Molecules in focus: indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is a heme enzyme that initiates the oxidative degradation of the least abundant, essential amino acid, l-tryptophan, along the kynurenine pathway. The local cellular depletion of l-tryptophan that results may enable the host to inhibit the growth of various infectious pathogens in vivo. However, over the past decade, it has become increasingly apparent that IDO also represents an important immune control enzyme. Thus, cells expressing IDO, seemingly paradoxically, are capable of suppressing local T cell responses to promote immune tolerance under various physiological and pathophysiological conditions of medical importance, including infectious diseases, foetal rejection, organ transplantation, neuropathology, inflammatory and auto-immune disorders and cancer. In this review, we briefly outline the biochemical properties of IDO, its known and hypothetical functions and the medical implications for inhibition or induction of IDO and/or its downstream catabolites in health and disease.     

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of d-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of d-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary l-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from d-tryptophan and l-tryptophan in mice.     

Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase

T lymphocytes undergo proliferation arrest when exposed to tryptophan shortage, which can be provoked by indoleamine 2,3-dioxygenase (IDO), an enzyme that is expressed in placenta and catalyzes tryptophan degradation. Here we show that most human tumors constitutively express IDO. We also observed that expression of IDO by immunogenic mouse tumor cells prevents their rejection by preimmunized mice. This effect is accompanied by a lack of accumulation of specific T cells at the tumor site and can be partly reverted by systemic treatment of mice with an inhibitor of IDO, in the absence of noticeable toxicity. These results suggest that the efficacy of therapeutic vaccination of cancer patients might be improved by concomitant administration of an IDO inhibitor.     

NADH oxidase activity of indoleamine 2,3-dioxygenase

The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme.     

Tryptophan 2,3-dioxygenase activity in rat skin

We have found an enzyme system that catalyzes the conversion of L-tryptophan to L-kynurenine, presumably via L-formylkynurenine, in soluble and insoluble fractions of rat skin. The enzymatic activity was stimulated by hematin, ascorbate, and catalase, but not by methylene blue. Highest activity was located in the skin of the dorsal posterior region and lowest activity in the abdominal region. The activity in plucked (depilated) skin was only about 25% of that obtained from unplucked (depilated) tissue of the same region. D-Tryptophan, 5-hydroxytryptophan, and tryptamine were not degraded by the skin enzyme and the Km for L-tryptophan determined with the crude enzyme was 1 microM. The decycling activity of rat skin and liver for L-tryptophan began to be stimulated after birth and reached the highest level at 6 weeks. But, 1 week later, most of the skin activity suddenly disappeared and the low level continued at least until 12 weeks. In contrast, the hepatic enzyme did not change so drastically. These findings suggest that an enzyme that catalyzes L-tryptophan to L-kynurenine via L-formylkynurenine is present in rat skin.     

Molecular evolution of bacterial indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD⁺). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD⁺, like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K_m, V_max and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD⁺) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking the kynU gene suggests its IDO has a function similar to eukaryotic enzymes.     

Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin

We tested the hypothesis that hCG can upregulate human trophoblast indoleamine 2, 3-dioxygenase (INDO), which catalyzes the breakdown of tryptophan in villous circulation. The results revealed that it can. Treatment of human trophoblasts with hCG resulted in a time and dose dependent increase in INDO mRNA and protein levels and its enzyme activity. The hCG effect was hormone specific and required the dimer conformation of hCG. The hCG effect required its receptors and was mediated by a cAMP dependent, but protein kinase A independent, mitogen-activated protein kinase 3/1 (MAPK3/1) signaling mechanism. In summary, the present data demonstrate a novel hCG effect on human placental INDO, which probably plays a key role at maternal fetal interface in preventing fetal rejection.     

A myoglobin evolved from indoleamine 2,3-dioxygenase.

