Fig 1.
Simplified overview of the kynurenine pathway of tryptophan metabolism.
The three main enzymes: indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monooxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) are shown abbreviated in bold italics whilst the main metabolites measured in this study are written in bold.
Fig 2.
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.
Table 1.
Antibodies used in flow cytometry experiments.
Fig 3.
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.
Table 2.
Significance difference levels between control and treatment groups for IDO-1, KMO and QPRT expression (gMFI) for both lymphocytes and monocytes at each time-point tested.
Fig 4.
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).
Fig 5.
Correlations of IDO-1, KMO and QPRT expression (gMFI) with KYN:TRP ratio, QUIN concentration (nM) and neopterin levels (nM).
Data was log10 transformed to normalize distributions. Straight-line fits show least squares regression with the r2 value providing a summary of fit. P values were computed using ANOVA for a regression analysis of the total variation for each correlation (n = 24).