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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).

Objective: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces tolerance to host immune surveillance within the tumor microenvironment. The present study aimed to investigate IDO expression and its prognostic significance in invasive cervical cancer., Methods: Immunohistochemical expression of IDO in tumor tissues and its association with clinicopathological factors and survival were analyzed in 112 stage IB-IIB cervical cancer patients treated with radical hysterectomy and pelvic lymphadenectomy., Results: IDO was diffusely expressed in tumor cells in 29 (26%) cases and focally expressed at the invasive front in 29 (26%) cases, while the other 54 (48%) cases were IDO-negative. IDO expression was positively correlated with clinical stage, lymph node metastasis, and lymph-vascular space invasion, but not with histological type. Patients with diffuse IDO expression had significantly reduced overall survival (OS) and disease-free survival (DFS) compared to patients with no IDO expression. The 5-year OS/DFS rates for the IDO-negative, focally positive, and diffusely positive groups were 92.3%/84.9%, 89.5%/75.8%, and 65.5%/51.7%, respectively. When we analyzed patients with stage IB disease alone (n=67), the OS and DFS for the IDO-diffusely positive group were significantly lower than those for the IDO-negative group. In multivariate analysis, diffuse IDO expression was found to be an independent prognostic factor for impaired OS and DFS., Conclusions: Diffuse expression of IDO in the tumor obtained from Stage IB-IIB cervical cancer patients who underwent radical hysterectomy was correlated with an unfavorable clinical outcome. These findings suggest that IDO may be a novel post-operative prognostic indicator for stage IB-IIB cervical cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)

The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase (IDO) has emerged as an important driver of immune escape in a growing number of cancers and cancer-associated chronic infections. In this study, we define novel immunotherapeutic applications for the heme precursor compound zinc protoporphyrin IX (ZnPP) based on our discovery that it is a potent small-molecule inhibitor of IDO. Inhibitory activity was determined using in vitro and in-cell enzyme assays as well as a novel in vivo pharmacodynamic system. An irreversible mechanism of inhibition was documented, consistent with competition for heme binding in newly synthesized cellular protein. siRNA methodology and an IDO-deficient mouse strain were used to verify the specificity of ZnPP as an IDO inhibitor. In a preclinical model of melanoma, ZnPP displayed antitumor properties that relied on T-cell function and IDO integrity. ZnPP also phenocopied the known antitumor properties of IDO inhibitors in preclinical models of skin and breast carcinoma. Our results suggest clinical evaluation of ZnPP as an adjuvant immunochemotherapy in chronic infections and cancers in which there is emerging recognition of a pathophysiologic role for IDO dysregulation.

Efforts to improve cancer care in the developing world will benefit from the identification of simple, inexpensive, and broadly applicable medical modalities based on emergent innovations in treatment, such as targeting mechanisms of tumoral immune tolerance. In this report, we offer preclinical evidence that the low-cost, anti-inflammatory agent ethyl pyruvate elicits a potent immune-based antitumor response through inhibition of indoleamine 2,3-dioxygenase (IDO), a key tolerogenic enzyme for many human tumors. Consistent with its reported ability to interfere with NF-kappaB function, ethyl pyruvate blocks IDO induction both in vitro and in vivo. Antitumor activity was achieved in mice with a noncytotoxic dosing regimen of ethyl pyruvate shown previously to protect against lethality from sepsis. Similar outcomes were obtained with the functional ethyl pyruvate analogue 2-acetamidoacrylate. Ethyl pyruvate was ineffective at suppressing tumor outgrowth in both athymic and Ido1-deficient mice, providing in vivo corroboration of the importance of T-cell-dependent immunity and IDO targeting for ethyl pyruvate to achieve antitumor efficacy. Although ethyl pyruvate has undergone early-phase clinical testing, this was done without consideration of its possible applicability to cancer. Our findings that IDO is effectively blocked by ethyl pyruvate treatment deepen emerging links between IDO and inflammatory processes. Further, these findings rationalize oncologic applications for this agent by providing a compelling basis to reposition ethyl pyruvate as a low-cost immunochemotherapy for clinical evaluation in cancer patients.

Inflammation-induced activation of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) causes depressive-like behavior in mice following acute activation of the innate immune system by lipopolysaccharide (LPS). Here we investigated the mechanism of IDO expression induced by LPS in primary cultures of microglia derived from neonatal C57BL/6J mice. LPS (10 ng/ml) induced IDO transcripts that peaked at 8h and enzymatic activity at 24h, resulting in an increase in extracellular kynurenine, the catabolic product of IDO-induced tryptophan catabolism. This IDO induction by LPS was accompanied by synthesis and secretion of the proinflammatory cytokines TNFalpha and IL-6, but without detectable IFNgamma expression. To explore the mechanism of LPS-induced IDO expression, microglia were pretreated with the c-Jun-N-terminal kinase (JNK) inhibitor SP600125 for 30 min before LPS treatment. We found that SP600125 blocked JNK phosphorylation and significantly decreased IDO expression induced by LPS, which was accompanied by a reduction of LPS-induced expression of TNFalpha and IL-6. Collectively, these data extend to microglia the property that LPS induces IDO expression via an IFNgamma-independent mechanism that depends upon activation of JNK. Inhibition of the JNK pathway may provide a new therapy for inflammatory depression., (Copyright 2009 Elsevier Inc. All rights reserved.)

A well-known traditional Chinese medicinal prescription, Oren-gedoku-to (OGT), has been used in clinical therapies for many types of dementia in China and Japan. Additionally, it ameliorates the age-related deterioration of learning and memory in an Alzheimer's disease (AD) rat model. Indoleamine 2, 3-dioxygenase (IDO-1) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism, which ultimately leads to the production of the excitotoxin quinolinic acid (QUIN). IDO-1 has recently been established as one of the key players involved in the pathogenesis of AD. OGT is indicated to prevent cholinergic dysfunction and reduce oxidative stress; however, the exact mechanism underlying its ability to improve cognitive ability remains elusive. Here we present a novel mechanism of OGT's therapeutic potential in AD. We demonstrated that OGT significantly inhibited recombinant human IDO-1 (rhIDO-1) activity in vitro, and its four main constituents (i.e., berberine, palmatine, jatrorrhizine, and baicalein) were potent IDO-1 inhibitors. IC50 values, obtained from a cell-based assay, of HEK 293 cells and an enzymatic assay were much lower than the most commonly used IDO-1 inhibitor, 1-methyl tryptophan (1-MT). Berberine was the best inhibitor and had IC50 values of 7 μM (cell-based assay) and 9.3 μM (enzymatic assay). Jatrorrhizine and palmatine exhibited irreversible inhibition of rhIDO-1, whereas berberine and baicalein behaved as uncompetitive, reversible inhibitors with Ki values of 8 μM and 215 μM, respectively. In conclusion, constituents of OGT show strong IDO-1 inhibitory activity and may have significant therapeutic potential for AD.

Previous cancer vaccination approaches have shown some efficiency in generating measurable immune responses, but they have rarely led to tumor regression. It is therefore possible that tumors emerge with the capacity to down-regulate immune counterparts, through the local production of immunosuppressive molecules, such as IDO. Although it is known that IDO exerts suppressive effects on T cell functions, the mechanisms of IDO regulation in tumor cells remain to be characterized. Here, we demonstrate that activated T cells can induce functional IDO expression in breast and kidney tumor cell lines, and that this is partly attributable to IFN-gamma. Moreover, we found that IL-13, a Th2 cytokine, has a negative modulatory effect on IDO expression. Furthermore, we report IDO expression in the majority of breast and kidney carcinoma samples, with infiltration of activated Th1-polarized T cells in human tumors. These findings demonstrate complex control of immune activity within tumors. Future immune therapeutic interventions should thus include strategies to counteract these negative mechanisms.

