Intracellular Indoleamine 2,3-dioxygenase Research Articles

Developing a flow cytometry-based quantitative ido assay to measure immune potency of mesenchymal stromal cells product for phase i clinical trial

Background & Aim Establishment of a potency assay in the manufacturing of clinical grade mesenchymal stromal cells (MSCs) has been a challenge due to the issues of variability in starting cellular material, complex mechanisms of action, and lack of reference cells or materials. Indoleamine 2, 3-dioxygenase (IDO) is one of the most important factors for human MSC-mediated immunosuppression. IDO is expressed in IFN-γ-licensed MSC, but not in resting MSC in vitro. This intracellular enzyme catalyzes tryptophan degradation toward kynurenine and depletion of tryptophan suppresses T cells proliferation. Methods, Results & Conclusion In this study, we have developed a flow cytometry-based IDO assay for quantitatively measuring the IDO expression level per cell, the IPBV (Intracellular Protein Binding Value) of the cells represents the relative quantity of IDO expressed in the MSC. IPBV is defined as the number of anti-IDO fluorescent antibody molecules bound to intracellular IDO and standardized by fluorescence intensity of beads. Two kinds of in-house quality control reference cells were running in parallel with product to ensure that the assay is performing as expected and the level of IDO expression. Validation of the quantitative IDO assay, including accuracy, precision, specificity, linearity, range and robustness, were presented in this study. Moreover, we presented the application of the quantitative IDO assay for analysis of MSC product stability, comparability, and compatibility in the manufacturing of clinical grade MSC-based product- RegStem® ASC. In this study, we demonstrated the flow cytometry-based quantitative MSC IDO assay correlates with MSC's inhibition of autologous T cells proliferation, as well as the correlation with preliminary clinical effects in the phase I autologous MSC-based clinical trial for the treatment of Osteoarthritis. As the results, we believe that our novel flow cytometry-based quantitative IDO assay can be a potential potency assay candidate for advanced phase clinical trial of MSCs products.

The role of B7 family costimulatory molecules and indoleamine 2,3-dioxygenase in primary Sjögren's syndrome and systemic sclerosis.

B7 costimulatory molecules are present on antigen-presenting cells (APCs) and influence intracellular expression of indoleamine 2,3-dioxygenase (IDO), a molecule with important immunoregulatory functions. We determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2 molecules, and that of CD3+ and CD4+ T cells expressing the corresponding CD28, CTLA-4, PD-1, and ICOS receptors in peripheral blood samples of 20 healthy adults and 9 SSc and 15 pSS patients using flow cytometry. We also examined the intracellular expression of IDO. The expression of CD28 was lower in both SSc and pSS patients. The frequency of CTLA-4 was increased in pSS. The expression of ICOS, a stimulator of T cell activation, was elevated in pSS, but not in SSc, while that of its corresponding costimulatory molecule, B7-H2, was strongly decreased in SSc compared to controls. The frequency of PD-1 expressing T lymphocytes was decreased in both pSS and SSc. The frequency of IDO-expressing APCs, as well as intracellular IDO content in T cells was higher in pSS than in controls. Our investigation identified a number of differences in B7 costimulation between SSc and pSS patients which may play a role in the distinct pathogenesis and clinical features of these autoimmune disorders.

O75. B7 Costimulation and intracellular indoleamine-2,3-dioxygenase expression in peripheral blood of healthy pregnant and preeclamptic women

