IDO Activity Research Articles

The correlation of inflammation, tryptophan-kynurenine pathway, and suicide risk in adolescent depression.

Accumulating evidence suggests a role for the tryptophan-kynurenine pathway (TKP) in the psychopathology of major depressive disorder (MDD). Abnormal inflammatory profile and production of TKP neurotoxic metabolites appear more pronounced in MDD with suicidality. Progress in understanding the neurobiology of MDD in adolescents lags significantly behind that in adults due to limited empirical evidence. Aims of this study was to investigate the association between inflammation, TKP, and suicidality in adolescent depression. Seventy-three adolescents with MDD were assessed for serum levels of interleukin (IL)-1β, IL-6, IL-18, IL-10, tumor necrosis factor-α (TNF-α), tryptophan (TRP), kynurenine (KYN), 3-hydroxykynurenine (3-HK), and kynurenine acid (KA). Correlations between cytokines and TKP measures were examined. Patients were divided into high- (n = 42) and non-high-suicide-risk groups (n = 31), and serum levels of cytokines and TKP metabolites were compared. Significant negative correlations were found between TRP and IL-8 (r = - 0.27, P < 0.05) and IL-10 (r = - 0.23, P < 0.05), while a significant positive correlation was observed between 3-HK and IL-8 (r = 0.39, P < 0.01) in depressed adolescents. The KYN/TPR (index of indoleamine 2,3-dioxygenase, IDO) was positively correlated with IL-1β (r = 0.34), IL-6 (r = 0.32), IL-10 (r = 0.38) and TNF-α (r = 0.35) levels (P < 0.01); and 3-HK/KYN (index of kynurenine3-monooxidase, KMO) was positively correlated with IL-8 level (r = 0.31, P < 0.01). Depressed adolescents at high suicide risk exhibited significantly higher levels of IL-1β (Z = 2.726, P < 0.05), IL-10 (Z = 2.444, P < 0.05), and TNF-α (Z = 2.167, P < 0.05) and lower levels of 3-HK (Z = 2.126, P < 0.05) compared to their non-high suicide risk counterparts. Our findings indicated that serum inflammatory cytokines were robustly associated with IDO and KMO activity, along with significantly decreased serum level of TRP, increased level of 3-HK, and higher suicide risk in adolescent depression.

Transformable self-delivered supramolecular nanomaterials combined with anti-PD-1 antibodies alleviate tumor immunosuppression to treat breast cancer with bone metastasis

Breast cancer is the most common malignant tumor that threatens women’s life and health, and metastasis often occurs in the advanced stage of breast cancer, leading to pathological bone destruction and seriously reducing patient quality of life. In this study, we coupled chlorin e6 (Ce6) with mono-(6-amino-6-deoxy)-beta-cyclodextrin (β-CD) to form Ce6-CD, and combined ferrocene with the FFVLG3C peptide and PEG chains to form the triblock molecule Fc-pep-PEG. In addition, the IDO-1 inhibitor NLG919 was loaded with Ce6-CD and Fc-pep-PEG to construct the supramolecular nanoparticle NLG919@Ce6-CD/Fc-pep-PEG (NLG919@CF). After laser irradiation, Ce6 produced robust reactive oxidative species to induce tumor cell apoptosis. Simultaneously, ferrocene became charged, and Fc-pep-PEG dissociated from the spherical nanoparticles, enabling their transformation into nanofibers, which increased both the retention effect and the induction of ferroptosis. The released NLG919 reduced the number of regulatory T cells (Tregs) and restored the function of cytotoxic T lymphocytes (CTLs) by inhibiting the activity of IDO-1. Moreover, combined administration with an anti-PD-1 antibody further relieved immune suppression in the tumor microenvironment. This article presents a new strategy for the clinical treatment of breast cancer with bone metastasis and osteolysis.Graphical

Platelet Ido1 expression is induced during Plasmodium yoelii infection, altering plasma tryptophan metabolites

AbstractPlatelets are immune responsive in many diseases as noted by changes in platelet messenger RNA in conditions such as sepsis, atherosclerosis, COVID-19, and many other inflammatory and infectious etiologies. The malaria causing Plasmodium parasite is a persistent public health threat and significant evidence shows that platelets participate in host responses to infection. Using a mouse model of nonlethal/uncomplicated malaria, Plasmodium yoelii XNL (PyNL)-infected but not control mouse platelets expressed Ido1, a rate limiting enzyme in tryptophan metabolism that increases kynurenine at the expense of serotonin. Interferon-γ (IFN-γ) is a potent inducer of Ido1 and mice treated with recombinant IFN-γ had increased platelet Ido1 and IDO1 activity. PyNL-infected mice treated with anti-IFN-γ antibody had similar platelet Ido1 and metabolic profiles to that of uninfected controls. PyNL-infected mice become thrombocytopenic by day 7 after infection and transfusion of platelets from IFN-γ–treated wild-type mice but not Ido1−/− mice increased the plasma kynurenine-to-tryptophan ratio, indicating that platelets are a source of postinfection IDO1 activity. We generated platelet-specific Ido1 knockout mice to assess the contribution of platelet Ido1 during PyNL infection. Platelet-specific Ido1−/− mice had increased death and evidence of lung thrombi, which were not present in infected wild-type mice. Platelet Ido1 may be a significant contributor to plasma kynurenine in IFN-γ-driven immune processes and the loss of platelets may limit total Ido1, leading to immune and vascular dysfunction.

