Chronic stress or inflammation increases tryptophan metabolism along the kynurenine pathway (KP), and the generation of neuroactive kynurenine metabolites contributes to subsequent depressive-like behaviors. Microglia regulate KP balance by preferentially producing oxidative metabolites, including quinolinic acid. Research has focused on the interplay between cytokines and HPA axis-derived corticosteroids in regulating microglial activity and effects of KP metabolites directly on neurons; however, the potential role that KP metabolites have directly on microglial activity is unknown. Here, murine microglia were stimulated with lipopolysaccharide(LPS). After 6 h, mRNA expression of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α and inducible nitric oxide synthase(iNOS) was dose-dependently increased along with the rate-limiting enzymes for oxidative KP metabolism, indoleamine-2,3-dioxygenase(IDO)-1 and kynurenine 3-monooxygenase(KMO). By 24 h post-LPS, kynurenine and quinolinic acid in the media was elevated. Inhibiting KMO with Ro 61-8048 during LPS challenge attenuated extracellular nitrite accumulation and expression of KMO and TNF-α in response to LPS. Similarly, primary microglia isolated from KMO-/- mice exhibited a significantly reduced pro-inflammatory response to LPS compared to WT controls. To determine whether the substrate (kynurenine) or end product (quinolinic acid) of KMO-dependent metabolism modulates the LPS response, microglia were treated with increasing concentrations of L-kynurenine or quinolinic acid in combination with LPS or saline. Interestingly, quinolinic acid did not impact the microglial LPS response. However, L-kynurenine had dose-dependent inhibitory effect on the LPS response. These data are the first to show an anti-inflammatory effect of KMO inhibition on microglia during immune challenge and suggest that KP metabolic balance may play a direct role in regulating microglia activity.

Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP), KYN and α-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.

Glutamate receptors sensitive to N-methyl-D-aspartate (NMDA) are involved in embryonic brain development but their activity may be modulated by the kynurenine pathway of tryptophan metabolism which includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Our previous work has shown that prenatal inhibition of the pathway produces abnormalities of brain development. In the present study kynurenine and probenecid (both 100mg/kg, doses known to increase kynurenic acid levels in the brain) were administered to female Wistar rats on embryonic days E14, E16 and E18 of gestation and the litter was allowed to develop to post-natal day P60. Western blotting revealed no changes in hippocampal expression of several proteins previously found to be altered by inhibition of the kynurenine pathway including the NMDA receptor subunits GluN1, GluN2A and GluN2B, as well as doublecortin, Proliferating Cell Nuclear Antigen (PCNA), sonic hedgehog and unco-ordinated (unc)-5H1 and 5H3. Mice lacking the enzyme kynurenine-3-monoxygenase (KMO) also showed no changes in hippocampal expression of several of these proteins or the 70-kDa and 100-kDa variants of Disrupted in Schizophrenia-1 (DISC1). Electrical excitability of pyramidal neurons in the CA1 region of hippocampal slices was unchanged, as was paired-pulse facilitation and inhibition. Long-term potentiation was decreased in the kynurenine-treated rats and in the KMO(-/-) mice, but galantamine reversed this effect in the presence of nicotinic receptor antagonists, consistent with evidence that it can potentiate glutamate at NMDA receptors. It is concluded that interference with the kynurenine pathway in utero can have lasting effects on brain function of the offspring, implying that the kynurenine pathway is involved in the regulation of early brain development.

The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by approximately 50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.

