Abstract
The kynurenine pathway is a fundamental mechanism of immunosuppression and peripheral tolerance. It is increasingly recognized as playing a major role in the pathogenesis of a wide variety of inflammatory, neurodegenerative and malignant disorders. However, the temporal dynamics of kynurenine pathway activation and metabolite production in human immune cells is currently unknown. Here we report the novel use of flow cytometry, combined with ultra high-performance liquid chromatography and gas chromatography-mass spectrometry, to sensitively quantify the intracellular expression of three key kynurenine pathway enzymes and the main kynurenine pathway metabolites in a time-course study. This is the first study to show that up-regulation of indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monoxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) is lacking in lymphocytes treated with interferon gamma. In contrast, peripheral monocytes showed a significant elevation of kynurenine pathway enzymes and metabolites when treated with interferon gamma. Expression of IDO-1, KMO and QPRT correlated significantly with activation of the kynurenine pathway (kynurenine:tryptophan ratio), quinolinic acid concentration and production of the monocyte derived, pro-inflammatory immune response marker: neopterin. Our results also describe an original and sensitive methodological approach to quantify kynurenine pathway enzyme expression in cells. This has revealed further insights into the potential role of these enzymes in disease processes.
Highlights
The kynurenine pathway (KP) is the major route for the catabolism of the essential amino acid tryptophan, which leads to production of several potent neuroactive and immunomodulatoryPLOS ONE | DOI:10.1371/journal.pone.0131389 June 26, 2015Kynurenine Pathway in Lymphocytes and Monocytes the Ultra High Performance Liquid Chromatography (UHPLC) and Gas Chromatography-Mass Spectrometry (GC-MS) equipment used in the study
This is the first study to show that up-regulation of indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monoxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) is lacking in lymphocytes treated with interferon gamma
We consistently observed no significant increase in the expression of IDO-1, KMO or QPRT in lymphocyte populations stimulated with IFN-γ at all doses tested
Summary
The kynurenine pathway (KP) is the major route for the catabolism of the essential amino acid tryptophan, which leads to production of several potent neuroactive and immunomodulatory. IDO-1 has been shown to exert potent immunosuppressive effects in a range of contexts These include the prevention of foetal rejection during pregnancy [24], suppression of autoimmune disease [25, 26] and expression in tumours to evade immune attack [27]. Doi:10.1371/journal.pone.0131389.g001 interleukin-4 (IL-4) was found to inhibit the induction of IDO-1 mRNA and enzymatic activity in human monocytes [47] Despite these early studies, and the potential clinical relevance of KP activation in immune cells, the characterization of KP enzyme expression and metabolite production remains poorly defined in lymphocytes and monocytes. This has important implications for cellular damage or protection if at different time-points distinct arms of the KP predominate This is given further significance in light of recent findings that some KP products antagonize each other, along with the potential toxic effects of sustained KP activation. We describe here for the first time the expression of IDO-1, KMO and QPRT in human peripheral blood mononuclear cells (PBMCs) untreated (control) or treated with varying doses of IFN-γ over different time-points
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