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Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases., (© 2024. The Author(s).)

Background: Chronic systemic inflammation reduces the bioavailability of circulating endothelial progenitor cells (EPCs). Indoleamine 2,3-dioxygenase 1 (IDO1), a key enzyme of immune tolerance catalyzing the initial step of tryptophan degradation along the so-called l-kynurenine (l-kyn) pathway, that is induced by inflammatory stimuli and exerts anti-inflammatory effects. A specific relationship between IDO1 activity and circulating EPC numbers has not yet been investigated., Methods: In this study, circulating EPCs were examined in mice treated with low doses of lipopolysaccharide (LPS) to mimic low-grade inflammation. Moreover, the association between IDO1 activity and circulating EPCs was studied in a cohort of 277 patients with variable systemic low-grade inflammation., Results: Repeated low doses of LPS caused a decrease in circulating EPCs and l-kyn supplementation, mimicking IDO1 activation, significantly increased EPC numbers under homeostatic conditions preventing EPC decline in low-grade endotoxemia. Accordingly, in patients with variable systemic low-grade inflammation, there was a significant interaction between IDO1 activity and high-sensitivity C-reactive protein (hs-CRP) in predicting circulating EPCs, with high hs-CRP associated with significantly lower EPCs at low IDO1 activity but not at high IDO1 activity., Interpretation: Overall, these findings demonstrate that systemic low-grade inflammation reduces circulating EPCs. However, high IDO1 activity and l-kyn supplementation limit circulating EPC loss in low-grade inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mannarino, Bianconi, Scalisi, Franceschini, Manni, Cucci, Bagaglia, Mencarelli, Giglioni, Ricciuti, Figorilli, Pieroni, Cosentini, Padiglioni, Colangelo, Fuchs, Puccetti, Follenzi, Pirro, Gargaro and Fallarino.)

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene ( Ido1 WT ) or a mutated variant lacking the catalytic, but not signaling activity ( Ido1 H350A ). As compared to the Ido1 WT -transfected counterpart (B16 WT ), B16-F10 cells expressing Ido1 H350A (B16 H350A ) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16 H350A cells, also detectable in vivo , were found to be accompanied by a reduction in tumor-infiltrating CD8 + T cells and an increase in Foxp3 + regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule., Competing Interests: The authors report no conflict of interest., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The Aryl hydrocarbon receptor (AhR) is a critical regulator of both innate and adaptive immune responses, with potent immunomodulatory effects that makes this receptor an attractive molecular target for novel therapeutics. Accumulating evidence indicates that diverse-both host's and microbial-tryptophan metabolites profoundly regulate the immune system in the host via AhR, promoting either tolerance or immunity, largely as a function of the qualitative and quantitative nature of the metabolites being contributed by either source. Additional findings indicate that host and microbiota-derived tryptophan metabolic pathways can influence the outcome of immune responses to tumors. Here, we review recent studies on the role and modalities of AhR activation by various ligands, derived from either host-cell or microbial-cell tryptophan metabolic pathways, in the regulation of immune responses. Moreover, we highlight potential implications of those ligands and pathways in tumor immunotherapy, with particular relevance to checkpoint-blockade immune intervention strategies.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

L-Tryptophan (Trp) metabolism through the kynurenine pathway (KP) is involved in the regulation of immunity, neuronal function and intestinal homeostasis. Imbalances in Trp metabolism in disorders ranging from cancer to neurodegenerative disease have stimulated interest in therapeutically targeting the KP, particularly the main rate-limiting enzymes indoleamine-2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan-2,3-dioxygenase (TDO) as well as kynurenine monooxygenase (KMO). However, although small-molecule IDO1 inhibitors showed promise in early-stage cancer immunotherapy clinical trials, a phase III trial was negative. This Review summarizes the physiological and pathophysiological roles of Trp metabolism, highlighting the vast opportunities and challenges for drug development in multiple diseases.

A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.

