Indoleamine 2,3-dioxygenase Expression Levels Research Articles

Increased IDO expression and regulatory T cells in acute myeloid leukemia: implications for immune escape and therapeutic targeting

PurposeThis study aimed to determine the frequency of regulatory T cells (Tregs) (CD4+/FOXP3+) and indoleamine 2,3-dioxygenase (IDO) expression in patients with acute myeloid leukemia (AML).MethodsThis cross-sectional case–control study was conducted between Jan 2022 and Dec 2023. Bone marrow samples were collected from 20 healthy individuals and 15 patients with AML. Flow cytometry, real-time polymerase chain reaction (PCR), and western blotting were used to evaluate the frequency of Treg and IDO expression levels.ResultsThe Treg percentage among total lymphocytes was lower in the AML group than that in the normal group. However, Treg percentage among T-helper (Th) lymphocytes was significantly higher in the AML group than that in the normal group (p < 0.05). The mean IDO expression in the AML group was significantly higher than that in the normal group (p = 0.004). A significant relationship was observed between IDO expression and Treg percentage among Th lymphocytes in the AML group (correlation = 0.637; p = 0.003). Moreover, western blot analysis showed a significant increase in IDO protein intensity in the AML group compared with that in the control group (p < 0.001). A significant difference was observed between the IDO concentrations in the AML group and that in the control group (p < 0.001). In addition, a significant difference between TGF-β levels in the AML group and those in the control group (p < 0.01) was observed.ConclusionIDO inhibition using novel IDO inhibitors along with chemotherapy is a promising approach to overcome the immune escape mechanisms in patients with AML, who exhibit increased levels of IDO expression and Tregs.

Effects of the kynurenine pathway on the osteogenic differentiation of periodontal ligament stem cells

Objective: To explore the effect of kynurenine pathway on the osteogenic differentiation of periodontal ligament stem cells (PDLSC). Methods: Unstimulated saliva samples were collected from 19 patients with periodontitis (periodontitis group) and 19 periodontally healthy individuals (health group) in Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Nanjing University from June to October of 2022. Contents of kynurenine and the metabolites in saliva samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The expressions of indoleamine 2, 3-dioxygenase (IDO) and aryl hydrocarbon receptor (AhR) were further detected by immunohistochemistry in gingival tissues. The PDLSC used in this study were isolated from extracted teeth for orthodontic treatment in Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Nanjing University from July to November of 2022. Experiments were then conducted using the cells by incubating with (kynurenine group) or without kynurenine (control group) in vitro. Seven days later, alkaline phosphatase (ALP) staining and assays of ALP activity were performed. Real-time fluorescence quantitative PCR (RT-qPCR) was utilized to detect the expressions of osteogenic related genes ALP, osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), collagen type-Ⅰ (COL-Ⅰ) as well as the kynurenine pathway-associated genes AhR, cytochrome P450 family (CYP) 1A1, CYP1B1. Western blotting was used to detect the expression levels of RUNX2, osteopontin (OPN) and AhR proteins on day 10 and alizarin red staining was performed to observe the formation of mineral nodules on day 21 in control group and kynurenine group. Results: Salivary concentrations of kynurenine [8.26 (0, 19.60) nmol/L] and kynurenic acid [11.4 (3.34, 13.52) nmol/L] were significantly higher in the periodontitis group than in the health group [0.75(0, 4.25) nmol/L, 1.92(1.34, 3.88) nmol/L] (Z=-2.84, P=0.004; Z=-3.61, P<0.001). The expression levels of IDO (18.33±2.22) and AhR (44.14±13.63) in gingival tissues of periodontitis patients were significantly higher than that of the health group (12.21±2.87, 15.39±5.14) (t=3.38, P=0.015; t=3.42, P=0.027). In vitro, the ALP activity of PDLSC in the kynurenine group (291.90±2.35) decreased significantly compared with the control group (329.30±19.29) (t=3.34, P=0.029). The mRNA expression levels of ALP, OCN and RUNX2 in the kynurenine group (0.43±0.12, 0.78±0.09, 0.66±0.10) were decreased compared with the control group (1.02±0.22, 1.00±0.11, 1.00±0.01) (t=4.71, P=0.003; t=3.23, P=0.018; t=6.73, P<0.001), while the levels of AhR and CYP1A1 were increased in the kynurenine group (1.43±0.07, 1.65±0.10) compared with those in the control group (1.01±0.12, 1.01±0.14) (t=5.23, P=0.006; t=6.59, P<0.001). No significant difference was observed in COL-Ⅰ and CYP1B1 mRNA levels between groups. The protein levels of OPN, RUNX2 (0.82±0.05, 0.87±0.03) were reduced and that of AhR (1.24±0.14) was increased in the kynurenine group compared with those in the control group (1.00±0.00, 1.00±0.00, 1.00±0.00) (t=6.79, P=0.003; t=7.95, P=0.001; t=3.04, P=0.039). Conclusions: Over-activated kynurenine pathway in periodontitis patients can promote upregulation of AhR and suppress the osteogenic differentiation of PDLSC.

