Indoleamine 2, 3-dioxygenase （IDO） is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.     

IL-17 and therapeutic kynurenines in pathogenic inflammation to fungi

Largely viewed as proinflammatory, innate responses combine with adaptive immunity to generate the most effective form of antifungal resistance, and T cells exercise feedback control over diverse effects of inflammation on infection. Some degree of inflammation is required for protection, particularly in mucosal tissues, during the transitional response occurring between the rapid innate and slower adaptive response. However, progressive inflammation worsens disease and ultimately prevents pathogen eradication. IDO, tryptophan catabolites ("kynurenines"), and regulatory T cells help to tame overzealous and exaggerated inflammatory responses. In this context, IL-23 and the Th17 pathway, which down-regulate tryptophan catabolism, may instead favor pathology and serve to accommodate the seemingly paradoxical association of chronic inflammation with fungal persistence. Recent data support a view in which IL-23/IL-17 antagonistic strategies, including the administration of synthetic kynurenines, could represent a new means of harnessing progressive or potentially harmful inflammation.     

Indoleamine 2,3 dioxygenase and regulation of T cell immunity

Regulation of adaptive immune responses is critically important to allow the adaptive immune system to eradicate infections while causing minimal collateral damage to infected tissues, as well as preventing autoimmune disease mediated by self-reactive lymphocytes. Tumors and pathogens that cause persistent infections can subvert immunoregulatory processes to protect themselves from destruction by T cells, to the detriment of patients. A growing body of evidence supports the hypothesis that specialized subsets of dendritic cells expressing indoleamine 2,3 dioxygenase (IDO), which catalyzes oxidative catabolism of tryptophan, play critical roles in regulation of T cell-mediated immune responses. IDO-dependent T cell suppression by dendritic cells suggests that biochemical changes due to tryptophan catabolism have profound effects on T cell proliferation, differentiation, effector functions, and viability. This has critical implications for immunotherapeutic manipulations designed for patients with cancer and chronic infectious diseases. In this review, I focus on dendritic cells that can express IDO, and which acquire potent T cell regulatory functions as a consequence.     

Blockade of IDO inhibits nasal tolerance induction

The amino acid tryptophan is essential for the proliferation and survival of cells. Modulation of tryptophan metabolism has been described as an important regulatory mechanism for the control of immune responses. The enzyme IDO degrades the indole moiety of tryptophan, not only depleting tryptophan but also producing immunomodulatory metabolites called kynurenines, which have apoptosis-inducing capabilities. In this study, we show that IDO is more highly expressed in nonplasmacytoid dendritic cells of the nose draining lymph nodes (LNs), which form a unique environment to induce tolerance to inhaled Ags, when compared with other peripheral LNs. Upon blockade of IDO during intranasal OVA administration, Ag-specific immune tolerance was abrogated. Analysis of Ag-specific T cells in the LNs revealed that inhibition of IDO resulted in enhanced survival at 48 h after antigenic stimulation, although this result was not mediated through alterations in apoptosis or cell proliferation. Furthermore, no differences were found in CD4(+) T cells expressing FoxP3. Our data suggest that the level of IDO expression in dendritic cells, present in nose draining LNs, allows for the generation of a sufficient number of regulatory T cells to control and balance effector T cells in such a way that immune tolerance is induced, whereas upon IDO blockade, effector T cells will outnumber regulatory T cells, leading to immunity.     

Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice

Indoleamine 2,3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. In cultured cells, the induction of IDO leads to depletion of tryptophan and tryptophan starvation. Recent studies suggest that modulation of tryptophan concentration via IDO plays a fundamental role in innate immune responses. Induction of IDO by interferon-γ in macrophages and dendritic cells results in tryptophan depletion and suppresses the immune-mediated activation of fibroblasts and T, B, and natural killer cells. To assess the role of IDO in collagen-induced arthritis (CIA), a model of rheumatoid arthritis characterized by a primarily Th1-like immune response, activity of IDO was inhibited by 1-methyl-tryptophan (1-MT) in vivo. The results showed significantly increased incidence and severity of CIA in mice treated with 1-MT. Activity of IDO, as determined by measuring the levels of kynurenine/tryptophan ratio in the sera, was increased in the acute phase of arthritis and was higher in collagen-immunized mice that did not develop arthritis. Treatment with 1-MT resulted in an enhanced cellular and humoral immune response and a more dominant polarization to Th1 in mice with arthritis compared with vehicle-treated arthritic mice. The results demonstrated that development of CIA was associated with increased IDO activity and enhanced tryptophan catabolism in mice. Blocking IDO with 1-MT aggravated the severity of arthritis and enhanced the immune responses. These findings suggest that IDO may play an important and novel role in the negative feedback of CIA and possibly in the pathogenesis of rheumatoid arthritis.     

