Epacadostat stabilizes the apo-form of IDO1 and signals a pro-tumorigenic pathway in human ovarian cancer cells.

The tryptophan-degrading enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a plastic immune checkpoint molecule that potently orchestrates immune responses within the tumor microenvironment (TME). As a heme-containing protein, IDO1 catalyzes the conversion of the essential amino acid tryptophan into immunoactive metabolites, called kynurenines. By depleting tryptophan and enriching the TME with kynurenines, IDO1 catalytic activity shapes an immunosuppressive TME. Accordingly, the inducible or constitutive IDO1 expression in cancer correlates with a negative prognosis for patients, representing one of the critical tumor-escape mechanisms. However, clinically trialed IDO1 catalytic inhibitors disappointed the expected anti-tumor efficacy. Interestingly, the non-enzymatic apo-form of IDO1 is still active as a transducing protein, capable of promoting an immunoregulatory phenotype in dendritic cells (DCs) as well as a pro-tumorigenic behavior in murine melanoma. Moreover, the IDO1 catalytic inhibitor epacadostat can induce a tolerogenic phenotype in plasmacytoid DCs, overcoming the catalytic inhibition of IDO1. Based on this recent evidence, IDO1 plasticity was investigated in the human ovarian cancer cell line, SKOV-3, that constitutively expresses IDO1 in a dynamic balance between the holo- and apo-protein, and thus potentially endowed with a dual function (i.e., enzymatic and non-enzymatic). Besides inhibiting the catalytic activity, epacadostat persistently stabilizes the apo-form of IDO1 protein, favoring its tyrosine-phosphorylation and promoting its association with the phosphatase SHP-2. In SKOV-3 cells, both these early molecular events activate a signaling pathway transduced by IDO1 apo-protein, which is independent of its catalytic activity and contributes to the tumorigenic phenotype of SKOV-3 cells. Overall, our findings unveiled a new mechanism of action of epacadostat on IDO1 target, repositioning the catalytic inhibitor as a stabilizer of the apo-form of IDO1, still capable of transducing a pro-tumorigenic pathway in SKOV-3 tumor. This mechanism could contribute to clarify the lack of effectiveness of epacadostat in clinical trials and shed light on innovative immunotherapeutic strategies to tackle IDO1 target.

Membrane Localization and Phosphorylation of Indoleamine 2,3-Dioxygenase 2 (IDO2) in A549 Human Lung Adenocarcinoma Cells: First Steps in Exploring Its Signaling Function.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a paralog of Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan-degrading enzyme producing immunomodulatory molecules. However, the two proteins are unlikely to carry out the same functions. IDO2 shows little or no tryptophan catabolic activity and exerts contrasting immunomodulatory roles in a context-dependent manner in cancer and autoimmune diseases. The recently described potential non-enzymatic activity of IDO2 has suggested its possible involvement in alternative pathways, resulting in either pro- or anti-inflammatory effects in different models. In a previous study on non-small cell lung cancer (NSCLC) tissues, we found that IDO2 expression revealed at the plasma membrane level of tumor cells was significantly associated with poor prognosis. In this study, the A549 human cell line, basally expressing IDO2, was used as an in vitro model of human lung adenocarcinoma to gain more insights into a possible alternative function of IDO2 different from the catalytic one. In these cells, immunocytochemistry and isopycnic sucrose gradient analyses confirmed the IDO2 protein localization in the cell membrane compartment, and the immunoprecipitation of tyrosine-phosphorylated proteins revealed that kinase activities can target IDO2. The different localization from the cytosolic one and the phosphorylation state are the first indications for the signaling function of IDO2, suggesting that the IDO2 non-enzymatic role in cancer cells is worthy of deeper understanding.