Background: Tumor-specific cytotoxic T cells and infiltrating lymphocytes are frequently found in tumor tissues in patients with nasopharyngeal carcinoma (NPC). Most patients with NPC, however, especially those with advanced stages, have a poor clinical prognosis despite conventional immunotherapy. The aim of this work was to examine the effect of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme, on the lymphocyte function in NPC., Methods: The NPC cell line CNE2 was treated by interferon-gamma (IFNgamma) and the levels of IDO expression was analyzed by Western blotting and reverse phase high-performance liquid chromatography (HPLC). Lymphocytes from health human exposed to the milieu created by IDO-positive CNE2 cells and the lymphocyte cytotoxicity to target tumor cells was analyzed by standard lactate dehydrogenase (LDH) release assay. Additionally, expression of IDO was determined by Immunohistochemical assay in the tumor tissues form clinically evaluated NPC., Results: IDO expression was acutely induced in the NPC cell line CNE2 by low dose interferon-gamma (IFNgamma) or by co-incubation with activated lymphocytes. Exposure to the milieu created by IDO-positive CNE2 cells did not promote lymphocyte death, but lymphocyte cytotoxicity against target tumor cells was impaired. The suppression of lymphocyte cytotoxic function was fully restored when the conditioned medium was replaced by fresh medium for 24 h. In additionally, the IDO-positive cells were found scattered in the tumor tissues from patients with NPC., Conclusion: Altogether, these findings suggest that IDO-mediated immunosuppression may be involved in the tumor immune evasion, and that blocking IDO activity in tumor cells may help to re-establish an effective anti-tumor T cell response in NPC.

Objective: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance. The purpose of the present study is to investigate IDO expression and its functional role in ovarian cancer cells in vitro and in vivo., Methods: IDO expression was immunohistochemically scored in surgically-resected ovarian cancer tissues (n=60), and its association with tumor-infiltrating lymphocyte (TIL) count or patient survival was analyzed. Next, IDO cDNA was transfected into the human ovarian carcinoma cell line SKOV3, establishing stable clones of IDO-overexpressing cells (SK-IDO). SK-IDO cells were characterized in vitro as well as in vivo using a nude mouse xenograft model., Results: High IDO expression in tumor cells was found in 34 (56.7%) cases and was correlated with a reduced number of CD8+ TIL. Patients with high IDO expression had significantly impaired overall and progression-free survival compared to patients with no or low IDO expression. There were no significant differences in in vitro cell proliferation, migration, invasion, or chemosensitivity to paclitaxel between the SK-IDO and control vector-transfected (SK-pcDNA) cells. However, tumor peritoneal dissemination was significantly increased in SK-IDO-xenografted mice compared to SK-pcDNA-xenografted mice. This tumor-progressive effect in SK-IDO-xenografted mice was abrogated by oral administration of the IDO inhibitor 1-methyl-tryptophan (1-MT). Finally, treatment with weekly i.p. paclitaxel combined with daily administration of 1-MT significantly prolonged the survival of the SK-IDO-xenografted mice compared to treatment with paclitaxel alone., Conclusions: These results suggest that IDO is involved in ovarian cancer progression in vivo and may be a promising therapeutic target for advanced ovarian cancer.

Cyclophosphamide, methotrexate and 5-fluorouracile (CMF)-based chemotherapy for adjuvant treatment of breast cancer reduces the risk of relapse. In this exploratory study, we tested the feasibility of identifying molecular markers of recurrence in CMF-treated patients. Using Affymetrix U133A GeneChips, RNA samples from 19 patients with primary breast cancer who had been uniformly treated with adjuvant CMF chemotherapy were analyzed. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. An additional independent validation set of 51 patients and 21 genes were analyzed by quantitative RT-PCR. Applying different algorithms to evaluate our microarray data, we identified two gene expression signatures of 21 and 12 genes containing eight overlapping genes, that predict recurrence in 19 cases with high accuracy (94%). Quantitative RT-PCR demonstrated that six genes from the combined signatures (CXCL9, ITSN2, GNAI2, H2AFX, INDO, and MGC10986) were significantly differentially expressed in the recurrence versus the non-recurrence group of the 19 cases and the independent breast cancer patient cohort (n = 51) treated with CMF. High expression levels of CXCL9, ITSN2, and GNAI2 were associated with prolonged disease-free survival (DFS) (P = 0.029, 0.018 and 0.032, respectively). When patients were stratified by combined CXCL9/ITSN2 or CXCL9/FLJ22028 tumor levels, they exhibited significantly different disease-free survival curves (P = 0.0073 and P = 0.005, respectively). Finally, the CXCL9/ITSN2 and CXCL9/FLJ22028 ratio was an independent prognostic factor (P = 0.034 and P = 0.003, respectively) for DFS by multivariate Cox analysis in the 70-patient cohort. Our data highlight the feasibility of a prognostic assay that is applicable to therapeutic decision-making for breast cancer. Whether the biomarker profile is chemotherapy-specific or whether it is a more general indicator of bad prognosis of breast cancer patients remains to be explored.

Infection with Chlamydia pneumoniae, a human respiratory pathogen, has been associated with various chronic diseases such as asthma and atherosclerosis, possibly because the pathogen can exist in a persistent form. C. pneumoniae persistently infect DCs in a TNF-alpha dependent manner. The present study investigated whether C. pneumoniae infection can induce indoleamine 2,3-dioxygenase (IDO) activity in dendritic cells, and whether the restriction of chlamydial growth in the DCs by TNF-alpha is IDO dependent. Our data indicate that infection of DCs with C. pneumoniae resulted in the induction of IDO expression. Reporting on our use of anti-TNF-alpha antibody adalimumab and varying concentrations of TNF-alpha, we further demonstrate that IDO induction following infection of DCs with C. pneumoniae is TNF-alpha dependent. The anti-chlamydial activity induced by TNF-alpha and the expression of chlamydial 16S rRNA gene, euo, groEL1, ftsk and tal genes were correlated with induction of IDO. Addition of excess amounts of tryptophan to the DC cultures resulted in abrogation of the TNF-alpha-mediated chlamydial growth restriction. These findings suggest that infection of DCs by C. pneumoniae induces production of functional IDO, which subsequently causes depletion of tryptophan. This may represent a potential mechanism for DCs to restrict bacterial growth in chlamydial infections.

Dendritic cells (DCs) express the immunoregulatory enzyme IDO in response to certain inflammatory stimuli, but it is unclear whether DCs express this enzyme under steady-state conditions in vivo. In this study, we report that the DCs in mesenteric lymph nodes (MLNs) constitutively express functional IDO, which metabolizes tryptophan to kynurenine. In line with a previous report that regulatory T cells (Tregs) can induce IDO in DCs via the CTLA-4/B7 interaction, a substantial proportion of the MLN DCs were located in juxtaposition to Tregs, whereas this tendency was not observed for splenic DCs, which do not express IDO constitutively. When CTLA-4 was selectively deleted in Tregs, the frequency of IDO-expressing DCs in MLNs decreased significantly, confirming CTLA-4's role in IDO expression by MLN DCs. We also found that the MLN DCs produced CCL22, which can attract Tregs via CCR4, and that the phagocytosis of autologous apoptotic cells induced CCL22 expression in CCL22 mRNA-negative DCs. Mice genetically deficient in the receptor for CCL22, CCR4, showed markedly reduced IDO expression in MLN-DCs, supporting the involvement of the CCL22/CCR4 axis in IDO induction. Together with our previous observation that MLN DCs contain much intracytoplasmic cellular debris in vivo, these results indicate that reciprocal interactions between the DCs and Tregs via both B7/CTLA-4 and CCL22/CCR4 lead to IDO induction in MLN DCs, which may be initiated and/or augmented by the phagocytosis of autologous apoptotic cells by intestinal DCs. Such a mechanism may help induce the specific milieu in MLNs that is required for the induction of oral tolerance.

