Higher Kynurenine Research Articles

Targeted metabolomics: new insights into pathobiology of retained placenta in dairy cows and potential risk biomarkers

A targeted quantitative metabolomics approach was used to study temporal changes of serum metabolites in cows that normally released their fetal membranes and those that retained the placenta. We identified and measured serum concentrations of 128 metabolites including amino acids, acylcarnitines, biogenic amines, glycerophospholipids, sphingolipids and hexose at −8 and −4 weeks before parturition, during the week of retained placenta (RP) diagnosis, and at +4 and +8 weeks after parturition. In addition, we aimed at identifying metabolite signatures of pre-RP in the serum that might be used as predictive biomarkers for risk of developing RP in dairy cows. Results revealed major alterations in the metabolite fingerprints of pre-RP cows starting as early as −8 weeks before parturition and continuing as far as +8 weeks after calving. Biomarker candidates found in this study are mainly biomarkers of inflammation which might not be specific to RP. Therefore, the relevance of serum Lys, Orn, acetylornithine, lysophophatidylcholine LysoPC a C28:0, Asp, Leu and Ile as potential serum biomarkers for prediction of risk of RP in dairy cows will have to be tested in the future. In addition, lower concentrations of LysoPCs, Trp, and higher kynurenine in the serum during prepartum and the week of occurrence of RP suggest involvement of inflammation in the pathobiology of RP.

Abstract P6-02-08: Modulation of indoleamine 2, 3-dioxygenase (IDO1) expression in breast cancer cells by activated CD8+ T cells is controlled by DNA promoter methylation

Abstract Tumor infiltrating lymphocytes (TILs) play a critical role in regulating the immunomodulatory properties of triple negative breast cancer (TNBC). However, the specific adaptations that TNBC tumors undergo when challenged by lymphocyte infiltration remain unclear. In order to address this gap in knowledge, we conducted an immuno-phenotype comparison using mRNA sequencing between the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were co-cultured with activated human T-cells. Although the cytokine-induced immune signature of the two cell lines were similar, MDA-MD-231 cells were able to transcribe the tryptophan catabolizing enzyme IDO1 at a significantly higher level than MCF7 cells. Stimulation with IFNg was able to differentially induce IDO protein expression and enzymatic activity in ER- cell lines compared to ER+ cell lines, though no differences were observed in upstream JAK/STAT1 signaling or IDO1 mRNA stability between the two cell lines. Further experiments showed that treatment with the demethylating agent 5-aza-deoxycytidine was able to reverse suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation serves as a potential determinant in IDO1 induction. Analysis of TCGA and other previously published breast cancer datasets revealed subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal-like subtypes exhibiting lower promoter methylation and higher mRNA expression than ER+/luminal subtypes. Bisulfite pyrosequencing validated the subtype-specific association of decreased promoter methylation with increased IDO1 expression in breast cancer cell lines and an independent cohort of primary breast tumors. In addition, decreased IDO1 promoter methylation and elevated IDO1 expression in basal-like breast tumors was found to be associated with increased levels of kynurenine, the metabolic product of IDO1, as well as higher numbers of CD8+ TILs. Furthermore, high kynurenine levels in breast tumors were associated with worse patient survival. Taken together, these findings suggest that subtype-specific IDO1 promoter methylation regulates the ability of breast tumors to escape from antitumor immune responses driven by CD8+ TILs and could be used as a predictive biomarker for IDO inhibitor-based immunotherapy. Citation Format: Gu F, Noonepalle SK, Lee E-J, Choi J-H, Shull AY, Pei L, Sreekumar A, Ambs S, Shi H. Modulation of indoleamine 2, 3-dioxygenase (IDO1) expression in breast cancer cells by activated CD8+ T cells is controlled by DNA promoter methylation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-08.

High Kynurenine (a Tryptophan Metabolite) Predicts Remission in Patients with Major Depression to Add-on Treatment with Celecoxib.

Signs of an inflammatory process have been described in major depression. In a double-blind, randomized study of celecoxib or placebo add-on to reboxetine in 40 depressed patients, celecoxib treatment has beneficial effects. In order to evaluate the tryptophan/kynurenine metabolism and to identify predictors for remission, tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), and quinolinic acid (QUIN) were estimated in the serum of 32 patients before and after treatment and in a group of 20 healthy controls. KYN levels were significantly lower in patients (p = 0.008), and the QUIN/KYN ratios were significantly higher (p = 0.028). At baseline, the higher KYN/TRP ratio was predictive for remission during celecoxib add-on treatment (p = 0.04) as well as for remission in the overall patient group (p = 0.01). In the placebo group, remitters showed a higher KYNA/QUIN ratio (p = 0.032). In the overall group, remitters showed lower KYNA/KYN (p = 0.035) and QUIN/KYN (p = 0.011) ratios. The lower the formation of downstream metabolites, especially QUIN, the better the treatment outcome. The high KYN/TRP ratio predicted remission after treatment with celecoxib in this small sample of depressed patients. Eventually, the KYN/TRP ratio might be a marker for those patients, which benefit from an additional anti-inflammatory treatment.