Expression of tolerogenic dendritic cells in the small intestinal tissue of patients with celiac disease

Tolerogenic dendritic cells (tolCDs) play an important role in the regulation of inflammation in autoimmune diseases such as celiac disease (CeD). Dendritic cells express CD207, CD11c, and CD103 on their surface. In addition to the receptors mentioned above, tolCDs can express the immune-regulating enzyme indoleamine 2,3-dioxygenase (IDO). This study aimed to determine the mRNA and protein expression of CD11c, CD103 and CD207 markers, and also IDO gene expression in intestinal tissues of CeD patients in comparison to the healthy individuals. Duodenal biopsies were collected from 60 CeD patients and 60 controls. Total RNA was extracted and gene expression analysis was performed using Real-time PCR SYBR® Green method. Additionally, biopsy specimens were paraffinized and protein expression was evaluated using immunohistochemistry (IHC) for expression of CD11c+, CD207+and CD103+. Gene expression levels of CD11c (P = 0.045), CD103 (P < 0.001), CD207 (P < 0.001) and IDO (P = 0.01) were significantly increased in CeD patients compared to the control group. However, only CD103 protein expression was found to be significantly higher in CeD patients in comparison to the control group (P < 0.001). The result of this study showed that the expresion levels of CD11c, CD103, CD207 and IDO markers were higher in CeD patients compared to the controls, indicating the effort of dendritic cells to counterbalance the gliadin-triggered abnormal immune responses in CeD patients.

The immunosuppressive role of indoleamine 2, 3-dioxygenase in glioblastoma: mechanism of action and immunotherapeutic strategies.

Glioblastoma multiforme (GBM) is a fatal brain tumor in adults with a bleak diagnosis. Expansion of immunosuppressive and malignant CD4 + FoxP3 + GITR + regulatory T cells is one of the hallmarks of GBM. Importantly, most of the patients with GBM expresses the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). While IDO1 is generally not expressed at appreciable levels in the adult central nervous system, it is rapidly stimulated and highly expressed in response to ongoing immune surveillance in cancer. Increased levels of immune surveillance in cancer are thus related to higher intratumoral IDO expression levels and, as a result, a worse OS in GBM patients. Conversion of the important amino acid tryptophan into downstream catabolite known as kynurenines is the major function of IDO. Decreasing tryptophan and increasing the concentration of immunomodulatory tryptophan metabolites has been shown to induce T-cell apoptosis, increase immunosuppressive programming, and death of tumor antigen-presenting dendritic cells. This observation supported the immunotherapeutic strategy, and the targeted molecular therapy that suppresses IDO1 activity. We review the current understanding of the role of IDO1 in tumor immunological escape in brain tumors, the immunomodulatory effects of its primary catabolites, preclinical research targeting this enzymatic pathway, and various issues that need to be overcome to increase the prospective immunotherapeutic relevance in the treatment of GBM malignancy.

Indoleamine 2,3-dioxygenase in melanoma progression and BRAF inhibitor resistance

Indoleamine 2,3-dioxygenase (IDO) is associated with the progression of many types of tumors, including melanoma. However, there is limited information about IDO modulation on tumor cell itself and the effect of BRAF inhibitor (BRAFi) treatment and resistance. Herein, IDO expression was analyzed in different stages of melanoma development and progression linked to BRAFi resistance. IDO expression was increased in primary and metastatic melanomas from patients’ biopsies, especially in the immune cells infiltrate. Using a bioinformatics approach, we also identified an increase in the IDO mRNA in the vertical growth and metastatic phases of melanoma. Using in silico analyses, we found that IDO mRNA was increased in BRAFi resistance. In an in vitro model, IDO expression and activity induced by interferon-gamma (IFNγ) in sensitive melanoma cells was decreased by BRAFi treatment. However, cells that became resistant to BRAFi presented random IDO expression levels. Also, we identified that treatment with the IDO inhibitor, 1-methyltryptophan (1-MT), was able to reduce clonogenicity for parental and BRAFi-resistant cells. In conclusion, our results support the hypothesis that the decreased IDO expression in tumor cells is one of the many additional outcomes contributing to the therapeutic effects of BRAFi. Still, the IDO production changeability by the BRAFi-resistant cells reiterates the complexity of the response arising from resistance, making it not possible, at this stage, to associate IDO expression in tumor cells with resistance. On the other hand, the maintenance of 1-MT off-target effect endorses its use as an adjuvant treatment of melanoma that has become BRAFi-resistant.