Immunosuppressive factors derived from the tumor and nontumor cells present in the tumor microenvironment contribute to tumor escape from host immune attack. Recently, the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) derived from both the tumor cells and surrounding nontumor cells was found to function as a critical immunosuppressive factor. While the expression of IDO is intensively under investigation in many types of cancers, little information is available in esophageal squamous cell carcinomas (ESCC) thus far. In this study, we have therefore investigated the cellular localization of IDO in 45 ESSCs and ten morphologically normal esophageal tissues; the correlation of IDO with clinicopathological parameters was also analyzed. Immunohistochemistry (IHC) analysis revealed that the density of IDO-positive cells was increased in ESCCs relative to controls (P < 0.01). These cells were distributed as clusters and formed a patchy pattern in both the cancerous epithelium and the surrounding noncancerous cells. Double IHC further confirmed that many IDO-positive cells in the tumor stroma were smooth-muscle-actin-alpha-positive myofibroblasts, CD68-positive macrophages, and S100-positive dendritic cells. Statistical analysis showed that the densities of IDO-positive cells were not significantly correlated with tumor clinical parameters (tumor invasion depth, node metastasis, and TNM stages) and lymphocytic infiltration. Our current findings suggested that the increased IDO expression in ESCCs is from a mixed cellular source (both cancer cells and noncancerous cells). Further studies on immune cell functional analysis are required in the future.

Interferon (IFN)-gamma is a pleiotropic cytokine with a central role in innate and adaptive immunity. As a potent pro-inflammatory and antitumor cytokine, IFN-gamma is conventionally thought to be responsible for driving cellular immune response. On the other hand, accumulating evidence suggests that IFN-gamma also has immunosuppressive activity. An important role for IFN-gamma in inhibiting graft-versus-host disease (GVHD) has been demonstrated in murine models, despite IFN-gamma being one of the key factors amplifying T cell activation during the process of acute GVHD (aGVHD), the major complication and cause of post-transplant mortality in allogeneic bone marrow transplantation (BMT). At the same time, IFN-gamma facilitates graft-versus-leukemia (GVL) activity. Dissociation of GVL effects from GVHD has been the ultimate goal of allogeneic BMT in the treatment of hematologic malignancies. This paradoxic role of IFN-gamma makes modulating its activity a promising strategy to maximize GVL while minimizing GVHD and improve clinical outcomes in BMT. In this review, the effects of IFN-gamma on GVHD and GVL are discussed with consideration of the mechanism of IFN-gamma action.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of I kappaB alpha. Hb-induced IDO expression was inhibited by inhibitors of PI3-kinase (PI3K), PKC and nuclear factor (NF)-kappaB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O(2) (-), H(2)O(2), and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-kappaB through the PI3K-PKC-ROS and PI3K-Akt pathways is required for the Hb-induced IDO expression in BMDCs., ((c) 2009 Wiley-Liss, Inc.)

Neospora caninum is an apicomplexan parasite closely related to Toxoplasma gondii. In nature this parasite is found especially in dogs and cattle, but it may also infect other livestock. The growth of N. caninum, which is an obligate intracellular parasite, is controlled mainly by the cell-mediated immune response. During infection the cytokine gamma interferon (IFN-gamma) plays a prominent role in regulating the growth of N. caninum in natural and experimental disease. The present study showed that induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) is responsible for the inhibition of parasite growth that is mediated by IFN-gamma-activated bovine fibroblasts and endothelial cells. This antiparasite effect could be abrogated by addition of tryptophan, as well as by the IDO-specific inhibitor 1-L-methyltryptophan. In conclusion, our data show that human and bovine cells use the same effector mechanism to control the growth of N. caninum.

Human immunodeficiency virus (HIV) infection is often complicated by the development of acquired immunodeficiency syndrome (AIDS) dementia complex (ADC). Implications of kynurenine pathway (KP) are suggested in ADC and other inflammatories brain diseases. The first and regulatory enzyme of the KP is the indoleamine-2,3-dioxygenase (IDO). IDO activation is known to contribute to the modulation of the immune response during various infectious diseases particularly in AIDS. HIV and viral proteins can activate IDO in immune cells leading to an increase catabolism of tryptophan through the KP; the consequence being the production of immuno-modulative and neuroactive metabolites. This mechanism is likely to favour HIV persistence. The present study analysed concomitantly several parameters involved in IDO regulation and activity associated with HIV-1-infection. We investigated relevant intracellular and extracellular mechanisms involved in the regulation of IDO expression and activity during the HIV infection and replication in human monocyte-derived macrophages (MdM). Using a complementary set of in vitro experiments, we found that HIV-1/Ba-L infection induces IDO expression and increases its activity in MdM. We also showed that IDO activation by HIV-1 is likely to be a direct effect of the infection and seems to be independent of IFN-gamma production.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of the kynurenine pathway of tryptophan metabolism, ultimately leading to production of the excitotoxin quinolinic acid (QUIN) by monocytic cells. In the Tg2576 mouse model of Alzheimer's disease, systemic inflammation induced by lipopolysaccharide leads to an increase in IDO expression and QUIN production in microglia surrounding amyloid plaques. We examined whether the IDO over-expression in microglia could be mediated by brain proinflammatory cytokines induced during the peripheral inflammation using THP-1 cells and peripheral blood mononuclear cells (PBMC) as models for microglia. THP-1 cells pre-treated with 5-25 muM amyloid beta peptide (Abeta) (1-42) but not with Abeta (1-40) or Abeta (25-35) became an activated state as indicated by their morphological changes and enhanced adhesiveness. IDO expression was only slightly increased in the reactive cells but strongly enhanced following treatment with proinflammatory cytokine interferon-gamma (IFN-gamma) but not with interleukin-1beta, tumor necrosis factor-alpha, or interleukin-6 at 100 U/mL. The concomitant addition of Abeta (1-42) with IFN-gamma was totally ineffective, indicating that Abeta pre-treatment is prerequisite for a high IDO expression. The priming effect of Abeta (1-42) for the IDO induction was also observed for PBMC. These findings suggest that IFN-gamma induces IDO over-expression in the primed microglia surrounding amyloid plaques.

Background: The discovery of indoleamine 2,3-dioxygenase (IDO) as a modulator for the maintenance of fetomaternal immuno-privileged state has been heralded as a significant step in further defining the role of IDO in immunobiology. IDO is an IFN-inducible, intracellular enzyme that catalyzes the initial and rate-limiting step in the degradation of the essential amino acid, tryptophan. It has been suggested that IDO has the capacity to regulate the immune system via two discrete mechanisms; firstly the deprivation of tryptophan, which is essential for T cell proliferation and via the cytotoxic effects of tryptophan metabolites on T(H)1 cell survival., Methods: The sources of information used to prepare the paper are published work on Pubmed/Medline. In this review, we examine the therapeutic role of modulating IDO activity a variety of disease states including tumour tolerance, chronic infection, transplant rejection, autoimmunity and asthma. We propose that IDO represents a novel therapeutic target for the treatment of these diseases. We also explore the diverse strategies which are being employed, either to augment or to inhibit IDO activity in order to modify various disease processes. The limitations associated with these strategies are also scrutinized.