Hypoxia-Specific Metal-Organic Frameworks Augment Cancer Immunotherapy of High-Intensity Focused Ultrasound.

As a noninvasive treatment modality, high-intensity focused ultrasound (HIFU)-induced antitumor immune responses play a vital role in surgery prognosis. However, limited response intensity largely hinders postoperative immunotherapy. Herein, a hypoxia-specific metal-organic framework (MOF) nanosystem, coordinated by Fe3+, hypoxic-activated prodrug AQ4N, and IDO-1 signaling pathway inhibitor NLG919, is developed for the potentiating immunotherapy of HIFU surgery. The loaded AQ4N enhances the photoacoustic imaging effects to achieve accurate intraoperative navigation. Within the HIFU-established severe hypoxic environment, AQ4N is activated sequentially, following which it cooperates with Fe3+ to effectively provoke immunogenic cell death. In addition, potent NLG919 suppresses IDO-1 activity and degrades the immunosuppressive tumor microenvironment aggravated by postoperative hypoxia. In vivo studies demonstrate that the MOF-mediated immunotherapy greatly inhibits the growth of primary/distant tumors and eliminates lung metastasis. This work establishes a robust delivery platform to improve immunotherapy and the overall prognosis of HIFU surgery with high specificity and potency.

Artemisia argyi essential oil alleviates asthma by regulating 5-LOX-CysLTs and IDO-1-KYN pathways: Insights from metabolomics

Ethnopharmacological relevanceArtemisia argyi essential oil (AAEO) is a traditional herbal remedy for asthma. However, the potential effect of AAEO on asthma has not been elucidated. Aim of the studyTo investigate the protective properties of AAEO upon asthma and elucidate its mechanism. Materials and methodsThe effects of AAEO in asthma were assessed by histology and biochemical analysis. Then, we integrated real-time reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry and metabolomics analysis to reveal its mechanism. ResultsIn vivo, AAEO reduced the counts of white blood cells (WBCs) and cytokines in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of OVA-sIgE and muc5ac. Metabolomics results showed that AAEO can exert therapeutic effects on asthmatic mice by regulating disordered arachidonic acid metabolism and tryptophan metabolism. Further studies shown that AAEO inhibited the expression of 5-LOX and reduced the accumulation of CysLTs in mice. Meanwhile, AAEO promoted the activity of IDO-1, facilitated the conversion of tryptophan to kynurenine, and regulated the imbalance of Treg/Th17 immunity. Immunohistochemical results showed that AAEO promoted the expression of IDO-1. RT-qPCR results showed that AAEO promoted the expression of IL-10 and Foxp3 mRNA, and inhibited the expression of IL-17A and RORγt mRNA, thus regulated the imbalance of Treg/Th17 immunity and exerted its therapeutic effects. ConclusionAAEO treatment not only attenuates the clinical symptoms of asthma but is also involved in regulating lung tissue metabolism. The anti-asthmatic activity of AAEO may be achieved by reprogramming 5-LOX-CysLTs and IDO-1-KYN pathways.

A metabolic dependency of EBV can be targeted to hinder B cell transformation.

After infection of B cells, Epstein-Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates nicotinamide adenine dinucleotide (NAD) de novo biosynthesis by driving expression of the metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match adenosine triphosphate (ATP) production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is therefore a druggable metabolic vulnerability of EBV-driven B cell transformation, opening therapeutic possibilities for EBV-related diseases.

Self‐Carrier Nanoparticles for Delivery of Paclitaxel and IDO Inhibitor to Boost Antitumor Chemo‐Immunotherapy

AbstractThe combination of chemotherapy and immunotherapy holds great potential in clinical treatment of advanced cancers. Whereas, the therapeutic outcome of chemotherapeutic and immune regulator is suboptimal due to their poor tumor availability and severe off‐target toxicity. Herein, self‐carrier nanoparticles (PSMT NPs) integrating a paclitaxel (PTX) prodrug and indoleamine 2,3‐dioxygenase 1 (IDO) inhibitor (1‐methyl‐tryptophan, 1MT) for tumor‐specific chemo‐immunotherapy is constructed. After internalization by cancer cells, PSMT NPs can respond to endogenous redox stimulus, and release PTX and 1MT. The released PTX can not only promote cell apoptosis via the intervention of cell mitosis but also initiate immunogenic cell death to facilitate the recruitment and activation of tumor‐infiltrating cytotoxic T lymphocytes. The concomitant 1MT can inhibit the IDO activity to exhaust regulatory T cells, thereby synergistically activating cytotoxic T lymphocytes. PSMT NPs exhibit potentiated antitumor output toward triple‐negative breast cancer and negligible systemic toxicity. This facile and versatile nanoplatform provides a promising strategy to cooperatively activate antitumor immunity for potentiated chemo‐immunotherapy.

