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Original article
Activity of the kynurenine pathway and its interplay with immunity in patients with pulmonary arterial hypertension
  1. Malgorzata Jasiewicz1,
  2. Marcin Moniuszko2,3,
  3. Dariusz Pawlak4,
  4. Malgorzata Knapp1,
  5. Malgorzata Rusak5,
  6. Remigiusz Kazimierczyk1,
  7. Wlodzimierz Jerzy Musial1,
  8. Milena Dabrowska5,
  9. Karol Adam Kaminski1,6
  1. 1Department of Cardiology, Medical University of Bialystok, Bialystok, Poland
  2. 2Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, Bialystok, Poland
  3. 3Department of Allergology and Internal Medicine, Medical University of Bialystok, Bialystok, Poland
  4. 4Department of Pharmacodynamics, Medical University of Bialystok, Bialystok, Poland
  5. 5Department of Haematological Diagnostics, Medical University of Bialystok, Bialystok, Poland
  6. 6Department of Population Medicine and Prevention of Civilization Diseases, Medical University of Bialystok, Bialystok, Poland
  1. Correspondence to Professor Karol Adam Kaminski, Department of Cardiology, Medical University of Bialystok, Ul. M. Sklodowskiej-Curie 24A, Bialystok 15-276, Poland, fizklin{at}wp.pl

Abstract

Objective We evaluated blood concentrations of kynurenine pathway metabolites, natural and induced regulatory T cells (nTreg, iTreg), and Th17 cells in order to examine the activity of the kynurenine pathway and its relation to immune status in patients with pulmonary arterial hypertension (PAH).

Methods Plasma concentrations of tryptophan, kynurenine, kynurenic acid, anthranilic acid, and 3-hydroxykynurenine were quantified in 26 patients with PAH (vs 30 healthy controls) at baseline and after 6 months, and assessed them in relation to clinical parameters, frequencies of lymphocyte subsets, and outcome.

Results The PAH group presented higher concentrations of tryptophan (52.9 (IQR 46.3–57.5) vs 40.3 (35.2–46.3) µmol/L, p=0.00003), kynurenine 2.8 (2.4−3.4) vs 1.9 (1.5–2.3) µmol/L, p=0.000007), kynurenine/tryptophan ratio (0.051 (0.044–0.064) vs 0.043 (0.039–0.055), p=0.03), iTreg frequencies (10.5 (8.8–13.9)% vs 6.8 (5.2–9.5)%, p=0.002) and iTreg/Th17 (1.73 (1.2–2.8) vs 0.93 (0.61–1.27), p=0.003) together with lower ratios of kynurenic acid/kynurenine, 3-hydroxykynurenine/kynurenine, and anthranilic acid/kynurenine. Kynurenine concentrations and kynurenine/tryptophan ratio correlated positively with iTreg/Th17, and inversely with Th17 subsets, whereas kynurenic acid/kynurenine and anthranilic acid/kynurenine ratios correlated positively with Th17. Adverse outcomes occurred in 9 of 26 patients and they showed higher baseline concentrations of kynurenine (3.6 (2.8–4.3) vs 2.7 (2.1–3.2) µmol/L, p=0.033). Median kynurenine values ≥3.4 µmol/L (67% sensitivity, 94% specificity) identified patients with a worse clinical course.

Conclusions PAH is characterised by upregulated tryptophan metabolism and enhanced biosynthesis of kynurenine. Elevated kynurenine concentration is associated with an adverse clinical course. Dysregulated immunity, delineated by Treg-Th17 imbalance, is directly related to diverse activation of the kynurenine pathway, indicating the potential interplay between kynurenines and the immune system in PAH.

