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It is well-known that the pregnane X receptor (PXR)/Nr1i2 is a critical xenobiotic-sensing nuclear receptor enriched in liver and intestine and is responsible for drug-drug interactions, due to its versatile ligand binding domain (LBD) and target genes involved in xenobiotic biotransformation. PXR can be modulated by various xenobiotics including pharmaceuticals, nutraceuticals, dietary factors, and environmental chemicals. Microbial metabolites such as certain secondary bile acids (BAs) and the tryptophan metabolite indole-3-propionic acid (IPA) are endogenous PXR activators. Gut microbiome is increasingly recognized as an important regulator for host xenobiotic biotransformation and intermediary metabolism. PXR regulates and is regulated by the gut-liver axis. This review summarizes recent research advancements leveraging pharmaco- and toxico-metagenomic approaches that have redefined the previous understanding of PXR. Key topics covered in this review include: (1) genome-wide investigations on novel PXR-target genes, novel PXR-DNA interaction patterns, and novel PXR-targeted intestinal bacteria; (2) key PXR-modulating activators and suppressors of exogenous and endogenous sources; (3) novel bidirectional interactions between PXR and gut microbiome under physiologic, pathophysiological, pharmacological, and toxicological conditions; and (4) modifying factors of PXR-signaling including species and sex differences and time (age, critical windows of exposure, and circadian rhythm). The review also discusses critical knowledge gaps and important future research topics centering around PXR. SIGNIFICANCE STATEMENT: This review summarizes recent research advancements leveraging O'mics approaches that have redefined the previous understanding of the xenobiotic-sensing nuclear receptor pregnane X receptor (PXR). Key topics include: (1) genome-wide investigations on novel PXR-targeted host genes and intestinal bacteria as well as novel PXR-DNA interaction patterns; (2) key PXR modulators including microbial metabolites under physiological, pathophysiological, pharmacological, and toxicological conditions; and (3) modifying factors including species, sex, and time.

Objective The aryl hydrocarbon receptor (AHR) plays a key role in obesity. In vitro studies revealed that the tryptophan metabolite kynurenine (Kyn) activates AHR signaling in cultured hepatocytes. The objective of this study was to determine whether Kyn activated the AHR in mice to induce obesity. Methods Mice were fed a low-fat diet and the same diet supplemented with Kyn. Body mass, liver status, and the expression of identified relevant genes were determined. Results Kyn caused mice to gain significant body mass, develop fatty liver and hyperglycemia, and increase expression levels of cytochrome P450 1B1 and stearoyl-CoA desaturase 1. The hyperglycemia was accompanied with decreased insulin levels, which may have been due to the repression of genes involved in insulin secretion. Kyn plasma concentrations and BMI were measured in female patients, and a significant association was observed between Kyn and age in patients with obesity but not in patients who were lean. Conclusions Results show that (1) Kyn or a metabolite thereof is a ligand responsible for inducing AHR-based obesity, fatty liver, and hyperglycemia in mice; (2) plasma Kyn levels increase with age in women with obesity but not in lean women; and (3) an activated AHR is necessary but not sufficient to attain obesity, a status that also requires fat in the diet.

Several risk factors are known to be involved in the initiation and development of gastric cancer. Among them, H. pylori is one of the most prominent with multiple virulence factors contributing to its pathogenicity. In this study, we have discussed an interesting immunological cycle exploring the interplay between H. pylori, aryl hydrocarbon receptor (AHR), tryptophan, arginine, and the metabolites of these two amino acids in the development of gastric cancer. AHR is a ligand-activated transcription factor which acts as a regulator for a diverse set of genes and has various types of exogenous and endogenous ligands. The tryptophan metabolite, kynurenine, is one of these ligands that can interact with AHR, leading to immune suppression and subsequently, susceptibility to gastric cancer. On the other hand, H. pylori downregulates the expression of AHR and AHR repressor (AHRR), leading to increased inflammatory cytokine production. A metabolite of the kynurenine pathway, xanthurenic acid, is a potent inhibitor of a terminal enzyme in the synthetic pathway of tetrahydrobiopterin (BH4). BH4, itself, is a cofactor in the process of nitric oxide (NO) production from arginine that has been shown to have immune-enhancing properties. Arginine has also been evidenced to have anti-tumoral function through inducing apoptosis in gastric cell lines; however, controversy exists regarding the anti-tumor role of arginine and BH4, since they are also associated with increased NO production, subsequently promoting tumor angiogenesis. Hence, although several synergistic connections result in immunity improvement, these correlations can also act as a double-edged sword, promoting tumor development. This emphasizes on the need for further investigations to better understand this complex interplay.

Tryptophan and metabolites have important biological functions in humans. Milk is an important source of tryptophan intake. In this study, we developed a method to detect levels of tryptophan and 12 metabolites in milk. The analytes were extracted by using the QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure and analyzed by liquid chromatography-tandem mass spectrometry with electrospray ionization. The proposed method resulted in suitable accuracy (standard deviation ≤10.31%) and high sensitivity (the limits of quantification were between 0.05 and 5 ng/mL). Recoveries were in the range of 44 to 126%. Finally, the developed method was successfully applied to compare the content of tryptophan and metabolites in 4 milk products produced by different processes: pasteurized milk, UHT milk, milk powder, and yogurt. The results of partial least squares-discriminant analysis (PLS-DA) showed that different types of processed milk could be distinguished clearly according to the method used here. The determined tryptophan and metabolites levels in milk can provide a new reference for evaluation of milk.

Recent advances in our understanding of the pathophysiology of atopic dermatitis (AD) have revealed that skin microbiome dysbiosis plays an important role in the disease. In this review, we describe how changes in the structure and function of the microbiome are involved in the pathogenesis of AD. We highlight recent data showing that differential changes in microbial diversity, both within and across communities from different body habitats (including the skin, gut, and oral mucosa), are associated with the development and severity of AD. We also describe recent evidence demonstrating that the metabolic activity of the skin microbiome can act as a regulator of inflammation, with alterations in the level of a skin microbiome-derived tryptophan metabolite, indole-3-aldehyde (IAId), being shown to play a role in AD. The various mechanisms by which interactions between the microbiome and components of the non-histaminergic pathway result in itch in AD are also discussed.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is refractory to available treatments. Delineating the regulatory mechanisms of metabolic reprogramming, a key event in pancreatic cancer progression, may identify candidate targets with potential therapeutic significance. We hypothesized that inflammatory signaling pathways regulate metabolic adaptations in pancreatic cancer. Metabolic profiling of tumors from PDAC patients with a high‐ (>median, N=31) and low‐NOS2 (inducible nitric oxide synthase) (•(nitric oxide)‐mediated signaling pathway was elucidated. The level of kynurenine, a tryptophan metabolite, was associated with high NOS2 expression, and a higher level of kynurenine predicted poor survival in patients (N=63,P= 0.01). Gene expression analysis in PDAC tumors (N=63) showed a positive correlation between the expression of NOS2 and the tryptophan/kynurenine pathway genes, including indoleamine‐2,3‐dioxygenase 1 (IDO1) and several aryl hydrocarbon receptor (AHR)‐target genes includingNFE2L2(NRF2),SERPINB2, IL1b,IL6andIL8, which are implicated in pancreatic cancer. Consistently, treatment of pancreatic cancer cell lines with NO•donor induced IDO1, kynurenine production, and the expression of AHR‐target genes. Furthermore, kynurenine treatment enhanced spheroid growth and invasive potential of pancreatic cancer cell lines. Mechanistically, NO•‐induced IDO1/Kynurenine/AHR signaling was mediated by RUNX3 transcription factor. Our findings identified a novel NO•/RUNX3/Kynurenine metabolic axis, which enhances disease aggressiveness in pancreatic cancer and may have potential translational significance in improving disease outcome.

