ABSTRACT
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.
Subject(s)
Arginase/immunology , Dendritic Cells/immunology , Immune Tolerance/physiology , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Signal Transduction/immunology , Animals , Arginase/metabolism , Arginine/immunology , Arginine/metabolism , Blotting, Western , Dendritic Cells/metabolism , Female , Gene Expression Profiling , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Transcriptome , Tryptophan/immunology , Tryptophan/metabolismABSTRACT
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.
Subject(s)
Inflammation , Leukocytes, Mononuclear/metabolism , Membrane Proteins/genetics , Mutation , Wolfram Syndrome/metabolism , Child , Cytokines/genetics , Cytokines/metabolism , Female , Gene Expression Regulation , Humans , Leukocytes, Mononuclear/immunology , Sequence Analysis, DNA , Wolfram Syndrome/genetics , Wolfram Syndrome/immunology , Wolfram Syndrome/physiopathologyABSTRACT
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.
Subject(s)
Adaptive Immunity , Dendritic Cells , Immune Tolerance , Indoleamine-Pyrrole 2,3,-Dioxygenase , Signal Transduction/immunology , Transforming Growth Factor beta/immunology , Adaptive Immunity/drug effects , Animals , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/enzymology , Dendritic Cells/immunology , Humans , Hypersensitivity/immunology , Immune Tolerance/drug effects , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Interferon-gamma/immunology , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta/pharmacology , Tryptophan/metabolismABSTRACT
Polyamines (i.e., putrescine, spermidine, and spermine) are bioactive polycations capable of binding nucleic acids and proteins and modulating signaling pathways. Polyamine functions have been studied most extensively in tumors, where they can promote cell transformation and proliferation. Recently, spermidine was found to exert protective effects in an experimental model of multiple sclerosis (MS) and to confer immunoregulatory properties on dendritic cells (DCs), via the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme. IDO1 converts l-tryptophan into metabolites, collectively known as kynurenines, endowed with several immunoregulatory effects via activation of the arylhydrocarbon receptor (AhR). Because AhR activation increases polyamine production, the emerging scenario has identified polyamines and kynurenines as actors of an immunoregulatory circuitry with potential implications for immunotherapy in autoimmune diseases and cancer.
Subject(s)
Autoimmune Diseases , Immunomodulation , Kynurenine , Multiple Sclerosis , Polyamines , Animals , Autoimmune Diseases/immunology , Disease Models, Animal , Humans , Immunomodulation/immunology , Kynurenine/immunology , Multiple Sclerosis/enzymology , Multiple Sclerosis/immunology , Polyamines/immunology , Signal TransductionABSTRACT
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.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/chemistry , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Allosteric Regulation , Allosteric Site , Animals , Biocatalysis , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Female , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Kynurenine/metabolism , Leukocytes, Mononuclear/metabolism , Male , Mice, Knockout , Multiple Sclerosis/enzymology , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , Serotonin/analogs & derivatives , Serotonin/chemistry , Serotonin/metabolism , Tryptophan/metabolismABSTRACT
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.
Subject(s)
Indoleamine-Pyrrole 2,3,-Dioxygenase , Phosphatidylinositol 3-Kinases , Dendritic Cells/metabolism , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Inflammation , Phosphatidylinositol 3-Kinases/genetics , Signal TransductionABSTRACT
Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.
