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
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
Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1α-mediated transcriptional regulation of stress response genes with anti-inflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , COVID-19/metabolism , Endoplasmic Reticulum Stress/drug effects , NF-kappa B/metabolism , SARS-CoV-2 , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , COVID-19/virology , Caspase 9/metabolism , Chlorocebus aethiops , Cytopathogenic Effect, Viral/drug effects , Humans , MAP Kinase Signaling System/drug effects , Models, Biological , Nelfinavir/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Unfolded Protein Response/drug effects , Vero CellsABSTRACT
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
Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre-pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2 O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione-S-transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK-ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co-administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2 O2 levels, was observed both at 5 and 10 µM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd-induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK-ERK1/2 activity upregulation was associated with these effects at 5 µM Cd, whereas glutathione biosynthesis and efflux were involved at 10 µM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2 O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.
Subject(s)
Melatonin , NF-E2-Related Factor 2 , Animals , Cadmium/pharmacology , Cystine/metabolism , Cystine/pharmacology , Glutathione/metabolism , Male , Melatonin/metabolism , Melatonin/pharmacology , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Proto-Oncogene Proteins c-akt/metabolism , Sertoli Cells/metabolism , SwineABSTRACT
The field of targeted protein degradation, through the control of the ubiquitin-proteasome system (UPS), is progressing considerably; to exploit this new therapeutic modality, the proteolysis targeting chimera (PROTAC) technology was born. The opportunity to use PROTACs engaging of new E3 ligases that can hijack and control the UPS system could greatly extend the applicability of degrading molecules. To this end, here we show a potential application of the ELIOT (E3 LIgase pocketOme navigaTor) platform, previously published by this group, for a scaffold-repurposing strategy to identify new ligands for a novel E3 ligase, such as TRIM33. Starting from ELIOT, a case study of the cross-relationship using GRID Molecular Interaction Field (MIF) similarities between TRIM24 and TRIM33 binding sites was selected. Based on the assumption that similar pockets could bind similar ligands and considering that TRIM24 has 12 known co-crystalised ligands, we applied a scaffold-repurposing strategy for the identification of TRIM33 ligands exploiting the scaffold of TRIM24 ligands. We performed a deeper computational analysis to identify pocket similarities and differences, followed by docking and water analysis; selected ligands were synthesised and subsequently tested against TRIM33 via HTRF binding assay, and we obtained the first-ever X-ray crystallographic complexes of TRIM33α with three of the selected compounds.
Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin-Protein Ligases , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Ligands , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolismABSTRACT
Neuroblastoma survivors show signs of immunosenescence early after therapy in CD8+ T cell compartment and elevated plasma TNF-α but in later follow-up immune recovery comes into play. Whether the recovery phenotype is long lasting or transient remains to be elucidated, however, late adverse effects often occur in childhood cancer survivors.
Subject(s)
Immunosenescence/immunology , Neuroblastoma/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Survivors , Humans , Risk Factors , Survivors , Tumor Necrosis Factor-alpha/immunologyABSTRACT
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 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
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory, demyelinating disease of the CNS that mimics human multiple sclerosis (MS), and it is thought to be driven by Th1 and Th17 myelin-reactive cells. Although adaptive immunity is clearly pivotal in the pathogenesis of EAE, with an essential role of CD4+ T cells, little is known of early, innate responses in this experimental setting. CpG-rich oligodeoxynucleotides (ODNs), typically found in microbial genomes, are potent activators of TLR9 in plasmacytoid dendritic cells (pDCs). In this study, we compared the effects of two types of CpG, namely, type A and type B, on EAE. We found that treatment with CpG type A ODN (CpG-A), known to induce high amounts of IFN-α in pDCs, significantly reduced disease severity in EAE, relative to controls (12.63 ± 1.86 versus 23.49 ± 1.46, resp.; p = 0.001). Treatment also delayed onset of neurological deficits and reduced spinal cord demyelination, while increasing the percentage of splenic regulatory (Foxp3+ CD4+) T cells. CpG-A likewise reduced the levels of IL-17 and IFN-γ in the CNS. Mechanistic insight into those events showed that CpG-A promoted a regulatory phenotype in pDCs. Moreover, adoptive transfer of pDCs isolated from CpG-A-treated mice inhibited CNS inflammation and induced disease remission in acute-phase EAE. Our data thus identify a link between TLR9 activation by specific ligands and the induction of tolerance via innate immunity mechanisms.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/metabolism , Immunity, Innate , Oligodeoxyribonucleotides/metabolism , Animals , Dendritic Cells/cytology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/immunology , Endotoxins/metabolism , Female , Inflammation , Ligands , Mice , Mice, Inbred C57BL , Multiple Sclerosis , Phenotype , Signal Transduction , Spleen/metabolism , T-Lymphocytes, Regulatory/cytologyABSTRACT
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
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.
