Indoleamine 2,3-dioxygenase mRNA Research Articles

ASOptimizer: Optimizing antisense oligonucleotides through deep learning for IDO1 gene regulation

Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered undruggable; however, manually designing optimal ASO sequences can be labor intensive and time consuming, which potentially limits their broader application. To address this challenge, we introduce a platform, the ASOptimizer, a deep-learning-based framework that efficiently designs ASOs at a low cost. This platform not only selects the most efficient mRNA target sites but also optimizes the chemical modifications for enhanced performance. Indoleamine 2,3-dioxygenase 1 (IDO1) promotes cancer survival by depleting tryptophan and producing kynurenine, leading to immunosuppression through the aryl-hydrocarbon receptor (Ahr) pathway within the tumor microenvironment. We used ASOptimizer to identify ASOs that target IDO1 mRNA as potential cancer therapeutics. Our methodology consists of two stages: sequence engineering and chemical engineering. During the sequence-engineering stage, we optimized and predicted ASO sequences that could target IDO1 mRNA efficiently. In the chemical-engineering stage, we further refined these ASOs to enhance their inhibitory activity while reducing their potential cytotoxicity. In conclusion, our research demonstrates the potential of ASOptimizer for identifying ASOs with improved efficacy and safety.

ARID5A stabilizes Indoleamine 2,3-dioxygenase expression and enhances CAR T cell exhaustion in colorectal cancer

Resistance to chimeric antigen receptor (CAR) T-cell therapy remains a significant challenge in the treatment of solid tumors. This resistance is attributed to various factors, including antigen loss, immunosuppressive tumor microenvironment, and upregulated checkpoint molecules. Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that promotes immune escape in tumors. In this study, we investigated the role of ARID5A (AT-rich interactive domain 5A) in resistance to CAR-T cell therapy. Our findings revealed that ARID5A upregulation in tumor cells induces T cell exhaustion and immune evasion. Mechanistically, ARID5A plays a crucial role in resistance to CAR-T cell therapy by stabilizing IDO1 mRNA, leading to upregulation of IDO1 expression. Elevated IDO1 expression facilitates the conversion of tryptophan to kynurenine, which contributes to CAR-T cell exhaustion. Moreover, kynurenine accumulation within CAR-T cells activates the aryl hydrocarbon receptor (AhR), further exacerbating the exhaustion phenotype. Importantly, we demonstrated that targeting the ARID5A-IDO1-AhR axis using AhR or IDO1 inhibitors effectively alleviated T cell exhaustion induced by ARID5A. These findings suggest that modulating the ARID5A-IDO1-AhR axis may represent a promising therapeutic strategy to overcome CAR T-cell therapy resistance in solid tumors and enhance treatment efficacy.

Antidepressant effects of Peiyuan Jieyu formula in a mouse model of chronic stress in conjunction with lipopolysaccharide-induced depression

ObjectiveTo explore the mechanism of the Peiyuan Jieyu formula in treating depression by assessing its impact on a lipopolysaccharide-induced (LPS-induced) depression mouse model. MethodsWe created a mouse model of depression by exposing mice that had previously received chronic stress to intraperitoneal LPS injections. The mice were divided into the following groups: control, model, fluoxetine, Tiansi Yin, Sini powder, and low-, medium-, and high-dose Peiyuan Jieyu formula groups. Forced swim and tail suspension tests were used to assess the efficacy of the depression (despair) model, and weight gain rates were also measured. Furthermore, serum levels of various depression and inflammation-associated molecules, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), tryptophan, 5-hydroxytryptamine, kynurenine (KYN), and kynurenic acid (KA) were assessed. Furthermore, the expression levels of ionic calcium-binding adaptor molecule-1 (IBA-1) and indoleamine 2,3-dioxygenase (IDO) mRNA in hippocampal microglia were measured. ResultsThe model group displayed greater despair-associated immobility, which was shortened in response to various doses of Peiyuan Jieyu formula. Furthermore, formula administration significantly reduced serum TNF-α levels and hippocampal IDO mRNA expression. The high formula dose also reduced IFN-γ and IBA-1 levels, the latter was also decreased in response to the medium formula dose. However, the low formula dose reduced serum KYN level and KYN/tryptophan (TRP) and KYN/KA ratios. ConclusionThe Peiyuan Jieyu formula holds immense potential in treating depression in a mouse model, potentially inhibiting inflammation and improving TRP-KYN metabolic disorders.