Tumor immune escape mechanisms are being regarded as suitable targets for tumor therapy. Among these, tryptophan catabolism plays a central role in creating an immunosuppressive environment, leading to tolerance to potentially immunogenic tumor antigens. Tryptophan catabolism is initiated by either indoleamine 2,3-dioxygenase (IDO-1/-2) or tryptophan 2,3-dioxygenase 2 (TDO2), resulting in biostatic tryptophan starvation and l-kynurenine production, which participates in shaping the dynamic relationship of the host's immune system with tumor cells. Current immunotherapy strategies include blockade of IDO-1/-2 or TDO2, to restore efficient antitumor responses. Patients who might benefit from this approach are currently identified based on expression analyses of IDO-1/-2 or TDO2 in tumor tissue and/or enzymatic activity assessed by kynurenine/tryptophan ratios in the serum. We developed a monoclonal antibody targeting l-kynurenine as an in situ biomarker of IDO-1/-2 or TDO2 activity. Using Tissue Micro Array technology and immunostaining, colorectal and breast cancer patients were phenotyped based on l-kynurenine production. In colorectal cancer l-kynurenine was not unequivocally associated with IDO-1 expression, suggesting that the mere expression of tryptophan catabolic enzymes is not sufficiently informative for optimal immunotherapy.

Dendritic cells (DCs) are specialized antigen-presenting cells with a bipolar nature. Depending on environmental factors, DCs will promote either inflammatory or anti-inflammatory effects. Lipopolysaccharide (LPS), a ligand of Toll-like receptor (TLR)4 and a most potent proinflammatory stimulus, is responsible for complex signaling events in different cell types, including DCs. LPS effects range from protective inflammation-capable of counteracting growth and dissemination of gram-negative bacteria - to hyperacute detrimental responses, as it occurs in endotoxic shock. Consistent with the plasticity of TLR4 signaling, a low dosage of LPS will induce a regulatory response capable of protecting mice against a subsequent, otherwise lethal challenge ('endotoxin tolerance'). By examining CD11c(+) DCs ('conventional' DCs, or cDCs), we investigated whether DC flexibility in promoting either inflammation or tolerance can be differentially affected by single vs. repeated exposure to LPS in vitro. cDCs stimulated twice with LPS expressed high levels of indoleamine 2,3-dioxygenase 1 (IDO1) - one of the most effective mediator of anti-inflammatory activity by DCs - and of TGF-β, an immunoregulatory cytokine capable of upregulating IDO1 expression and function. In contrast, a single exposure to LPS failed to upregulate IDO1, and it was instead associated with high-level production of IL-6, a cytokine that promotes inflammation and proteolysis of IDO1. When adoptively transferred in vivo, only cDCs on double endotoxin exposure greatly improved the outcome of an otherwise lethal LPS challenge. The protective effect required that the transferred cDCs be fully competent for IDO1 and the host for TGF-β production. Thus cDCs, conditioned by LPS in vitro to mimic an endotoxin-tolerant state, can protect recipients from endotoxic shock, pointing to adoptive transfer of tolerance as a new option for controlling potentially harmful responses to TLR4 signaling., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The aryl hydrocarbon receptor (AhR) is a nuclear receptor regulating a wide range of biological and toxicological effects. Metabolites of L-tryptophan are able to bind and activate AhR, providing a link between tryptophan catabolism and a novel mechanism of protective tolerance, referred to as "disease tolerance". The notion that pharmacologic modulation of genes associated with endotoxin tolerance would be beneficial in clinical settings dominated by acute hyperinflammatory responses to infection thrusts AhR into the limelight as an interesting druggable target. Combining homology modeling, docking studies, and molecular dynamic simulations with mutagenesis experiments and gene profiling, in this work we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and two different L-tryptophan metabolites, namely L-Kynurenine and FICZ (6-formylindolo[3,2-b]carbazole), are able to bind to mAhR, exploiting different key interactions with distinct set of fingerprint residues. As a result, they stabilize different conformations of mAhR that, in turn, selectively regulate downstream signaling and transcription of specific target genes. Collectively, these results open new avenues for the design and development of selective AhR modulators that, by targeting specific receptor conformations associated with specific AhR functions, may offer novel therapeutic opportunities in infectious diseases and other morbidity that may be associated with the receptor.