Abnormal Expression of Indoleamine 2, 3-Dioxygenase in Human Recurrent Miscarriage

Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that mediates the conversion of tryptophan to kynurenine, was shown to play a key role in placental development during normal pregnancy. However, little is known about the pattern of IDO expression in the endometrium and its attendant functional significance in pregnancies complicated with recurrent miscarriage (RM). Immunohistochemical studies of IDO, Foxp3, CD56, and CD163 expression were performed in endometrial samples from women with RM and healthy fertile controls. Our study found that IDO was localized in glandular epithelial cells, surface epithelial cells, and a small number of cells within the stromal compartment (including stromal cells and leukocytes) in endometrium. Indoleamine 2, 3-dioxygenase expression in the RM group was significantly lower than control group. The Foxp3 and CD56 expression were significantly increased with the elevated IDO expression in controls but not in RM. The percentage of Foxp3 + Tregs was significantly correlated with the level of IDO expression in the control group. Comparatively, no correlation was found between the percentage of CD56 + cells, CD163 + cells, and the level of IDO expression, no matter in controls and RM patients. This study demonstrated that the downregulation of IDO expression and noncoordinated association between IDO and other endometrial immune cells were associated with RM. Our findings provide insights into the contribution of IDO in immune regulation to maintain normal pregnancy, which could be used to develop potential therapeutic methods for RM.

Differential Expression and Clinicopathological Significance of HER2, Indoleamine 2,3-Dioxygenase and PD-L1 in Urothelial Carcinoma of the Bladder.

Purpose: Evasion of the immune system by cancer cells allows for the progression of tumors. Antitumor immunotherapy has shown remarkable effects in a diverse range of cancers. The aim of this study was to determine the clinicopathological significance of human epidermal growth factor receptor 2 (HER2), indoleamine 2,3-dioxygenase (IDO), and programmed death ligand-1 (PD-L1) expression in urothelial carcinoma of the bladder (UCB). Materials and Methods: We retrospectively studied 97 patients with UCB. We performed an immunohistochemical study to measure the expression levels of HER2, IDO, and PD-L1 in UCB tissue from these 97 patients. Results: In all 97 cases, the PD-L1 expression of tumor-infiltrating immune cells (ICs) was significantly correlated with higher pathologic tumor stage (pT). In pT2–pT4 cases (n = 69), higher levels of HER2 and IDO expression in invasive tumor cells (TCs) were associated with shorter periods of disease-free survival (DFS). Conclusion: These results imply that the expression of PD-L1 in ICs of the UCB microenvironment is associated with cancer invasion and the expression of HER2 or IDO in the invasive cancer cell and suggestive of the potential for cancer recurrence. We suggest that the expression levels of IDO, HER2, and PD-L1 could be useful as targets in the development of combined cancer immunotherapeutic strategies.

Tyrosine kinase inhibitors modulate dendritic cell activity via confining c-Kit signaling and tryptophan metabolism

Dendritic cell (DC)-based vaccine has been established in tumor immunotherapy. Importantly, the efficiency of anti-tumor T-cells in draining lymph nodes is dependent on the status of DCs surrounding in tumors. It has been shown that Indoleamine 2,3-dioxygenase (IDO) plays a key role to induce tolerogenic DCs in tumor microenvironment, and tyrosine kinase inhibitors (TKIs) can suppress the function of IDO in DCs. However, the stimulatory effect of TKI-modified DCs on T cells remains unclear. In this report, we found that one type of TKI-dasatinib can modify DCs to increasing the activation of allogenic T cells. These TKI-modified DCs delayed the onset of B16 melanoma progression in mice. In mechanistic studies, TKIs did not increase the maturation but reduce the expression and phosphorylation levels of IDO and IDO mediated tryptophan metabolism in DCs. In addition, the suppressive effect of TKIs on tryptophan metabolism may be caused by blocking c-Kit pathway in DCs. Furthermore, the increased phosphorylation of general control nonderepressible (GCN2) and decreased expression of aryl hydrocarbon receptor (AhR)/aryl hydrocarbon receptor nuclear translocator (ARNT) were observed in the T cells activated by TKI-modified DCs, suggesting the enhancement of effector function of T cells. These results indicate that TKI could be used to modulate DC immunogenic activity and may potentially be applied in DC-based cancer immunotherapy.