The Roles of Aryl Hydrocarbon Receptor in T Cells at IDO-Positive Tumor Microenvironment

Introduction:Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor well known to generate biological responses to dioxins. A recent study indicated that kynurenine was an endogenous ligand of AhR and was associated with suppression of the antitumor response. The essential amino acid tryptophan metabolism proceeds via the enzymes IDO, which generates the immunosuppressive metabolites, such as kynurenine. Accumulation of these tryptophan derivatives block antigen-specific T-cell proliferation and induces T-cell death through the AhR. IDO is known to be produced by tumor cells and by some immune cells, such as dendritic cells and macrophages, which reside in tumor-draining lymph nodes or are recruited to tumors. IDO is overexpressed in several human cancers, including prostate, breast, brain, and hematologic malignancies.We previously showed that both IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in non-Hodgkin lymphoma patients. We also revealed that tumor IDO activity can inhibit CD19-specific chimeric antigen-receptor (CAR) T-cells (CARTs) therapy through the action of tryptophan metabolites (Blood. 2015; 125(25): 3905-3916). Therefore, we focused on the AhR in T-cells at IDO-positive tumor microenvironment.Methods and Results:We collected PBMCs from healthy blood donors, and activated the cells with anti-CD3 and anti-CD28 Abs, which mimic physiological T-cell activation. We measured the expression of AhR in activated T-cells using by flow cytometry, and found that the range of expression were from 4.5% to 38%. We then studied the effect of kynurenine on the proliferation of T-cells. The proliferation was assessed by MTS assay. We found that kynurenine significantly inhibited the proliferation of T-cells with high expression of AhR. The percentage inhibition with kynurenine 25µM was 33%. While T-cells with low expression of AhR were not inhibited with even 50µM of kynurenine. To investigate potential mechanisms of T-cell inhibition by kynurenine, we assessed the effect of kynurenine on T-cell apoptosis. We found that kynurenine was also associated with increased the apoptosis of T-cells with high AhR, as assessed by Annexin-V staining (percent Annexin-V positive and 7-AAD negative cells in 0 and 25 µM kynurenine was 11.8% and 36%, respectively). To evaluate the effect of inhibiting AhR in T-cells, we used AhR inhibitor (CH-223191). We found that CH-223191 could blocked the inhibitory effects of kynurenine on T-cells. The percent Annexin-V positive and 7-AAD negative cells in 25 µM kynurenine plus CH-223191 was 22.5%. Next, to assess the functional effects of tryptophan metabolites on the T-cells, we cocultured T-cells (1x106) with GFP-transduced wild-type Raji cells or IDO-transduced Raji cells (1x106). After 72 hours, cells were stained with CD8 antibody to distinguish tumor (GFP+ CD8-) and T-cells (GFP- CD8+). We found that the percentage of T-cells with high AhR in culture with IDO negative and positive Raji cells was 46.7% and 61.4%, respectively, while the percentage of T-cells with low AhR was 41.6% and 39.4%. These data showed that only T-cells with high AhR were inhibited at IDO-positive tumor microenvironment. Next, bone marrow (BM) samples were obtained from multiple myeloma (MM) patients at diagnosis. We could generated the activated T-cells from 10 of 12 BM samples. The expression of AhR in T-cells was ranged from 3.3% to 26.6%. We found that there was a positive correlation between the expression of AhR and the proportion of plasma cells in BM (r=0.76, P=0.04).Discussion:The expression of AhR in activated T cells play an important role in the effect of kynurenine, which is a metabolite produced by IDO. A few clinical trials are evaluating the effects of IDO inhibitors that competitively block the degradation of tryptophan to kynurenine by the enzyme. Because anti-PD-1 and anti-CTLA-4 therapies, which block directly the inhibitory signal in T-cells, have been getting some clinical benefits against such as melanoma and Hodgkin lymphoma. Therefore, AhR in T cells might be a target for IDO-positive hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

Response of glioblastoma cells to activation of the G-protein coupled estrogen receptor, GPER1/GPR30, and possible crosstalk with the aryl hydrocarbon receptor.

