Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease

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.     

Endogenous neuroprotection in chronic neurodegenerative disorders: with particular regard to the kynurenines

Parkinson's disease (PD) and Huntington's disease (HD) are progressive chronic neurodegenerative disorders that are accompanied by a considerable impairment of the motor functions. PD may develop for familial or sporadic reasons, whereas HD is based on a definite genetic mutation. Nevertheless, the pathological processes involve oxidative stress and glutamate excitotoxicity in both cases. A number of metabolic routes are affected in these disorders. The decrease in antioxidant capacity and alterations in the kynurenine pathway, the main pathway of the tryptophan metabolism, are features that deserve particular interest, because the changes in levels of neuroactive kynurenine pathway compounds appear to be strongly related to the oxidative stress and glutamate excitotoxicity involved in the disease pathogenesis. Increase of the antioxidant capacity and pharmacological manipulation of the kynurenine pathway are therefore promising therapeutic targets in these devastating disorders.     

Tryptophan metabolism and oxidative stress in patients with Huntington's disease

Abnormalities in the kynurenine pathway may play a role in Huntington's disease (HD). In this study, tryptophan depletion and loading were used to investigate changes in blood kynurenine pathway metabolites, as well as markers of inflammation and oxidative stress in HD patients and healthy controls. Results showed that the kynurenine : tryptophan ratio was greater in HD than controls in the baseline state and after tryptophan depletion, indicating increased indoleamine dioxygenase activity in HD. Evidence for persistent inflammation in HD was provided by elevated baseline levels of C-reactive protein, neopterin and lipid peroxidation products compared with controls. The kynurenate : kynurenine ratio suggested lower kynurenine aminotransferase activity in patients and the higher levels of kynurenine in patients at baseline, after depletion and loading, do not result in any differences in kynurenic acid levels, providing no supportive evidence for a compensatory neuroprotective role for kynurenic acid. Quinolinic acid showed wide variations in blood levels. The lipid peroxidation data indicate a high level of oxidative stress in HD patients many years after disease onset. Levels of the free radical generators 3-hydroxykynurenine and 3-hydroxyanthranilic acid were decreased in HD patients, and hence did not appear to contribute to the oxidative stress. It is concluded that patients with HD exhibit abnormal handling of tryptophan metabolism and increased oxidative stress, and that these factors could contribute to ongoing brain dysfunction.     

Systemic metabolism of tryptophan and its catabolites,kynurenine and 3-HAA,in mice with inflammatory arthritis

Tryptophan is an essential amino acid. The liver is primary organ involved the oxidative catabolism of tryptophan. However, in the immune system, tryptophan and its catabolites, kynurenine and 3-hydroxyanthranilic acid (3-HAA), play an anti-inflammatory role. Rheumatoid arthritis (RA) is an autoimmune disease. Collagen induced arthritis (CIA) is an animal model of RA. Therefore, it was of interest to measure concentration of tryptophan, kynurenine and 3-HAA in mice with CIA. Concentration of tryptophan and 3-HAA was measured with HPLC methods. Concentration of kynurenine was measured with colorimetric test. mRNA expression for the kynurenine pathway genes was assessed using qRT-PCR. It has been found that in sera from diseased mice concentration of tryptophan was not changed. Concentration of kynurenine and 3-HAA was decreased. Moreover, in the livers from mice with CIA, concentration of tryptophan and kynurenine was decreased. These observations coincided with decreased mRNA expression for Ido2 and Afm and increased mRNA expression for Kynureninase in the liver. It has been also shown that in CIA the concentration of 3-HAA was increased in the kidneys.     

Purine,kynurenine, neopterin and lipid peroxidation levels in inflammatory bowel disease

The kynurenine metabolites of tryptophan may be involved in the regulation of neuronal activity and thus gut motility and secretion. We have now performed a pilot study to measure serum concentrations of purines and kynurenines in patients with mild inflammatory bowel disease, as well as in sex- and age-matched control subjects. For some analyses, the patients were subdivided into subgroups of those with Crohn's disease and those with ulcerative colitis. The analyses indicated an increased activity in one branch of the kynurenine pathway. While there was no demonstrable difference in neopterin levels in either of the patient groups compared with controls, indicating that the disorders were in an inactive quiescent phase, both groups showed significantly higher levels of lipid peroxidation products. This suggests the presence of increased oxidative stress even during relative disease inactivity. The increased level of kynurenic acid may represent either a compensatory response to elevated activation of enteric neurones or a primary abnormality which induces a compensatory increase in gut activity. In either case, the data may indicate a role for kynurenine modulation of glutamate receptors in the symptoms of inflammatory bowel disease.     