Introduction B7 costimulatory molecules are expressed on antigen presenting cells (APCs) and are important regulators of T cell activation. We investigated their role in the development of the systemic maternal immune tolerance during healthy pregnancy (HP) and preeclampsia (PE). Objectives We also aimed to investigate the intracellular expression of indoleamine-2,3-dioxygenase (IDO) and plasma levels of tryptophane (TRP), kynurenine (KYN) and kynurenic acid (KYNA), important molecules with immunoregulatory properties, in order to describe their potential contribution to the pregnancy-specific maternal immune tolerance. Methods We determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2, and that of T cells and CD4+ T helper cells expressing CD28, CTLA-4, PD-1, and ICOS in peripheral blood samples of 20 PE, 20 HP and 14 non-pregnant (NP) women using flow cytometry. We also examined the intracellular expression of IDO applying flow cytometry and plasma levels of TRP, KYN and KYNA using high-performance liquid chromatography. Results A significant increase in the prevalence of CD28+ T cells was observed in HP compared to NP women. At the same time a decrease was shown in the expression of CTLA-4 on these cells. The frequency of B7-1 and B7-2 expressing monocytes and that of IDO expressing T lymphocytes was lower in PE than in HP. The prevalence of IDO-expressing T cells and monocytes was higher in HP compared to NP women. Plasma KYN, KYNA and TRP levels were lower, while at the same time, the KYN/TRP ratio was higher in HP than in NP women. We found a positive correlation between the expression of B7-2 and IDO within activated monocytes. Conclusions Costimulation via CD28 may not contribute to the immunosuppressive environment, at least in the third trimester of pregnancy. The development of the pregnancy-specific immune tolerance in the mechanism of B7 costimulation may be more related to the altered expression of B7 proteins on APCs rather than that of their receptors on T cells. The elevated intracellular IDO expression in monocytes and T cells, as well as higher plasma enzymatic IDO activity are likely to contribute to the systemic immunosuppressive environment in the third trimester characteristic for healthy gestation. Lower expression of B7-1 and B7-2 proteins on peripheral monocytes in PE might indicate a secondary regulatory mechanism in response to the ongoing systemic maternal inflammation. Immunosuppression by IDO may play an important role in the pregnancy-specific immune tolerance, and might be a contributing factor in the pathogenesis of PE.

A Phase II Study to Determine the Safety and Efficacy of the Oral Inhibitor of Indoleamine 2,3-Dioxygenase (IDO) Enzyme INCB024360 in Patients with Myelodysplastic Syndromes

Introduction:Recent studies emphasized the role of immune dysregulation and innate immune activation in the pathogenesis of myelodysplastic syndromes (MDS). The expansion of T-regulatory cells, namely effector memory cells (Treg EM), in MDS correlates with worse outcome. The expansion of inflammatory hematopoietic suppressive cells called myeloid-derived suppressor cells (MDSC) is sufficient to perturb hematopoiesis and result in the development of MDS in a mouse model. INCB024360 is an oral inhibitor of the enzyme indoleamine 2,3-dioxygenase (IDO) which catalyzes the degradation of tryptophan (Trp) to Kynurenine (Kyn). Increased expression of IDO1 was an independent prognostic variable for survival in patients with acute myeloid leukemia (AML). Preclinical data suggests that IDO1 inhibition by INCB024360 will increase