Glioblastomas are almost invariably fatal brain tumors. Glioblastoma incidence in men is 1.5 times that of pre-menopausal women and this difference decreases post-menopause. Therefore estrogen is thought to play a protective role, although the mechanism is not well understood. Glioblastomas produce and respond to a tryptophan derivative, kynurenine, that binds and activates the aryl hydrocarbon receptor (AHR). Promiscuity of ligand binding can result in cross talk between AHR and nuclear estrogen receptors and both receptor types are known to use the p300 acetyltransferase as a cofactor. We demonstrated that inhibition of p300 resulted in a reduction of proliferation of CH157-MN meningioma cells. Treatment with β-estradiol or kynurenine partially abrogated this effect, suggesting both ligands may act through another receptor that we hypothesize to be the G-protein linked estrogen receptor, GPER1/GPR30. We verified the expression of GPER1, estrogen receptor β; some estrogen receptor β isoforms and AHR by CH157-MN cells and U-87 and A-172 glioblastoma cells. Inhibition of p300 with C646 reduced proliferation of both U87 and A172 cells. In U87 cells, this effect was abrogated by co-treatment with β-estradiol, the xenobiotic AHR agonist 2’,3’,7,8’-tetrachlorodibenzo-p-dioxin, and the GPER1 agonist G-1. Exogenous kynurenine did not abrogate the effect of p300 inhibition. However, in A172 cells, none of the agonists abrogated the effect of p300 inhibition, possibly due to the higher kynurenine production in A172 cells. Treatment of A172 and U87 cells with G-1 resulted in decreased cell proliferation which was abrogated by co-treatment with the GPER1 antagonist G-36.

Plasma Zonulin and its Association with Kidney Function, Severity of Heart Failure, and Metabolic Inflammation.

The tight junction regulator zonulin has attracted clinical attention as a biomarker of increased gastrointestinal permeability. Recent work also suggests zonulin to represent a general regulator of tissue barriers and a player in metabolic inflammation. Here, we investigated the associations of zonulin with chronic heart failure (CHF), kidney function, and metabolic inflammation. Using multiple linear regression (Generalized Linear Model), this study determined the association of plasma zonulin with different laboratory and clinical parameters in 225 patients carrying automatic implantable cardioverters/defibrillators (AICD) for primary or secondary prevention. In another 115 patients with diastolic or systolic CHF, we investigated a possible relationship between zonulin and CHF severity. In the AICD cohort, zonulin associated inversely with serum creatinine (p = 0.013), carboxymethyl-lysine calprotectin (p < 0.001), and kynurenine (p = 0.009) and positively with homoarginine (p < 0.001). In the subgroup with type-2 diabetes (T2D) (n = 51), zonulin increased significantly with high-sensitivity CRP (p = 0.014). In the CHF cohort, we found a highly significant rise of NT-proBNP, but not of zonulin with NYHA functional classes I-IV or other parameters of CHF severity. The inverse associations of zonulin with creatinine and markers of cardio-vascular risk (high CMLcalprotectin and kynurenine, low homoarginine) are novel findings that need further experimental and clinical clarification. Our study indicates zonulin involvement in metabolic inflammation in T2D, but no association with disease status in CHF.

Mouse chronic social stress increases blood and brain kynurenine pathway activity and fear behaviour: Both effects are reversed by inhibition of indoleamine 2,3-dioxygenase

Psychosocial stress is a major risk factor for mood and anxiety disorders, in which excessive reactivity to aversive events/stimuli is a major psychopathology. In terms of pathophysiology, immune-inflammation is an important candidate, including high blood and brain levels of metabolites belonging to the kynurenine pathway. Animal models are needed to study causality between psychosocial stress, immune-inflammation and hyper-reactivity to aversive stimuli. The present mouse study investigated effects of psychosocial stress as chronic social defeat (CSD) versus control-handling (CON) on: Pavlovian tone–shock fear conditioning, activation of the kynurenine pathway, and efficacy of a specific inhibitor (IDOInh) of the tryptophan–kynurenine catabolising enzyme indoleamine 2,3-dioxygenase (IDO1), in reversing CSD effects on the kynurenine pathway and fear. CSD led to excessive fear learning and memory, whilst repeated oral escitalopram (antidepressant and anxiolytic) reversed excessive fear memory, indicating predictive validity of the model. CSD led to higher blood levels of TNF-α, IFN-γ, kynurenine (KYN), 3-hydroxykynurenine (3-HK) and kynurenic acid, and higher KYN and 3-HK in amygdala and hippocampus. CSD was without effect on IDO1 gene or protein expression in spleen, ileum and liver, whilst increasing liver TDO2 gene expression. Nonetheless, oral IDOInh reduced blood and brain levels of KYN and 3-HK in CSD mice to CON levels, and we therefore infer that CSD increases IDO1 activity by increasing its post-translational activation. Furthermore, repeated oral IDOInh reversed excessive fear memory in CSD mice to CON levels. IDOInh reversal of CSD-induced hyper-activity in the kynurenine pathway and fear system contributes significantly to the evidence for a causal pathway between psychosocial stress, immune-inflammation and the excessive fearfulness that is a major psychopathology in stress-related neuropsychiatric disorders.

