IDO Expression Research Articles

The Two Sides of Indoleamine 2,3-Dioxygenase 2 (IDO2).

Indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2 originated from gene duplication before vertebrate divergence. While IDO1 has a well-defined role in immune regulation, the biological role of IDO2 remains unclear. Discovered in 2007, IDO2 is located near the IDO1 gene. Because of their high sequence similarity, IDO2 was initially thought to be a tryptophan (Trp)-degrading enzyme like IDO1. Differently from what expected, IDO2 displays extremely low catalytic activity toward Trp. Nevertheless, many studies, often contradictory, have tried to demonstrate that IDO2 modulates immune responses by catabolizing Trp into kynurenine, an unconvincing hypothesis linked to an incomplete understanding of IDO2's activity. In this study, we review IDO2's functional role beyond Trp metabolism. IDO2's evolutionary persistence across species, despite being almost inactive as an enzyme, suggests it has some relevant biological importance. IDO2 expression in human normal cells is poor, but significant in various cancers, with two prevalent SNPs. Overall, the comparison of IDO2 to IDO1 as a Trp-degrading enzyme may have led to misunderstandings about IDO2's true physiological and pathological roles. New insights suggest that IDO2 might function more as a signaling molecule, particularly in cancer contexts, and further studies could reveal its potential as a target for cancer therapy.

IMMU-48. CRACKING THE ROLE OF ONCOMETABOLITES IN VIROIMMUNOTHERAPY FOR PEDIATRIC MALIGNANT BRAIN TUMORS

Abstract Diffuse midline gliomas (DMGs) are among the most frequent and lethal pediatric tumors. The standard of care for DMGs is palliative, resulting in a median survival of 12 months. Viroimmunotherapy is an emerging alternative treatment for these tumors. Our group developed a platform of oncolytic adenoviruses that was translated to the clinic. Specifically, Delta-24-RGD was recently tested in a Phase I clinical trial for patients with newly diagnosed DMG (NCT03178032) with encouraging results. To further stimulate the immune arm of this therapy, we developed Delta-24-RGDOX, expressing the T-cell activator OX40L (NCT03714334, NCT04714983). Using bulk RNAseq, we showed that treatment with Delta-24-RGDOX upregulated the immunosuppressive immunometabolic IDO-AhR pathway. Of importance, treatment with Th1-related cytokine IFNg resulted in the upregulation of IDO1 expression in a panel of human and murine DMG cells. In agreement with these data, we showed synergy between the oncolytic virus and several IDO1-AhR inhibitors in tumor and immune cell co-cultures, as indicated by T-cell activation and proliferation. To further dissect the reshaping of the TME in clinically relevant DMG models treated with Delta-24-RGD or Delta-24-RGDOX, we performed scRNA seq analysis. We showed a pronounced T-cell infiltration in virus-treated animals versus control-treated mice, further confirmed by flow cytometry. Additionally, Th1 and CD8 effector memory subsets were preferentially enriched in the Delta-24-RGDOX group, indicating an enhanced proinflammatory shift. Importantly, we detected, for the first time, upregulation of IDO-AhR downstream effectors in the myeloid compartment in the virus-treated groups. Interestingly, we detected IL4I1 in myeloid cells, a recently discovered checkpoint upstream of AhR never described in the context of virotherapy. Preliminary data using a combination of Delta-24-RGDOX and AhR inhibitors prolongs the survival of glioma-bearing mice. Collectively, our in vivo and in vitro data support the rationale to propel a future clinical trial combining Delta-24-RGDOX with AhR inhibitors for DMG.

Initial WNT/β-Catenin or BMP Activation Modulates Inflammatory Response of Mesodermal Progenitors Derived from Human Induced Pluripotent Stem Cells.

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines, we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers, while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing.

Investigation of IDO1 and TDO2 expression in breast tumors by immunohistochemistry and real-time polymerase chain reaction methods

Although diagnostic and therapeutic advances have been made in the treatment of breast cancer, the challenge of effectively controlling tumor progression persists. The objective of this study was to determine the relationship between IDO1 and TDO2 expression in breast cancer by immunohistochemistry and real-time polymerase chain reaction, hormone receptor status, Ki67 proliferation index, molecular classification, metastasis, and to investigate whether IDO1 and TDO2 expression can be used in combination with targeted therapy or as a marker to increase treatment efficacy in selected cases. The study included 74 cases of breast cancer and 14 cases of normal breast tissue as controls. All cases were analyzed for IDO1 and TDO2 by both immunohistochemistry and real-time polymerase chain reaction. The immunohistochemical analysis revealed that IDO1 immunoreactivity was significantly higher in tumor tissue compared to normal breast tissue. A statistically significant correlation was observed between IDO1 immunoreactivity and histologic subtypes. Furthermore, IDO1 gene expression was correlated with IDO1 immunoreactivity. TDO2 immune reactivity did not differ between tumor and non-tumor tissues and no correlation was found between histological subtypes. There was no correlation between TDO2 immunoreactivity and gene expression. The significant increase in IDO1 levels in tumor tissue and high positivity in age, HER2 positive and triple negative cases compared to other cases suggest that IDO1 inhibitors may be suitable for the target patient group in treatment selection.