Subject(s)
Enzyme Inhibitors , Indoleamine-Pyrrole 2,3,-Dioxygenase , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Ligands , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Regulatory T (Treg) cells are crucial mediators of immune tolerance suppressing self-reactive T cells and preventing autoimmune diseases. Besides activation of the T cell receptor (TCR), empowerment of Treg cell functions requires co-accessory signals, such as those released by the TNF receptor superfamily (TNFRSF) that, however, can also promote immunostimulatory responses when engaged by effector T cells. In this issue of European Journal of Immunology, Lubrano di Ricco et al. [Eur. J. Immunol. 2020. 50: 972-985] have taken a closer look at the important question of the functional meaning of TNFRSF-activated signaling pathways in Treg cells. They have demonstrated that costimulation by TNFR2, 4-1BB, GITR, DR3, but not OX40 in mouse Foxp3+ Treg cells activates the same and unique signaling pathway, i.e., canonical NF-κB, which in turn leads to Foxp3 gene upregulation, cell expansion in vitro and in vivo, and suppressive activity in an experimental model of colitis. Moreover, induction of markers of T helper 2 (Th2) and Th17 as well as of genes encoding proteins involved in noncanonical NF-κΒ was also observed. We here discussed how these findings further highlight the emerging concept of Treg cell plasticity in immune tolerance.
Subject(s)
Immune Tolerance , Receptors, Tumor Necrosis Factor/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Humans , Mice , T-Lymphocytes, Regulatory/cytologyABSTRACT
The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates numerous cellular responses. Originally investigated in toxicology because of its ability to bind environmental contaminants, AhR has attracted enormous attention in the field of immunology in the last 20 years. In addition, the discovery of endogenous and plant-derived ligands points to AhR also having a crucial role in normal cell physiology. Thus, AhR is emerging as a promiscuous receptor that can mediate either toxic or physiologic effects upon sensing multiple exogenous and endogenous molecules. Within this scenario, several factors appear to contribute to the outcome of gene transcriptional regulation by AhR, including the nature of the ligand as such and its further metabolism by AhR-induced enzymes, the local tissue microenvironment, and the presence of coregulators or specific transcription factors in the cell. Here, we review the current knowledge on the array of transcription factors and coregulators that, by interacting with AhR, tune its transcriptional activity in response to endogenous and exogenous ligands.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Drug Discovery , Ligands , Receptors, Aryl Hydrocarbon/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Carrier Proteins , Drug Discovery/methods , Gene Expression Regulation/drug effects , Humans , Immunomodulation , Protein Binding , Receptors, Aryl Hydrocarbon/genetics , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Myelodysplastic Syndromes/genetics , Proteasome Endopeptidase Complex/metabolism , DNA Mutational Analysis , Exons/genetics , HEK293 Cells , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Molecular Dynamics Simulation , Mutation, Missense , Myelodysplastic Syndromes/immunology , ProteolysisABSTRACT
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.
Subject(s)
Disease Resistance/genetics , Disease Resistance/immunology , Receptors, Aryl Hydrocarbon/metabolism , Animals , Bacterial Infections/immunology , Bacterial Infections/metabolism , Disease Resistance/drug effects , Endotoxemia/genetics , Endotoxemia/immunology , Endotoxemia/metabolism , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Inflammation/enzymology , Inflammation/genetics , Inflammation/metabolism , Kynurenine/metabolism , Lipopolysaccharides/pharmacology , Mice , Phosphorylation , Receptors, Aryl Hydrocarbon/genetics , Signal Transduction , Tryptophan Oxygenase/metabolism , src-Family Kinases/metabolismABSTRACT
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.
Subject(s)
Arginase/immunology , Dendritic Cells/immunology , Immune Tolerance/immunology , Interleukins/immunology , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , Apyrase/immunology , Apyrase/metabolism , Arginase/genetics , Arginase/metabolism , Dendritic Cells/metabolism , Female , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Immune Tolerance/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Interleukin-2 Receptor alpha Subunit/immunology , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-4/genetics , Interleukin-4/immunology , Interleukin-4/metabolism , Interleukins/genetics , Interleukins/metabolism , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolismABSTRACT
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.