The catalytic inhibitor epacadostat can affect the non-enzymatic function of IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme chronically activated in many cancer patients and its expression and activity correlate with a poor prognosis. In fact, it acts as an immune regulator and contributes to tumor-induced immunosuppression by determining tryptophan deprivation and producing immunosuppressive metabolites named kynurenines. These findings made IDO1 an attractive target for cancer immunotherapy and small-molecule inhibitors, such as epacadostat, have been developed to block its enzymatic activity. Although epacadostat was effective in preclinical models and in early phase trials, it gave negative results in a metastatic melanoma randomized phase III study to test the benefit of adding epacadostat to the reference pembrolizumab therapy. However, the reason for the epacadostat failure in this clinical trial has never been understood. Our data suggest that a possible explanation of epacadostat ineffectiveness may rely on the ability of this drug to enhance the other IDO1 immunoregulatory mechanism, involving intracellular signaling function. These findings open up a new perspective for IDO1 inhibitors developed as new anticancer drugs, which should be carefully evaluated for their ability to block not only the catalytic but also the signaling activity of IDO1.

Optimization of Ultrasound-Assisted Extraction of Chlorogenic Acid from Potato Sprout Waste and Enhancement of the In Vitro Total Antioxidant Capacity.

Potato sprouts, an underutilized by-product of potato processing, could be exploited for the recovery of caffeoyl-quinic acids (CQAs), a family of polyphenols with well-recognized biological activities. In this work, the predominant compound of this class, 5-CQA, was extracted by Ultrasound-Assisted Extraction (UAE) under conditions optimized by an Experimental Design. The investigated variables solid/solvent ratio (1:10-1:50 g/mL), water content in ethanol (30-100% v/v) and UAE time (5-20 min) highlighted a critical influence of the last two factors on the extraction efficiency: extracts richer in 5-CQA were obtained with lower water content (30%) and time (5 min). The addition of ascorbic acid (1.7 mM) as anti-browning agent to the extraction solvent improved the extraction efficiency of 5-CQA compared to acetic and citric acids (3158.71 µg/mL, 1766.71 µg/mL, 1468.20 µg/mL, respectively). A parallel trend for the three acids and an increase in 5-CQA recovery was obtained with the use of freeze-dried sprouts (4980.05 µg/mL, 4795.62, 4211.25 µg/mL, respectively). Total antioxidant capacity (TAC) in vitro demonstrated UAE being a more valuable technique than conventional maceration. Furthermore, three-times-higher values of TPC (7.89 mg GAE/g) and TAC (FRAP: 24.01 mg TE/g; DPPH: 26.20 mg TE/g; ABTS 26.72 mg TE/g) were measured for the optimized extract compared to the initial one. An HPLC-DAD method was applied to monitor 5-CQA recovery, while an LC-HRMS/MS investigation allowed us to perform analyte identity confirmation along with detection of the glycoalkaloids α-solanine and α-chaconine. This evidence underlines the necessity to develop purification strategies in order to maximize the potential of potato sprout waste as a source of 5-CQA.

In-depth characterization of phenolic profiling of Moraiolo extra-virgin olive oil extract and initial investigation of the inhibitory effect on Indoleamine-2,3-Dioxygenase (IDO1) enzyme.

In this research, the phenolic extract from Moraiolo extra-virgin olive oil (EVOO) was thoroughly characterized. A reversed-phase HPLC method with a photodiode detector allowed to measure a total phenol content higher than 500 mg/kg EVOO, with elevated amounts of oleocanthal, oleacein, and oleouropein aglycone (131.2, 213.5, and 158.4 mg/kg EVOO, respectively). Appreciable amounts of (+)-pinoresinol and (+)- 1-acetoxy-pinoresinol, 3.2 and 12.5 mg/kg EVOO respectively, were measured. High-resolution mass spectrometry with Orbitrap mass analyzer technology was used to confirm the identity of the analytes. Afterwards, the extract was tested, for the first time, for its activity on Indoleamine-2,3-Dioxygenase (IDO1). This enzyme appears as a promising target for the modulation of the neuroinflammatory-oxidative processes relying on the pathogenesis of several neurodegenerative diseases. The extract showed an inhibitory effect on the catalytic activity of both human and murine IDO1, with a good safety at the concentrations of 15 and 30 µg/mL.

