Indoleamine 2,3-dioxygenase 1 Induction Research Articles

Uterine Commensal Peptostreptococcus Species Contribute to IDO1 Induction in Endometrial Cancer via Indoleacrylic Acid.

Microbial dysbiosis has an increasingly appreciated impact on carcinogenesis, and the cervicovaginal microbiome plays a critical role in microenvironmental inflammation. Here, we investigated the involvement of the female genital tract Peptostreptococcus species in gynecological cancer via indoleacrylic acid (IAA). IAA production from Peptostreptococcus species and the effect of bacterial culture on tumor growth in vivo were examined. The impact of IAA on cytokine production and indoleamine-2,3-dioxygenase 1 (IDO1) expression in an endometrial cancer (EC) cell line, as well as their effect on Treg and Teff cells, and M1 and M2 macrophage populations were examined in EC patients and tumor-grafted mice. Clinically, Peptostreptococcus species abundance, IAA, and IDO1 expression were verified in EC patients. The results showed that IAA production was induced in the uteri of BALB/c nude mice by Peptostreptococcus species transplantation, and the intratumoral injection of a conditioned medium from Peptostreptococcus cultures into tumor-grafted mice promoted tumor growth. IL-10 expression was upregulated by IAA; IFN-γ expression was increased by IL-10. IFN-γ induced IDO1 expression in the EC cell line. The co-culture of IDO1-expressing EC cells with peripheral blood mononuclear cells upregulated the Treg proportion and decreased the M1/M2 ratio. Clinically, P. anaerobius was more abundant amongst the uterine microbiota of EC patients than the control. The IAA, IDO1, and kynurenine/tryptophan ratios were all higher in EC tissue, and the M1/M2 ratio was lower. Our study sheds light on the link between IDO1 induction and uterine Peptostreptococcus dysbiosis and provides a potential rationale for the role of Peptostreptococcus species in immune tolerance induction in type I endometrial cancer.

IL-1β mediates the induction of immune checkpoint regulators IDO1 and PD-L1 in lung adenocarcinoma cells

IntroductionInflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators.MethodsTo determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines (A549, H1792, H1838, H2347, H2228, HCC364 and HCC827) grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry.ResultsUpon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells.ConclusionsOur studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion.AWNWNjEXZEagXxNY6iY8VPVideo

T cells in pancreatic cancer stroma: Tryptophan metabolism plays an important role in immunoregulation.

Several studies have shown that the immune system is highly regulated by tryptophan metabolism, which serves as an immunomodulatory factor. The indoleamine 2,3-dioxygenase 1 (IDO1), as an intracellular enzyme that participates in metabolism of the essential amino acid tryptophan in the kynurenine pathway, is an independent prognostic marker for pancreatic cancer (PC). First, overexpression of IDO1 inhibits the maturation of dendritic cells and T-cell proliferation in the liver and spleen. Second, the high expression of kynurenine induces and activates the aryl hydrocarbon receptor, resulting in upregulated programmed cell death protein 1 expression. Third, the induction of IDO1 can lead to loss of the T helper 17 cell/regulatory T cell balance, mediated by the proximal tryptophan catabolite from IDO metabolism. In our study, we found that overexpression of IDO1 upregulated CD8+ T cells and reduced natural killer T cells in pancreatic carcinoma in mice. Hence, it may be essential to pay more attention to tryptophan metabolism in patients, especially those who are tolerant to immunotherapy for PC.

