Osamu Takikawa

Lipopolysaccharide induction of indoleamine 2,3‐dioxygenase is mediated dominantly by an IFN‐γ‐independent mechanism

Indoleamine 2,3‐dioxygenase (IDO) is a rate‐limiting enzyme in the L‐tryptophan‐kynurenine pathway, which converts an essential amino acid, L‐tryptophan, to N‐formylkynurenine. It has been speculated that IFN‐γ is a dominant IDO inducer in vivo. The present study used IFN‐γ or TNF‐α gene‐disrupted mice and IFN‐γ antibody‐treated mice to demonstrate that lipopolysaccharide (LPS)‐induced systemic IDO is largely dependent on TNF‐α rather than IFN‐γ. IFN‐γ‐independent IDO induction was also demonstrated in vitro with LPS‐stimulated monocytic THP‐1 cells. These findings clearly indicate that there is an IFN‐γ‐independent mechanism of IDO induction in addition to the IFN‐γ‐dependent mechanism.

Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase in early concepti

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.

Nitric Oxide-Mediated Regulation of Gamma Interferon-Induced Bacteriostasis: Inhibition and Degradation of Human Indoleamine 2,3-Dioxygenase

ABSTRACT Tryptophan depletion resulting from indoleamine 2,3-dioxygenase (IDO) activity within the kynurenine pathway is one of the most prominent gamma interferon (IFN-γ)-inducible antimicrobial effector mechanisms in human cells. On the other hand, nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) serves a more immunoregulatory role in human cells and thereby interacts with tryptophan depletion in a number of ways. We investigated the effects of NO on IDO gene transcription, protein synthesis, and enzyme activity as well as on IDO-mediated bacteriostasis in the human epithelial cell line RT4. IFN-γ-stimulated RT4 cells were able to inhibit the growth of Staphylococcus aureus in an IDO-mediated fashion, and this bacteriostatic effect was abolished by endogenously produced NO. These findings were supported by experiments which showed that IDO activity in extracts of IFN-γ-stimulated cells is inhibited by the chemical NO donors diethylenetriamine diazeniumdiolate, S-nitroso-l-cysteine, and S-nitroso-N-acetyl-d,l-penicillamine. Furthermore, we found that both endogenous and exogenous NO strongly reduced the level of IDO protein content in RT4 cells. This effect was not due to a decrease in IDO gene transcription or mRNA stability. By using inhibitors of proteasomal proteolytic activity, we showed that NO production led to an accelerated degradation of IDO protein in the proteasome. This is the first report, to our knowledge, that demonstrates that the IDO is degraded by the proteasome and that NO has an effect on IDO protein stability.

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue

Abstract An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood–brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.

Interferon-Gamma-Dependent/Independent Expression of Indoleamine 2,3-Dioxygenase

The role of IFN-gamma in the expression of indoleamine 2,3-dioxygenase (IDO), a tryptophan oxidizing enzyme, in mouse tissues under physiological and pathological conditions was investigated using IFN-gamma-knockout mice. The results revealed that i) the expression of IDO in the large intestine or in the cecum is mediated by IFN-gamma, ii) for the systemic IDO induction under endotoxin shock, IFN-gamma is a dominant inducer but not essential, and an IFN-gamma-independent mechanism is also operative, iii) the systemic induction of IDO caused by IL-12 or Pokeweed mitogen is mediated by IFN-gamma, and iv) the constitutive IDO expression in the epididymis is IFN-gamma-independent.

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.

Inhibition of indoleamine 2,3‐dioxygenase in human macrophages inhibits interferon‐γ‐induced bacteriostasis but does not abrogate toxoplasmastasis

Induction of indoleamine 2,3‐dioxygenase (IDO) by IFN‐γ results in growth inhibition of Toxoplasma and Chlamydia spp. as well as tumor cells. This is caused by the degradation, and therefore depletion, of L‐tryptophan necessary for cell protein synthesis. Human macrophages stimulated with IFN‐γ express IDO and inhibit the growth of intracellular toxoplasma and chlamydia as well as that of extracellular bacteria such as group B streptococci. Here we describe experiments in which the L‐tryptophan analog, 6‐chloro‐DL‐tryptophan (CDLT) caused a dose‐dependent inhibition in the IFN‐γ‐induced IDO‐mediated L‐tryptophan degradation in monocyte‐derived macrophages and glioblastoma cells. An inhibition of IDO activity of up to 80u2009% was observed at concentrations of CDLT of 750 μM. Expression of IDO at this concentration, as shown by Northern blot analysis, was unimpaired. This inhibition of IDO was coupled in glioblastoma cells by a complete abrogation of the IFN‐γ‐induced toxoplasmastasis in these cells. IDO inhibition by CDLT in human macrophages resulted in a complete abrogation of the IFN‐γ‐induced growth inhibition of streptococci and staphylococci. In contrast to this, IFN‐γ‐induced toxoplasmastasis was not inhibited in human macrophages by CDLT‐mediated IDO inhibition.