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Introduction

The chronic inflammatory process has been widely recognised, among others, as a prominent pathogenic component of pulmonary arterial hypertension (PAH). In addition, there is a large body of evidence showing that dysregulation of the immune system plays a significant role in disease development and progression by promoting inflammatory cell recruitment, autoimmune responses, and proliferation and migration of pulmonary vascular cells. Activation and perivascular infiltration of adaptive and innate immune cells, comprising macrophages, T and B lymphocytes, dendritic cells and mast cells, accompanied by elevated circulating or local concentrations of various cytokines and chemokines, have been documented in patients with PAH.1–3

Special attention has recently focused on regulatory T (Treg) cells. Treg cells are implicated in the maintenance of self-tolerance and control of autoimmunity by suppressing autoreactive responses. A dynamic balance between Treg cells and effector T cells regulates immune homeostasis. In contrast, Th17 cells, a subset of IL-17-producing effector T cells, have a highly proinflammatory nature and are broadly implicated in inflammatory and autoimmune diseases.4 When the dynamic balance between effector T cells and Treg cells is altered, immune reactions are shifted toward the predominance of immune-soothing or excessive ones. Recent studies in the field of Treg cells in PAH are equivocal, showing that idiopathic PAH (IPAH) is associated either with higher numbers of circulating Treg cells than controls or reduced local numbers of Tregs in lung tissue or its aberrant activity.5 ,6 The involvement of Th17 cells in the pathogenesis of PAH has been poorly examined.

Tryptophan (TRP) catabolism has recently emerged as a powerful mechanism of peripheral immune tolerance by preventing autoimmunity or uncontrolled immune responses. The major non-protein route for the oxidative degradation of TRP (95%) is the formation of kynurenine (KYN) and downstream metabolites, collectively referred to as kynurenines (figure 1). KYN is catalysed by the enzymes indoleamine 2,3-dioxygenase (IDO) 1 and 2 and TRP 2,3-dioxygenase (TDO). IDO 1 is expressed is many tissues and cells, including lungs, monocytes, macrophages and endothelial cells, and is activated by pro-inflammatory mediators. TDO, highly expressed in the liver, is inducible by stress hormones and by a substrate, TRP.7 The kynurenine pathway is involved in several conditions including cancer, inflammatory disorders, and neurological and neurodegenerative diseases. IDO is frequently expressed within human tumour cells where it is involved in tumour survival and self-tolerance by suppression of T cell mediated immune responses, even in the absence of any inflammation.8 In the studies of patients with end-stage renal disease,9 ,10 activation of the kynurenine pathway was associated with increased oxidative stress, inflammation, haemostatic disturbances, and prevalence of cardiovascular disease. In PAH, to date, the clinical and experimental data focusing on the kynurenine pathway are lacking.

Figure 1

Kynurenine pathway of tryptophan metabolism. IDO, indoleamine-2,3-dioxygenase; KAT, kynurenine aminotransferase; KMO, kynurenine-3-monooxygenase; KYNU, kynureninase; TDO, tryptophan 2,3-dioxygenase.

Taking discussed data into consideration, the objective of this cross-sectional study was to assess plasma concentrations of TRP and downstream metabolites─KYN, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HKYN) and anthranilic acid (AA)─in patients with PAH and to examine the relationship between the studied molecules and the clinical status of these patients. Moreover, we assessed circulating T lymphocyte subsets that have been implicated in dysregulated immunity in PAH: Treg, both natural thymus-derived (nTreg, delineated by CD4+CD25+CD127− phenotype) and putative adaptive, developing at the periphery (also referred to as induced iTreg, characterised by CD4+CD25−CD127− phenotype), together with Th17 cells which are their counter-interacting effector T-helper subsets. We aimed to examine if there is any relation between the activity of the kynurenine pathway and the immune status in our PAH population and, if so, attempted to describe the potential relationship.

Methods

We conducted a prospective single-centre study that included 26 patients with PAH. The study group comprised the complete population of these patients in north-eastern Poland, referred to and treated in the University Hospital of Bialystok in 2010–2013. The diagnosis of PAH was based on established criteria11 and confirmed by right-sided heart catheterisation, using standard haemodynamic measurements: mean pulmonary artery pressure and pulmonary artery occlusion pressure. The reference cohort consisted of 30 healthy volunteers (from outpatient clinics and general practice) matched for age, sex, bodyweight and comorbidities with the PAH subjects. Patients with acute infection, malignancy, and chronic obstructive pulmonary disease were not included in the study. None of participants received corticosteroids or immunosuppressive drugs and all were on a regular diet.