Background: There is evidence that the immune systems of patients with breast cancer are dysfunctional. Regulatory T cells (Tregs), and IDO, an immunosuppressive enzyme, are associated with more advanced disease in some cancers and may promote immunologic tolerance to tumors. Our aim was to assess whether expression of Foxp3, a marker of Tregs, and IDO were linked with nodal metastasis in breast cancer patients. Inhibitors of IDO are available and could potentially demonstrate utility in breast cancer if IDO drives progression of disease., Methods: Sentinel lymph nodes (SLN) of 47 breast cancer patients with varying degrees of nodal disease and 10 controls were evaluated for expression of Foxp3 and IDO using immunohistochemistry. Positively stained cells were quantified and their distribution within the SLN noted., Results: The proportion of Foxp3+ cells was higher in SLN of cancer patients than controls (19% v. 10%, p < 0.001). Specifically, there were more Foxp3+ cells in SLN with metastasis than tumor-free SLN (20% v. 14%, p = 0.02). The proportion IDO+ cell in SLN of cancer patients was not statistically different than controls (4.0% v. 1.6%, p = 0.08). In order to demonstrate the combined immunosuppressive effect of Foxp3 and IDO, we categorized each SLN as positive or negative for Foxp3 and IDO. The Foxp3+/IDO+ group almost exclusively consisted of cancer patients with node positive disease., Conclusion: In conclusion, our study shows that Foxp3+ cells are associated with more advanced disease in breast cancer, a finding that is proving to be true in many other cancers. As IDO has been found to promote differentiation of Tregs, IDO may become a suitable target to abrogate the development of T-cell tolerance and to promote an effective immune response to breast cancer. Our results about the combined expression of IDO and Foxp3 in metastastic SLN support this assumption.

The mechanism by which malignant melanoma (MM) cells survive in lymph nodes is poorly understood. One possible mechanism by which MM cells can escape immune surveillance is through upregulation of immunomodulatory enzymes such as indoleamine 2,3-dioxygenase (IDO). In this study, 25 cases of MM lymph node metastases from patients with long and short survival were evaluated for expression of IDO and the number of Forkhead box p3 (FOXP3)-expressing regulatory T cells. Moderate to strong cytoplasmic IDO expression was present in all (15/15) MM lymph node metastases in patients with poor survival. Eight of 10 patients with metastatic MM and long survival were negative or only weakly positive for IDO. Upregulation of IDO in metastatic MM cells was associated with an increased number of regulatory T cells (Tregs). There was a statistically significant association between shorter survival and both a stronger IDO expression (p = 0.0019) and a higher number of FOXP3 expressing Tregs (p < 0.001). Using RT-PCR analysis, we showed that IDO expression in MM cells is induced by interferon-gamma. These data support the notion that metastatic MM cells select for expression of IDO to evade immunologic detection. Therefore, inhibition of IDO in MM patients may be a useful treatment strategy.

The present study investigated an immunotherapeutic strategy for rearranged during transfection proto-oncogene (ret)-associated carcinomas in a transgenic MT/ret 304/B6 mouse model in which spontaneous tumors develop due to overexpression of the ret gene. A Ret peptide vaccine comprising an extracellular fragment of Ret protein and Th1-polarized immunoregulator CpG oligonucleotide (1826) induced strong and specific cellular and humoral immune responses in wild-type C57BL/6 mice, showing that the Ret peptide has a strong immunogenic potential as part of an antitumor vaccine. In MT/ret 304/B6 mice, however, the vaccine was only modestly effective as an inducer of the humoral immune response, and it failed to elicit a T-cell response. An immunohistochemical analysis revealed marked indoleamine 2,3-dioxygenase expression after immunization with Ret peptide vaccine in the lymph nodes and spleens of MT/ret 304/B6 mice. The systemic administration of the potent inhibitor of indoleamine 2,3-dioxygenase 1-methyl tryptophan (1MT) along with Ret vaccine produced a significant increase in tumor-specific cytotoxic activity. A delay in spontaneous tumor development was also observed in the MT/ret 304/B6 mice to which the Ret vaccine and 1MT were administered. These results indicate that an improved Ret vaccine composed of Ret peptide plus CpG oligonucleotide plus 1MT is a potential therapeutic strategy for treatment of ret-associated carcinomas.

Infection by multiple lentiviral strains is recognized as a major driving force in the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, but the neuropathogenic consequences of multivirus infections remain uncertain. Herein, we investigated the neurovirulence and underlying mechanisms of dual lentivirus infections with distinct viral strains. Experimental feline immunodeficiency virus (FIV) infections were performed using cultured cells and an in vivo model of AIDS neuropathogenesis. Dual infections were comprised of two FIV strains (FIV-Ch and FIV-PPR) as copassaged or superinfected viruses, with subsequent outcome analyses of host immune responses, viral load, neuropathological features, and neurobehavioral performance. Dual infections of feline macrophages resulted in greater IL-1beta (interleukin-1beta), TNF-alpha (tumor necrosis factor alpha), and IDO (indoleamine 2,3-dioxygenase) expression and associated neurotoxic properties. FIV coinfection and sequential superinfection in vivo also induced greater IL-1beta, TNF-alpha, and IDO expression in the basal ganglia (BG) and cortex (CTX), compared to the monovirus- and mock-infected groups, although viral loads were similar in single virus- and dual virus-infected animals. Immunoblot analyses disclosed lower synaptophysin immunoreactivity in the CTX resulting from FIV super- and coinfections. Cholinergic and GABAergic neuronal injury was evident in the CTX of animals with dual FIV infections. With increased glial activation and neuronal loss in dual FIV-infected brains, immunohistochemical analysis also revealed elevated detection of cleaved caspase-3 in dysmorphic neurons, which was associated with worsened neurobehavioral abnormalities among animals infected with the copassaged viruses. Dual lentivirus infections caused an escalation in neuroinflammation and ensuing neurodegeneration, underscoring the contribution of infection by multiple viruses to neuropathogenesis.

Previous studies have demonstrated that infection with HIV-1 clades might differentially contribute to the neuropathogenesis of HIV-1-associated dementia (HAD). HIV-1 transactivator regulatory protein (Tat) plays a major role in the process of disruption of neuronal function. It is not well understood how these HIV-1 subtypes exert different neuropathogenic effects. Activation of indoleamine-2,3-dioxygenase (IDO), the rate-limiting enzyme of the kynurenine pathway, leads to increased tryptophan catabolism and the generation of neurotoxins such as kynurenine (KYN). It is known that KYN plays a crucial role in the neuropathogenesis of HAD. We hypothesize that HIV-1 clade B and C Tat proteins might exert differential effects on human primary astrocytes by the upregulation of the IDO gene and protein expression as well as its activity and production of the neurotoxin KYN. RNA extracted from human primary astrocytes treated with either HIV-1 clade B and C Tat proteins was reverse transcribed and analyzed by quantitative real-time PCR to determine IDO gene expression. In addition, the enzymatic activity of IDO and the concentration of KYN were measured in cell lysates and culture supernatants. Our results indicate that HIV-1 clade B Tat protein significantly upregulated the IDO gene and protein expression, IDO enzyme activity, as well as KYN concentration compared to HIV-1 clade C Tat protein. Thus, our studies for the first time demonstrate that HIV-1 clade B Tat protein in human primary astrocytes appears to increase the level of neuropathogenic agents, such as IDO and KYN, as compared to HIV-1 clade C Tat protein. These results provide further evidence that the prevalence of HAD may be correlated with the difference in clades of HIV-1.