Combined Toxoplasma gondii seropositivity and high blood kynurenine – Linked with nonfatal suicidal self-directed violence in patients with schizophrenia

Toxoplasma gondii (T. gondii) chronic infection and elevated kynurenine (KYN) levels have been individually associated with non-fatal suicidal self-directed violence (NF-SSDV). We aimed to test the hypothesis that the association between T. gondii seropositivity and history of NF-SSDV would be stronger in schizophrenia patients with high plasma KYN levels than in those with lower KYN levels. We measured anti-T. gondii IgG antibodies and plasma KYN in 950 patients with schizophrenia, and used logistic regression to evaluate the relationship between NF-SSDV and KYN in patients who were either seropositive or seronegative for T. gondii. For those with KYN levels in the upper 25th percentile, the unadjusted odds ratio for the association between NF-SSDV history and KYN in T. gondii seropositive patients was 1.63 (95% CI 1.01 to 2.66), p = 0.048; the adjusted odds ratio was 1.95 (95% CI 1.15 to 3.30), p = 0.014. Plasma KYN was not associated with a history of NF-SSDV in T. gondii seronegative patients. The results suggest that T. gondii and KYN may have a nonlinear cumulative effect on the risk of NF-SSDV among those with schizophrenia. If confirmed by future longitudinal studies, this result is expected to have both theoretical and clinical implications for the prevention and treatment of suicidal behavior.

Tryptophan pathway alterations in the postpartum period and in acute postpartum psychosis and depression

ObjectivesWomen are at very high risk for the first onset of acute and severe mood disorders the first weeks after delivery. Tryptophan breakdown is increased as a physiological phenomenon of the postpartum period and might lead to vulnerability for affective psychosis (PP) and severe depression (PD). The aim of the current study was to investigate alterations in tryptophan breakdown in the physiological postpartum period compared to patients with severe postpartum mood disorders. MethodsWe included 52 patients (29 with PP, 23 with PD), 52 matched healthy postpartum women and 29 healthy non-postpartum women. Analyzes of serum tryptophan metabolites were performed using LC–MS/MS system for tryptophan, kynurenine, 3-hydroxykynurenine, kynurenic acid and 5-hydroxyindoleacetic acid. ResultsThe first two months of the physiological postpartum period were characterized by low tryptophan levels, increased breakdown towards kynurenine and a downstream shift toward the 3-OH-kynurenine arm, away from the kynurenic acid arm. Kynurenine was significantly lower in patients with PP and PD as compared to healthy postpartum women (p=0.011 and p=0.001); the remaining tryptophan metabolites demonstrated few differences between patients and healthy postpartum women. LimitationLow prevalence of the investigated disorders and strict exclusion criteria to obtain homogenous groups, resulted in relatively small sample sizes. ConclusionThe high kynurenine levels and increased tryptophan breakdown as a phenomenon of the physiological postpartum period was not present in patients with severe postpartum mood disorders. No differences were observed in the levels of the ‘neurotoxic’ 3-OH-kynurenine and the ‘neuroprotective’ kynurenic acid arms between patients and healthy postpartum women.

Tryptophan Breakdown in Patients with HCV Infection is Influenced by IL28B Polymorphism.

Until recently, the standard treatment of chronic hepatitis C virus (HCV) infection was a combination therapy with PEG-IFN-α plus ribavirin. Previous studies have proven that several markers predict the outcome of such therapy, e.g., pretreatment plasma levels of interferon inducible protein IP-10, HCV RNA and IL28B-related single nucleotide polymorphisms (SNP). Altered activity of tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been also shown in patients suffering from HCV infection. In this study, we investigated whether IL28B SNP in patients infected with HCV is related to the tryptophan breakdown rate. Before therapy, serum tryptophan and kynurenine concentrations were determined in 25 patients with established HCV infection and the kynurenine to tryptophan ratio (KYN/TRP) was calculated as an estimate of the tryptophan breakdown rate. In parallel, neopterin and nitrite concentrations were determined. A significant difference of serum KYN/TRP existed between the three IL28B polymorphism groups: C/C genotype had the highest and T/T genotype had the lowest KYN/TRP (p < 0.05). Likewise, C/C genotype was associated with higher KYN/TRP than non-C/C genotype (p = 0.01). There was a smaller difference between the three groups regarding the absolute kynurenine concentrations, the C/C genotype being associated with higher kynurenine concentrations. None of the other comparisons revealed any statistical significance. In conclusion, patients with C/C genotype presented with the highest tryptophan breakdown rate already before antiretroviral therapy with IFN-α/ribavirin. The differences in tryptophan metabolism might relate to HCV clearance and also to side effects of IFN-α therapy.