All patients underwent the same diagnostic assessment: a medical interview with initial determination of the WHO functional class, physical examination, transthoracic echocardiography, cardiopulmonary exercise test (CPET), 6 min walk test with distance assessment, and fasting venous blood tests. The same assessment of PAH patients was performed 6 months after the study enrolment, additionally noting the end point of clinical deterioration (WHO class change, the need for escalation of therapy) or death.

Echocardiography was performed to assess morphology and function of the right heart as well as to exclude any significant heart abnormalities in the control group. Quantification of two-dimensional and Doppler echocardiography data, including right ventricle and atrium dimensions, degree of tricuspid regurgitation, and estimation of systolic pulmonary artery pressure, was performed in a standard manner. Systolic function of the right ventricle was assessed by measuring the tricuspid annular plane systolic excursion (TAPSE) and percent fractional area change (FAC). Only patients with normal morphology and function of the right ventricle and with preserved left ventricular ejection fraction were included in the control group.

CPET was performed using maximum, symptom limited treadmill exercise with ramp protocol in order to assess exercise capacity and gas exchange parameters. Of the numerous variables, peak oxygen uptake (peak VO2), ventilation equivalents VE/VO2, VE/VCO2, the slope of the VE/VCO2 relationship from the initiation to peak exercise (VE/VCO2 slope), and peak end-tidal partial pressure of CO2 (PetCO2) were used for analysis.

Blood sampling and assays

Fasting peripheral venous blood samples were obtained from the patients with PAH as well as controls. Plasma aliquots of 1.5 mL were stored at −80°C for future analysis. In order to estimate kynurenine concentrations, the plasma was deproteinised with 2 M HClO4 and centrifuged at 12 000 g for 15 min at 4°C. The supernatant fluid was passed through a WATERS 0.45 μM filter. TRP and downstream metabolites were determined by high-performance liquid chromatography as previously described by Pawlak et al.12

Flow cytometric analysis: EDTA anticoagulated fresh whole blood samples were stained with 5 μL of the following monoclonal antibodies: anti-CD4 fluorescein isothiocyanate (FITC) (BD Biosciences), anti-CD127 PE (Beckman Coulter), anti-CD25 PE-Cy5 (BD Biosciences), anti-CD161 APC (BD Bioscience), and anti-CD196 PerCP-Cy5.5 (BD Bioscience); the samples were then incubated for 30 min at room temperature, in the dark. Additionally, appropriate unstained and FMO (fluorescence-minus-one) controls were prepared according to the procedures described above in order to separate the positive signals from the background. Flow cytometry data were acquired on FACSCalibur flow cytometer (BD Biosciences, San Jose, California, USA) and analysed using FlowJo software (TreeStar Inc, Ashland, Oregon, USA). Two populations of Treg cells were distinguished on the basis of differential expression of analysed markers as described previously.13 ,14 Natural regulatory T cells (nTreg) were identified by CD4+CD25+CD127− phenotype while induced regulatory T cells were characterised by CD4+CD25−CD127− phenotype. Th17 cells enriched in IL-17-expressing cells were delineated by the CD4+CD161+CD196+ phenotype. Data are presented as percentages of studied lymphocyte subsets.

All molecules and cells were assessed from the blood samples collected at the same time point. Blood samples were also analysed for B-type natriuretic peptide and other routine biochemical and hematological parameters in the local laboratory of the University Hospital of Bialystok.

The study was approved by the institutional medical ethics committee. All patients gave written informed consent for participating in the study, including the taking and storage of blood samples. The study complied with the declaration of Helsinki.

Statistical analysis

The distribution of all variables was verified with the Kolmogorov-Smirnov test. Data are expressed as mean±SD or median values with IQR as appropriate. Statistical analysis was performed using Student t test or Mann-Whitney U test for continuous data depending on distribution, and χ2 test for categorical variables. Spearman's or Pearson's correlation coefficient was used to examine the relationship between two continuous variables. Receiver operating characteristics (ROC) curves were used for establishing optimal values for differentiation of analysed subgroups. The distribution of the time-to-event variables was estimated using the Kaplan–Meier method with log-rank testing. A value of p<0.05 was deemed statistically significant. The statistical software package Statistica V.10 (USA) was used for analysis.

