Abstract
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.
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Acknowledgements
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.
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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)
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Jackman, K.A., Brait, V.H., Wang, Y. et al. Vascular expression, activity and function of indoleamine 2,3-dioxygenase-1 following cerebral ischaemia–reperfusion in mice. Naunyn-Schmiedeberg's Arch Pharmacol 383, 471–481 (2011). https://doi.org/10.1007/s00210-011-0611-4
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DOI: https://doi.org/10.1007/s00210-011-0611-4