IL-1β mediates the induction of immune checkpoint regulators IDO1 and PD-L1 in lung adenocarcinoma cells

IntroductionInflammation plays a significant role in various cancers, including lung cancer, where the inflammatory cytokine IL-1β is often elevated in the tumor microenvironment. Patients with lung adenocarcinoma show higher levels of serum IL-1β compared to healthy individual. Moreover, IL-1β blockade reduces the incidence and mortality of lung cancer. Our prior studies revealed that alveolar type-II cells, the precursors for lung adenocarcinoma, display an induction in the expression of the enzyme tryptophan 2,3-dioxygenase (TDO2) during normal lung development. This induction of TDO2 coincides with an increase in IL-1β levels and is likely caused by IL-1β. Given that cancer cells can co-opt developmentally regulated pathways, we hypothesized that IL-1β may exert its pro-tumoral function by stimulating TDO2 and indoleamine 2, 3-dioxygenase-1 (IDO1), parallel enzymes involved in the conversion of tryptophan (Trp) into the immune-suppressive oncometabolite kynurenine (Kyn). Our goal was to determine whether IL-1β is a common upstream regulator of immune checkpoint regulators.MethodsTo determine whether IL-1β regulates IDO1, TDO2, PD-L1, and PD-L2, we measured mRNA and protein levels in lung adenocarcinoma cells lines (A549, H1792, H1838, H2347, H2228, HCC364 and HCC827) grown in 2D or 3D and in immortalized normal lung epithelial cells (HBEC3-KT and HSAEC1-KT). To determine the importance of the NFκB pathway in mediating IL-1β -regulated cellular effects, we used siRNA to knockdown RelA/p65 in IL-1β treated cells. The levels of Trp and Kyn in the IL-1β-treated cells and media were measured by mass spectrometry.ResultsUpon IL-1β stimulation, lung adenocarcinoma cells exhibited significant increases in IDO1 mRNA and protein levels, a response that depended on the NFκB pathway. Interestingly, this induction was more pronounced in 3D spheroid cultures compared to monolayer cultures and was not observed in normal immortalized lung epithelial cells. Furthermore, the conversion of Trp to Kyn increased in cells exposed to IL-1β, aligning with the heightened IDO1 expression. Remarkably, IL-1β also upregulated the expression of programmed death ligand-1 (PD-L1) and PD-L2 in multiple cell lines, indicating that IL-1β triggers parallel immune-suppressive mechanisms in lung adenocarcinoma cells.ConclusionsOur studies demonstrate that lung adenocarcinoma cells, but not normal immortalized lung epithelial cells, respond to IL-1β signaling by inducing the expression of parallel immune checkpoint proteins that have the potential to promote immune evasion.AWNWNjEXZEagXxNY6iY8VPVideo

IDO-Kynurenine pathway mediates NLRP3 inflammasome activation-induced postoperative cognitive impairment in aged mice

Aim Postoperative cognitive dysfunction (POCD) is a common postoperative complication, especially in elderly patients. It extends hospital stay, increases the mortality rate and are heavy burdens to the family and society. Accumulating research has indicated that overactivation of pyrin domain-containing protein 3 (NLRP3) inflammasomes is related to POCD andplays a critical role in activating pro-inflammatory cytokines. According to existing studies, indoleamine 2,3-dioxygenase (IDO) is potently up-regulated by inflammatory factors, tryptophan in brain is mainly catalyzed by IDO to kynurenine (KYN), KYN metabolism may contribute to the development of depressive disorder and memory deficits. Hence, this study elucidated whether IDO-Kynurenine pathway mediates NLRP3 inflammasome activation-induced postoperative cognitive impairment in aged mice. Material and Methods POCD model was established in aged C57BL/6J mice by exploratory laparotomy under isoflurane anesthesia. Learning and memory were determined using Morris water maze. Results The data showed that IDO and kynurenine aminotransferase-II (KAT-II) mRNA in hippocampus was up-regulated, and NLRP3, caspase recruitment domain (ASC), interleukin-1b (IL-1b) and IDO overexpressed, KYN levels increased after anesthesia and surgery. NLRP3 inflammasome inhibitor (MCC950) reversed NLRP3, ASC, IL-1b and IDO overexpression, and the elevation of KYN levels. To clarify the role of IDO-Kynurenine pathway in postoperative cognitive impairment, IDO inhibitor (1-methyl-Ltryptophan 1-MT) reduced the elevation of KYN and kynurenic acid (KYNA) levels, reduction of tryptophan (TRP), as well as improved learning and memory abilities. Finally, KAT-II inhibitor (PF-04859989) reduced brain KYNA levels and restored the cognitive impairment. Conclusion These results reveal that IDO-Kynurenine pathway mediates NLRP3 inflammasome activation-induced postoperative cognitive impairment.