Results

The PAH group comprised patients with IPAH (46.2%, n=12) and PAH associated with connective tissue disease or congenital heart disease (26.9%, n=7 for both groups). Of these, 61.5% (n=16) were prevalent cases with specific treatment applied, and the rest (38.5%, n=10) were incident cases. Most PAH patients had WHO class III functional limitations, and were predominantly women. They manifested severely limited exercise capacity as well as significant ventilatory inefficiency during exercise. Detailed characteristics of the groups are summarised in tables 1 and 2.

Table 1

Baseline characteristics: biochemical, echocardiographic and functional parameters of PAH and control group

Table 2

Haemodynamic data and targeted treatment in the PAH group

The PAH patients were characterised by significantly higher median TRP and KYN concentrations together with a higher KYN/TRP ratio compared to control subjects (figure 2 and table 3). Both groups did not differ in KYNA, 3HKYN and AA concentrations, but the ratios of KYNA/KYN, 3HKYN/KYN and AA/KYN were significantly lower in the study group. The same pattern of concentrations was observed after 6 months of follow-up if compared to controls, but there was no dynamics in the changes of kynurenines concentrations reported in PAH group during this observation period (figure 2 and table 3).

Table 3

Concentrations of downstream TRP metabolites with product to substrate ratios and frequencies of induced regulatory T cells with its ratio to Th17 cells in PAH (baseline and after 6 months) and control group

Figure 2

Concentrations of tryptophan (TRP), kynurenine (KYN) and the ratio of kynurenine to tryptophan (KYN/TRP) in the pulmonary arterial hypertension (PAH) group (baseline and after 6 months) and in the control group. TRP and KYN concentrations and KYN/TRP ratio are constantly elevated in the PAH population and higher when compared to controls. ‡p=0.00003 PAH (baseline) vs control group. ‡‡p=0.00001 PAH (after 6 months) vs control group. #p=0.000007 PAH (baseline) vs control group. ##p=0.00001 PAH (after 6 months) vs control group. *p=0.03 PAH (baseline) vs control group. **p=0.04 PAH (after 6 months) vs control group.

There were no differences in the baseline plasma concentrations of the studied molecules between the incident and prevalent cases and according to WHO class. However, 3HKYN concentrations and the KYN/TRP ratio differed between the groups of patients with different aetiologies of PAH: connective tissue disease and idiopathic PAH (33 (29.8–51.1) vs 19.7 (16.6–26.1) nmol/L, p=0.046; and 0.073 (0.047–0.095) vs 0.049 (0.039–0.06), p=0.03) (figure 3).

Figure 3

3-hydroxykynurenine concentration and kynurenine to tryptophan (KYN/TRP) ratio in patients with different aetiologies of pulmonary arterial hypertension (PAH): connective tissue disease (CTD)-associated PAH, congenital heart disease (CHD)-associated PAH, and idiopathic PAH. #p=0.046 CTD-associated PAH vs idiopathic PAH. *p=0.03 CTD-associated PAH vs idiopathic PAH.

Moreover, we report significantly higher iTreg frequencies and iTreg/Th17 ratio in the PAH group compared to controls (table 3). Both groups did not differ in nTreg and Th17 percentages or in nTreg/Th17 ratio. Importantly, in the PAH population Th17 subsets correlated inversely with KYN concentrations and KYN/TRP ratio, and positively with KYNA/KYN and AA/KYN ratios (figure 4A–D). Furthermore, nTreg/Th17 and iTreg/Th17 ratios correlated positively with KYN concentrations and KYN/TRP ratio (figure 4E–H). We did not observe correlations of studied lymphocyte populations with other biochemical and functional parameters in PAH patients.