Macrophages/Microglia Represent the Major Source of Indolamine 2,3-Dioxygenase Expression in Melanoma Metastases of the Brain.

The manifestation of brain metastases in patients with advanced melanoma is a common event that limits patient's survival and quality of life. The immunosuppressive properties of the brain parenchyma are very different compared to the rest of the body, making it plausible that the current success of cancer immunotherapies is specifically limited here. In melanoma brain metastases, the reciprocal interplay between immunosuppressive mediators such as indoleamine 2, 3-dioxygenase (IDO) or programmed cell death-ligand 1 (PD-L1) in the context of neoplastic transformation are far from being understood. Therefore, we analyzed the immunoreactive infiltrate (CD45, CD3, CD8, Forkhead box P3 [FoxP3], CD11c, CD23, CD123, CD68, Allograft Inflammatory factor 1[AIF-1]) and PD-L1 with respect to IDO expression and localization in melanoma brain metastases but also in matched metastases at extracranial sites to correlate intra- and interpatient data with therapy response and survival. Comparative tissue analysis identified macrophages/microglia as the major source of IDO expression in melanoma brain metastases. In contrast to the tumor infiltrating lymphocytes, melanoma cells per se exhibited low IDO expression levels paralleled by cell surface presentation of PD-L1 in intracranial metastases. Absolute numbers and pattern of IDO-expressing cells in metastases of the brain correlated with recruitment and localization of CD8+ T cells, implicating dynamic impact on the regulation of T cell function in the brain parenchyma. However, paired analysis of matched intra- and extracranial metastases identified significantly lower fractions of cytotoxic CD8+ T cells in intracranial metastases while all other immune cell populations remain unchanged. In line with the already established clinical benefit for PD-L1 expression in extracranial melanoma metastases, Kaplan-Meier analyses correlated PD-L1 expression in brain metastases with favorable outcome in advanced melanoma patients undergoing immune checkpoint therapy. In summary, our data provide new insights into the landscape of immunosuppressive factors in melanoma brain metastases that may be useful in the implication of novel therapeutic strategies for patients undergoing cancer immunotherapy.

IDO inhibitor synergized with radiotherapy to delay tumor growth by reversing T cell exhaustion

Previous studies suggest that radiotherapy (RT) can induce immune activation, which not only reduces the progression of tumors, but also causes the release of tumor antigens. The combination of RT and immune checkpoint blockade, such as the inhibition of programmed cell death 1 (PD‑1) and programmed cell death ligand 1 (PD‑L1), has been demonstrated to yield impressive response rates. However, a substantial proportion of patients develop resistance such therapies. Previous studies have shown that indoleamine 2,3‑dioxygenase (IDO) causes T cell exhaustion and increased formation of regulatory T cells (Tregs), upregulating the expression of inhibitory receptors and ligands. Therefore, the application of IDO inhibitors combined with RT may have a synergistic effect by relieving immunosuppression. Lewis lung cancer cell‑bearing mice were treated with the IDO inhibitor 1‑methyl‑tryptophan (1MT) and/or 10Gy RT. Tumor size was measured every day, flow cytometry was performed to measure the expression of dendritic cell (DC) maturation markers, inhibitory receptors, ligands, cytotoxic T cells and Treg phenotypic markers. Reverse transcription‑quantitative PCR was used to evaluate the mRNA expression levels of IDO, PD‑L1, PD‑1, T cell immunoglobulin domain and mucin domain 3 (TIM‑3), B‑ and T‑lymphocyte attenuator (BTLA) and galectin‑9. Compared with the control group, treatment with 1MT or RT reduced tumor growth, however, the combination therapy was more effective than either treatment alone. Flow cytometry showed the upregulation of CD80, CD86 and the major histocompatibility complex II in spleen DCs and the concurrent downregulation of CD4, CD25 and forkhead box protein P3 in lymphocytes in the treatment groups. Compared with the control group, inhibitory receptors and ligands that affect DCs and T cells were observed, expression levels of PD‑L1, PD‑1, TIM‑3, BTLA and galectin‑9 are decreased in treatment group compared with control. IDO inhibition synergized with RT to downregulate Tregs, inhibitory receptors and ligands to prevent T cell exhaustion. By activating DCs and T cells, this combination therapy may strongly enhance antitumor immunity and inhibit tumor progression.