GROWTH INHIBITION OF MULTIRESISTANT ENTEROCOCCI BY INTERFERON-GAMMAACTIVATED HUMAN URO-EPITHELIAL CELLS

Nosocomial infections with enterococci are an increasing problem in modern medical practice due to the development of resistance to a wide range of antibiotics, including the glycopeptides vancomycin and teicoplanin. An increasing number of vancomycin-resistant enterococci (VRE) have been cultured from clinical specimens -- especially from patients undergoing immunosuppressive therapy -- and bacteraemia caused by these VRE, subsequent to colonisation of epithelial surfaces, is a significant cause of mortality in such patients. Recent evidence showed that the induction of indoleamine 2,3 dioxygenase (IDO) by interferon-gamma (IFN-gamma) inhibited growth of group B streptococci by depleting the essential amino acid L-tryptophan. This study describes the IFN-gamma-induced expression of IDO -- shown at a transcriptional level by Northern blot analysis, at translational level by Western blot and also at a functional level by L-tryptophan degradation to L-kynurenine -- in the uro-epithelial cell line RT4. The depletion of L-tryptophan resulted in growth inhibition of enterococci, and this was confirmed by abrogation of the inhibitory effect by re-supplementation with excess L-tryptophan. Multiresistant enterococci, including vancomycin-resistant strains resistant to all commercially available antibiotics, were inhibited by the IFN-gamma-induced expression of IDO and subsequent L-tryptophan degradation. This may be an important mechanism in the local restriction of colonisation of the urinary tract by endogenous enterococci and in inhibiting the spread of the bacteria beyond the epithelial barrier.

Age-Related Nuclear Cataract and Indoleamine 2,3-Dioxygenase-Initiated Tryptophan Metabolism in the Human Lens

Tryptophan-derived UV filters (kynurenine and 3-hydroxylkynurenine glucoside) have recently been shown to bind to human lens proteins. These UV filter adducts increase in amount with age and appear to be mainly responsible for the yellowing of the lens in man. On the basis of research performed in other tissues, it has been assumed that indoleamine 2,3-dioxygenase (IDO) may be the first and probably rate-limiting enzyme in UV filter biosynthesis. In this study, 25 human lenses were examined by a reliable and sensitive assay method with a monoclonal antibody specific for IDO. IDO activity was detected in all lenses ranging from 26 to 80 years, and there was no clear relationship of IDO activity with age. The mean activity was 0.85 + 0.49 nmol of kynurenine/h/lens. The level in the iris/ciliary body was negligible (<0.05 nmol of kynurenine/h). The lens IDO activity is consistent with UV filter turnover values obtained previously. These findings indicate that IDO is the first enzyme in the UV filter pathway and that UV filter biosynthesis is active even in aged lenses. Yellowing of the aged lens may therefore be preventable by drug-induced suppression of IDO activity.

Regulation of indoleamine 2,3-dioxygenase, the first enzyme in UV filter biosynthesis in the human lens. Relevance for senile nuclear cataract.

3-Hydroxykynurenine (3OHKyn), the precursor of UV filters in human lens, is highly autooxidizable, generates H2O2, and binds to lens proteins, yielding a tanned/yellow product resembling senile nuclear cataractous materials. Thus, if 3OHkyn can be shown to be the causative agent in cataract, it may be possible to prevent the disease by lowering the level of 3OHKyn. To this end, indoleamine 2,3-dioxygenase, the first enzyme in UV filter synthesis, was studied using lens epithelial cell lines. The results indicated that the IDO expression is mediated by IFN-gamma. Immuno-suppressants which inhibit production of IFN-gamma may act as anti-cataract agents. Another way to lower the level of 3OHKyn is to use specific inhibitors for IDO. A recombinant human IDO was expressed to develop the inhibitors.