Elevated serum levels of kynurenine pathway metabolites in Behçet's patients

doi:10.21203/rs.3.rs-987705/v1

Behçet's disease (BD) is a inflammatory, multisystemic vasculitis of unknown etiopathogenesis. However, innate and adaptive immune system involvement and immune-mediated networks play a key role in the inflammatory cascade. Studies have shown that both indoleamine 2,3-dioxygenase enzyme, which is induced in inflammatory conditions and catalyzes the first and rate-limiting step in the tryptophan (TRP) metabolism by the kynurenine pathway (KP), and downstream metabolites have immunomodulatory properties. The study was aimed to measure KP metabolites levels in patients with BD and to investigate the relationship between disease activity and clinical findings with these metabolites. The study included 120 patients with BD and 120 healthy volunteers. Serum TRP, kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3HAA), 3-hydroxykynurenine (3HK), quinolinic acid (QUIN) levels were analyzed with tandem mass spectrometric method. Demographic data, clinical manifestations and disease activity score (BDCAF) were recorded. Serum KYN, KYNA, 3HK, 3HAA, QUIN levels and KYN/TRP ratio were higher (p<0.05) in patients with BD compared to the control group, while TRP levels were lower (p<0.05). KYN/TRP ratio and QUIN levels were significantly higher in the presence of neurobehçet's while serum KYN levels were significantly higher in the presence of arthritis (p<0.05). Also, serum QUIN levels were significantly higher in the presence of thrombosis (p<0.05). BDCAF score positively correlated with KYN/TRP ratio and QUIN levels. Our findings showed that serum KP metabolite levels were elevated in patients with BD and there is a relationship between these metabolites with disease activity, clinical findings and inflammatory burden.

Allergy & Immune Disorders

Immunology

General Microbiology

Behçet’s disease

kynurenine pathway

immune regulation

inflammation

Behçet's disease (BD) is a chronic, autoimmune, inflammatory multisystemic vasculitis characterized by recurrent oral aphthae, genital ulcers, ocular and cutaneous lesions [1]. It can also effects the central nervous system, gastrointestinal tract, joints, and pulmonary system [2]. BD is less prevelant in Europe and the Unites States, while it is more common in regions along the historical Silk Road such as Turkey, Israel, Iran, Japan, and China. Turkey has the highest prevelance with the rate of 70–420/100,000 among these countries [3]. The age of onset of the disease is often between 20 and 40 years [4]. BD affects both genders equally, but there are regional variations between male and female ratios [5]. Although the etiology of BD is unknown, genetic and environmental factors are thought to play an important role in the onset and progression of the disease. The strongest genetic risk factor identified for BD is the presence of human leukocyte antigen (HLA) B51, and it has been reported that it can increase the risk of BD from 1.5 to 16 times in different ethnic groups populations [6]. There is no specific laboratory test for the diagnosis of BD, so it is commonly diagnosed based on clinical criteria following the elimination of alternative diagnoses, but detection of BD is more difficult especially in non-symptomatic individuals [7]. Therefore, it is essential to identify new sensitive and specific markers for the diagnosis of BD. Various studies have reported that cytokine-producing cells play a key role in the immunopathogenesis of the inflammatory process in BD. In particular, the altered cytokine production profile of T helper 1 (Th1), Th2, or Th17 cell types is important in the immune response in BD [8]. Th1 and Th17-related cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6 and IL-17 in patients with BD increased levels have been reported [9–11]. The kynurenine pathway (KP) is the main route (~95%) of tryptophan catabolism, resulting in the formation of nicotinamide adenine dinucleotide (NAD⁺) and various biologically active intermediates such as kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HAA) and quinolinic acid (QUIN) [12]. TRP is converted to KYN by tryptophan 2,3-dioxygenasedioxygenase (TDO) in the liver, while it is metabolized by indoleamine 2,3-dioxygenase 1 (IDO-1), indoleamine 2,3-dioxygenase 2 (IDO-2), in extrahepatic tissues such as immune system cells, brain and this is the first and rate-limiting step of KYN synthesis. Proinflammatory cytokines upregulate IDO-1 expression and increase flux during KP [13]. The most important inducer of IDO-1 is interferon-γ (IFN-γ), but also proinflammatory cytokines such as tumor necrosis factor (TNF-α), and interleukins (IL) 6 (IL-6) and 1 (IL-1) induce IDO-1 activity [14, 15]. In recent years, it has been recognized that KP plays an important role in the regulation of the innate and adaptive immune system and the inflammatory response. It has been shown that IDO-1 has a strong immunosuppressive effect and plays an important role in the suppression of autoimmune diseases. These immunosuppressive effects on T lymphocytes are thought to be related to the depletion of tryptophan and the direct effects of KP metabolites. Some of KP metabolites such as QUIN and 3HAA may suppress T cell proliferation by selectively targeting activated immune cells. In addition, KYN has been shown to be involved in immune system regulation by its ligand function on the aryl hydrocarbon receptor (AhR) [16]. In the light of all this information, the aim of this study is to measure the KP metabolite levels in patients with BD and to investigate the relationship between disease activity and clinical findings with these metabolites.

Study design

Patients

The study included 120 patients who were diagnosed with BD according to 1990 the International Study Group (ISG) criteria and were admitted to the Rheumatology outpatient clinic of Selcuk University Medical Faculty Hospital, and 120 healthy volunteers who did not have any chronic disease, had normal physical examination and routine test results and applied to our hospital for routine health control [17]. Subjects who smoke, use alcohol, have chronic kidney, liver disease, cardiovascular diseases, endocrine disorders, acute or chronic infections, other autoimmune or inflammatory diseases, neurodegenerative or psychiatric diseases, and malignancies were excluded from the study.

