Tryptophan-catabolizing Enzyme Research Articles

Marked IDO2 expression and activity related to autophagy and apoptosis in brain tissue of fatal tuberculous meningitis

In about 1% of tuberculosis (TB) patients, Mycobacterium tuberculosis (M. tuberculosis) can disseminate to the meninges, causing tuberculous meningitis (TBM) with mortality rate up to 60%.Chronic granulomatous inflammation (non-necrotizing and necrotizing) in the brain is the histological hallmark of TBM. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and the generated kynurenine metabolites exert major effector functions relevant to TB granuloma functioning. Here we have assessed immunohistochemically IDO1 expression and activity and its effector function and that of its isoform, IDO2, in post-mortem brain tissue of patients that demised with neurotuberculosis. We also related these findings to brain tissue of fatal/severe COVID-19. In this study, IDO1 and IDO2 were abundantly expressed and active in tuberculoid granulomas and were associated with the presence of M. tuberculosis as well as markers of autophagy and apoptosis. Like in fatal/severe COVID-19, IDO2 was also prominent in specific brain regions, such as the inferior olivary nucleus of medulla oblongata and cerebellum, but not associated with granulomas or with M. tuberculosis. Spatially associated apoptosis was observed in TBM, whereas in fatal COVID-19 autophagy dominated. Together, our findings highlight IDO2 as a potentially relevant effector enzyme in TBM, which may relate to the symptomology of TBM.

TDO2 deficiency attenuates the hepatic lipid deposition and liver fibrosis in mice with diet-induced non-alcoholic fatty liver disease

PurposeNon-alcoholic fatty liver disease (NAFLD) represents an increasingly prevalent set of liver diseases. Tryptophan 2,3-dioxygenase 2 (TDO2) is the major enzyme of tryptophan catabolism and is abnormally expressed in liver cancer, but the function of TDO2 in NAFLD remains unclear. The current study was designed to probe into the effect and mechanism of TDO2 on NAFLD. MethodsC57BL/6 mice and TDO2-knockout (KO) mice were fed with a high-fat diet for 16 weeks to construct the NAFLD model in vivo; primary hepatocytes isolated from TDO2-KO mice were exposed to palmitate (PA) to establish the NAFLD model in vitro. The expression of TDO2 was determined using Western blot. The function and mechanism of TDO2 were evaluated by enzyme-linked immunosorbent assay, hematoxylin-eosin staining, Oil Red O staining, immunohistochemical assay, and Western blot. ResultsThe expression of TDO2 in the liver tissue of NAFLD mice was more than three times that in the control group. Functionally, TDO2 knockout reduced hepatic lipid deposition and liver fibrosis in NAFLD mice in vivo and primary hepatocytes induced by 200 μM PA in vitro. Mechanistically, the loss of TDO2 restrained hepatic lipid deposition and expression levels of fibrosis-related markers in PA-treated primary hepatocytes, and these trends were partially reversed by 10 ng/ml receptor activator of the nuclear factor kappa-B ligand (RANKL, an activator of the NF-κB pathway). ConclusionKnocking out TDO2 repressed hepatic lipid deposition and liver fibrosis in mice with NAFLD, and reduced hepatic lipid deposition and expressions of fibrosis-related markers in PA-treated primary hepatocytes by inactivating the NF-κB pathway.

Upregulation of Indoleamine 2,3-Dioxygenase 1 in Tumor Cells and Tertiary Lymphoid Structures is a Hallmark of Inflamed Non-Small Cell Lung Cancer.