Diagnoses and new therapeutic strategy focused on physiological alteration of tryptophan metabolism

The monoamine hypothesis has been common hypotheses for the pathophysiology of major depressive disorder (MDD). Since mainstream antidepressants are selective serotonin (5-HT) reuptake inhibitors, hypo-serotonergic function has been implicated in the MDD. However, one-third of patients are refractory to the treatment with antidepressants. Tryptophan (TRP) is metabolized via the kynurenine (KYN) and 5-HT pathways. Indoleamine 2,3-dioxygenase 1 (IDO1) is the first metabolizing enzyme in the TRP-KYN pathway which is inducible by pro-inflammatory cytokines, involved depression-like behavior via 5-HT depletion due to decreased level of TRP in the 5-HT pathway. Kynurenine 3-monooxygenase (KMO) is the enzyme in the metabolism of KYN to 3-hydroxykynurenine. KMO deficiency increases level of kynurenic acid (KA), a KYN metabolite by kynurenine aminotransferases (KATs) and induces depression-like behavior. Interestingly, Chronic unpredictable mild stress (CUMS) is associated with a disruption of the hypothalamus-pituitary-adrenocortical (HPA) system and increases KA level with decreased KMO expression in the prefrontal cortex. The decrease of KMO may be related to the reduction in expression of microglia, since KMO is mainly found in microglia in the nervous system. CUMS increases KA level via alternation of enzymes from KMO to KAT. KA is α7 nicotinic acetylcholine receptor (α7nAChR) antagonist. Activation of α7nAChR by nicotine or galantamine attenuates CUMS-induced depression-like behaviors. Taken together, depletion of 5-HT by induction of IDO1 and α7nAChR antagonism by KA via decreased KMO expression cause depression-like behavior, suggesting that metabolic alterations in TRP-KYN pathway are highly involved in the pathophysiology of MDD. Therefore, TRP-KYN pathway is expected to be an attractive target for the development of novel diagnosis of MDD and antidepressants.

A novel pathologic marker, indoleamine 2,3-dioxygenase 1, for the cholangiopathy of immune checkpoint inhibitors-induced immune mediated hepatotoxicity as adverse events and the prediction of additional ursodeoxycholic acid treatment.

Immune-related adverse events (irAE) has been clarified according the usage of immune checkpoint inhibitors(ICI). We primarily found indoleamine 2,3-dioxygenase 1(IDO-1) as a histologic biomarker for the cholangiopathy of primary biliary cholangitis(PBC). In this study, we evaluated the utility of IDO-1 in identifying ICI-induced immune-mediated hepatotoxicity(IMH). Immunostaining for IDO-1 using liver sections of PBC, ICI-induced IMH and controls, revealed that IDO-1 expression in bile ducts is mostly restricted in PBC and ICI-induced IMH. In ICI-induced IMH, IDO-1-positive bile ducts is found in 2/2 cases of cholangitis type and also positive/focal ducts in 11/15 cases of hepatitis type. Moreover, in 8/13 positive/focal cases, ursodeoxycholic acid as well as steroids were needed to improve liver dysfunction, but just one case (1/4) in IDO-1-negative cases. One IDO-1 positive case of hepatitis type did not receive additional UDCA, but biliary enzymes worsen. In vitro study using cultured human biliary epithelial cells revealed that IDO-1 induction was found with the stimulation of IFN-γ. In conclusion, the presence of IDO-1-positive cells is found in bile ducts in hepatitic type as well as sclerosing cholangitis of ICI-induced IMH. IDO-1 is surely a valuable pathologic marker for diagnosing ICI-induced IMH and also for predicting an additional need of UDCA in clinical practice.

Kynurenine Pathway-An Underestimated Factor Modulating Innate Immunity in Sepsis-Induced Acute Kidney Injury?

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, and it accounts for about half of the cases of acute kidney injury (AKI). Although sepsis is the most frequent cause of AKI in critically ill patients, its pathophysiological mechanisms are not well understood. Sepsis has the ability to modulate the function of cells belonging to the innate immune system. Increased activity of indoleamine 2,3-dioxygenase 1 (IDO1) and production of kynurenines are the major metabolic pathways utilized by innate immunity cells to maintain immunological tolerance. The activation of the kynurenine pathway (KP) plays a dual role in sepsis—in the early stage, the induction of IDO1 elicits strong proinflammatory effects that may lead to tissue damage and septic shock. Afterwards, depletion of tryptophan and production of kynurenines contribute to the development of immunosuppression that may cause the inability to overpower opportunistic infections. The presented review provides available data on the various interdependencies between elements of innate immunity and sepsis-induced AKI (SAKI) with particular emphasis on the immunomodulatory significance of KP in the above processes. We believe that KP activation may be one of the crucial, though underestimated, components of a deregulated host response to infection during SAKI.