A total of 6 cc blood samples from the participants were taken into serum separator gel tubes and EDTA tubes. Serum samples were separated by centrifugation at 2000 g for 10 minutes than they were stored at -80°C until analysis. This study was approved by Selcuk University Faculty of Medicine Ethics Committee (Number: 2020/226, Date: 03.06.2020).

Clinical Evaluation

Demographic data of the patients (age, gender, duration of disease), mucocutaneous involvement such as oral and genital ulcers, joint involvement, eye involvement, intestinal involvement, neurological involvement, cardiovascular involvement, disease activity score, pathergy test and medical treatments were evaluated and recorded by clinicians. Organ involvements were assessed according to clinical symptoms and with the diagnostic tests. BD activity score was evaluated using Behcet's Disease Current Activity Form (BDCAF). The BDCAF was filled in by the clinicians considering the patient's admission day and the last 4 weeks, and a score ranging from 0 to 12 was obtained by scoring the active findings in the last 4 weeks. Active BD was considered as BDCAF score ≥2 while a score <2 was accepted as inactive BD.

Demographical characteristics and clinical manifestations of BD were showed in Table 1.

Routine laboratory analysis

Hemoglobin (HGB), white blood cell count (WBC), neutrophil (NEU), monocyte (MONO), lymphocyte (LYM), platelet (PLT), mean platelet volume (MPV), Red Cell Distribution Width (RDW), and plateletcrit (PCT) counts in whole blood samples drawn into EDTA tubes from participants were analyzed Beckman Coulter LH 780 analyzer (Beckman Coulter, Miami, FL, USA) according to the manufacturer's instructions. CRP levels and erythrocyte sedimentation rate (ESR) were measured by an immunolephelometric method with IMMAGE 800 (Beckman Coulter, Brea, USA) immunochemistry system and by a capillary photometry method with Alifax (Padova, Italy) analyzer.

Tandem mass spectrometric analysis

Chemicals

L-TRP (CAS Number: 73-22-3), L-KYN (CAS Number 2922-83-0), KYNA (CAS Number 492-27-3), 3HAA (CAS Number 548-93-6), 3HK (CAS Number 484-78-6), QUIN (CAS Number 89-00-9), HPLC grade water (CAS Number: 7732-18-5), acetonitrile (CAS Number 75-05-8), formic acid (CAS Number: 64-18-6) were obtained from Sigma Aldrich Sigma Aldrich (St. Louis, MO, USA) and L-KYN- d₄ trifluoroacetic acid salt (Catalog No: K661007) was obtained from Toronto Research Chemicals (North York, ON, Canada).

Instrumentation

Chromatographic separation of analytes was performed by a Shimadzu HPLC system (Kyoto, Japan) and Phenomenex C18 column (50 mm x 4.6 mm, 5µm, 100 Å). KP metabolites were detected by ABSciex API 3200 tandem mass spectrometer (Applied Biosystems/MDS Sciex) in positive electrospray ionization mode. HPLC grade water containing 0.1% formic acid (v/v%) (A) and acetonitrile containing the same ratio of formic acid (v/v%) (B) were applied by gradient elution as mobile phase. Total analysis time was 5 minutes for each sample. The precursor to product ion transitions (m/z) for TRP, KYN, KYNA, 3HAA, 3HK, QUIN and internal standard (KYN-d4) were set as 205.2/146.2, 209.1/94.1, 190.2/144.0, 154.0/136.0, 225.1/110.0, 168.0/124.0 and 213.1/140.1, respectively. The ion-source was operated in positive electrospray ionization mode and parameters were as follows: ionspray voltage, 5000 V; source temperature, 350°C; curtain, 20 psi; ion source (GS1), 50 psi; and ion source (GS2), 50 psi, respectively. The intra- and inter-assay imprecisions were < 12% for all analytes.

Sample preparation

Serum KP metabolite concentrations were measured with the modification of the method developed by Tong et al. [18]. Briefly; 300 µL of serum sample, 100 µL of internal standard (KYN-d₄) and 1000 µl of acetonitrile containing 1% formic acid (v/v%) were taken into eppendorf tubes and vortexed for 30 seconds. Than, the mixture was centrifuged at 2000xg during 10 minutes. 1000 µl of supernatants were taken into clean reaction tubes and evaporated with the nitrogen gas at 40°C. The residues were dissolved in 200 µl of acetonitrile containing 0.1% formic acid: water (25:75, v/v%) mixture. 30 µL was injected into the system.

Statistical analysis

Statistical evaluation was performed with SPSS statistical software package version 21.0. One-Sample Kolmogorov-Smirnov test was used to determine the distribution of data. The means of parametric data were compared with Student's t test, while the medians of nonparametric data were compared with Mann-Whitney U test. Gender of the groups were compared using the chi-square test. Spearmen correlation analysis was performed. P<0.05 was considered as statistically significant.

The mean ages of patients with BD and control groups were 41.50 ± 9.78 and 41.41 ± 8.92 years, respectively. The patient and control groups had similar age (p=0.944) and gender distribution (p=0.870). The median disease duration of patients with BD was 3 years and ranged from 1 to 28 years.

