Quinolinate as a Marker for Kynurenine Metabolite Formation and the Unresolved Question of NAD+ Synthesis During Inflammation and Infection.

Quinolinate (Quin) is a classic example of a biochemical double-edged sword, acting as both essential metabolite and potent neurotoxin. Quin is an important metabolite in the kynurenine pathway of tryptophan catabolism leading to the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As a precursor for NAD+, Quin can direct a portion of tryptophan catabolism toward replenishing cellular NAD+ levels in response to inflammation and infection. Intracellular Quin levels increase dramatically in response to immune stimulation [e.g., lipopolysaccharide (LPS) or pokeweed mitogen (PWM)] in macrophages, microglia, dendritic cells, and other cells of the immune system. NAD+ serves numerous functions including energy production, the poly ADP ribose polymerization (PARP) reaction involved in DNA repair, and the activity of various enzymes such as the NAD+-dependent deacetylases known as sirtuins. We used highly specific antibodies to protein-coupled Quin to delineate cells that accumulate Quin as a key aspect of the response to immune stimulation and infection. Here, we describe Quin staining in the brain, spleen, and liver after LPS administration to the brain or systemic PWM administration. Quin expression was strong in immune cells in the periphery after both treatments, whereas very limited Quin expression was observed in the brain even after direct LPS injection. Immunoreactive cells exhibited diverse morphology ranging from foam cells to cells with membrane extensions related to cell motility. We also examined protein expression changes in the spleen after kynurenine administration. Acute (8 h) and prolonged (48 h) kynurenine administration led to significant changes in protein expression in the spleen, including multiple changes involved with cytoskeletal rearrangements associated with cell motility. Kynurenine administration resulted in several expression level changes in proteins associated with heat shock protein 90 (HSP90), a chaperone for the aryl-hydrocarbon receptor (AHR), which is the primary kynurenine metabolite receptor. We propose that cells with high levels of Quin are those that are currently releasing kynurenine pathway metabolites as well as accumulating Quin for sustained NAD+ synthesis from tryptophan. Further, we propose that the kynurenine pathway may be linked to the regulation of cell motility in immune and cancer cells.

A review of the immunomodulatory role of dietary tryptophan in livestock and poultry.

Tryptophan, a nutritionally essential amino acid, is active in the regulation of immune responses in animals. The products of tryptophan metabolism, such as indoleamine 2,3-dioxygenase, kynurenine, quinolinic acid, and melatonin, may improve immunity in an organism and induce anti-inflammatory responses. The immune tolerance processes mediated by tryptophan metabolites are not well understood. Recent studies have reported that the enzymes that break down tryptophan through the kynurenine metabolic pathway are found in numerous cell types, including immunocytes. Moreover, some tryptophan metabolites have been shown to play a role in the inhibition of T lymphocyte proliferation, elevation of immunoglobulin levels in the blood, and promotion of antigen-presenting organization in tissues. This review summarizes the effects and mechanisms of tryptophan and metabolites in immune functions in livestock and poultry. It also highlights the areas in which our understanding of the role(s) of tryptophan is incomplete and suggests possible future research that might prove of benefit to livestock and poultry producers.

Tryptophan Metabolism Along the Kynurenine Pathway Downstream of Toll-like Receptor Stimulation in Peripheral Monocytes.

Tryptophan degradation along the kynurenine pathway is of central importance for the immune function. Toll-like receptors (TLRs), representing the first line of immune defence against pathogens, are expressed in various cell types. The most abundant expression is found on monocytes, macrophages and dendritic cells. The aim of this study was to investigate whether stimulation with different TLR ligands induces the kynurenine pathway in human peripheral monocytes. Cell supernatants were analysed using a liquid chromatography/mass spectrometry to measure kynurenine, kynurenic acid (KYNA), quinolinic acid (QUIN) and tryptophan. Stimulation of TLR-2, TLR-3, TLR-4, TLR-7/8 and TLR-9 was found to induce the production of kynurenine, but only stimulation of TLR-3 increased levels of further downstream metabolites, such as KYNA and QUIN. Stimulation of TLR-1, TLR-5 and TLR-6 did not induce the kynurenine pathway. Taken together, this study provides novel evidence demonstrating that TLR activation induces a pattern of downstream tryptophan degradation along the kynurenine pathway in monocytes. The results of this study may implicate that TLRs can be used as new drug targets for the regulation of aberrant tryptophan metabolism along this pathway, a potential therapeutic strategy that may be of importance in several disorders.