Enhanced tryptophan-kynurenine metabolism via indoleamine 2,3-dioxygenase 1 induction in dermatomyositis

ObjectivesExtrahepatic tryptophan (Trp)-kynurenine (Kyn) metabolism via indoleamine 2,3-dioxygenase 1 (IDO1) induction was found to be associated with intrinsic immune regulation. However, the Trp-Kyn metabolism–associated immune regulation in dermatomyositis (DM) remains unknown. Therefore, we aimed to investigate the clinical relevance of the Trp-Kyn metabolism via IDO1 induction in DM.MethodsLiquid chromatography-mass spectrometry (HPLC–MS) was used to examine the serum Kyn and Trp concentrations in DM. In addition, we used X-tile software to determine the optimal cutoff value of the Kyn/Trp ratio, a surrogate marker for Trp-Kyn metabolism. Spearman analysis was performed to evaluate the association of Trp-Kyn metabolism with muscle enzymes and inflammatory markers.ResultsDM patients had significantly higher serum Kyn/Trp ratio (× 10−3) when compared with the healthy controls. The serum Kyn/Trp ratio was positively correlated with the levels of muscle enzymes and inflammatory markers. In addition, the serum Kyn/Trp ratio significantly decreased (36.89 (26.00–54.00) vs. 25.00 (18.00–37.00), P = 0.0006) after treatment. DM patients with high serum Kyn/Trp ratio had a significantly higher percentage of muscle weakness symptoms (62.5% vs. 20.0%, P = 0.019) and higher levels of LDH (316.0 (236.0–467.0) vs. 198.0 (144.0–256.0), P = 0.004) and AST (56.5 (35.0–92.2) vs. 23.0 (20.0–36.0), P = 0.002)) than those with low serum Kyn/Trp ratio. Multiple Cox regression analyses identified ln(Kyn/Trp) (HR 4.874, 95% CI 1.105–21.499, P = 0.036) as an independent prognostic predictor of mortality in DM.ConclusionsDM patients with enhanced Trp-Kyn metabolism at disease onset are characterized by more severe disease status and poor prognosis. Intrinsic immune regulation function via enhanced Trp-Kyn metabolism by IDO1 induction may be a potential therapeutic target in DM.Key Points• HPLC–MS identified increased serum Kyn/Trp ratio in DM patients, which positively correlated with levels of muscle enzymes and inflammatory markers and was downregulated upon treatment.• Cox regression analyses identified ln(Kyn/Trp) as an independent prognostic predictor of mortality in DM.• Monitoring intrinsic immune regulation function should be considered a potential therapeutic target in DM patients.