Figure 4

(A–H) Correlations of kynurenines and lymphocyte subsets. (A) Th17 and kynurenine (KYN); (B) Th17 and kynurenine to tryptophan (KYN/TRP) ratio; (C) Th17 and kynurenic acid (KYNA)/KYN ratio; (D) Th17 and anthranilic acid (AA)/KYN ratio; (E) nTreg/Th17 ratio and KYN; (F) nTreg/Th17 and KYN/TRP ratios; (G) iTreg/Th17 ratio and KYN; (H) iTreg/Th17 and KYN/TRP ratios. (I–M) Correlations of TRP and clinical parameters. (I) TRP and uric acid; (J) TRP and EQ O2; (K) TRP and EQ CO2; (L) TRP and peak PetCO2; (M) TRP and VE/VCO2 slope.

Regarding the relationship of kynurenines with other parameters describing the PAH population, we reported statistically significant correlations between TRP concentrations and uric acid and parameters of ventilatory efficiency during exercise (figure 4J–M). The KYN concentrations correlated with uric acid (r=0.62, p=0.0006) and glomerular filtration rate estimated by the Cockcroft-Gault formula (eGFR) (r=−0.43, p=0.03), and the KYN/TRP ratio with uric acid (r=0.48, p=0.01).

During 6 months of observation, clinical deterioration occurred in nine patients (including two deaths), while 17 patients remained stable. Importantly, patients with clinical deterioration presented significantly higher baseline concentrations of KYN (3.6 (2.8–4.3) vs 2.7 (2.1–3.2) µmol/L, p=0.03), but not the other kynurenines, compared with stable subjects. ROC curve analysis demonstrated that a KYN cut-off value of 3.4 µmol/L separated patients with a better and worse clinical course with a sensitivity of 67% and a specificity of 94% (area under the curve (AUC) 0.76, 95% CI 0.55 to 0.97). Kaplan–Meier analysis showed a statistically significant event-free survival difference between patients with a baseline KYN concentration below and above 3.4 µmol/L (p=0.0009) (figure 5).

Figure 5

Kaplan–Meyer curves illustrating the probability of event-free survival (clinical deterioration, death) in the pulmonary arterial hypertension (PAH) population during 6 months of observation, depending on baseline concentrations of kynurenine (KYN); cut-off value of 3.4 µmol/L (p=0.0009, log-rank test).

Discussion

In the present study we describe for the first time the elevated plasma concentrations of TRP and KYN in patients with PAH. Moreover, KYN/TRP ratio, the potential marker of inflammation and the indicator of IDO activity, was also significantly elevated. These findings were accompanied by lower ratios of KYNA/KYN, 3HKYN/KYN and AA/KYN, reflecting diminished activity of downstream enzymes. To the best of our knowledge these downstream TRP metabolites have not been previously evaluated in a PAH population. Collectively, our results indicate that PAH pathobiology is characterised by enhanced biosynthesis and decreased degradation of KYN. In our PAH population, elevated KYN concentration was associated with an adverse clinical course. In addition, we showed that iTreg cells and iTreg/Th17 ratios were elevated in PAH patients, indicating that the balance between these T cells is impaired and shifted toward adaptive responses. Importantly, we highlighted the potential interplay between kynurenines and the immune system in PAH, as the Treg–Th17 imbalance remained in relation to the altered activation of the kynurenine pathway. Thus, for the first time we provide evidence that the kynurenine axis might be involved in PAH pathobiology and that kynurenines might be, among others, a link between inflammation, metabolism and immunity in PAH.