The immune effects of TRYCATs (tryptophan catabolites along the IDO pathway): relevance for depression - and other conditions characterized by tryptophan depletion induced by inflammation.

Immune activation is accompanied by induction of indoleamine (2,3)-dioxygenase (IDO), an enzyme which degrades tryptophan, a phenomenon which plays a role in the pathophysiology of major depression and post-natal depression and anxiety states. TRYCATs - tryptophan catabolites along the IDO pathway - such as kynurenine, kynurenic acid, xanthurenic acid, and quinolinic acid, have multiple effects, e.g. apoptotic, anti- versus pro-oxidant, neurotoxic versus neuroprotective, and anxiolytic versus anxiogenic effects. The aim of the present study was to study the immune effects of the above TRYCATS. Toward this end we examined the effects of the above TRYCATs on the LPS + PHA-induced production of interferon-gamma (IFNgamma), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNFalpha) in 18 normal volunteers. We found that the production of IFNgamma was significantly decreased by all 4 catabolites. Xanthurenic acid and quinolinic acid decreased the production of IL-10. Kynurenine, kynurenic acid, and xanthurenic acid, decreased the IFNgamma/IL-10 production ratio, whereas quinolinic acid increased this ratio. Kynurenic acid significantly reduced the stimulated production of TNFalpha. It is concluded that kynurenine, kynurenic acid, and xanthurenic acid have anti-inflammatory effects trough a reduction of IFNgamma, whereas quinolinic acid has pro-inflammatory effects in particular via significant decreases in IL-10. Following inflammation-induced IDO activation, some TRYCATs, i.e. kynurenine, kynurenic acid, and xanthurenic acid, exert a negative feedback control over IFNgamma production thus downregulating the initial inflammation, whereas an excess of quinolinic acid further aggravates the initial inflammation.

Tryptophan and the immune response.

The immune system continuously modulates the balance between responsiveness to pathogens and tolerance to non-harmful antigens. The mechanisms that mediate tolerance are not well understood, but recent findings have implicated tryptophan catabolism through the kynurenine metabolic pathway as one of many mechanisms involved. The enzymes that break down tryptophan through this pathway are found in numerous cell types, including cells of the immune system. Some of these enzymes are induced by immune activation, including the rate limiting enzyme present in macrophages and dendritic cells, indoleamine 2,3-dioxygenase (IDO). It has recently been found that inhibition of IDO can result in the rejection of allogenic fetuses, suggesting that tryptophan breakdown is necessary for maintaining aspects of immune tolerance. Two theories have been proposed to explain how tryptophan catabolism facilitates tolerance. One theory posits that tryptophan breakdown suppresses T cell proliferation by dramatically reducing the supply of this critical amino acid. The other theory postulates that the downstream metabolites of tryptophan catabolism act to suppress certain immune cells, probably by pro-apoptotic mechanisms. Reconciling these disparate views is crucial to understanding immune-related tryptophan catabolism and the roles it plays in immune tolerance. In this review we examine the issue in detail, and offer additional insight provided by studies with antibodies to quinolinate, a tryptophan catabolite which is also necessary for nicotinamide adenine dinucleotide (NAD +) production. In addition to the immunomodulatory actions of tryptophan catabolites, we discuss the possible involvement of quinolinate as a means of replenishing NAD + in leucocytes, which is depleted by oxidative stress during an immune response.

Temporal and spatial changes of quinolinic acid immunoreactivity in the gerbil hippocampus following transient cerebral ischemia.

Quinolinic acid (QUIN) is an endogenous neurotoxin which originates from the kynurenine pathway of tryptophan metabolism. An increase of brain QUIN level occurs in several degenerative and inflammatory disorders, but the cellular source of QUIN is still a matter of controversy. In the present study, the gerbil model of transient global ischemia was used to investigate the time course and the cellular localization of QUIN immunoreactivity. Neurodegeneration was evident in the subiculum and in the CA1 area of the hippocampus 4, 7 and 14 days after ischemia. QUIN positive cells, with microglia-like morphology, appeared in the subiculum and in the CA1, 4 days after ischemia. At 7 days post-ischemia they extended to the whole CA1, disappearing at 14 days. Neither neurodegeneration nor QUIN positive cells could be detected in ischemic gerbils sacrificed at 1 and 2 days after ischemia and in sham-operated animals. These findings suggest that microglia-like cells infiltrating the degenerating areas of the hippocampus represent the major source of QUIN following transient ischemia in the gerbil. Thus, in situ production of QUIN in vulnerable brain regions may contribute to the pathophysiological mechanisms of delayed brain injury.

