Control of Tissue Fibrosis by 5-Methoxytryptophan, an Innate Anti-Inflammatory Metabolite

Tissue fibrosis causes debilitating human diseases such as liver cirrhosis, heart failure, chronic kidney disease and pulmonary insufficiency. It is a dynamic process orchestrated by specific subsets of monocyte-macrophages, fibroblasts, pericytes and hepatic stellate cells. Fibrosis is linked to tissue inflammation. Pro-inflammatory macrophages promote fibrosis by driving myofibroblast differentiation and macrophage myofibroblast transition. Myofibroblasts express α-smooth muscle cell actin (α-SMA) and secrete extracellular matrix (ECM) proteins notably collagen I and III. Deposition of ECM proteins at injury sites and interstitial tissues distorts normal structure and impairs vital functions. Despite advances in the mechanisms of fibrosis at cellular, molecular and genetic levels, prevention and treatment of fibrotic diseases remain poorly developed. Recent reports suggest that 5-methoxytryptophan (5-MTP) is effective in attenuating injury-induced liver, kidney, cardiac and pulmonary fibrosis. It inhibits macrophage activation and blocks fibroblast differentiation to myofibroblasts. Furthermore, it inhibits hepatic stellate cell differentiation into myofibroblasts. As 5-MTP is an endogenous molecule derived from tryptophan catabolism via tryptophan hydroxylase pathway, it is well-suited as a lead compound for developing new anti-fibrotic drugs. This article provides an overview of 5-MTP synthesis, and a critical review of its anti-fibrotic activities. Its mechanisms of actions and potential therapeutic value will be discussed.

Interferon-gamma Impairs Human Coronary Artery Endothelial Glucose Metabolism via Tryptophan Catabolism and Activates Fatty Acid Oxidation

Background: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. Interferon-gamma (IFN- γ )-producing CD4 + and CD8 + T-lymphocytes have been identified as the predominant pathologic cell subsets in human atherosclerotic plaques. While the immunological consequences of these cells have been extensively evaluated, their IFN- γ -mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN- γ , on human coronary artery endothelial cells (HCAEC). Methods: The metabolic effects of IFN- γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cyclic guanosine monophosphate (cGMP) content, wound healing capacity, and adhesion molecule expression. Results: IFN- γ exposure inhibited basal glycolysis of quiescent primary HCAEC by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal hypoxia inducible factor 1α (HIF1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN- γ -induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated aryl hydrocarbon receptor (AHR). Additionally, IFN- γ resulted in a 23% depletion of intracellular NAD + in HCAEC. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by over 20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a pro-inflammatory state. Conclusions: IFN- γ impairs endothelial glucose metabolism via altered tryptophan catabolism destabilizing HIF1, depletes NAD + , and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathologic T-lymphocyte-endothelial interactions in atherosclerosis mediated by IFN- γ , linking endothelial glucose, tryptophan, and fatty acid metabolism with NAD(H) and ATP generation, and their adverse endothelial functional consequences.

L. rhamnosus improves the immune response and tryptophan catabolism in laying hen pullets

In mammals, early-life probiotic supplementation is a promising tool for preventing unfavourable, gut microbiome-related behavioural, immunological, and aromatic amino acid alterations later in life. In laying hens, feather-pecking behaviour is proposed to be a consequence of gut-brain axis dysregulation. Lactobacillus rhamnosus decreases stress-induced severe feather pecking in adult hens, but whether its effect in pullets is more robust is unknown. Consequently, we investigated whether early-life, oral supplementation with a single Lactobacillus rhamnosus strain can prevent stress-induced feather-pecking behaviour in chickens. To this end, we monitored both the short- and long-term effects of the probiotic supplement on behaviour and related physiological parameters. We hypothesized that L. rhamnosus would reduce pecking behaviour by modulating the biological pathways associated with this detrimental behaviour, namely aromatic amino acid turnover linked to neurotransmitter production and stress-related immune responses. We report that stress decreased the proportion of cytotoxic T cells in the tonsils (P = 0.047). Counteracting this T cell depression, birds receiving the L. rhamnosus supplementation significantly increased all T lymphocyte subset proportions (P < 0.05). Both phenotypic and genotypic feather peckers had lower plasma tryptophan concentrations compared to their non-pecking counterparts. The probiotic supplement caused a short-term increase in plasma tryptophan (P < 0.001) and the TRP:(PHE + TYR) ratio (P < 0.001). The administration of stressors did not significantly increase feather pecking in pullets, an observation consistent with the age-dependent onset of pecking behaviour. Despite minimal changes to behaviour, our data demonstrate the impact of L. rhamnosus supplementation on the immune system and the turnover of the serotonin precursor tryptophan. Our findings indicate that L. rhamnosus exerts a transient, beneficial effect on the immune response and tryptophan catabolism in pullets.