RATIONALE: Stress is related to cognitive impairments which are observed in most major brain diseases. Prior studies showed that the brain concentration of the tryptophan metabolite kynurenic acid (KYNA) is modulated by stress, and that changes in cerebral KYNA levels impact cognition. However, the link between these phenomena has not been tested directly so far. OBJECTIVES: To investigate a possible causal relationship between acute stress, KYNA and fear discrimination. METHODS: Adult rats were exposed to one of three acute stressors - restraint, predator odor, or inescapable foot shocks (ISS) - and KYNA in the prefrontal cortex was measured using microdialysis. Corticosterone was analyzed in a subset of rats. Another cohort underwent a fear discrimination procedure immediately after experiencing stress. Different auditory conditioned stimuli (CSs) were either paired with foot shock (CS+) or non-reinforced (CS−). One week later, fear was assessed by re-exposing rats to each CS. Finally, to test whether stress-induced changes in KYNA causally impacted fear discrimination, a group of rats that received ISS were pre-treated with the selective KYNA synthesis inhibitor PF-04859989. RESULTS: ISS caused the greatest increase in circulating corticosterone levels and raised extracellular KYNA levels by ~85%. The two other stressors affected KYNA much less (

In allergen-specific immunotherapy for asthma, antigens attached to dendritic cells increase the tryptophan metabolism in these cells and alter the Th17/Treg balance in the airways. Tryptophan metabolism has long been suggested to be relevant in the pathophysiology of allergic disorders, including asthma. Our study investigated whether tryptophan metabolites are responsible for the changes in Th17/Treg balance and decreases in airway hyperreactivity and inflammation seen during allergen-specific immunotherapy in an asthma model. Ovalbumin was injected intraperitoneally into mice to establish an asthma model, and then high dose ovalbumin allergen-specific immunotherapy was administered to induce immune tolerance. Airway hyperreactivity and serum ovalbumin-specific immunoglobulin E were measured to assess whether the animal model was successfully established. We then examined the influence of inhibition of tryptophan metabolism and the addition of tryptophan metabolites on allergen-specific immunotherapy-induced changes in the Th17/Treg balance and decreases in airway inflammation and inflammatory cytokines. Production of tryptophan metabolites was partly responsible for the allergen-specific immunotherapy-induced increase in Tregs, decrease in airway inflammation, and decrease in inflammatory cytokines. Ovalbumin-specific immunoglobulin E and airway hyperreactivity were not affected. In the context of asthma, an increase in tryptophan metabolites is one of the mechanisms by which allergen-specific immunotherapy achieves immune tolerance.

Tryptophan is an essential amino acid for the human body, whose intake is through the diet. Several studies support the theory that microbiota-derived tryptophan metabolite played a crucial role in maintaining the balance between gut microbiota and the mucosal immune system. Previously, we selected the Lactobacillus plantarum KLDS 1.0386 strain with high tryptophan-metabolic activity after the screening of 16 Lactobacillus strains. The current study aimed to assess the effects of L. plantarum KLDS 1.0386 combination with tryptophan in improving ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) and the potential mechanisms involved. Our results showed that L. plantarum KLDS 1.0386 combined with tryptophan (LAB + Trp) decreased DAI score, MPO level, and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) concentration. It also increased anti-inflammatory cytokine (IL-10) production, tight junction proteins (claudin-1, occludin, and ZO-1), and mucin (MUC1 and MUC2) mRNA expressions. The level of indole-3-acetic acid (IAA), an important tryptophan metabolite in the liver, serum, and colon, was elevated after LAB + Trp treatment, which further upregulated aryl hydrocarbon receptor (AHR) mRNA expression to activate the IL-22/STAT3 signaling pathway. Moreover, the supplementation with LAB + Trp modulated gut microbiota composition. The present study provided novel insights that can be used to reduce the number of UC patients by employing a method utilizing tryptophan-catabolizing Lactobacillus strains.

Tryptophan and kynurenine pathway (KP) metabolites are implicated in the pathophysiology of depression. We aimed to investigate their plasma concentrations in medicated patients with depression (n = 94) compared to age- and sex-matched healthy controls (n = 57), and in patients with depression after electroconvulsive therapy (ECT) in a real-world clinical setting, taking account of co-variables including ECT modality and heterogenous psychopathology. Depression severity was assessed using the Hamilton Depression Rating Scale (HAM-D24). Tryptophan (TRP) and kynurenine (KYN) metabolite concentrations [anthranilic acid (AA), 3-hydroxyanthranilic acid (3HAA), picolinic acid (PA), kynurenic acid (KYNA), and xanthurenic acid (XA)] and KYNA/KYN and KYNA/quinolinic acid (QUIN) ratios were lower in patients compared to controls. For the total group there was no significant change in KP metabolites post-ECT or correlations with mood ratings. However, improvements in mood score were correlated with increased KYN, 3-hydroxykynurenine (3HK), 3HAA, QUIN, and KYN/TRP in a subgroup of unipolar depressed patients. Additionally, in remitters baseline KYN, 3HK, and QUIN were associated with baseline HAM-D24 scores, and changes in 3HK and 3HAA concentrations post-ECT correlated with improvement in mood. KYN, KYNA, AA, 3HK, XA, PA, and QUIN were increased in a smaller 3-month follow-up group (n = 19) compared to pre-ECT concentrations. Overall, the results indicate that ECT mobilizes the KP, where a moderate association between selected metabolites and treatment response in unipolar depressed patients is evident.

The moust symptoms of piglets infected with Encephalomyocarditis virus (EMCV) are related to breeding difficulty, circulation insufficiency, depression and occurrence of high lethality. An increase of tryptophan metabolism in the periphery and in the central nervous system (CNS) in human and non-human subjects with inflammatory diseases has been suggested. We investigated an alterations of tryptophan metabolite i.e. kynurenic acid (KYNA) level in the serum of piglets after EMC virus infection. In addition, we investigated the markers of immune stimulation i.e. neopterin and β2-microglobulin.KYNA was determined by high performance liquid chromatography method, while neopterin and β2-microglobulin by ELISA method. Piglets with an age of 8 weeks were infected intranasal and orally with the EMC virus. Blood samples were collected before virus inoculation at day 0 (control) and at 1, 2, 3 and 4 days post inoculation (DPI) and piglets as control subjects were used, too.In EMCV infected piglets we observed a time dependent alteration of investigated parameters. KYNA level increased significantly and at 3 DPI was 341% of CO, p0.001 and at 4 DPI an enhancement was 242% of CO, p0.001, respectively. Neopterin increased moderately after EMCV infection and at 4 DPI was 130% of CO, p0.05. Serum β2-microglobulin was slightly lowered and at 4 DPI was 86% of CO, p0.05. Present data indicate an marked increase of kynurenine metabolism in the periphery after EMCV infection and an moderate activation of immune system.A marked increase of KYNA and a moderate enhancement of neopterin indicate sensibility of kynurenine metabolism to EMCV infection. Lowering of ß2-microgobulin might relate to development of events leading to the lethality. We suggest that due to viral infection an increase of KYNA might contribute to the inpairment of organs in the periphery and CNS function and might participate by sudden death.