T cell proliferation, and decrease T reg cells and will decrease MDSC suppressive activity. We report the results of a phase II clinical study with laboratory correlatives exploring the potential role of INCB024360 for the treatment of MDS patients.Methods:This was a phase II, 2-step design to explore the activity and pharmacodynamics of INCB024360 in previously treated MDS patients. All patients signed informed consent. All patients with WHO defined MDS and AML with a myeloblast percentage between 20-30% (RAEB-t by FAB) were included. All risk categories by international prognostic scoring systems (IPSS) were allowed. The study excluded patients with viral hepatitis, HIV infection, prior solid organ or hematopoietic stem cell transplant or active autoimmune disease. The primary endpoint was overall response rate by the International Working Group criteria (IWG 2006). The secondary endpoints included IDO suppression measured intracellular by flowcytometry, change in Treg EM % with treatment and the percentage of bone marrow MDSC change after treatment with INCB024360 . All patients were treated with 600 mg oral twice a day for 16 weeks unless clear evidence of disease progression or toxicity was evident. Descriptive statistics were used to report baseline characteristics and response rates. The paired t-test was used to compare means. The study was registered at clinicaltrials.gov NCT01822691.Results:Between August 2013 and January 2014, 15 patients were enrolled at Moffitt Cancer Center. The median age was 72 years the majority of whom were white (93%) and male gender (80%). Seven patients were RCMD WHO subtype, 3 RAEB-I and RAEB-II each, and 1 RARS and MDS/MPN each. The IPSS risk was low 27% (4), intermediate-1 47% (7) and intermediate-2 27% (4). By the revised IPSS, 5 patients were intermediate risk (33%), 2 (13%) high and very high risk respectively. Two thirds were intermediate-2 or high risk by global MD Anderson risk model. The median number of prior therapies was 3 (2-10) and all patients had prior azacitidine therapy. The best response was stable disease in 12 (80%); 3 (20%) patients experienced disease progression and no hematological improvement was observed. The median duration of follow up was 10 months, median duration on study treatment 3.9 months, and median overall survival was not reached. Two patients progressed to AML. The treatment was relatively well tolerated; no treatment-emergent grade 3/4 adverse events were reported. One patient developed hypothyroidism and adrenal insufficiency (grade 2) and one patient had a low testosterone level. Mean IDO expression measured intracellular in mononuclear bone marrow cells was 39% at baseline compared to 26% after treatment (n=9, p 0.4). Mean BFU-E recovery improved from 72 to 191 colonies/106 (n=5, p value 0.036) and mean CFU-GM from 62 to180 colonies/106 (n=6, p 0.5). The mean MDSC % (CD33Lin-HLA cells) was 29.5% at baseline compared to 27.6% after treatment (n=9, p 0.7) however most patients experienced some reduction in MDSC%. The mean T Reg EM cell % changed from 9.6% at screening to 7.4% at end of treatment. (n=14, p 0.8)Conclusions:INCB024360 was relatively well tolerated in MDS patients. At the current dosages tested no significant clinical activity was observed. No significant decrease in intracellular IDO expression was observed. No significant decrease in MDSC and T reg EM as potential mechanism of action were observed.. DisclosuresKomrokji:Incyte Corporation: Consultancy, Research Funding. Padron:Incyte: Honoraria, Research Funding. Tinsley:Incyte: Speakers Bureau.

