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Keywords:

  • autoimmunity;
  • IDO1;
  • IDO2;
  • kynurenine;
  • tryptophan;
  • rheumatoid arthritis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Tryptophan is an essential amino acid which influences a wide range of physiological processes, including mood, cognition, and immunity. In the autoimmune diseases, such as rheumatoid arthritis (RA), the induction of tryptophan catabolism may help to diminish exacerbated immune responses. In this study, using collagen-induced arthritis (CIA) in DBA/1 mice which is an animal model of RA, the endogenous activity of the kynurenine pathway in the immune system was monitored before and after onset of the disease. An increased rate of the initiation of tryptophan catabolism via the kynurenine pathway throughout CIA has been observed. However, decreased tryptophan concentration in the lymph nodes from pre-arthritic mice was not enough to prevent development of CIA. In contrast, resolution of inflammation coincided with the decreased concentration of tryptophan and accumulation of its catabolites: kynurenine, anthranilic acid, and 3-hydroxyanthranilic acid in lymph nodes but not in the spleen. In addition, the lack of the accumulation of kynurenine and its downstream metabolites in the pre-arthritic lymph nodes coincided with increased mRNA expression for genes involved in the catabolism of kynurenine (Kynureninase, kynurenine 3-monooxygenase, and 3-hydroxyanthranilate 3,4 dioxygenase). However, in the lymph nodes from mice with established CIA, mRNA expression for these genes was normalized. Hence, keeping in mind an exploratory character of the results, it can be postulated that an anti-inflammatory role of the kynurenine pathway reaches its full potential only when decreased concentration of tryptophan coincides with accumulation of kynurenines driven by metabolic regulation of gene expression on the kynurenine pathway. © 2012 IUBMB IUBMB Life, 64(12): 983–987, 2012

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Catabolism of tryptophan via the kynurenine pathway is thought to play an important immunosuppressive role (1, 2). In mice during an allogeneic pregnancy, inhibition of an enzyme, indoleamine 2,3 dioxygenase 1 (IDO1), which initiates catabolism of tryptophan, can evoke spontaneous abortion driven by an immunological reaction against the developing fetus (3). In addition, tolerogenic subset of the dendritic cells (DC) has been shown to express IDO1 (4). Hence, the decreased concentration of tryptophan may act as an endogenous and negative regulator of the immune response. On this basis it could be predicted that in autoimmunity, tryptophan metabolism via IDO1 would be increased, as an attempt to diminish deleterious immune responses. This hypothesis is strengthened by the observations showing DC (4–6) and macrophages (7–9) express enzymes required for tryptophan catabolism via the kynurenine pathway. These enzymes are: IDO1, IDO2, arylformamidase (AFM), kynurenine-3-monooxygenase (KMO), kynureninase (KYNU), and 3-hydroxyanthranilate 3,4 dioxygenase (HAAO) (10).

However, it is not clear yet how activity of the kynurenine pathway could impact the immune system. There are three possibilities. The decreased concentration of tryptophan on its own could reduce the rate of cell proliferation (9, 11) and ability to synthesize pro-inflammatory cytokines (12). Alternatively, toxic byproducts of tryptophan catabolism could impact the immune cells (13–15). It is also possible that the full anti-inflammatory action of the kynurenine pathway is achieved upon synergistic action of decreased tryptophan concentration and accumulation of its catabolites (16). This hypothesis is strengthened by the observations showing reduced activity of Th17 cells (17) and promoted conversion of naïve T cells to Treg upon decreased tryptophan concentration and accumulation of its catabolites in vitro (18, 19).

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with the progressive joint damage (20). Pathology is mostly driven by the infiltration of T cells into the joints and subsequent overproduction of pro-inflammatory cytokines, including interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 17 (IL-17) (21). Activated macrophages are a major source of TNF-α in RA (22). Non-tolerogenic DC propagate inflammation by the presentation of the antigens to T cells (23). Th17 cells have been shown to be pro-arthrogenic (24, 25) whereas regulatory T cells (Treg) suppress symptoms of RA (26, 27). It has been found that concentration of tryptophan was decreased in blood from patients with RA (28, 29), various cancers (30), and other autoimmune diseases, for example, antineutrophil cytoplasmic antibody-associated vasculitis (31).