A peripheral blood mononuclear cell-based in vitro model: A tool to explore indoleamine 2, 3-dioxygenase-1 (IDO1)

BackgroundProinflammatory cytokines powerfully induce the rate-limiting enzyme indoleamine 2, 3-dioxygenase-1 (IDO-1) in dendritic cells (DCs) and monocytes, it converts tryptophan (Trp) into L-kynurenine (KYN), along the kynurenine pathway (KP). This mechanism represents a crucial innate immunity regulator that can modulate T cells. This work explores the role of IDO1 in lymphocyte proliferation within a specific pro-inflammatory milieu. MethodsPeripheral blood mononuclera cells (PBMCs) were isolated from buffy coats taken from healthy blood donors and exposed to a pro-inflammatory milieu triggered by a double-hit stimulus: lipopolysaccharide (LPS) plus anti-CD3/CD28. The IDO1 mRNA levels in the PBMCs were measured by RT-PCR; the IDO1 activity was analyzed using the KYN/Trp ratio, measured by HPLC-EC; and lymphocyte proliferation was measured by flow cytometry. Trp and epacadostat (EP) were used as an IDO1 substrate and inhibitor, respectively. KYN, which is known to modulate Teffs, was tested as a positive control in lymphocyte proliferation. ResultsIDO1 expression and activity in PBMCs increased in an in vitro pro-inflammatory milieu. The lymphoid stimulus increased IDO1 expression and activity, which supports the interaction between the activated lymphocytes and the circulating myeloid IDO1-expressing cells. The addition of Trp decreased lymphocyte proliferation but EP, which abrogated the IDO1 function, had no impact on proliferation. Additionally, incubation with KYN seemed to decrease the lymphocyte proliferation. ConclusionIDO1 inhibition did not change T lymphocyte proliferation. We present herein an in vitro experimental model suitable to measure IDO1 expression and activity in circulating myeloid cells.

Quantitative Multiplexed Analysis of Indoleamine 2,3-Dioxygenase (IDO) and Arginase-1 (ARG1) Expression and Myeloid Cell Infiltration in Colorectal Cancer

Indoleamine 2,3-dioxygenase (IDO) and arginase-1 (ARG1) are amino acid–metabolizing enzymes, frequently highly expressed in cancer. Their expression may deplete essential amino acids, lead to immunosuppression, and promote cancer growth. Still, their expression patterns, prognostic significance, and spatial localization in the colorectal cancer microenvironment are incompletely understood. Using a custom 10-plex immunohistochemistry assay and supervised machine learning–based digital image analysis, we characterized IDO and ARG1 expression in monocytic cells, granulocytes, mast cells, and tumor cells in 833 colorectal cancer patients. We evaluated the prognostic value and spatial arrangement of IDO- and ARG1-expressing myeloid and tumor cells. IDO was mainly expressed not only by monocytic cells but also by some tumor cells, whereas ARG1 was predominantly expressed by granulocytes. Higher density of IDO+ monocytic cells was an independent prognostic factor for improved cancer-specific survival both in the tumor center (Ptrend = .0002; hazard ratio [HR] for the highest ordinal category Q4 [vs Q1], 0.51; 95% CI, 0.33-0.79) and the invasive margin (Ptrend = .0015). Higher density of granulocytes was associated with prolonged cancer-specific survival in univariable models, and higher FCGR3+ARG1+ neutrophil density in the tumor center also in multivariable analysis (Ptrend = .0020). Granulocytes were, on average, located closer to tumor cells than monocytic cells. Furthermore, IDO+ monocytic cells and ARG1− granulocytes were closer than IDO− monocytic cells and ARG1+ granulocytes, respectively. The mRNA expression of the IDO1 gene was assessed in myeloid and tumor cells using publicly available single-cell RNA sequencing data for 62 colorectal cancers. IDO1 was mainly expressed in monocytes and dendritic cells, and high IDO1 activity in monocytes was associated with enriched immunostimulatory pathways. Our findings provided in-depth information about the infiltration patterns and prognostic value of cells expressing IDO and/or ARG1 in the colorectal cancer microenvironment, highlighting the significance of host immune response in tumor progression.

Association between altered tryptophan metabolism, plasma aryl hydrocarbon receptor agonists, and inflammatory Chagas disease.