Indoleamine 2,3-dioxygenase 1 (IDO1): an up-to-date overview of an eclectic immunoregulatory enzyme.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the initial rate-limiting step in the degradation of the essential amino acid tryptophan along the kynurenine pathway. When discovered more than 50 years ago, IDO1 was thought to be an effector molecule capable of mediating a survival strategy based on the deprivation of bacteria and tumor cells of the essential amino acid tryptophan. Since 1998, when tryptophan catabolism was discovered to be crucially involved in the maintenance of maternal T-cell tolerance, IDO1 has become the focus of several laboratories around the world. Indeed, IDO1 is now considered as an authentic immune regulator not only in pregnancy, but also in autoimmune diseases, chronic inflammation, and tumor immunity. However, in the last years, a bulk of new information-including structural, biological, and functional evidence-on IDO1 has come to light. For instance, we now know that IDO1 has a peculiar conformational plasticity and, in addition to a complex and highly regulated catalytic activity, is capable of performing a nonenzymic function that reprograms the expression profile of immune cells toward a highly immunoregulatory phenotype. With this state-of-the-art review, we aimed at gathering the most recent information obtained for this eclectic protein as well as at highlighting the major unresolved questions.

Antioxidant Power on Dermal Cells by Textiles Dyed with an Onion (Allium cepa L.) Skin Extract.

In this study, the phenol loading and antioxidant activity of wool yarn prepared with the aqueous extract of onion (Allium cepa L.) skin was enhanced by implementing the dyeing process with the use of alum as a mordant. Spectrophotometric and chromatographic methods were applied for the characterization of polyphenolic substances loaded on the wool yarn. The antioxidant/anti-inflammatory properties were evaluated by determining the level of intra- and extra-cellular reactive oxygen species (ROS) production in keratinocytes and dermal fibroblasts pre-treated with lipopolysaccharide put in contact with artificial sweat. An elevated dye uptake on wool was observed for the pre-mordanted sample, as demonstrated by high absorbance values in the UV-Visible spectral range. Chromatographic results showed that protocatechuic acid and its glucoside were the main phenolic acid released in artificial sweat by the wool yarns, while quercetin-4'-glucoside and its aglycone quercetin were more retained. The extract released from the textile immersed in artificial sweat showed a significant reducing effect on the intra-and extracellular ROS levels in the two cell lines considered. Cytofluorimetric analyses demonstrated that the selected mordant was safe at the concentration used in the dyeing procedure. Therefore, alum pre-mordanted textiles dyed with onion-skin extracts may represent an interesting tool against skin diseases.

Current Challenges for IDO2 as Target in Cancer Immunotherapy.

Immune checkpoint inhibitors have revolutionized the clinical approach of untreatable tumors and brought a breath of fresh air in cancer immunotherapy. However, the therapeutic effects of these drugs only cover a minority of patients and alternative immunotherapeutic targets are required. Metabolism of l-tryptophan (Trp) via the kynurenine pathway represents an important immune checkpoint mechanism that controls adaptive immunity and dampens exaggerated inflammation. Indoleamine 2,3-dioxygenase 1 (IDO1), the enzyme catalyzing the first, rate-limiting step of the pathway, is expressed in several human tumors and IDO1 catalytic inhibitors have reached phase III clinical trials, unfortunately with disappointing results. Although much less studied, the IDO1 paralog IDO2 may represent a valid alternative as drug target in cancer immunotherapy. Accumulating evidence indicates that IDO2 is much less effective than IDO1 in metabolizing Trp and its functions are rather the consequence of interaction with other, still undefined proteins that may vary in distinct inflammatory and neoplastic contexts. As a matter of fact, the expression of IDO2 gene variants is protective in PDAC but increases the risk of developing tumor in NSCLC patients. Therefore, the definition of the IDO2 interactome and function in distinct neoplasia may open innovative avenues of therapeutic interventions.

A novel mutation of indoleamine 2,3-dioxygenase 1 causes a rapid proteasomal degradation and compromises protein function.

