An investigation of the kynurenine pathway in experimental arthritis

Abstract

The kynurenine pathway is a catabolic biochemical pathway responsible for degradation of tryptophan, an essential amino acid. As a consequence, biologically active molecules, kynurenines, are produced. These chemical entities can influence immune responses. Previously, it has been shown that pharmacological inhibition of the initial step on the pathway increases the severity of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. In contrast, treatment with kynurenine, a major by product of tryptophan degradation, effectively ameliorated the disease. This project was based around the hypothesis that tryptophan metabolism via the kynurenine pathway represents an endogenous regulatory mechanism that is activated in response to inflammation. To test this hypothesis, I carried out a comprehensive analysis of the kynurenine pathway in the immune system in CIA as well as in the liver or kidneys, organs in which kynurenine pathway is the most active under normal conditions. In this study, the endogenous activity of the kynurenine pathway in the immune system (lymph nodes and spleen), inflamed paws, liver, and kidneys was monitored during the induction phase of CIA (day 14 after immunisation) and during the period of disease resolution (day 10 after disease onset). In addition, the concentration of tryptophan, kynurenine and its selected catabolites anthranilic acid (AA) and 3-hydroxyanthranilic acid (3-HAA) was determined in the sera. All results were compared with naive tissues.Increased expression of all enzymes along the kynurenine pathway was observed locally in draining lymph nodes during the pre-arthritic phase of arthritis and this was accompanied by reduced levels of tryptophan. In contrast, during the resolution phase of arthritis not only was there decreased tryptophan concentration, but also there was an accumulation of the downstream tryptophan metabolites, kynurenine, AA, and 3-HAA in lymph nodes. In addition, the accumulation of kynurenine and its downstream metabolites observed during the resolution of arthritis was accompanied by reduced expression of enzymes involved in kynurenine catabolism (kynureninase, kynurenine 3-monooxygenase, and 3-hydroxyanthranilate 3,4 dioxygenase) towards the levels found in naïve mice. These findings provide for the first time evidence of an association between resolution of arthritis and the local accumulation of kynurenines in lymph nodes. However, in the paws and spleens of mice with CIA, there was no evidence of activation of the kynurenine pathway. Surprisingly, however, kynurenine catabolism was increased in the kidneys and liver during CIA which may explain why in sera from mice with CIA, the tryptophan concentration was not changed, whereas levels of kynurenine, AA, and 3-HAA actually decreased, despite the increased levels found in lymph nodes at the same time points. Based on these findings I assessed the potential therapeutic effect of exogenous administration of AA and 3-HAA in mice with established CIA and in order to facilitate this study I established a novel method of assessment of bone integrity based on 3-dimensional imaging using micro-computed tomography. Using in vivo observations, micro-computed tomography and histological sectioning with hematoxylin and eosin staining, I showed that neither AA nor 3-HAA treatment was effective in established CIA. However, treatment with etanercept, a potent inhibitor of TNF, profoundly reduced the severity of bone and cartilage damage. I also confirmed previous findings that tranilast, a derivative of 3-HAA which exhibits kynurenine-like activity and has a longer half-life than naturally occurring tryptophan metabolite, was effective in established CIA. Thus, taken together, activation of the kynurenine pathway in the lymph nodes may constitute a fine tuning mechanism involved in resolution of inflammation. However, exogenous administration of naturally occurring kynurenines is unlikely to be an effective therapeutic strategy to reduce inflammation in arthritis, possibly because of their rapid clearance from the circulation