Serum KYN, KYNA, 3HK, 3HAA, QUIN levels and KYN/TRP ratio in patients with BD were statistically significantly higher than the control group, while serum TRP levels were lower (p<0.05). Also, the levels of inflammation markers including CRP, ESR, RDW, NEU, MONO, MLO and PCT were significantly higher in patients with BD (p<0.05).

Serum KP metabolite levels and other laboratory parameters of patients with BD and control group were expressed in Table 2.

When evaluated in terms of clinical findings, all patients with BD had oral aphthae 120 (%100). Of 120 patients, 70 (58.3%) had genital ulcer, 64 (53.3%) skin lesions, 48 (45%) arthritis, 53 (44.2%) uveitis, 35 (29.1%) thrombosis, 14 (11.7%) had neuro-Behcet's disease, 5 (4.2%) entero-Behcet's disease, 55 (45.8%) pathergy test positivity and 57 (47.5%) had HLA-B51 positivity. The median BDCAF score was 4.5 years and ranged from 2 to 8. The comparison of the BD group according to clinical manifestations showed that the KYN/TRP ratio and QUIN levels were significantly higher in patients with neurobehçet than in other BD patients. Serum QUIN levels were also significantly elevated in the presence of thrombosis while serum KYN levels were significantly higher in BD patients with arthritis.

The comparison results of demographic and laboratory findings according to manifestations of BD were expressed in Table 3.

The correlations of serum KP metabolites, inflammatory markers and disease activity were shown in Table 4.

IDO-1 catalyzes the first and rate-limiting step in the catabolism of tryptophan by the KP and is expressed primarily by antigen-presenting cells in the placenta, some immune cell subsets, mucosal tissues, pancreas, and the eye [19]. IDO-1 is expressed at low levels in healthy individuals, while its expression increases dramatically in infection or inflammation induced by lipopolysaccharide, proinflammatory cytokines, or other agents [20]. It is consider that IDO-1 inhibit the proliferation of microbes, tumor cells and activated T lymphocytes through the catabolism of tryptophan, an essential amino acid. Recent studies have revealed that it plays an important role in the induction of immune tolerance during infections, pregnancy, malignancies, autoimmune disorders and transplantation. IDO-1 upregulation is induced by IFN-γ, which is also responsible for the activation of T cells. Therefore, IDO-1 activation and immunosuppressive effect in inflammatory conditions are thought to be a feedback mechanism against T cell activation. T cells are particularly sensitive to tryptophan depletion. At low tryptophan levels, the cell cycle is arrested in the mid-G1 phase. With the restoration of tryptophan to these cells, the cell cycle induces. So, the immunosuppressant effect of IDO-1 based on tryptophan depletion [21]. The blood KYN/TRP ratio is often used to reflect or express IDO-1 enzyme activity. This ratio is more commonly used instead of measuring IDO-1 activity, which is low in immune system cells or other cells under basal conditions, but increases dramatically during immune activation or inflammation [22]. Our findings showed that serum TRP levels in patients with BD were statistically significantly lower than in the control group, while kynurenine levels and KYN/TRP ratio were significantly higher. BD is a chronic, autoimmune, inflammatory disease involving innate and adaptive immunity, and numerous studies have demonstrated important roles of Th cells such as Th1, Th17 and elevated serum levels of proinflammatory cytokines such as IFN-γ, IL-6, IL-1, and various inflammation markers in patients with BD [9–11]. Our findings suggested increased inflammation and immune activation in patients with BD. Systemic inflammation markers such as NLR, PLO, MLO, RDW, acute phase reactants CRP and ESR levels, and PCT which have recently been shown to be a potential marker of inflammation, were statistically significantly higher in patients with BD compared to the control group [23, 24]. There was a positive correlation between KYN/TRP ratio with RDW, NLR, PLR, CRP, ESR and PCT levels. Therefore, increased KYN/TRP ratio in patients with BD is an indicator of increased IDO-1 activity as a feedback mechanism due to increased inflammation and immune system activation in patients with BD. However, catabolism of TRP by KP results in the formation of a number of biologically active metabolites called kynurenines such as KYN, 3HK, KYNA, 3HAA, QUIN, and these metabolites themselves inhibit T cell activation and proliferation. KP metabolites have a proapoptotic effect especially on activated immune system cells and Th1 lymphocytes [25].

KYNA is the pleotropic product of a branch of KP pathway and it has an endogenous competitive antagonistic activity for all ionotropic excitatory amino acid receptors, including N-methyl-D-aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA). In addition, KYNA is an α7 nicotinic receptor antagonist. KYNA is referred to as the neuroprotective branch of KP because of its effects on these receptors [26]. However, alterations in KYNA levels have been reported in various inflammatory conditions. Recently, KYNA has been shown to have anti-inflammatory properties through its agonistic activity on the orphan G-protein-coupled receptor (GPR35), regulation of cAMP production, and inhibition of N-type Ca2+ channels of sympathetic neurons and astrocytes. It has also been reported to modulate the immune response by its agonistic effect on the AhR [27]. Elevated serum KYNA levels have been reported in various chronic inflammatory diseases such as type 2 diabetes, multiple sclerosis, and inflammatory bowel disease. Therefore, considering the immunosuppressive and anti-inflammatory properties of KYNA, it is thought that increased KYNA levels in inflammatory diseases may be a compensation mechanism [28]. Our findings showed increased serum KYNA levels in patients with BD. Moreover, there was a positive correlation between serum KYNA levels and inflammation markers PLR, CRP and ESR levels. Therefore, increased KYNA levels in patients with BD may be considered as a compensation mechanism against inflammation and immune system activation in BD.