The tryptophan metabolite kynurenic acid (KYNA) may play an important role in normal and abnormal cognitive processes, most likely by interfering with α7 nicotinic and NMDA receptor function. KYNA is formed from its immediate precursor kynurenine either by non-enzymatic oxidation or through irreversible transamination by kynurenine aminotransferases. In the mammalian brain, kynurenine aminotransferase II (KAT II) is the principal enzyme responsible for the neosynthesis of rapidly mobilizable KYNA, and therefore constitutes an attractive target for pro-cognitive interventions. N-acetylcysteine (NAC), a brain-penetrant drug with pro-cognitive efficacy in humans, has been proposed to exert its actions by increasing the levels of the anti-oxidant glutathione (GSH) in the brain. We report here that NAC, but not GSH, inhibits KAT II activity in brain tissue homogenates from rats and humans with IC50 values in the high micromolar to low millimolar range. With similar potency, the drug interfered with the de novo formation of KYNA in rat brain slices, and NAC was a competitive inhibitor of recombinant human KAT II (Ki: 450 μM). Furthermore, GSH failed to S-glutathionylate recombinant human KAT II treated with the dithiocarbamate drug disulfiram. Shown by microdialysis in the prefrontal cortex of rats treated with kynurenine (50 mg/kg, i.p.), peripheral administration of NAC (500 mg/kg, i.p., 120 and 60 min before the application of kynurenine) reduced KYNA neosynthesis by ∼50%. Together, these results suggest that NAC exerts its neurobiological effects at least in part by reducing cerebral KYNA formation via KAT II inhibition.

The thermal and photochemical reactivity of kynurenine (KN), a tryptophan metabolite found in human lenses, has been studied in aqueous solution. The decarboxylation reaction of KN, resulting in the formation of 4-hydroxyquinoline, is reported for the first time. Rate constants for KN deamination and decarboxylation were determined in the temperature range 50-90 degrees C. The quantum yields for KN photodecomposition under argon were measured to be Phi Ar=(2.0+/-0.2) x 10(-5) and under oxygen Phi O2=(1.1+/-0.1) x 10(-4).

Psoriasis is a common dermatosis which affects the patient's skin and general well-being because of its link to diseases such as depression, kidney disease and metabolic syndrome. Pathogenesis remains unknown; however, genetic, environmental and immunological factors seem to play a role in the development of the disease. Due to a lack of complete understanding of the psoriasis pathology, effective treatment is yet to be developed. The kynurenine pathway is one of the ways amino acid tryptophan is metabolised. In comorbidities typical for psoriasis such as chronic kidney disease, depression and atherosclerotic alterations in the activation of the kynurenine pathway were observed, which were mainly characterised by higher activity compared to that in healthy individuals. However, the kynurenine pathway has not been thoroughly studied among patients with psoriasis even though increased levels of L-kynurenine, one of the enzymes in the kynurenine pathway, were found in psoriatic skin lesions. Given the unknown pathogenesis of the disease, this finding seems to be a potential new field of study and shows a possible link between psoriasis and its comorbidities that could also lead to novel effective treatment for this chronic condition.

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer's disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases.

Neurosurgical procedures result in surgically induced brain injury (SBI) that causes postoperative complications including brain edema and neuronal apoptosis in the surrounding brain tissue. SBI leads to the release of cytokines that indirectly cause the stimulation of kynurenine 3-monooxygenase (KMO) and the release of neurotoxic quinolinic acid (QUIN). This study tested a KMO inhibitor, RO 61-8048, to prevent postoperative brain edema and consequent neuronal apoptosis in an in vivo model of SBI. A rodent model of SBI was utilized which involves partial resection of the right frontal lobe. A total of 127 Sprague-Dawley male rats (weight 275-325 g) were randomly divided into the following groups: Sham surgical group, SBI, SBI + DMSO, SBI + RO 61-8048 (10 mg/kg), SBI + RO 61-8048 (40 mg/kg), and SBI + RO 61-8048 (40 mg/kg) + KAT II inhibitor PF-04859989 (5 mg/kg). RO 61-8048 was administered by intraperitoneal injection after SBI. Postoperative assessment at different time points included brain water content (brain edema), neurological scoring, and western blot. SBI increased brain water content (ipsilateral frontal lobe), decreased neurological function, and increased apoptotic markers compared with sham animals. Treatment with RO 61-8048 (40 mg/kg) reduced brain water content and improved long-term neurological function after SBI. RO 61-8048 increased the expression of kynurenic acid while reducing QUIN and apoptotic markers in the surrounding brain tissue after SBI. These neuroprotective effects were reversed by PF-04859989. This study suggests KMO inhibition via RO 61-8048 as a potential postoperative therapy following neurosurgical procedures.