Collagen-induced arthritis (CIA) is an animal model of RA (32). In CIA, a pre-arthritic stage of the disease usually lasts for 14 days after immunization. In contrast, 10 days after first onsets of the disease become apparent, inflammation undergoes self-resolution (33). It has been previously shown that expression of Ido1 mRNA was increased in the inguinal lymph nodes (iLN) of DBA/1 mice with CIA (34). The same authors have also observed that pharmacological inhibition of IDO1 resulted in the increased incidence and severity of the disease (34). In addition, Ido1−/− mice developed exacerbated joint destruction compared to wild-type controls (34). Conversely, increased tryptophan catabolism, following administration of IDO-containing exosomes, has been shown to ameliorate CIA (35). Thus, taken together, in order to establish the potential disease modifying role of the kynurenine pathway in CIA, concentration of tryptophan and its by-products on the kynurenine pathway was measured in iLN and spleen. In addition, mRNA expression for genes involved in the kynurenine pathway was assessed in CIA.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Animals, CIA Development, and Tissue Harvesting

All experimental procedures were approved by the UK Home Office. Adult DBA/1 mice (aged 10–12 weeks) were used in experiments (Charles River, UK). CIA was induced as previously described (32). Mice were humanely sacrificed. Samples were taken 14 days after immunization and referred to as a pre-arthritic material. Animals with established CIA were sacrificed 10 days after first symptoms of CIA were spotted. After death of animals, lymph nodes and spleens were immediately frozen and kept at −80 °C.

HPLC Analysis and Kynurenine Measurements

Concentration of tryptophan, anthranilic acid (AA), and 3 hydroxyanthranilic acid (3-HAA) was determined with high-pressure liquid chromatography (HPLC) method. The HPLC system (UltiMate 3000) was provided by Dionex, UK. All chromatographic procedures were performed at 37 °C, with C18 column (Acclaim 120, Dionex, UK) 3 μm, 120 Å; 4.6 × 150 mm, and injection volume of 10 μL. Tryptophan concentration was determined by HPLC with fluorescence detection (excitation λ = 284 nm; emission λ = 365 nm). The mobile phase (1 mL/min flow rate) consisted of 50 mM acetic acid, 100 mM zinc acetate, and 3% acetonitrile). Concentration of AA and 3-HAA was determined by HPLC with fluorescence detection (excitation λ = 320 nm; emission λ = 420 nm). The mobile phase (1 mL/min of flow rate) consisted of 25 mM sodium acetate (Sigma, Gillingham, UK) and 1 mM acetic acid (pH 5.5). Kynurenine concentration was assessed by colorimetric assay as it has been applied by other authors (34, 36).

RNA Isolation Preparation of cDNA and Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)

RNA was extracted using RNA-Stat60 reagent (AMS Biotechnology, Abingdon, UK) according to the manufacturer's instructions. cDNA was transcribed using the Applied Biosystems Reverse Transcription System. The total volume of qRT-PCR reaction was 10 μL. TaqMan primer probes were provided by Applied Biosystems, UK. List of primers is shown in the Supporting Information Table 2. PCR reaction was performed in a Corbett Rotor-gene 6000 thermocycler (Corbett Lifesciences, Sydney). mRNA expression was assessed by the ΔΔCt method.

Statistical Analysis

Data were statistically analyzed using Prism 4.03 software. One-way ANOVA with Dunnett's multiple comparison post-test was used to compare three or more groups and the two tailed unpaired T-test was used to compare two groups.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In iLN isolated from pre-arthritic mice and animals with established CIA, the mean concentration of tryptophan was significantly decreased in comparison with naïve samples (Table 1). In the spleens isolated from pre-arthritic mice and animals with established CIA, mean concentration of tryptophan was not significantly decreased between samples taken from naive, pre-arthritic animals, and mice with established CIA (Supporting Information Table 1). Next, concentration of kynurenine, a major by-product of tryptophan catabolism via the kynurenine pathway, was measured. It was found that kynurenine was only accumulated in the iLN only taken from mice with established CIA (Table 1). Catabolites of kynurenine, AA, and 3-HAA, have been also accumulated during the established phase of CIA but not in the pre-arthritic stage of the disease (Table 1).