Chronic inflammation activates the tryptophan-degrading enzyme IDO, which is well known to impair T cell proliferation. We have previously established that bacille Calmette-Guérin (BCG), an attenuated form of Mycobacterium bovis, is associated with persistent activation of IDO in the brain and chronic depressive-like behavior, but a causative role has not been established. In these experiments we used both pharmacologic and genetic approaches to test the hypothesis that IDO activation is responsible for the development of chronic depression that follows BCG infection. BCG induced TNF-alpha, IFN-gamma, and IDO mRNA steady-state transcripts in the brain as well as the enzyme 3-hydroxyanthranilic acid oxygenase (3-HAO) that lies downstream of IDO and generates the neuroactive metabolite, quinolinic acid. Behaviors characteristic of depression were apparent 1 wk after BCG infection. Pretreatment with the competitive IDO inhibitor 1-methyltryptophan fully blocked BCG-induced depressive-like behaviors. Importantly, IDO-deficient mice were completely resistant to BCG-induced depressive-like behavior but responded normally to BCG induction of proinflammatory cytokines. These results are the first to prove that the BCG-induced persistent activation of IDO is accompanied by the induction of 3-hydroxyanthranilic acid oxygenase and that IDO is required as an initial step for the subsequent development of chronic depressive-like behavior.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step in the degradation of tryptophan and is strongly induced in interferon-gamma (IFNgamma)-stimulated dendritic cells (DCs). IDO has recently been established as a key enzyme in T-cell suppression-mediated immune tolerance to tumors. STAT1 phosphorylation appears to play an important role in the control of IDO expression by IFNgamma, but the precise regulatory mechanism remains obscure. Here we present a novel mechanism of IFNgamma-induced IDO expression in bone marrow-derived dendritic cells. In addition, we demonstrate that curcumin, an active component of turmeric, significantly inhibited the induction of IDO expression and activity by IFNgamma. We found that curcumin suppressed STAT1 activation by directly inhibiting Janus-activated kinase 1/2 and protein kinase Cdelta phosphorylation in bone marrow-derived DCs, suppressing the subsequent translocation and binding of STAT1 to the GAS element of the IRF-1 promoter. Coincident with these inhibitory effects on IFNgamma-induced IDO expression, curcumin reversed IDO-mediated suppression of T-cell responses. Our results, thus, suggest that down-regulation of IDO in DCs is an important immunomodulatory property of curcumin that may be exploited therapeutically in the control of cancers.

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. In this study, two different approaches were used to examine the role of indoleamine 2,3-dioxygenase-1 (IDO-1) and its metabolites in the development of murine CM. Mice genetically deficient in IDO-1 were not protected against CM, but partial protection was observed in C57BL/6 mice treated with Ro 61-8048, an inhibitor of kynurenine-3-hydroxylase. This protection was associated with suppressed levels of picolinic acid (PA) within the brain, but not with changes in the levels of kynurenic acid (KA) or quinolinic acid (QA). These data suggest that although IDO-1 is not directly involved in the pathogenesis of CM in C57BL/6 mice, the production of the kynurenine pathway metabolite PA may contribute to the development of murine CM.

The aim of this study was to evaluate the ability of local overexpression of indoleamine dioxygenase (IDO) to abrogate rat liver transplant rejection by the use of an adeno-associated virus vector [recombinant adeno-associated virus 2/8 (rAAV2/8)] to deliver the transgene to the allograft prior to transplantation. A green fluorescent protein (GFP)-expressing vector [recombinant adeno-associated virus 2/8-liver-specific promoter 1-enhanced green fluorescent protein (rAAV2/8-LSP1-eGFP)] was used to examine the kinetics of expression and optimal dosing for transduction of Piebald Virol Glaxo (PVG) rat livers. A vector encoding the rat IDO gene (rAAV2/8-LSP1-rIDO) was constructed and tested by its ability to induce tryptophan catabolism and kynurenine production in vitro and in vivo. PVG donor rats were injected, via the portal vein, with rAAV2/8-LSP1-rIDO 2 weeks before transplantation into PVG strain isograft or Lewis (LEW) strain allograft recipients. With the enhanced GFP vector, 29.5% and 47.4% of hepatocytes were found to express GFP at 3 and 6 weeks after injection, respectively. In untransplanted PVG animals, the rAAV2/8-LSP1-rIDO vector induced, 3 weeks after administration, a 1.8-fold increase (P = 0.0161) in liver IDO activity, which was associated with a fall in serum tryptophan to 0.5 times the baseline level (P < 0.001). PVG recipients of PVG liver isografts pretreated with the IDO-expressing vector had a 45% lower level of serum tryptophan than recipients of isografts pretreated with the GFP-expressing vector (P = 0.03). LEW recipients of PVG liver allografts pretreated with the rat IDO vector had a median survival time of 12 days, whereas recipients of allografts pretreated with rAAV2/8-LSP1-eGFP had a median survival time of 13 days (P = 0.38). Both groups displayed similar histological features of acute cellular rejection. In conclusion, rAAV2/8 vectors produce highly efficient, though delayed, hepatocyte transduction in vivo and provide a useful gene delivery tool for transplantation models. However, gene delivery using IDO was unsuccessful in prolonging rat liver allograft survival., ((c) 2009 AASLD.)

Background: Indoleamine 2, 3-dioxygenase (IDO), a tryptophan-degrading enzyme in dendritic cells (DCs), mediates an immunosuppressive effect on activated T lymphocytes. However, little is known about the effect of Der p 1 on IDO in human DCs., Objective: The aim was to investigate the effect of Der p 1 on the expression and activity of IDO in monocyte-derived DCs from house dust mite (HDM)-sensitive patients with asthma., Methods: Using real-time RT-PCR and HPLC, the expression and activity of IDO were assessed in TNF-alpha-induced mature DCs from HDM-sensitive and nonatopic patients with asthma in response to Der p 1 exposure ex vivo. We also monitored the alteration of IDO activity in Der p 1-pulsed DCs after the coincubation with autologous T cells., Results: With a reliance on its protease activity, Der p 1 suppressed functional IDO in DCs from HDM-sensitive patients with asthma but enhanced IDO activity in DCs from nonatopic patients with asthma. This suppression was maintained by the reciprocally induced IL-4 from the coculturing autologous HDM-sensitive T cells. Conversely, the upregulation of IDO activity in Der p 1-pulsed DCs was maintained by IFN-gamma released from autologous nonatopic T cells and the regulatory T-cell subset. Der p 1 pulsation to sensitive DCs failed to raise regulatory T cells but raised progenitor fractions from cloned HDM-sensitive CD4(+) cells through direct contact and soluble mediators., Conclusion: House dust mite-sensitive DCs exposed to Der p 1 downregulated IDO activity and tipped the T(H)1/T(H)2 cytokine balance toward IL-4, resulting in sustainable IDO suppression.

Mature dendritic cells (DCs) are potent stimulators of T cells that recognize antigens presented by the DCs. In this chapter we describe mature DCs that suppress T cell responses to antigens they present due to expression of the intracellular enzyme indoleamine 2,3 dioxygenase (IDO). IDO-competent DCs are a subset of plasmacytoid DCs that can be induced to express IDO under certain inflammatory conditions in humans and mice. Though rare, IDO-expressing DCs acquire potent T cell suppressor activity that may predominate over the T cell stimulatory functions of all other antigen-presenting cells in physiologic environments due in part, to cooperation with regulatory T cells. Thus, IDO-expressing DCs are critical regulators of adaptive immunity that contribute to a wide range of inflammatory disease processes. As such, manipulating IDO expression in DCs using IDO inhibitors or IDO inducers offers considerable opportunities to improve immunotherapies in a range of clinically-significant disease syndromes.