Objective: Lithium is a cornerstone treatment option in bipolar disorder. Currently, only clinical findings are used to predict the response of lithium treatment but reliable biological marker has not yet been identified. With this background, it was aimed to compare tryptophan metabolite levels between bipolar patients and healthy control group and to investigate the relationship between lithium treatment response and tryptophan metabolite levels of euthymic bipolar patients(BP). Materials and Methods: 52 euthymic BP patients on lithium were included in the study. Sociodemographic and clinical information were recorded and lithium treatment response status was determined according to the Alda scale. Age and gender matched Healthy controls(HC) were selected from the people who admitted to Selcuk University Medical Faculty Hospital Health Board. Plasma tryptophan metabolite levels were compared between BP and HC. The correlation and association between the lithium response levels of the patients and the plasma tryptophan metabolites were evaluated. Results: Plasma tryptophan(TRP), kynurenine(KYN), kynurenine/tryptophan ratio(KYN/TRP), 3-hydroxy-kynurenine(3-HK) and kynurenic acid(KYNA) levels were significantly lower in BP than HC. In the regression analysis, only plasma KYNA concentrations were found to be significantly lower in the responding well to lithium treatment and TRP levels. None of the tryptophan metabolites were found to be associated well response to lithium treatment after the logistic regression analysis. Conclusion: Our findings indicate that KYNA levels might be used as a screening test for discriminating bipolar patients from the HC. TRP levels might be used to predict lithium treatment response in spite of low specifity and low sensitivity levels. Key words: Tryptophan metabolites, lithium treatment response, bipolar disorder

The cellular and molecular mechanisms underlying loss of muscle mass with age (sarcopenia) are not well-understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is a ligand of the aryl hydrocarbon receptor (Ahr). Here, we tested the hypothesis that KYN activation of Ahr plays a role in muscle loss with aging. Results indicate that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) 2-fold and KYN treatment in vivo reduced muscle size and strength and increased muscle lipid peroxidation in young mice. PCR array data indicate that muscle fiber size reduction with KYN treatment reduces protein synthesis markers whereas ubiquitin ligase gene expression is not significantly increased. KYN is generated by the enzyme indoleamine 2,3-dioxygenase (IDO), and aged mice treated with the IDO inhibitor 1-methyl-D-tryptophan showed an increase in muscle fiber size and muscle strength. Small-molecule inhibition of Ahr in vitro, and Ahr knockout in vivo, did not prevent KYN-induced increases in ROS, suggesting that KYN can directly increase ROS independent of Ahr activation. Protein analysis identified very long-chain acyl-CoA dehydrogenase as a factor activated by KYN that may increase ROS and lipid peroxidation. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the prevention of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.

The neurotoxic activity of the tryptophan metabolite 3-hydroxykynurenine (3OHKyn) in neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, is related to oxidative stress and 3OHKyn interaction with cellular proteins. The pattern of protein modification induced by 3OHKyn involves the nucleophilic side chains of Cys, His, and Lys residues, similarly to the one promoted by dopamine and other catecholamines. In the present work, we have analyzed the reactivity of 3OHKyn toward the neuronal targets alpha-synuclein (and its N-terminal fragments 1-6 and 1-15) and amyloid-beta peptides (1-16 and 1-28) and characterized the resulting conjugates through spectrometric (LCMS/MS) and spectroscopic (UV-vis, fluorescence, NMR) techniques. The amino acid residues of alpha-synuclein and amyloid-beta peptides involved in derivatizations by 3OHKyn and its autoxidation products (belonging to the xanthommatin family) are Lys and His, respectively. The pattern of protein modification is expanded in the conjugates obtained in the presence of the metal ions copper(II) or iron(III), reflecting a more oxidizing environment that in addition to adducts with protein/peptide residues also favors the fragmentation of the protein. These results open the perspective to using the 3OHKyn-protein/peptide synthetic conjugates to explore their competence to activate microglia cell cultures as well as to unravel their role in neuroinflammatory conditions.

Polybrominated diphenyl ethers (PBDEs) are persistent environmental toxicants associated with increased risk for metabolic syndrome. Intermediary metabolism is influenced by the intestinal microbiome. To test the hypothesis that PBDEs reduce host-beneficial intermediary metabolites in an intestinal microbiome–dependent manner, 9-week old male conventional (CV) and germ-free (GF) C57BL/6 mice were orally gavaged once daily with vehicle, BDE-47, or BDE-99 (100 μmol/kg) for 4 days. Intestinal microbiome (16S rDNA sequencing), liver transcriptome (RNA-Seq), and intermediary metabolites in serum, liver, as well as small and large intestinal contents (SIC and LIC; LC-MS) were examined. Changes in intermediary metabolite abundances in serum, liver, and SIC, were observed under basal conditions (CV vs. GF mice) and by PBDE exposure. PBDEs altered the largest number of metabolites in the LIC; most were regulated by PBDEs in GF conditions. Importantly, intestinal microbiome was necessary for PBDE-mediated decreases in branched-chain and aromatic amino acid metabolites, including 3-indolepropionic acid, a tryptophan metabolite recently shown to be protective against inflammation and diabetes. Gene-metabolite networks revealed a positive association between the hepatic glycan synthesis gene α-1,6-mannosyltransferase (Alg12) mRNA and mannose, which are important for protein glycosylation. Glycome changes have been observed in patients with metabolic syndrome. In LIC of CV mice, 23 bacterial taxa were regulated by PBDEs. Correlations of certain taxa with distinct serum metabolites further highlight a modulatory role of the microbiome in mediating PBDE effects. In summary, PBDEs impact intermediary metabolism in an intestinal microbiome–dependent manner, suggesting that dysbiosis may contribute to PBDE-mediated toxicities that include metabolic syndrome.

Recent research has shown significant health benefits deriving from high-dietary fiber or microbiome-accessible carbohydrate consumption. Compared with native starch (NS), dietary resistant starch (RS) is a high microbiome-accessible carbohydrate that significantly alters the gut microbiome. The aim of this study was to determine the systemic metabolic effects of high microbiome-accessible carbohydrate. Male C57BL/6 mice were divided into 2 groups and fed either NS or RS for 18 wk (n = 20/group). Metabolomic analyses revealed that plasma levels of numerous metabolites were significantly different between the RS-fed and NS-fed mice, many of which are microbiome-derived. Most strikingly, we observed a 22-fold increase in gut microbiome-derived tryptophan metabolite indole-3-propionate (IPA), which was positively correlated with several gut microbiota, including Allobaculum, Bifidobacterium, and Lachnospiraceae, with Allobaculum having the most consistently increased abundance of all the IPA-associated taxa across all RS-fed mice. In addition, major changes were observed for metabolites solely or primarily metabolized in the gut (e.g., trimethylamine-N-oxide), metabolites that have a significant entero-hepatic circulation (i.e., bile acids), lipid metabolites (e.g., cholesterol sulfate), metabolites indicating increased energy turnover (e.g., tricarboxylic acid cycle intermediates and ketone bodies), and increased antioxidants such as reduced glutathione. Our findings reveal potentially novel mediators of high microbiome-accessible carbohydrate-derived health benefits.-Koay,Y. C., Wali. J. A., Luk, A. W. S., Macia, L., Cogger, V. C., Pulpitel, T. J., Wahl, D., Solon-Biet, S. M., Holmes, A., Simpson, S. J., O'Sullivan, J. F. Ingestion of resistant starch by mice markedly increases microbiome-derived metabolites.

The contribution of gut-liver signaling to the development of non-alcoholic hepatic steatosis (NHS) in non-diabetic adults remains unclear. We therefore performed comprehensive 16S ribosomal RNA sequencing and fecal metabolomics analyses in 32 controls and 59 non-diabetic adults with NHS and performed fecal microbiota transplantation into germ-free mice using controls and NHS patients as donors. Compared to controls, the abundance of the genera Collinsella and Acinetobacter were higher, while that of Lachnospira was lower, in NHS subjects. Fecal metabolomics analysis showed decreased L-tryptophan levels and increased abundance of the tryptophan metabolite kynurenine in individuals with NHS. Correlation analysis showed that kynurenine levels positively associated with the abundance of Collinsella and Acinetobacter. ROC analysis demonstrated that the combination of tryptophan and kynurenine could discriminate NHS patients from controls with good statistical power [P < 0.05; AUC = 0.833 (95% CI, 0.747 to 0.918)]. Supporting a key role of dysbiotic gut microbiota in NHS development, incipient hepatic steatosis and increased kynurenine levels were observed in GF mice colonized with samples from NHS patients. These results indicate that enhanced kynurenine production resulting from altered gut microbiota composition contributes to NHS in nondiabetic adults and suggest the relevance of tryptophan metabolites as diagnostic biomarkers.