Epigenetic mechanisms can establish and maintain mitotically stable patterns of gene expression while retaining the DNA sequence. These mechanisms can be affected by environmental factors such as nutrients. The importance of intracellular dosages of nutrient metabolites such as acetyl coenzyme A and S-adenosylmethionine, which are utilized as donors for post-translational modifications, is well-known in epigenetic regulation; however, the significance of indirect metabolites in epigenetic regulation is not clear. In this study, we screened for metabolites that function as epigenetic modulators. Because the expression of genes related to hypothalamic function is reportedly affected by nutritional conditions, we used a neural cell culture system and evaluated hypothalamic-linked loci. We supplemented the culture medium with 129 metabolites separately during induction of human-iPS-derived neural cells and used high-throughput ChIP-qPCR to determine the epigenetic status at 37 hypothalamus-linked loci. We found three metabolites (kynurenine, 3-OH-kynurenine, and anthranilate) from tryptophan pathways that increased H3K4 trimethylation and H2AS40 O-GlcNAcylation, resulting in upregulated gene expression at most loci, except those encoding pan-neural markers. Dietary supplementation of these three metabolites and the resulting epigenetic modification were important for stability in gene expression. In conclusion, our findings provide a better understanding of how nutrients play a role in epigenetic mechanisms.

Cutaneous sclerosis, resembling that seen in subcutaneous morphea, is a feature of eosinophilic fasciitis and eosinophilia-myalgia syndrome, two conditions in which the kynurenine pathway is known to be activated. To investigate the possibility of activation of the kynurenine pathway in morphea, skin biopsies were taken from involved and non-involved sites in a series of three patients with morphea. Immunohistochemical stains for quinolinic acid and indoleamine 2,3-dioxygenase (IDO) were performed.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. The enzyme indoleamine-2,3-dioxygenase (IDO), which participates in the rate-limiting step of tryptophan catabolism through the kynurenine pathway (KP), is associated with poor prognosis in patients with GBM. The metabolites produced after tryptophan oxidation have immunomodulatory properties that can support the immunosuppressor environment. In this study, mRNA expression, protein expression, and activity of the enzyme kynurenine monooxygenase (KMO) were analyzed in GBM cell lines (A172, LN-18, U87, U373) and patient-derived astrocytoma samples. KMO mRNA expression was assessed by real-time RT-qPCR, KMO protein expression was evaluated by flow cytometry and immunofluorescence, and KMO activity was determined by quantifying 3-hydroxykynurenine by HPLC. Heterogenous patterns of both KMO expression and activity were observed among the GBM cell lines, with the A172 cell line showing the highest KMO expression and activity. Higher KMO mRNA expression was observed in glioma samples than in patients diagnosed with only a neurological disease; high KMO mRNA expression was also observed when using samples from patients with GBM in the TCGA program. The KMO protein expression was localized in GFAP+ cells in tumor tissue. These results suggest that KMO is a relevant target to be explored in glioma since it might play a role in supporting tumor metabolism and immune suppression.

The up-regulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol conjugates and saturated metabolites. Kyn-CKA induces NFE2 like bZIP transcription factor 2- and aryl hydrocarbon receptor-regulated genes and inhibits nuclear factor κB- and NLR family pyrin domain containing 3-dependent proinflammatory signaling. Sickle cell disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both transgenic SCD mice and patients with SCD exhibit increased kynurenine and Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is up-regulated during pathogenic vascular stress. Administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in lung injury development. These findings demonstrate that the up-regulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.

Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes.