Increased expression of IDO1 is associated with improved survival and increased number of TILs in patients with high-grade serous ovarian cancer

BackgroundThe enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a crucial role in regulating the immune system's response to tumors, but its exact role in cancer, especially in high-grade serous ovarian cancer (HGSOC), remains controversial. We aimed to investigate the prognostic impact of IDO1 expression and its correlation with tumor-infiltrating lymphocytes (TILs) in HGSOC. MethodsImmunohistochemical (IHC) staining and bioimage analysis using the QuPath software were employed to assess IDO1 protein expression in a well-characterized cohort of 507 patients with primary HGSOC. Statistical evaluation was performed using SPSS, and in silico validation considering IDO1 mRNA expression in bulk and single-cell gene expression datasets was conducted. Additionally, IDO1 expression in interferon-gamma (IFNG) stimulated HGSOC cell lines was analyzed. ResultsOur findings revealed that IDO1 protein and mRNA expression serve as positive prognostic markers for overall survival (OS) and progression-free survival (PFS) in HGSOC. High IDO1 expression was associated with a significant improvement in OS by 21 months (p < 0.001) and PFS by 6 months (p = 0.016). Notably, elevated IDO1 expression correlated with an increased number of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ TILs (p < 0.001). Furthermore, high IDO1 mRNA expression and protein level were found to be associated with enhanced responsiveness to pro-inflammatory cytokines, particularly IFNG. ConclusionsOur study provides evidence that IDO1 expression serves as a positive prognostic marker in HGSOC and is associated with an increased number of CD3+, CD4+ and CD8+ TILs. Understanding the intricate relationship between IDO1, TILs, and the tumor microenvironment may hold the key to improving outcomes in HGSOC.

Promotion of skin wound healing using hypoimmunogenic epidermal cell sheets

ObjectiveThe physiological process of wound healing is dynamic, continuous, and intricate. Nowadays, full-thickness burn wounds are treated by autologous skin transplantation. Unfortunately, when substantial burns develop, there are fewer donor sites accessible, making it difficult to satisfy the requirement for large-scale skin transplants and increasing the risk of patient mortality. This study investigated the possibility of using a newly created hypoimmunogenic epidermal cell sheet to heal skin wounds. MethodsTransfection with lentivirus was used to generate Keratinocytes (KCs) that overexpress Indoleamine 2,3-Dioxygenase (IDO). Western blotting and quantitative polymerase chain reaction were used to measure IDO levels. To evaluate the function of IDO+ keratinocytes, CCK-8 and Transwell assays were performed. In cell sheet induction media, KCs and Fibroblasts (FBs) were cultured to yield epidermal cell sheets. The full-thickness skin excisions of BALB/c mice were transplanted with epidermal cell sheets. To assess the tumorigenicity of IDO+ keratinocytes, BALB/c nude mouse xenograft models were also used. CD3 and CD31 immunofluorescence labeling of wound tissue on day 12 to identify T lymphocyte infiltration and capillary development. ELISA measurement of IL-1 and TNF-α concentrations. ResultsIDO + keratinocytes dramatically enhanced the expression levels of IDO mRNA and protein, as well as the amount of kynurenine in the conditioned media of IDO+ keratinocytes, compared to the Control and NC groups. CD8+ T cell apoptosis was considerably greater in the IDO group than in the Control and NC groups. Nevertheless, the proliferation and migratory capabilities of IDO+ keratinocytes were not substantially different from those of the Control and NC groups. In vitro cultivation of the hypoimmunogenic epidermal cell sheet was effective. In vivo transplantation experiments demonstrated that IDO+ epidermal cell sheets can effectively promote wound healing without tumorigenicity, and IDO+ epidermal cell sheets may promote wound healing by decreasing the expression levels of inflammatory factors (TNF and IL-1) in wound tissue, decreasing CD3+ T lymphocytes, and increasing infiltration and new capillaries in wound tissue. ConclusionIn this study, we successfully constructed the hypoimmunogenic epidermal cell sheet and demonstrated that the hypoimmunogenic epidermal cell sheet could accelerate wound healing.