The metabolic syndrome (MetSyn) is a disorder characterized by a cluster of diseases where the regulation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor endowed with ligand- and context-dependent activities, can have a major therapeutic relevance. We have recently discovered a tryptophan metabolite of microbial origin, indole-3-carboxaldehyde (3-IAld), able to regulate intestinal mucosal homeostasis by acting as a ligand of AhR. This makes 3-IAld a potential candidate to treat MetSyn related ailments. Herein, we provide a proof of concept on the efficacy and safety of 3-IAld encapsulated in enteric microparticles (MP) in vivo in a MetSyn murine model. In particular, we showed that 3-IAld: i) is released from MPs in the intestine and potentially metabolized; ii) is able to activate AhR locally; iii) prevents the metabolic complications and the intestinal epithelial barrier dysfunction; iv) is not associated with toxic events. This study does not only extend the biological activity of 3-IAld in vivo, but also provides hints on the therapeutic potential of 3-IAld delivered by enteric MP in MetSyn related diseases.

We found that changes in the concentrations of tryptophan metabolites in the blood serum and in the intestinal contents are one of the mechanisms for the formation of metabolic coupling in the system "macroorganism-intestinal microbiota", which undergoes significant changes in the development of obesity. Although blood kynurenine remained basically unchanged in obese children we found an increase in some of its serum metabolites: anthranilic, kynurenic and xanthurenic acids. It is noteworthy that in the analysis of fecal matter in obese children, revealed a 2-fold increase in the level of kynurenine while the concentration of kynurenine pathway metabolites corresponded to the level of the group of healthy children. This may indicate the metabolic activation of the microbiota associated with the intestinal mucosa. This is also supported by the absence of statistically significant differences in the concentration of indole in healthy children and in obese children in fecal analyses, and a significant increase in the concentration of indole-3-lactate and indole-3-acetate in the blood serum of obese children.Nami ustanovleno, chto izmeneniia kontsentratsiĭ metabolitov obmena triptofana v syvorotke krovi i v soderzhimom kishechnika iavliaiutsia odnim iz mekhanizmov formirovaniia metabolicheskogo sopriazheniia v sisteme “makroorganizm-mikrobiota kishechnika”, kotoryĭ preterpevaet znachitel'nye izmeneniia pri razvitii ozhireniia. U deteĭ s ozhireniem net statisticheski znachimogo povysheniia urovnia kinurenina v syvorotke krovi, no pri étom proiskhodit povyshenie nekotorykh ego syvorotochnykh metabolitov: antranilovoĭ, kinurenovoĭ i ksanturenovoĭ kislot. Pri analize kala u deteĭ s ozhireniem vyiavlen povyshennyĭ v dva raza uroven' kinurenina. Poskol'ku kontsentratsiia metabolitov kinureninovogo puti sootvetstvuet urovniu gruppy zdorovykh deteĭ, éto mozhet svidetel'stvovat' o metabolicheskoĭ aktivatsii mikrobioty assotsiirovannoĭ so slizistoĭ obolochkoĭ kishechnika. Otsutstvie statisticheski znachimykh otlichiĭ dlia kontsentratsii indola u zdorovykh deteĭ i u deteĭ s ozhireniem v analizakh kala i znachimoe uvelichenie kontsentratsii indol-3-laktata i indol-3-atsetata v syvorotke krovi u deteĭ s ozhireniem tak zhe svidetel'stvuet v pol'zu étogo predpolozheniia.

Blockade of the immunosuppressive tryptophan catabolism mediated by indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) holds enormous promise for sensitising cancer patients to immune checkpoint blockade. Yet, only IDO1 inhibitors had entered clinical trials so far, and those agents have generated disappointing clinical results. Improved understanding of molecular mechanisms involved in the immune-regulatory function of the tryptophan catabolism is likely to optimise therapeutic strategies to block this pathway. The immunosuppressive role of tryptophan metabolite kynurenine is becoming increasingly clear, but it remains a mystery if tryptophan exerts functions beyond serving as a precursor for kynurenine. Here we hypothesise that tryptophan acts as a rheostat of kynurenine-mediated immunosuppression by competing with kynurenine for entry into immune T-cells through the amino acid transporter called System L. This hypothesis stems from the observations that elevated tryptophan levels in TDO-knockout mice relieve immunosuppression instigated by IDO1, and that the vacancy of System L transporter modulates kynurenine entry into CD4+ T-cells. This hypothesis has two potential therapeutic implications. Firstly, potent TDO inhibitors are expected to indirectly inhibit IDO1 hence development of TDO-selective inhibitors appears advantageous compared to IDO1-selective and dual IDO1/TDO inhibitors. Secondly, oral supplementation with System L substrates such as leucine represents a novel potential therapeutic modality to restrain the immunosuppressive kynurenine and restore anti-tumour immunity.

Inflammatory bowel diseases (IBD) are kind of recurrent inflammatory issues that occur in the gastrointestinal tract, and currently clinical treatment is still unideal due to the complex pathogenesis of IBD. Basically, gut barrier dysfunction is triggered by gut microbiota dysbiosis that is closely associated with the development of IBD, we thus investigated the therapeutic capacity of berberine (BBR) to improve the dysregulated gut microbiota, against IBD in rats, using a combinational strategy of targeted metabolomics and 16 s rDNA amplicon sequencing technology. Expectedly, our data revealed that BBR administration could greatly improve the pathological phenotype, gut barrier disruption, and the colon inflammation in rats with dextran sulfate sodium (DSS)-induced colitis. In addition, 16S rDNA-based microbiota analysis demonstrated that BBR could alleviate gut dysbiosis in rats. Furthermore, our targeted metabolomics analysis illustrated that the levels of microbial tryptophan catabolites in the gastrointestinal tract were significantly changed during the development of the colitis in rats, and BBR treatment can significantly restore such changes of the tryptophan catabolites accordingly. At last, our in vitro mechanism exploration was implemented with a Caco-2 cell monolayer model, which verified that the modulation of the dysregulated gut microbiota to change microbial metabolites coordinated the improvement effect of BBR on gut barrier disruption in the colitis, and we also confirmed that the activation of AhR induced by microbial metabolites is indispensable to the improvement of gut barrier disruption by BBR. Collectively, BBR has the capacity to treat DSS-induced colitis in rats through the regulation of gut microbiota associated tryptophan metabolite to activate AhR, which can greatly improve the disrupted gut barrier function. Importantly, our finding elucidated a novel mechanism of BBR to improve gut barrier function, which holds the expected capacity to promote the BBR derived drug discovery and development against the colitis in clinic setting.