IL-33 Induces a Switch in Intestinal Metabolites Revealing the Tryptophan Pathway as a Target for Inducing Allograft Survival.

IL-33, a pleiotropic cytokine, has been associated with a plethora of immune-related processes, both inflammatory and anti-inflammatory. T regulatory (Treg) cells, the main leukocyte population involved in immune tolerance, can be induced by the administration of IL-33, the local microbiota, and its metabolites. Here, we demonstrate that IL-33 drastically induces the production of intestinal metabolites involved on tryptophan (Trp) metabolism. naïve mice were treated with IL-33 for 4 days and leukocyte populations were analyzed by flow cytometry, and feces were processed for microbiota and intestinal metabolites studies. Using a murine skin transplantation model, the effect of Kynurenic acid (KA) on allograft survival was tested. Under homeostatic conditions, animals treated with IL-33 showed an increment in Treg cell frequencies. Intestinal bacterial abundance analysis indicates that IL-33 provokes dysbiosis, demonstrated by a reduction in Enterobacteria and an increment in Lactobacillus genera. Furthermore, metabolomics analysis showed a dramatic IL-33 effect on the abundance of intestinal metabolites related to amino acid synthesis pathways, highlighting molecules linked to Trp metabolism, such as kynurenic acid (KA), 5-Hydroxyindoleacetic acid (5-HIAA), and 6-Hydroxynicotinic acid (6-HNA), which was supported by an enhanced expression of Ido and Kat mRNA in MLN cells, which are two enzymes involved on KA synthesis. Interestingly, animals receiving KA in drinking water and subjected to skin transplantation showed allograft acceptance, which is associated with an increment in Treg cell frequencies. Our study reveals a new property for IL-33 as a modulator of the intestinal microbiota and metabolites, especially those involved with Trp metabolism. In addition, we demonstrate that KA favors Tregs in vivo, positively affecting skin transplantation survival.

Differential Host Gene Expression in Response to Infection by Different Mycobacterium tuberculosis Strains-A Pilot Study.

Tuberculosis (TB) represents a global public health threat and is a leading cause of morbidity and mortality worldwide. Effective control of TB is complicated with the emergence of multidrug resistance. Yet, there is a fundamental gap in understanding the complex and dynamic interactions between different Mycobacterium tuberculosis strains and the host. In this pilot study, we investigated the host immune response to different M. tuberculosis strains, including drug-sensitive avirulent or virulent, and rifampin-resistant or isoniazid-resistant virulent strains in human THP-1 cells. We identified major differences in the gene expression profiles in response to infection with these strains. The expression of IDO1 and IL-1β in the infected cells was stronger in all virulent M. tuberculosis strains. The most striking result was the overexpression of many interferon-stimulated genes (ISGs) in cells infected with the isoniazid-resistant strain, compared to the rifampin-resistant and the drug-sensitive strains. Our data indicate that infection with the isoniazid-resistant M. tuberculosis strain preferentially resulted in cGAS-STING/STAT1 activation, which induced a characteristic host immune response. These findings reveal complex gene signatures and a dynamic variation in the immune response to infection by different M. tuberculosis strains.

Sirtuin 1 regulates the phenotype and functions of dendritic cells through Ido1 pathway in obesity

Sirtuin 1 (SIRT1) is a class III histone deacetylase (HDAC3) that plays a crucial role in regulating the activation and differentiation of dendritic cells (DCs) as well as controlling the polarization and activation of T cells. Obesity, a chronic inflammatory condition, is characterized by the activation of immune cells in various tissues. We hypothesized that SIRT1 might influence the phenotype and functions of DCs through the Ido1 pathway, ultimately leading to the polarization towards pro-inflammatory T cells in obesity. In our study, we observed that SIRT1 activity was reduced in bone marrow-derived DCs (BMDCs) from obese animals. These BMDCs exhibited elevated oxidative phosphorylation (OXPHOS) and increased extracellular acidification rates (ECAR), along with enhanced expression of class II MHC, CD86, and CD40, and elevated secretion of IL-12p40, while the production of TGF-β was reduced. The kynurenine pathway activity was decreased in BMDCs from obese animals, particularly under SIRT1 inhibition. SIRT1 positively regulated the expression of Ido1 in DCs in a PPARγ-dependent manner. To support these findings, ATAC-seq analysis revealed that BMDCs from obese mice had differentially regulated open chromatin regions compared to those from lean mice, with reduced chromatin accessibility at the Sirt1 genomic locus in BMDCs from obese WT mice. Gene Ontology (GO) enrichment analysis indicated that BMDCs from obese animals had disrupted metabolic pathways, including those related to GTPase activity and insulin response. Differential expression analysis showed reduced levels of Pparg and Sirt1 in BMDCs from obese mice, which was challenged and confirmed using BMDCs from mice with conditional knockout of Sirt1 in dendritic cells (SIRT1∆). This study highlights that SIRT1 controls the metabolism and functions of DCs through modulation of the kynurenine pathway, with significant implications for obesity-related inflammation.

