Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.

Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.

Novel mutations in the WFS1 gene are associated with Wolfram syndrome and systemic inflammation.

Mutations in the WFS1 gene, encoding wolframin (WFS1), cause endoplasmic reticulum (ER) stress and are associated with a rare autosomal-recessive disorder known as Wolfram syndrome (WS). WS is clinically characterized by childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus and neurological signs. We identified two novel WFS1 mutations in a patient with WS, namely, c.316-1G > A (in intron 3) and c.757A > T (in exon 7). Both mutations, located in the N-terminal region of the protein, were predicted to generate a truncated and inactive form of WFS1. We found that although the WFS1 protein was not expressed in peripheral blood mononuclear cells (PBMCs) of the proband, no constitutive ER stress activation could be detected in those cells. In contrast, WS proband's PBMCs produced very high levels of proinflammatory cytokines (i.e. TNF-α, IL-1ß, and IL-6) in the absence of any stimulus. WFS1 silencing in PBMCs from control subjects by means of small RNA interference also induced a pronounced proinflammatory cytokine profile. The same cytokines were also significantly higher in sera from the WS patient as compared to matched healthy controls. Moreover, the chronic inflammatory state was associated with a dominance of proinflammatory T helper 17 (Th17)-type cells over regulatory T (Treg) lymphocytes in the WS PBMCs. The identification of a state of systemic chronic inflammation associated with WFS1 deficiency may pave the way to innovative and personalized therapeutic interventions in WS.

Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells.

Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-ß (TGF-ß) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-ß-driven tolerance in noninflammatory contexts.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

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 immunosuppression pathway of tumor-associated macrophages is controlled by heme oxygenase-1 in glioblastoma patients.

The immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) is mainly driven by tumor-associated macrophages (TAMs). We explored whether their sustained iron metabolism and immunosuppressive activity were correlated, and whether blocking the central enzyme of the heme catabolism pathway, heme oxygenase-1 (HO-1), could reverse their tolerogenic activity. To this end, we investigated iron metabolism in bone marrow-derived macrophages (BMDMs) isolated from GBM specimens and in in vitro-derived macrophages (Mφ) from healthy donor (HD) blood monocytes. We found that HO-1 inhibition abrogated the immunosuppressive activity of both BMDMs and Mφ, and that immunosuppression requires both cell-to-cell contact and soluble factors, as HO-1 inhibition abolished IL-10 release, and significantly reduced STAT3 activation as well as PD-L1 expression. Interestingly, not only did HO-1 inhibition downregulate IDO1 and ARG-2 gene expression, but also reduced IDO1 enzymatic activity. Moreover, T cell activation status affected PD-L1 expression and IDO1 activity, which were upregulated in the presence of activated, but not resting, T cells. Our results highlight the crucial role of HO-1 in the immunosuppressive activity of macrophages in the GBM TME and demonstrate the feasibility of reprogramming them as an alternative therapeutic strategy for restoring immune surveillance.

Class IA PI3Ks regulate subcellular and functional dynamics of IDO1.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

Decoding the Complex Crossroad of Tryptophan Metabolic Pathways.

Among the 20 amino acids needed for protein synthesis, Tryptophan (Trp) is an aromatic amino acid fundamental not only for the synthesis of the major components of living cells (namely, the proteins), but also for the maintenance of cellular homeostasis [...].

Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

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.

Aryl hydrocarbon receptor control of a disease tolerance defence pathway.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Bioadhesive Polymeric Films Based on Red Onion Skins Extract for Wound Treatment: An Innovative and Eco-Friendly Formulation.

The onion non-edible outside layers represent a widely available waste material deriving from its processing and consumption. As onion is a vegetable showing many beneficial properties for human health, a study aiming to evaluate the use of extract deriving from the non-edible outside layers was planned. An eco-friendly extraction method was optimized using a hydroalcoholic solution as solvent. The obtained extract was deeply characterized by in vitro methods and then formulated in autoadhesive, biocompatible and pain-free hydrogel polymeric films. The extract, very soluble in water, showed antioxidant, radical scavenging, antibacterial and anti-inflammatory activities, suggesting a potential dermal application for wounds treatment. In vitro studies showed a sustained release of the extract from the hydrogel polymeric film suitable to reach concentrations necessary for both antibacterial and anti-inflammatory activities. Test performed on human keratinocytes showed that the formulation is safe suggesting that the projected formulation could be a valuable tool for wound treatment.

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.

Wolfram syndrome, a rare neurodegenerative disease: from pathogenesis to future treatment perspectives.