BackgroundBreast cancer is the most common malignancy among women in the United States, with more than 250,000 cases diagnosed each year. Metabolomics, which reflect the aggregate effects of genetics and the environment on an individual’s metabolic state, can shed light on biochemical pathways involved in susceptibility to breast cancer. We investigated associations between pre-diagnostic circulating amino acids-related metabolites and subsequent risk of breast cancer among predominantly premenopausal women.MethodsIn 1996-1999, 29,611 women (average age, 44 years) in the Nurses’ Health Study II donated blood samples. Between blood collection and June 2011, 1057 women were diagnosed with breast cancer (average of 8 years after blood collection). Women were predominately premenopausal at the time of blood collection. 207 amino acid and amino acid-related metabolites were profiled with LC-MS/MS. Conditional logistic regression (CLR) was used to estimate odds ratios (ORs) of breast cancer and 95% confidence intervals (CIs). Multivariable analyses evaluating the joint association of all metabolites with breast cancer risk were based on CLR with a lasso penalty (Lasso), CLR with an elastic net penalty (Elastic Net), and Random Forests. We used FDR to account for testing multiple hypotheses.ResultsEleven metabolites were associated with breast cancer risk in CLR models, after adjustment for multiple comparisons (p value < 0.05 and q value < 0.20; creatine had q value > 0.20), 6 of which remained significant after adjustment for breast cancer risk factors (p-valuesimple(95%CI) = 0.85 (0.78-0.93); ORadjusted(95%CI)=0.84 (0.77-0.92)). Higher levels of asparagine, creatine and 3 lipids (C20:1 LPC, C34:3 PC plasmalogen, C40:7 PE plasmalogen) were associated with increased breast cancer risk (e.g., C40:7 PE plasmalogen ORsimple(95%CI) = 1.14 (1.05-1.25); ORadjusted(95%CI) = 1.11 (1.01-1.22)). Piperine, 2-aminohippuric acid, C40:7 PE plasmalogen and creatine were also selected in multivariable modeling approaches (Lasso, Elastic Net, and Random Forests).ConclusionsTwo diet-related metabolites, piperine (responsible for the pungency of pepper) and 2-aminohippuric acid (the glycine conjugate of the tryptophan metabolite anthranilic acid) were inversely associated, while C40:7 PE plasmalogen (a highly unsaturated glycerophospholipid and key component of the lipid bilayer of cells) was positively associated with breast cancer risk among predominately premenopausal women, independent of established breast cancer risk factors. Further validation of the specific metabolite associations with breast cancer risk in independent cohorts is warranted.

Commensal microbiota-dependent tryptophan catabolism within the gastrointestinal tract is known to exert profound effects upon host physiology, including the maintenance of epithelial barrier and immune function. A number of abundant microbiota-derived tryptophan metabolites exhibit activation potential for the aryl hydrocarbon receptor (AHR). Gene expression facilitated by AHR activation through the presence of dietary or microbiota-generated metabolites can influence gastrointestinal homeostasis and confer protection from intestinal challenges. Utilizing untargeted mass spectrometry-based metabolomics profiling, combined with AHR activity screening assays, we identify four previously unrecognized tryptophan metabolites, present in mouse cecal contents and human stool, with the capacity to activate AHR. Using GC/MS and LC/MS platforms, quantification of these novel AHR activators, along with previously established AHR-activating tryptophan metabolites, was achieved, providing a relative order of abundance. Using physiologically relevant concentrations and quantitative gene expression analyses, the relative efficacy of these tryptophan metabolites with regard to mouse or human AHR activation potential is examined. These data reveal indole, 2-oxindole, indole-3-acetic acid and kynurenic acid as the dominant AHR activators in mouse cecal contents and human stool from participants on a controlled diet. Here we provide the first documentation of the relative abundance and AHR activation potential of a panel of microbiota-derived tryptophan metabolites. Furthermore, these data reveal the human AHR to be more sensitive, at physiologically relevant concentrations, to tryptophan metabolite activation than mouse AHR. Additionally, correlation analyses indicate a relationship linking major tryptophan metabolite abundance with AHR activity, suggesting these cecal/fecal metabolites represent biomarkers of intestinal AHR activity.

Reactive oxygen species (ROS) that are induced upon pathogen infection plays an important role in host defence. The rickettsial pathogen Anaplasma phagocytophilum, which is primarily transmitted by Ixodes scapularis ticks in the United States, has evolved many strategies to escape ROS and survive in mammalian cells. However, little is known on the role of ROS in A. phagocytophilum infection in ticks. Our results show that A. phagocytophilum and hemin induce activation of l-tryptophan pathway in tick cells. Xanthurenic acid (XA), a tryptophan metabolite, supports A. phagocytophilum growth in tick cells through inhibition of tryptophan dioxygenase (TDO) activity leading to reduced l-kynurenine levels that subsequently affects build-up of ROS. However, hemin supports A. phagocytophilum growth in tick cells by inducing TDO activity leading to increased l-kynurenine levels and ROS production. Our data reveal that XA and kynurenic acid (KA) chelate hemin. Furthermore, treatment of tick cells with 3-hydroxyl l-kynurenine limits A. phagocytophilum growth in tick cells. RNAi-mediated knockdown of kynurenine aminotransferase expression results in increased ROS production and reduced A. phagocytophilum burden in tick cells. Collectively, these results suggest that l-tryptophan pathway metabolites influence A. phagocytophilum survival by affecting build up of ROS levels in tick cells.

5-methoxytryptophan (5-MTP) is an endothelial factor with anti-inflammatory properties. It is synthesized from L-tryptophan via two enzymatic steps: tryptophan hydroxylase-1 (TPH-1) and hydroxyindole O-methyltransferase. Lipopolysaccharide (LPS) and pro-inflammatory cytokines suppress endothelial 5-MTP production by inhibiting TPH-1 expression. 5-MTP protects endothelial barrier function and promotes endothelial repair, while it blocks vascular smooth muscle cell migration and proliferation by inhibiting p38 MAPK activation. 5-MTP controls macrophage transmigration and activation by inhibiting p38 MAPK and NF-κB activation. 5-MTP administration attenuates arterial intimal hyperplasia, defends against systemic inflammation and prevents renal fibrosis in relevant murine models. Serum 5-MTP level is depressed in human sepsis as well as in mice with sepsis-like disorder. It is reduced in chronic kidney disease and acute myocardial infarction in humans. The reported data suggest that serum 5-MTP may be a theranostic biomarker. In summary, 5-MTP represents a new class of tryptophan metabolite which defends against inflammation and inflammation-mediated tissue damage and fibrosis. It may be a valuable lead compound for developing new drugs to treat complex human inflammatory disorders.

Background Total glucosides of peony (TGP), extracted from the root and rhizome of Paeonia lactiflora Pall, has well-confirmed immunomodulatory efficacy in the clinic. However, the mechanism and active ingredients remain largely unclear. Hypothesis/Purpose Our previous study revealed a low systemic exposure but predominant gut distribution of TGP components. The aim of this study was to investigate involvement of the gut microbiota in the immunoregulatory effects and identify the active component. Methods Mice received 3% DSS to establish a model of colitis. The treatment group received TGP or single paeoniflorin (PF) or albiflorin (AF). Body weight, colon length, inflammatory and histological changes were assessed. Gut microbiota structure was profiled by 16s rRNA sequencing. Antibiotic treatment and fecal transplantation were used to explore the involvement of gut microbiota. Metabolomic assay of host and microbial metabolites in colon was performed. Results TGP improved colonic injury and gut microbial dysbiosis in colitis mice, and PF was responsible for the protective effects. Fecal microbiota transfer from TGP-treated mice conferred resilience to colitis, while antibiotic treatment abrogated the protective effects. Both TGP and PF decreased colonic indole-3-lactate (ILA), a microbial tryptophan metabolite. ILA was further identified as an inhibitor of epithelial autophagy and ILA supplementation compromised the benefits of TGP. Conclusion Our findings suggest that TGP acts in part through a gut microbiota-ILA-epithelial autophagy axis to alleviate colitis.