Deficient tryptophan catabolism along the kynurenine pathway reveals that the epididymis is in a unique tolerogenic state

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1(-/-)-deficient mice. In the caput epididymis of Ido1(-/-) animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.     

Psychological Stress-Induced,IDO1-Dependent Tryptophan Catabolism: Implications on Immunosuppression in Mice and Humans

It is increasingly recognized that psychological stress influences inflammatory responses and mood. Here, we investigated whether psychological stress (combined acoustic and restraint stress) activates the tryptophan (Trp) catabolizing enzyme indoleamine 2,3-dioxygenase 1(IDO1) and thereby alters the immune homeostasis and behavior in mice. We measured IDO1 mRNA expression and plasma levels of Trp catabolites after a single 2-h stress session and in repeatedly stressed (4.5-days stress, 2-h twice a day) naïve BALB/c mice. A role of cytokines in acute stress-induced IDO1 activation was studied after IFNγ and TNFα blockade and in IDO1^−/− mice. RU486 and 1-Methyl-L-tryptophan (1-MT) were used to study role of glucocorticoids and IDO1 on Trp depletion in altering the immune and behavioral response in repeatedly stressed animals. Clinical relevance was addressed by analyzing IDO1 activity in patients expecting abdominal surgery. Acute stress increased the IDO1 mRNA expression in brain, lung, spleen and Peyer''s patches (max. 14.1±4.9-fold in brain 6-h after stress) and resulted in a transient depletion of Trp (−25.2±6.6%) and serotonin (−27.3±4.6%) from the plasma measured 6-h after stress while kynurenine levels increased 6-h later (11.2±9.3%). IDO1 mRNA up-regulation was blocked by anti-TNFα and anti-IFNγ treatment. Continuous IDO1 blockade by 1-MT but not RU486 treatment normalized the anti-bacterial defense and attenuated increased IL-10 inducibility in splenocytes after repeated stress as it reduced the loss of body weight and behavioral alterations. Moreover, kynurenic acid which remained increased in 1-MT treated repeatedly stressed mice was identified to reduce the TNFα inducibility of splenocytes in vitro and in vivo. Thus, psychological stress stimulates cytokine-driven IDO1 activation and Trp depletion which seems to have a central role for developing stress-induced immunosuppression and behavioral alteration. Since patients showed Trp catabolism already prior to surgery, IDO is also a possible target enzyme for humans modulating immune homeostasis and mood.     

Apicomplexan parasite, Eimeria falciformis, co-opts host tryptophan catabolism for life cycle progression in mouse

The obligate intracellular apicomplexan parasites, e.g. Toxoplasma gondii and Plasmodium species, induce an IFNγ-driven induction of host indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of tryptophan catabolism in the kynurenine pathway. Induction of IDO1 supposedly depletes cellular levels of tryptophan in host cells, which is proposed to inhibit the in vitro growth of auxotrophic pathogens. In vivo function of IDO during infections, however, is not clear, let alone controversial. We show that Eimeria falciformis, an apicomplexan parasite infecting the mouse caecum, induces IDO1 in the epithelial cells of the organ, and the enzyme expression coincides with the parasite development. The absence or inhibition of IDO1/2 and of two downstream enzymes in infected animals is detrimental to the Eimeria growth. The reduced parasite yield is not due to a lack of an immunosuppressive effect of IDO1 in the parasitized IDO1(-/-) or inhibitor-treated mice because they did not show an accentuated Th1 and IFNγ response. Noticeably, the parasite development is entirely rescued by xanthurenic acid, a by-product of tryptophan catabolism inducing exflagellation in male gametes of Plasmodium in the mosquito mid-gut. Our data demonstrate a conceptual subversion of the host defense (IFNγ, IDO) by an intracellular pathogen for progression of its natural life cycle. Besides, we show utility of E. falciformis, a monoxenous parasite of a well appreciated host, i.e. mouse, to identify in vivo factors underlying the parasite-host interactions.     

Foxp3+CD25+ T regulatory cells stimulate IFN-gamma-independent CD152-mediated activation of tryptophan catabolism that provides dendritic cells with immune regulatory activity in mice unresponsive to staphylococcal enterotoxin B