Expression of Tryptophan 2,3-Dioxygenase and Production of Kynurenine Pathway Metabolites in Triple Transgenic Mice and Human Alzheimer's Disease Brain

To assess the role of the kynurenine pathway in the pathology of Alzheimer''s disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.     

Kynurenine metabolism in health and disease

Kynurenine is a small molecule derived from tryptophan when this amino acid is metabolised via the kynurenine pathway. The biological activity of kynurenine and its metabolites (kynurenines) is well recognised. Therefore, understanding the regulation of the subsequent biochemical reactions is essential for the design of therapeutic strategies which aim to interfere with the kynurenine pathway. However, kynurenine concentration in the body may not only be determined by the efficiency of kynurenine synthesis but also by the rate of kynurenine clearance. In this review, current knowledge about the mechanisms of kynurenine production and routes of its clearance is presented. In addition, the involvement of kynurenine and its metabolites in the biology of different T cell subsets (including Th17 cells and regulatory T cells) and neuronal cells is discussed.     

Kynurenine Metabolism and Alzheimer’s Disease: The Potential Targets and Approaches

l-tryptophan, an essential amino acid, regulates protein homeostasis and plays a role in neurotransmitter-mediated physiological events. It also influences age-associated neurological alterations and neurodegenerative changes. The metabolism of tryptophan is carried majorly through the kynurenine route, leading to the production of several pharmacologically active enzymes, substrates, and metabolites. These metabolites and enzymes influence a variety of physiological and pathological outcomes of the majority of systems, including endocrine, haemopoietic, gastrointestinal, immunomodulatory, inflammatory, bioenergetic metabolism, and neuronal functions. An extensive literature review of PubMed, Medline, Bentham, Scopus, and EMBASE (Elsevier) databases was carried out to understand the nature of the extensive work done on the kynurenine metabolites that influence cellular redox potential, immunoregulatory mechanisms, inflammatory pathways, cell survival channels, and cellular communication in close association with several neurodegenerative changes. The imbalanced state of kynurenine pathways has found a close association to several pathological disorders, including HIV infections, cancer, autoimmune disorders, neurodegenerative and neurological disorders including Parkinson’s disease, epilepsy and has found special attention in Alzheimer's disease (AD). Kynurenine pathway (KP) is intricately linked to AD pathogenesis owing to the influence of kynurenine metabolites on excitotoxic neurotransmission, oxidative stress, uptake of neurotransmitters, and modulation of neuroinflammation, amyloid aggregation, microtubule disruption, and their ability to induce a state of dysbiosis. Pharmacological modulation of KP pathways has shown encouraging results, indicating that it may be a viable and explorable target for the therapy of AD.

     

Synthesis,accumulation and excretion of kynurenine during the pupal and adult stages of Papilio xuthus

Changes of kynurenine levels in fat body, haemolymph and wings during the pupal stage of Papilio xuthus were determined. In fat body and haemolymph, free kynurenine begins to increase at the time of the onset of eye pigmentation, reaches a maximum at the stage of, or shortly after red spot appearance in the wings and then rapidly decreases. The bound form of kynurenine seems not to be present. In the wings, on the contrary, the bound form of kynurenine begins to increase shortly before the appearance of red spot and accumulates up to the time of emergence.Tryptophan oxygenase activity is greatest in the fat body, with less in the wings and haemolymph did not show any activity.At the emergence of the butterfly, a considerable amount of kynurenine is excreted in the meconium. Empty pupal cases also contain some kynurenine. At the same time in the adult, the yellow scales contain the largest amounts of kynurenine, and the adult body (minus yellow scales) also contain a considerable amount of kynurenine.All these results suggest the possibility that kynurenine is synthesized in the fat body, is transported through the haemolymph and accumulates as the bound form in the yellow scales. But the possibility cannot be ruled out that some kynurenine is synthesized in the wings.     