Circulating Mir-153 Participates in Occurrence of Acute Graft-Versus-Host Disease By Down-Regulating Indoleamine-2,3-Dioxygenase

Background Indoleamine-2,3-dioxygenase (IDO) is able to catalyze the first and rate-limiting step in the catabolism of tryptophan, which is the essential amino acid for T cell proliferation. Many previous studies have proved that IDO is able to play important roles in immunosuppression-associated with tumor immunity, autoimmunity and chronic infection. Since donor T cells will be activated, proliferate, differentiate and finally lead to tissue injury, growing research paid more attention to the immunoregulatory role of IDO in the process of aGVHD. Our previous work also demonstrated that plasma IDO level was correlated with the severity of aGVHD. Increased IDO expression would serve as a protective molecular during aGVHD. However, the exact regulatory mechanism of IDO is still unclear. MicroRNAs (miRNAs) are a class of small non-coding RNA that negatively regulate gene expression by translational repression or induction of mRNA degradation. Recently, circulating miRNAs have been reported to be the promising biomarkers for various kinds of diseases. We speculated that miRNA that might regulate those GVHD-related molecules/cytokines expression including IDO, and the circulating miRNA that directed at IDO would serve as new potential biomarker for aGVHD. This study is to find the potential regulatory miRNA of IDO and investigate the correlation between plasma miRNA expression and aGVHD.Methods The potential regulatory miRNA for IDO was identified through bioinformatics analysis using TargetScan and Miranda. The regulatory function of target miRNA on IDO protein and gene was confirmed by Western Blot analysis and luciferase assay. Human plasma samples from 70 patients who underwent allo-HSCT from Sep 2012 to Jun 2013 were prospectively collected at +7 day(d), +14d, +21d, +30d, +45d, +60d, +90d and the occurrence of aGVHD from Peking University Institute of Hematology. All patients provided informed consent. This study was approved by the institutional review board at the Peking University Institute of Hematology (Protocol number 2013-42). Real-time quantitative PCR (RQ-PCR) analysis was performed to examine the miRNA expression of plasma at different time points posttransplant. Receiver operating characteristic (ROC) analysis was employed to determine the optimal cut-off value for predict the occurrence of aGVHD.Results Through bioinformatics analysis, we found the predicted target sites in its 3’UTR binding miRNA-153. The intracellular IDO protein would be reduced when miRNA-153 was transfected into interferon-gamma-treated Hela cells. Surprisingly, the mRNA expression level of IDO was also affected in miRNA-153 transfected cells obviously (P<0.001). In addition, luciferase assay in 293T cells showed that miRNA-153 was able to bind directly to the promoter region of IDO gene and affect its transcription. Luciferase activity dropped around 44% when wild-type construct was co-transfected into 293T cells with miRNA-153 (P=0.009). However, when the potential binding sites were mutated, this phenomenon could not be observed. This indicates that the IDO 3’UTR is normally targeted by endogenous miR-153. Among the 70 patients who underwent allo-HSCT, 35 patients developed into aGVHD and 35 patients did not. A significant difference in miRNA-153 expression levels between the two groups was confirmed at +7d (P<0.001). Furthermore, we used ROC analysis to evaluate the diagnostic accuracy. Plasma levels of miRNA-153 at day 7 could predict an impending aGVHD with a sensitivity of 38.71% and a specificity of 100%, and the area under the curve (AUC) was 0.898 (95%CI: 0.821-0.974, P<0.0001).Conclusions Our study suggested that miRNA-153 might participate in occurrence of aGVHD by down regulating IDO and might be a putative target for novel aGVHD therapy. The plasma level of miRNA-153 at +7d after allo-HSCT would be a good biomarker for predicting the occurrence of aGVHD. DisclosuresNo relevant conflicts of interest to declare.

B7 costimulation and intracellular indoleamine-2,3-dioxygenase (IDO) expression in peripheral blood of healthy pregnant and non-pregnant women.

BackgroundB7 costimulatory molecules are expressed on antigen presenting cells (APCs) and are important regulators of T cell activation. We investigated the role of the B7 family of costimulatory molecules in the development of the systemic maternal immune tolerance during healthy pregnancy (HP). We also aimed to investigate the intracellular expression of indoleamine-2,3-dioxygenase (IDO) and plasma levels of tryptophane (TRP), kynurenine (KYN) and kynurenic acid (KYNA), important molecules with immunoregulatory properties, in order to describe their potential contribution to the pregnancy-specific maternal immune tolerance.MethodsWe determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2, and that of T cells and CD4+ T helper cells expressing CD28, CTLA-4, PD-1, and ICOS in peripheral blood samples of healthy pregnant (HP) and non-pregnant (NP) women using flow cytometry. We also examined the intracellular expression of IDO applying flow cytometry and plasma levels of TRP, KYN and KYNA using high-performance liquid chromatography.ResultsA significant increase in the prevalence of CD28+ T cells was observed in HP compared to NP women. At the same time a decrease was shown in the expression of CTLA-4 on these cells. The frequency of CD80+ monocytes was lower in HP women. The prevalence of IDO-expressing T cells and monocytes was higher in HP compared to NP women. Plasma KYN, KYNA and TRP levels were lower, while at the same time, the KYN/TRP ratio was higher in HP than in NP women.ConclusionsCostimulation via CD28 may not contribute to the immunosuppressive environment, at least in the third trimester of pregnancy. The development of the pregnancy-specific immune tolerance in the mechanism of B7 costimulation may be more related to the altered expression of B7 proteins on APCs rather than that of their receptors on T cells. The elevated intracellular IDO expression in monocytes and T cells, as well as higher plasma enzymatic IDO activity are likely to contribute to the systemic immunosuppressive environment in the third trimester characteristic for healthy gestation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2393-14-306) contains supplementary material, which is available to authorized users.