Table 1. Concentration of tryptophan and its catabolites in iLN during CIA
CompoundNaïve (nmol/g of wet tissue)Pre-arthritic (nmol/g of wet tissue)Established CIA (nmol/g of wet tissue)
  • Small molecules were isolated from iLN from naïve mice (n = 3), pre-arthritic (n = 5), and from mice with established CIA (n = 5). Concentration of tryptophan, AA, and 3-HAA was measured with HPLC, whereas concentration of kynurenine was determined using colorimetric assay.

  • *

    P < 0.05;

  • **

    P < 0.01.

Tryptophan75.86 ± 9.2253.92 ± 8.47*40.74 ± 8.47**
Kynurenine3.62 ± 1.539.86 ± 4.6915.28 ± 6*
AA0.742 ± 0.1750.712 ± 0.0831.081 ± 0.083*
3-HAA0.013 ± 0.0020.0195 ± 0.0080.0298 ± 0.008**

Changes in the concentration of tryptophan and kynurenines could be driven by non-metabolic factors, for example, increased size of the iLN upon T cell proliferation and/or slower diffusion of small molecules between proliferating cells. Thus, it was of interest to test whether mRNA expression for the subsequent genes on the kynurenine pathway could be affected by CIA. mRNA expression for Ido1 was significantly (P < 0.05) increased in the pre-arthritic phase of CIA and in the established CIA in comparison with iLN taken from naive mice (Fig. 1A). mRNA expression for Ido2 and Afm was found significantly (P < 0.01) increased in both experimental groups, pre-arthritic iLN and tissues taken from animals with established CIA (Figs. 1B and 1C). Hence, decreased tryptophan concentration could be driven by increased mRNA expression for Ido1 and Ido2 genes, whereas mRNA expression for Afm is not a limiting factor in the anabolism of kynurenine. In the spleen, mRNA expression for the initial enzymes on the kynurenine pathway was not significantly affected by CIA (Supporting Information Figs. 1A–1C).

thumbnail image

Figure 1. mRNA expression for the kynurenine pathway genes in iLN during CIA. CIA was induced in DBA/1 mice and iLN were taken from mice (n = 4) 14 days after immunization (a pre-arthritic stage of CIA) and animals (n = 5) with established CIA (10 days after first onset of the disease become apparent). Results were compared with naive iLN (n = 5). mRNA expression for (A) IDO1, (B) IDO2, (C) AFM (D) KMO, (E) KYNU, and (F) HAAO was measured with qRT-PCR technique on cDNA prepared with total RNA isolated from iLN. Expression of these genes was normalized to hypoxanthine guanine phosphoribosyltransferase (Hprt1) transcripts. Values represent arbitrary units with SEM provided as result of gene expression analysis with ΔΔCt method. Results were statistically assessed using one-way ANOVA with Dunnetts multiple comparison test to compare experimental groups. *P < 0.05, **P < 0.01, ***P < 0.001.

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Next, mRNA expression for the subsequent genes involved in the catabolism of kynurenines (Kmo, Kynu, and Haao) was assessed in iLN. In the pre-arthritic iLN, mRNA expression for all three genes: Kmo, Fig. 1D; Kynu, Fig. 1E; and Haao, Fig. 1F; followed the same pattern. In the pre-arthritic iLN, mRNA expression for all three genes was significantly (P < 0.01) increased in comparison with naive tissues and organs isolated from mice with established arthritis (Figs. 1C–1E).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Activation of the kynurenine pathway during CIA was found to be restricted to the iLN and in the spleen this metabolic pathway is not affected by the disease. In fact, it has been previously found that IDO1 dependent tryptophan catabolism is increased by the blood borne pathogens (37, 38). When animals were immunized by the subcutaneous antigen administration (like in CIA), activation of the kynurnine pathway was confined to lymph nodes (38). Moreover, mechanisms responsible for organ specific activation of the kynurenine pathway upon immune challenge are poorly characterized.