Overexpression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been reported in several tumor types, including non-small cell lung cancer (NSCLC), and has been shown to promote tumor-immune evasion and inhibit T-cell activation through increased tryptophan degradation and the production of several immunosuppressive metabolites collectively known as kynurenines. However, it remains unclear whether IDO1 expression by tumor cells is detrimental specifically in the context of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis blockade. We analyzed the transcriptome of 891 NSCLC tumor samples from patients enrolled in two large randomized clinical trials investigating the safety and activity of atezolizumab, a humanized IgG1 mAb that targets PD-L1, versus docetaxel in patients with advanced NSCLC. We complemented these transcriptomics results at the protein level by using multiplex immunofluorescence and at the functional level with in vitro experiments. The increased expression of the tryptophan-catabolizing enzyme IDO1 was significantly associated with improved objective response, progression-free survival, and overall survival in patients treated with PD-L1 inhibitors, but not in those treated with chemotherapy. Strikingly, inflamed tumors had higher levels of IDO1, and IDO1 was also expressed in tertiary lymphoid structures (TLS) by mature follicular dendritic cells. L-kynurenine impaired the differentiation of antibody-producing B cells induced by follicular helper T (Tfh)/B-cell interactions, a hallmark process within TLS. IDO1 pathway in NSCLC is driven by the immune system rather than by tumor cells. Targeting IDO1 in combination with anti-PD-1/PD-L1 might be beneficial only in patients with inflamed tumors and particularly in those bearing TLS.

Exploring the Potential of Plant-Based CTB-INS Oral Vaccines in Treating Type 1 Diabetes

The 19th century saw the development of vaccines, which were biological preparations designed to enhance immunity against specific diseases. Edible vaccines function by stimulating both systemic and mucosal immune responses against foreign pathogens, and they may potentially protect the host from autoimmunity. The mucosal surfaces provide a convenient and rapid route for delivering therapeutic small molecules. This is due to their large surface areas and easy administration. The effectiveness of mucosal immunization relies on the fact that mucous membranes represent the body’s largest immunogenic organ. Within this interface, there is a well-organized lymphatic structure known as MALT (mucosa-associated lymphoid tissue), which includes both T and B cells and encompasses the adaptive arms of the immune system. Oral vaccines specifically stimulate immune responses in the gut-associated lymphoid tissue (GALT), which consists of lymph nodes, Payer’s patches (where B cells make up about 75% of the population and T cells account for approximately 20%), and isolated lymphoid follicles within the gastrointestinal tract (GIT). However, a significant challenge in developing vaccines is the rapid degradation of antigens within the harsh environment of the digestive tract, which hampers effective protein delivery to the GIT. In light of recent proteomic analysis revealing strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in DCs inoculated with the Cholera toxin B-subunit-Insulin fusion protein vaccine (CTB-INS), we are interested in investigating the effects of transgene integration into a selected plant cell as an edible vaccine.

Regulation of IDO2 by the Aryl Hydrocarbon Receptor (AhR) in Breast Cancer.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a tryptophan-catabolizing enzyme and a homolog of IDO1 with a distinct expression pattern compared with IDO1. In dendritic cells (DCs), IDO activity and the resulting changes in tryptophan level regulate T-cell differentiation and promote immune tolerance. Recent studies indicate that IDO2 exerts an additional, non-enzymatic function and pro-inflammatory activity, which may play an important role in diseases such as autoimmunity and cancer. Here, we investigated the impact of aryl hydrocarbon receptor (AhR) activation by endogenous compounds and environmental pollutants on the expression of IDO2. Treatment with AhR ligands induced IDO2 in MCF-7 wildtype cells but not in CRISPR-cas9 AhR-knockout MCF-7 cells. Promoter analysis with IDO2 reporter constructs revealed that the AhR-dependent induction of IDO2 involves a short-tandem repeat containing four core sequences of a xenobiotic response element (XRE) upstream of the start site of the human ido2 gene. The analysis of breast cancer datasets revealed that IDO2 expression increased in breast cancer compared with normal samples. Our findings suggest that the AhR-mediated expression of IDO2 in breast cancer could contribute to a pro-tumorigenic microenvironment in breast cancer.

Endothelial Indoleamine-2,3-Dioxygenase-1 is not Critically Involved in Regulating Antitumor Immunity in the Central Nervous System.