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.

Abnormal Expression of Indoleamine 2, 3-Dioxygenase in Human Recurrent Miscarriage.

Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that mediates the conversion of tryptophan to kynurenine, was shown to play a key role in placental development during normal pregnancy. However, little is known about the pattern of IDO expression in the endometrium and its attendant functional significance in pregnancies complicated with recurrent miscarriage (RM). Immunohistochemical studies of IDO, Foxp3, CD56, and CD163 expression were performed in endometrial samples from women with RM and healthy fertile controls. Our study found that IDO was localized in glandular epithelial cells, surface epithelial cells, and a small number of cells within the stromal compartment (including stromal cells and leukocytes) in endometrium. Indoleamine 2, 3-dioxygenase expression in the RM group was significantly lower than control group. The Foxp3 and CD56 expression were significantly increased with the elevated IDO expression in controls but not in RM. The percentage of Foxp3+ Tregs was significantly correlated with the level of IDO expression in the control group. Comparatively, no correlation was found between the percentage of CD56+ cells, CD163+ cells, and the level of IDO expression, no matter in controls and RM patients. This study demonstrated that the downregulation of IDO expression and noncoordinated association between IDO and other endometrial immune cells were associated with RM. Our findings provide insights into the contribution of IDO in immune regulation to maintain normal pregnancy, which could be used to develop potential therapeutic methods for RM.

Expression of indoleamine 2, 3-dioxygenase and cytoplasmic polyadenylation element binding protein 4 in gastric carcinoma and their clinical significance

Objective To investigate the expression of indoleamine 2, 3-dioxygenase (IDO) and cytoplasmic polyadenylation element binding protein 4 (CPEB4) in gastric carcinoma and their clinical significance. Methods A total of 92 cases of gastric carcinoma from January 2015 to December 2017 were collected. The immunohistochemical method was used to detect and analyze the expression levels of IDO and CPEB4 in gastric carcinoma and adjacent gastric tissues. Results The expression rate of IDO was 65.22% (60/92) in gastric carcinoma tissues, and 38.04% (35/92) in non-cancerous adjacent gastric tissues (t= 13.60, P<0.01). The expressions levels of IDO were related gastric carcinoma to TNM stage and lymph node metastasis (t=7.64, 5.37 respectively, P<0.05). The expression rate of CPEB4 was 57.61% (53/92) in gastric carcinoma tissues, and 32.61% (30/92) in non-cancerous adjacent gastric tissues (t=11.61, P<0.01). The expression levels of CPEB4 were related gastric carcinoma to histology grade, TNM stage and lymph node metastasis (t= 5.58, 6.85, 7.35 respectively, P<0.05). Conclusion The expression levels of IDO and CPEB4 may be related to the carcinogenesis and progression of gastric carcinoma. Key words: Indoleamine 2, 3-dioxygenase; Cytoplasmic polyadenylation element binding protein 4; Gastric carcinoma; Immunohistochemical method

High Expression of Indoleamine 2, 3-Dioxygenase in Adenosquamous Lung Carcinoma Correlates with Favorable Patient Outcome.

Background: Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan (Trp) metabolism, is generally considered to be an immunosuppressive molecule. The prognostic role of IDO expression in tumor has not been well studied in non-small cell lung cancer (NSCLC) and even not been reported in adenosquamous lung carcinoma (AdSqLC). Herein, the aim of this study is to investigate the prognostic significance of IDO expression in patients with AdSqLC.Patients and Methods: We conducted immunohistochemical analyses of IDO expression, as well as CD3 and CD8 expression, in 183 primary tumor tissue samples from patients with AdSqLC treated at our institution between July 1999 and September 2014. Patients' clinicopathological characteristics were retrospectively reviewed. Survival analysis was performed in the entire cohort of patients and those who received radical resection, respectively.Results: IDO was expressed in 146 (79.8%) tumor samples. A higher level of IDO expression was significantly associated with increased CD8+ tumor-infiltrating lymphocytes (TILs) in tumor tissues (P<0.001). Surprisingly, overall survival (OS) was significantly better for patients with high IDO expression (hazard ratio (HR)= 0.505; confidence interval (CI)= 0.329-0.775; P=0.002) for the entire cohort. In patients who were unable to be treated with radical resection, IDO expression had no effect on OS (P=0.598). In contrast, a significant, independent association between high expression of IDO and better OS (HR=0.469; CI=0.290-0.758; P=0.002) was identified in patients who received radical resection.Conclusions: IDO is expressed in most AdSqLC tissues, with a higher level of IDO expression associated with an occurrence of CD8+ TILs. Moreover, IDO expression in tumor promises to serve as a strongly independent favorable prognostic factor, particularly in patients who received radical resection.