SARS-CoV2 Infection Alters Tryptophan Catabolism and Phospholipid Metabolism

Coronavirus disease 2019 (COVID-19) has so far infected hundreds of million individuals, with several million deaths worldwide. The lack of understanding of the disease pathophysiology and the host’s immune response has resulted in this rapid spread of the disease on a global scale. In this respect, we employed UPLC-MS to compare the metabolites in the serum from COVID-19-positive patients and COVID-19-recovered subjects to determine the metabolic changes responsible for an infection. Our investigations revealed significant increase in the levels of serum phospholipids including sphingomyelins, phosphatidylcholines and arachidonic acid in the serum of COVID-19-positive patients as compared to COVID-19-recovered individuals. We further show increased levels of tryptophan and its metabolites in the serum of COVID-19-positive patients thus emphasizing the role of tryptophan metabolism in the disease pathogenesis of COVID-19. Future studies are required to determine the changes in the lipid and tryptophan metabolism at various stages of COVID-19 disease development, progression and recovery to better understand the host–pathogen interaction and the long-term effects of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection in humans.

1007Vitamin B6 intake, its active form pyridoxal 5’-phosphate, and markers of B6 activity and catabolism

Background Several biological pathways implicated in cancer risk rely on vitamin B6, which can be measured in its active form pyridoxal 5’-phosphate (PLP). Functional markers of B6 enzymatic activity have been proposed, including the homocysteine:cysteine ratio (Hcy:Cys, a marker of transsulfuration), 3-hydroxykynurenine ratio (HKr, a marker of tryptophan catabolism), and the 4-pyridoxic acid ratio (PAr, a marker of B6 catabolism). We investigated the extent to which these markers are associated with B6 intake. Methods Data from 4,750 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) study were included. We estimated the expected percentage change in each of the markers (PLP, Hcy:Cys, HKr, and PAr) for a doubling in B6 intake using log-linear Bayesian hierarchical regression models with log-transformed intake and biomarker data. Results The percent change (posterior mean [95% Credible Interval (CrI)]) for a doubling of B6 intake was 61.0 [51.2, 71.8] for PLP, -12.7 [-15.2, -9.9] for Hcy:Cys, -12.9 [-15.7, -9.9] for HKr, and 1.3 [-3.5, 6.2] for PAr. Conclusions B6 intake is most strongly associated with PLP, but is also associated with functional markers of transsulfuration and tryptophan catabolism, in the direction of increased activity in these pathways. There is no evidence of a linear association between vitamin B6 intake and catabolism. Key messages Our results show differing sensitivity of PLP, markers of tryptophan catabolism and transsulfuration, and vitamin B6 catabolism to B6 intake.

Kynurenic Acid and Its Synthetic Derivatives Protect Against Sepsis-Associated Neutrophil Activation and Brain Mitochondrial Dysfunction in Rats

Background and AimsThe systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood–brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis.MethodsSprague–Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1β, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI–CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions.ResultsSepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1β were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI–CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues.ConclusionThis study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.

Kynurenine Monooxygenase Expression and Activity in Human Astrocytomas

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.