Breast cancer progression and kynurenine pathway enzymes are induced by hexachlorobenzene exposure in a Her2-positive model.

Breast cancer is one of the leading cancers among women worldwide. Given the evidence that pesticides play an important role in breast cancer, interest has grown in pesticide impact on disease progression. Hexachlorobenzene (HCB), an aryl hydrocarbon receptor (AhR) ligand, promotes triple-negative breast cancer cell migration and invasion. Estrogen receptor β (ERβ) inhibits cancer motility, while G protein-coupled ER (GPER) modulates the neoplastic transformation. Tryptophan is metabolized through the kynurenine pathway by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), with kynurenine signaling activation often predicting worse prognosis in cancer. In this context, we examined the HCB (0.005; 0.05; 0.5 and 5μM) effect on LM3 cells, a human epidermal growth factor receptor 2 (HER2)-positive breast cancer model. Results show that HCB increases IDO and TDO mRNA levels and promotes cell viability, proliferation and migration through the AhR pathway. Moreover, HCB boosts mammosphere formation, vascular endothelial growth factor and cyclooxygenase-2 expression and reduces IL-10 levels. For some parameters, U-shaped or inverted U-shaped dose-response curves are shown. HCB alters ER levels, reducing ERβ while increasing GPER. These results demonstrate that exposure to environmentally relevant concentrations of HCB up-regulates the kynurenine pathway and dysregulates ERβ and GPER levels, collaborating in HER2-positive breast cancer progression.

Tryptophan degradation enzymes expression in the placenta and the Kynurenine/Tryptophan ratio in maternal plasma after elective cesarean section

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) metabolize tryptophan in the kynurenine pathway. We evaluated these enzymes’ mRNA expression in maternal and fetal sides of the placenta of uncomplicated, unlabored full-term pregnancies after elective cesarean section and compared it with that of placentas obtained from vaginal delivery. Tryptophan and kynurenine plasmatic levels after cesarean section were measured, to investigate their possible correlation with IDO1 and TDO mRNA (TDO2) expression. The results suggested that IDO1 and TDO2 expression was higher in the maternal side of the placenta and that labor significantly affects TDO2 expression and the plasma Kynurenine/Tryptophan ratio.

Effect of apigenin on tryptophan metabolic key enzymes expression in lipopolysaccharide-induced microglial cells and its mechanism

[Aims] Flavonoid apigenin (API) has a wide range of biological functions, particularly anti-inflammation. Indoleamine 2,3-dioxygenase (IDO) and 2-Amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) are important tryptophan metabolic enzymes that play pivotal roles in the production of toxic metabolite quinolinic acid. However, the relationship between inflammation and ACMSD remains unclear. The present study investigated the relationship between inflammation and tryptophan metabolic key enzymes. Similarly, the anti-inflammatory effect of API on important tryptophan metabolic enzymes was examined in lipopolysaccharide (LPS)-treated microglial cells.[Main methods] MG6 cells were exposed to LPS with or without API treatment for 24–48 h. IDO and ACMSD mRNA expression and production of inflammatory mediators were analyzed. Activation of inflammatory signaling pathways, such as mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB), was also examined to study the mechanism of API in the inflammatory state.[Key findings] LPS suppressed ACMSD expression and enhanced IDO expression. However, API elevated ACMSD mRNA expression and suppressed IDO mRNA expression in LPS-treated MG6 cells. Furthermore, API suppressed interleukin-6 and nitric oxide production, whereas overproduction of inflammatory mediators enhanced IDO expression and assisted tryptophan degradation. API also inhibited activation of extracellular signal-regulated kinase (Erk) and jun N-terminal kinase (JNK) MAPK, and degradation of IκBα.[Significance] These results indicate alteration of ACMSD expression under inflammatory conditions. Moreover, API recovers expression of tryptophan metabolic key enzymes, which may be mediated by inhibition of proinflammatory mediator production via inactivation of Erk, JNK MAPK, and NF-κB pathways in LPS-stimulated microglial cells.