Early Maternal Deprivation Induces Microglial Activation, Alters Glial Fibrillary Acidic Protein Immunoreactivity and Indoleamine 2,3-Dioxygenase during the Development of Offspring Rats.

Maternal deprivation (MD) induces behavioral changes and impacts brain circuits that could be associated with the pathophysiology of depression. This study investigated the markers of microglia and astrocyte activation as well as indoleamine 2,3-dioxygenase (IDO) expression in developmental programming after early life MD (on postnatal days (PNDs) 20, 30, 40, and 60). On PND 60, the rats that were subjected to MD displayed depressive-like behavior. On PND 10, it was found that there was a decrease in the level of glial fibrillary acidic protein (GFAP) immunopositive cells, a decrease in the level of IDO expression, and an increase in the level of Iba-1 (microglial marker) in the hippocampus of rats that were subjected to MD. On PND 20, levels of GFAP were also found to have decreased in the hippocampus, and there was an increase in the level of Iba-1 in the hippocampus. AIF-1 (microglial marker) expression was observed in the PFC following MD. On PND 30, the levels of Iba-1 remained elevated. On PND 40, the levels of GFAP were found to have increased in the hippocampus of rats that were subjected to MD. On PND 60, the levels of GFAP and AIF-1 remained elevated following MD. These results suggest that early life stress induces negative developmental programming in rats, as demonstrated by depressive-like behavior in adult life. Moreover, MD increases microglial activation in both early and late developmental phases. The levels of GFAP and IDO decreased in the early stages but were found to be higher in later developmental periods. These findings suggest that MD could differentially affect the expression of the IDO enzyme, astrocytes, and microglial activation depending on the neurodevelopmental period. The onset of an inflammatory state from resident brain cells could be associated with the activation of the kynurenine pathway and the development of depressive behavior in adulthood.

High Indoleamine 2,3-Dioxygenase Is Correlated With Microvessel Density and Worse Prognosis in Breast Cancer.

Indoleamine 2,3-dioxygenase (IDO), which catalyzes the breakdown of the essential amino acid tryptophan into kynurenine, is understood to have a key role in cancer immunotherapy. IDO has also received more attention because of its non-immune functions including regulating angiogenesis. The purpose of this study was to investigate the effects of IDO on microvessel density (MVD), and to explore its prognostic role in breast cancer. We showed IDO expression was positively correlated with MVD labeled by CD105 (MVD-CD105) rather than MVD labeled by CD31 (MVD-CD31) in breast cancer specimens. Both IDO expression and MVD-CD105 level were associated with initial TNM stage, histological grade, and tumor-draining lymph nodes (TDLNs) metastasis in breast cancer. In the prognostic analysis, TDLNs metastasis, an advanced TNM stage (III) and high histological grade (III) significantly predicted shorter survival in univariate analysis. Concentrating on IDO and MVD, the patients with IDO expression or high MVD level had poorer prognosis compared with no IDO expression [P = 0.047 for progress-free survival (PFS)] and low MVD level (P = 0.019 for OS); the patients with IDO expression and high MVD level had a tendency with shorter overall survival when compared with non IDO expression, low MVD level, or both (P = 0.062 for OS). In multivariate analysis, an advanced TNM stage (III) was significantly associated with shorter 5-year survival rate of PFS (HR: 0.126, 95% CI: 0.024–0.669, P = 0.015). In order to verify the phenomenon of IDO promoting angiogenesis, we contained the study in vitro. We detected the expression of IDO mRNA in breast cancer cell lines and measured the concentration of tryptophan and kynurenine in the supernatants of MCF-7 by high performance liquid chromatography. The ratio of Kyn and trp (kyn/trp) was calculated to estimate IDO-enzyme activity. MCF-7 cells, which produce high level of IDO and metabolize tryptophan, promoted human umbilical vein endothelial cells (HUVEC) proliferation significantly in co-culture system. Meanwhile IDO could upregulate the expression of CD105 in HUVEC, which was downregulated after adding IDO inhibitor, 1-methyl-d-trytophan. These results suggest that IDO could promote angiogenesis in breast cancer, providing a novel, potentially effective molecular or gene therapy target for angiogenesis inhibition in the future.

