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 . 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 . 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 . 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 . 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 .
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 . 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 . 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 . 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 . 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 . It has also been shown that QUIN has an immunosuppressive effect by inhibiting T cell proliferation . 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 .
3HK and 3HAA are other KP metabolites that have been reported to have immunomodulatory properties . 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 . 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 . 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 . 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 . 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) . 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 . 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 . 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.