Increased quinolinate immunoreactivity in the peripheral blood monocytes/macrophages from SIV-infected monkeys.

Quinolinate (QUIN), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that is thought to act through the NMDA receptor system, was localized in cultured peripheral blood monocytes/macrophages from SIV-infected monkeys using a recently developed immunohistochemical method. Significant increases in QUIN immunoreactive (IR) cells were detected in all five SIV-infected monkeys examined. Multinucleated giant cells, a hallmark of lentiviral infection, were visible in selected samples. Treatment with the QUIN precursors, tryptophan and kynurenine, increased the number of QUIN-IR cells in both the control and SIV-infected preparations, perhaps by a mass action mechanism. We hypothesize that in SIV-infected monkeys, infiltrating monocytes/macrophages contribute to the high level of brain QUIN and associated neuropathology.

Temporal and spatial changes of quinolinic acid immunoreactivity in the immune system of lipopolysaccharide-stimulated mice.

Quinolinic acid (Quin), a metabolite of tryptophan, is a neurotoxin that has been implicated in a variety of neuropathologic disorders that have immune components. The goal of this study was to characterize the changes in the cellular localization of Quin immunoreactivity in a paradigm of immune stimulation with lipopolysaccharide (LPS) in vivo to provide a basis for further studies on the physiological role of Quin in the immune system. Intraperitoneal LPS injection significantly increased Quin immunoreactivity (IR) in lymphoid tissues within 24 h. Spatial changes in splenic Quin-IR demonstrated a shift from the periarterial lymphoid sheaths to the follicles before returning to control levels by 72 h post-LPS. The strongly Quin-IR cells were tentatively identified as interdigitating dendritic cells and macrophages. Only minimal Quin-IR was detected in liver and lung, even under conditions of LPS stimulation combined with tryptophan loading. These data emphasize the temporally and spatially specific nature of Quin-IR changes in lymphoid tissues under conditions of immune stimulation and raise the possibility that Quin may have an immunomodulatory function.

Antibodies to quinolinic acid and the determination of its cellular distribution within the rat immune system.

Antibodies to quinolinic acid were produced in rabbits with protein-conjugated and gold particle-adsorbed quinolinic acid. Quinolinic acid immunoreactivity was below detection limits in carbodiimide-fixed rat brain. In contrast, strong quinolinic acid immunoreactivity was observed in spleen cells with variable, complex morphology located predominantly in the periarterial lymphocyte sheaths. In the thymus, quinolinic acid immunoreactivity was observed in cells with variable morphology, located almost exclusively in the medulla. Lymph nodes and gut-associated lymphoid tissue contained many, strongly stained cells of similar complex morphology in perifollicular areas. Immunoreactivity in liver and lung was restricted to widely scattered, perivascular cells and alveolar cells respectively. Additional stained cells with complex morphology were observed in bronchus-associated lymphoid tissue, in skin, and in the lamina propria of intestinal villi. Follicles in all secondary lymphoid organs were diffusely stained, ranging from mildly to moderately immunoreactive in spleen, to intensely immunoreactive in gut-associated lymphoid tissue. These results suggest that quinolinic acid is an immune system-specific molecule. Two hypothetical schemes are proposed to account for high levels of quinolinic acid in specific cells of the immune system.

Antibodies to quinolinic acid reveal localization in select immune cells rather than neurons or astroglia.

Polyclonal antibodies were produced against quinolinic acid. No immunoreactivity was observed in any cell type in carbodiimide-fixed brain tissue from control rats. When the antibodies were applied to carbodiimide-fixed spleen tissue, strong quinolinic acid immunoreactivity was observed in some cells with the appearance of macrophages and dendritic cells. These findings indicate an immune system origin for quinolinic acid, and implicate immune cells in excitotoxic CNS pathologies. These findings also raise the possibility that quinolinic acid is a unique cytokine in immune system signal transmission.