Indoleamine 2,3‐dioxygenase expression is correlated to prognosis in localized prostate cancer.

BackgroundIndoleamine 2,3‐dioxygenase (IDO) is an enzyme that acts as an immunomodulatory molecule. It is found in several cancer types where it seems to be associated with tumor escape due to its immunosuppressive mechanisms. Some studies have reported a correlation between carcinogenesis and changes in IDO expression. However, the role of IDO expression in prostate cancer (PC) is unclear. The aim of our study was to evaluate the expression of IDO in localized PC, and to correlate its expression with the classic prognostic factor and recurrence after surgical treatment.MethodsFor this case‐control study, we retrospectively evaluated surgical specimens from 111 patients with localized PC, who underwent radical prostatectomy. Recurrence was defined as a prostate specific antigen (PSA) level exceeding 0.2 ng/ml postoperatively, and the median follow‐up was 123 months. IDO expression was evaluated by immunohistochemistry in 72 cases of which 42 (58%) had biochemical recurrence.ResultsWe found that lower IDO expression was associated with higher Gleason score (p = 0.022) and PSA levels (p = 0.042). The multivariate analyses revealed that the loss of IDO expression and higher PSA levels were independently associated with biochemical recurrence. The chance of recurrence was increased by 85% in patients with lower IDO expression (OR = 0.15; p = 0.009 CI 95% [0.038–0.633]) and increased by 5.5 times in patients with higher PSA levels (OR = 5.51; p = 0.012 CI 95% [1.435–21.21]). The recurrence‐free survival curve also demonstrates that lower IDO expression (p = 0.0004) was associated with lower time to biochemical recurrence.ConclusionThe loss of IDO expression was associated with increased chance of biochemical recurrence, higher PSA, and a Gleason score in localized PC.Support or Funding InformationThe study was supported by São Paulo Research Foundation (FAPESP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Photosensitizer Micelles Together with IDO Inhibitor Enhance Cancer Photothermal Therapy and Immunotherapy.

The therapeutic outcome of photothermal therapy (PTT) remains impeded by the transparent depth of light. Combining PTT with immunotherapy provides strategies to solve this problem. Regulating metabolism‐related enzymes is a promising strategy to stimulate immune response. Here, a nanosystem (NLG919/IR780 micelles) with the properties of photothermal conversion and regulation of the tryptophan metabolic pathway is used to suppress the growth of the tumor margin beyond effective PTT and promote tumor PTT and immunotherapy. It is revealed that mild heat treatment promotes the growth of the tumor margin beyond effective PTT for the upregulation of heat shock protein (HSP), indoleamine 2,3‐dioxygenase (IDO), and programmed death‐ligand 1 (PD‐L1). The NLG919/IR780 micelles can effectively inhibit the activity of IDO but do not affect the level of IDO expression. NLG919/IR780 micelles can effectively accumulate in the tumor and can migrate to lymph nodes and the lymphatic system. In vivo antitumor studies reveal that NLG919/IR780 micelles effectively suppress the growth of tumor margin following PTT in primary tumors. NLG919/IR780 micelle‐mediated PTT and IDO inhibition further stimulate the activation of T lymphocytes, inhibiting the growth of distal tumors (abscopal effect). The results demonstrate that the NLG919/IR780 micelles combine PTT and immunotherapy and suppress the tumor margin as well as distal tumor growth post photothermal therapy.

