Kynurenine pathway activation and deviation to anthranilic and kynurenic acid in fibrosing chronic graft-versus-host disease

Abstract

Fibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogeneic stem cell transplantation (alloSCT). A driver of fibrosis is the kynurenine (Kyn) pathway, and Kyn metabolism patterns and cytokines may influence cGVHD severity and manifestation (fibrosing versus gastrointestinal [GI] cGVHD). Using a liquid chromatography-tandem mass spectrometry approach on sera obtained from 425 patients with allografts, we identified high CXCL9, high indoleamine-2,3-dioxygenase (IDO) activity, and an activated Kyn pathway as common characteristics in all cGVHD subtypes. Specific Kyn metabolism patterns could be identified for non-severe cGVHD, severe GI cGVHD, and fibrosing cGVHD, respectively. Specifically, fibrosing cGVHD was associated with a distinct pathway shift toward anthranilic and kynurenic acid, correlating with reduced activity of the vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGVHD and provides a rationale for translational trials on prophylactic vitamin B2/B6 supplementation for cGVHD prevention.

Keywords: CXCL9; IL-18; anthranilic acid; fibrosing chronic graft-versus-host disease; indoleamine-2,3-dioxygenase; kynurenic acid; kynurenine; tryptophan; vitamin B2; vitamin B6.

Graphical abstract

Figure 1

Kynurenine pathway The kynurenine pathway (KP) constitutes the main route of tryptophan (Trp) degradation. More than 95% of dietary Trp is metabolized via the KP, and only 1% is converted to serotonin. The rate-limiting first step of the KP is the opening of the indole ring by either Trp-2,3-dioxygenase (TDO; liver enzyme) or indoleamine-2,3-dioxygenase (IDO; many cell types, induced by IFN-γ). IL-18 is an inducer of IFN-γ, and IFN-γ induces CXCL9. l-kynurenine (Kyn) is the key compound metabolized by three different enzymes in mammalian tissues: (1) kynurenin-3-hydroxylase (or -monooxygenase [KMO]), which forms 3-hydroxykynurenine (3-HK), quinolinic and picolinic acid, and finally nicotinamide adenine dinucleotide⁺ (NAD⁺); (2) kynureninase (KYNU), which forms anthranilic acid (AA) and 3-hydroxy anthranilic acid (3-HAA); and (3) kynurenine aminotransferases (KAT I and KAT II), which form kynurenic acid (KA). KMO metabolization of Kyn to 3-HK is enhanced by vitamin B2, whereas the conversion of 3-HK to 3-HAA is enhanced by vitamin B6.

Figure 2

Differences in cGVHD subtypes (A) Comparison of the onset of mild symptoms (left panel) and the onset of severe symptoms (right panel) in patients with severe fibrosing cGVHD and severe gastro-intestinal (GI) cGVHD. Left: all patients who develop first mild, and then severe, cGVHD during the observation period are included. Start of follow-up: day 0 of alloSCT; event, onset of mild symptoms, no censoring. Median time until onset of mild symptoms, fibrosing: 6.5 months (n = 37), GI: 3.9 months (n = 15). Right: included are all patients who develop severe cGVHD during observation. Start of follow-up: day 0 of alloSCT; event, onset of severe symptoms, no censoring. Median time until onset of severe symptoms, fibrosing: 16.5 months (n = 61), GI: 6.1 months (n = 38). (B) Boxplots of the ratio of 3HAA/AA and the concentration of KA at the onset of cGVHD, n = 161.The median is indicated by a horizontal line. The upper and lower border of the box correspond to the 3^rd and 1^st quartiles. The whiskers correspond to the most extreme data points, which are no more than 1.5 times the interquartile range away from the upper and lower border of the box. (C and D) Cumulative incidence of severe cGVHD after onset of mild symptoms, stratified by the Kyn metabolite score (0, 1, or 2 risk factors [RFs]). Included are all patients who developed mild cGVHD symptoms. Follow-up starts at the time of onset of mild cGVHD symptoms. (C) Event: onset of severe fibrosing cGVHD (27 events), censored at last follow-up without severe fibrosing cGVHD or at death without severe fibrosing cGVHD. Cox regression two RFs versus < 2 RFs: HR, 3.92; 95% CI, 1.83–8.37; p < 0.001. (D) Event: onset of severe GI cGVHD (eight events), censored at last follow-up without severe GI cGVHD or at death without severe GI cGVHD. Cox regression two RFs versus < 2 RF: n.s. RF: ratio of 3HAA/AA < median, and KA > median.

Figure 3

Influence of KMO, vitamin B6, CXCL9, and IL-18 on the kynurenine pathway (A) Low KMO activity and low vitamin B6 (VitB6) correlate with a low ratio HAA/AA but not with KA (for details, see Figure S3). (B) High CXCL9 (but not IL-18) correlates with IDO activity (for details, see Figures S5A and S5B). (C) High IL-18 correlates with high AA and with high KA, and there is no effect of CXCL9 on KA (for details, see Figures S6 and S7). (D) Triple-hit model of fibrosing cGVHD (hypothesis): severe fibrosing cGVHD is associated with strong CXCL9-induced activation of IDO. The result of this activation is influenced by reduced KMO activity, either because of genetic polymorphisms or because of the lack of vitamin B2, and is further aggravated by a lack of vitamin B6. The resulting metabolic deviation increases AA concentrations and reduces the 3-HAA/AA ratio. Finally, high IL-18 is associated with increased serum KA. These three hits result in a metabolic situation with high KA and a low ratio of 3-HAA/AA, which predicts a risk of fibrosing cGVHD. The number of replicates depend on the available measurements of the respective metabolites (for details, see Figures S3–S7).