Aberrant Upregulation of Indoleamine 2,3-Dioxygenase 1 Promotes Proliferation and Metastasis of Hepatocellular Carcinoma Cells via Coordinated Activation of AhR and β-Catenin Signaling.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide. Chronic liver inflammation due to hepatitis virus infection and other major effectors is a major risk factor of HCC. Indoleamine 2,3-dioxygenase 1 (IDO1), a heme enzyme highly expressed upon stimulation with proinflammatory cytokines such as interferon-γ (IFN-γ), is activated to modulate the tumor microenvironment and potentially crucial in the development of certain cancer types. Earlier studies have majorly reported an immunomodulatory function of IDO1. However, the specific role of IDO1 in cancer cells, particularly HCC, remains to be clarified. Analysis of The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) dataset in the current study revealed a significant correlation between IDO1 expression and HCC. We further established inducible IDO1-expressing cell models by coupling lentivirus-mediated knockdown and IFN-γ induction of IDO1 in normal and HCC cells. In functional assays, proliferation and motility-related functions of HCC cells were compromised upon suppression of IDO1, which may partially be rescued by its enzymatic product, kynurenine (KYN), while normal hepatocytes were not affected. Aryl hydrocarbon receptor (AhR), a reported endogenous KYN receptor, is suggested to participate in tumorigenesis. In mechanistic studies, IDO1 activation promoted both AhR and β-catenin activity and nuclear translocation. Immunofluorescence staining and co-immunoprecipitation assays further disclosed interactions between AhR and β-catenin. In addition, we identified a Src-PTEN-PI3K/Akt-GSK-3β axis involved in β-catenin stabilization and activation following IDO1-mediated AhR activation. IDO1-induced AhR and β-catenin modulated the expression of proliferation- and EMT-related genes to facilitate growth and metastasis of HCC cells. Our collective findings provide a mechanistic basis for the design of more efficacious IDO1-targeted therapy for HCC.

Cell based functional assays for IDO1 inhibitor screening and characterization.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a new immune-oncology target and its inhibitors have shown promise in the clinic especially in combination with other immune-stimulating agents. Here we describe two robust cell-based assays for screening IDO1 inhibitors. Both assays can be easily adopted by most laboratories and utilized for screening of IDO1 inhibitors. Endogenous IDO1 expression is induced in a cancer cell line with interferon gamma and its activity is assessed by measuring kynurenine secreted into the media. The effect of cancer cell IDO1 induction and inhibition on T cell activation is evaluated in a co-culture assay using Jurkat T cell line. Additional readouts assessing cell viability are employed for early detection of false positive IDO1 inhibitors and toxic compounds. Clinical candidates epacadostat and BMS-986205 were evaluated in the assays as control compounds, the former can completely inhibit IDO1 activity while the maximum effect of the later is limited (to about 80% in our system) consistent with the differences in their interaction with IDO1. Nanomolar concentrations of both compounds rescued IDO1 mediated inhibition of T cell activation. However, treatment with micromolar concentrations of BMS-986205 blocked Jurkat T cell activation and after prolonged incubation induced cell death.

Endothelial indoleamine 2,3-dioxygenase-1 regulates the placental vascular tone and is deficient in intrauterine growth restriction and pre-eclampsia

Indoleamine 2,3-dioxygenase-1 (IDO1) mediates the degradation of L-tryptophan (L-Trp) and is constitutively expressed in the chorionic vascular endothelium of the human placenta with highest levels in the microvasculature. Given that endothelial expression of IDO1 has been shown to regulate vascular tone and blood pressure in mice under the condition of systemic inflammation, we asked whether IDO1 is also involved in the regulation of placental blood flow and if yes, whether this function is potentially impaired in intrauterine growth restriction (IUGR) and pre-eclampsia (PE). In the large arteries of the chorionic plate L-Trp induced relaxation only after upregulation of IDO1 using interferon gamma and tumor necrosis factor alpha. However, ex vivo placental perfusion of pre-constricted cotyledonic vasculature with L-Trp decreases the vessel back pressure without prior IDO1 induction. Further to this finding, IDO1 protein expression and activity is reduced in IUGR and PE when compared to gestational age–matched control tissue. These data suggest that L-Trp catabolism plays a role in the regulation of placental vascular tone, a finding which is potentially linked to placental and fetal growth. In this context our data suggest that IDO1 deficiency is related to the pathogenesis of IUGR and PE.

RNAScope as a platform to detect IDO1 expression in tumor tissue sections.