The activation of the kynurenine pathway in cancer progression and metastasis through immunomodulatory pathways has drawn attention to the potential for kynurenine pathway inhibition. The activation of the kynurenine pathway, which results in the production of kynurenine metabolites through the degradation of tryptophan, promotes the development of intrinsically malignant properties in cancer cells while facilitating tumour immune escape. In addition, kynurenine metabolites act as biologically active substances to promote cancer development and metastasis.

The kynurenine pathway (KP) is the principal route of L-tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), the excitotoxin, quinolinic acid (QUIN) and the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2,3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. IDO-1 and TDO-2 induction in tumors are crucial mechanisms implicated to play pivotal roles in suppressing anti-tumor immunity. Here, we report the first comprehensive characterisation of the KP in 1) cultured human glioma cells and 2) plasma from patients with glioblastoma (GBM). Our data revealed that interferon-gamma (IFN-γ) stimulation significantly potentiated the expression of the KP enzymes, IDO-1 IDO-2, kynureninase (KYNU), kynurenine hydroxylase (KMO) and significantly down-regulated 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) and kynurenine aminotransferase-I (KAT-I) expression in cultured human glioma cells. This significantly increased KP activity but significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. KP activation (KYN/TRP) was significantly higher, whereas the concentrations of the neuroreactive KP metabolites TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlight a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumors, aimed at restoring anti-tumor immunity and reducing the capacity for malignant cells to produce NAD(+), which is necessary for energy production and DNA repair.

Recently, neuroinflammation and the immune-kynurenine pathway have received increased attention in the psychoimmunology field of major depressive disorder (MDD), while studies related to anxiety disorders have been very limited.

Regulatory and functional aspects of the kynurenine (K) pathway (KP) of tryptophan (Trp) degradation are reviewed. The KP accounts for ~95% of dietary Trp degradation, of which 90% is attributed to the hepatic KP. During immune activation, the minor extrahepatic KP plays a more active role. The KP is rate-limited by its first enzyme, Trp 2,3-dioxygenase (TDO), in liver and indoleamine 2,3-dioxygenase (IDO) elsewhere. TDO is regulated by glucocorticoid induction, substrate activation and stabilization by Trp, cofactor activation by heme, and end-product inhibition by reduced nicotinamide adenine dinucleotide (phosphate). IDO is regulated by IFN-γ and other cytokines and by nitric oxide. The KP disposes of excess Trp, controls hepatic heme synthesis and Trp availability for cerebral serotonin synthesis, and produces immunoregulatory and neuroactive metabolites, the B3 "vitamin" nicotinic acid, and oxidized nicotinamide adenine dinucleotide. Various KP enzymes are undermined in disease and are targeted for therapy of conditions ranging from immunological, neurological, and neurodegenerative conditions to cancer.

Several enzymes and metabolites of the kynurenine pathway (KP) have immunomodulatory effects. Modulation of the activities and levels of these molecules might be of particular importance under disease conditions when the amelioration of overreacting immune responses is desired. Results obtained by the use of animal and tissue culture models indicate that by eliminating or decreasing activities of key enzymes of the KP, a beneficial shift in disease outcome can be attained. This review summarizes experimental data of models in which IDO, TDO, or KMO activity modulation was achieved by interventions affecting enzyme production at a genomic level. Elimination of IDO activity was found to improve the outcome of sepsis, certain viral infections, chronic inflammation linked to diabetes, obesity, aorta aneurysm formation, and in anti-tumoral processes. Similarly, lack of TDO activity was advantageous in the case of anti-tumoral immunity, while KMO inhibition was found to be beneficial against microorganisms and in the combat against tumors, as well. On the other hand, the complex interplay among KP metabolites and immune function in some cases requires an increase in a particular enzyme activity for the desired immune response modulation, as was shown by the exacerbation of liver fibrosis due to the elimination of IDO activity and the detrimental effects of TDO inhibition in a mouse model of autoimmune gastritis. The relevance of these studies concerning possible human applications are discussed and highlighted. Finally, a brief overview is presented on naturally occurring genetic variants affecting immune functions via modulation of KP enzyme activity.