Despite the accumulating evidence from in vitro and animal experiments supporting the role of kynurenine (a tryptophan metabolite) in a number of degenerative age-related changes, the relationship between kynurenine and frailty in older adults is not well understood. We collected blood samples from 73 participants who underwent a comprehensive geriatric assessment, measuring kynurenine levels using liquid chromatography-tandem mass spectrometry. We assessed the phenotypic frailty and the deficit accumulation frailty index using widely validated approaches proposed by Fried et al. and Rockwood et al., respectively. After adjusting for sex, age, and body mass index, the frail participants presented 52.9% and 34.3% higher serum kynurenine levels than those with robustness and prefrailty, respectively (P = 0.005 and 0.014, respectively). Serum kynurenine levels were positively associated with the frailty index, time to complete 5 chair stands, and patient health questionnaire-2 score and inversely associated with grip strength and gait speed (P = 0.042 to

Background Skin commensal bacteria play important roles in skin homeostasis. Langerhans cells (LCs) are epidermis-resident dendritic cells that sense environmental stimuli and are critical in the induction of immune tolerance to allergen and bacterial skin flora. However, response of LCs to the metabolites of the skin microbiota is not clear. Objective To explore the effects of the skin microbial metabolites on LCs activation. Methods LCs derived from CD34+ hematopoietic stem cells in the cord blood were treated with a microbial metabolite of tryptophan, indole-3-aldehyde (IAId). Activation aryl hydrocarbon receptor (AhR) signaling, production of IL-10, and expression of receptor activator of NF-κB (RANK) / receptor activator of NF-κB ligand (RANKL) in LCs or keratinocytes were analyzed using quantitative PCR, western blotting and flow cytometry. LCs maturation induced by IAId and CD4+ T cell response induced by IAId-conditioned LCs were also investigated. Results IAId induced the production of indoleamine 2,3-dioxygenase (IDO) and IL-10 in LCs through the activation of AhR. IAId promoted the expression of RANK and RANKL on LCs and keratinocytes in an AhR-dependent manner respectively, which might result in activation of NF-κB signaling and production of IL-10. Moreover, a mature phenotype of LCs was induced by IAId, and IAId-activated LCs inhibited CD4+ T cell proliferation and induced IL-10 secretion. Conclusions Our study revealed a negatively regulatory function of a tryptophan metabolite on LCs through the activation of AhR, and the microbial metabolites could be utilized in future treatment for inflammatory skin diseases.

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.

Background The neurochemical background of the evolution of headache disorders, still remains partially undiscovered. Accordingly, our aim was to further explore the neurochemical profile of Complete Freund’s adjuvant (CFA)-induced orofacial pain, involving finding the shift point regarding small molecule neurotransmitter concentrations changes vs. that of the previously characterized headache-related neuropeptides. The investigated neurotransmitters consisted of glutamate, γ-aminobutyric acid, noradrenalin and serotonin. Furthermore, in light of its influence on glutamatergic neurotransmission, we measured the level of kynurenic acid (KYNA) and its precursors in the kynurenine (KYN) pathway (KP) of tryptophan metabolism. Methods The effect of CFA was evaluated in male Sprague Dawley rats. Animals were injected with CFA (1 mg/ml, 50 μl/animal) into the right whisker pad. We applied high-performance liquid chromatography to determine the concentrations of the above-mentioned compounds from the trigeminal nucleus caudalis (TNC) and somatosensory cortex (ssCX) of rats. Furthermore, we measured some of these metabolites from the cerebrospinal fluid and plasma as well. Afterwards, we carried out permutation t-tests as post hoc analysis for pairwise comparison. Results Our results demonstrated that 24 h after CFA treatment, the level of glutamate, KYNA and that of its precursor, KYN was still elevated in the TNC, all diminishing by 48 h. In the ssCX, significant concentration increases of KYNA and serotonin were found. Conclusion This is the first study assessing neurotransmitter changes in the TNC and ssCX following CFA treatment, confirming the dominant role of glutamate in early pain processing and a compensatory elevation of KYNA with anti-glutamatergic properties. Furthermore, the current findings draw attention to the limited time interval where medications can target the glutamatergic pathways.

5-Methoxytryptophan (5-MTP) is a tryptophan metabolite with recently discovered anti-inflammatory and tumor-suppressing activities. Its synthesis is catalyzed by a hydroxyindole O-methyltransferase (HIOMT)-like enzyme. However, the exact identity of this HIOMT in human cells remains unclear. Human HIOMT exists in several alternatively spliced isoforms, and we hypothesized that 5-MTP–producing HIOMT is a distinct isoform. Here, we show that human fibroblasts and cancer cells express the HIOMT298 isoform as contrasted with the expression of the HIOMT345 isoform in pineal cells. Sequencing analysis of the cloned isoforms revealed that HIOMT298 is identical to the sequence of a previously reported truncated HIOMT isoform. Of note, HIOMT298 expression was reduced in cancer cells and tissues. Stable transfection of A549 cancer cells with HIOMT298 restored HIOMT expression to normal levels, accompanied by 5-MTP production. Furthermore, HIOMT298 transfection caused a tryptophan-metabolic switch from serotonin to 5-MTP production. To determine the in vivo relevance of this alteration, we compared growth and lung metastasis of HIOMT298-transfected A549 cells with those of vector- or untransfected A549 cells as controls in a murine xenograft model. Of note, the HIOMT298-transfected A549 cells exhibited slower growth and lower metastasis than the controls. Our findings provide insight into the crucial role of HIOMT298 in 5-MTP production in cells and in inhibiting cancer progression and highlight the potential therapeutic value of 5-MTP for managing cancer.

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100–1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

A tryptophan metabolite of microbial origin, indole-3-carboxaldehyde (3-IAld), has been recently identified as a Janus molecule that, acting at the host-pathogen interface and activating the aryl hydrocarbon receptor, can result as a potential candidate to treat infections as well as diseases with an inflammatory and/or immune component. In this work, an inhaled dry powder of 3-IAld was developed and evaluated for its efficacy, compared to oral and intranasal administration using an aspergillosis model of infection and inflammation. The obtained inhalable dry powder was shown to: i) be suitable to be delivered for pulmonary administration, ii) possess good toxicological safety, and iii) be superior to other administration modalities (oral and intranasal) in reducing disease scores by acting on infection and inflammation. This study supports the use of 3-IAld inhalable dry powders as a potential novel therapeutic tool to target inflammation and infection in pulmonary diseases.

COVID-19 Sex affects immune responses to severe acute respiratory syndrome coronavirus 2 infection, with male sex being a risk factor for mortality, particularly among older individuals. Cai et al. identified 17 serum metabolites associated with COVID-19 infection. In male COVID-19 patients only, the amount of the tryptophan metabolite kynurenic acid correlated with age, inflammation, and disease outcome. Kynurenic acid inhibits glutamate release, and glutamate levels were reduced in patients who deteriorated. Thus, kynurenic acid may be linked in a sex-specific manner to immune responses and clinical outcomes in COVID-19. Sci. Signal. 14 , eabf8483 (2021).

Context: We have previously shown that an antidepressant-like effect of probiotics in rats was associated with a higher plasma level of the microbial tryptophan metabolite indole-3-propionic acid (IPA). Objective: We therefore wanted to study the isolated effect of IPA on behaviour and glucose metabolism in rats. Methods: Male Sprague–Dawley rats were fed control or IPA-enriched diet for six weeks (n = 12 per group) and assessed in the elevated plus maze, open field and forced swim test. Blood glucose, metabolic hormones and the white blood cell (WBC) composition were analysed. Results: IPA (mean intake 27.3 mg/kg/day) significantly lowered fasting blood glucose level by 0.42 mM (95% CI 0.11–0.73). Similarly, fasting plasma insulin levels and the homeostatic model assessment (HOMA) index of insulin resistance were reduced, whereas plasma metabolic hormones, behaviour and WBC composition remained unaffected by IPA. Conclusions: Our findings highlight IPA as a promising candidate for treatment of metabolic disorders associated with insulin resistance

Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. μCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.