Expression of programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO) in the tumor microenvironment and in tumor-draining lymph nodes of breast cancer

Programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase (IDO) are both immunosuppressive proteins. Here, we investigated the relationship between PD-1 and IDO in the tumor microenvironment (TME) and in tumor-draining lymph nodes (TDLNs) in breast cancer patients. First, the protein and mRNA expression levels of PD-1 and IDO in 20 frozen tissues were examined using Western blotting and real-time polymerase chain reaction. Second, 151 paraffin-embedded breast samples and 52 lymph node samples were analyzed by immunohistochemistry. Third, correlation and survival data for PD-1 and IDO in 963 breast tumor patients were mined using the cBio Cancer Genomics Portal. We found that the protein expression level of IDO was significantly increased in frozen tumor tissues (P = .005). From paraffin-embedded samples in the TME, PD-1+ cells were only located in the stroma, while IDO was expressed in myoepithelial, stromal, and tumor cells. PD-1 and stromal IDO in the TME showed increased expression in tumors (P< .001 and P < .001, respectively). In TDLNs, PD-1+ cells were primarily located in the germinal centers (GCs), and IDO+ cells were primarily located in the paracortex. Normal lymph nodes expressed PD-1 and IDO at the same level as non-metastatic and metastatic lymph nodes (P = .151 and P = .812, respectively). According to cBioPortal, the correlation analysis showed that IDO and PD-1 had high correlation coefficients (r = 0.83). These findings suggest that there is a positive correlation between the expression of PD-1 and IDO and that blocking both PD-1 and IDO pathways may represent an attractive therapeutic strategy in breast cancer treatment.

Abstract A46: Upregulation of IDO in bacterial vaginosis: Role in HPV coinfection

Abstract Introduction: Bacterial vaginosis, a modification of the vaginal microbiota, is positively associated with the acquisition of certain genital infections, including cervical human papillomavirus (HPV) infection. In turn, HPV infection is the chief etiologic agent in the development of cervical cancer. Although HPV infection is widely prevalent, only few infected women will develop cervical cancer, suggesting that environmental or host factors contribute to malignancy. In this study, we are interested in identifying if the enzyme indoleamine-2,3-dioxygenase (IDO) has a role in the mechanism by which BV contributes to HPV infection and cervical lesion. IDO catalyzes the oxidation of the essential amino acid tryptophan (Trp) to kynurenine. IDO expression is induced by IFN-gamma and has been linked to T-cell immunosuppression and immune tolerance. IDO also plays an important role in host–pathogen interactions during viral and bacterial infections and on immune-escape of cancer cells. Methods: This cross-sectional study included 296 women attending the Women's Hospital - CAISM/UNICAMP. The case group was composed of women who had a confirmed histologic diagnosis of cervical intraepithelial neoplasia (CIN) grade 1, 2, or 3 or squamous cell carcinoma (SCC). The control group was composed of women who presented at least two previous normal Pap smears. The levels of IDO expression were analyzed by immunocytochemistry in liquid-based cytology samples from 196 women. Score 1 and 2 (0 to 50% of DAB stained squamous cells) were considered as mild expression and Score 3 and 4 (51 to 100% of DAB stained squamous cells) were considered as strong expression. Immunocytochemical analysis considered both atypical and typical squamous in the counting. The Gram-stained slides were analyzed by Nugent criteria. We used the Linear Array Genotyping HPV kit for detection of 37 types of low- and high-risk HPVs. Multiplex PCR was performed for the diagnosis of Chlamydia trachomatis (Ct), Neisseria gonorrhoeae (Ng), and Trichomonas vaginalis. Study variables were evaluated by odds ratio (OR) with a 95% confidence interval (CI) and Qui-square test and significance level was defined at 5%. Results: BV was positively associated with genital infection by HPV (p = 0.02), Trichomonas vaginalis (p=0.001) and endocervicitis by Ct and Ng (p= 0.000003). We found a higher frequency of strong expression of IDO (74%, 31/42) in women with BV when compared with women without BV (44%, 60/137 and p= 0.0007). Although the expression of IDO was slightly greater for HPV infection associated with BV (62%, 15/24), and women with CIN1 and CIN3 had a slightly higher frequency of IDO expression, these associations were not statistically significant. No correlation was found for IDO and CIN2, CIN3, or SCC. Conclusion: Here we show that IDO is upregulated in BV but is not associated with the severity of cervical lesion. We also found that BV is positively associated with genital infection by HPV. We suggest that upregulation of IDO in BV could be one of the cofactors that lead to an increased risk of HPV coinfection, whose importance is attenuated during cervical lesion progression. Ongoing studies are focused on the analysis of IDO expression, specifically in the atypical cells of cervical lesions. Citation Format: Paloma Almeida Venancio, Silvya Stuchi Maria-Engler, Marcia Edilaine Lopes Consolaro, Luisa Lina Villa, Sophie Françoise Derchain, Ana Campa, Michelle Garcia Discacciati. Upregulation of IDO in bacterial vaginosis: Role in HPV coinfection [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A46.