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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

Expression of IDO-1, KMO and QPRT in lymphocytes and monocytes treated with IFN-γ.Flow cytometry histogram plots are based on forward scatter (FSC) and side scatter (SSC) gating for lymphocyte and monocyte populations. This was validated using CD14 and CD3 staining in some experiments. A representative example of the gating strategy is shown in (A). For histogram plots the mean fluorescence intensity (MFI) of expression for IDO-1, KMO and QPRT is represented on the x axis and normalized event numbers on the y axis at 24 hours (B), 48 hours (C) and 72 hours (D) of IFN-γ treatment. Untreated (control) cells are shown as the filled distribution, 50 IU/mL IFN-γ solid lines, 100 IU/mL IFN-γ dotted lines and 500 IU/mL IFN-γ dashed lines. Data is representative of at least 5 independent experiments.
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pone.0131389.g002: Expression of IDO-1, KMO and QPRT in lymphocytes and monocytes treated with IFN-γ.Flow cytometry histogram plots are based on forward scatter (FSC) and side scatter (SSC) gating for lymphocyte and monocyte populations. This was validated using CD14 and CD3 staining in some experiments. A representative example of the gating strategy is shown in (A). For histogram plots the mean fluorescence intensity (MFI) of expression for IDO-1, KMO and QPRT is represented on the x axis and normalized event numbers on the y axis at 24 hours (B), 48 hours (C) and 72 hours (D) of IFN-γ treatment. Untreated (control) cells are shown as the filled distribution, 50 IU/mL IFN-γ solid lines, 100 IU/mL IFN-γ dotted lines and 500 IU/mL IFN-γ dashed lines. Data is representative of at least 5 independent experiments.

Mentions: Following fixation, PBMCs were washed in buffer containing 0.1% Tween20 (Sigma-Aldrich) to permeabilize the cell membrane and stained for 30 minutes in the dark at 4°C with Alexa Fluor 488 conjugated anti-human IDO-1, anti-human KMO or anti-human QPRT and the corresponding isotype control and secondary antibodies (Table 1). Gating for lymphocyte and monocyte populations was based on forward scatter and side scatter profiles. This was validated by surface staining cells with anti-CD3 and anti-CD14 antibodies in some experiments (Fig 2A). Following gating on these populations IDO-1, KMO and QPRT expression was visualized with histogram plots using FlowJo software (vX.0.6 Treestar). Geometric mean fluorescence intensity (gMFI) was computed for untreated lymphocytes/monocytes and IFN-γ (500 IU/mL) treated lymphocytes/monocytes. These values were corrected, in each independent experiment, for background fluorescence by subtracting the gMFI of cells treated with isotype control or secondary antibodies alone from the untreated and treated expression values.


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)

Expression of IDO-1, KMO and QPRT in lymphocytes and monocytes treated with IFN-γ.Flow cytometry histogram plots are based on forward scatter (FSC) and side scatter (SSC) gating for lymphocyte and monocyte populations. This was validated using CD14 and CD3 staining in some experiments. A representative example of the gating strategy is shown in (A). For histogram plots the mean fluorescence intensity (MFI) of expression for IDO-1, KMO and QPRT is represented on the x axis and normalized event numbers on the y axis at 24 hours (B), 48 hours (C) and 72 hours (D) of IFN-γ treatment. Untreated (control) cells are shown as the filled distribution, 50 IU/mL IFN-γ solid lines, 100 IU/mL IFN-γ dotted lines and 500 IU/mL IFN-γ dashed lines. Data is representative of at least 5 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131389.g002: Expression of IDO-1, KMO and QPRT in lymphocytes and monocytes treated with IFN-γ.Flow cytometry histogram plots are based on forward scatter (FSC) and side scatter (SSC) gating for lymphocyte and monocyte populations. This was validated using CD14 and CD3 staining in some experiments. A representative example of the gating strategy is shown in (A). For histogram plots the mean fluorescence intensity (MFI) of expression for IDO-1, KMO and QPRT is represented on the x axis and normalized event numbers on the y axis at 24 hours (B), 48 hours (C) and 72 hours (D) of IFN-γ treatment. Untreated (control) cells are shown as the filled distribution, 50 IU/mL IFN-γ solid lines, 100 IU/mL IFN-γ dotted lines and 500 IU/mL IFN-γ dashed lines. Data is representative of at least 5 independent experiments.
Mentions: Following fixation, PBMCs were washed in buffer containing 0.1% Tween20 (Sigma-Aldrich) to permeabilize the cell membrane and stained for 30 minutes in the dark at 4°C with Alexa Fluor 488 conjugated anti-human IDO-1, anti-human KMO or anti-human QPRT and the corresponding isotype control and secondary antibodies (Table 1). Gating for lymphocyte and monocyte populations was based on forward scatter and side scatter profiles. This was validated by surface staining cells with anti-CD3 and anti-CD14 antibodies in some experiments (Fig 2A). Following gating on these populations IDO-1, KMO and QPRT expression was visualized with histogram plots using FlowJo software (vX.0.6 Treestar). Geometric mean fluorescence intensity (gMFI) was computed for untreated lymphocytes/monocytes and IFN-γ (500 IU/mL) treated lymphocytes/monocytes. These values were corrected, in each independent experiment, for background fluorescence by subtracting the gMFI of cells treated with isotype control or secondary antibodies alone from the untreated and treated expression values.

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