Endogenous tryptophan (Trp) metabolites have an important role in mammalian gut immune homeostasis, yet the potential contribution of Trp metabolites from resident microbiota has never been addressed experimentally. Here, we describe a metabolic pathway whereby Trp metabolites from the microbiota balance mucosal reactivity in mice. Switching from sugar to Trp as an energy source (e.g., under conditions of unrestricted Trp availability), highly adaptive lactobacilli are expanded and produce an aryl hydrocarbon receptor (AhR) ligand-indole-3-aldehyde-that contributes to AhR-dependent Il22 transcription. The resulting IL-22-dependent balanced mucosal response allows for survival of mixed microbial communities yet provides colonization resistance to the fungus Candida albicans and mucosal protection from inflammation. Thus, the microbiota-AhR axis might represent an important strategy pursued by coevolutive commensalism for fine tuning host mucosal reactivity contingent on Trp catabolism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Control of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile regulator of innate and adaptive immune responses. In acute reactions, the otherwise inflammatory cytokine interferon γ (IFN-γ) acts in a feedback fashion to induce IDO's enzymatic function--and thus prevent potentially harmful, exaggerated responses--through the combined effects of tryptophan starvation and tryptophan catabolites acting via the aryl hydrocarbon receptor of T cells. IDO, however, is also involved in the maintenance of stable tolerance to self in noninflammatory contexts, thus restraining autoimmunity. Exposure, indeed, of mouse plasmacytoid DCs (pDCs) to transforming growth factor β (TGF-β) provides IDO with regulatory effects that are distinct, in nature, from its enzymic activity. Once phosphorylated, IDO mediates signaling events culminating in self-amplification and maintenance of a stably regulatory condition in pDCs. Therefore, IDO has dual immunoregulatory functions driven by distinct cytokines. Firstly, the IFN-γ-IDO axis is crucial in generating and sustaining the function of regulatory T cells. Secondly, a nonenzymic function of IDO--as a signaling molecule--contributes to TGF-β-driven tolerance. The latter function is part of a regulatory circuit in pDCs whereby--in response to TGF-β--the kinase Fyn mediates tyrosine phosphorylation of IDO-associated immunoreceptor tyrosine-based inhibitory motifs, resulting in downstream effects that regulate gene expression and preside over a proper, homeostatic balance between immunity and tolerance. All these aspects are covered in this review., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.

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.

Plasmacytoid dendritic cells (pDCs) represent a specialized cell population that produces large amounts of type I interferons. Recently, murine and human pDCs have been credited with a unique ability to express the inducible, tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO) as an effector means of regulatory T cell function and thus mediate immunosuppression in specific settings. Here we review and further clarify specific features of the relative efficacy and mechanisms of IDO induction in pDCs after 'ligand' engagement by the soluble forms of molecules present on regulatory T cell surface ('reverse signaling'). We found that splenic pDCs do not express IDO and are not tolerogenic under steady-state conditions. B7 engagement by the two different ligands CTLA-4-Ig and CD28-Ig resulted in opposing outcomes on IDO function-i.e., activation by the former (through autocrine IFN-gamma) and inhibition by the latter (via autocrine IL-6 and SOCS3). In addition, engagement of GITR ligand by GITR-Ig in pDCs also resulted in IDO activity via type I IFNs and noncanonical NF-kappaB signaling. Therefore, although pDCs do not express IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by those cells.

Tryptophan catabolism occurring in dendritic cells (DCs) and initiated by indoleamine 2,3-dioxygenase (IDO) is an emerging major mechanism of peripheral tolerance. Here we provide evidence that: 1) tryptophan conversion to kynurenines is activated in DCs by cytotoxic T lymphocyte antigen 4, both in a soluble form or anchored to the regulatory T cell (Treg) membrane; 2) an increased IDO-dependent tolerogenesis correlates with the inhibition of DAP12 functions, an adapter molecule associated with activating receptors; 3) a tolerogenic phenotype can be acquired by DCs lacking functional IDO through the paracrine production of kynurenines by IDO-competent DCs; 4) the suppressive effect of Treg generated in a microenvironment with low tryptophan concentration and a mixture of kynurenines can protect mice in an experimental model of fulminant diabetes. Altogether, these data indicate that, in addition to tryptophan starvation induced by IDO activity, the paracrine production of kynurenines by enzymes downstream of IDO can also contribute to tolerogenesis in DCs, independently of tryptophan deprivation.