Subject(s)
Dendritic Cells/immunology , Diabetes Mellitus, Type 1/immunology , Immunity, Cellular/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Skin/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Blotting, Western , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Kynurenine/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Nude , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Skin/cytology , Skin/metabolismABSTRACT
Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-ß-dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3+ T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.
Subject(s)
Dendritic Cells/immunology , Immune Tolerance/genetics , Oligodeoxyribonucleotides/pharmacology , Animals , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/enzymology , Female , Humans , Immune Tolerance/drug effects , Indoleamine-Pyrrole 2,3,-Dioxygenase/biosynthesis , Interferon-beta/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/genetics , Oligodeoxyribonucleotides/genetics , Protein Structure, Tertiary , Receptors, Interleukin-1/physiology , Signal Transduction/immunology , Toll-Like Receptor 7/physiology , Transcription, Genetic/immunology , Transforming Growth Factor beta/physiologyABSTRACT
Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4(+) T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.
Subject(s)
Dexamethasone/pharmacology , Hypersensitivity/prevention & control , Hypersensitivity/physiopathology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Receptors, Nerve Growth Factor/physiology , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/physiology , Animals , Dendritic Cells/immunology , Disease Models, Animal , Enzyme Activation/drug effects , Glucocorticoid-Induced TNFR-Related Protein , Humans , Mice , Receptors, Nerve Growth Factor/drug effects , Receptors, Tumor Necrosis Factor/drug effects , Spleen/immunology , Tumor Necrosis Factors/physiologyABSTRACT
Half a century ago, chronic granulomatous disease (CGD) was first described as a disease fatally affecting the ability of children to survive infections. Various milestone discoveries have since been made, from an insufficient ability of patients' leucocytes to kill microbes to the underlying genetic abnormalities. In this inherited disorder, phagocytes lack NADPH oxidase activity and do not generate reactive oxygen species, most notably superoxide anion, causing recurrent bacterial and fungal infections. Patients with CGD also suffer from chronic inflammatory conditions, most prominently granuloma formation in hollow viscera. The precise mechanisms of the increased microbial pathogenicity have been unclear, and more so the reasons for the exaggerated inflammatory response. Here we show that a superoxide-dependent step in tryptophan metabolism along the kynurenine pathway is blocked in CGD mice with lethal pulmonary aspergillosis, leading to unrestrained Vgamma1(+) gammadelta T-cell reactivity, dominant production of interleukin (IL)-17, defective regulatory T-cell activity and acute inflammatory lung injury. Although beneficial effects are induced by IL-17 neutralization or gammadelta T-cell contraction, complete cure and reversal of the hyperinflammatory phenotype are achieved by replacement therapy with a natural kynurenine distal to the blockade in the pathway. Effective therapy, which includes co-administration of recombinant interferon-gamma (IFN-gamma), restores production of downstream immunoactive metabolites and enables the emergence of regulatory Vgamma4(+) gammadelta and Foxp3(+) alphabeta T cells. Therefore, paradoxically, the lack of reactive oxygen species contributes to the hyperinflammatory phenotype associated with NADPH oxidase deficiencies, through a dysfunctional kynurenine pathway of tryptophan catabolism. Yet, this condition can be reverted by reactivating the pathway downstream of the superoxide-dependent step.
Subject(s)
Granulomatous Disease, Chronic/metabolism , Granulomatous Disease, Chronic/pathology , Inflammation/metabolism , Kynurenine/metabolism , Tryptophan/metabolism , Animals , Aspergillosis/complications , Aspergillosis/immunology , Aspergillosis/microbiology , Aspergillosis/pathology , Aspergillus fumigatus/physiology , Chronic Disease , Disease Models, Animal , Granulomatous Disease, Chronic/complications , Granulomatous Disease, Chronic/drug therapy , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Inflammation/complications , Inflammation/drug therapy , Inflammation/pathology , Interferon-gamma/immunology , Interferon-gamma/therapeutic use , Interleukin-17/deficiency , Interleukin-17/metabolism , Kynurenine/therapeutic use , Lung/immunology , Lung/pathology , Lung Diseases, Fungal/complications , Lung Diseases, Fungal/immunology , Lung Diseases, Fungal/microbiology , Lung Diseases, Fungal/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/deficiency , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism , Receptors, Antigen, T-Cell, gamma-delta/immunology , Superoxides/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
RATIONALE: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator affect the innate epithelial immune function of the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pathogenic fungi. The appreciation of whether such fungi are primarily responsible for or a consequence of ineffective airway inflammation is important for future therapeutics development. OBJECTIVES: To characterize the impact of the tryptophan/kynurenine pathway on pathogenic airway inflammation preventing effective fungal clearance in CF. METHODS: We studied the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the kynurenine pathway of tryptophan degradation, in human and murine CF, the impact of IDO on lung inflammation and immunity in murine CF, and the potential role of tryptophan catabolism in pathogenesis and therapy of fungus-associated lung inflammation. MEASUREMENTS AND MAIN RESULTS: IDO was defective in murine and human CF. Genetic and transcriptional regulatory mechanisms contributed to dysfunctional IDO activity that, in turn, correlated with imbalanced Th17/Treg-cell responses to Aspergillus fumigatus in murine CF. Treatments enhancing IDO function or preventing pathogenic Th17-cell activation restored protective immunity to the fungus and improved lung inflammation in murine CF. CONCLUSIONS: This study provides a link between tryptophan catabolism and lung immune homeostasis in murine CF, representing a proof-of-concept that targeting pathogenic inflammation via IDO-mimetic drugs may benefit patients with CF.