Tryptophan degradation in mice initiated by indoleamine 2,3-dioxygenase

Tryptophan degradation in mice initiated by indoleamine 2,3-dioxygenase was characterized, taking advantage of its induction by bacterial lipopolysaccharide. Our results demonstrated that in various tissues, N-formylkynurenine produced by the dioxygenase from tryptophan was rapidly hydrolyzed into kynurenine by a kynurenine formamidase, but it was not further metabolized. The localization in the liver and kidney of the kynurenine-metabolizing enzymes suggested that kynurenine thus formed was transported by the bloodstream to those two organs to be metabolized. In fact, the plasma kynurenine level increased in parallel with the induction of the dioxygenase by lipopolysaccharide, and kinetic analysis indicated that at the maximal induction of the enzyme there was a 3-fold increase in the kynurenine production. The major metabolic route of kynurenine was excretion in urine as xanthurenic acid. This increase in the kynurenine production was not explained by L-tryptophan 2,3-dioxygenase in the liver, because during the induction of indoleamine 2,3-dioxygenase, the hepatic enzyme level was substantially suppressed. These findings indicated that indoleamine 2,3-dioxygenase actively oxidized tryptophan in mice and that its induction resulted in an increase in tryptophan degradation.     

The concentration of kynurenine in rat model of asthma

Asthma is a chronic inflammatory disease that involves the immune system activation. Evidence is accumulating about the role of kynurenine pathway in the immune system regulation. The kynurenine pathway includes several metabolites of tryptophan, among others kynurenine (KYN). To study the immunological system regulation in asthma a simple and sensitive models of asthma are required. In the present study we induced rat model of asthma using ovalbumin (OVA) sensitization followed by challenge with OVA. The development of asthma has been confirmed by plasma total IgE measurement and the histological examination. The concentration of KYN has been determined in plasma, lungs and liver by high-performance liquid chromatography (HPLC). In OVA sensitized rats the concentration of total IgE was statistically significantly increased as compared to VEH sensitized control groups (437.6 +/- 97.7 kU/l vs 159.2 +/- 22.7 kU/l, respectively; p< 0.01). In asthmatic animals, the number of eosinophils, neutrophils and mast cells increased considerably, and epithelial lesion and the increase in airway epithelium goblet cells and edema of bronchial mucosa were present. We did not observe any significant changes in the concentration of KYN in plasma, lungs or liver between studied groups. In conclusion, the concentration of KYN remains unchanged in asthmatic animals as compared to control groups. Further studies using rat model of asthma are warranted to establish the role of kynurenine pathway regulation in asthma.     

Determination of kynurenine by a simple gas—liquid chromatographic method applicable to urine,plasma, brain and cerebrospinal fluid

A simple, sensitive and specific method for the determination of kynurenine is described. This is based on alkaline cleavage of kynurenine, followed by solvent extraction, trifluoroacetylation and gas—liquid chromatography with electron capture detection. Using this method kynurenine has been determined in urine and plasma, and for the first time in brain and cerebrospinal fluid. Increases in kynurenine in brain, plasma and urine are demonstrated following tryptophan administration to man and rat.     

The kynurenine pathway of tryptophan metabolism detected in the nervous system of Drosophila melanogaster

Isolated thoracic ganglia were incubated in physiological solution containing 14C-tryptophan. After this procedure, they were homogenized in a 1 per cent solution of HCL in methanol, and supernatant was subjected to two-dimensional thin layer chromatography in the presence of tryptophan, kynurenine, 3-hydroxy kynurenine, as well as kynurenic, anthranilic and xanthurenic acids. The spots were cut out and counted by liquid scintillation technique. Except tryptophan, only kynurenine and 3-hydroxy kynurenine spots contained notable radioactivity. Therefore, at least the initial stages of kynurenine pathway operate in the nervous system of Drosophila melanogaster. This finding is in accordance with observations of the effects of kynurenines on insect behaviour.     

