Hepatocyte-intrinsic type I interferon signaling reprograms metabolism and reveals a novel compensatory mechanism of the tryptophan-kynurenine pathway in viral hepatitis.

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.

A defect in tryptophan catabolism impairs tolerance in nonobese diabetic mice.

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.

IDO, TDO, and AHR overexpression is associated with poor outcome in diffuse large B-cell lymphoma patients in the rituximab era.

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.

The immunological basis of glutamatergic disturbance in schizophrenia: towards an integrated view.

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.

Insulin modulates rat liver glucocorticoid receptor.

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.

Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function.

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.

Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages.

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.

Do molluscs possess indoleamine 2,3-dioxygenase?

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.

Tumoral Immune Resistance Mediated by Enzymes That Degrade Tryptophan.

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).

Exclusion of the 5-HT1A serotonin neuroreceptor and tryptophan oxygenase genes in a large British kindred multiply affected with Tourette's syndrome, chronic motor tics, and obsessive-compulsive behavior.

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.

Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.

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.

Tryptophan catabolism prevents maternal T cells from activating lethal anti-fetal immune responses.

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.

Regulation of prostaglandin synthesis and cell adhesion by a tryptophan catabolizing enzyme.

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.

A crucial role for tryptophan catabolism at the host/Candida albicans interface.

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.

Intercellular adhesion molecule-1/LFA-1 cross talk is a proximate mediator capable of disrupting immune integration and tolerance mechanism at the feto-maternal interface in murine pregnancies.

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.

Inhibition of Chlamydia pneumoniae replication in human aortic smooth muscle cells by gamma interferon-induced indoleamine 2, 3-dioxygenase activity.

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.

The role of indoleamine 2,3-dioxygenase (IDO) in the pathophysiology of interferon-alpha-induced depression.

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.

Central nervous system activation of the indoleamine-2,3-dioxygenase pathway in human T cell lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis.

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.