Vascular expression, activity and function of indoleamine 2,3-dioxygenase-1 following cerebral ischaemia–reperfusion in mice

  • Katherine A. Jackman
  • Vanessa H. Brait
  • Yutang Wang
  • Ghassan J. Maghzal
  • Helen J. Ball
  • Gavin Mckenzie
  • T. Michael De Silva
  • Roland Stocker
  • Christopher G. Sobey


Indoleamine 2,3-dioxygenases-1 (Ido1) and -2 initiate the kynurenine pathway of tryptophan metabolism. In addition to the established immune regulatory effects of Ido1 and the ability of nitric oxide to regulate Ido1 activity, it is now also known that Ido1-mediated metabolism of tryptophan to kynurenine can modulate vascular tone. Ido activity is reportedly elevated in stroke patients and correlates with increased risk of death. Thus, the present goals were to test whether, following cerebral ischaemia, Ido activity and cerebrovascular Ido1 expression are altered and whether expression of Ido1 contributes to stroke outcome. Transient cerebral ischaemia was induced in wild-type and Ido1 gene-deficient (Ido1 −/−) mice. Mice were pre-treated with vehicle, the Ido1 inhibitor, 1-methyl-D-tryptophan (1-MT; 50 mg/kg i.p.) or the inducible nitric oxide synthase (Nos2) inhibitor, aminoguanidine (AG, 100 mg/kg i.p.). At 24 h, neurological function, brain infarct size and swelling were assessed. In addition, Ido activity was estimated by plasma kynurenine and tryptophan, and Ido1 expression was examined in cerebral arterioles. Cerebral ischaemia–reperfusion in wild-type mice increased Ido activity and its expression in cerebral arterioles. Ido1 −/− and 1-MT-treated wild-type mice had lower Ido activity but similar post-stroke neurological function and similar total brain infarct volume and swelling, relative to control mice. Inhibition of Nos2 with AG also did not affect Ido activity or outcome following stroke. This study provides molecular and pharmacological evidence that the expression and the activity of Ido1 increase following stroke. However, such Ido1 expression does not appear to affect overall outcome following acute ischaemic stroke, and furthermore, a regulatory role of Nos2-derived nitric oxide on Ido activity following cerebral ischaemia–reperfusion appears unlikely.


Aminoguanidine Indoleamine 2,3-dioxygenase Kynurenine Stroke Tryptophan Vascular 



This study was supported by funds from a project grant from the National Health and Medical Research Council of Australia (NHMRC; ID 570861). KAJ was supported by an NHMRC Dora Lush Biomedical Research Scholarship and VHB was supported by a Monash Graduate Scholarship. TMD was supported by an Australian Postgraduate Award and RS was supported by a NHMRC Senior Principal Research Fellowship, a University of Sydney Professorial Fellowship, and the University of Sydney Medical Foundation. CGS is a Senior Research Fellow of the NHMRC.

