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Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease.

Jones SP, Franco NF, Varney B, Sundaram G, Brown DA, de Bie J, Lim CK, Guillemin GJ, Brew BJ - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Peter Duncan Neurosciences Research Unit, St Vincent's Centre for Applied Medical Research, Sydney, Australia; St Vincent's Clinical School, Faculty of Medicine, UNSW, Sydney, Australia.

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.

No MeSH data available.


Related in: MedlinePlus

Geometric mean fluorescence intensity (gMFI) for IDO-1, KMO and QPRT expression.A summary of gMFI expression for the three KP enzymes in lymphocytes (A-C) and monocytes (D-F). Data is from 5 different biological samples with time-point (24, 48 and 72 hours) shown on the x axis. Black bars represent control experiments (untreated) and grey bars represent treated (500 IU/mL IFN-γ). The y axis scale for gMFI has been normalized to improve comparison of expression for IDO-1, KMO and QPRT between lymphocytes (upper row) and monocytes (lower row). Data is presented as mean±SEM, ns = no significant difference, *p<0.01, **p<0.001.
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pone.0131389.g003: Geometric mean fluorescence intensity (gMFI) for IDO-1, KMO and QPRT expression.A summary of gMFI expression for the three KP enzymes in lymphocytes (A-C) and monocytes (D-F). Data is from 5 different biological samples with time-point (24, 48 and 72 hours) shown on the x axis. Black bars represent control experiments (untreated) and grey bars represent treated (500 IU/mL IFN-γ). The y axis scale for gMFI has been normalized to improve comparison of expression for IDO-1, KMO and QPRT between lymphocytes (upper row) and monocytes (lower row). Data is presented as mean±SEM, ns = no significant difference, *p<0.01, **p<0.001.

Mentions: We validated our forward scatter and side scatter gating strategy using CD14 and CD3 staining in some experiments (Fig 2A). The monocyte gate was consistently 80–85% CD14+ and negative for CD3. The lymphocyte gate 60–70% CD3+ and negative for CD14 (Fig 2A). Furthermore, we saw no significant differences in the expression of KP enzymes when comparing lymphocyte gate to CD3+ cells and when comparing our monocyte gate to CD14+ cells (data not shown). 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. This is shown in the representative flow cytometry histograms for the lymphocyte gate in Fig 2B–2D, as well as the graphs depicting the lymphocyte gate gMFI values for 5 independent biological samples (Fig 3).


Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease.

Jones SP, Franco NF, Varney B, Sundaram G, Brown DA, de Bie J, Lim CK, Guillemin GJ, Brew BJ - PLoS ONE (2015)

Geometric mean fluorescence intensity (gMFI) for IDO-1, KMO and QPRT expression.A summary of gMFI expression for the three KP enzymes in lymphocytes (A-C) and monocytes (D-F). Data is from 5 different biological samples with time-point (24, 48 and 72 hours) shown on the x axis. Black bars represent control experiments (untreated) and grey bars represent treated (500 IU/mL IFN-γ). The y axis scale for gMFI has been normalized to improve comparison of expression for IDO-1, KMO and QPRT between lymphocytes (upper row) and monocytes (lower row). Data is presented as mean±SEM, ns = no significant difference, *p<0.01, **p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482723&req=5

pone.0131389.g003: Geometric mean fluorescence intensity (gMFI) for IDO-1, KMO and QPRT expression.A summary of gMFI expression for the three KP enzymes in lymphocytes (A-C) and monocytes (D-F). Data is from 5 different biological samples with time-point (24, 48 and 72 hours) shown on the x axis. Black bars represent control experiments (untreated) and grey bars represent treated (500 IU/mL IFN-γ). The y axis scale for gMFI has been normalized to improve comparison of expression for IDO-1, KMO and QPRT between lymphocytes (upper row) and monocytes (lower row). Data is presented as mean±SEM, ns = no significant difference, *p<0.01, **p<0.001.
Mentions: We validated our forward scatter and side scatter gating strategy using CD14 and CD3 staining in some experiments (Fig 2A). The monocyte gate was consistently 80–85% CD14+ and negative for CD3. The lymphocyte gate 60–70% CD3+ and negative for CD14 (Fig 2A). Furthermore, we saw no significant differences in the expression of KP enzymes when comparing lymphocyte gate to CD3+ cells and when comparing our monocyte gate to CD14+ cells (data not shown). 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. This is shown in the representative flow cytometry histograms for the lymphocyte gate in Fig 2B–2D, as well as the graphs depicting the lymphocyte gate gMFI values for 5 independent biological samples (Fig 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Peter Duncan Neurosciences Research Unit, St Vincent's Centre for Applied Medical Research, Sydney, Australia; St Vincent's Clinical School, Faculty of Medicine, UNSW, Sydney, Australia.

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.

No MeSH data available.


Related in: MedlinePlus