QUIN is one of the most controversial KP metabolites, functioning as both an essential metabolite and a potent neurotoxic. It is well known that QUIN has a neurotoxic effect by inducing NMDA receptors and reactive oxygen species (ROS) / reactive nitrogen species (RNS)-dependent mechanisms [29]. However, QUIN is an essential metabolite of KP that plays a role as a precursor in the synthesis of NAD⁺. In cases such as inflammation and infection, the NAD⁺ requirements of certain cells increase to provide the necessary immune cell response. QUIN can direct some of the tryptophan catabolism to replenish cellular NAD⁺ levels in response to inflammatory conditions and infections. As a matter of fact, in conditions characterized by inflammation and immune system activation, intracellular QUIN levels increase significantly in immune system cells such as macrophages and microglia [30]. It has also been shown that QUIN has an immunosuppressive effect by inhibiting T cell proliferation [31]. Moffet et al. developed antibodies against QUIN and demonstrated elevated QUIN levels in leukocytes by immunohistochemistry technique. Researchers have shown that, unlike rat liver, leukocytes store large amounts of QUIN to ensure adequate production of NAD⁺, whose consumption is increased due to oxidative stress and inflammatory conditions during the immune response. During the inflammatory response and immune system activation, blood QUIN levels increase significantly, and it may reaches µM levels. Therefore, considering the blood levels of QUIN, it is thought that it may be the main immunosuppressant among the KP metabolites [32, 33]. Our findings showed that serum QUIN levels in patients with BD were statistically significantly increased compared to the control group. In addition, QUIN levels were positively correlated with PCT, CRP and ESR levels. Therefore, it can be thought that increased QUIN levels are a result of IDO activity induced in inflammatory conditions and it is a defense mechanism of the body against immune system activation. However, QUIN, which is produced in high amounts in inflammatory conditions, causes a toxic effect by various mechanisms by triggering the activation of destructive enzymatic pathways such as protein kinases, phospholipases, nitric oxide (NO) synthase and proteases via NMDA receptor activation, and lipid peroxidation [34].

3HK and 3HAA are other KP metabolites that have been reported to have immunomodulatory properties [30]. However, 3HK and 3HA have dual pro-oxidant/antioxidant properties. Studies investigating the oxidant/antioxidant role of 3HAA reported that it functions as a key antioxidant by scavenging free radicals in the absence of metal ions, has a peroxyl radical scavenging effect in both lipid and aqueous solutions, while it exhibits pro-oxidant behavior in the presence of metal ions [35]. Similarly, 3HK is thought to have dual activity. 3HK has been reported to have antioxidant behavior under normal conditions, while it has been shown to act as a pro-oxidant by binding to peptide chains under oxidant conditions. It has also been shown that reactive oxygen derivatives produced during the oxidation of 3HK cause oxidation in lipids, proteins and nucleic acids [36]. Our findings showed that serum 3HK and 3HAA levels increased in BD patients compared to the control group, in parallel with the activation of IDO-1 and thus the depletion of tryptophan through KP. The positive correlation between the KYN/TRP ratio and these metabolites supported our findings. In addition, there was a positive correlation between 3HK levels with CRP and MPV levels and 3HAA levels with PLR, PCT and ESR levels. However, increased 3HK and 3HAA in patients with BD may trigger oxidative stress and cause tissue damage.

When the relationship between disease activity, clinical findings and KP metabolites was evaluated, the KYN/TRP ratio was significantly higher in the subgroup with neurobehçet's than in patients with BD who have not these manifestations while serum KYN levels were higher in presence of arthritis. Serum QUIN levels were statistically significantly higher in the presence of thrombosis and neurobehçet's manifestations. In addition, BDCAF score were positively correlated with NEU, WBC counts, MLO, CRP, KYN/TRP ratio and QUIN levels. It has been reported that QUIN promotes atherosclerosis by triggering oxidative stress in endothelial and vascular smooth muscle cells and leukocytes in individuals with chronic kidney disease [37]. Experimental studies show that increased QUIN levels are especially associated with carotid atherosclerosis and endothelial dysfunction, so it considered that QUIN is involved in the pathogenesis of cardiovascular diseases [38, 39]. A contradictory set of biological activities of QUIN in patients with chronic renal failure has been described recently, including its contribution to hypercoagulation or hyperfibrinolysis [40–42]. In the study conducted by Leszczyńska et al. with green fluorescent protein (GFP)-expressing transgenic mice, it was shown that a single dose of IV QUIN administration had antithrombotic activity, while 14-day administration of QUIN played a prothrombotic role by increasing platelet aggregation [37]. Our findings showed that QUIN was elevated in BD patients with thrombosis, consistent with previous studies. Overproduction of QUIN in patients with BD due to inflammatory conditions and immune system activation may have played a role for vascular involvement by inducing oxidative damage and endothelial dysfunction in these patients. However, further studies are needed to elucidate the exact mechanism between QUIN and vascular involvement in patients with BD.

There is increasing evidence that KYN impairs osteogenesis and increases age-related bone loss. KYN affects bone resorption by increasing the number of osteoclasts covering bone surfaces and has direct osteoclastic activity through its effects on nuclear factor kappa-B ligand (RANKL) [43]. It has been explained that accumulated KYN negatively affects bone marrow stromal cells by impairing osteogenic differentiation, autophagy and bioenergy of bone marrow cells and has negative effects on bone metabolism [44]. Our findings showed that KYN levels were increased in patients with BD with joint involvement. Therefore, overproduction of KYN as a result of accelerated metabolism of TRP with inflammatory stimuli in patients with BD may be responsible for joint involvement in patients with BD.