Indoleamine 2,3-dioxygenase gene expression and kynurenine to tryptophan ratio correlation with nasopharyngeal carcinoma progression and survival.

IntroductionIndoleamine 2,3‐dioxygenase (IDO) is an immunosuppressive tryptophan‐depleting enzyme expressed in nasopharyngeal carcinoma (NPC) tissue. However, IDO has not been reported in the peripheral blood of NPC patients. The aim of this study was to analyze, IDO1 and IDO2 messenger RNA (mRNA) expression, the kynurenine (Kyn) and tryptophan (Trp) plasma levels, their clinical values and their relationship with cytokine levels in NPC.MethodsWe evaluated IDO1 and IDO2 mRNA expression in peripheral blood mononuclear cells (PBMC) by quantitative real‐time PCR, plasma Trp and Kyn levels by HPLC, and cytokine levels by ELISA in 75 NPC patients and 51 healthy controls.ResultsCompared to controls, IDO1 mRNA expression was significantly upregulated and IDO2 mRNA expression was significantly downregulated in PBMC of patients. Also compared to controls, plasma Kyn levels and Kyn/Trp ratio were significantly higher in patients. At the time of diagnosis, the plasma Kyn/Trp ratio was associated with advanced cancer status and was an independent prognostic factor for worse disease‐specific survival. According to cancer stages, IDO1 mRNA expression was positively correlated with plasma Kyn/Trp ratio in patients with earlier stages (I–II–III) but negatively correlated in patients with the late‐stage cancer (IV). Tumor necrosis factor‐α, interleukin (IL)‐6 and IL‐10 levels were significantly higher in patients compared to controls. Moreover, and despite treatment, patients simultaneously carrying high plasma Kyn/Trp ratio and high plasma IL‐6 and IL‐10 levels at diagnosis died approximately 1 year after first diagnosis.ConclusionMeasuring blood IDO mRNA expression and Kyn/Trp ratio at diagnosis could be a potential marker to evaluate NPC progression and predict survival outcome.

Xenotransplantation of cryopreserved human clumps of mesenchymal stem cells/extracellular matrix complexes pretreated with IFN-γ induces rat calvarial bone regeneration.

IntroductionThree-dimensional (3D) clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes, composed with cells and self-produced intact ECM, can be grafted into defect areas without artificial scaffold to induce successful bone regeneration. Moreover, C-MSCs pretreated with IFN-γ (C-MSCsγ) increased the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) expression and thereby inhibited T cell activity. Xenotransplantation of human C-MSCsγ suppressed host T cell immune rejection and induced bone regeneration in mice. Besides, we have also reported that C-MSCs retain the 3D structure and bone regenerative property even after cryopreservation. To develop the “off-the-shelf” cell preparation for bone regenerative therapy that is promptly provided when needed, we investigated whether C-MSCsγ can retain the immunosuppressive and osteogenic properties after cryopreservation.MethodsConfluent human MSCs that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The round cell clumps were incubated with a growth medium for 3 days, and then C-MSCs were obtained. To generate C-MSCsγ, after 2 days’ culture, C-MSCs were stimulated with 50 ng/ml of IFN-γ. Both C-MSCs and C-MSCsγ were cryopreserved for 2 days and then thawed to obtain Cryo-C-MSCs and Cryo-C-MSCsγ, respectively. The biological properties of those cell clumps were assessed in vitro. In addition, to test whether human Cryo-C-MSCsγ attenuates immune rejection to induce bone regeneration, a xenograft study using a rat calvarial defect was performed.ResultsBoth IFN-γ pretreatment and cryopreservation process did not affect the 3D structure and cell viability in all human cell clumps. Interestingly, Cryo-C-MSCsγ showed significantly increased IDO mRNA expression equivalent to C-MSCsγ. More importantly, xenotransplantation of human C-MSCsγ and Cryo-C-MSCsγ induced rat calvarial bone regeneration by suppressing rat T cells infiltration and the grafted human cells reduction in the grafted area. Finally, there were no human donor cells in the newly formed bone, implying that the bone reconstruction by C-MSCsγ and Cryo-C-MSCsγ can be due to indirect host osteogenesis.ConclusionThese findings implied that Cryo-C-MSCsγ can be a promising bone regenerative allograft therapy that can be certainly and promptly supplied on demand.