TRP, the essential amino acid in the human diet, is an important precursor of several neurotransmitters and metabolic regulators. On the other hand TRP is listed in the classification for drug- and toxin-induced PAH as a drug of likely association with PAH development.15 In the 1990s, the eosinophilia-myalgia syndrome and pulmonary hypertension (PH) were epidemiologically associated with ingestion of preparations containing TRP.16 When open lung biopsy was performed, the specimens showed arteriopathy compatible with other forms of PAH.17 We report significantly elevated circulating TRP values in our PAH population, although all groups were on a regular diet and did not use any TRP containing preparations. This observation is strengthened by the TRP measurements both at baseline and after a 6-month period. Moreover, TRP concentrations correlated with exercise ventilatory parameters and uric acid concentration—indicators of disease severity and prognosis. We hypothesise that patients with PAH may have, for some reason, an increased demand and ability to uptake TRP, which is further metabolised in early stages of the kynurenine pathway; this hypothesis, however, requires further investigation. TRP might be chronically elevated to maintain the production of biologically active metabolites because, together with elevated TRP concentrations, we reported elevated KYN values and KYN/TRP ratio, a marker of IDO activity. IDO also reflects up-regulation of the kynurenine pathway associated with inflammatory conditions. Importantly, TRP is required for T lymphocyte effector functions. T lymphocytes are extremely sensitive to TRP shortage, which causes their arrest in the G1 phase of the cell cycle.18 However, TRP derivatives also exert immunoregulatory functions because they can influence the differentiation of regulatory T cells.19 ,20 In this regard, IDO activation may not be the only trigger for up-regulation of the TRP metabolic cascade; thus the potential role of TDO should not be neglected.

TDO is responsible for the metabolism of dietary TRP and regulation of TRP blood concentrations. It has also been described as possessing the immunosuppressive function involved, for example, in tumour progression.21 In contrast to IDO, which is activated by pro-inflammatory mediators, TDO is both activated and substantially up-regulated by the substrate, TRP.22 Thus, the immunoregulatory effects of TDO are probably mediated mostly by TRP derivatives, whereas the immunosuppressive effect of IDO is related to TRP depletion, which is needed to impair T cell mediated immune responses. With respect to PAH, we assume that enhanced KYN biosynthesis might result both from the setting of a chronic inflammatory condition as well as from elevated TRP concentrations. TRP might be globally elevated but depleted locally due to complex but partly mutual interactions of the KYN axis with the immune system. This may be in agreement with the observations of reduced local numbers of Tregs in lung tissue6 as well as with the systemic increase of iTreg frequencies observed in our population. In addition, the notion of complex interactions between the KYN axis-immune system is strengthened by our observations of a positive correlation of KYN and KYN/TRP ratio with iTreg/Th17 and nTreg/Th17, and a negative correlation with Th17. This immune-regulatory network might also indirectly indicate, maybe specific for some way in PAH, the susceptibility to maintain the production of KYN.

It is difficult to delineate the specific role of KYN. It was reported that KYN might be responsible for the immunosuppressive phenotype of dendritic cells23 as well as for the inhibition of dendritic cell maturation. These actions promote self-tolerance, but may also lead to the reduction of tumour recognition, for example. Importantly, immature dendritic cells have been recognised to accumulate in the wall of remodelled pulmonary vessels in PAH.24 On the other hand, KYN has been identified as a potent, nitric oxide (NO)-independent, vasodilator.25 In the context of endothelial dysfunction in PAH, which is a key hallmark of the disease, KYN may exert protective effects on the vasculature. Nevertheless we showed that elevated KYN concentrations were associated with an adverse clinical course, which may indicate that the potentially protective effects of KYN are insufficient and that the primary immune compensatory mechanisms have failed in PAH. Unfortunately, in light of the current state of knowledge on PAH, it is particularly difficult to delineate or even speculate whether dysregulated immunity, up-regulated TRP metabolism or its interrelation are a cause or effect of the disease.

To date, there are only a few reports addressing the mechanisms of interplay between Th17 and Treg cells and TRP metabolites in the context of PAH. Notably, our data on Th17 and Treg cells contrast with previously published works. Recently, increased Th17 and decreased Treg cell concentrations were found in patients with connective tissue disease-associated PAH.26 As we did not observe a similar pattern in our study, this may suggest that the role of both Th17 and Treg cells can vary between different types of PAH. In concert with this notion is the recent work of Hashimoto-Kataoka and collaborators who did not find any beneficial effect of IL-17 axis blockade on hypoxia-induced PAH.27 In another animal study on hypoxia-induced PAH, natural Tregs were shown to protect against the development and progression of PAH.28 In our clinical samples, we did not find any increase in natural Treg cells, in contrast to putative induced Treg cells delineated by CD4+CD25−CD127− phenotype. Quite surprisingly, to our knowledge the role of induced Treg cells in PAH was never studied. Our finding of increased concentrations of induced Tregs in PAH patients indicates that performing further mechanistic studies involving administration and/or blockade of induced Tregs could provide novel cellular targets for the treatment of PAH. Interestingly, we demonstrated a significant negative correlation between Th17 cell frequencies and KYN concentrations. This suggests either the inhibitory effects of KYN are exerted on Th17 cells, or that Th17 cells could play a role in suppressing the intensity of TRP metabolism. The relationships between Th17 cells and TRP derivatives can be specific only in the setting of PAH, as contrasting patterns were observed in candidaemic patients wherein IL-17A and kynurenine concentrations were positively correlated with each other.29 Regardless of the specificity of the mechanism, these intriguing interactions suggest that experimental therapies targeting Th17-related mechanisms could exert a potential modulating effect on the kynurenine pathway.