Human plasmacytoid dendritic cells (PDCs) can drive naive, allogeneic CD4(+)CD25(-) T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). However, the intracellular mechanism or mechanisms underlying PDC-induced Treg generation are unknown. In this study, we show that human PDCs express high levels of IDO, an intracellular enzyme that catabolizes tryptophan degradation. Triggering of TLR 9 with CpG oligodeoxynucleotides activates PDCs to up-regulate surface expression of B7 ligands and HLA-DR Ag, but also significantly increases the expression of IDO and results in the generation of inducible Tregs from CD4(+)CD25(-) T cells with potent suppressor cell function. Blocking IDO activity with the pharmacologic inhibitor 1-methyl-D-tryptophan significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine, the immediate downstream metabolite of tryptophan, bypasses the 1-methyl-D-tryptophan effect and restores PDC-driven Treg generation. Our results demonstrate that the IDO pathway is essential for PDC-driven Treg generation from CD4(+)CD25(-) T cells and implicate the generation of kynurenine pathway metabolites as the critical mediator of this process.

Objectives: Indoleamine 2,3-dioxygenase (IDO), which catalyzes the breakdown of tryptophan into kyneurenine, has immunologic significance for the induction of maternal tolerance and liver allograft tolerance by inhibiting T-cell activation. In the present study, we compared survival of syngeneic or allogeneic hepatocytes in livers with or without hepatectomy. Subsequently, we investigated gene expression and localization of IDO in the recipient liver., Methods: DA and Fisher 344 rats were used in the following experimental groups: group 1, DA hepatocytes transplanted into hepatectomized Fisher 344 rats; group 2, Fisher 344 hepatocytes transplanted into hepatectomized Fisher 344 rats; group 3, DA hepatocytes transplanted into nonhepatectomized Fisher 344 rats; and group 4, Fisher 344 hepatocytes transplanted into nonhepatectomized Fisher 344 rats. After transplantation, the surviving cells were evaluated on day 5. The IDO signal of the recipient liver was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry., Results: In the hepatectomized groups subjected to allogeneic or syngeneic hepatocyte transplantation, the number of surviving hepatocytes was greater than in the nonhepatectomized group after transplantation. The IDO signals (RT-PCR) in the hepatectomized groups were stronger than those in the nonhepatectomized groups. Immunohistochemistry demonstrated that the IDO signal is located in liver antigen-presenting cells, such as Kupffer cells or dendritic cells, and not expressed in hepatocytes., Conclusions: Our results demonstrated that IDO is induced in antigen-presenting cells of hepatectomized livers by which subsequently transplanted cells may be protected from rejection by inhibiting indirect or direct recognition of donor antigen and further T-cell activation.

In contrast to the role of dendritic cells (DC) in immunity and tolerance, little is known about their possible role in the resolution of inflammatory processes. In addition to the reduction in the number of infiltrating immune cells, the elimination of effector T cells already present at the inflammatory site represents an essential step toward resolution. Recently, lipid mediators such as the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and their metabolites, including resolvin E1 (RvE1), have been shown to accumulate in inflammatory foci during the resolution phase. RvE1 has been reported to reduce immune cell infiltration and proinflammatory cytokine production. In this study we report that DC exposed to RvE1, especially during differentiation, acquire the capacity to induce apoptosis of activated T cells through the induction and activity of indoleamine 2,3-dioxygenase. To our knowledge, this study is the first to report on an omega-3 fatty acid derivative inducing indoleamine 2,3-dioxygenase expression in DC. RvE1-exposed DC maintain an immature chemokine receptor expression pattern even following TLR stimulation, with high CCR5 and no CCR7 expression. This effect implies that DC exposed to RvE1 and pathogens remain at the inflammatory site, instead of migrating to lymph nodes, and induce apoptosis in effector T cells infiltrating the inflammatory site. To our knowledge, the DC described in this study represent a new functional DC subtype, whose essential function resides in the resolution of inflammation.

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.

The glycolipid alpha-galactosylceramide (alpha-GalCer), when presented on CD1 molecules by antigen presenting cells (APCs) to invariant NKT (iNKT cells), is a potent immunomodulator. Indoleamine 2,3-dioxygenase (IDO), an enzyme catalyzing the catabolism of L-tryptophan along the kynurenine pathway, is inducible in APC and represents one of the main endogenous mechanisms of T cell homeostasis, peripheral tolerance and immunosuppression. No data have been published yet on the effect of alpha-GalCer on IDO in APC. We aimed to determine if: (1) alpha-GalCer modulates IDO in APC; (2) the alpha-GalCer-induced effect on IDO correlates with the production by APC of active compounds; (3) the medium from alpha-GalCer-treated APC is able to stimulate iNKT cells. From our results alpha-GalCer alone did not modify IDO expression (RT-PCR) in APC, but when human peripheral blood mononuclear cells (PBMC), monocytes, and monocytic cell lines (THP-1), expressing high levels of CD1d, were treated with interferon-gamma (IFN-gamma) plus alpha-GalCer a significant potentiation of IDO transcription was measured. This effect was not induced by increased IFN-gamma release by APC, and it was functionally correlated with increased L-kynurenine (L-KYN) release by alpha-GalCer-treated CD1d-transfected THP-1 cells. The medium of these cells stimulated iNKT hybridoma cells to release interleukin (IL)-2, while alpha-GalCer alone resulted ineffective. The data demonstrate that alpha-GalCer: (1) does not induce IFN-gamma release by APC; (2) potentiates IFN-gamma-induced IDO expression and function in APC; (2) requires CD1d molecules for inducing these effects; (3) induces the release by APC of compounds active in stimulating iNKT cells.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs), which can differentiate into several lineages, have immunomodulatory properties similar to those of bone marrow-derived MSCs. However, the specific mechanism by which the immunomodulatory effect of MSCs occurs is not clear. In this study, we isolated canine AD-MSCs (cAD-MSCs) and induced their development into adipocyte, osteocyte, and neuron-like cells. We then investigated their phenotype and cytokine expression to determine whether they were able to exert an immunomodulatory effect and what the underlying mechanisms of this effect were. cAD-MSCs expressed CD44, CD90, and MHC class I and were also partially positive for the expression of CD34; however, they did not express CD14 and CD45. In addition, they expressed the mRNA of transforming growth factor beta (TGF-beta), IL-6, IL-8, CCL2, CCL5, vascular endothelial growth factor, hepatocyte growth factor (HGF), tissue inhibitor metalloproteinase-1/2, and cyclooxygenase-2 but not that of IL-10. Further, leukocyte proliferation induced by mitogens was suppressed when they were cocultured with irradiated cAD-MSCs, as well as with culture supernatants of cAD-MSCs alone. Moreover, TNF-alpha production significantly decreased, whereas TGF-beta, IL-6, and interferon-gamma production significantly increased in cAD-MSCs that were cocultured with leukocytes. Finally, immonomodulatory factors of MSCs, such as TGF-beta, HGF, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in cAD-MSCs that were cocultured with leukocytes; however, the production of PGE2 and IDO showed different kinetics, and leukocyte proliferation was effectively restored by PGE2 and IDO inhibitors. Taken together, these results indicate that the immunomodulatory effects of cAD-MSCs are associated with soluble factors (TGF-beta, HGF, PGE2, and IDO). Therefore, it is suggested that cAD-MSCs have a potential therapeutic use in the treatment of immune-mediated disease.

Indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan to N-formyl kynurenine and has a proapoptotic role in renal tubular epithelial cells (TEC) in response to IFN-gamma and TNF-alpha in vitro. TEC produce abundant amounts of IDO in vitro in response to inflammation but a pathological role for IDO in renal injury remains unknown. We investigated the role of IDO in a mouse model of renal ischemia-reperfusion injury (IRI). IRI was induced by clamping the renal pedicle of C57BL/6 mice for 45 min at 32 degrees C. Here, we demonstrate upregulation of IDO in renal tissue at 2 h after reperfusion which reached maximal levels at 24 h. Inhibition of IDO following IRI prevented the increase in serum creatinine observed in vehicle-treated mice (86.4 +/- 25 micromol/l, n = 11) compared with mice treated with 1-methyl-D-tryptophan, a specific inhibitor of IDO (33.7 +/- 8.7 micromol/l, n = 10, P = 0.031). The role of IDO in renal IRI was further supported by results in IDO-KO mice which maintained normal serum creatinine levels (32.5 +/- 2.0 micromol/l, n = 6) following IRI compared with wild-type mice (123 +/- 30 micromol/l, n = 9, P = 0.008). Our data suggest that attenuation of IDO expression within the kidney may represent a novel strategy to reduce renal injury as a result of ischemia reperfusion.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine pathway that converts L-tryptophan to L-kynurenine. Transient forebrain ischemia initiates a series of cellular events that lead to the delayed neuronal degeneration of several brain regions. The goal of this study was to determine the localization of IDO in gerbil brain, and analyze the spatiotemporal expression of IDO in a transient forebrain ischemic model. Expression of IDO in the normal gerbil brain was observed by using immunohistochemistry. Time-course of the expression of IDO following transient forebrain ischemic gerbils was examined by immunohistochemistry, combined with hematoxylin and eosin staining for morphological analysis, and in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method. In normal gerbils, IDO immunostaining was observed in thalamus, hypothalamus and amygdaloid nucleus. IDO expression was negative in the cingulate cortex, hippocampal CA1 region and parietal cortex. Following transient ischemia, we observed a time-dependent increase of IDO expression in CA1, cingulate cortex and hypothalamus. The peak of IDO expression in CA1 and cingulate cortex occurred at 48 h after ischemic insult and diminished by 2 weeks. TUNEL staining was observed only in the CA1 region at 72 and 96 h after transient ischemia. Thus, IDO protein is present in specific regions in gerbil brain, and dynamic changes of IDO expression was observed in some neurons following transient ischemia.

To elucidate the role of innate immunity in susceptibility to the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we induced EAE by immunization with spinal cord homogenate (SCH) plus complete Freund adjuvant or carbonyl iron in 3 inbred rat strains. Lewis are considered "susceptible," PVG/c-Rt7a (PVG) as "semisusceptible," and Brown Norway (BN) as "resistant" to EAE. Immunization with SCH-carbonyl iron resulted in clinical disease in all 3 strains, but the pathologic features of EAE in the resistant BN and the semisusceptible PVG rats differed from those in the Lewis and PVG model of EAE induced with SCH-complete Freund adjuvant. In BN and PVG rats, there were numerous inflammatory lesions with prominent involvement of microglia and, to a lesser extent, perivascular macrophages. These data suggest that different levels of activation of the innate immune system by different adjuvants determine whether EAE will or will not develop. Accordingly, the widely accepted scale of susceptibility to EAE development (Lewis > PVG > BN) should be revised because it does not take into account the important contribution of the composition of the adjuvant to the quality and quantity of the innate immune response and, consequently, to the generation and extent of the pathogenic T-cell-mediated, that is, adaptive, autoimmune disease.

Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte-derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T-cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS- or necrotic cell-induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS-induced CD4+ T-cell proliferation. Reduced levels of interleukin-12 (IL-12), IL-10, IL-6, tumour necrosis factor-alpha and interferon-gamma (IFN-gamma) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN-gamma expression by DC in association with apoptotic environments. The specific generation of IFN-gamma by DC within apoptotic environments is suggestive of an anti-inflammatory role by the induction of indoleamine 2,3-dioxygenase (IDO). Both neutralization of IFN-gamma and IDO blockade demonstrated a role for IFN-gamma and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN-gamma-dependent. Blocking transforming growth factor-beta (TGF-beta) also produced a partial release in T-cell proliferation. Our study strongly suggests that apoptosis-induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN-gamma-induced IDO and TGF-beta.

Background: The mechanism by which pancreatic ductal adenocarcinoma (PDA) cells escape immune detection and survive in lymph nodes is poorly understood. One possible mechanism by which PDA cells can escape immune detection is through upregulation of indoleamine 2,3-dioxygenase (IDO), an enzyme that can starve T lymphocytes of tryptophan., Study Design: Seventeen cases of PDA were evaluated by immunohistochemistry for expression of IDO in tumor cells and the number of Forkhead box p3-expressing regulatory T cells. To validate IDO protein expression, Western blot analysis for IDO was performed on primary pancreatic cancer cell-line protein lysates., Results: Upregulation of IDO in metastatic PDA cells was associated with an increased number of regulatory T cells. Cytoplasmic IDO expression was present in all tumors (primary and metastatic) from patients with lymph node metastases. Intensity of staining was stronger in the corresponding metastatic foci when compared with the primary tumor. Three nonmetastatic PDAs were negative or only focally positive for IDO. Additionally, IDO expression in PDA was independent of tumor histologic grade. Forkhead box p3 regulatory T cells were increased in lymph nodes containing metastatic tumor cells expressing IDO. Using Western blot and reverse transcriptase polymerase chain reaction analysis, we validated that IDO expression in pancreatic cancer cells is induced by interferon-gamma as reported previously., Conclusions: These data support the notion that metastatic PDA cells select for overexpression of IDO to evade immunologic detection. Future studies will define whether IDO expression in PDA patients with lymph node-positive metastases correlates with decreased survival. In addition, inhibition of IDO in PDA patients can be useful to enhance immunotherapeutic strategies.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first step in the degradation of tryptophan, an essential amino acid. During inflammation IDO can be induced in different cell types resulting in local tryptophan depletion. This inhibits T cell proliferation and may induce apoptosis. High expression of IDO was previously found in inflammatory bowel disease and is thought to represent a mechanism for downregulation of the local immune response. Our aim is to investigate the expression pattern of IDO in normal and inflamed murine and human intestinal mucosa. Immunohistochemical staining for IDO was performed on paraffin sections of colon of two mouse models for colitis and their controls and on paraffin sections of human ileum and colon in normal and two different inflammatory conditions, namely inflammatory bowel disease and diverticulitis. IDO immunohistochemistry showed similar results in murine and human tissue. In normal, as well as in inflamed mucosa, some mononuclear cells, fibroblasts and endothelial cells were positive for IDO. In inflamed mucosa a specific expression pattern of epithelial IDO was found where epithelial cells flanking ulcers or bordering crypt abscesses showed high IDO expression. Moreover, in human intestinal inflammation, IDO was expressed in ulcer associated cell lineage. Since bacterial invasion is more pronounced in erosions and in crypt abscesses and since IDO activity and the resulting local tryptophan depletion can cause growth arrest of several tryptophan-dependent microorganisms, IDO expression in the vicinity of interruptions of the epithelial barrier may point to a role for IDO as a local anti-infectious agent. Furthermore, expression of IDO at the margin of ulcerations and in the reparative ulcer-associated cell lineage suggests involvement of IDO in repair processes.

Joining chain (J-chain) is well known as an integrated component of dimeric immunoglobulin A (IgA) and pentameric IgM. We show here that the J-chain protein is also expressed in a subset of CD11c+ dendritic cells (DC) in C57BL/6 mice. J-chain knockout mice (J-/- mice) had a reduced fraction of CD4-/CD8alpha+ and mPDCA-1+ DC in the spleen. J-/- mice also had reduced levels of RNA for the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in the spleen. Furthermore, in lymph nodes from C57BL/6 mice the majority of J-chain-expressing CD11c+ cells also expressed IDO, while the number of IDO-expressing cells in lymph nodes and the amount of IDO protein in splenic CD11c+ cells were reduced in J-/- mice. Also, J-/- mice had a lower ratio of kynurenine/tryptophan in serum compared to C57BL/6 mice, indicating a lower overall IDO activity in J-/- mice. We also show that J-/- mice are less susceptible to tolerance induction than C57BL/6 mice. In conclusion, our data show that J-chain protein is expressed outside the B-cell compartment in a subset of immunoregulatory DC that are compromised in animals that cannot express J-chain.