Subject(s)
Neurodegenerative Diseases/diagnosis , Neurodegenerative Diseases/therapy , Wolfram Syndrome/diagnosis , Wolfram Syndrome/therapy , Adolescent , Adult , Child , Child, Preschool , Clinical Trials as Topic , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 1/etiology , Diabetes Mellitus, Type 1/therapy , Disease Progression , Drug Development , Drug Repositioning , Endoplasmic Reticulum/metabolism , Female , Genes, Recessive , Humans , Infant , Interdisciplinary Communication , Male , Membrane Proteins/genetics , Neurodegenerative Diseases/complications , Neurodegenerative Diseases/etiology , Prognosis , Quality of Life , Wolfram Syndrome/complications , Wolfram Syndrome/etiology , Young AdultABSTRACT
IFN-α prevents Ag-induced arthritis (AIA), and in this study we investigated the role of IDO1 and TGF-ß signaling for this anti-inflammatory property of IFN-α. Arthritis was induced by methylated BSA (mBSA) in mBSA-sensitized wild-type (WT), Ido1-/-, or Ifnar-/- mice, treated or not with IFN-α or the IDO1 product kynurenine (Kyn). Enzymatic IDO1 activity, TGF-ß, and plasmacytoid dendritic cells (pDC) were neutralized by 1-methyltryptophan and Abs against TGF-ß and pDC, respectively. IDO1 expression was determined by RT-PCR, Western blot, and FACS, and enzymatic activity by HPLC. Proliferation was measured by 3H-thymidine incorporation and TGF-ß by RT-PCR and ELISA. WT but not Ido1-/- mice were protected from AIA by IFN-α, and Kyn, the main IDO1 product, also prevented AIA, both in WT and Ifnar-/- mice. Protective treatment with IFN-α increased the expression of IDO1 in pDC during AIA, and Ab-mediated depletion of pDC, either during mBSA sensitization or after triggering of arthritis, completely abrogated the protective effect of IFN-α. IFN-α treatment also increased the enzymatic IDO1 activity (Kyn/tryptophan ratio), which in turn activated production of TGF-ß. Neutralization of enzymatic IDO1 activity or TGF-ß signaling blocked the protective effect of IFN-α against AIA, but only during sensitization and not after triggering of arthritis. Likewise, inhibition of the IDO1 enzymatic activity in the sensitization phase, but not after triggering of arthritis, subdued the IFN-α-induced inhibition of mBSA-induced proliferation. In conclusion, presence of IFN-α at Ag sensitization activates an IDO1/TGF-ß-dependent anti-inflammatory program that upon antigenic rechallenge prevents inflammation via pDC.
Subject(s)
Arthritis, Experimental/immunology , Dendritic Cells/physiology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Receptor, Interferon alpha-beta/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cell Proliferation , Cells, Cultured , Gene Expression Regulation , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Kynurenine/administration & dosage , Mice , Mice, 129 Strain , Mice, Knockout , Receptor, Interferon alpha-beta/genetics , Serum Albumin, Bovine/immunology , Signal TransductionABSTRACT
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.
Subject(s)
Immunosuppressive Agents/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Tyrosine/immunology , Animals , Cytokines/immunology , Dendritic Cells/immunology , Female , Half-Life , Immune Tolerance/immunology , Kynurenine/immunology , Mice , Mice, Inbred C57BL , Phosphorylation/immunology , Protein Domains/immunology , Signal Transduction/immunology , Suppressor of Cytokine Signaling Proteins/immunology , Tryptophan/immunologyABSTRACT
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.