BACKGROUND: Wolfram syndrome (WS), a rare genetic disorder, is considered the best prototype of endoplasmic reticulum (ER) diseases. Classical WS features are childhood-onset diabetes mellitus, optic atrophy, deafness, diabetes insipidus, neurological signs, and other abnormalities. Two causative genes (WFS1 and WFS2) have been identified. The transmission of the disease takes place in an autosomal recessive mode but autosomal dominant mutations responsible for WS-related disorders have been described. Prognosis is poor, death occurs at the median age of 39 years with a major cause represented by respiratory failure as a consequence of brain stem atrophy and neurodegeneration. The aim of this narrative review is to focus on etiology, pathogenesis and natural history of WS for an adequate patient management and for the discussion of future therapeutic interventions. MAIN BODY: WS requires a multidisciplinary approach in order to be successfully treated. A prompt diagnosis decreases morbidity and mortality through prevention and treatment of complications. Being a monogenic pathology, WS represents a perfect model to study the mechanisms of ER stress and how this condition leads to cell death, in comparison with other prevalent diseases in which multiple factors interact to produce the disease manifestations. WS is also an important disease prototype to identify drugs and molecules associated with ER homeostasis. Evidence indicates that specific metabolic diseases (type 1 and type 2 diabetes), neurodegenerative diseases, atherosclerosis, inflammatory pathologies and also cancer are closely related to ER dysfunction. CONCLUSIONS: Therapeutic strategies in WS are based on drug repurposing (i.e., investigation of approved drugs for novel therapeutic indications) with the aim to stop the progression of the disease by reducing the endoplasmic reticulum stress. An extensive understanding of WS from pathophysiology to therapy is fundamental and more studies are necessary to better manage this devastating disease and guarantee the patients a better quality of life and longer life expectancy.

Urinary l-kynurenine quantification and selective extraction through a molecularly imprinted solid-phase extraction device.

l-Kynurenine is an endogenous metabolite generated by the catabolic pathway of l-tryptophan and it could be a potential biomarker to test the efficacy of several checkpoint inhibitors that have already reached the clinical trials in the antitumor therapy. Thus, a molecularly imprinted polymer specific for the recognition of this metabolite was synthesized and used as innovative system in solid-phase extraction technique for the specific extraction and quantification of l-kynurenine in human urine. The off-line system was firstly tested on l-kynurenine standard solutions, allowing recoveries up to 97.7% (relative standard deviation = 2.2%) and then applied to fortified and deproteinated human urine samples, where a recovery of 84.1% (relative standard deviation = 3.1%) was obtained. The method was validated and it revealed a good linearity in the range of 0.157-20 µg/mL (r2  = 0.9992). The optimized procedure demonstrated a good feasibility on biological samples, allowing a ready quantification of l-kynurenine in the human urine, where the metabolite was found at a very low concentration (0.80 µg/mL). The extraction system developed could attract attention of pharmaceutical industries for l-kynurenine production as potential drug in the treatment of autoimmune disorders through its extraction and purification from biological matrixes.

Distinct roles of immunoreceptor tyrosine-based motifs in immunosuppressive indoleamine 2,3-dioxygenase 1.

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the initial, rate-limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1-dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine-based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP-1 and SHP-2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine-phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half-life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM-associated tyrosines with phospho-mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 - but not ITIM2 mutant - did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen-specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1-dependent events will occur in a local microenvironment.

Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase1.

Although human amniotic fluid does contain different populations of foetal-derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second-trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)-γ, including induction of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN-γ-treated fHASCs caused significantly decreased T-cell proliferation and increased frequency in CD4(+)  CD25(+)  FOXP3(+) regulatory T cells. Both effects required an intact IDO1 function and were cell contact-independent. An unprecedented finding in our study was that purified vesicles from IFN-γ-treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC-like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4(+)  CD25(+)  Foxp3(+) T cells in graft-draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.

Cytokines in systemic juvenile idiopathic arthritis and haemophagocytic lymphohistiocytosis: tipping the balance between interleukin-18 and interferon-γ.

OBJECTIVES: To study the role of IFN-γ in the pathogenesis of systemic JIA (sJIA) and haemophagocytic lymphohistiocytosis (HLH) by searching for an IFN-γ profile, and to assess its relationship with other cytokines. METHODS: Patients with inactive (n = 10) and active sJIA (n = 10), HLH [n = 5; of which 3 had sJIA-associated macrophage activation syndrome (MAS)] and healthy controls (n = 16) were enrolled in the study. Cytokines and IFN-γ-induced genes and proteins were determined in plasma, in patient peripheral blood mononuclear cells (PBMCs) and in lymph node biopsies of one patient during both sJIA and MAS episodes. IFN-γ responses were investigated in healthy donor PBMCs, primary fibroblasts and endothelial cells. RESULTS: Plasma IFN-γ, IL-6 and IL-18 were elevated in active sJIA and HLH. Levels of IFN-γ and IFN-γ-induced proteins (IP-10/CXCL-10, IL-18BP and indoleamine 2,3-dioxygenase) in HLH were much higher than levels in active sJIA. Free IL-18 and ratios of IL-18/IFN-γ were higher in active sJIA compared with HLH. HLH PBMCs showed hyporesponsiveness to IFN-γ in vitro when compared with control and sJIA PBMCs. Endothelial cells and fibroblasts expressed IFN-γ-induced proteins in situ in lymph node staining of a MAS patient and in vitro upon stimulation with IFN-γ. CONCLUSION: Patients with active sJIA and HLH/MAS show distinct cytokine profiles, with highly elevated plasma levels of IFN-γ and IFN-γ-induced proteins typically found in HLH/MAS. In addition to PBMCs, histiocytes, endothelial cells and fibroblasts may contribute to an IFN-γ profile in plasma. Increasing levels of IFN-γ compared with IL-18 may raise suspicion about the development of MAS in sJIA.

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.