Indoleamine 2,3-dioxygenase 1 (IDO1) - the enzyme catalyzing the rate-limiting step of tryptophan catabolism along the kynurenine pathway - belongs to the class of inhibitory immune checkpoint molecules. Such regulators of the immune system are crucial for maintaining self-tolerance and thus, when properly working, preventing autoimmunity. A dysfunctional IDO1 has recently been associated with a specific single nucleotide polymorphism (SNP) and with the occurrence of autoimmune diabetes and multiple sclerosis. Many genetic alterations of IDO1 have been proposed being related with dysimmune disorders. However, the molecular and functional meaning of variations in IDO1 exomes as well as the promoter region remains a poorly explored field. In the present study, we identified a rare missense variant (rs751360195) at the IDO1 gene in a patient affected by coeliac disease, thyroiditis, and selective immunoglobulin A deficiency. Molecular and functional studies demonstrated that the substitution of lysine (K) at position 257 with a glutamic acid (E) results in an altered IDO1 protein that undergoes a rapid protein turnover. This genotype-to-phenotype relation is produced by peripheral blood mononuclear cells (PBMCs) of the patient bearing this variation and is associated with a specific phenotype (i.e., impaired tryptophan catabolism and defective mechanisms of immune tolerance). Thus decoding functional mutations of the IDO1 exome may provide clinically relevant information exploitable to personalize therapeutic interventions.

Indoleamine 2,3-Dioxygenase 2 Immunohistochemical Expression in Resected Human Non-small Cell Lung Cancer: A Potential New Prognostic Tool.

Indoleamine 2,3-dioxygenase 2 (IDO2) is an analog of the tryptophan degrading and immunomodulating enzyme indoleamine 2,3-dioxygenase 1 (IDO1). Although the role of IDO1 is largely understood, the function of IDO2 is not yet well-elucidated. IDO2 overexpression was documented in some human tumors, but the linkage between IDO2 expression and cancer progression is still unclear, in particular in non-small cell lung cancer (NSCLC). Immunohistochemical expression and cellular localization of IDO2 was evaluated on 191 formalin-fixed and paraffin-embedded resected NSCLC. Correlations between IDO2 expression, clinical-pathological data, tumor-infiltrating lymphocytes (TILs), immunosuppressive tumor molecules (IDO1 and programmed cell death ligand-1 - PD-L1 -) and patients' prognosis were evaluated. IDO2 high expression is strictly related to high PD-L1 level among squamous cell carcinomas group (p = 0.012), to either intratumoral or mixed localization of TILs (p < 0.001) and to adenocarcinoma histotype (p < 0.001). Furthermore, a significant correlation between IDO2 high expression and poor non-small cell lung cancer prognosis was detected (p = 0.011). The current study reaches interesting knowledge about IDO2 in non-small cell lung cancer. The close relationship between IDO2 expression, PD-L1 increased levels, TILs localization and NSCLC poor prognosis, assumed IDO2 as a potential prognostic biomarker to be exploited for optimizing innovative combined therapies with immune checkpoint inhibitors.

Engagement of Nuclear Coactivator 7 by 3-Hydroxyanthranilic Acid Enhances Activation of Aryl Hydrocarbon Receptor in Immunoregulatory Dendritic Cells.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step in the kynurenine pathway of tryptophan (Trp) degradation that produces several biologically active Trp metabolites. L-kynurenine (Kyn), the first byproduct by IDO1, promotes immunoregulatory effects via activation of the Aryl hydrocarbon Receptor (AhR) in dendritic cells (DCs) and T lymphocytes. We here identified the nuclear coactivator 7 (NCOA7) as a molecular target of 3-hydroxyanthranilic acid (3-HAA), a Trp metabolite produced downstream of Kyn along the kynurenine pathway. In cells overexpressing NCOA7 and AhR, the presence of 3-HAA increased the association of the two molecules and enhanced Kyn-driven, AhR-dependent gene transcription. Physiologically, conventional (cDCs) but not plasmacytoid DCs or other immune cells expressed high levels of NCOA7. In cocultures of CD4+ T cells with cDCs, the co-addition of Kyn and 3-HAA significantly increased the induction of Foxp3+ regulatory T cells and the production of immunosuppressive transforming growth factor ß in an NCOA7-dependent fashion. Thus, the co-presence of NCOA7 and the Trp metabolite 3-HAA can selectively enhance the activation of ubiquitary AhR in cDCs and consequent immunoregulatory effects. Because NCOA7 is often overexpressed and/or mutated in tumor microenvironments, our current data may provide evidence for a new immune check-point mechanism based on Trp metabolism and AhR.