Chagas disease causes a cardiac illness characterized by immunoinflammatory reactions leading to myocardial fibrosis and remodeling. The development of Chronic Chagas Cardiomyopathy (CCC) in some patients while others remain asymptomatic is not fully understood, but dysregulated inflammatory responses are implicated. The Aryl hydrocarbon receptor (AhR) plays a crucial role in regulating inflammation. Certain tryptophan (Trp) metabolites have been identified as AhR ligands with regulatory functions. We investigated AhR expression, agonist response, ligand production, and AhR-dependent responses, such as IDO activation and regulatory T (Treg) cells induction, in two T. cruzi-infected mouse strains (B6 and Balb/c) showing different polymorphisms in AhR. Furthermore, we assessed the metabolic profile of Trp catabolites and AhR agonistic activity levels in plasma samples from patients with chronic Chagas disease (CCD) and healthy donors (HD) using a luciferase reporter assay and liquid chromatography-mass spectrophotometry (LC-MS) analysis. T. cruzi-infected B6 mice showed impaired AhR-dependent responses compared to Balb/c mice, including reduced IDO activity, kynurenine levels, Treg cell induction, CYP1A1 up-regulation, and AhR expression following agonist activation. Additionally, B6 mice exhibited no detectable AhR agonist activity in plasma and displayed lower CYP1A1 up-regulation and AhR expression upon agonist activation. Similarly, CCC patients had decreased AhR agonistic activity in plasma compared to HD patients and exhibited dysregulation in Trp metabolic pathways, resulting in altered plasma metabolite profiles. Notably, patients with severe CCC specifically showed increased N-acetylserotonin levels in their plasma. The methods and findings presented here contribute to a better understanding of CCC development mechanisms and may identify potential specific biomarkers for T. cruzi infection and the severity of associated heart disease. These insights could be valuable in designing new therapeutic strategies. Ultimately, this research aims to establish the AhR agonistic activity and Trp metabolic profile in plasma as an innovative, non-invasive predictor of prognosis for chronic Chagas disease.

A theoretical study on the activity and selectivity of IDO/TDO inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO) is a tryptophan (Trp) metabolic enzyme along the kynurenine (NFK) pathway. Under pathological conditions, IDO overexpressed by tumor cells causes depletion of tryptophan and the accumulation of metabolic products, which inhibit the local immune response and form immune escape. Therefore, the suppression of IDO activity is one of the strategies for tumor immunotherapy, and drug design for this target has been the focus of research for more than two decades. Apart from IDO, tryptophan dioxygenase (TDO) of the same family can also catalyze the same biochemical reaction in the human body, but it has different tissue distribution and substrate selectivity from IDO. Based on the principle of drug design with high potency and low cross-reactivity to specific targets, in this subject, the activity and selectivity of IDO and TDO toward small molecular inhibitors were studied from the perspective of thermodynamics and kinetics. The aim was to elucidate the structural requirements for achieving favorable biological activity and selectivity of IDO and TDO inhibitors. Specifically, the interactions of inhibitors from eight families with IDO and TDO were initially investigated through molecular docking and molecular dynamics simulations, and the thermodynamic data for binding of inhibitors were predicted by the molecular mechanics/generalized Born surface area (MM/GBSA) method. Secondly, we explored the free energy landscape of JKloops, the kinetic control element of IDO/TDO, using temperature replica exchange molecular dynamics (T-REMD) simulations and elucidated the connection between the rules of IDO/TDO conformational changes and the inhibitor selectivity mechanism. Furthermore, the binding and dissociation processes of the C1 inhibitor (NLG919) were simulated by the adaptive steering molecular dynamics (ASMD) method, which not only addressed the possible stable, metastable, and transition states for C1 inhibitor-IDO/TDO interactions, but also accurately predicted kinetic data for C1 inhibitor binding and dissociation. In conclusion, we have constructed a complete process from enzyme (IDO/TDO) conformational activation to inhibitor binding/dissociation and used the thermodynamic and kinetic data of each link as clues to verify the control mechanism of IDO/TDO on inhibitor selectivity. This is of great significance for us to understand the design principles of tumor immunotherapy drugs and to avoid drug resistance of immunotherapy drugs.