The vascular niche of malignant gliomas is a key compartment that shapes the immunosuppressive brain tumor microenvironment (TME). The blood-brain-barrier (BBB) consisting of specialized endothelial cells (ECs) and perivascular cells forms a tight anatomical and functional barrier critically controlling transmigration and effector function of immune cells. During neuroinflammation and tumor progression, the metabolism of the essential amino acid tryptophan (Trp) to metabolites such as kynurenine has long been identified as an important metabolic pathway suppressing immune responses. Previous studies have demonstrated that indoleamine-2,3-dioxygenase-1 (IDO1), a key rate-limiting enzyme in tryptophan catabolism, is expressed within the TME of high-grade gliomas. Here, we investigate the role of endothelial IDO1 (eIDO1) expression for brain tumor immunity. Single-cell RNA sequencing data revealed that in human glioma tissue, IDO1 is predominantly expressed by activated ECs showing a JAK/STAT signaling pathway-related CXCL11+ gene expression signature. In a syngeneic experimental glioma model, eIDO1 is induced by low-dose tumor irradiation. However, cell type-specific ablation of eIDO1 in experimental gliomas did not alter frequency and phenotype of tumor-infiltrating T cells nor tumor growth. Taken together these data argue against a dominant role of eIDO1 for brain tumor immunity.

Aberrant NAD synthetic flux in podocytes under diabetic conditions and effects of indoleamine 2,3-dioxygenase on promoting de novo NAD synthesis

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme in the kidney. The first step in de novo NAD synthesis is regulated by indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme. Here, we investigated NAD synthetic flux and NAD levels in podocytes under diabetic conditions. We also studied the effects of IDO overexpression on NAD synthetic flux and high glucose (HG)-induced podocyte injury. NAD synthetases in the de novo, Preiss-Handler and salvage pathways were analyzed using in vivo single-nucleus RNA sequencing datasets (GSE131882) of control and diabetic kidney disease (DKD). The mRNA levels of these NAD synthetases were measured in vitro in HG-treated podocytes. The effects of IDO on NAD synthesis were examined by transducing cultured podocytes with an adenovirus encoding IDO, and apoptosis, podocyte markers and mobility were investigated. Cellular transcriptome analysis revealed that control podocytes had relatively low levels of NAD synthetases. In DKD podocytes, de novo NAD synthetase levels were further downregulated. IDO levels were virtually undetectable and did not increase in DKD. In vitro experiments confirmed aberrant de novo NAD synthetic flux and decreased IDO levels in HG-treated podocytes. Overexpression of IDO promoted NAD de novo synthesis, reduced NAD-bypass metabolic enzyme, increased NAD content and recovered podocyte injury markers under diabetic conditions. Taken together, our findings suggest that the de novo NAD synthetic flux is aberrant in DKD, and IDO promotes de novo NAD synthesis and NAD levels, as well as alleviates injury in HG-treated podocytes.

The exploitation of enzyme-based cancer immunotherapy.

Cancer immunotherapy utilizes the immune system and its wide-ranging components to deliver anti-tumor responses. In immune escape mechanisms, tumor microenvironment-associated soluble factors and cell surface-bound molecules are mainly accountable for the dysfunctional activity of tumor-specific CD8+ T cells, natural killer (NK) cells, tumor associated macrophages(TAMs) and stromal cells. The myeloid-derived suppressor cells (MDSCs) and Foxp3+regulatory T cells (Tregs), are also key tumor-promoting immune cells. These potent immunosuppressive networks avert tumor rejection at various stages, affecting immunotherapies' outcomes. Numerous clinical trials have elucidated that disruption of immunosuppression could be achieved via checkpoint inhibitors. Another approach utilizes enzymes that can restore the body's potential to counter cancer by triggering the immune system inhibited by the tumor microenvironment. These immunotherapeutic enzymes can catalyze an immunostimulatory signal and modulate the tumor microenvironment via effector molecules. Herein, we have discussed the immuno-metabolic roles of various enzymes like ATP-dephosphorylating ectoenzymes, inducible Nitric Oxide Synthase, phenylamine, tryptophan, and arginine catabolizing enzymes in cancer immunotherapy. Understanding the detailed molecular mechanisms of the enzymes involved in modulating the tumor microenvironment may help find new opportunities for cancer therapeutics.

