could produce IDO which could potentially regulate T cell proliferation. Northern blot analysis of RNA from cultured monocyte-derived human DC revealed that IDO mRNA was induced upon activation with CD40 ligand and IFN-gamma. IDO produced from activated DCs was functionally active and capable of metabolizing tryptophan to kynurenine. Activated T cells were also capable of inducing IDO production by DCs, which was inhibited by a neutralizing Ab against IFN-gamma. DC production of IDO resulted in inhibition of T cell proliferation, which could be prevented using the IDO inhibitor 1-methyl-dl -tryptophan. These results suggest that activation of DCs induces the production of functional IDO, which causes depletion of tryptophan and subsequent inhibition of T cell proliferation. This may represent a potential mechanism for DCs to regulate the immune response.

The most prominent gamma interferon (IFN-gamma)-induced antimicrobial effector mechanisms are the induction of nitric oxide (NO) synthase (NOS) and of indoleamine 2,3-dioxygenase (IDO) activity. We have recently found that human glioblastoma cells and human macrophages inhibit the growth of group B streptococci after stimulation with IFN-gamma. In this report, we show that in addition, human RT4 (uroepithelial) cells can inhibit the growth of enterococci. Murine macrophages (RAW cells) are unable to inhibit bacterial growth after IFN-gamma stimulation. Stimulation of human glioblastoma cells, macrophages, and RT4 cells with human IFN-gamma results in a strong expression of IDO activity; however, NO production remains undetectable. In strong contrast, murine RAW cells produce large amounts of NO when stimulated with murine IFN-gamma and IDO activity is not detectable. Interleukin-1 (IL-1) induces NO synthase in human RT4 cells when the cells are costimulated with IFN-gamma. We found that IL-1 inhibits IFN-gamma-stimulated IDO activity and antimicrobial effects in RT4 cells, while in human glioblastoma cells, which lack detectable NO synthase activity, neither of these effects was altered by costimulation with IFN-gamma and IL-1. The IL-1-mediated inhibition of IDO activity and of subsequent antibacterial effect is due to the production of NO. This conclusion was supported by evidence that N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible NOS activity, is able to block the inhibitory action of IL-1 on IFN-gamma-induced bacteriostasis. We therefore conclude that NO production does not inhibit the growth of enterococci but might be involved in the regulation of IDO activity in some human cells.

An in vitro model of human CMV infection of primary retinal pigment epithelial (RPE) cells was used to study the effects of cytokines on CMV replication in these cells, which are targets of CMV infection in vivo. IFN-gamma and IFN-beta were potent inhibitors of CMV replication in RPE cells, while TNF-alpha, IL-1beta, or TGF-beta2 did not affect viral replication. Inhibition by IFN-gamma, and to a lesser extent IFN-beta, was almost completely reversed by addition of L-tryptophan to the culture medium, strongly implicating the indoleamine 2,3 dioxygenase (IDO) pathway. Polyadenylated IDO mRNA accumulation was detected as early as 2 h after IFN stimulation. Furthermore, CMV blocked the production of nitric oxide by the inducible form of nitric oxide synthase. This inhibition depended on a functional viral genome. However, exogenous nitric oxide significantly inhibited viral protein expression in RPE cells. Thus, CMV infection blocks the inducible nitric oxide synthase pathway activated by IFN-gamma and IL-1beta, but cannot counteract the IFN-induced IDO pathway, which ultimately controls its replication in primary human RPE cells.

Cycloheximide (20 mg/kg body wt, given intraperitoneally at-1 and 3 h after withdrawal of an ethanol-containing liquid diet) prevents the activation of liver tryptophan pyrrolase, the consequent inhibition of synthesis of brain 5-hydroxytryptamine, and the audiogenic seizures observed at 7 h after alcohol withdrawal. We suggest that a rapidly-turning-over protein mediates the alcohol withdrawal syndrome and discuss the possible role of liver tryptophan pyrrolase.

Toxoplasma gondii, an obligate intracellular parasite, is able to replicate in human brain cells. We recently showed that interferon (IFN)-gamma-activated cells from glioblastoma line 86HG39 were able to restrict Toxoplasma growth. The effector mechanism responsible for this toxoplasmostatic effect was shown by us to be the IFN-gamma-mediated activation of indolamine 2,3-dioxygenase (IDO), resulting in the degradation of the essential amino acid tryptophan. In contrast, glioblastoma 87HG31 was unable to restrict Toxoplasma growth after IFN-gamma activation, and IFN-gamma-mediated IDO activation was weak. We observed that tumor necrosis factor (TNF)-alpha alone is unable to activate IDO or to induce toxoplasmostasis in any glioblastoma cell line tested. Interestingly, we found that TNF-alpha and IFN-gamma were synergistic in the activation of IDO in glioblastoma cells 87HG31, 86HG39 and U373MG and in native astrocytes. This was shown by the measurement of enzyme activity as well as by the detection of IDO mRNA in TNF-alpha + IFN-gamma activated cells. This IDO activity results in a strong toxoplasmostatic effect mediated by glioblastoma cells activated simultaneously by both cytokines. Antibodies directed against TNF-alpha or IFN-gamma were able to inhibit IDO activity as well as the induction of toxoplasmostasis in glioblastoma cells stimulated with both cytokines. Furthermore, it was found that the addition of L-tryptophan to the culture medium completely blocks the antiparasitic effect. We therefore conclude that both TNF-alpha and IFN-gamma may be involved in the defense against cerebral toxoplasmosis by inducing IDO activity as an antiparasitic effector mechanism in brain cells.