IFNγ Primed MSCs Suppress Stimulated T Cells By Inducing Perturbations of Redox Homeostasis

Acute Graft versus host disease (GVHD) remains the primary cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation (HCT). Despite the current standard prophylaxis, approximately 50% of patients will develop acute GVHD. the outcome of patients with steroid-refractory and/or severe GVHD is poor; hence, more effective prophylaxis reducing the risk of these scenarios is greatly needed. In recent years, considerable attention has been directed toward mesenchymal stromal cells (MSCs) as prophylaxis and therapy for acute GVHD. Even though the immunosuppressive and immunomodulatory properties of MSCs are enhanced by proinflammatory cytokines such as interferon γ (γMSCs), relatively less attention has been focused on these cells. Our laboratory has shown human γMSCs are effective in GVHD prophylaxis in a fully mismatched murine HCT/GVHD model. When infused IV on day +1 after HCT, γMSCs traffic to the spleen, liver, and gut-associated lymphoid tissue (GALT) which is essential for effective prophylaxis. This may be a key element of the mechanism of prophylaxis as the gut and GALT are thought to be paramount for the development of GVHD. We now sought to understand the mechanism(s) of γMSC suppression of alloreactive T cells suppressive activity in this specific clinical and biological setting. In vivo, control mice transplanted with a fully MHC mismatched graft developed GVHD in 6-8 days (0% GVHD-free survival) while human γMSCs administration at day +1 completely prevented GVHD in the first 17 days (100% GVHD survival) but the animals developed acute GVHD in 21-28 days. Hence, a single dose of γMSCs is 100% effective early but is insufficient for overall GVHD prevention. The mechanism is highly effective but of relatively short duration, and seemingly reversible. In vitro, the reversible T cell suppressive activity was entirely contact-independent, while cell-to-cell contact could induce T cell death. Moreover, γMSC IDO1 expression, alone, was sufficient to suppress T cells. After 24 hours of T cell activation or γMSC suppression in vitro, we found the T cells were arrested in G0 inconsistent with most cell cycle regulation which would arrest cells in late G1 at the so-called restriction point. In scRNA Seq, we identified 16,478 expressed genes in both groups but only 98 genes were significantly differentially expressed (≥20% FC, Padj ≤ 10 -4). ROS response pathways were significantly enriched in the suppressed cells suggesting oxidative stress. Paradoxically, flow cytometric analysis of CM-H2DCFDA stained T cells revealed less ROS in suppressed cells compared to controls. Additionally, total NAD is elevated and the NAD/NADH ratio was 10-fold less in suppressed T cells compared to activated cells indicating an excess of reducing equivalents in the suppressed cells. Collectively these data define reductive stress. On this note, the upregulation of ROS response genes is also consistent with reductive stress. IDO1 activity seems to have induced reductive stress. The IDO1 catalytic activity converts tryptophan (Trp) to kynurenine (Kyn). We then found the addition of Trp (10 µM) to γMSC CM abolished suppression; supplementing fresh media with 15-fold excess Kyn had no effect. Moreover, RPMI without Trp and with dialyzed FBS also conferred suppression to activated T cells. These data suggest that among the many metabolites consumed or generated in γMSC CM, the lack of Trp induces T cell suppression. Trp supplementation of γMSC CM showed the expected level of ROS in activated T cells suggesting the Trp deficiency caused the reduced ROS. Supplementing γMSC CM with a graded concentration of hydrogen peroxide, we found that 100 nM hydrogen peroxide was not toxic to control cells, had ROS similar to activated T cells, and abolished suppression indicating the reduced ROS directly caused suppression. Kyn, nutritional deficiency, or an integrated stress response did not seem to play a measurable role in this setting. Collectively, our data suggest that IDO1-mediated Trp deficiency perturbs redox homeostasis (reductive stress) which suppresses activated T cells. We postulate that once redox homeostasis is restored, T cell activation proceeds. Future efforts will be directed toward identifying the link between Trp deficiency and redox homeostasis in this setting, and the genomic changes that directly mediate the lack of T cell proliferation.

CSIG-33. A 2ND GENERATION IDO1 PROTEIN DEGRADER TO OVERCOME NON-ENZYME-MEDIATED IDO1-DEPENDENT IMMUNE SUPPRESSION FOR GLIOBLASTOMA

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive rate-limiting enzyme that metabolizes the essential amino acid, tryptophan (Trp), into the downstream catabolite, kynurenine. IDO1 is expressed in &amp;gt;90% of patient-resected glioblastoma (GBM). We recently validated that IDO1 can suppress the anti-brain tumor immune response and ablate immunotherapeutic treatment efficacy through non-metabolic IDO1 activity. To-date, most IDO1-targeted therapy has focused on inhibiting tryptophan metabolism. This class of drugs has failed to improve the overall survival of patients with cancer. To overcome this failure, we developed a therapeutic agent that inhibits both IDO1 enzyme- and non-enzyme-activities in the form of an IDO1-Proteolysis Targeting Chimera (PROTAC). We previously published the characterization of a 1st generation lead compound (Bollu et al., 2022; J. Med. Chem) with a DC50 for IDO1 of 0.329 µM in human GBM cells. In a more recent investigation, we discovered a 2nd generation IDO1-PROTAC, NU227326, with 30× more potency and a DC50 of 10.4 nM in human GBM cells. IDO1-expressing U87, U138, and patient derived xenograft (PDX) GBM43 were treated with either IDO1-PROTAC, IDO1-mutant-PROTAC, IDO1 enzyme inhibitor, or IDO1 siRNA. Transcriptomic analysis revealed differentially expressed genes that were commonly regulated after treatment with the IDO1-PROTAC as compared to treatment with the IDO1-mutant-PROTAC or IDO1 enzyme inhibitor groups in U87, U138, and GBM43 cells. Mass spectrometry discovered 34 unique proteins that were differentially expressed inside of human GBM cells and an additional 20 unique proteins that were identified in the supernatant of cultured human GBM cells after IDO1-PROTAC treatment. Meta-analysis of the transcriptomic and proteomic analyses identified a novel factor that was unique to IDO1-PROTAC treatment. IDO1-PROTAC treatment-enriched GO terms were associated with nucleoside kinase and metallocarboxypeptidase activities. This study discovered multiple new pathways that immunosuppressive IDO1 non-metabolically regulates in human glioblastoma cells.