Indoleamine 2,3-dioxygenase-expressing peripheral cells in rheumatoid arthritis and systemic lupus erythematosus: a cross-sectional study

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme which suppresses T lymphocyte activity and induces Foxp3+ CD4+ regulatory T cells (Tregs) polarisation. The aim of this study was to evaluate the expression of IDO in freshly isolated peripheral cells as well as to enumerate Tregs and Th17 subpopulation in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients. The percentage of IDO-expressing cells as well as Tregs and Th17 was evaluated in 14 active RA- (aRA), 13 inactive RA- (iRA), 7 aSLE-, 12 iSLE-treated patients and 11 healthy donors (controls). Intracellular IDO was analysed by flow cytometry in CD14+, CD8α+, CD16+ and CD123+ cell subpopulations. Tregs and Th17 were assessed by intracellular of Foxp3 and IL17A detection in CD4+ CD14- cells. A total of 50,000 events were recorded for each sample. The amounts of CD14+/CD16-/IDO+, CD14-/CD16+/IDO+ and CD14+/CD16+/IDO+-expressing peripheral cells were slightly lower in inactive vs. active disease in RA and SLE patients. Notwithstanding, only inactive patients had statistically significant lower percentages when compared to controls. aRA and iRA showed a statistically significant decrease in CD8α+/CD123+/IDO+ vs. controls. Meanwhile, only iSLE patients had lower CD8α+/CD123+/IDO+ cells vs. aSLE patients and controls. Th17 subset was present in higher amounts in aRA and iRA patients vs. controls. Tregs showed an increase in aRA patients vs. controls. A decreased percentage of IDO-expressing peripheral cells were determined in iRA and iSLE compared to controls. It could play a critical role in tolerance loss in these diseases.

High Levels of IDO-Expressing CD16+ Peripheral Cells, and Tregs in Graft Biopsies From Kidney Transplant Recipients Under Belatacept Treatment

Background Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme that suppresses T-lymphocyte activity. Costimulation blockade through CTLA4lg increases IDO in antigen-presenting cells. The suppressive effect of IDO is thought to be mediated by Foxp3+CD4+CD25+ regulatory T-cells (Tregs). Objective In this descriptive study, we evaluated the percentage of IDO-expressing peripheral cell subpopulations as well as Tregs in 27 stable kidney transplant recipients receiving either belatacept (LEA29Y), a daughter compound of abatacept (CTLA4lg; n = 19) or cyclosporine (n = 8). Methods Blood samples were obtained at 24 ± 2 months (belatacept) and 23 ± 6 months (cyclosporine) of treatment. Intracellular IDO was analyzed by flow cytometry in CD14+, CD11c+, CD16+, CD56+, and CD8+ cell subpopulations. Tregs were assessed by intracellular Foxp3 detection in CD4+CD25+ cells. CD3+, CD4+, CD8+, CD20+, CD68+, IDO+, and Foxp3+ cells were evaluated by immunohistochemistry on graft biopsies obtained preimplantation, at 12 months posttransplant, and in subjects with dysfunction during the first 12 months. Results Only percentages of CD16+/IDO+-expressing peripheral monocytes were significantly increased among the group receiving belatacept. No differences were observed in peripheral Tregs between the groups. In contrast, higher percentages of Tregs, CD4+, CD8+, and CD68+ cells were noted in dysfunction and at 12 months vs baseline among graft biopsies in subjects receiving belatacept, and also among dysfunction cohorts of belatacept vs Cyclosporine treatment. Conclusion Patients receiving belatacept showed greater amounts of peripheral blood CD16+/IDO+ cells and Tregs on graft biopsies than those under cyclosporine treatment.