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that its effects include the emergence of a regulatory phenotype in naive CD4(+)CD25(-) cells via the general control non-depressing 2 (GCN2) protein kinase mediated induction of the forkhead transcription factor Foxp3. These cells are capable of effective control of diabetogenic T cells in vivo.

Thymosin alpha1 (Talpha1), a naturally occurring thymic peptide, primes dendritic cells (DCs) for antifungal T-helper type 1 resistance through Toll-like receptor 9 (TLR9) signaling. As TLR9 signaling also activates the immuno-suppressive pathway of tryptophan catabolism via indoleamine 2,3-dioxygenase (IDO), we examined Talpha1 for possible induction of DC-dependent regulatory effects. Talpha1 affected T-helper cell priming and tolerance induction by human and murine DCs and induced IDO expression and function in the latter cells. IDO activation by Talpha1 required TLR9 and type I interferon receptor signaling and resulted in interleukin-10 production and generation of regulatory T cells. In transfer experiments, functionally distinct subsets of differentiated DCs were required for priming and tolerance to a fungal pathogen or alloantigens. In contrast, Talpha1-primed DCs fulfilled multiple requirements, including the induction of T-helper type 1 immunity within a regulatory environment. Thus, instructive immunotherapy with Talpha1 targeting IDO-competent DCs could allow for a balanced control of inflammation and tolerance.

Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that the short-term, combined effects of tryptophan deprivation and tryptophan catabolites result in GCN2 kinase-dependent down-regulation of the TCR zeta-chain in murine CD8+ T cells. TCR zeta down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25- T cells via TGF-beta induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69-, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN-gamma produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-beta, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germinating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

A series of recent studies, including an article in this issue of the European Journal of Immunology, have demonstrated that the administration of CpG-rich oligodeoxynucleotides (CpG-ODN) in experimental settings may lead to the activation of the immunosuppressive pathway of tryptophan catabolism, depending on several factors, including the route of CpG-ODN administration. These studies call attention to the need for a careful evaluation of the modalities of inclusion of CpG-ODN in vaccines for human use. At the same time, these studies may offer novel opportunities for use of CpG-ODN as immunosuppressive agents and may also lead to an improved understanding of the cellular events mediated by Toll-like receptor 9 signaling.

DAP12 is an immunoreceptor tyrosine-based activation motif-bearing membrane adapter molecule expressed by different cell types. Although several receptors associate with DAP12 in murine dendritic cells (DC), the function of these receptors is as yet unknown. Here we report that splenic mature DC with DAP12 overexpression are characterized by an impaired tolerogenic potential. In contrast, inhibition of DAP12 function results in enhanced tolerogenesis and constitutive expression of immunosuppressive tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO). Increased resistance to experimental encephalomyelitis is observed in DAP12 knockin mice, which is dependent on IDO expression. Therefore, DAP12-related receptors act as negative regulators of IDO-mediated tolerance in vivo.

By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-gamma- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-gamma may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.

In this study, using a soluble CD200-Ig fusion protein, we provide evidence that murine dendritic cells (DCs) possess a functional CD200R, whose engagement results in the reinforcement or appearance of immunosuppressive properties in these cells. In particular, the plasmacytoid subset (CD11c+B220+120G8+) of splenic DCs (pDCs) is induced by CD200-Ig to express the enzyme IDO, which initiates the tolerogenic pathway of tryptophan catabolism. As a result, pDCs are capable of suppressing Ag-specific responses in vivo when transferred into recipient hosts after treatment with CD200-Ig. IDO induction in pDCs through CD200R engagement requires type I IFNR signaling. Although the release of IFN-alpha may contribute to the full expression of CD200-Ig activity, autocrine IFN-alpha is unlikely to mediate alone the effects of CD200R engagement. These data prospect novel functions for both pDCs and the CD200-CD200R pair in the mouse. At the same time, these data underscore the possible unifying role of the IDO mechanism in immune tolerance., (Copyright 2004 The American Association of Immunologists, Inc.)