In addition, our findings showed that serum KYN/TRP and QUIN levels were higher in neurobehçet patients compared to other BD patients. It has been reported that peripheral TRP, KYN and 3HK cross the blood-brain barrier, while QUIN and KYNA cannot cross the blood-brain barrier. It indicates that peripheral activation of KP in inflammatory conditions may translocated to the central nervous system and increase neurotoxicity [45]. KYN, which is increased in inflammatory conditions in patients with BD, may pass through the blood-brain barrier and turn into neurotoxic metabolites such as 3HK and QUIN, and may have induced neuronal damage in these patients. However, considering the potential prothrombotic role of increased QUIN in these patients, we think that it may be involved in neurobehçet's pathogenesis by inducing cerebral venous thrombosis. However, further studies are needed to elucidate the exact mechanism.

The positive correlation between BDCAF score with KYN/TRP ratio, QUIN levels and inflammation markers supported that these metabolites were directly related to the inflammatory burden and BD progression.

Our findings showed that tryptophan degradation and serum levels of KP metabolites including KYN, 3HK, 3HAA, QUIN, KYNA were elevated in patients with BD. There was a correlation between serum KP metabolite levels, inflammation markers and disease activity score. In addition, there were altered serum KYN, QUIN levels and KYN/TRP ratio in the presence of arthritis, neurobehçet's and thrombosis. Therefore, our findings suggest that KP metabolites can be valuable markers for diagnosis and progression of BD and in monitoring of clinical manifestations. The study was superior in terms of it was the first to investigate KP metabolite levels, disease activity, and the relationship between clinical indicators and these markers in patients with BD. However, current study has some disadvantages in terms of the limited patient population, lack of measurement of IDO-1 enzyme activity, other KP metabolites such as anthranilic acid and picolinic acid and further studies in larger populations are needed.

Acknowledgements

The authors would like to thank the Selcuk University for this study.

Author’s contribution

Each named author has substantially contributed to conducting the research and drafting the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Ethics approval