Mice made unresponsive by repeated injection of staphylococcal enterotoxin B (SEB) contained SEB-specific CD25(+)CD4(+)TCRBV8(+) T cells that were able to transfer their state of unresponsiveness to primary-stimulated T cells. About one-half of these cells stably up-regulated the expression of CD152. We undertook the present study to determine whether CD152(high) cells seen in this system were T regulatory cells responsible for suppression or whether they represented SEB-activated CD4(+) T effector cells. Our results show that, among SEB-specific TCRBV8(+) T cells isolated from unresponsive mice, all CD152(high)CD25(+)CD4(+) T cells expressed Foxp3, the NF required for differentiation and function of natural T regulatory cells. Moreover, suppression by CD25(+)CD4(+)TCRBV8(+) T cells was fully inhibited by anti-CD152 Abs. Following stimulation by soluble CD152-Ig, dendritic cells (DC) isolated from unresponsive mice strongly increased the expression and the function of indoleamine-2,3-dioxygenase (IDO), the enzyme responsible for the catabolism of tryptophan. This capacity to activate IDO was independent of IFN-gamma production by DC because CD152-Ig stimulation of DC isolated from SEB-treated IFN-gamma-deficient animals activated IDO expression and function. Finally, adding 1-methyl-tryptophan, an inhibitor of tryptophan catabolism, increased substantially the capacity of DC from unresponsive animals to stimulate primary T cell response toward SEB. Thus, we conclude that IFN-gamma-independent CD152-mediated activation of tryptophan catabolism by Foxp3(+)CD25(+) T regulatory cells provides DC with immune regulatory activity in mice unresponsive to SEB.     

Indoleamine 2,3-Dioxygenase Controls Fungal Loads and Immunity in Paracoccidioidomicosis but is More Important to Susceptible than Resistant Hosts

Background

Paracoccidioidomycosis, a primary fungal infection restricted to Latin America, is acquired by inhalation of fungal particles. The immunoregulatory mechanisms that control the severe and mild forms of paracoccidioidomycosis are still unclear. Indoleamine 2,3-dioxygenase (IDO), an IFN-γ induced enzyme that catalyzes tryptophan metabolism, can control host-pathogen interaction by inhibiting pathogen growth, T cell immunity and tissue inflammation.

Methodology/Principal Findings

In this study, we investigated the role of IDO in pulmonary paracoccidioidomycosis of susceptible and resistant mice. IDO was blocked by 1-methyl-dl-tryptophan (1MT), and fungal infection studied in vitro and in vivo. Paracoccidioides brasiliensis infection was more severe in 1MT treated than untreated macrophages of resistant and susceptible mice, concurrently with decreased production of kynurenines and IDO mRNA. Similar results were observed in the pulmonary infection. Independent of the host genetic pattern, IDO inhibition reduced fungal clearance but enhanced T cell immunity. The early IDO inhibition resulted in increased differentiation of dendritic and Th17 cells, accompanied by reduced responses of Th1 and Treg cells. Despite these equivalent biological effects, only in susceptible mice the temporary IDO blockade caused sustained fungal growth, increased tissue pathology and mortality rates. In contrast, resistant mice were able to recover the transitory IDO blockade by the late control of fungal burdens without enhanced tissue pathology.

Conclusions/Significance

Our studies demonstrate for the first time that in pulmonary paracoccidioidomycosis, IDO is an important immunoregulatory enzyme that promotes fungal clearance and inhibits T cell immunity and inflammation, with prominent importance to susceptible hosts. In fact, only in the susceptible background IDO inhibition resulted in uncontrolled tissue pathology and mortality rates. Our findings open new perspectives to understand the immunopathology of paracoccidioidomycosis, and suggest that an insufficient IDO activity could be associated with the severe cases of human PCM characterized by inefficient fungal clearance and excessive inflammation.     

Regulation of indoleamine 2,3-dioxygenase expression in simian immunodeficiency virus-infected monkey brains

The human immunodeficiency virus type 1-associated cognitive-motor disorder, including the AIDS dementia complex, is characterized by brain functional abnormalities that are associated with injury initiated by viral infection of the brain. Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in tryptophan catabolism in extrahepatic tissues, can lead to neurotoxicity through the generation of quinolinic acid and immunosuppression and can alter brain chemistry via depletion of tryptophan. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we demonstrate that cells of the macrophage lineage are the main source for expression of IDO in the SIV-infected monkey brain. Animals with SIV encephalitis have the highest levels of IDO mRNA, and the level of IDO correlates with gamma interferon (IFN-gamma) and viral load levels. In vitro studies on mouse microglia reveal that IFN-gamma is the primary inducer of IDO expression. These findings demonstrate the link between IDO expression, IFN-gamma levels, and brain pathology signs observed in neuro-AIDS.     