T Lymphocyte Infiltration in Association with IDO1 Expression in Resected Lung Adenocarcinoma and Normal Adjacent Lung Tissues

Background Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step of tryptophan catabolism in the kynurenine (Kyn) pathway. IDO1 downregulates natural killer cell receptors, and by mechanism, tumor cells escape immune surveillance. Methods IDO1 protein and mRNA were assessed by immunohistochemistry, immunoblotting, and PCR in the 68 resected lung adenocarcinomas at stages I–III as well as adjacent normal lung tissues. Infiltration of CD3, CD8, and CD4 lymphocytes in the tumor and adjacent normal lung tissues was assessed by immunohistochemical staining. Results IDO1 protein and mRNA were detected in various stages of lung adenocarcinoma with highest expression at stage III. In contrast, biomarkers of T cell subset, CD3, CD4, and CD8, were highly expressed in the normal lung tissues and stage I adenocarcinoma tissues but significantly reduced in the stage II and III tumor tissues. Conclusions The current study demonstrated that the higher level of IDO1 expression in the lung adenocarcinoma was, the less infiltration of T lymphocytes was found in the tumors. Findings of this study indicated that IDO1 may contribute to the reduction of T lymphocyte infiltration into the lung adenocarcinoma.

Assessment of interferon gamma and indoleamine 2,3-dioxygenase 1 analysis during respiratory syncytial virus infection in infants in Italy: an observational case–control study

ObjectivesThe aim of this study was to measure interferon gamma (IFN-γ) and indoleamine 2,3-dioxygenase 1 (IDO1) values in the White blood cells of infants during respiratory tract infections and to...

Optimization of Human Mesenchymal Stem Cells for Rheumatoid Arthritis: Implications for Improved Therapeutic Outcomes.

ObjectiveSeropositive rheumatoid arthritis (RA) is a chronic autoimmune disease that is rarely “cured.” Human mesenchymal stem cells (hMSCs) are known to reduce inflammation and restore immune homeostasis. However, methods for predicting therapeutic hMSC potency have not been established. The goal of these studies was to use and refine an ex vivo functional assay that determines potency of hMSCs and can then be validated in clinical trials as a potency measure of hMSCs used therapeutically to treat RA.MethodsAllogeneic hMSCs were cytokine‐stimulated, and a conditioned medium (CM) was harvested. The CM was tested for the potential to attenuate RA CD4+ T cell proliferation using suppression assays. Indoleamine 2, 3‐dioxygenase (IDO) mRNA, and protein were quantified in hMSCs as a measure to compare hMSCs across (prior) studies.ResultsTo mimic a proinflammatory environment that resembles that in RA, interleukin‐1(IL1β), tumor necrosis factor α (TNFα), and interferon γ (IFNγ) (alone or in combination) were used to precondition hMSCs. Treating hMSCs with a combination of these cytokines generated a CM “secretome” that suppressed T cell proliferation between 70 and 83%. Forty‐eight hours of cytokine preconditioning hMSCs was required to maximize this effect. T cell suppression positively correlated with increases in hMSC cellular IDO mRNA and protein.ConclusionBy standardizing assays to measure hMSC effects, their potency on T cell suppression can be quantified. These studies demonstrate that hMSCs can be compared functionally to identify optimal preparation(s) for therapeutic use in RA and that the potency of hMSC‐dependent T cell suppression may differ between hMSC donors. Clinical studies are warranted to validate the hypothesis that ex vivo potency in suppressing T cells will positively correlate with a reduction in RA disease activity and increase in immunological quiescence.