Supplementary material

210_2011_611_MOESM1_ESM.ppt (342 kb)
Supplementary Fig. 1 The Ido1 antibody from transgenic does not detect mouse Ido2. Lysate was prepared from untransfected HEK 293T cells and HEK 293T cells transfected with either pDEST26-mouse Ido1 or pDEST26-mouse Ido2. Both constructs express Ido proteins with an N-terminal 6× histidine (His) tag. Protein lysate (10 μL) was loaded in triplicate on a 10% denaturing polyacrylamide gel as follows: M—protein ladder (Pageruler, Fermentas), 1—untransfected cell lysate, 2—Ido1-transfected cell lysate and 3—Ido2-transfected cell lysate. Wells were left empty between the cell lysates to prevent contamination. Protein was transferred to Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences) that was then divided into three for the antibody hybridisation. Details of expression constructs, lysate preparation, generation of Ido2 antibody and electrophoresis and transfer conditions can be found in Ball et al. (2007). The membranes were blocked for 30 min in Odyssey Blocking buffer (OBB) (Licor) and the primary incubations were performed overnight in OBB + 0.1% Tween 20 as outlined below. a The membrane was simultaneously incubated with the Ido1 antibody used elsewhere in this study (5 μg/mL, transgenic) and an anti-His antibody (2 μg/mL, Invitrogen). b The membrane was simultaneously incubated with a mouse Ido2-specific antibody (1 μg/mL) and the anti-His antibody. c The membrane was incubated with an anti-β-tubulin antibody (1 μg/mL, Cell Signalling Technology). Membranes were washed five times in PBS + 0.1% Tween 20. This was followed by a 1-h incubation with goat anti-mouse IR680 CW antibody and donkey anti-rabbit IR800 CW antibody (LiCor) in OBB + 0.1% Tween 20 + 0.01% SDS. After washing, the membranes were scanned on an Odyssey InfraRed imaging system (LiCor). The anti-His antibody was generated in mice whilst the other antibodies were generated in rabbits. Therefore, the Ido and tubulin proteins are visible in the 800-nM channel and the His-tagged proteins are detected in the 700-nM channel. The molecular weight markers are also visible in the 700-nM channel. His-tagged proteins of the predicted size for mouse Ido1 and mouse Ido2 were detected in the transfected cell lysates (a and b, lower images, lanes 2 and 3, respectively). The two His-tagged proteins were expressed at similar levels in the lysates. Only the His-tagged protein from the Ido1-transfected cells was detected with the transgenic Ido1 antibody (a, upper image, lane 2) indicating that this antibody does not cross-react with mouse Ido2. Similarly, an Ido2 antibody only detected the His-tagged protein in the lysate from mouse Ido2-transfected cells (b, upper image, lane 3). The tubulin expression indicates total protein levels were consistent between the lysate preparations (c, lanes 1–3) (PPT 342 kb)


  1. Andre C, O’Connor JC, Kelley KW, Lestage J, Dantzer R, Castanon N (2008) Spatio-temporal differences in the profile of murine brain expression of proinflammatory cytokines and indoleamine 2, 3-dioxygenase in response to peripheral lipopolysaccharide administration. J Neuroimmunol 200:90–99PubMedCrossRefGoogle Scholar
  2. Austin CJ, Mailu BM, Maghzal GJ, Sanchez-Perez A, Rahlfs S, Zocher K, Yuasa HJ, Arthur JW, Becker K, Stocker R, Hunt NH, Ball HJ (2010) Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2, 3-dioxygenase-2. Amino Acids 39:565–578PubMedCrossRefGoogle Scholar
  3. Ball HJ, Sanchez-Perez A, Weiser S, Austin CJD, Astelbauer F, Miu J, McQuillan JA, Stocker R, Jermiin LS, Hunt NH (2007) Characterization of an indoleamine 2, 3-dioxygenase-like protein found in humans and mice. Gene 396:203–213PubMedCrossRefGoogle Scholar