The limitations of this study include its cross-sectional design, the relatively small number of subjects in each group, the heterogeneity of both the patient population and the disease-modifying therapies, and the inclusion of both incident and prevalent cases. Also, the study design was limited to the examination of systemic circulating concentrations of kynurenines and immune cells, not its local expression or IDO activity in particular cell subsets. It remains to be established whether elevated plasma concentrations of TRP and KYN reflect causal pathways or only epiphenomena of disease development. Thus, due to the exploratory character of the study, the results are still of interest and provide direction for future work. Interestingly, we observed the correlation of KYN values with parameters of renal function in PAH. The role of renal insufficiency in TRP metabolism has been documented in various clinical and experimental studies.9 ,10 ,12 Although renal function was only mildly impaired in very few PAH cases (probably related to the disease itself or the implementation of diuretic treatment), and the potential impact of this condition on our results seems to be rather weak, we cannot exclude it. This issue needs further investigation.

Conclusions

We have reported, for the first time, the potential contribution of TRP metabolism in PAH pathobiology and highlighted the potential interplay between kynurenines and the immune system in PAH. Our results support the growing observations that metabolic and inflammatory mechanisms overlap in PAH and may potentiate each other in triggering or amplifying pulmonary vascular remodelling in PAH. Thus, our data provide epidemiological support for further studies linking the KYN pathway to PAH pathobiology. This may be of particular value because the KYN axis can eventually be targeted by medical interventions such as IDO or TDO inhibitors.

Key messages

What is already known on this subject?

  • There is a large body of evidence showing that dysregulation of the immune system plays a significant role in the development and progression of pulmonary arterial hypertension (PAH). Particular attention has recently focused on regulatory T cells. Tryptophan catabolism via the kynurenine pathway has emerged as a powerful mechanism of peripheral immune tolerance. To date, clinical and experimental data focusing on the kynurenine pathway have been lacking for patients with PAH.

What might this study add?

  • We report for the first time the potential contribution of tryptophan metabolism in PAH pathobiology and highlight the potential interplay between kynurenines and the immune system in PAH.

How might this impact on clinical practice?

  • Our data provide epidemiological support to further studies linking the kynurenine pathway to PAH pathobiology. This may be of particular value because the kynurenine axis can eventually be targeted by medical intervention.

References

Footnotes

  • Contributors MJ: oversaw all activities related to the conduct of the study and contributed to the study idea, data collection, statistical analysis, discussion and writing of the manuscript. MM: contributed to the data collection, flow cytometry and writing of the manuscript. DP: contributed to the assays and discussion. MK: contributed to the data collection. MR: contributed to assays and flow cytometry. RK: contributed to data collection and statistical analysis. WJM: contributed to the editing of the manuscript. MD: contributed to the discussion. KAK: oversaw all activities related to the conduct of the study and contributed to the study idea, statistical analysis, discussion and editing of the manuscript. All authors read and approved the final manuscript.

  • Funding The study was supported by statutory grants from the Medical University of Bialystok, including funds from the National Leading Research Centre in the Medical University of Bialystok as well as by the National Science Centre grant (2011/01/N/ NZ5/04361, to MJ).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval Medical Ethics Committee, Medical University of Bialystok.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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