Longitudinal study of tryptophan degradation during and after pregnancy

In mice, activation of indoleamine-2,3-dioxygenase (IDO), an enzyme converting tryptophan to N-formyl-kynurenine, was found to be necessary requirement to achieve immunotolerance against the fetus and thus uncomplicated pregnancy. In plasma from 20 healthy pregnant women with singleton pregnancies we consecutively analyzed kynurenine and tryptophan concentrations during pregnancy (1 specimen at each trimester of gestation) and postpartum (week 6). None of the women had any signs of infection at the time of plasma sampling, but the study population was otherwise unselected. The kynurenine to tryptophan ratio (kyn/trp) was calculated as an estimate of IDO activity, and data were compared to concentrations of neopterin and 55kD soluble tumor necrosis factor receptor (sTNF-R55), two indicators of immune activation, and to alanineaminotransferase (ALT) levels. Increasing kynurenine and decreasing tryptophan concentrations were found during pregnancy, data suggesting significant degradation of tryptophan. In parallel, increasing concentrations of immune activation markers neopterin and sTNF-R55 were observed, correlating significantly to kyn/trp. The data point to an involvement of cytokine-induced IDO activation in the degradation of tryptophan observed during pregnancy. After pregnancy, sTNF-R55 and also neopterin concentrations declined, whereas tryptophan concentrations increased, indicating that immune activation and activation-induced tryptophan degradation returned to baseline. By contrast, still increased kynurenine concentrations and also increased kyn/trp point to continuing catabolism of tryptophan. Postpartum elevation of liver enzyme ALT may suggest that increased activity of hepatic tryptophan pyrrolase could be involved in increased conversion of tryptophan despite low degree of immune activation. We conclude that IDO is activated in pregnancy and that the decrease of tryptophan might be related to immune activation phenomena. Sustained increase of kynurenine postpartum seems independent from immune activation process.     

Different partners, opposite outcomes: a new perspective of the immunobiology of indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO), a metabolic enzyme that catalyzes tryptophan conversion into kynurenines, is a crucial regulator of immunity. Altered IDO activity is often associated with pathology, including neoplasia and autoimmunity. IDO is highly expressed in dendritic cells (DCs) that exploit the enzyme's activity and the production of tryptophan catabolites to regulate immune responses by acting on several cell types, including T lymphocytes, of which they promote a regulatory phenotype. IDO also contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that, once bound by distinct molecular partners, will either promote degradation or initiate signaling activity and self-maintenance of the enzyme. We here discuss how ITIM-dependent molecular events can affect the functional plasticity of IDO by modifying the protein half-life and its enzymic and nonenzymic functions.     

Physiologic control of IDO competence in splenic dendritic cells

Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.     

Suppression of cytotoxic and proliferative xenogeneic T-cell responses by transgenic expression of indoleamine 2,3-dioxygenase

Tryptophan catabolism initiated by the enzyme indoleamine 2,3-dioxygenase (IDO) has been postulated to be a natural regulatory mechanism for T cells. In this study, we generated a pig endothelial cell line expressing full-length human IDO (P-HuIDO) to serve as a simple model of a cellular xenogeneic graft. Splenocytes from mice primed to P-HuIDO cells were found to be as responsive to secondary stimulation as splenocytes from mice primed to parental cells. However, in T-cell proliferation assays using P-HuIDO cells as stimulators, a significant inhibition of both naive and memory xenogeneic proliferative responses was noted. Furthermore, the production of interferon-gamma and cytotoxic T lymphocyte function were also affected. When severe combined immunodeficiency mice were grafted with P-HuIDO cells, then challenged with primed splenocytes from BALB/c mice, cellular infiltration to the graft was delayed. Our findings suggest that transgenic expression of IDO in xenografts contributes to prolonged graft survival.     

Suppression of memory CD8 T cell generation and function by tryptophan catabolism

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of T(CM) cells or T(CM) cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, T(CM) cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of T(EM) cells or T(EM) cell-mediated allograft rejection, suggesting that T(EM) cells, unlike T(CM) cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.     

FcepsilonRI induces the tryptophan degradation pathway involved in regulating T cell responses

FcepsilonRI is suspected to play a pivotal role in the pathophysiology of atopic disorders such as atopic dermatitis. In search for genes differentially regulated by FcepsilonRI on APCs, a differential cDNA bank of receptor-stimulated and unstimulated monocytes was established. By means of suppression subtractive hybridization, we identified kynurenine 3-monooxygenase and subsequently indoleamine 2,3-dioxygenase (IDO) to be overexpressed in FcepsilonRI-activated monocytes. IDO is the rate-limiting enzyme in the catabolism of the essential amino acid tryptophan. We show that cross-linking of FcepsilonRI on monocytes results in low tryptophan concentrations associated with impaired T cell stimulatory capacity. Importantly, T cell suppression could be prevented by the addition of tryptophan or inhibition of IDO. Moreover, stimulation of T cells by FcepsilonRI-activated monocytes was increased compared with T cell stimulation by nonactivated monocytes if exogenous supply of tryptophan was available. We speculate that the expression of IDO by FcepsilonRI(+) APCs in vivo allows these cells to regulate T cell responses in atopic disorders by inhibiting or stimulating T cell proliferation, depending on the metabolic environment.