Prednisolone Intake Differentially Regulates IL12p40 and IL18 - Impact on Severe Chronic Gvhd, Relapse and Mortality

Background: Chronic graft-versus-host disease (cGVHD) is an immune-mediated complication after allogeneic stem cell transplantation (alloSCT). It is associated with graft-versus-leukemia effects and lower relapse rates, but in its severe form also with increased morbidity and mortality. The early interferon-gamma (IFNγ)-response marker CXCL9 is increased in mild and severe cGVHD and strongly correlates with IDO activity (1). Interleukin (IL)-18 is an innate, IFNγ inducing cytokine that associates with poor outcome of allografted patients (2), and with severe fibrosing cGVHD (1). The adaptive immunological arm of IFNγ induction relies on IL12 that is produced during cognate interactions between dendritic cells and T cells. IL12 kinetics is poorly characterized after alloSCT. We hypothesized that adaptive (IL12p40) and innate (IL18) immune mediators and their modulation by immunosuppressive drugs (ATG, prednisolone) impact cGVHD severity and outcome. Methods: Patients before alloSCT were included in an observational study allowing collection of clinical data and blood before and for one year after alloSCT. The training cohort included 350 patients transplanted before 2018 at our institution. The validation cohort consisted of 205 patients transplanted after 2018. Patients were eligible if they were not relapsed and did not receive donor leukocyte infusions (DLI) until d180, and did not develop refractory acute GVHD. Cytokines that belonged to the IFNγ pathway (IFNγ, CXCL9, CXCL10, IL12p40, IL12EBI3, IL17, IL18, IL23) or to innate inflammatory pathways (TNFa, IFNa, IL1b, activin, follistatin) were measured by R&amp;D ELISAs. Cumulative (equivalent) prednisolone dosages between d0 and d100 after alloSCT were calculated from electronic patient records. Flow cytometry of PBMC of 100 patients on day+180/onset cGVHD were performed using 24-gate multicolor staining. Mild, severe fibrosing and severe gastro-intestinal (GI) cGVHD were diagnosed according to simplified NIH criteria (training cohort only). Overall survival, non-relapse mortality and time to relapse from time of blood sampling were calculated by multivariable Cox regression analyses. Results: Median time of blood sampling at onset of cGVHD (n=180) was day +168 (range 41-723). Control patients (n=169) were collected on day +180 (171-183). Mild cGVHD was recorded in 133 patients, severe fibrosing cGVHD in 39 patients, and isolated GI-cGVHD in 8 patients. Receiver-operator characteristic (ROC) curves showed that only CXCL9, IL18 and IL12p40 were significantly linked to severe cGVHD. IL18 was associated with severe fibrosing cGVHD, whereas CXCL9 and IL12p40 were correlated to any grade cGVHD. In multivariable Cox analyses including IL12p40, IL18, age, mismatch, recipient and donor gender, disease stage, ATG, MTX, and diagnosis, IL18 associated with poor OS (HR per log2 increase 1.81 (1.19-2.74, p=0.005), whereas IL12p40 associated with superior OS (HR 0.53 (0.35-0.81), p=0.003). This could be validated in the independent cohort. Time course analyses of serum levels of CXCL9, IL12p40 and IL18 revealed a strong increase of CXCL9 and IL12p40 toward onset of cGVHD, but no stimulation of IL18 levels. Cumulative prednisolone intake&amp;gt;100 mg until day+100 significantly reduced IL12p40 serum levels on d180/at onset of cGVHD, without influencing IL18 production (Figure 1). In contrast, ATG reduced both, IL18 and IL12p40 at day180/onset of cGVHD. This might explain the reduced incidence of severe fibrosing cGVHD in patients receiving ATG prophylaxis (days-3 to -1 before alloSCT, 4% vs. 23%, p&amp;lt;0.001, n=343), whereas no effect of early prednisolone intake was noticed (p=0.916, n=137). Multicolor flow cytometry showed that IL12p40, but not IL18 correlated with blood counts of T-lymphocyte populations, including CD4+, CD8+ and γδ-Tcells. IL12p40 and γδ-Tcells were associated with reduced relapse rates. In summary, CXCL9, IL12p40 and IL18 are associated with severe cGVHD. IL18 correlated with severe fibrosing cGVHD and increased mortality, whereas IL12p40 was associated with reduced NRM, reduced relapse risk, improved lymphocyte recovery and γδ-T-cells, and was affected by prednisolone intake but not ATG. Conclusion: These results may help to understand and potentially separate the GVL effects from mortality/morbidity that associate with cGVHD. 1)PMID34755129 2)PMID31680001