The antiparasitic activity of 3-hydroxykynurenine (3-HK), one of the major tryptophan catabolites of the kynurenine pathway, against both Trypanosoma cruzi evolutive forms that are important for human infection, trypomastigotes (Tps) and amastigotes (Am), possible targets in the parasite and the drug toxicity to mammalian cells have been investigated. 3-HK showed a potent activity against Am with IC50 values in the μM concentration range while the IC50 values to cause Tps death was ∼6000-times higher, indicating that the replicative form present in the vertebrate hosts is much more susceptible to 3-HK than bloodstream Tps. In addition, 3-HK showed activity against Tps and Am, at concentrations which did not exhibit toxicity to mammalian cells. Ultrastructural analysis and flow cytometry studies indicated that Am and Tps mitochondrion and nuclei contain 3-HK targets. The potency and selectivity of 3-HK, which is generated during T. cruzi infection in human and mice, suggest that 3-HK may be a suitable candidate for drug research and development for Chagas disease. Fil: Knubel, Carolina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Insfran, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Martinez, Fernando Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Díaz Luján, Cintia María. Universidad Nacional de Cordoba. Facultad de Medicina. Instituto de Biologia Celular; Argentina Fil: Fretes, Ricardo Emilio. Universidad Nacional de Cordoba. Facultad de Medicina. Instituto de Biologia Celular; Argentina Fil: Theumer, Martín Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Cervi, Laura Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Motran, Claudia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Aims/hypothesis The tryptophan metabolite kynurenine has potent immune modulatory and vasoactive properties. Experimental data implicate kynurenine in obesity-related morbidities. Epidemiological studies are, however, sparse. We evaluated associations of the plasma and urine kynurenine:tryptophan ratio (KTR) to incident type 2 diabetes. Methods We followed 2519 individuals with coronary artery disease (CAD; 73.1% men) without diabetes at baseline for a median of 7.6 years, during which 173 (6.9%) new incidences of type 2 diabetes were identified. Multivariate Cox regression analyses were applied to investigate the prospective relationships of plasma and urine KTR with new onset type 2 diabetes. Results At inclusion, mean (SD) age was 61.3 (10.4) years, BMI was 25.9 (3.71) kg/m2 and median (interquartile range) HbA1c was 5.6% (5.0%–6.0%) (38 [31–42] mmol/mol). Plasma KTR was not significantly related to type 2 diabetes risk. By contrast, urine KTR showed a strong positive association. Comparing quartile 4 with quartile 1, the HRs (95% CIs) were 2.59 (1.56, 4.30) and 2.35 (1.39, 3.96) in the age- and sex-adjusted and multivariate models, respectively. Conclusions/interpretation Urine KTR is a strong predictor of incident type 2 diabetes in individuals with CAD. Potential clinical implications and possible pathogenic roles of renal kynurenine excretion in type 2 diabetes development should be further elucidated. Electronic supplementary material The online version of this article (doi:10.1007/s00125-017-4329-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO ) derived from the AAPH (2,2′-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO ), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals ( f ) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction ( f ri ) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction ( f T ) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes ( f syn ) and homogenates ( f hom ), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO . Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.

The antioxidant and pro-oxidant behavior of 3-hydroxyanthranilic acid was investigated using density functional theory. In the absence of metal ions 3-hydroxyanthranilic acid acts as an excellent antioxidant by scavenging free radicals. It was found to be an excellent peroxyl radical scavenger in both lipid and aqueous solution, reacting with ˙OOH faster than Trolox. Moreover, the gathered kinetic data support the idea that 3-hydroxyanthranilic acid significantly contributes to the antioxidant activity usually attributed to tryptophan. In contrast, in the presence of metal ions (at physiological pH) it exhibits pro-oxidant behavior. This behavior arises from the Cu(II) reducing ability of the anionic fractions of this compound, which would contribute to producing Cu(I) and consequently promote ˙OH production via the Fenton reaction. Accordingly, the environmental factor identified to be crucial for ruling the dual behavior of 3-hydroxyanthranilic acid is the presence of metal ions. In addition, the pH is also predicted to influence the pro-oxidant effects of this compound.

Background Tryptophan (trp) is an essential aminoacid, required for de novo NAD+ synthesis. Our previous work showed that the microenvironment in classical Hodgkin Lymphoma is characterized by dysfunctional neutrophils and myeloid derived suppressor cells that produce a trp-degrading enzyme indoelamine deoxygenase (IDO-1), lowering the amount of trp. Aims To detect metabolomics changes in human cHL cell lines exposed in vitro to tryptophan deprivation, an amino acid involved in immune dysregulation and generation of anergic and tolerogenic T- cells. Methods In order to better understand the impact of extra-cellular IDO1 increase on the metabolome of human cHL cells, three human cHL cell lines (L428, L540 and KM-H2) were individually cultured with customized complete media or media lacking tryptophan (W0), +10% dyalised fetal bovum serum, in six independent experiments. After 48 hours of culture the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc. Following normalization to DNA concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (Table 1). Results The lack of tryptophan in media had a profound effect on the cell metabolome in 2 cell lines, KMH2 and L428 cells, while L540 cell line was pretty resilient (Table 1). In all cell lines, the removal of tryptophan from the media resulted in significantly lower levels of tryptophan. Kynurenine, the metabolic product of IDO-1 action on tryptophan, was lower in all cells, but did not reach significance in the L540 cells, whereas it trended lower in L428 and was significantly lower in the KMH2 compared to controls. Indolelactate, another major tryptophan metabolite was also significantly lower in the L428 and KMH2 cells lines compared to controls. Glucose uptake and aerobic glycolysis are frequently upregulated in tumor cells to support energy needs and provide biosynthetic precursors (e.g. pentose phosphate pathway intermediates for nucleotide synthesis). Known as the Warburg effect, this process of reliance on glucose for energy results in high levels of lactate production. We found that trp deficiency lead to lower levels of the hexose diphosphates (fructose 1,6-diphosphate/glucose 1,6-diphosphate/myo-inositol diphosphates isobar) and dihydroxyacetone phosphate, suggesting the revert of Warburg effect due to reduced bio-energetic requirements for proliferation. In line with this observation, culture in trp deficient media resulted in increased levels of long chain saturated fatty acids and long chain polyunsaturated fatty acids (PUFAs), suggesting that specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media, to preserve membrane dynamics. Since prolonged trp deprivation (up to 10 days) delayed cell cycle length without affecting proliferation or changes in intracellular amount of NAD+, we investigated changes in mitochondrial membranes network to explain these findings. Trp deprivation induced the rearrangement of the mitochondrial network at 48 hours, with more fission than fusion, as suggested by increased expression of Fis1 and Drp1 and reduced expression of Tfam and Opa1, without affecting significantly mitochondrial mass and depolarization. This adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the L540 and KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Conclusions The removal of trp from L428 and KMH2 resulted in changes in the specific-amino acid related metabolites. The adaptive response to trp-depleted microenvironment can revert the Warburg effect, promoting a shift in the glucose use in the futile attempt to preserve mitochondrial function, and increase oxidative stress. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Puglisi: Amgen:Honoraria.Di Raimondo:Takeda:Consultancy, Honoraria;GILEAD, Incyte:Research Funding;Amgen, Takeda, Novartis:Honoraria;Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;GSK:Consultancy, Honoraria;Amgen:Consultancy, Honoraria.Romano:Takeda:Honoraria;Novartis:Honoraria.

Background The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise question regarding the unclarified heterogeneity of disease pathomechanisms. While biomarkers for the differentiation of Crohn’s disease (CD) vs. ulcerative colitis (UC) have been suggested, specific markers for a subclassification of CD phenotypes are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterise potential biomarkers focusing on the downstream metabolites of kynurenine metabolism. The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise question regarding the unclarified heterogeneity of disease pathomechanisms. While biomarkers for the differentiation of Crohn’s disease (CD) vs. ulcerative colitis (UC) have been suggested, specific markers for a subclassification of CD phenotypes are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterise potential biomarkers focusing on the downstream metabolites of kynurenine metabolism. Methods Using immunohistochemical staining, we analysed and compared the mucosal tryptophan immune metabolism in biotic samples from patients with UC (n = 11), CD (n = 11) and healthy control (n = 12). Localisation-specific quantification of immune cell infiltration, tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. Results As expected, we found generally increased immune cell infiltrates in the tissue of all patients with IBD. However, in patients with CD, significant differences were found between regulatory T-cell markers in the ileum compared with the colon. In line with this finding, we identified kynureninase as a modulator of immunosuppressive kynurenine levels specifically in the ileum of patients with CD. Correspondingly, significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in CD ileum samples. Conclusion Highlighting the heterogeneity of the different phenotypes of CD, we identified 3-hydroxyanthranilic acid as a potential mucosal biomarker allowing the localisation-specific differentiation of ileum or colon inflammation in patients with CD. Moreover, we characterised the kynurenine-degrading enzyme kynureninase as a modulator of immunosuppression and chronic inflammation with potential therapeutic relevance.