Hemoglobins and myoglobins are some of the best studied proteins. They are distributed in animals, plants and bacteria, and the characteristic two intron-three exon structure is widely conserved in animal globin genes (Jhiang et al., 1988). To date, all of the hemoglobins and myoglobins are believed to have a common origin, and so they are considered to be homologous. We have isolated a completely new type of myoglobin from the red muscle of the abalone Sulculus diversicolor aquatilis. The myoglobin consists of an unusual 41 kDa polypeptide chain, contains one heme per chain and forms a homodimer under physiological conditions. The cDNA-derived amino acid sequence of Sulculus myoglobin showed no significant homology with any other globins, but, surprisingly, showed high homology (35% identity) with human indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. This clearly indicates that Sulculus myoglobin evolved from a gene for indoleamine dioxygenase, but not from a globin gene. Sulculus myoglobin lacks the enzyme activity of indoleamine dioxygenase. However, in the presence of tryptophan, the autoxidation rate of oxymyoglobin was greatly accelerated, suggesting that a tryptophan binding site remains near or in the heme cavity as a relic of the molecular evolution.     

Interactions between nitric oxide and indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Similar to inducible nitric oxide synthase (iNOS), IDO is induced by interferon-gamma and lipopolysaccharide in the inflammatory response. In vivo studies indicate that the nitric oxide (NO) produced by iNOS inhibits IDO activity by directly interacting with it and by promoting its degradation through the proteasome pathway. In this work, the molecular mechanisms underlying the interactions between NO and human recombinant IDO (hIDO) were systematically studied with optical absorption and resonance Raman spectroscopies. Resonance Raman data show that the heme prosthetic group in the NO-bound hIDO is situated in a unique protein environment and adopts an out-of-plane deformed geometry that is sensitive to L-Trp binding. Under mildly acidic conditions, the proximal heme iron-His bond is prone to rupture, resulting in a five-coordinate (5C) NO-bound species. The bond breakage reaction induces significant conformational changes in the protein matrix, which may account for the NO-induced inactivation of hIDO and its enhanced proteasome-linked degradation in vivo. Moreover, it was found that the NO-induced bond breakage reaction occurs more rapidly in the ferrous protein than in the ferric protein and is fully inhibited by L-Trp binding. The spectroscopic data presented here not only provide the first glimpse of the possible regulatory mechanism of hIDO by NO in the cell at the molecular level, but they also suggest that the NO-dependent regulation can be modulated by cellular factors, such as the NO abundance, pH, redox environment, and L-Trp availability.     

Post-translational regulation of human indoleamine 2,3-dioxygenase activity by nitric oxide

The heme protein indoleamine 2,3-dioxygenase (IDO) is induced by the proinflammatory cytokine interferon-gamma (IFNgamma) and plays an important role in the immune response by catalyzing the oxidative degradation of L-tryptophan (Trp) that contributes to immune suppression and tolerance. Here we examined the mechanism by which nitric oxide (NO) inhibits human IDO activity. Exposure of IFNgamma-stimulated human monocyte-derived macrophages (MDM) to NO donors had no material impact on IDO mRNA or protein expression, yet exposure of MDM or transfected COS-7 cells expressing active human IDO to NO donors resulted in reversible inhibition of IDO activity. NO also inhibited the activity of purified recombinant human IDO (rhIDO) in a reversible manner and this correlated with NO binding to the heme of rhIDO. Optical absorption and resonance Raman spectroscopy identified NO-inactivated rhIDO as a ferrous iron (Fe(II))-NO-Trp adduct. Stopped-flow kinetic studies revealed that NO reacted most rapidly with Fe(II) rhIDO in the presence of Trp. These findings demonstrate that NO inhibits rhIDO activity reversibly by binding to the active site heme to trap the enzyme as an inactive nitrosyl-Fe(II) enzyme adduct with Trp bound and O2 displaced. Reversible inhibition by NO may represent an important mechanism in controlling the immune regulatory actions of IDO.     

Induction of indoleamine 2,3-dioxygenase in primary human macrophages by HIV-1

Abstract

Increased kynurenine pathway metabolism has been implicated in the aetiology of the AIDS dementia complex (ADC). The rate limiting enzyme for this pathway is indoleamine 2,3- dioxygenase (IDO). We tested the efficacy of different strains of HIV-1 (HIV1-BaL, HIV1-JRFL and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain derived HIV-1 isolates, laboratory adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the high replicating macrophage tropic LA strain, HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day-8 post-infection. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent oxidative tryptophan catabolism in MDM.     