Insensitive Effects of Inflammatory Cytokines on the Reference Genes of Synovial Fluid Resident-Mesenchymal Stem Cells Derived from Rheumatoid Arthritis Patients.

Mesenchymal stem cells derived from rheumatoid arthritis patients (RA-MSCs) provide an understanding of a variety of cellular and immunological responses within the inflammatory milieu. Sustained exposure of MSCs to inflammatory cytokines is likely to exert an influence on genetic variations, including reference genes (RGs). The sensitive effect of cytokines on the reference genes of RA-SF-MSCs may be a variation factor affecting patient-derived MSCs as well as the accuracy and reliability of data. Here, we comparatively evaluated the stability levels of nine RG candidates, namely GAPDH, ACTB, B2M, EEF1A1, TBP, RPLP0, PPIA, YWHAZ, and HPRT1, to find the most stable ones. Alteration of the RG expression was evaluated in MSCs derived from the SF of healthy donors (H-SF-MSCs) and in RA-SF-MSCs using the geNorm and NormFinder software programs. The results showed that TBP, PPIA, and YWHAZ were the most stable RGs for the normalization of H-SF-MSCs and RA-SF-MSCs using RT-qPCR, whereas ACTB, the most commonly used RG, was less stable and performed poorly. Additionally, the sensitivity of RG expression upon exposure to proinflammatory cytokines (TNF-α and IL-1β) was evaluated. RG stability was sensitive in the H-SF-MSCs exposed to TNF-α and IL-1β but insensitive in the RA-SF-MSCs. Furthermore, the normalization of IDO expression using ACTB falsely diminished the magnitude of biological significance, which was further confirmed with a functional analysis and an IDO activity assay. In conclusion, the results suggest that TBP, PPIA, and YWHAZ can be used in SF-MSCs, regardless of their exposure to inflammatory cytokines.

Analyzing serum tryptophan metabolites in patients with gestational diabetes mellitus

Abstract Introduction: Although pregnancy is a physiological condition, the secretion of diabetogenic hormones such as growth hormone, corticotropin-releasing hormone, placental lactogen hormone, prolactin and progesterone from the placenta could lead to insulin resistance (IR). In Metabolic Syndrome, obesity and Type1&amp;2 diabetes, a shift in the kynurenine pathway (KP) towards IDO activation is observed. The activation of the IDO leads to the activation of the Aryl hydrocarbon receptor (AhR) and Interleukin-6 (IL-6) also, which may also induce some effects like insulin resistance, β-cell disfunction and increased gluconeogenesis. We hypothesized that the overactivation of IDO and some KP enzymes would be observed in GDM patients, in a similar manner to metabolic syndrome, prediabetes, and diabetes patients. Methods: 50 patients and 50 controls, who applied to the Endocrinology outpatient clinic of Selcuk University Faculty of Medicine were included. Serum triptophan metabolite levels were measured with liquid chromatography tandem mass spectrometry. Results: Tryptophan and KYNA values was found to be lower in the patient group diagnosed with GDM (p&lt;0.001 and p&lt;0.001, respectively). The levels of KYN, 3-OH AA, 3-OH-KYN and KTR were significantly higher in the patient group compared to the control group (p=0.008, p&lt;0.001, p=0.05 and p&lt;0.001, respectively). Conclusions: Understanding the changes that occur in this pathway in GDM patients may provide insight into the development of the disease. Also these tests could be used as supplementary tests in gestational diabetes, which could assist in diagnosis and patient follow-up.

Development and Optimization of a Target Engagement Model of Brain IDO Inhibition for Alzheimer's Disease.

Indoleamine 2,3-dioxygenase (IDO1) inhibition is a promising target as an Alzheimer's disease (AD) Disease-modifying therapy capable of downregulating immunopathic neuroinflammatory processes. To aid in the development of IDO inhibitors as potential AD therapeutics, we optimized a lipopolysaccharide (LPS) based mouse model of brain IDO1 inhibition by examining the dosedependent and time-course of the brain kynurenine:tryptophan (K:T) ratio to LPS via intraperitoneal dosing. We determined the optimal LPS dose to increase IDO1 activity in the brain, and the ideal time point to quantify the brain K:T ratio after LPS administration. We then used a brain penetrant tool compound, EOS200271, to validate the model, determine the optimal dosing profile and found that a complete rescue of the K:T ratio was possible with the tool compound. This LPS-based model of IDO1 target engagement is a useful tool that can be used in the development of brain penetrant IDO1 inhibitors for AD. A limitation of the present study is the lack of quantification of potential clinically relevant biomarkers in this model, which could be addressed in future studies.