Human immunodeficiency virus type 1 clade B and C Tat differentially induce indoleamine 2,3-dioxygenase and serotonin in immature dendritic cells: Implications for neuroAIDS

Human immunodeficiency virus type 1 (HIV-1) is commonly associated with immune dysfunctions and the suppression of antigen-presenting cells. This results in immune alterations, which could lead to impaired neuronal functions, such as neuroAIDS. The neurotoxic factor kynurenine (KYN), the rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO), serotonin (5-HT), and serotonin transporter (5-HTT) may play a role in tryptophan deficiency and serotogenic dysfunction in neuroAIDS. HIV-1 transactivator regulatory protein (Tat) is known to play a major role in immune dysfunction. Previous studies suggest that HIV-1 B and C clades differentially manifest neuronal dysfunctions in the infected host. In the present study we examine the effect of HIV-1 B and C clade-derived Tat on IDO and 5-HTT gene and protein expressions by dendritic cells as studied by quantitative polymerase chain reaction (qPCR) and Western blot. In addition, the intracellular IDO expression, IDO enzyme activity, and the levels of 5-HT and KYN were also measured. Results indicate that HIV-1 clade B Tat up-regulates IDO and down-regulates 5-HTT gene and protein expressions. Further, HIV-1 clade B Tat caused a reduction of 5-HT with simultaneous increase in KYN levels as compared to HIV-1 clade C Tat. These studies suggest that HIV-1 clade B and C Tat proteins may play a differential role in the neuropathogenesis of HIV-associated dementia (HAD) or HIV-associated neurocognitive disorder (HAND).

Binding immunoglobulin protein‐treated peripheral blood monocyte‐derived dendritic cells are refractory to maturation and induce regulatory T‐cell development

Binding immunoglobulin protein (BiP) has been shown previously to have immunomodulatory functions. Herein we investigated whether BiP could affect the differentiation of monocytes into dendritic cells (DCs) and thence the development of regulatory T cells. Peripheral blood monocyte-derived DCs were matured with lipopolysaccharide in the presence or absence of BiP. DC development and T-cell changes were monitored by flow cytometry and regulatory T-cell function was measured by uptake of tritiated thymidine. More BiP-treated DCs (DC((BiP))s) expressed amounts of intracellular indoleamine 2,3-dioxygenase (IDO) and cell surface leucocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), retained CD14 expression but down-regulated expression of human leucocyte antigen (HLA)-DR and CD86, and produced copious amounts of interleukin (IL)-10, when compared with control DCs. T cells co-cultured with DC((BiP))s developed regulatory function with increased surface expression of CD4(+) CD25(hi) CD27(hi) but with no concomitant increase in forkhead box P3 (Foxp3). These T cells also showed significantly higher levels of intracellular cytotoxic T-lymphocyte antigen (CTLA)-4. The latter could be inhibited by the presence of the IDO inhibitor 1 methyl tryptophan. The addition of neutralizing anti-IL-10 antibody or the specific mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 reversed the inhibition of DC differentiation by BiP. In conclusion, BiP is an immunomodulator able to arrest inflammation through induction of tolerogenic DCs and subsequent generation of T regulatory cells.

Indoleamine 2,3-Dioxygenase (IDO): The Antagonist of Type I Interferon-Driven Skin Inflammation?

Recent studies have provided evidence that a type I interferon (IFN)-driven immune response might play an important role in the pathogenesis of lichen planus (LP), an inflammatory disorder of the skin of unclear etiology. Plasmacytoid dendritic cells in affected skin from LP have been proposed to produce IFN-alpha/beta locally, which leads to the expression of IFN-inducible chemokines such as IP10/CXCL10 in the epidermis. This chemokine recruits chemokine receptor CXCR3-expressing T-lymphocytes into the skin via CXCR3/IP10 interactions. Indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan and suppresses T-cell proliferation, is induced by IFNs and other inflammatory cytokines. We show that type I IFN-mediated skin disorders, such as LP, strongly express IDO in lesional skin. This expression closely correlates to the expression of the highly specific type I IFN marker MxA. We further demonstrate that the IDO+ cells in LP are large myeloid CD11c+S100+CD68(-) dendritic cells. Accordingly, CD11c+ antigen-presenting cells significantly up-regulate IDO gene expression and intracellular IDO protein expression after stimulation with IFN-alpha in vitro. These findings reveal that both proinflammatory and counterregulatory mechanisms are operative in cutaneous lesions of LP. We propose that the balance of these mechanisms may be involved in the pathogenesis of this disorder.