T Cells Expressing CD19-Specific Chimeric Antigen Receptors Are Inhibited By Indoleamine 2,3-Dioxygenase in Tumors

Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that mediates the metabolism of tryptophan to kynurenines, which have an inhibitory activity on immune cells. IDO-positive tumors thus establish a microenvironment in which NK and T cells are inactivated, and high IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in diffuse large B-cell lymphoma patients.CD19-specific chimeric antigen receptor T cell (CART) therapy is a promising new approach against B-cell malignancies but not every tumor responds. To determine whether the presence of IDO in tumors affects CART activity, we first investigated the expression of IDO by the human B-cell lymphoma cell lines Raji, Daudi, BJAB and Jeko-1. We found that only Jeko-1 expresses IDO and produces kynurenine natively. IDO was not expressed by the other cell lines, even after exposure to IFNγ, a known IDO inducer. Based on these results, we chose Raji as a baseline IDO-negative cell line and made an IDO-positive Raji clone by retroviral transduction with human IDO cDNA (Raji-IDO); a clone transduced with an empty vector served as control (Raji-control). We injected SCID mice subcutaneously in opposite flanks with luciferase-transduced Raji-control and Raji-IDO cells. Seven days later, we injected human non-transduced T cells (NTs) or CARTs intravenously. In the NT group, tumors had similar growth on both sides. In contrast, in the CART group, Raji-control tumors had significantly slower growth than Raji-IDO tumors (3.1 ± 1.1×108 and 20 ± 7.3×108 bioluminescence units [BU] at day 7, respectively, P = 0.03). We also found that CARTs significantly inhibited Raji-control tumor growth (NT: 27 ± 6.8×108 vs CART: 3.1 ± 1.1×108 BU at day 7, P = 0.02), but did not affect Raji-IDO tumors (NT: 24 ± 5.4×108 vs CART: 20 ± 7.3×108 BU at day 7, P = 0.35). In another experiment, Raji-IDO cells were injected subcutaneously, and mice were treated with an IDO inhibitor (1-methyl-tryptophan, 1-MT), CARTs, or both. The combination treatment produced significantly better tumor control than either single therapy (1-MT: 45 ± 6.8×108 and CART: 22 ± 4.6×108 vs both: 8.2 ± 3×108 BU at day 7, P = 0.001 and 0.04, respectively). Thus, the IDO inhibitor protects CARTs against the deleterious effects of IDO-positive tumors.To investigate potential mechanisms for CART inhibition by IDO, we assessed the effect of kynurenine and found that even low concentrations (12.5 µM) inhibited CART proliferation in response to IL-2, IL-7, IL-15 or CD19-positive targets, although there was no effect on proliferation of B-cell lymphoma cell lines at this kynurenine concentration. CART apoptosis was also increased by kynurenine (8.6 ± 0.6%, 13.9 ± 2.2%, and 33.5 ± 10.6% annexin V-positive cells with 0, 12.5, or 25 µM kynurenine). In coculture of CARTs with wild-type Raji cells, the latter were eliminated by day 6 in the absence of kynurenine, but increased in numbers (in parallel with a decrease in CARTs) in its presence. Kynurenine also inhibited the release of IFNγ (13,143 ± 848 pg/mL vs 2,663 ± 1,873 pg/mL, P = 0.02) and IL-2 (718 ± 355 pg/mL and 199 ± 165 pg/mL, P = 0.03) by CARTs. Expression of granzyme B, PD-1 and CTLA-4 on CARTs was not significantly affected.Fludarabine has been reported to downregulate IDO expression in tumors and this drug is used in many lymphodepleting regimens that are administered before CART infusion in an effort to augment the efficacy of these therapies. However, the beneficial mechanism of lymphodepleting chemotherapy drugs is not fully understood. Therefore, we also measured the effect of fludarabine and mafosfamide (a cyclophosphamide analog) on IDO expression by Jeko-1 cells. We found that both drugs downregulate IDO expression by Jeko-1, even in the presence of IFNγ.In summary, expression of IDO by tumor cells inhibits CART activity, likely because kynurenine is produced and has negative effects on proliferation and cytokine secretion by CARTs. Fludarabine and cyclophosphamide downregulate IDO expression in tumors and this effect may contribute to the benefits of lymphodepletion before CART therapy. Direct IDO inhibitors may further improve clinical CART activity against IDO-positive tumors. DisclosuresNo relevant conflicts of interest to declare.

Role of Human Corneal Endothelial Cells in T-Cell–Mediated Alloimmune Attack In Vitro

Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death. Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry. Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 μM. Concentrations of ≥400 μM Kyn were required to suppress T-cell proliferation. Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

Indoleamine 2,3-Dioxygenase 1 (IDO1) Is Up-Regulated in Thyroid Carcinoma and Drives the Development of an Immunosuppressant Tumor Microenvironment

Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it appears to exert an immunosuppressive function as part of an acquired mechanism of immune escape mediated by the inhibition of lymphocyte proliferation and survival and by the induction of FoxP3+ T regulatory cells. The objective of the study was to evaluate IDO1 expression in thyroid carcinoma and demonstrate its immunosuppressive function in the context of thyroid tumors. IDO1 expression was evaluated by quantitative PCR in 105 papillary thyroid carcinomas (PTCs), 11 medullary thyroid carcinomas, six anaplastic thyroid carcinomas, and five thyroid carcinoma cell lines (TCCLs), by immunohistochemistry in 55 PTCs and by Western blotting in five TCCLs. FoxP3+ Treg lymphocyte density was evaluated by immunohistochemistry in 29 PTCs. IDO1 inhibitory effect on lymphocyte proliferation was tested in coculture experiments of TCCLs and activated lymphocytes. IDO1 mRNA expression resulted significantly higher in all the analyzed thyroid carcinoma histotypes compared with normal thyroid. Interestingly, an increase of IDO1 mRNA expression magnitude could be observed with gain of aggressiveness (PTCs and medullary thyroid carcinomas ≪ anaplastic thyroid carcinomas). In PTCs, IDO1 mRNA expression magnitude correlated with IDO1 immunostaining intensity in cancer cells and with FoxP3+ Treg lymphocyte density in the tumor microenvironment. IDO1 was expressed in human thyroid cancer cell lines in vitro, and FTC-133 cells showed high kynurenine concentration in the conditioned medium and a strong suppressive action on the proliferation of activated lymphocytes in coculture experiments. For the first time, this study demonstrates a pivotal role of IDO1 in the suppression of lymphocyte function in thyroid carcinoma microenvironment.

Combination of Indoleamine 2,3-Dioxygenase Expression and L-Kynurenine Level Can Identify Truly Poor Prognosis Patients with Diffuse Large B-Cell Lymphoma

Abstract 2639 INTRODUCTION:Recent studies have suggested that one mechanism of immune escape could be catabolism of the essential amino acid tryptophan, via indoleamine 2,3-dioxygenase (IDO) along the kynurenine pathway. Some metabolites derived from tryptophan by IDO, such as L-kynurenine, block antigen-driven specific T-cell proliferation and induce T-cell death. IDO activity thus plays an important role in regulating immune responses exerted by antigen-presenting cells (APCs) and also provides transformed cells with a potent tool to help escape from assault by the immune system. We have recently been able to clarify the utility of either serum L-kynurenine or tissue IDO expression as prognostic factors for DLBCL patients treated using R-CHOP. The present study therefore examined the serum level of L-kynurenine and tissue IDO expression simultaneously in DLBCL patients in an attempt to identify patients showing truly poor prognosis. MATERIALS AND METHODS:The study protocol used a retrospective case series design that was approved by our institutional review board. We investigated DLBCL patients who had been histologically diagnosed according to the WHO classification of hematopoietic tumors between December 2003 and November 2006. All serum samples were obtained on admission before starting any treatments. Informed consent was obtained from all patients for the use of samples in this study. Tumor tissue was obtained from every patient by lymph node biopsy before initiation of therapy. Patients were assigned to receive six to eight cycles of R-CHOP or R-THP-COP therapy. Each regimen consisted of rituximab (375 mg/m2 on day 1), cyclophosphamide (750 mg/m2 on day 3), doxorubicin (DOX) or tetrahydropyranyl-adriamycin (THP) (50 mg/m2, given as a 30-min infusion on day 3), vincristine (1.4 mg/m2 on day 3, with a maximal dose of 2.0 mg), and prednisolone (100 mg/day on days 3–7). We measured serum concentrations of L-kynurenine by high-performance liquid chromatography (HPLC), and serum levels of sTNF-Rs (p55, TNF-R1; p75, TNF-R2) by ELISA. And we examined IDO expression in DLBCL tissues by immunohistochemical staining in a total of 119 patients. RESULTS:Median serum levels of L-kynurenine were significantly higher in DLBCL patients (1.598 mM; range, 0.489–5.066 mM) than in healthy volunteers (1.133 mM; range, 0.752–1.388 mM). And we found that lymphoma cells expressed IDO in 32% cases. We therefore classified cases as IDO-positive DLBCL or IDO-negative DLBCL based on whether lymphoma cells themselves expressed IDO. No significant correlation was identified between serum L-kynurenine level and IDO expression. Serum L-kynurenine level showed a significant positive correlation with concentrations of sTNF-R1 and sTNF-R2 in IDO-negative DLBCL patients. CR rates with IDO-positive and IDO-negative DLBCL were 42% and 75%, respectively (p=0.012). Three-year OS rates were 55% and 78% for patients with L-kynurenine ≥1.5 and <1.5 mM, respectively (p=0.027), and 44% and 75% for patients with IDO-positive and IDO-negative DLBCL, respectively (p=0.0017). Univariate Cox analysis identified the following prognostic factors: IDO expression, serum kynurenine, PS, and multiple site extranodal involvement. Multivariate analysis of these four parameters showed that positive IDO expression and serum kynurenine ≥1.5 mM were significant prognostic factors. We analyzed patients’ outcome according to serum concentration of L-kynurenine and IDO expression of lymphoma cells at diagnosis. Patients with low kynurenine and IDO-negative showed the best outcomes, while patients with high kynurenine and IDO-positive showed the poorest outcome. This combination distinguishes 3 separate prognostic groups of “very good”, “good”, and “poor”, with 3-year OS ranging from 84.1% to 31.1% (p<0.001, Figure). CONCLUSION:IDO activity might play an important role in the disease activity of DLBCL. The combination of tissue IDO expression and serum level of L-kynurenine is a useful tool to predict the prognosis of patients with DLBCL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Indoleamine 2,3-dioxygenase 1 (IDO1) activity correlates with inflammation in systemic sclerosis (115.5)