Effect of radiation fractionation on IDO1 via the NF-κB/COX2 axis in non-small cell lung cancer

Radiotherapy (RT) is the mainstay treatment modality for lung cancer. We recently reported that conventionally fractionated radiotherapy (CRT) with daily fractionation of 2Gy significantly increased the activity of indoleamine 2,3-dioxygenase (IDO1), a known immune checkpoint, which predicted poorer long-term survival in patients with non-small cell lung cancer (NSCLC), while stereotactic body radiotherapy (SBRT) using fractionation size of 10Gy did not increase IDO1 activity and had better survival. Here we hypothesized that the hypofractionated SBRT kind of dose fraction stimulates host antitumor immunity via downregulating IDO1 in which CRT could not. We tested this hypothesis in vitro and in vivo using 10Gyx1 and 2Gyx8 fractionations in the laboratory. The results demonstrated that, although there was an initial downregulation after RT, the expression of IDO1 was ultimately upregulated by both fractionation regimens. The 10Gyx1 regimen had minimum upregulation, while the 2Gyx8 regimen significantly increased in IDO1 expression which was positively correlated with the elevated expressions of p-NF-κB and COX2. Pharmacological inhibition of COX2 abolished RT-induced IDO1 expression. Furthermore, the IDO1 inhibitor, D-1-methyl-tryptophan (D-1MT), exerted RT-related tumor-killing effects in the NSCLC cell lines and mouse models. These findings suggest that, in addition to being an immune suppressor, IDO1 may serve as an adaptive resistance factor in RT. Furthermore, an unappreciated mechanism may exist, where a larger fraction size might be superior to conventional sizes in cancer treatment. This study may provide a rationale for future research in using IDO1 as a biomarker to personalize RT dose fractionation and COX2 inhibitor to decrease radiation immune suppression from CRT.

THE KYNURENINE PATHWAY AND ITS ROLE IN GLIOBLASTOMA CELL MIGRATION

Abstract AIMS The kynurenine (Kyn) pathway plays an important role in the pathogenesis of many cancers including glioblastomas (GBMs). The enzymes, indoleamine-2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO2), regulate the first and rate-limiting step resulting in the formation of N-formyl-kynurenine, which is then readily converted into Kyn by kynurenine formaldehyde. Cancers expressing the IDO1 and TDO2 enzymes can produce immunosuppressive tryptophan metabolites that compromise the host immune system leading to poor patient prognosis. The inhibitors TD12, TD18 and TD34 were recently developed due to the lack of TDO2 and IDO1/TDO2 inhibitors, of which TD34 can block both IDO1 and TDO2 activity. Here, we investigated the ability of these novel inhibitors to also target cell migration in GBM as part of our ongoing research interest in this area. METHOD Two established brain tumour cell lines, U87 and U251 and one patient-derived cell line, E55, were used to determine IC50 values and anti-migratory concentrations by MTT for application in 2D and 3D migration/invasion assays. RESULTS Cell migration in all cell lines was significantly reduced in the presence of the TD12, TD18 and TD34 inhibitors. E55 appeared to be 100-fold more sensitive to drug activity; immunofluorescence analysis indicates changes in the actin cytoskeleton and cell adhesion CONCLUSIONS Initial results suggest that TDO2 and the IDO1 enzymes play also a role in cell migration in GBM and provide further evidence for the involvement of the Kyn pathway in the pathology of GBMs. This also supports the application of inhibitors targeting TDO2 and IDO1 as novel complementary treatment options in GBM.

The kynurenine pathway in HIV, frailty and inflammaging.

Kynurenine (Kyn) is a circulating tryptophan (Trp) catabolite generated by enzymes including IDO1 that are induced by inflammatory cytokines such as interferon-gamma. Kyn levels in circulation increase with age and Kyn is implicated in several age-related disorders including neurodegeneration, osteoporosis, and sarcopenia. Importantly, Kyn increases with progressive disease in HIV patients, and antiretroviral therapy does not normalize IDO1 activity in these subjects. Kyn is now recognized as an endogenous agonist of the aryl hydrocarbon receptor, and AhR activation itself has been found to induce muscle atrophy, increase the activity of bone-resorbing osteoclasts, decrease matrix formation by osteoblasts, and lead to senescence of bone marrow stem cells. Several IDO1 and AhR inhibitors are now in clinical trials as potential cancer therapies. We propose that some of these drugs may be repurposed to improve musculoskeletal health in older adults living with HIV.