Design, synthesis and biological evaluation of erlotinib-based IDO1 inhibitors.

Erlotinib is a highly specific and reversible epidermal growth factor receptor tyrosine kinase inhibitor for the targeted therapy of non-small-cell lung cancer (NSCLC) However, the efficacy of erlotinib is limited because the development of drug resistance during chemotherapy. Indoleamine 2,3-dioxygenase-1 (IDO1) is a rate-limiting tryptophan catabolic enzyme that is activated in many human cancers. In this study, we designed a series of erlotinib-based 1,2,3-triazole compounds by combining erlotinib with phenyl or benzyl azide. Attentive FP prediction model was used to predict the bioactivity of those compounds. We discovered that most of the erlotinib-based 1,2,3-triazole compounds are capable of suppressing IDO1 activities in vitro experiments. Among them, compound 14b (IC50 = 0.59 ± 0.05 μM) had the strongest inhibitory effect on IDO1. In addition, compound 14b significantly inhibited tumor growth comparable to the antitumor activity of erlotinib and the IDO1 inhibitor epacadostat in murine tumor models.

Wheatgerm Supplementation Reduces Gut Inflammation and Epithelial Barrier Dysfunction in IL-10 KO Mice Fed Atherogenic Diet

Abstract Objectives Wheat germ (WG) contains many bioactive compounds with the potential to maintain an anti-inflammatory gut environment. This study investigated the effects of WG supplementation on gut inflammation and integrity in high-fat fed interleukin (IL)-10 KO mice. Methods Eight-wk-old female B6.129P2-Il10tm1Cgn/J (IL-10KO) and C57BL/6 (WT) mice (n = 10/group) were randomly assigned to diets: WT fed a control diet (WTCO; AIN93-M) and IL-10 KO mice fed control (KOCO), high-fat with high-cholesterol (HFHC; 45% fat kcal, 1% cholesterol), or HFHC + 10% WG (HFWG) for 3 m. Disease activity indices (fecal blood, ruffled fur, stool softness, and rectal prolapse) were monitored twice a week. Fecal indole and short chain fatty acids (SCFAs) concentration were assessed at the beginning and end of study. Proinflammatory cytokines were assessed in the serum and ileum. Ileal and colonic protein expression of transcription factors (STAT3, p-STAT3, PPARg, FoxP3, and AhR), tight junction proteins (ZO-1, occludin), and tryptophan catabolizing enzyme (IDO-1) were assessed by immunoblotting. Relative ileal and colonic gene expression of IL-22 and antimicrobial peptides (Reg3b and Reg3g) were assessed using qRT-PCR. P < 0.05 was considered statistically significant. Results WG increased (P = 0.003) colon length compared to the HFHC group. Weight loss (12.2% in HFHC vs WTCO) was not prevented by WG, but disease activity indices were significantly reduced in the WG vs HFHC group. WG also increased fecal indole, total SCFAs and acetate accompanied by an increase in colonic protein expression of PPARg (P < 0.0001) and FoxP3 (P = 0.001). Ileal STAT3 phosphorylation was reduced (P = 0.0076) due to WG supplementation. An increased colon and ileal protein expression of IDO-1 in the HFHS group was reduced by WG, while also increasing the expression of AhR, ZO-1, and occludin. The relative gene expression of the antimicrobial peptides (Reg3b and Reg3g) was increased (P < 0.05) while serum and ileal tissue concentration of the proinflammatory cytokine, IL-17 was reduced (P = 0.0165 and p = 0.0248 respectively) by WG. Conclusions WG modulated changes that are associated with HF-feeding in IL-10 KO mice, and might be a promising regimen for ameliorating the effects of gut inflammation. Funding Sources Oklahoma Agriculture Experiment Station, Jim and Lynn Williams Professorship, and Barbara K. Pass Grant.