This study was performed according to guidelines established by the 2013 Helsinki Declaration and protocol was approved by Selcuk University Faculty of Medicine Ethics Committee (Number: 2020/226, Date: 03.06.2020).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Coutinho, I., D. Silva, I. Ferreira, B. Grima, M. Lisboa, M. Bernardo, and I. Prieto. 2017. Behçet disease: our experience. Revista Brasileira de Oftalmologia 76: 210–212.
Sun, F., H. Cao, F. Wang, and G. Cao. 2020. Behçet's disease with invasive pulmonary aspergillosis and Aspergillus auriculatus infection: A case report. Medicine 99: e18938.
Mumcu, G., F. Alibaz Öner, T. Ergun, and R. H. Direskeneli. 2020. Decreasing incidence and severity of Behçet’s disease: a changing trend in epidemiological spectrum possibly associated with oral health. Turkish Journal of Medical Sciences 50: 1587–1590.
Leccese, P., and E. Alpsoy. 2019. Behçet's Disease: An Overview of Etiopathogenesis. Frontiers in Immunology 10: 1067.
Esatoglu, S. N., Z. Kutlubay, D. Ucar, I. Hatemi, U. Uygunoglu, A. Siva, and G. Hatemi. 2017. Behçet's syndrome: providing integrated care. Journal of Multidisciplinary Healthcare 10: 309–319.
Rokutanda, R., M. Kishimoto, and M. Okada. 2014. Update on the diagnosis and management of Behçet's disease. Open Access Rheumatology 7: 1–8.
Alpsoy, E. 2016. Behçet's disease: A comprehensive review with a focus on epidemiology, etiology and clinical features, and management of mucocutaneous lesions. The Journal of Dermatology 43: 620–632.
Tong, B., X. Liu, J. Xiao, and G. Su. 2019. Immunopathogenesis of Behcet's Disease. Frontiers in immunology 10: 665–665.
Gholijani, N., M. R. Ataollahi, A. Samiei, E. Aflaki, S. Shenavandeh, and E. Kamali-Sarvestani. 2017. An elevated pro-inflammatory cytokines profile in Behcet's disease: A multiplex analysis. Immunology Letters 186: 46–51.
Aktaş-Çetin, E., F. Cosan, A. Cefle, and G. Deniz. 2014. IL-22-secreting Th22 and IFN-γ-secreting Th17 cells in Behçet's disease. Modern rheumatology / the Japan Rheumatism Association 24.
Türkçüoğlu, P., Y. O. Arat, E. Kan, E. K. Kan, I. A. Chaudhry, S. Koca, Ü. Çeliker, and N. İlhan. 2016. Association of Disease Activity with Serum and Tear IL-2 Levels in Behçet Disease. Ocular Immunology and Inflammation 24: 313–318.
Badawy, A. A. 2017. Kynurenine Pathway of Tryptophan Metabolism: Regulatory and Functional Aspects. International Journal of Tryptophan Research 10: 1178646917691938.
Savitz, J. 2020. The kynurenine pathway: a finger in every pie. Molecular Psychiatry 25: 131–147.
Pallotta, M. T., S. Rossini, C. Suvieri, A. Coletti, C. Orabona, A. Macchiarulo, C. Volpi, and U. Grohmann. 2021. Indoleamine 2,3-dioxygenase 1 (IDO1): an up-to-date overview of an eclectic immunoregulatory enzyme. Febs journal.
Jovanovic, F., K. D. Candido, and N. N. Knezevic. 2020. The Role of the Kynurenine Signaling Pathway in Different Chronic Pain Conditions and Potential Use of Therapeutic Agents. International Journal of Molecular Sciences 21.
Jones, S. P., N. F. Franco, B. Varney, G. Sundaram, D. A. Brown, J. de Bie, C. K. Lim, G. J. Guillemin, and B. J. Brew. 2015. Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease. PLoS One 10: e0131389.
International Study Group for Behçet's Disease. 1990. Criteria for diagnosis of Behçet's disease. Lancet 335: 1078–1080.
Tong, Q., J. Song, G. Yang, L. Fan, W. Xiong, and J. Fang. 2018. Simultaneous determination of tryptophan, kynurenine, kynurenic acid, xanthurenic acid and 5-hydroxytryptamine in human plasma by LC-MS/MS and its application to acute myocardial infarction monitoring. Biomedical Chromatogrography 32.
Tang, K., Y.-H. Wu, Y. Song, and B. Yu. 2021. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors in clinical trials for cancer immunotherapy. Journal of Hematology & Oncology 14: 68.
Lanser, L., P. Kink, E. M. Egger, W. Willenbacher, D. Fuchs, G. Weiss, and K. Kurz. 2020. Inflammation-Induced Tryptophan Breakdown is Related With Anemia, Fatigue, and Depression in Cancer. Frontiers in immunology 11: 249–249.
Curti, A., S. Trabanelli, V. Salvestrini, M. Baccarani, and R. M. Lemoli. 2009. The role of indoleamine 2,3-dioxygenase in the induction of immune tolerance: focus on hematology. Blood 113: 2394–2401.
Badawy, A. A., and G. Guillemin. 2019. The Plasma [Kynurenine]/[Tryptophan] Ratio and Indoleamine 2,3-Dioxygenase: Time for Appraisal. International Jornal of Tryptophan Research 12: 1178646919868978.
Işık, M., H. Şahin, and E. Hüseyin. 2014. New platelet indices as inflammatory parameters for patients with rheumatoid arthritis. European Journal of Rheumatology 1: 144–146.
Coskun, E., A. Alidris, M. Temel, S. Akbayram, and S. Hizli. 2019. Plateletcrit: A possible biomarker of inflammation in hepatitis A infection. Nigerian journal of clinical practice. 22: 727–730.
Zaher, S. S., C. Germain, H. Fu, D. F. P. Larkin, and A. J. T. George. 2011. 3-hydroxykynurenine suppresses CD4+ T-cell proliferation, induces T-regulatory-cell development, and prolongs corneal allograft survival. Invest Ophthalmol Vis Sci 52: 2640–2648.
Mor, A., A. Tankiewicz-Kwedlo, A. Krupa, and D. Pawlak. 2021. Role of Kynurenine Pathway in Oxidative Stress during Neurodegenerative Disorders. Cells 10.
Muneer, A. 2020. Kynurenine Pathway of Tryptophan Metabolism in Neuropsychiatric Disorders: Pathophysiologic and Therapeutic Considerations. Clinical Psychopharmacology and Neuroscience 18: 507–526.
Wirthgen, E., A. Hoeflich, A. Rebl, and J. Günther. 2018. Kynurenic Acid: The Janus-Faced Role of an Immunomodulatory Tryptophan Metabolite and Its Link to Pathological Conditions. Frontiers in Immunology 8.
Bansal, Y., R. Singh, I. Parhar, A. Kuhad, and T. Soga. 2019. Quinolinic Acid and Nuclear Factor Erythroid 2-Related Factor 2 in Depression: Role in Neuroprogression. Frontiers in Pharmacology 10: 452.
Moffett, J. R., P. Arun, N. Puthillathu, R. Vengilote, J. A. Ives, A. A. Badawy, and A. M. Namboodiri. 2020. Quinolinate as a Marker for Kynurenine Metabolite Formation and the Unresolved Question of NAD(+) Synthesis During Inflammation and Infection. Frontiers in Immunology 11: 31.
Badawy, A. A. 2018. Hypothesis kynurenic and quinolinic acids: The main players of the kynurenine pathway and opponents in inflammatory disease. Med Hypotheses 118: 129–138.
Moffett, J. R., and M. A. Namboodiri. 2003. Tryptophan and the immune response. Immunology & Cell Biology 81: 247–265.
Moffett, J. R., M. G. Espey, and M. A. Namboodiri. 1994. Antibodies to quinolinic acid and the determination of its cellular distribution within the rat immune system. Cell and Tissue Research 278: 461–469.
Pérez-De La Cruz, V., P. Carrillo-Mora, and A. Santamaría. 2012. Quinolinic Acid, an endogenous molecule combining excitotoxicity, oxidative stress and other toxic mechanisms. International Journal of Tryptophan Research 5: 1–8.
Pérez-González, A., J. Alvarez-Idaboy, and A. Galano. 2017. Dual Antioxidant / Pro-Oxidant Behavior of the Tryptophan Metabolite 3-Hydroxyanthranilic Acid: A Theoretical Investigation on Reaction Mechanisms and Kinetics. New Journal of Chemistry 41.
Colín-González, A. L., P. D. Maldonado, and A. Santamaría. 2013. 3-Hydroxykynurenine: an intriguing molecule exerting dual actions in the central nervous system. Neurotoxicology 34: 189–204.
Leszczyńska, A., T. Misztal, N. Marcińczyk, T. Kamiński, K. Kramkowski, E. Chabielska, and D. Pawlak. 2019. Effect of quinolinic acid - A uremic toxin from tryptophan metabolism - On hemostatic profile in rat and mouse thrombosis models. Advances in Medical Sciences 64: 370–380.
Pawlak, K., S. Brzosko, M. Mysliwiec, and D. Pawlak. 2009. Kynurenine, quinolinic acid--the new factors linked to carotid atherosclerosis in patients with end-stage renal disease. Atherosclerosis 204: 561–566.
Pawlak, K., T. Domaniewski, M. Mysliwiec, and D. Pawlak. 2009. Kynurenines and oxidative status are independently associated with thrombomodulin and von Willebrand factor levels in patients with end-stage renal disease. Thrombosis Research 124: 452–457.
Pawlak, K., M. Mysliwiec, and D. Pawlak. 2009. Hypercoagulability is independently associated with kynurenine pathway activation in dialysed uraemic patients. Thrombosis and Haemostasis 102: 49–55.
Pawlak, K., M. Mysliwiec, and D. Pawlak. 2010. Haemostatic system, biochemical profiles, kynurenines and the prevalence of cardiovascular disease in peritoneally dialyzed patients. Thrombosis Research 125: e40–e45.
Pawlak, K., A. Buraczewska-Buczko, M. Mysliwiec, and D. Pawlak. 2010. Hyperfibrinolysis, uPA/suPAR system, kynurenines, and the prevalence of cardiovascular disease in patients with chronic renal failure on conservative treatment. The American Journal of the Medical Sciences 339: 5–9.
Eisa, N. H., S. V. Reddy, A. M. Elmansi, G. Kondrikova, D. Kondrikov, X. M. Shi, C. M. Novince, M. W. Hamrick, M. E. McGee-Lawrence, C. M. Isales, S. Fulzele, and W. D. Hill. 2020. Kynurenine Promotes RANKL-Induced Osteoclastogenesis In Vitro by Activating the Aryl Hydrocarbon Receptor Pathway. International Journal of Molecular Science 21.
Patel, D., M. Potter, J. M. Anaya, M. E. McGee-Lawrence, M. W. Hamrick, W. D. Hill, C. M. Isales, and S. Fulzele. 2021. Kynurenine induces an age-related phenotype in bone marrow stromal cells. Mechanisms of Ageing and Development 195: 111464.
Joisten, N., M. Schumann, A. Schenk, D. Walzik, N. Freitag, A. Knoop, M. Thevis, W. Bloch, and P. Zimmer. 2020. Acute hypertrophic but not maximal strength loading transiently enhances the kynurenine pathway towards kynurenic acid. European Journal of Applied Physiology 120.