IL-35Ig-expressing dendritic cells induce tolerance via Arginase 1.

The cytokine interleukin IL-35 is known to exert strong immunosuppressive functions. Indoleamine 2,3-dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL-35 and the activity of those enzymes. We transfected a single chain IL-35Ig gene construct in murine splenic DCs (DC35 ) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL-35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC35 , and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed-type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC35 was associated with the detection of CD25+ CD39+ , rather than Foxp3+ , regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC35 appears to be an early event in IL-35Ig-mediated immunosuppression.

Amino acid metabolism as drug target in autoimmune diseases.

In mammals, amino acid metabolism has evolved to control immune responses. Autoimmune diseases are heterogeneous conditions that involve the breakdown of tolerogenic circuitries and consequent activation of autoreactive immune cells. Therefore, critical enzymes along amino acid degradative pathways may be hijacked to keep in check autoimmunity. We examined here current knowledge of indoleamine 2,3-dioxygenase 1 (IDO1) and arginase 1 (ARG1), the main enzymes catabolizing tryptophan and arginine, respectively, in organ-specific and systemic autoimmune diseases as well as in the development of autoantibodies to therapeutic proteins. At variance with neoplastic contexts, in which it is known to act as a pure immunosuppressive molecule, ARG1 exhibited a protective or pathogenetic profile, depending on the disease. In contrast, in several autoimmune conditions, the bulk of data indicated that drugs capable of potentiating IDO1 expression and activity may represent valuable therapeutic tools and that IDO1-based immunotherapeutic protocols could be more effective if tailored to the genetic profile of individual patients.

Onion (Allium cepa L.) Skin: A Rich Resource of Biomolecules for the Sustainable Production of Colored Biofunctional Textiles.

The aqueous extract of dry onion skin waste from the 'Dorata di Parma' cultivar was tested as a new source of biomolecules for the production of colored and biofunctional wool yarns, through environmentally friendly dyeing procedures. Specific attention was paid to the antioxidant and UV protection properties of the resulting textiles. On the basis of spectrophotometric and mass spectrometry analyses, the obtained deep red-brown color was assigned to quercetin and its glycoside derivatives. The Folin⁻Ciocalteu method revealed good phenol uptakes on the wool fiber (higher than 27% for the textile after the first dyeing cycle), with respect to the original total content estimated in the water extract (78.50 ± 2.49 mg equivalent gallic acid/g onion skin). The manufactured materials showed remarkable antioxidant activity and ability to protect human skin against lipid peroxidation following UV radiation: 7.65 ± 1.43 (FRAP assay) and 13.60 (ORAC assay) mg equivalent trolox/g textile; lipid peroxidation inhibition up to 89.37%. This photoprotective and antioxidant activity were therefore ascribed to the polyphenol pool contained in the outer dried gold skins of onion. It is worth noting that citofluorimetric analysis demonstrated that the aqueous extract does not have a significative influence on cell viability, neither is capable of inducing a proapoptotic effect.

Amino-acid sensing and degrading pathways in immune regulation.

Indoleamine 2,3-dioxygenases (IDOs) - belonging in the heme dioxygenase family and degrading tryptophan - are responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As such, they are expressed by a variety of invertebrate and vertebrate species. In mammals, IDO1 has remarkably evolved to expand its functions, so to become a prominent homeostatic regulator, capable of modulating infection and immunity in multiple ways, including local tryptophan deprivation, production of biologically active tryptophan catabolites, and non-enzymatic cell-signaling activity. Much like IDO1, arginase 1 (Arg1) is an immunoregulatory enzyme that catalyzes the degradation of arginine. Here, we discuss the possible role of amino-acid degradation as related to the evolution of the immune systems and how the functions of those enzymes are linked by an entwined pathway selected by phylogenesis to meet the newly arising needs imposed by an evolving environment.