  4. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472–476PubMedGoogle Scholar
  5. Brait VH, Jackman KA, Walduck A, Selemidis S, Diep H, Mast AE, Broughton BRS, Drummond GR, Sobey CG (2010) Mechanisms contributing to cerebral infarct size after stroke—gender, reperfusion, T lymphocytes and Nox2-derived superoxide. J Cereb Blood Flow Metab 30:1306–1317PubMedCrossRefGoogle Scholar
  6. Darlington LG, Mackay GM, Forrest CM, Stoy N, George C, Stone TW (2007) Altered kynurenine metabolism correlates with infarct volume in stroke. Eur J Neurosci 26:2211–2221PubMedCrossRefGoogle Scholar
  7. Eastman CL, Guilarte TR (1989) Cytotoxicity of 3-hydroxykynurenine in a neuronal hybrid cell line. Brain Res 495:225–231PubMedCrossRefGoogle Scholar
  8. Freitag S, Schachner M, Morellini F (2003) Behavioral alterations in mice deficient for the extracellular matrix glycoprotein tenascin-R. Behav Brain Res 145:189–207PubMedCrossRefGoogle Scholar
  9. Gigler G, Szenasi G, Simo A, Levay G, Harsing LG Jr, Sas K, Vecsei L, Toldi J (2007) Neuroprotective effect of l-kynurenine sulfate administered before focal cerebral ischemia in mice and global cerebral ischemia in gerbils. Eur J Pharmacol 564:116–122PubMedCrossRefGoogle Scholar
  10. Guillemin GJ, Kerr SJ, Smythe GA, Smith DG, Kapoor V, Armati PJ, Croitoru J, Brew BJ (2001) Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection. J Neurochem 78:842–853PubMedCrossRefGoogle Scholar
  11. Guillemin GJ, Smythe G, Takikawa O, Brew BJ (2005) Expression of indoleamine 2, 3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons. Glia 49:15–23PubMedCrossRefGoogle Scholar
  12. Guillemin GJ, Cullen KM, Lim CK, Smythe GA, Garner B, Kapoor V, Takikawa O, Brew BJ (2007) Characterization of the kynurenine pathway in human neurons. J Neurosci 27:12884–12892PubMedCrossRefGoogle Scholar
  13. Haber R, Bessette D, Hulihan-Giblin B, Durcan MJ, Goldman D (1993) Identification of tryptophan 2, 3-dioxygenase RNA in rodent brain. J Neurochem 60:1159–1162PubMedCrossRefGoogle Scholar
  14. Hansen AM, Driussi C, Turner V, Takikawa O, Hunt NH (2000) Tissue distribution of indoleamine 2, 3-dioxygenase in normal and malaria-infected tissue. Redox Rep 5:112–115PubMedCrossRefGoogle Scholar
  15. Hansen AM, Ball HJ, Mitchell AJ, Miu J, Takikawa O, Hunt NH (2004) Increased expression of indoleamine 2, 3-dioxygenase in murine malaria infection is predominantly localised to the vascular endothelium. Int J Parasitol 34:1309–1319PubMedCrossRefGoogle Scholar
  16. Hirata F, Hayaishi O (1975) Studies on indoleamine 2, 3-dioxygenase. I. Superoxide anion as substrate. J Biol Chem 250:5960–5966PubMedGoogle Scholar
  17. Hoshi M, Saito K, Murakami Y, Taguchi A, Fujigaki H, Tanaka R, Takemura M, Ito H, Hara A, Seishima M (2009) Marked increases in hippocampal neuron indoleamine 2, 3-dioxygenase via IFN-gamma-independent pathway following transient global ischemia in mouse. Neurosci Res 63:194–198PubMedCrossRefGoogle Scholar
  18. Hou DY, Muller AJ, Sharma MD, DuHadaway J, Banerjee T, Johnson M, Mellor AL, Prendergast GC, Munn DH (2007) Inhibition of indoleamine 2, 3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses. Cancer Res 67:792–801PubMedCrossRefGoogle Scholar
  19. Iadecola C, Zhang F, Casey R, Nagayama M, Ross ME (1997) Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. J Neurosci 17:9157–9164PubMedGoogle Scholar