Impact of PD-L Ligands and Indoleamine 2,3-Dioxygenase on Prognosis and Survival of Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin B-cell lymphoma and one of the most chemosensitive malignant diseases. Modern risk-adapted treatment regimens lead to a good control of the disease and high cure rate. However, 30-40% of patients are resistant to standard and salvage chemotherapeutic regimens. New therapeutic methods are needed to improve the outcome. Currently, a lot of studies are aimed to identify the interplay between immune system and tumor for some newly revealed potential biomarkers.Goals. We assessed the impact of PD-L1/2 and IDO expression on clinical outcome in patients with primary DLBCL.Methods: 31 untreated patients were included in the study (median age: 48, range: 23-74 years; males: 9, females: 22). 13 patients were diagnosed with I-II stages and 18 patients with III-IV stages of DLBCL. All patients received R-CHOP/CHOP-like regimens as a 1st-line therapy. PD-L1, PD-L2 and IDO mRNA expression levels were analyzed in fresh pre-treatment lymph node biopsies using qRT-PCR.Results: The overall response rate in the group was 56.4% (22 patients). Five patients had relapses and six patients had refractory disease (follow-up period range: 7-58 months; median duration - 32 months). All patients with refractory disease and three patients with CR/PR had high expression of PD-L ligands. In order to assess the prognostic relevance of PD-L ligands in DLBCL, Kaplan Meier curves were generated. High expression levels of PD-L1 and PD-L2 in tumors were associated with significantly poorer 3-year event free survival (EFS) compared to tumors with low expression (56% vs. 66% and 60% vs .73%, respectively, p<0.05). DLBCL patients with high expression (>0.0006) level of PD-L1 had a significantly higher risk relapse (p=0.03 by Cox' test).ROC analysis revealed that the high expression (>0.6878) of PD-L2 in tumor is an important marker that increases the relapse rate for DLBCL patients (Se=41.6 %; Sp=100 %; AUC=0.71, p=0.007).The mRNA expression of indoleamine 2,3-dioxygenase (IDO) was found in 15 cases (48.4%) and further this group was considered as IDO-positive. The presence of IDO expression was significantly associated with advanced stage of disease (p<0.05), ABC subtype and progression of DLBCL (17.4% vs. 4.3%, p<0.05). Patients with the absence of IDO expression (IDO-negative) tended to have a better response to the 1st line chemotherapy comparing to patients with IDO-positive expression. The overall response rate was achieved in 46.6% (7/15) and 68.7% (11/16) of IDO-positive and IDO-negative cases, respectively (p<0.05).3-year EFS for patients with IDO-positive expression was lower compared to patients with IDO-negative expression (46% vs .70%, p<0.05).Summary: The obtained results suggest the high level of PD-L ligands are associated with unfavorable prognosis for patients with DLBCL. Positive expression of IDO could be considered as an additional unfavorable risk factor for this group of patients. It may represent a promising therapeutic target for DLBCL patients with resistance to chemotherapy. Further studies on a larger group of patients are required in order to evaluate the impact of PD-L ligands and indoleamine 2,3-dioxygenase on prognosis and survival of Diffuse Large B-cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Tryptophan catabolism is dysregulated in leiomyomas

To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma. Experimental study. Academic research laboratory. Women undergoing hysterectomy for leiomyoma. Blockade of IDO1 and TDO2. Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix. Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids. These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas.

RETRACTED ARTICLE: Indoleamine 2,3-Dioxygenase 1 (IDO1) Promotes Cardiac Hypertrophy via a PI3K-AKT-mTOR-Dependent Mechanism

Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme for tryptophan metabolism, involved in immune cell differentiation/maturation and cancer biology. IDO1 is also expressed in cardiomyocytes, but its roles in the cardiovascular system are not fully understood. Here, we reported the functions of IDO1 during cardiac hypertrophy. Quantitative real-time PCR and Western blot experiments demonstrated the upregulation of IDO1 mRNA and protein levels in human and hypertrophic mouse hearts, as well as in angiotensin II (Ang II)-induced hypertrophic rat cardiomyocytes. IDO1 activity and metabolite product kynurenine were upregulated in rodent hypertrophic hearts and cardiomyocytes. Inhibition of IDO1 activity with PF-06840003 reduced Ang II-induced cardiac hypertrophy and rescued cardiac function in mice. siRNA-mediated knockdown of Ido1 repressed Ang II-induced growth in cardiomyocyte size and overexpression of hypertrophy-associated genes atrial natriuretic peptide (Anp or Nppa), brain natriuretic peptide (Bnp or Nppb), β-myosin heavy chain (β-Mhc or Myh7). By contrast, adenovirus-mediated rat Ido1 overexpression in cardiomyocytes promoted hypertrophic growth induced by Ang II. Mechanism analysis showed that IDO1 overexpression was associated with PI3K-AKT-mTOR signaling to activate the ribosomal protein S6 kinase 1 (S6K1), which promoted protein synthesis in Ang II-induced hypertrophy of rat cardiomyocytes. Finally, we provided evidence that inhibition of PI3K with pictilisib, AKT with perifosine, or mTOR with rapamycin, blocked the effects of IDO1 on protein synthesis and cardiomyocyte hypertrophy in Ang II-treated cells. Collectively, our findings identify that IDO1 promotes cardiomyocyte hypertrophy partially via PI3K-AKT-mTOR-S6K1 signaling.

Association of IDO immune suppression with brain metastasis in non-small cell lung cancer.

e21215 Background: Indoleamine 2, 3-dioxygenase (IDO), a known immunoinhibitory enzyme, plays an important role on tumor metastasis through manipulation of host immune status. We have demonstrated that IDO level has prognostic value and low IDO level is associated with low risk of distant metastasis in patients with non-small cell lung cancer (NSCLC). However, the association between IDO immune status in patients with brain metastasis (BrM) is unknown. We hypothesized that the IDO1 activity are different in the NSCLC patients of various stages and in patients with or without BrM and the IDO mRNA expression in brain metastatic lesion differ from the primary tumor or metastasized regional lymph nodes. Methods: This was part of a prospective study of blood immune biomarkers for prognosis and prediction. Newly diagnosed or recurrent NSCLC patients were eligible. Blood samples were obtained before treatment start and plasma were used for the Kynurenine (Kyn) and tryptophan (Trp) measurement by the high-performance chromatography. Kyn and trp was detected with more than 95% re-productivity. IDO activity was defined as ratio of kyn/Trp. Student T-test and One-way anova were applied for group comparison. CI: confidence interval. P &lt; 0.05 was considered as statistical significance. The IDO cellular expression was analyzed by the http://ureca-singlecell.kr/ website tool using the GEO dataset GSE131907. Results: Between July 2019 and Dec 2020, a total of 121 patients with NSCLC were eligible. The mean concentration of Kyn was 1.69 uM in patients with stage IV (n = 60, CI: 1.38-2.00), compared with 1.57 in patients with stage I (n = 38, CI: 1.03-2.10), 1.63 in stage II (n = 13, CI: 1.03-2.23) and 2.01 in stage III (n = 10, CI: 0.72-3.30, mean = 2.01). The mean ratio of Kyn:Trp was 0.10 in stage IV (n = 60, CI: 0.07-0.12), compared with 0.13 in patients with stage I (n = 38, CI: 0.06-0.20), 0.15 in stage II (n = 13, CI: 0.03-0.29) and 0.15 in stage III (n = 10, CI: 0.12-0.29). In patients with stage IV, there was no significant difference in the kyn concentration in patients with BrM (n = 13) and those without BrM (n = 47) (mean: 1.74, CI: 0.47-2.41 v.s 2.03 mean: 1.12-3.08; p = 0.45). The IDO activity in the patients with BrM was not significantly different from that of patients without BrM (mean: 0.11, CI: 0.03-0.20 v.s mean: 0.13, CI: 0.08-0.17; p = 0.74). Interestingly, GEO dataset analysis of the IDO1 mRNA expressions in 44 patients showed enrichments in myeloid cells in primary lung cancer tumor, natural killer cells in metastasized lymph nodes, and B cells in brain metastatic lesion. Conclusions: This study demonstrated no significant differences in circulating IDO expressions in patients with brain metastasis but differential IDO patterns of cellular expression in brain metastasis from that of primary tumor in NSCLC patients. This finding suggests the need of new strategy of research for immune status in the brain metastatic process of non-small cell lung cancer.

Interactions of human mesenchymal stromal cells with peripheral blood mononuclear cells in a Mitogenic proliferation assay

BackgroundImmunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs. MethodsPooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events. ResultsPBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations. ConclusionsA bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.