Induction of Indoleamine 2,3-Dioxygenase by Interferon-γ in Human Islets

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial, rate-limiting step of tryptophan (Trp) catabolism along the kynurenine (KYN) pathway, and its induction in cells of the immune system in response to cytokines has been implicated in the regulation of antigen presentation and responses to cell-mediated immune attack. Microarray and quantitative PCR analyses of isolated human islets incubated with interferon (IFN)-gamma for 24 h revealed increased expression of IDO mRNA (>139-fold) and Trp-tRNA synthase (WARS) (>17-fold) along with 975 other transcripts more than threefold, notably the downstream effectors janus kinase (JAK)2, signal transducer and activator of transcription (STAT)1, IFN-gamma regulatory factor-1, and several chemokines (CXCL9/MIG, CXCL10/IP10, CXCL11/1-TAC, CCL2, and CCL5/RANTES) and their receptors. IDO protein expression was upregulated in IFN-gamma-treated islets and accompanied by increased intracellular IDO enzyme activity and the release of KYN into the media. The response to IFN-gamma was countered by interleukin-4 and 1alpha-methyl Trp. Immunohistochemical localization showed IDO to be induced in cells of both endocrine, including pancreatic duodenal homeobox 1-positive beta-cells, and nonendocrine origin. We postulate that in the short term, IDO activation may protect islets from cytotoxic damage, although chronic exposure to various Trp metabolites could equally lead to beta-cell attrition.

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-gamma showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-gamma-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-gamma-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-gamma alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-gamma. Coculture of eosinophils with an IFN-gamma-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase gene expression.

Alterations in the rate of cellular tryptophan metabolism are involved in mediating important biological activities associated with cytokines and growth factors. Indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase are enzymes of tryptophan metabolism whose expression in a variety of cells and tissues is highly inducible by interferon-gamma (IFN-gamma). Transforming growth factor-beta (TGF-beta) antagonizes many cellular responses to IFN-gamma. The interaction of these two cytokines plays an important role in maintaining homeostasis during inflammation and repair. In human skin and synovial fibroblasts in vitro, TGF-beta caused time- and dose-dependent abrogation of IFN-gamma-stimulated expression of IDO and tryptophanyl-tRNA synthetase mRNAs. The inhibition was selective and did not appear to be due to down-regulation of IFN-gamma signaling by TGF-beta. In parallel with its effect on IDO mRNA expression, TGF-beta caused a marked reduction in intracellular IDO protein levels and abrogated IDO activity and tryptophan catabolism in these cells induced by IFN-gamma. IFN-gamma caused a rapid and striking increase in the amount of IDO heterogeneous nuclear pre-mRNA and induced transcription of the IDO gene, as demonstrated by transient transfection assays. TGF-beta partially reversed this stimulation. IFN regulatory factor (IRF)-1 and stat1 are cellular intermediates in IFN signaling. Both are implicated in activation of IDO transcription in response to IFN-gamma. The stimulation by IFN-gamma of IRF-1 protein and mRNA expression was not prevented by treatment of fibroblasts with TGF-beta. Furthermore, gel mobility shift assays indicated that TGF-beta did not inhibit the induction of stat1 and IRF-1 binding activity to their cognate DNA recognition sites in the IDO gene promoter. In contrast, the stability of IDO mRNA transcripts was reduced in fibroblasts treated with TGF-beta, as shown by determination of mRNA half-lives following blockade of transcription with 5,6-dichlorobenzimidazole riboside. The findings indicate that TGF-beta prevents the induction of IDO and tryptophanyl-tRNA synthetase gene expression in fibroblasts. The repression of IDO expression by TGF-beta is mediated at both transcriptional and posttranscriptional levels. These results implicate TGF-beta in the negative regulation of tryptophan metabolism, provide evidence for the molecular basis of this regulation, and indicate that cellular tryptophan metabolism is under tight immunological control.