In iLN from pre-arthritic mice, tryptophan concentration was decreased, whereas its catabolites were not accumulated and the disease progressed. In contrast, 10 days after the first symptoms of the disease become apparent, not only was tryptophan concentration decreased but also its toxic catabolites were accumulated in iLN. CIA is an acute disease for around 10 days since the first symptoms of the disease are spotted (33). In the next stage, the disease enters self-remitting stage (33). Hence, based on data presented here it can be concluded that the full anti-arthritic potential of the kynurenine pathway is achieved only upon coincidence between the decreased tryptophan concentration and accumulation of its biologically active catabolites. However, due to the exploratory character of this work further studies are needed highlighting the role of the kynurenine pathway in RA. For example, in the context of result presented in this article it would be very interesting to test whether pharmacological inhibition of the downstream enzymes on the kynurenine pathway in the pre-arthritic mice would interrupt progression of CIA.

Investigation of mRNA expression for genes encoding enzymes on the kynurenine pathway has provided some interesting data. In CIA, genes encoding initial enzymes of the kynurenine pathway (IDO1, IDO2, and AFM) seem to exhibit similar expression pattern with each other. mRNA expression for these genes was simultaneously increased in the pre-arthritic iLN as well as in those taken from mice with established CIA. In contrast, mRNA expression for the downstream genes on the kynurenine pathway (Kmo, Kynu, and Haao) was simultaneously increased in the iLN taken from pre-arthritic mice, whereas in iLN isolated from mice with established CIA their expression was normalized almost to the control level allowing accumulation of tryptophan catabolites. Thus, it could be that in vivo mRNA expression for the initial as well as downstream genes on the kynurenine pathway is regulated by the set of similar but not overlapping mechanisms which determines metabolic flux through the kynurenine pathway during inflammation (39). However, it is very important to keep in mind that increased mRNA expression does not automatically correspond to the increased enzyme activity. Hence, it would be very interesting to measure activities of the enzymes on the kynurenine pathway during CIA. Nonetheless, data showing changes in the concentration of tryptophan and its catabolites support the notion that changes in the mRNA expression for the genes on the kynurenine pathway upon inflammation are of biological importance.

Desvignes and Ernst have demonstrated that an equimolar mixture of kynurenines could inhibit production of IL-17 in a dose-dependent manner (12). In addition, it has been shown that kynurenines were able to abrogate the Th17-promoting capacity of IL-23 in Th17 cells (12). In addition, Fallarino et al. have also shown that in a long-term cell culture (7 days), low tryptophan concentration (35 μM), and an equimolar mixture of kynurenine, AA, 3-HK, 3-HAA, and QA promote conversion of naive CD4+ T cells into CD25+ Foxp3+ regulatory T cells (Treg) (40). Here, it is shown that in iLN from mice with established CIA the mean concentration of tryptophan was in a similar micro molar (μM) range as previously applied by Fallarino et al. (40). However, it is difficult to extrapolate data showing values of IC50 for small molecules tested in vitro with biological effects in vivo. In a petri dish cells have been freely exposed to the action of a given compound. In contrast, in the intact organ, the concentration of a given metabolite may not be particularly high, whereas in the local tissue environment, for example, the immunological synapse concentration of a given molecule can reach very high concentration required to exhibit biological effect.

Keeping in mind an exploratory character of work presented here, results presented in this article may rise new opportunities for pharmacological intervention into RA. It might be beneficial to develop chemical activators of IDO enzymes and introduce therapy of RA with inhibitors of the downstream enzymes on the kynurenine pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The author would like to thank KIR Trustees and Arthritis Research Campaign UK for their financial support. Author thanks Dr. Ewa Paleolog and Dr. Richard O Williams for teaching CIA model. The author declare no competing of interests.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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