116 RNAScope is a sensitive, specific platform to detect IDO1 expression in tumor tissue sections. M Pratta, M Rupar, P Waeltz, T Burn, G Hollis, M Covington, M Smith, and R Newton. Incyte Corp. Wilm. DE. Background: Indoleamine 2,3-Dioxygenase 1 (IDO1) catalyzes the primary and rate-limiting step in tryptophan catabolism to generate N-formyl-kynurenine (Kyn). Through a combination of local depletion of tryptophan and an increase in Kyn concentrations, IDO-1 activity can result in the suppression of antitumor immune responses. Because IDO-1 inhibitors are now in the clinic for treatment of multiple tumor types, immunohistological approaches are employed to demonstrate IDO1 expression in tumor biopsies. However, using a commercially available antibody to detect IDO1 by immunohistochemistry (IHC), the level of sensitivity was inadequate. Methods: In order to improve the sensitivity of IDO1 detection, we evaluated in situ hybridization (ISH) using RNAScope technology and digital quantitation by HALO analysis in collaboration with Advanced Cell Diagnostics (ACD). The technology was cross-validated using IDO1 qRT-PCR, Western blot, and activity analysis and compared with standard IHC. We initially evaluated IDO1 expression in HeLa cells stimulated with various concentrations of IFNγ, and then extended the observations using tissue sections from multiple tumor types. Results: In the HeLa cell model, IFNγ induced a time- and concentration-dependent increase of IDO1 at the mRNA, protein, and activity level. Although IDO1 was successfully detected in the HeLa cell samples by IHC, comparison of the platforms indicated IFNγ EC50 values were in strong agreement between RNAScope (193.8 pg/ml) and Western blot analysis (170.8 pg/ml), but was much higher by IHC analysis (2206 pg/ml). A strong positive correlation (*p < 0.0001) between RNAScope and Western blot analysis was observed, suggesting a highly coordinated induction of IDO1 by IFNγ at both the mRNA and protein levels. FFPE tumor tissue from melanoma, HNSCC, bladder, renal, ovarian, and lung cancers visualized by RNAScope all show varying levels of IDO1 expression. Conclusions: These data support the use of RNAScope for the analysis of IDO1 expression in clinical trials.

Suppression of indoleamine-2,3-dioxygenase 1 expression by promoter hypermethylation in ER-positive breast cancer

ABSTRACTKynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor α (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between IDO1 and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the IDO1 promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an IDO1 promoter construct reduced IDO1 expression, suggesting that enhanced methylation of the IDO1 promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as IDO1 was not suppressed by IDO1 promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

Chemotherapy-Dependent ATP Release from Leukemia Dying Cells Induces Indoleamine 2,3-Dioxygenase 1 in Dendritic Cells

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML.METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells.RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell.CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. DisclosuresMartinelli:Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII.

The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein.

Involvement of the kynurenine pathway in human glioma pathophysiology.

The kynurenine pathway (KP) is the principal route of L-tryptophan (TRP) catabolism leading to the production of kynurenine (KYN), the neuroprotectants, kynurenic acid (KYNA) and picolinic acid (PIC), the excitotoxin, quinolinic acid (QUIN) and the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+). The enzymes indoleamine 2,3-dioxygenase-1 (IDO-1), indoleamine 2,3-dioxygenase-2 (IDO-2) and tryptophan 2,3-dioxygenase (TDO-2) initiate the first step of the KP. IDO-1 and TDO-2 induction in tumors are crucial mechanisms implicated to play pivotal roles in suppressing anti-tumor immunity. Here, we report the first comprehensive characterisation of the KP in 1) cultured human glioma cells and 2) plasma from patients with glioblastoma (GBM). Our data revealed that interferon-gamma (IFN-γ) stimulation significantly potentiated the expression of the KP enzymes, IDO-1 IDO-2, kynureninase (KYNU), kynurenine hydroxylase (KMO) and significantly down-regulated 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) and kynurenine aminotransferase-I (KAT-I) expression in cultured human glioma cells. This significantly increased KP activity but significantly lowered the KYNA/KYN neuroprotective ratio in human cultured glioma cells. KP activation (KYN/TRP) was significantly higher, whereas the concentrations of the neuroreactive KP metabolites TRP, KYNA, QUIN and PIC and the KYNA/KYN ratio were significantly lower in GBM patient plasma (n = 18) compared to controls. These results provide further evidence for the involvement of the KP in glioma pathophysiology and highlight a potential role of KP products as novel and highly attractive therapeutic targets to evaluate for the treatment of brain tumors, aimed at restoring anti-tumor immunity and reducing the capacity for malignant cells to produce NAD+, which is necessary for energy production and DNA repair.