The Proteasome Inhibitor Bortezomib Controls Indoleamine 2,3-Dioxygenase 1 Breakdown and Restores Immune Regulation in Autoimmune Diabetes.

Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease.

Advances in indoleamine 2,3-dioxygenase 1 medicinal chemistry.

Indoleamine 2,3-dioxygenase 1 (IDO1) mediates multiple immunoregulatory processes including the induction of regulatory T cell differentiation and activation, suppression of T cell immune responses and inhibition of dendritic cell function, which impair immune recognition of cancer cells and promote tumor growth. On this basis, this enzyme is widely recognized as a valuable drug target for the development of immunotherapeutic small molecules in oncology. Although medicinal chemistry has made a substantial contribution to the discovery of numerous chemical classes of potent IDO1 inhibitors in the past 20 years, only very few compounds have progressed in clinical trials. In this review, we provide an overview of the current understanding of structure-function relationships of the enzyme, and discuss structure-activity relationships of selected classes of inhibitors that have shaped the hitherto few successes of IDO1 medicinal chemistry. An outlook opinion is also given on trends in the design of next generation inhibitors of the enzyme.

Forced IDO1 expression in dendritic cells restores immunoregulatory signalling in autoimmune diabetes.

Indoleamine 2,3-dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. We have recently demonstrated that IDO1 does not merely degrade tryptophan and produce immunoregulatory kynurenines, but it also acts as a signal-transducing molecule, independently of its enzymic function. IDO1 signalling activity is triggered in plasmacytoid dendritic cells (pDCs) by transforming growth factor-ß (TGF-ß), an event that requires the non-canonical NF-κB pathway and induces long-lasting IDO1 expression and autocrine TGF-ß production in a positive feedback loop, thus sustaining a stably regulatory phenotype in pDCs. IDO1 expression and catalytic function are defective in pDCs from non-obese diabetic (NOD) mice, a prototypic model of autoimmune diabetes. In the present study, we found that TGF-ß failed to activate IDO1 signalling function as well as up-regulate IDO1 expression in NOD pDCs. Moreover, TGF-ß-treated pDCs failed to exert immunosuppressive properties in vivo. Nevertheless, transfection of NOD pDCs with Ido1 prior to TGF-ß treatment resulted in activation of the Ido1 promoter and induction of non-canonical NF-κB and TGF-ß, as well as decreased production of the pro-inflammatory cytokines, interleukin 6 (IL-6) and tumour necrosis factor-α (TNF-α). Overexpression of IDO1 in TGF-ß-treated NOD pDCs also resulted in pDC ability to suppress the in vivo presentation of a pancreatic ß-cell auto-antigen. Thus, our data suggest that a correction of IDO1 expression may restore its dual function and thus represent a proper therapeutic manoeuvre in this autoimmune setting.

High doses of CpG oligodeoxynucleotides stimulate a tolerogenic TLR9-TRIF pathway.

CpG-rich oligodeoxynucleotides activate the immune system, leading to innate and acquired immune responses. The immune-stimulatory effects of CpG-rich oligodeoxynucleotides are being exploited as a therapeutic approach. Here we show that at high doses, CpG-rich oligodeoxynucleotides promote an opposite, tolerogenic response in mouse plasmacytoid dendritic cells in vivo and in a human in vitro model. Unveiling a previously undescribed role for TRIF and TRAF6 proteins in Toll-like receptor 9 (TLR9) signalling, we demonstrate that physical association of TLR9, TRIF and TRAF6 leads to activation of noncanonical NF-κB signalling and the induction of IRF3- and TGF-ß-dependent immune-suppressive tryptophan catabolism. In vivo, the TLR9-TRIF circuit--but not MyD88 signalling--was required for CpG protection against allergic inflammation. Our findings may be relevant to an increased understanding of the complexity of Toll-like receptor signalling and optimal exploitation of CpG-rich oligodeoxynucleotides as immune modulators.