Over the past 50 y, tryptophan has been ingested in amounts well in excess of its dietary requirement. This use is based on extensive findings that ingesting tryptophan increases brain tryptophan concentrations, which stimulates the synthesis and release of the neurotransmitter serotonin, from which it is derived. Such increases in serotonin function may improve mood and sleep. However, tryptophan ingestion has other effects, such as increasing serotonin production in the gut, increasing serotonin concentrations in blood, stimulating the production of the hormone melatonin (a tryptophan metabolite), stimulating tryptophan metabolism via the kynurenine pathway, and possibly stimulating the production of tryptophan metabolites in the gut microbiome. Several of the kynurenine metabolites have actions on excitatory glutamate receptors in the gut and brain and on cells of the immune system. In addition, metabolites of tryptophan produced by colonic bacteria are reported to cause adverse effects in some species. This review examines each of these tryptophan pathways to determine if any of the metabolites increase after tryptophan ingestion, and if so, whether effects are seen on target body functions. In this regard, recent research suggests that it may be useful to examine kynurenine pathway metabolites and some microbial tryptophan metabolites to determine whether supplemental tryptophan consumption increases their concentrations in the body and amplifies their actions.

Excessive alcohol (ethanol) use has long been known to affect human health negatively. However, the underlying molecular basis is incompletely understood. Moreover, consumption of alcohol is often mixed with kynurenic acid (KYNA), an abundant tryptophan metabolite produced during fermentation. The combined effect of ethanol and KYNA on host gene expression has not been investigated. The current study used mice as the model to interrogate the impact of ethanol and/or KYNA on global gene transcription. Adult male mice were administered with 2 g/kg ethanol and/or 0.1 mg/kg KYNA by gavage once a day for a week. Three organs: brain, kidney, and liver were collected and their total RNAs extracted for transcriptome sequencing and quantitative real-time PCR. Gene ontology, Kyoto encyclopedia of genes, and genomes pathway analyses revealed that alcohol affects the three organs differentially. Furthermore, the gene expression profile from alcohol and KYNA co-administration was significantly different from that of alcohol or KYNA administration alone. Strikingly, Indolamine 2,3-dioxygenase 1, a rate-limiting enzyme in tryptophan metabolism, was significantly increased in the brain after a combined exposure of alcohol and KYNA, suggesting that Trp metabolism was skewed towards the kynurenine pathway in the brain. Our systemic analysis provides new insights into the mechanism whereby alcohol and KYNA affects organ functions.

Indoleamine 2, 3‐dioxygenase (IDO)‐mediated regulation of tryptophan metabolism plays an important role in immune tolerance in transplantation, but it has not been elucidated which mechanism specifically induces the occurrence of immune tolerance. Our study revealed that IDO exerts immunosuppressive effects through two pathways in mouse heart transplantation, ‘tryptophan depletion’ and ‘tryptophan metabolite accumulation’. The synergism between IDO+DC and TC (tryptophan catabolic products) has stronger inhibitory effects on T lymphocyte proliferation and mouse heart transplant rejection than the two intervention factors alone, and significantly prolong the survival time of donor‐derived transplanted skin. This work demonstrates that the combination of IDO+DC and TC can induce immune tolerance to a greater extent, and reduce the rejection of transplanted organs.

Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide, particularly among older adults. Despite the advent of medical technology, restenosis is still an issue after interventional procedures. Tryptophan metabolite 5-methoxytryptophan (5-MTP) has recently been shown to protect against systemic inflammatory responses. This study aimed to investigate the function and mechanisms of 5-MTP in interventional procedure-induced restenosis. We found that after mouse femoral artery denudation with a guide wire, 5-MTP accelerated recovery of endothelium in the denuded area and reduced vascular leakage and intimal thickening. 5-MTP increased endothelial cell proliferation in the denuded arteries and rescued TNF-α-reduced endothelial cell proliferation and migration, likely via maintaining vascular endothelial growth factor receptor 2 activation. In contrast, 5-MTP preserved differentiated phenotype of medial vascular smooth muscle cells (VSMCs) and decreased VSMC proliferation and migration. Furthermore, 5-MTP maintained expression levels of critical transcription factors for VSMC marker gene expressions via attenuated activation of p38 MAPK and NFκB-p65. Our findings uncover a novel protective mechanism of 5-MTP in restenosis. In response to denudation injury, 5-MTP attenuates intimal hyperplasia via concerted but opposing actions on endothelial cells and VSMCs. Taken together, our results suggest that 5-MTP is a valuable therapeutic target for arterial injury-induced restenosis.

The intestinal microbiota in allogeneic bone marrow transplant (allo-BMT) recipients modulates graft-versus-host disease (GVHD), a systemic inflammatory state initiated by donor T cells that leads to colitis, a key determinant of GVHD severity. Indole or indole derivatives produced by tryptophan metabolism in the intestinal microbiota limit intestinal inflammation caused by diverse stressors, so we tested their capacity to protect against GVHD in murine major histocompatibility complex–mismatched models of allo-BMT. Indole effects were assessed by colonization of allo-BMT recipient mice with tryptophanase positive or negative strains of Escherichia coli, or, alternatively, by exogenous administration of indole-3-carboxaldehyde (ICA), an indole derivative. Treatment with ICA limited gut epithelial damage, reduced transepithelial bacterial translocation, and decreased inflammatory cytokine production, reducing GVHD pathology and GVHD mortality, but did not compromise donor T-cell-mediated graft-versus-leukemia responses. ICA treatment also led to recipient-strain-specific tolerance of engrafted T cells. Transcriptional profiling and gene ontology analysis indicated that ICA administration upregulated genes associated with the type I interferon (IFN1) response, which has been shown to protect against radiation-induced intestinal damage and reduce subsequent GVHD pathology. Accordingly, protective effects of ICA following radiation exposure were abrogated in mice lacking IFN1 signaling. Taken together, these data indicate that indole metabolites produced by the intestinal microbiota act via type I IFNs to limit intestinal inflammation and damage associated with myeloablative chemotherapy or radiation exposure and acute GVHD, but preserve antitumor responses, and may provide a therapeutic option for BMT patients at risk for GVHD.

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor and N-methyl-D-aspartic acid receptor at endogenous brain concentrations. Recent studies have suggested that increases of brain KYNA levels are involved in psychiatric disorders such as schizophrenia and depression, and regulation of KYNA production has become a new target for treatment of these diseases. Kynurenine (KYN), the immediate precursor of KYNA, is transported into astrocytes via large neutral amino acid transporters (LATs). In the present study, the effect of LATs regulation on KYN uptake and KYNA production was investigated in vitro and in vivo using an LATs inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). In the in vitro study, cortical slices of rat brain were incubated with a physiological concentration of KYN and 3 µmol/L-3 mmol/L BCH. BCH inhibited KYNA production and KYN uptake in a dose-dependent manner, and their IC50 values were 90.7 and 97.4 µmol/L, respectively. In the in vivo study, mice were administered KYN (50 mg/kg BW) orally and BCH (200 mg/kg BW) intravenously. Administration of KYN increased brain KYN and KYNA levels compared with the mice treated with vehicle, whereas additional administration of BCH suppressed KYN-induced elevations in KYN and KYNA levels to 50 and 70 % in the brain. These results suggest that inhibition of LATs prevented the increase of KYNA production via blockade of KYN uptake in the brain in vitro and in vivo. LATs can be a target to modulate brain function by regulation of KYNA production in the brain.