An epigenetic mechanism for high, synergistic expression of indoleamine 2,3-dioxygenase 1 (IDO1) by combined treatment with zebularine and IFN-γ: Potential therapeutic use in autoimmune diseases

IDO1 can be induced by interferon gamma (IFN-γ) in multiple cell types. We have earlier described that the DNA methyltransferase inhibitor zebularine also induces IDO1 in several rat cell clones. We now describe a synergistic induction of IDO1 expression by IFN-γ and zebularine. To elucidate the mechanism of the IDO1 induction we have studied the methylation status in the promoter region of the IDO1 gene from both human monocytic THP-1 cells and H1D2 rat colon cancer cells. Interestingly, the IDO1 promoter is hypermethylated and IFN-γ is shown to induce a significant demethylation. The synergism in effect of zebularine and IFN-γ on IDO1 expression is paralleled by a similar synergistic effect on expression of two other IFN-γ-responsive genes, the transcription factors STAT1 and IRF1 with binding sites in the IDO1 promoter region. The demonstrated synergistic activation of IDO1 expression has implications in relation to therapeutic induction of immunosuppression in autoimmunity and chronic inflammation.

Toll-Like Receptor Engagement Enhances the Immunosuppressive Properties of Human Bone Marrow-Derived Mesenchymal Stem Cells by Inducing Indoleamine-2,3-dioxygenase-1 via Interferon-β and Protein Kinase R

Mesenchymal stem cells (MSC) display unique suppressive properties on T-cell immunity, thus representing an attractive vehicle for the treatment of conditions associated with harmful T-cell responses such as organ-specific autoimmunity and graft-versus-host disease. Toll-like receptors (TLR) are primarily expressed on antigen-presenting cells and recognize conserved pathogen-derived components. Ligation of TLR activates multiple innate and adaptive immune response pathways to eliminate and protect against invading pathogens. In this work, we show that TLR expressed on human bone marrow-derived MSC enhanced the immunosuppressive phenotype of MSC. Immunosuppression mediated by TLR was dependent on the production of immunosuppressive kynurenines by the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase-1 (IDO1). Induction of IDO1 by TLR involved an autocrine interferon (IFN)-beta signaling loop, which was dependent on protein kinase R (PKR), but independent of IFN-gamma. These data define a new role for TLR in MSC immunobiology, which is to augment the immunosuppressive properties of MSC in the absence of IFN-gamma rather than inducing proinflammatory immune response pathways. PKR and IFN-beta play a central, previously unidentified role in orchestrating the production of immunosuppressive kynurenines by MSC.

Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) leads to immune suppression associated with the induction of regulatory T cells (T reg) expressing the transcription factor Foxp3. The immunological mechanisms of suppression are not well understood however dendritic cells (DC) are considered a key target for AhR-mediated immune suppression. Here we show that activation of AhR by TCDD induces DC indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase-like protein (IDO2). Induction of IDO1 and IDO2 was also found in lung and spleen associated with an increase of the T reg marker Foxp3 in spleen of TCDD-treated C57BL/6 mice, which is suppressed by inhibition of IDO. These data indicate that AhR-activation is an important signaling pathway for IDO expression and suggest a critical role of IDO in the mechanism leading to the generation of T reg that mediates the immune suppression through activation of AhR.