MHC-mismatched DBA/2 renal allografts are spontaneously accepted by C57BL/6 mice by poorly understood mechanisms, but both immune regulation and graft acceptance develop without exogenous immune modulation. Previous studies have shown that this model of spontaneous renal allograft acceptance is associated with TGF-beta-dependent immune regulation, suggesting a role for T regulatory cells. The current study shows that TGF-beta immune regulation develops 30 days posttransplant, but is lost by 150 days posttransplant. Despite loss of detectable TGF-beta immune regulation, renal allografts continue to function normally for >200 days posttransplantation. Because of its recently described immunoregulatory capabilities, we studied IDO expression in this model, and found that intragraft IDO gene expression progressively increases over time, and that IDO in "regulatory" dendritic cells (RDC) may contribute to regulation associated with long-term maintenance of renal allografts. Immunohistochemistry evaluation confirms the presence of both Foxp3+ T cells and IDO+ DCs in accepted renal allografts, and localization of both cell types within accepted allografts suggests the possibility of synergistic involvement in allograft acceptance. Interestingly, at the time when RDCs become detectable in spleens of allograft acceptors, approximately 30% of these mice challenged with donor-matched skin allografts accept these skin grafts, demonstrating progression to "true" tolerance. Together, these data suggest that spontaneous renal allograft acceptance evolves through a series of transient mechanisms, beginning with TGF-beta and T regulatory cells, which together may stimulate development of more robust regulation associated with RDC and IDO.

Several inducers of chlamydial persistence have been described, including interferon-gamma (IFN-gamma), IFN-alpha, IFN-beta, and tumour necrosis factor-alpha (TNF-alpha) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-gamma, IFN-alpha, and TNF-alpha are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-beta, IFN-gamma, indoleamine 2,3-dioxygenase, lymphotoxin-alpha and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C(6)-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.

Immune evasion and T cell tolerance induction have been associated both with malignant disease and chronic infection. In recent years, increasing evidence has been accumulated that antigen-presenting cells such as dendritic cells (DC) play a major role in immune regulation. They are not only involved in the induction of immunity but also can inhibit immune responses. Interesting parallels for major molecular mechanisms involved in turning DC from stimulatory to regulatory cells have been uncovered between malignant disease and chronic infection. Apparently, not only inhibitory cytokines such as IL-10 seem to play a role, but also metabolic mechanisms dysregulating tryptophan metabolism, thereby, leading to inhibition of T cells and pathogens. We focus here on recent findings establishing the tryptophan catabolizing enzyme indoleamine-pyrrole 2,3 dioxygenase (IDO) as a central feature of DC with regulatory function both in cancer and chronic infection. Induction of enzymatically active IDO can be triggered by various soluble and membrane-bound factors, and in general, require interferon (IFN) signaling. In addition, based on the most recently established link between tumor necrosis factor alpha (TNFalpha), prostaglandin E2 and IDO, a new model of regulation of IDO in context of cancer and infection is proposed. In light of the increasing use of anti-TNFalpha drugs, these findings are also of great interest to the clinician scientist.

Purpose: The inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) participates in immune tolerance and tumor immune escape processes by degradation of the essential amino acid tryptophan and formation of toxic catabolites. Here, we analyzed the role of IDO in tumor growth and disease progression in patients with clear cell renal cell carcinoma (RCC)., Experimental Design: Expression of IDO mRNA was analyzed by quantitative reverse transcription-PCR in 55 primary and 52 metastatic RCC, along with 32 normal kidneys. Western blot and immunohistochemistry analyses were used to semiquantitatively determine IDO proteins in a subset of tumor samples, in RCC cell lines, and microvessel endothelial cells. IDO expression was correlated with expression of the proliferation marker Ki67 in tumor cells and survival of patients with tumor., Results: More than 75% of the clear cell RCC in comparison to normal kidney contained elevated levels of IDO mRNA, which correlated with their IDO protein content. Low IDO mRNA levels in primary tumors represented an unfavorable independent prognostic factor (hazard ratio, 3.8; P = 0.016). Unexpectedly, immunohistochemical analyses revealed that IDO is nearly exclusively expressed in endothelial cells of newly formed blood vessels and is virtually absent from tumor cells, although RCC cells could principally synthesize IDO as shown by in vitro stimulation with IFN-gamma. A highly significant inverse correlation between the density of IDO-positive microvessels and the content of proliferating Ki67-positive tumor cells in primary and metastatic clear cell RCC was found (P = 0.004)., Conclusions: IDO in endothelial cells might limit the influx of tryptophan from the blood to the tumor or generate tumor-toxic metabolites, thus restricting tumor growth and contributing to survival.

Problem: The semi-allogeneic fetus is usually tolerated by the maternal immune system. This was proposed to be modulated by CD4+CD25+foxp3+ regulatory T cells (Treg). We aimed to determine the kinetics of Treg during murine gestation and investigate whether changes in Treg levels respond to hormonal variations during pregnancy or generated changes in the local indolamine dioxygenase (IDO) expression., Method of Study: We included in our studies the well-known CBA/JxDBA/2J abortion-prone combination using CBA/JxBALB/c as controls. CBA/JxC57/BL6 and BALB/cxC57/BL6 were included as further controls. Animals were killed on days 0, 2, 5, 8, 10, and 12 of pregnancy to measure the levels of Treg, pregnancy-related hormones and IDO expression., Results: A Treg augmentation in normal pregnancy combinations could be observed on day 2 in several organs contrary to the observations made in abortion-prone mice. No differences in hormonal levels could be seen among all groups. IDO was expressed exclusively in placenta starting from day eight, showing no variations among the groups., Conclusion: Differences in Treg levels and pregnancy outcome do not correlate with changes in hormonal levels. In addition, as Treg augmentation takes place early and it is observed mainly in the decidual component of the fetal-maternal interface, IDO does not seem to be the pathway underlying Treg protective activity as proposed for humans.

High levels of viral replication occur in gut-associated lymphoid tissue (GALT) and other lymphoid tissues (LT) since the early phase of human/simian immunodeficiency virus (HIV/SIV) infection. Regulatory T cells (T(reg)), a subset of immunosuppressive T cells expressing CTLA-4 and the FoxP3 transcription factor, accumulate in LT during HIV/SIV infection. Here we show that FoxP3 and CTLA-4 mRNA are increased in leukocytes from the spleens, lymph nodes (LN), and mucosal sites of chronically SIV-infected macaques with high viremia (SIV(HI)) compared to animals with low viremia (SIV(LO)). FoxP3 and CTLA-4 correlated with SIV RNA levels in tissues; SIV virus levels in the spleen, inguinal LN, mesenteric LN, colon, and jejunum directly correlated with the plasma virus level. Importantly, CTLA-4 and FoxP3 mRNA were predominantly increased in the CD25(-) subpopulation of leukocytes from SIV(HI), further challenging the classical definition of T(reg) as CD4(+) CD25(+) T cells. Similar to CTLA-4 and FoxP3, expression of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme induced by T(reg) in antigen-presenting cells, was increased in the spleens, mesenteric LN, colons, and jejuna from SIV(HI) compared to SIV(LO) and directly correlated to SIV RNA in the same tissues. Accordingly, plasma kynurenine/tryptophan, a marker for IDO enzymatic activity, was significantly higher in SIV(HI) compared to SIV(LO) and correlated with plasma viral levels. Increased T(reg) and IDO in LT of SIV-infected macaques may be the consequence of increased tissue inflammation and/or may favor virus replication during the chronic phase of SIV infection.