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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

Concentrations of KP metabolites in the supernatants of IFN-γ activated PBMCs.Levels of KP metabolites: tryptophan (TRP) and kynurenine (KYN)-shown as TRP:KYN ratio (A); 3-hydroxykynurenine (3-HK) (B); 3-hydroxyanthranillic acid (3-HAA) (C); anthranilic acid (AA) (D); quinolinic acid (QUIN) (E); picolinic acid (PIC) (F) and the pro-inflammatory marker: neopterin (G) were measured by UHPLC or GC-MS (QUIN and PIC only) in the supernatants of PBMCs untreated (control-black bars) or treated with IFN-γ (500 IU/mL-grey bars) at 24, 48 and 72 hours of culture. Values represent the mean±SEM of 4 independent biological repeats (ns = no significant difference, *p<0.05, **p<0.01, **p<0.001).
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pone.0131389.g004: Concentrations of KP metabolites in the supernatants of IFN-γ activated PBMCs.Levels of KP metabolites: tryptophan (TRP) and kynurenine (KYN)-shown as TRP:KYN ratio (A); 3-hydroxykynurenine (3-HK) (B); 3-hydroxyanthranillic acid (3-HAA) (C); anthranilic acid (AA) (D); quinolinic acid (QUIN) (E); picolinic acid (PIC) (F) and the pro-inflammatory marker: neopterin (G) were measured by UHPLC or GC-MS (QUIN and PIC only) in the supernatants of PBMCs untreated (control-black bars) or treated with IFN-γ (500 IU/mL-grey bars) at 24, 48 and 72 hours of culture. Values represent the mean±SEM of 4 independent biological repeats (ns = no significant difference, *p<0.05, **p<0.01, **p<0.001).

Mentions: We assessed the levels of major KP metabolites in the supernatant of PBMC either untreated (control) or treated with 500 IU/mL IFN-γ. The ratio of kynurenine to tryptophan (KYN:TRP, a sensitive measure of IDO-1 activation) was significantly increased at all time-points (p<0.0001). This was largely due to an increase in KYN production not a decrease in TRP, which remained relatively constant (Fig C in S1 File). The highest KYN:TRP ratio was seen at 72 hours (Fig 4A). Concentrations of 3-HK and 3-HAA were not significantly elevated in cultures of IFN-γ treated PBMC compared to control cultures at any time-point (Fig 4B & 4C). However, whilst not statistically significant, there was a clear trend for 3-HAA to be elevated in treated cultures at 72 hours (Fig 4C). Similarly, there was a strong trend for AA levels to be elevated in IFN-γ treated PBMC cultures with progressive increase over time, although this did not reach statistical significance (Fig 4D).


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)

Concentrations of KP metabolites in the supernatants of IFN-γ activated PBMCs.Levels of KP metabolites: tryptophan (TRP) and kynurenine (KYN)-shown as TRP:KYN ratio (A); 3-hydroxykynurenine (3-HK) (B); 3-hydroxyanthranillic acid (3-HAA) (C); anthranilic acid (AA) (D); quinolinic acid (QUIN) (E); picolinic acid (PIC) (F) and the pro-inflammatory marker: neopterin (G) were measured by UHPLC or GC-MS (QUIN and PIC only) in the supernatants of PBMCs untreated (control-black bars) or treated with IFN-γ (500 IU/mL-grey bars) at 24, 48 and 72 hours of culture. Values represent the mean±SEM of 4 independent biological repeats (ns = no significant difference, *p<0.05, **p<0.01, **p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131389.g004: Concentrations of KP metabolites in the supernatants of IFN-γ activated PBMCs.Levels of KP metabolites: tryptophan (TRP) and kynurenine (KYN)-shown as TRP:KYN ratio (A); 3-hydroxykynurenine (3-HK) (B); 3-hydroxyanthranillic acid (3-HAA) (C); anthranilic acid (AA) (D); quinolinic acid (QUIN) (E); picolinic acid (PIC) (F) and the pro-inflammatory marker: neopterin (G) were measured by UHPLC or GC-MS (QUIN and PIC only) in the supernatants of PBMCs untreated (control-black bars) or treated with IFN-γ (500 IU/mL-grey bars) at 24, 48 and 72 hours of culture. Values represent the mean±SEM of 4 independent biological repeats (ns = no significant difference, *p<0.05, **p<0.01, **p<0.001).
Mentions: We assessed the levels of major KP metabolites in the supernatant of PBMC either untreated (control) or treated with 500 IU/mL IFN-γ. The ratio of kynurenine to tryptophan (KYN:TRP, a sensitive measure of IDO-1 activation) was significantly increased at all time-points (p<0.0001). This was largely due to an increase in KYN production not a decrease in TRP, which remained relatively constant (Fig C in S1 File). The highest KYN:TRP ratio was seen at 72 hours (Fig 4A). Concentrations of 3-HK and 3-HAA were not significantly elevated in cultures of IFN-γ treated PBMC compared to control cultures at any time-point (Fig 4B & 4C). However, whilst not statistically significant, there was a clear trend for 3-HAA to be elevated in treated cultures at 72 hours (Fig 4C). Similarly, there was a strong trend for AA levels to be elevated in IFN-γ treated PBMC cultures with progressive increase over time, although this did not reach statistical significance (Fig 4D).

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