Abstract Background. A contributory effect of IDO1 to inflammatory autoimmune pathologies such as rheumatoid arthritis and systemic lupus erythematosus has been recently described. We aimed to address whether IDO1-driven kynurenine (KYN) production correlates with degree of inflammation and disease extent in systemic sclerosis (SSc). Methods. Peripheral blood was obtained from 27 consecutive patients (median age 62 years, range 22-78; median disease duration = 10 months, range 1-35) with either limited cutaneous SSc (n=17) or diffuse cutaneous SSc (n=10). In 11 of the 27 patients, CD19+ B cells were magnetically purified to estimate IDO1 mRNA levels by quantitative PCR. Results. High serum KYN in SSc (3.17±0.82 μM compared with 1.82±0.37 μM in healthy controls; p&amp;lt;0.0001) positively correlated with serum CRP (r=0.42; p=0.03), an established marker of inflammation. We showed a trend towards higher KYN levels in diffuse SSc compared with limited SSc, although no correlations emerged between KYN release and serological features or lung fibrosis. Interestingly, purified CD19+ B cells expressed IDO1 mRNA, whose copy number correlated with serum CRP (r=0.58; p=0.048) and with the frequency of bona fide CD4+FoxP3+ Treg cells. Conclusions. The generation of KYN and expansion of Treg cells may reflect a counter-regulatory mechanism to limit excessive inflammation in SSc.

Exposure to Helicobacter pylori and Serum Kynurenine to Tryptophan Ratio in Patients with Gastric Cancer

Abstract In Helicobacter pylori seropositive individuals, the subsequent risk of non-cardia gastric cancer development is frequently attributed to the infection agent. Although the link between inflammation and gastric cancer is formally recognized, the molecular basis of evading mechanisms of Helicobacter pylori from host immune system remains unclear. The aim of this study was to determine the effect of systemic factors in gastric cancer patients such as serum kynurenine to tryptophan ratios, neopterin, nitric oxide and urinary biopterin levels which may sustain the persistence of Helicobacter pylori seropositivity. This prospective randomized study involved 95 adults with gastric cancer and 108 cancer free patients with extradigestive diseases. Each group was assigned into two subgroups according to Helicobacter pylori IgG seropositivity. Exposure to Helicobacter pylori was detected by serum IgG test (ELISA). Serum tryptophan, kynurenine, neopterin and nitrite concentrations were measured. In simultaneous urinary samples, neopterin, biopterin, and creatinine levels were assayed. Significant decreases were found in serum tryptophan levels of both Helicobacter pylori seronegative and seropositive gastric cancer patients versus controls without malignant disease. However, marked increase in kynurenine/tryptophan was observed only in Helicobacter pylori seropositive cancer patients. Highly significant correlation between the serum neopterin and kynurenine/tryptophan indicated interferon gamma stimulated tryptophan degradation. Decrease in serum nitrite levels, and a parallel increase in urinary biopterin concentrations in Helicobacter pylori seropositive cancer cases might result from the excessive nitric oxide consumption due to reactive oxygen radical overproduction. Significantly higher kynurenine to tryptophan ratio and its correlation with increased neopterin levels suggested a role of tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and that persistence of Helicobacter pylori seropositivity might participate in enhanced immune tolerance of cancer patients.

Blood levels of kynurenines, interleukin‐23 and soluble human leucocyte antigen‐G at different stages of Huntington’s disease

There is substantial evidence that abnormal concentrations of oxidised tryptophan metabolites, produced via the kynurenine pathway, contribute to progressive neurodegeneration in Huntington's disease. We have now examined the blood levels of these metabolites in patients at different stages of Huntington's disease, assessed both in terms of clinical disease severity and numbers of CAG repeats. Close relatives of the patients were included in the study as well as unrelated healthy controls. Levels of lipid peroxidation products, the pro-inflammatory cytokine interleukin (IL)-23 and the soluble human leucocyte antigen-G (sHLA-G) were also measured. There were lower levels of tryptophan and a higher kynurenine : tryptophan ratio, indicating activation of indoleamine-2,3-dioxygenase, in the most severely affected group of patients, with increased levels of IL-23 and sHLA-G. Marked correlations were noted between IL-23 and the patient severity group, anthranilic acid levels and the number of CAG repeats, and between anthranilic acid and IL-23, supporting our previous evidence of a relationship between anthranilic acid and inflammatory status. Tryptophan was negatively correlated with symptom severity and number of CAG repeats, and positively correlated with sHLA-G. The results support the proposal that tryptophan metabolism along the kynurenine pathway in Huntington's disease is related to the degree of genetic abnormality, to clinical disease severity and to aspects of immunopathogenesis.