  20. Ikegami S, Harada A, Hirokawa N (2000) Muscle weakness, hyperactivity, and impairment in fear conditioning in tau-deficient mice. Neurosci Lett 279:129–132PubMedCrossRefGoogle Scholar
  21. Jackman KA, Miller AA, De Silva TM, Crack PJ, Drummond GR, Sobey CG (2009) Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice. Brit J Pharmacol 156:680–688CrossRefGoogle Scholar
  22. Kanai M, Nakamura T, Funakoshi H (2009) Identification and characterization of novel variants of the tryptophan 2, 3-dioxygenase gene: differential regulation in the mouse nervous system during development. Neurosci Res 64:111–117PubMedCrossRefGoogle Scholar
  23. Maghzal GJ, Thomas SR, Hunt NH, Stocker R (2008) Cytochrome b5, not superoxide anion radical, is a major reductant of indoleamine 2, 3-dioxygenase in human cells. J Biol Chem 283:12014–12025PubMedCrossRefGoogle Scholar
  24. Mellor AL, Baban B, Chandler P, Marshall B, Jhaver K, Hansen A, Koni PA, Iwashima M, Munn DH (2003) Cutting edge: induced indoleamine 2, 3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J Immunol 171:1652–1655PubMedGoogle Scholar
  25. Metz R, Duhadaway JB, Kamasani U, Laury-Kleintop L, Muller AJ, Prendergast GC (2007) Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indoleamine 2, 3-dioxygenase inhibitory compound d-1-methyl-tryptophan. Cancer Res 67:7082–7087PubMedCrossRefGoogle Scholar
  26. Miller AA, Dusting GJ, Roulston CL, Sobey CG (2006a) NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke. Brain Res 1111:111–116PubMedCrossRefGoogle Scholar
  27. Miller CL, Llenos IC, Dulay JR, Weiss S (2006b) Upregulation of the initiating step of the kynurenine pathway in postmortem anterior cingulate cortex from individuals with schizophrenia and bipolar disorder. Brain Res 1073–1074:25–37PubMedCrossRefGoogle Scholar
  28. Minatogawa Y, Suzuki S, Ando Y, Tone S, Takikawa O (2003) Tryptophan pyrrole ring cleavage enzymes in placenta. Adv Exp Med Biol 527:425–434PubMedGoogle Scholar
  29. Ohnishi T, Hirata F, Hayaish O (1977) Indoleamine 2, 3-dioxygenase. Potassium superoxide as substrate. J Biol Chem 252:4643–4647PubMedGoogle Scholar
  30. Osada T, Ikegami S, Takiguchi-Hayashi K, Yamazaki Y, Katoh-Fukui Y, Higashinakagawa T, Sakaki Y, Takeuchi T (1999) Increased anxiety and impaired pain response in puromycin-sensitive aminopeptidase gene-deficient mice obtained by a mouse gene-trap method. J Neurosci 19:6068–6078PubMedGoogle Scholar
  31. Robinson CM, Shirey KA, Carlin JM (2003) Synergistic transcriptional activation of indoleamine dioxygenase by IFN-gamma and tumor necrosis factor-alpha. J Interferon Cytokine Res 23:413–421PubMedCrossRefGoogle Scholar
  32. Robinson CM, Hale PT, Carlin JM (2005) The role of IFN-gamma and TNF-alpha-responsive regulatory elements in the synergistic induction of indoleamine dioxygenase. J Interferon Cytokine Res 25:20–30PubMedCrossRefGoogle Scholar
  33. Robotka H, Sas K, Agoston M, Rozsa E, Szenasi G, Gigler G, Vecsei L, Toldi J (2008) Neuroprotection achieved in the ischaemic rat cortex with l-kynurenine sulphate. Life Sci 82:915–919PubMedCrossRefGoogle Scholar
  34. Roy EJ, Takikawa O, Kranz DM, Brown AR, Thomas DL (2005) Neuronal localization of indoleamine 2, 3-dioxygenase in mice. Neurosci Lett 387:95–99PubMedCrossRefGoogle Scholar
  35. Saito K, Nowak TS Jr, Markey SP, Heyes MP (1993a) Mechanism of delayed increases in kynurenine pathway metabolism in damaged brain regions following transient cerebral ischemia. J Neurochem 60:180–192PubMedCrossRefGoogle Scholar
  36. Saito K, Nowak TS Jr, Suyama K, Quearry BJ, Saito M, Crowley JS, Markey SP, Heyes MP (1993b) Kynurenine pathway enzymes in brain: responses to ischemic brain injury versus systemic immune activation. J Neurochem 61:2061–2070PubMedCrossRefGoogle Scholar