Investigating the tryptophan‐metabolizing enzyme AFMID (arylformamidase) in colon cancer

Tryptophan is an essential amino acid which is metabolized into catabolites necessary for physiological homeostasis. Of the various catabolic fates of tryptophan, the kynurenine pathway is of both basic and clinical interests due to its signature catabolite kynurenine which contributes to immune privilege, is a precursor of NAD +, and acts as an activating ligand of the transcription factor aryl hydrocarbon receptor (AHR.) Through elevated expression of tryptophan transporters and tryptophan‐catabolizing enzymes, cancer cells promote the production of kynurenine and downstream catabolites. Oncogene‐mediated upregulation of tryptophan transporters and tryptophan catabolizing enzymes IDO1, TDO2, and arylformamidase (AFMID) occur in several cancer types including breast, esophageal, liver, and colon cancers with correlation to poor prognosis. Targeting of the kynurenine pathway has therefore been a clinical focus; however, all initial clinical trials aiming to inhibit IDO1 failed. A new generation of clinical trials targeting IDO1, TDO2, and tryptophan transporters are underway, yet no preclinical evaluation of the enzyme (AFMID) has ever been conducted. AFMID acts immediately downstream of IDO1, IDO2, and TDO2 to catalyze the reaction that produces kynurenine itself. Unlike IDO1/2 and TDO2 which are functionally redundant, AFMID features a unique function. Despite its importance, no studies have comprehensively evaluated AFMID’s direct roles in metabolic reprogramming, cell‐autonomous proliferation, or microenvironment remodeling. We aim to investigate the cell autonomous and immune modulatory functions of AFMID in colon cancer and to define novel approaches to inhibit AFMID activity as strategy to block the kynurenine pathway.

Microbiota-mediated skewing of tryptophan catabolism modulates CD4+ T cells in lupus-prone mice

SummaryA skewed tryptophan metabolism has been reported in patients with lupus. Here, we investigated the mechanisms by which it occurs in lupus-susceptible mice, and how tryptophan metabolites exacerbate T cell activation. Metabolomic analyses demonstrated that tryptophan is differentially catabolized in lupus mice compared to controls and that the microbiota played a role in this skewing. There was no evidence for differential expression of tryptophan catabolic enzymes in lupus mice, further supporting a major contribution of the microbiota to skewing. However, isolated lupus T cells processed tryptophan differently, suggesting a contribution of T cell intrinsic factors. Functionally, tryptophan and its microbial product tryptamine increased T cell metabolism and mTOR activation, while kynurenine promoted interferon gamma production, all of which have been associated with lupus. These results showed that a combination of microbial and T cell intrinsic factors promotes the production of tryptophan metabolites that enhance inflammatory phenotypes in lupus T cells.

Abstract P025: Uncovering molecular actors of IDO-mediated T cell dysfunction with genome-wide CRISPR/Cas9 knockout screens

Abstract Introduction: Despite tremendous progress in cancer immunotherapy, most patients fail to benefit because of poorly characterized immune resistance mechanisms. Among these, expression of the tryptophan-catabolizing enzyme Indoleamine 2,3-dioxygenase (IDO) has been found in several tumors and associated with local immune suppression, notably by inhibiting T cell functions. However, the exact molecular pathways making T cells sensitive to IDO are still unclear. We propose to exploit the power of whole-genome single-guide RNA (sgRNA) CRISPR screens to uncover novel mechanisms of IDO-mediated T cell dysfunction. Methods: Cas9-expressing transgenic mice were crossed with mice expressing a transgenic T cell receptor (TCR) specifically recognizing a tumor antigen called P1A. Primary Cas9xTCRP1A CD8+ T cells were isolated from these mice and stimulated with P1A-expressing tumor cells. Cells were then transduced with the Teichmann retroviral genome wide CRISPR knockout library. Seven days after the first stimulation, CD8+ T cells were re-stimulated in a control (Tryptophanhigh, kynureninelow) or selective (Tryptophanlow, kynureninehigh) medium, mimicking the function of IDO in vitro. After four days of screening selection, genomic DNA was extracted from remaining living cells and sequencing libraries were prepared. sgRNA representation was then assessed by next-generation sequencing. Results: In vitro CRISPR knockout screening pipeline was successfully set up and validated. This includes: (1.) Successful transduction of the CRISPR knockout library, as well as efficient gene knockout in Cas9xTCRP1A primary CD8+ T cells. (2.) Confirming the strong inhibition of T cell proliferation and survival in the selective medium as compared to the control medium, thereby validating the screening selection strategy. (3.) Optimizing genomic DNA extraction procedure, as well as sequencing library preparation. Finally, the proposed in vitro CRISPR knockout screening was successfully launched, and analysis of potential candidate genes is ongoing. Conclusions and perspectives: Our study proposes to perform genome wide CRISPR knockout screens in T cells exposed to conditions mimicking IDO activity in vitro. Enriched sgRNA at the end of the selection should reveal genes whose inhibition improved T cell survival in Tryptophanlow/kynureninehigh medium. Resulting potential candidate targets will then be validated by knocking them out in T cells and assessing T cell functions under conditions mimicking IDO-mediated immune suppression in vitro. Similar validation of top candidate genes will also be performed in vivo in a model of adoptive cell transfer of target-knockout T cells in mice bearing IDO-expressing tumors. The identified genes/pathways involved in T cell sensitivity to Tryptophan shortage/Kynurenine enrichment should reveal new relevant mechanisms of IDO-mediated immune suppression in the tumor microenvironment. Citation Format: Raphaële Bombart, Jingjing Zhu, Benoit J. Van den Eynde. Uncovering molecular actors of IDO-mediated T cell dysfunction with genome-wide CRISPR/Cas9 knockout screens [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P025.