Accelerated Tryptophan Degradation in Trauma and Sepsis Patients is Related to Pro-inflammatory Response and to the Diminished in vitro Response of Monocytes

Abstract Immune system activation and inflammation accompanies immune dysfunction in trauma and sepsis patients. Tryptophan degradation via the kynurenine pathway by the cytokine-inducible enzyme indoleamine 2,3-dioxygenase (IDO) could contribute to the deficient responsiveness of immunocompetent cells. Activated IDO in patients after trauma and with sepsis is indicated by an increased kynurenine to tryptophan ratio (kyn/trp), which was found to be associated with poor outcome of patients. In this study, tryptophan and kynurenine concentrations in 18 patients post trauma or with sepsis during 12-14 days of follow up (up to 84 specimens were available) were compared to concentrations of neopterin and cytokines tumor necrosis factor-α (TNF-α) and interleukin- 6 (IL-6) and the in vitro response of lipopolysaccharide-stimulated monocytes. Compared to healthy controls, average kyn/trp and kynurenine, TNF-α, IL-6 and neopterin concentrations were increased in patients, and tryptophan concentrations were decreased. During follow-up, only kyn/trp, kynurenine and neopterin concentrations (all p &lt;0.001) were increasing, whereas the changes of tryptophan, TNF-α and IL-6 concentrations were not significant. Non-survivors presented not only with higher kyn/trp and with higher kynurenine and neopterin concentrations than survivors, but also TNF-α and IL-6 concentrations were higher. Kyn/trp correlated with neopterin (rs = 0.590; p &lt;0.001) and also with TNF-α (rs = 0.374; p &lt;0.01) and IL-6 concentrations (rs = 0.262; p &lt;0.05) and inversely with the in vitro response of stimulated monocytes. Data imply that increased tryptophan degradation in patients post trauma is due to IDO activation. Increased IDO activity most likely represents a result of host defence response in patients, and data support a possible role of IDO to diminish immunoresponsiveness in such patients.

Activated cellular immunity and decreased serum tryptophan in healthy pregnancy.

We analyzed plasma samples from 45 randomly selected healthy pregnant women and from 20 healthy nonpregnant female controls of corresponding age. Tryptophan and kynurenine concentrations were measured by reverse-phase high performance liquid chromatography. The kynurenine per tryptophan ratio was calculated to allow a more accurate estimate of tryptophan degradation. In addition, neopterin was measured by radioimmunoassay. There were significantly lower tryptophan concentrations in pregnant women compared to nonpregnant controls. Kynurenine per tryptophan ratios were increased in pregnant women in the 3rd trimester. Significant correlations existed between neopterin increase and tryptophan decrease as well as kynurenine increase and tryptophan decrease. Also significant correlations between week of pregnancy and lower tryptophan and higher kynurenine per tryptophan ratio were found (p < 0.01). From our data, cellular immune activation is likely to be the cause of enhanced tryptophan degradation during pregnancy.

Pleiotropism of wh and el locus in Habrobracon juglandis

The two autonomously manifesting mutations, eyeless (el) and white eyes (wh), of Habrobracon juglandis were compared phenotypically with the wild-type. Eyeless reduces the compound eyes in size. The penetrance is 100%, while expressivity ranges from point-shaped ommochrome crumbs over individual facets to a small eye with abundant facets. There is no correlation between the sizes of left and right eyes. The head capsule is more deformed when the eye is smaller. The homozygote ♀♀ of wild-type have smaller eyes than the hemizygote ♂♂. This gene dose effect is still stronger in el. wh causes white eye colour which remains throughout lifetime. With regard to the amount of eye pigment the three stocks can be arranged in the following sequence: wild type &gt;el&gt;wh. The concentrations of ommochrome precursors, however, do not agree with this sequence. The amount of 3-hydroxykynurenine in el is the same as in wild-type; although in pupal stage the peak in the former appears earlier. The kynurenine concentration in el increases at the time of pupation and induces the beginning of pigmentation earlier than in wild-type; further this process is terminated in el earlier. In wh and in wild-type the amount of kynurenine is also the same. However, there is much less 3-hydroxykynurenine in wh than in wild-type. This is presumably due to a lower kynurenine hydroxylase activity. During the entire postembryonal development there is more xanthurenic and kynurenic acid in wh; in pupal stage, even, there is a second peak. Pupae and adults of wh have a higher kynurenine and presumably also a higher 3-hydroxykynurenine transaminase activity than wild-type. The influence of the mutation wh on the differential rates of hydroxylation and transamination is discussed in relation to the results obtained with mutants of the white locus in Drosophila.