Induction of indoleamine 2,3-dioxygenase in primary human macrophages by HIV-1

Increased kynurenine pathway metabolism has been implicated in the aetiology of the AIDS dementia complex (ADC). The rate limiting enzyme for this pathway is indoleamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of HIV-1 (HIV1-BaL, HIV1-JRFL and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain derived HIV-1 isolates, laboratory adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the high replicating macrophage tropic LA strain, HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day-8 post-infection. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent oxidative tryptophan catabolism in MDM.     

The role of serine 167 in human indoleamine 2,3-dioxygenase: a comparison with tryptophan 2,3-dioxygenase

The initial step in the l-kynurenine pathway is oxidation of l-tryptophan to N-formylkynurenine and is catalyzed by one of two heme enzymes, tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO). Here, we address the role of the conserved active site Ser167 residue in human IDO (S167A and S167H variants), which is replaced with a histidine in other mammalian and bacterial TDO enzymes. Our kinetic and spectroscopic data for S167A indicate that this residue is not essential for O 2 or substrate binding, and we propose that hydrogen bond stabilization of the catalytic ferrous-oxy complex involves active site water molecules in IDO. The data for S167H show that the ferrous-oxy complex is dramatically destabilized in this variant, which is similar to the behavior observed in human TDO [Basran et al. (2008) Biochemistry 47, 4752-4760], and that this destabilization essentially destroys catalytic activity. New kinetic data for the wild-type enzyme also identify the ternary [enzyme-O 2-substrate] complex. The data reveal significant differences between the IDO and TDO enzymes, and the implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.     

Comparative effects of oxygen on indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase of the kynurenine pathway

Indoleamine 2,3-dioxygenase (IDO) reacts with either oxygen or superoxide and tryptophan (trp) or other indoleamines while tryptophan 2,3-dioxygenase (TDO) reacts with oxygen and is specific for trp. These enzymes catalyze the rate-limiting step in the kynurenine (KYN) pathway from trp to quinolinic acid (QA) with TDO in kidney and liver and IDO in many tissues, including brain where it is low but inducible. QA, which does not cross the blood-brain barrier, is an excitotoxin found in the CNS during various pathologies and is associated with convulsions. We proposed that HBO-induced convulsions result from increased flux through the KYN pathway via oxygen stimulation of IDO. To test this, TDO and IDO of liver and brain, respectively, of Sprague Dawley rats were assayed with oxygen from 0 to 6.2 atm HBO. TDO activity was appreciable at even 30 microM oxygen and rose steeply to a maximum at 40 microM. Conversely, IDO had almost no detectable activity at or below 100 microM oxygen and maximum activity was not reached until about 1150 microM. (Plasma contains about 215 microM oxygen and capillaries about 20 microM oxygen when rats breathe air.) KYN was 60% higher in brains of HBO-convulsed rats compared to rats breathing air. While the oxygen concentration inside cells of rats breathing air or HBO is not known precisely, it is clear that the rate-limiting, IDO-catalyzed step in the brain KYN pathway (but not liver TDO) can be greatly accelerated in rats breathing HBO.     

Identification of selective inhibitors of indoleamine 2,3-dioxygenase 2

The kynurenine pathway is responsible for the breakdown of the majority of the essential amino acid, tryptophan (Trp). The first and rate-limiting step of the kynurenine pathway can be independently catalysed by tryptophan 2,3-dioxygenase (Tdo2), indoleamine 2,3-dioxygenase 1 (Ido1) or indoleamine 2,3-dioxygenase 2 (Ido2). Tdo2 or Ido1 enzymatic activity has been implicated in a number of actions of the kynurenine pathway, including immune evasion by tumors. IDO2 is expressed in several human pancreatic cancer cell lines, suggesting it also may play a role in tumorigenesis. Although Ido2 was originally suggested to be a target of the chemotherapeutic agent dextro-1-methyl-tryptophan, subsequent studies suggest this compound does not inhibit Ido2 activity. The development of selective Ido2 inhibitors could provide valuable tools for investigating its activity in tumor development and normal physiology. In this study, a library of Food and Drug Administration-approved drugs was screened for inhibition of mouse Ido2 enzymatic activity. A number of candidates were identified and IC₅₀ values of each compound for Ido1 and Ido2 were estimated. The Ido2 inhibitors were also tested for inhibition of Tdo2 activity. Our results showed that compounds from a class of drugs used to inhibit proton pumps were the most potent and selective Ido2 inhibitors identified in the library screen. These included tenatoprazole, which exhibited an IC₅₀ value of 1.8 μM for Ido2 with no inhibition of Ido1 or Tdo2 activity detected at a concentration of 100 μM tenatoprazole. These highly-selective Ido2 inhibitors will be useful for defining the distinct biological roles of the three Trp-catabolizing enzymes.     

Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice

Indoleamine 2,3-dioxygenase activity was found to be ubiquitously distributed in various tissues of mice, such as brain, lung, stomach, intestine, and epididymis. The highest enzyme activity was detected in the alimentary canal and the epididymis. Developmental and daily rhythmic changes of indoleamine 2,3-dioxygenase activity and the effects of various regulatory factors were studied with the supernatant fractions derived from the small intestine and the epididymis. The enzyme activity in these two tissues was absent during the first 2 weeks (the weaning period). From the third week, there was a rapid increase in activities and a maximum was reached when the mice were 8 to 10 weeks of age (adolescence). The enzyme activity in the small intestine then gradually diminished to zero level at 30 weeks of age (prime) or later, while that in the epididymis remained at the high level throughout 69 weeks of age (senescence). The enzyme activity of the small intestine from mice fed during the hours 9:00–13:00 showed daily rhythmic changes; high in the daytime and low at night. Under night feeding (21:00–1:00), the enzyme activity was high at night and low in the daytime. The epididymal enzyme activity showed no daily fluctuations by either feeding schedule. With regard to the developmental and daily rhythmic changes, indoleamine 2,3-dioxygenase activity in the small intestine was similar to that of hepatic tryptophan 2,3-dioxygenase. However, in contrast to the hepatic tryptophan 2,3-dioxygenase activity, indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis was not affected by adrenalectomy or intraperitoneal administration of adrenal steroid or tryptophan.     

Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8(+) dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.     

Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.     

Specific induction of indoleamine 2,3-dioxygenase by bacterial lipopolysaccharide in the mouse lung

Indoleamine 2,3-dioxygenase activity in the supernatant fractions (30,000g, 30 min) from various tissues of mice increased almost linearly after a single intraperitoneal administration of bacterial lipopolysaccharide (5 to 20 μg/mouse). The most prominent effect was observed in the lung, where both specific and total enzyme activities increased 40 to 80-fold during the first 24 h. Significant (10- to 20-fold) stimulation was also observed in the seminal vesicle, coagulating gland, colon, and caecum, and severalfold in the trachea, stomach, heart, small intestine, and spleen. Lipid A fraction, the biologically active unit in the lipopolysaccharide complex, was as active as the lipopolysaccharide preparations from either Escherichia coli or Salmonella S and R mutant strains, whereas the polysaccharide fraction was inactive under identical experimental conditions. When mice were pretreated with a series of daily injections of bacterial lipopolysaccharide, enzyme induction was no longer evident, indicating that tolerance to this agent had developed and that enzyme induction was caused by lipopolysaccharide but not by possible contaminants in the preparations. The enzyme activities from normal and lipopolysaccharide-treated mice were exclusively found in the soluble fractions of mouse lung homogenates. Other enzyme activities in the lung such as lysosomal (acid phosphatase), microsomal (prostaglandin cyclooxygenase), mitochondrial (monoamine oxidase and superoxide dismutase), and soluble enzyme activities (lipooxygenase and superoxide dismutase) were not significantly altered by this treatment. This increase in the enzyme activity with the lipopolysaccharide treatment was abolished with a simultaneous administration of cycloheximide or actinomycin D, and an immunological analysis with antibody for mouse enzyme (rabbit IgG) demonstrated that the observed increment of the enzyme activity was essentially due to an increase in the enzyme protein.