T cell fat catabolism: A novel target for kynurenine?

T cell fat catabolism: A novel target for kynurenine?

Tryptophan, Kynurenine and Kynurenic Acid Concentrations in Milk and Serum of Dairy Cows with Prototheca Mastitis.

Simple SummaryBovine mastitis continues to be a leading cause of heavy economic losses in the dairy industry worldwide. Among a broad spectrum of infectious agents implicated in this pathology are unicellular, achlorophyllous microalgae of the genus Prototheca. The prevalence of mastitis due to Prototheca algae is currently increasing. The aim of this work was to explore a possible association of the kynurenine pathway of tryptophan metabolism with Prototheca mastitis. The authors were eager to know whether metabolites of tryptophan degradation can be used as markers of the protothecal bovine mastitis. Both tryptophan and its metabolite kynurenine occurred at a significantly lower level in the milk of cows with mastitis compared to healthy animals. Whereas the activity of indoleamine 2,3-dioxygenase, a key enzyme of tryptophan catabolism, was significantly higher in milk of mastitic cows compared to control animals. It was thus concluded that low values of tryptophan and kynurenine concentrations or elevated indoleamine 2,3-dioxygenase activity in milk samples can be used as markers of mastitis due to Prototheca spp.The aim of this work was to investigate serum and milk levels of tryptophan (TRP), kynurenine (KYN), and kynurenic acid (KYNA), as well as the activity of indoleamine 2,3-dioxygenase (IDO) in cows with mastitis due to Prototheca algae. The study was prompted by previous research showing a link between the KYN pathway of TRP metabolism and bovine mastitis of bacterial etiology. The study was carried out over a 2-year period (2018–2019) and included quarter milk and serum samples collected from six dairy herds in Poland. The samples were obtained from healthy cows and cows with Prototheca mastitis of either clinical and subclinical manifestation, as determined upon direct measurement of the somatic cell count or indirectly by performing a California Mastitis Test on suspected quarters. Both TRP and KYN concentrations were significantly lower in milk of mastitic cows compared to healthy animals (0.8 vs. 8.72 µM, p = 0.001; 0.07 vs. 0.32 µM, p = 0.001, respectively). The difference in TRP and KYN concentrations in the sera of the two animal groups was much less pronounced (25.55 vs. 27.57 µM, 3.03 vs. 3.56 nM, respectively). The concentration of KYNA was almost at the same level in milk (1.73 vs. 1.70 nM) and in serum (80.47 vs. 75.48 nM) of both mastitic and healthy cows. The data showed that the level of TRP and its metabolites in serum was conspicuously higher compared to milk in all cows under the study. The activity of IDO was significantly higher in milk of cows with Prototheca mastitis compared to healthy animals (71.4 vs. 40.86, p < 0.05), while in serum it was pretty much the same (135.94 vs. 124.98, p > 0.05). The IDO activity differed significantly between serum and milk both for mastitic (135.94 vs. 71.4, p < 0.05) and healthy cows (124.98 vs. 40.86, p < 0.001). In conclusion, low values of TRP and KYN concentrations or elevated IDO activity in milk samples might be used as markers of mastitis due to infectious causes, including Prototheca spp.