  37. Sas K, Csete K, Vecsei L, Papp JG (2003) Effect of systemic administration of l-kynurenine on corticocerebral blood flow under normal and ischemic conditions of the brain in conscious rabbits. J Cardiovasc Pharmacol 42:403–409PubMedCrossRefGoogle Scholar
  38. Sas K, Robotka H, Rozsa E, Agoston M, Szenasi G, Gigler G, Marosi M, Kis Z, Farkas T, Vecsei L, Toldi J (2008) Kynurenine diminishes the ischemia-induced histological and electrophysiological deficits in the rat hippocampus. Neurobiol Dis 32:302–308PubMedCrossRefGoogle Scholar
  39. Schwarcz R, Kohler C (1983) Differential vulnerability of central neurons of the rat to quinolinic acid. Neurosci Lett 38:85–90PubMedCrossRefGoogle Scholar
  40. Stone TW (1993) Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev 45:309–379PubMedGoogle Scholar
  41. Stone TW, Perkins MN (1981) Quinolinic acid: a potent endogenous excitant at amino acid receptors in CNS. Eur J Pharmacol 72:411–412PubMedCrossRefGoogle Scholar
  42. Sugimoto K, Iadecola C (2002) Effects of aminoguanidine on cerebral ischemia in mice: comparison between mice with and without inducible nitric oxide synthase gene. Neurosci Lett 331:25–28PubMedCrossRefGoogle Scholar
  43. Taguchi A, Hara A, Saito K, Hoshi M, Niwa M, Seishima M, Mori H (2008) Localization and spatiotemporal expression of Ido following transient forebrain ischemia in gerbils. Brain Res 1217:78–85PubMedCrossRefGoogle Scholar
  44. Taniguchi T, Sono M, Hirata F, Hayaishi O, Tamura M, Hayashi K, Iizuka T, Ishimura Y (1979) Indoleamine 2, 3-dioxygenase. Kinetic studies on the binding of superoxide anion and molecular oxygen to enzyme. J Biol Chem 254:3288–3294PubMedGoogle Scholar
  45. Tsuchiya D, Hong S, Kayama T, Panter SS, Weinstein PR (2003) Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice. Brain Res 970:131–139PubMedCrossRefGoogle Scholar
  46. Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M, Changsirivathanathamrong D, Wu BJ, Ball HJ, Thomas SR, Kapoor V, Celermajer DS, Mellor AL, Keaney JF Jr, Hunt NH, Stocker R (2010) Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med 16:279–285PubMedCrossRefGoogle Scholar
  47. Xia CF, Smith RS Jr, Shen B, Yang ZR, Borlongan CV, Chao L, Chao J (2006) Postischemic brain injury is exacerbated in mice lacking the kinin B2 receptor. Hypertension 47:752–761PubMedCrossRefGoogle Scholar
  48. Zhang F, Iadecola C (1998) Temporal characteristics of the protective effect of aminoguanidine on cerebral ischemic damage. Brain Res 802:104–110PubMedCrossRefGoogle Scholar
  49. Zhao X, Ross ME, Iadecola C (2003) l-Arginine increases ischemic injury in wild-type mice but not in iNOS-deficient mice. Brain Res 966:308–311PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Katherine A. Jackman
    • 1
  • Vanessa H. Brait
    • 1
  • Yutang Wang
    • 2
  • Ghassan J. Maghzal
    • 2
  • Helen J. Ball
    • 3
  • Gavin Mckenzie
    • 4
  • T. Michael De Silva
    • 1
  • Roland Stocker
    • 2
  • Christopher G. Sobey
    • 1
  1. 1.Vascular Biology and Immunology Group, Department of PharmacologyMonash UniversityClaytonAustralia
  2. 2.Centre for Vascular Research, School of Medical Sciences (Pathology) and Bosch Institute, Sydney Medical SchoolUniversity of SydneySydneyAustralia
  3. 3.School of Medical Sciences (Pathology) and Bosch InstituteSydney Medical School, University of SydneySydneyAustralia
  4. 4.School of Medical SciencesUniversity of New South WalesSydneyAustralia

Personalised recommendations