Investigation of the kynurenine pathway in Indoleamine 2, 3 dioxygenase deficient mice with inflammatory arthritis

Tryptophan is an essential amino acid involved in the protein synthesis, cognition, and immunity. Oxidative catabolism of tryptophan is executed by the sets of biochemical reactions collectively referred to as the kynurenine pathway. In the immune system, two distinct enzymes, Indoleamne 2,3 dioxygenase 1 (IDO1) and Indoleamine 2, 3 dioxygenase 2 (IDO2) can initiate metabolic flux through the kynurenine pathway. Rheumatoid arthritis is an autoimmune disease driven by the exacerbated immune response towards self antigens and characterized by the chronic inflammatory reaction of the diarthrodial joints. Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis. Using CIA in wild type (WT) and mice deficient with Indoleamine 2,3 dioxygenase (Ido1KO), it was of interest to test the impact of Ido1 deletion on the concentration of tryptophan and its catabolites as well as on mRNA expression for other genes on the kynurenine pathway. Here, when compared with samples taken from naïve WT animals and those with CIA, it was found that only in the inguinal lymph nodes (iLN) taken from Ido1KO mice with CIA tryptophan concentration was significantly increased. In contrast, mRNA expression for Ido2 was decreased in naïve as well as in the diseased iLN taken from Ido1KO mice. Deletion of Ido1 and reduced mRNA expression for Ido2 neither affected the concentration of the downstream metabolites of tryptophan nor mRNA expression for downstream genes on the kynurenine pathway in iLN. Moreover, the concentration of kynurenine in sera of mice with CIA was significantly decreased in Ido1KO mice with arthritis.     

Differential effects of kynurenine and tryptophan treatment on quinolinate immunoreactivity in rat lymphoid and non-lymphoid organs

Quinolinate is a tryptophan metabolite and an intermediary in nicotinamide adenine dinucleotide (NAD+) synthesis in hepatocytes. Kynurenine is an upstream metabolite in the same biochemical pathway. Under normal physiological conditions, kynurenine is thought to be produced primarily in the liver as an NAD+ precursor. However, during immune stimulation or inflammation, numerous extrahepatic tissues convert systemic tryptophan to kynurenine, and its concentration subsequently rises dramatically in blood. The fate and role of extrahepatic kynurenine are uncertain. In order to begin addressing this question, the present study was performed to determine which cell types can produce quinolinate from either systemic tryptophan or kynurenine. By using highly specific antibodies to protein-coupled quinolinate, we found that intraperitoneal injections of tryptophan led to increased quinolinate immunoreactivity primarily in hepatocytes, with moderate increases in tissue macrophages and splenic follicles. In contrast, intraperitoneal injections of kynurenine did not result in any significant increase in hepatocyte quinolinate immunoreactivity, but rather led to dramatic increases in immunoreactivity in tissue macrophages, splenic white pulp, and thymic medulla. These findings suggest that hepatocytes do not make significant use of extracellular kynurenine for quinolinate or NAD+ synthesis, and that, instead, extrahepatic kynurenine is preferentially metabolized by immune cells throughout the body. The possible significance of the preferential metabolism of kynurenine by immune cells during an immune response is discussed.     

GENETIC CONTROL OF CYTODIFFERENTIATION

The cells of the anterior region of the larval fatbody of Drosophila melanogaster accumulate kynurenine at the end of the third larval instar, whereas the cells of the posterior region are involved in pteridine metabolism. Through a series of transplantation experiments it has been demonstrated that the anterior fat cells synthesize kynurenine. The mutant vermilion lacks kynurenine, and the anterior fat cells of this mutant strain lack the autofluorescence characteristic of kynurenine. When the non-allelic suppressor gene is combined with vermilion, the synthesis of kynurenine is restored in the anterior fat cells, and some of the cells of the posterior region contain kynurenine as well. A similar extension in the number of cells containing kynurenine can be induced in the normal Ore-R strain by feeding the precursor tryptophan. It has been concluded that the absence of a physiological process in a differentiated cell does not necessarily represent a loss of the genetic potential for that process. The normal allele at the suppressor locus inhibits the occurrence of kynurenine in the posterior fat cells, whereas the mutant allele su²-s allows the expression of this potential. An inducer such as tryptophan can overcome this inhibition in the normal strain, and as a result the cells which are normally differentiated as "isoxanthopterin cells" may produce kynurenine as well.     

Kynurenine Pathway Measurements in Huntington''s Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid

Recent evidence suggests that there may be overactivation of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors in Huntington's disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16-sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p less than 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.