Table 1. Demographical characteristics and clinical manifestations of BD.
Characteristics	BD group (n=120)	Control group (n=120)	p
Age (years)	41.5±9.78	41.41±8.92	0.944
Gender (M/F)	55/63	51/67	0.870
Duration of disease (years)	3 (1-28)
BDCAF score	4.5 (2.0-8.0)
Clinical manifestations of BD
Oral aft	120 (100)
Genital ulcer n (%)	70 (58.3)
Cutaneous lesion n (%)	64 (53.3)
Arthritis n (%)	48 (45)
Uveitis n (%)	53 (44.2)
Thrombosis n (%)	35 (29.1)
Neuro-Behcet's disease n (%)	14 (11.7)
Entero-Behcet's disease n (%)	5 (4.2)
Pathergy test positive n (%)	55 (45.8)
HLA-B51 positive n (%)	57 (47.5)
Drugs n (%)
Colchicine	120 (100)
Azathioprine	55 (45.8)
Methotrexate	5 (4.2)
Asetil Salicylic Acid	120 (100)

Table 2. Laboratory parameters of patients with BD and control group.
Parameters	BD group (n=120)	Control group (n=120)	p
TRP (ng/mL)	7339.9 (2877.0-20220.0)	9563.7 (3705.0-20800.0)	<0.001
KYN (ng/mL)	421.91 (100.0-2180.0)	236.6 (78.5-570.0)	<0.001
KYN/TRP (ng/mL)	0.061 (0.01-0.18)	0.028 (0.01-0.09)	<0.001
KYNA (ng/mL)	4.14 (1.33-14.40)	2.56 (0.57-13.0)	<0.001
QUIN (ng/mL)	18.6 (3.0-68.0)	12.6 (8.0-37.7)	0.035
3HK (ng/mL)	2.75 (0.86-11.70)	1.51 (0.56-5.40)	<0.001
3HAA (ng/mL)	4.74 (0.98-16.20)	3.76 (1.01-13.60)	0.004
WBC (K/uL)	7.65 ± 2.48	7.32 ± 1.76	0.232
HGB (g/dL)	13.84 ± 2.09	14.27 ± 1.50	0.074
PLT (K/uL)	280.20 ± 81.08	264.16 ± 57.54	0.094
NEU (K/uL)	4.66 ± 2.09	4.20 ± 1.19	0.041
LYM (K/uL)	2.22 ± 0.72	2.24 ± 0.59	0.814
MONO (K/uL)	0.58 (0.30-1.60)	0.50 (0.30-1.0)	0.039
PLO	129.11 (60.80-350.0)	120.47 (43.93-238.96)	0.124
NLO	1.99 (1.08-14.63)	1.84 (0.63-4.54)	0.073
MLO	0.25 (0.11-1.33)	0.23 (0.09-0.62)	0.029
RDW (%)	15.06 ± 2.12	14.15 ± 3.11	<0.001
MPV (fL)	8.62 ± 0.86	8.41 ± 0.90	0.068
PCT (%)	0.24 ± 0.05	0.21 ± 0.03	0.011
CRP(mg/L)	3.0 (1.0-41.0)	1.7 (0.7-4.6)	<0.001
ESR (mm/h)	10.5 (2.0-105.0)	9.0 (1.1-31.0)	0.039