Subject(s)
Cystic Fibrosis/enzymology , Indoleamine-Pyrrole 2,3,-Dioxygenase/deficiency , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Kynurenine/metabolism , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/metabolism , Animals , Cystic Fibrosis/microbiology , Forkhead Transcription Factors/metabolism , Immunohistochemistry , In Situ Nick-End Labeling , Mice , Mice, Inbred C57BL , Up-Regulation/physiologyABSTRACT
Originally predicated on the recognition of an increasing prevalence of allergy, the hygiene hypothesis was later found to accommodate the contrasting epidemiologic trends in developed countries for infectious vs autoimmune diseases. Experimentally, reduced exposure to infections will increase the risk of disease in several models of experimental autoimmunity. Although TLRs were initially considered as stimulatory molecules capable of activating early defense mechanisms against invading pathogens, emerging data suggest that they can also exert a regulatory function. In the present study, we evaluated whether TLR3 and TLR9, recognizing microbial dsDNA and CpG-containing DNA sequences, respectively, play a role in the protection from experimental autoimmune diabetes induced in C57BL/6 mice by streptozotocin. In wild-type animals, the disease was accompanied by up-regulation of IDO in pancreatic lymph nodes and would be greatly exacerbated by in vivo administration of an IDO inhibitor. Conversely, administration of a CpG-containing oligodeoxynucleotide greatly attenuated the disease in an IDO-dependent fashion. TLR9-, but not TLR3-deficient mice developed a more robust disease, an event accompanied by lack of IDO induction in pancreatic lymph nodes. Thus, our data suggest that the TLR9-IDO axis may represent a valuable target in the prevention/therapy of type 1 diabetes.
Subject(s)
Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Type 1/immunology , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Insulin-Secreting Cells/immunology , Toll-Like Receptor 3/immunology , Toll-Like Receptor 9/immunology , Animals , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 1/enzymology , Diabetes Mellitus, Type 1/genetics , Female , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Insulin-Secreting Cells/enzymology , Interleukin-17/immunology , Interleukin-17/metabolism , Interleukin-6/immunology , Interleukin-6/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Oligodeoxyribonucleotides/pharmacology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolismABSTRACT
Despite their common ability to activate intracellular signaling through CD80/CD86 molecules, cytotoxic T lymphocyte antigen 4 (CTLA-4)-Ig and CD28-Ig bias the downstream response in opposite directions, the latter promoting immunity, and CTLA-4-Ig tolerance, in dendritic cells (DCs) with opposite but flexible programs of antigen presentation. Nevertheless, in the absence of suppressor of cytokine signaling 3 (SOCS3), CD28-Ig-and the associated, dominant IL-6 response-become immunosuppressive and mimic the effect of CTLA-4-Ig, including a high functional expression of the tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO). Here we show that forced SOCS3 expression antagonized CTLA-4-Ig activity in a proteasome-dependent fashion. Unrecognized by previous studies, IDO appeared to possess two tyrosine residues within two distinct putative immunoreceptor tyrosine-based inhibitory motifs, VPY(115)CEL and LLY(253)EGV. We found that SOCS3-known to interact with phosphotyrosine-containing peptides and be selectively induced by CD28-Ig/IL-6-would bind IDO and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. This event accounted for the ability of CD28-Ig and IL-6 to convert otherwise tolerogenic, IDO-competent DCs into immunogenic cells. Thus onset of immunity in response to antigen within an early inflammatory context requires that IDO be degraded in tolerogenic DCs. In addition to identifying SOCS3 as a candidate signature for mouse DC subsets programmed to direct immunity, this study demonstrates that IDO undergoes regulatory proteolysis in response to immunogenic stimuli.