Decidual IDO+ macrophage promotes the proliferation and restricts the apoptosis of trophoblasts

Indoleamine 2, 3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is essential in physiological immunoregulation. The present research was conducted to elucidate the expression and roles of IDO in decidual macrophages (dMφ) during early pregnancy. Here, we observed a remarkable decrease of IDO+ dMφ from patients with unexplained recurrent spontaneous abortion (URSA). IDO+ dMφ displayed M2 phenotype with higher CD206, CD209 and CD163, and lower CD86. Interestingly, treatment with 1-methyl-d-tryptophan (1-MT, an IDO pathway inhibitor) led to the M1 bias of dMφ. Further analysis of the cytokine array and the qPCR showed decreased levels of trophoblast proliferation or invasion-related molecules (e.g., CXCL12 and BMP2) in 1-MT-treated dMφ. The data of co-culture system showed that 1-MT-pretreated dMφ decreased the proliferation and the expression of Ki-67 and Bcl-2, and increased cell apoptosis of HTR-8/Snveo cells. Additionally, the expression of IDO in U937 cells was up-regulated by decidual stromal cells (DSC) and HTR-8/Snveo cells in vitro, as well as estradiol and medroxyprogesterone. These data suggest that endocrine environment, DSC and trophoblasts should contribute to the high level of IDO in dMφ, and IDO+ dMφ with M2 dominant phenotype promote the survival of trophoblasts during early pregnancy. The abnormal lower level of IDO should trigger the dysfunction of dMφ, further suppress the survival of trophoblasts and increase the risk of miscarriage.

Parallel discovery of selective and dual inhibitors of tryptophan dioxygenases IDO1 and TDO2 with a newly-modified enzymatic assay

The expression of tryptophan catabolising enzyme indoleamine 2,3-dioxygenase 1 (IDO1) or tryptophan 2,3-dioxygenase 2 (TDO2) in cancers is associated with suppressed immunity and poor patient prognosis. Results from human clinical trials of IDO1 inhibitors have been disappointing. There is now a strong interest in the development of TDO2-selective or dual IDO1/TDO2 inhibitors that may surpass IDO1 inhibitors by providing broader efficacy and blocking constitutively-expressed hepatic TDO2. To expedite the discovery of novel TDO2-specific and dual inhibitors, an assay that enabled the efficient and accurate measurement of the inhibitory activity of compounds against both IDO1 and TDO2 enzymes, concurrently in the same experiment was established to screen 5,682 compounds that included the National Cancer Institute Diversity set 5, for inhibition of IDO1 and TDO2 activity. This screen identified 82 compounds that inhibited either IDO1, TDO2 or both enzymes > 50% at 20 µM. Thirty Pan Assay Interference compounds were removed from the list and the IC50 of the remaining 52 compounds against IDO1 and TDO2 was subsequently determined using the newly-developed concurrent assay. Ten compounds were confirmed as dual IDO1/TDO2 inhibitors having IC50 values under 50 µM against both enzymes and within 2-fold of each other. Six compounds with IC50 values between 1.39 and 8.41 µM were identified as potential TDO2-selective leads. The use of this concurrent protocol is anticipated to expedite the discovery of novel leads for dual and selective inhibitors against IDO1 and or TDO2 and speed the evaluation of novel analogues that will ensue.

IDO1-Targeted Therapy Does Not Control Disease Development in the Eµ-TCL1 Mouse Model of Chronic Lymphocytic Leukemia.