Objective: Previous studies have demonstrated a relationship between obsessive-compulsive disorder or behavior and Gilles de la Tourette syndrome. It has been hypothesized that the serotonergic system is implicated in the etiology of obsessive-compulsive disorder. Therefore, the authors investigated whether genetic variation in a serotonergic receptor and a modifying enzyme were associated with Tourette's syndrome., Method: A linkage analysis using DNA and blood group markers was carried out in a large British kindred multiply affected with Tourette's syndrome, chronic motor tics, and obsessive-compulsive behavior., Results: There was no evidence to support the hypothesis that genetic variation in the serotonin 5-HT1A receptor and tryptophan oxygenase genes causes susceptibility to Tourette's syndrome and chronic multiple tics., Conclusions: The results eliminate two possible candidate genes from having a role in the pathophysiology of Tourette's syndrome.

We have studied the relationship between L-tryptophan metabolism and the response to human IFN-gamma in 3 human ovarian cancer xenografts growing in nude mice. During IFN-gamma therapy all 3 tumours showed a profound depletion in L-tryptophan and a corresponding rise in L-kynurenine. The microenvironment surrounding the tumours was also depleted of L-tryptophan. The IFN-gamma-inducible enzyme indoleamine dioxygenase, IDO, was induced in treated tumours. While there was a variability in IDO mRNA expression in the different xenografts tested, in situ hybridization showed that the gene was induced at all levels of the tumour, and not just the periphery. These results show that induction of IDO by IFN-gamma in vivo can metabolize L-tryptophan rapidly enough for it to become depleted, despite a continued supply of L-tryptophan from the host. The IDO mRNA and protein remained induced after the L-tryptophan levels had returned to normal, suggesting that the gene may be post-transcriptionally regulated and/or the IDO co-factor supply may be limited. Another IFN-gamma-inducible gene, tryptophanyl tRNA synthetase, was also induced in the tumour. It is possible that this enzyme, which is responsible for synthesizing tryptophanyl tRNA, acts in a compensatory manner by allowing protein synthesis to continue despite low free L-tryptophan concentrations. There was no correlation of the above parameters with the anti-tumour response to IFN-gamma, suggesting that other mechanisms must play a role. L-tryptophan depletion may be a contributor to a multifactorial growth inhibition of tumour cells following IFN-gamma treatment, but cannot on its own explain their growth inhibition.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts.

The role of indoleamine 2,3-dioxygenase (IDO) in IFN-gamma-mediated inhibition of intracellular parasite growth has been examined previously, although earlier work has been largely correlative. In this study, we defined more completely the role of IDO in the IFN-antimicrobial response. Two mutant cell lines, derived from ME180 cells and exhibiting reduced IDO activity (IR3B6A, IR3B6B) were characterized to determine if they retained the capacity to inhibit intracellular Chlamydia and Toxoplasma growth. Mutant cells treated with IFN-gamma exhibited reduced capacity to suppress pathogen growth. The expression of several IFN-regulated genes also was measured to confirm that the inability to inhibit pathogen growth was because of the lack of IDO. The expression of class II MHC, intracellular adhesion molecule-1, MxA, and P68 kinase genes was induced in the IFN-gamma-treated wild type ME180 cells, but was variable in the mutant cell lines, supporting the hypothesis that IFN-gamma-induced production of IDO is a key IFN-gamma-mediated antimicrobial mechanism.

The effect of intraperitoneal administration of phenobarbital (80 mg kg-1 body weight) and tryptophan (200 mg kg-1 body weight), separately or in combination, on the microsomal content of cytochrome P-450 and the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) in Wistar rat liver was determined at different time intervals after injection. There was an increase in the amount of cytochrome P-450 within 12 h of administration of a single dose of phenobarbital which was maintained over the next 12 h. Tryptophan had no effect on the amount of cytochrome P-450, but administration of tryptophan in combination with phenobarbital blocked the increase that was found after administration of phenobarbital alone. Both phenobarbital and tryptophan increased tryptophan 2,3-dioxygenase activity (total enzyme and holoenzyme) but had different effects on the rate of activation and the degree of saturation of the enzyme with haem. Administration of tryptophan and phenobarbital in combination invoked the same effect as tryptophan alone. Significant activation of the holoenzyme was found, when tryptophan was administered 2 h after phenobarbital administration. It is proposed that combined administration of phenobarbital and tryptophan leads to substrate stabilisation of tryptophan 2,3-dioxygenase, and that this is accompanied by the binding of the newly synthesised haem, thus making haem unavailable for formation of cytochrome P-450.