Table 3. Comparison of manifestations of BD according to demographic and laboratory findings.
Clinical manifestations of BD	Categories		p
Genital ulcer	No (n=50)	Yes (n=70)
Age (years)	44.78±13.15	39.27±10.73	0.015
WBC (K/uL)	7.06±2.18	8.06±2.60	0.031
MPV (fL)	8.47±0.88	8.85±0.77	0.021
BDCAF	4.0 (2.0-8.0)	5.0 (2.0-8.0)	0.019
CRP (mg/L)	2.5 (1.05-30.0)	3.3 (1.0-41.0)	0.029
ESR (mm/h)	9.0 (2.0-43.0)	13.0 (2.0-105.0)	0.011
Cutaneous lesion	No (n=56)	Yes (n=64)
BDCAF	4.0 (2.0-8.0)	5.0 (2.0-8.0)	<0.001
Arthritis	No (n=72)	Yes (n=48)
BDCAF	4.0 (2.0-8.0)	5.0 (2.0-8.0)	0.028
KYN (ng/mL)	318.0 (100.0-1312.0)	434 (122.0-2180.0)	0.014
PCT (%)	0.22 (0.12-0.41)	0.25 (0.15-0.41)	0.020

Uveitis	No (n=67)	Yes (n=53)
BDCAF	4.0 (2.0-8.0)	5.0 (2.0-8.0)	0.021
MONO (K/uL)	0.50 (0.30-1.60)	0.6 (0.30-1.60)	0.008
Thrombosis	No (n=85)	Yes (n=35)
BDCAF	4.0 (2.0-8.0)	5.0 (2.0-8.0)	0.027
CRP (mg/L)	2.8 (1.0-13.0)	4.51 (1.10-41.0)	0.003
MLO	0.24 (0.12-0.98)	0.29 (0.11-1.33)	0.033
QUIN (ng/mL)	12.0 (3.0-61.5)	20.5 (4.0-68.0)	0.012
Neuro-Behcet's disease	No (n=106)	Yes (n=14)
BDCAF	4.0 (2.0-8.0)	7.0 (2.0-8.0)	0.040
LYM (K/uL)	1.55±0.8	2.28±1.1	<0.001
RDW (%)	14.9±1.9	16.4±2.9	0.042
NLO	1.9 (1.1-8.0)	3.5 (1.6-14.6)	0.001
ESR (mm/h)	30.0 (3.2-105.0)	10.0 (2.0-45.6)	0.004
KYN/TRP	0.07 (0.01-0.18)	0.052 (0.01-0.15)	0.037
QUIN (ng/mL)	12.1 (3.0-61.5)	24.1 (14.0-68.0)	0.001
Entero-Behcet's disease	No (n=115)	Yes (n=5)
BDCAF	4.0 (2.0-8.0)	8.0 (2.0-8.0)	0.011

Table 4. Correlations between KP metabolites and inflammatory markers and disease activity.
	TRP		KYN		KYN/TRP		KYNA		QUIN		3HK		3HAA
	r	p	r	p	r	p	r	p	r	p	r	p	r	p
BDCAF	-0.139	0.177	0.176	0.058	0.256	0.006	0.078	0.403	0.195	0.038	-0.055	0.556	0.078	0.409
CRP	-0.196	0.003	0.253	<0.001	0.342	<0.001	0.190	0.004	0.16	0.016	0.248	<0.001	0.059	0.378
ESR	-0.149	0.035	0.193	0.006	0.239	0.001	0.157	0.028	0.279	<0.001	0.116	0.103	0.17	0.016
WBC	-0.780	0.237	0.034	0.604	0.077	0.240	-0.013	0.850	0.029	0.664	0.073	0.271	0.074	0.264
PCT	0.034	0.619	0.177	0.009	0.139	0.041	0.098	0.155	0.175	0.010	0.073	0.287	0.16	0.019
NLO	-0.010	0.889	0.237	<0.001	0.211	0.002	0.111	0.106	0.088	0.198	0.122	0.075	0.065	0.344
PLO	0.012	0.860	0.179	0.008	0.134	0.047	0.128	0.06	0.057	0.406	0.092	0.173	0.148	0.029
MLO	-0.019	0.776	0.065	0.326	0.056	0.394	0.0121	0.069	0.079	0.233	0.011	0.868	-0.014	0.827
MPV	-0.044	0.504	0.043	0.516	0.079	0.232	0.088	0.188	0.006	0.932	0.244	<0.001	0.047	0.486
RDW	-0.077	0.244	0.111	0.092	0.146	0.025	0.113	0.090	0.091	0.167	0.042	0.529	-0.060	0.364

Elevated serum levels of kynurenine pathway metabolites in Behçet's patients

Status:

Version 1

Abstract

Introduction

Materials And Methods

Study design

Patients

Clinical Evaluation

Routine laboratory analysis

Tandem mass spectrometric analysis

Chemicals

Instrumentation

Sample preparation

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Tables

Status:

Version 1