Simple SummaryThe tryptophan-catabolizing enzyme IDO1 and its metabolite kynurenine were shown to be enhanced in patients with chronic lymphocytic leukemia (CLL), and their involvement in T cell suppression and immune escape was suggested. As we have observed increased IDO1 expression and kynurenine serum levels in the Eµ-TCL1 mouse model of CLL, we evaluated the therapeutic potential of targeting IDO1 in preclinical treatment studies with two IDO1 inhibitors in mice developing CLL. As both studies revealed only minor effects of IDO1 inhibition on leukemia development and the immune compartment at early time points of treatment which disappeared over time, our data suggest that even though IDO1 might be involved in immunosuppressive mechanisms in CLL, its targeting is not sufficient for preventing immune escape. Thus, compensatory mechanisms beyond IDO1 seem to be of relevance to prevent clinically relevant benefits with IDO1-targeting drugs.Indoleamine-2,3-dioxygenase 1 (IDO1), a tryptophan (Trp)-catabolizing enzyme producing metabolites such as kynurenine (Kyn), is expressed by myeloid-derived suppressor cells (MDSCs) and associated with cancer immune escape. IDO1-expressing monocytic MDSCs were shown to accumulate in patients with chronic lymphocytic leukemia (CLL) and to suppress T cell activity and induce suppressive regulatory T cells (Tregs) in vitro. In the Eµ-TCL1 mouse model of CLL, we observed a strong upregulation of IDO1 in monocytic and granulocytic MDSCs, and a significantly increased Kyn to Trp serum ratio. To explore the potential of IDO1 as a therapeutic target for CLL, we treated mice after adoptive transfer of Eµ-TCL1 leukemia cells with the IDO1 modulator 1-methyl-D-tryptophan (1-MT) which resulted in a minor reduction in leukemia development which disappeared over time. 1-MT treatment further led to a partial rescue of the immune cell changes that are induced with CLL development. Similarly, treatment of leukemic mice with the clinically investigated IDO1 inhibitor epacadostat reduced the frequency of Tregs and initially delayed CLL development slightly, an effect that was, however, lost at later time points. In sum, despite the observed upregulation of IDO1 in CLL, its inhibition is not sufficient to control leukemia development in the Eµ-TCL1 adoptive transfer model.

Dexamethasone Induces the Expression and Function of Tryptophan-2-3-Dioxygenase in SK-MEL-28 Melanoma Cells.

Tryptophan-2,3-dioxygenase (TDO) is one of the key tryptophan-catabolizing enzymes with immunoregulatory properties in cancer. Contrary to expectation, clinical trials showed that inhibitors of the ubiquitously expressed enzyme, indoleamine-2,3-dioxygenase-1 (IDO1), do not provide benefits in melanoma patients. This prompted the hypothesis that TDO may be a more attractive target. Because the promoter of TDO harbors glucocorticoid response elements (GREs), we aimed to assess whether dexamethasone (dex), a commonly used glucocorticoid, modulates TDO expression by means of RT-PCR and immunofluorescence and function by assessing cell proliferation and migration as well as metalloproteinase activity. Our results show that, in SK-Mel-28 melanoma cells, dex up-regulated TDO and its downstream effector aryl hydrocarbon receptor (AHR) but not IDO1. Furthermore, dex stimulated cellular proliferation and migration and potentiated MMP2 activity. These effects were inhibited by the selective TDO inhibitor 680C91 and enhanced by IDO1 inhibitors. Taken together, our results demonstrate that the metastatic melanoma cell line SK-Mel-28 possesses a functional TDO which can also modulate cancer cell phenotype directly rather than through immune suppression. Thus, TDO appears to be a promising, tractable target in the management or the treatment of melanoma progression.

IDO1 scavenges reactive oxygen species in myeloid-derived suppressor cells to prevent graft-versus-host disease

Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1-/-) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1-/- BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1-/- Gr-1+CD11b+ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6ClowLy6Ghi subset, compared with the WT counterparts. Importantly, Ido1-/-Gr-1+CD11b+ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1-/-Gr-1+CD11b+ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1-/- BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1+CD11b+ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.