Subject(s)
Amniotic Fluid/cytology , Immunomodulation , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Stem Cells/immunology , Stem Cells/metabolism , Adult , Allografts/drug effects , Animals , Biomarkers/metabolism , Cell Communication/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Separation , Cell Shape/drug effects , Clone Cells , Embryoid Bodies/cytology , Graft Survival/drug effects , Humans , Immunomodulation/drug effects , Interferon-gamma/pharmacology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Phenotype , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Stem Cells/cytology , Stem Cells/drug effects , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunologyABSTRACT
Dendritic cells (DCs) are professional antigen presenting cells capable of orchestrating either stimulatory or regulatory immune responses mediated by T cells. Interleukin 35 (IL-35) is an immunosuppressive, heterodimeric cytokine belonging to the IL-12 family and known to be produced by regulatory T cells but not DCs. In this study, we explored the possible immunosuppressive effect of IL-35 ectopically expressed by splenic DCs from nonobese diabetic (NOD) mice, a prototypical model of autoimmune diabetes. After pulsing with the IGRP peptide (a dominant, diabetogenic autoantigen in NOD mice) and transfer in vivo, IL-35Ig- but not Ig-transfected DCs suppressed antigen specific, T cell-mediated responses in a skin test assay. More importantly, transfer of IL-35Ig-transfected, IGRP-pulsed DCs into prediabetic NOD mice induced a delayed and less severe form of diabetes, an effect accompanied by the increase of CD4(+)CD39(+) suppressive T cells in pancreatic lymph nodes. Our data therefore suggest that DCs overexpressing ectopic IL-35Ig might represent a powerful tool in negative vaccination strategies.
Subject(s)
Antibodies/genetics , Dendritic Cells/immunology , Diabetes Mellitus, Type 1/prevention & control , Interleukins/genetics , Recombinant Fusion Proteins/genetics , Amino Acid Sequence , Animals , Antibodies/immunology , Base Sequence , CD4-Positive T-Lymphocytes/immunology , Cell Line , Dendritic Cells/metabolism , Dendritic Cells/transplantation , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/therapy , Female , Genetic Therapy/methods , HEK293 Cells , Humans , Interleukins/biosynthesis , Interleukins/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Molecular Sequence Data , Pancreas/cytology , Pancreas/immunology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunologyABSTRACT
Modulation of immune responses is one of the main research aims in transplant immunology. In this study, we investigate the local immunomodulatory properties of soluble CD83 (sCD83) at the graft-host interface using the high-risk corneal transplantation model. In this model, which mimics the inflammatory status and the preexisting vascularization of high-risk patients undergoing corneal transplantation, allogeneic donor corneas are transplanted onto sCD83-treated recipient animals. This model allows the direct and precise application of the immune modulator at the transplantation side. Interestingly, sCD83 was able to prolong graft survival after systemic application as well as after topical application, which is therapeutically more relevant. The therapeutic effect was accompanied by an increase in the frequency of regulatory T cells and was mediated by the immune-regulatory enzyme IDO and TGF-ß. In vitro, sCD83 induced long-term IDO expression in both conventional and plasmacytoid dendritic cells via autocrine or paracrine production of TGF-ß, a cytokine previously shown to be an essential mediator of IDO-dependent, long-term tolerance. These findings open new treatment avenues for local immune modulation after organ and tissue transplantation.
Subject(s)
Antigens, CD/therapeutic use , Corneal Transplantation , Graft Enhancement, Immunologic , Immunoglobulins/therapeutic use , Immunologic Factors/therapeutic use , Indoleamine-Pyrrole 2,3,-Dioxygenase/physiology , Membrane Glycoproteins/therapeutic use , T-Lymphocytes, Regulatory/drug effects , Transplantation Tolerance/drug effects , Administration, Ophthalmic , Allografts , Animals , Antigens, CD/administration & dosage , Antigens, CD/immunology , Bone Marrow Cells/immunology , Cells, Cultured , Coculture Techniques , Dendritic Cells/immunology , Drug Evaluation, Preclinical , Enzyme Induction/drug effects , Female , Forkhead Transcription Factors/analysis , Graft Survival , Immunoglobulins/administration & dosage , Immunoglobulins/immunology , Immunologic Factors/administration & dosage , Immunologic Factors/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/biosynthesis , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Injections, Intraperitoneal , Membrane Glycoproteins/administration & dosage , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Premedication , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Solubility , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Transforming Growth Factor beta/administration & dosage , Transforming Growth Factor beta/physiology , Transforming Growth Factor beta/therapeutic use , CD83 AntigenABSTRACT
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.