Kynurenine metabolites predict survival in pulmonary arterial hypertension: A role for IL-6/IL-6Rα

Figures

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  Figure 1. KP metabolite profile in PAH patients and two animal models of PH. (A) Scheme of de novo NAD+ synthesis through the kynurenine pathway with changes observed in PAH patients shown as red arrows. KP metabolite profiles in: (B) PAH patients at baseline (n = 43) vs Healthy controls (n = 111) in the controls), (C) MCT-PH rats (n = 8) vs Control rats (n = 9), (D) SuHx-PH rats (n = 10) vs Control rats (n = 5). A KP metabolite profile similar to PAH patients was only found in the MCT-PH rats which show a severe inflammatory phenotype, indicating the potential link between inflammation and KP metabolism in PAH. When compared to the controls (n = 111), (E) Tryptophan was significantly lower in PAH at baseline (n = 43) and after therapy (6Mos, n = 32; 1Yr, n = 28), (F,J) Kynurenine and anthranilic acid were significantly higher in PAH at baseline and 6 months after PAH therapy, (H,I) 3-hydroxykynurenine and quinolinic acid were significantly higher in PAH at baseline and after therapy, (G) Kynurenic acid was only higher in PAH at baseline. Data are presented as dot plots with median (IQR). *P < 0.05, **P < 0.01, ***P < 0.001 vs Controls, Mann–Whitney Test. MCT monocrotaline, SuHx Sugen plus…

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  Figure 2. NAD+ levels in lung tissue are higher in rats with monocrotaline (MCT) induced PAH (n=6) as compared to Control rats (Ctrl, n=6). * P < 0.05.

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  Figure 3. IL-6/IL-6Rα stimulation in vitro mimicked the abnormal KP metabolite profile observed in PAH patients. (A) Stimulation with IL-6/IL-6Rα in human lung MVECs (n = 6, 2 donors), PASMCs (n = 6, 2 donors), and lung fibroblasts (n = 6, 2 donors) for 24 h in vitro induced the abnormal phenotype of KP metabolism that was seen in PAH patients with lower Trp, higher Kyn, 3-HK, QA, KA, and AA, while unchanged 3-HA. Stimulation with IL-6 (B), TNF-α (C), TGF-β1 (D), or Hypoxia (1% O2, E) for 24 h in human lung MVECs, PASMCs, or fibroblasts in vitro resulted in different KP metabolite profiles. Data are presented as mean ± SEM, fold change to control, *P < 0.05, **P < 0.01, ***P < 0.001, Student T Test. MVECs microvascular endothelial cells, PASMCs pulmonary artery smooth muscle cells, IL-6Rα interleukin-6 receptor α, IL-6 interleukin-6, TNF-α tumor necrosis factor α, TGF-β1 transforming growth factor beta-1, Trp tryptophan, Kyn kynurenine, 3-HK 3-hydroxy-kynurenine, 3-HA 3-hydroxykynurenic acid, QA quinolinic acid, KA kynurenic acid, AA anthranilic acid.

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  Figure 4. Treatment effects on KP metabolite levels in surviving vs non surviving PAH patients. A Mixed-effects model showed a decrease over time in Kyn (P = 0.010, C), 3-HK (P = 0.022, E), KA (P = 0.050, I), and with a trend toward significance for QA (P = 0.082, G). Wilcoxon matched-pairs signed rank test showed that Kyn (P = 0.005, C), 3-HK (P < 0.001, E), QA (P = 0.013, G), KA (P = 0.007, I) and AA (P = 0.027, K) were significantly decreased in survivors after one year but not six months of PAH therapy. Moreover, at baseline, only Kyn was significantly higher in non-survivors when compared with survivors (D), while at the latest measurement after PAH therapy, Kyn (D), 3-HK (F), QA (H), KA (J) and AA (L) were all significantly higher in non-survivors when compared with survivors. Data are presented as dot plots with median (IQR), dash-lines indicate the baseline median value of KP metabolites in all PAH patients. P < 0.05, **P < 0.01, ***P < 0.001, Wilcoxon matched-pairs signed rank test. †P < 0.05, ††P < 0.01, †††P < 0.001, Mann–Whitney Test. BL baseline, Mos months, Yr year, Trp tryptophan, Kyn kynurenine, 3-HK 3-hydroxy-kynurenine, QA quinolinic acid, KA kynurenic acid, AA…

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  Figure 5. Correlation analysis. Correlation between KP metabolites in healthy controls and PAH patients. (A) KP metabolites correlated with each other in healthy controls except for Trp and QA. (B) At baseline, Trp lost its correlations with other metabolites, while other metabolites still correlated with each other in PAH patients. Such correlations were generally maintained after PAH therapy for six months (C) and one year (D). Correlations of KP metabolite levels with baseline characteristics. (E) In healthy controls, KA was associated with age, Trp and Kyn were associated with Sex, Kyn was associated with BMI. (F) In PAH patients, Kyn and QA were associated with age, Trp was associated with Sex, 3-HK and 3-HA were associated with sBP, 3-HK was associated with HR. (G) In PAH patients, Kyn, 3-HK, 3-HA, and KA were positively associated with PVR, Kyn, 3-HK, QA, KA, and AA were positively associated with NYHA, while Kyn was inversely associated with CO, CI and 6MWD, QA was inversely associated with CI, KA was inversely associated with CO and CI. Data are presented as rainbow heat map for Spearman coefficients, *P < 0.05, †P < 0.01, ‡P < 0.001, §P = 0.05, Spearman's Rank correlation…

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  Figure 6. Survival analyses in PAH patients. PAH patients were stratified into 2 groups according to the median of baseline (left panels) or latest (right panels) KP metabolite levels. Using baseline metabolite levels, PAH patients with high levels of Kynurenine, 3-Hydroxykynurenine, or Quinolinic acid had worse transplant-free survival at short-term than patients with low levels (Breslow Test, P all < 0.05). Using the latest measurement, patients with high levels of Kynurenine, 3-Hydroxykynurenine, 3-Hydroxykynurenic acid, Quinolinic acid, or Kynurenic acid, had worse short- (Breslow Test, P all < 0.05) and long-term transplant-free survival (Log-rank Test, P all < 0.05) than patients with low levels, while patients with high anthranilic acid had worse short-term survival only.