Regulatory T (T(R)) cells manifest constitutive expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), but the function of CTLA-4 in mediating the regulatory function of T(R) cells is unclear. We show here that mouse CD4+CD25+ cells, either resting or induced to overexpress CTLA-4 by treatment with antibody to CD3, initiated tryptophan catabolism in dendritic cells through a CTLA-4-dependent mechanism. This process required B7 expression and cytokine production by the dendritic cells. In contrast, T(R) cells cultured in the presence of bacterial lipopolysaccharide induced tryptophan catabolism by dendritic cells in a CTLA-4-independent but cytokine-dependent way. Thus, regulation of immunosuppressive tryptophan catabolism in dendritic cells might represent a major mechanism of action of T(R) cells.

The predisposition of nonobese diabetic (NOD) mice to develop autoimmunity reflects deficiencies in both peripheral and central tolerance. Several defects have been described in these mice, among which aberrant antigen-presenting cell function and peroxynitrite formation. Prediabetes and diabetes in NOD mice have been targeted with different outcomes by a variety of immunotherapies, including interferon (IFN)-gamma. This cytokine may be instrumental in specific forms of tolerance by virtue of its ability to activate immunosuppressive tryptophan catabolism. Here, we provide evidence that IFN-gamma fails to induce tolerizing properties in dendritic cells from highly susceptible female mice early in prediabetes. This effect is associated with impaired tryptophan catabolism, is related to transient blockade of the Stat1 pathway of intracellular signaling by IFN-gamma, and is caused by peroxynitrite production. However, the use of a peroxynitrite inhibitor can rescue tryptophan catabolism and tolerance in those mice. This is the first report of an experimental autoimmune disease in which defective tolerance is causally linked to impaired tryptophan catabolism.

Autoimmune diseases including insulin-dependent diabetes mellitus (IDDM) are characterized by the loss of tolerance to self determinants, activation of autoreactive lymphocytes, and subsequent damage to target organs. Recent evidence suggests that the development of autoimmune diabetes in the nonobese diabetic mouse (NOD), an animal model of IDDM, is under the control of dendritic cells. The potent antigen-presenting capacity of dendritic cells can be strongly influenced by the cell maturation state and by the cytokine milieu, and in fact these cells may acquire disparate functional abilities, from immunity to tolerance. We have previously demonstrated that, in the DBA/2 mouse, IFN-gamma potentiates the tolerogenic potential of a subset of splenic dendritic cells via activation of the enzyme indoleamine 2,3-dioxygenase (IDO) and production of tryptophan catabolites capable of inducing apoptosis in T cells. In the present study, we wanted to examine whether dendritic cells from NOD mice could be subjected to regulation by proinflammatory cytokines in the same fashion as in conventional mice. We found that IFN-gamma does not potentiate the tolerogenic effects of dendritic cells from NOD mice at four weeks of age. This finding correlates with a low expression of IDO activity, thus suggesting that poor expression of IDO by dendritic cells may play a role in the development of diabetes.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) plays a critical role in peripheral tolerance. However, regulatory pathways initiated by the interactions of CTLA-4 with B7 counterligands expressed on antigen-presenting cells are not completely understood. We show here that long-term survival of pancreatic islet allografts induced by the soluble fusion protein CTLA-4-immunoglobulin (CTLA-4-Ig) is contingent upon effective tryptophan catabolism in the host. In vitro, we show that CTLA-4-Ig regulates cytokine-dependent tryptophan catabolism in B7-expressing dendritic cells. These data suggest that modulation of tryptophan catabolism is a means by which CTLA-4 functions in vivo and that CTLA-4 acts as a ligand for B7 receptor molecules that transduce intracellular signals.

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that, expressed by different cell types, has regulatory effects on T cells resulting from tryptophan depletion in specific local tissue microenvironments. Different mechanisms, however, might contribute to IDO-dependent immune regulation. We show here that tryptophan metabolites in the kynurenine pathway, such as 3-hydroxyanthranilic and quinolinic acids, will induce the selective apoptosis in vitro of murine thymocytes and of Th1 but not Th2 cells. T cell apoptosis was observed at relatively low concentrations of kynurenines, did not require Fas/Fas ligand interactions, and was associated with the activation of caspase-8 and the release of cytochrome c from mitochondria. When administered in vivo, the two kynurenines caused depletion of specific thymocyte subsets in a fashion qualitatively similar to dexamethasone. These data suggest that the selective deletion of T lymphocytes may be a major mechanism whereby tryptophan metabolism affects immunity under physiopathologic conditions.