IDO1-mediated catabolism of tryptophan in gastric tumors: Its potential role in the axis of histopathology, differentiation and metastasis

BackgroundIndoleamine 2,3-Dioxygenase 1 (IDO1)-mediated tryptophan degradation, which is the rate-limiting enzyme of tryptophan/kynurenine pathway, may cause immune suppression in the tumor microenvironment, while potentiating proliferative and metastatic activity in the tumor focus, Phase studies of IDO1 inhibitors are ongoing, and our study aims to evaluate the potential contribution of IDO1 gene expression to the tryptophan/kynurenine pathway in tumor and tumor microenvironment foci in gastric cancer (GC) on a clinicopathological axis, MethodIn the case-control study design, the determination of tryptophan and its metabolites in the serum of 51 GC and 49 healthy controls was made using High Pressure Liquid Chromatography-Fluorescence Detector (HPLC-FD). IDO1 expression in a total of 102 tissues with tumor and tumor microenvironment was detected by quantitative PCR (q-PCR). ResultsIn gastric tumors, 3,25-fold decreased expression of IDO1 was detected according to the tumor microenvironment (p=0,05), IDO1 expression was found to be more than 2 times higher in signet ring cell carcinoma (SRCC) and poorly differentiated tumors without distant organ metastasis (p<0,05), In GC, tryptophan level was found to be 1,6 times lower than in control (AUC:0889; cut off≤21,57; p<0001), Low tryptophan level was found in advanced tumor stage compared to early stage and in the presence of perineural invasion compared to its absence (p<0,05) The level of kynurenine was found to be approximately 1,8 times lower in SRCC (p=0,04), ConclusionIncreased tryptophan accumulation in the gastric tumor and its microenvironment, when catabolized via IDO1, exhibits histological type, tumor differentiation, and metastasis-promoting effects more prominently in aggressive subtypes such as SRCC,

Tumor immune microenvironment dynamics and outcomes of prognosis in non-muscle-invasive bladder cancer.

Agents that target PD-1 and PD-L1 have been developed in the treatment of bladder cancer (BC). However, the diversity of immune cell infiltration in non-muscle-invasive BC (NMIBC) and the dynamics of the microenvironment as it progresses to muscle-invasive/metastatic disease remains unknown. To assess tumor immune activity, hierarchical clustering was applied to 159 BC samples based on cellular positivity for the defined immune cellular markers (CD3/CD4/CD8/FOXP3/CD20/PD-1/PD-L1/LAG3/TIGIT), divided into two clusters. There was a "hot cluster" (25%) consisting of patients with a high expression of these markers and a "cold cluster" (75%) comprising those without. The expression of CD39, CD44, CD68, CD163, IDO1, and Ki67 was significantly higher in tumors in the hot cluster. Immunologically, high-grade T1 tumors were significantly hotter, whereas tumors that had progressed to muscle invasion turned cold. However, a certain number of high-grade NMIBC patients were in the cold cluster, and these patients had a significantly higher risk of disease progression. Using an externally available TCGA dataset, RB1 and TP53 alterations were more frequently observed in TCGA hot cluster; rather FGFR3, KDM6A, and KMT2A alterations were common in TCGA cold/intermediate cluster. Analyses of recurrent tumors after BCG therapy revealed that tumor immune activity was widely maintained before and after treatment, and high FGFR3 expression was detected after recurrence in tumors initially classified into the cold cluster. Collectively, we revealed the dynamics of the tumor microenvironment in BC as a whole and identified candidate molecules as therapeutic targets for recurrent NMIBC, e.g., after BCG therapy.

Clinicopathological and prognostic significance of indoleamine 2,3-dioxygenase (IDO) expression in head and neck squamous cell carcinoma: A systematic review and meta-analysis

ObjectiveIndoleamine 2,3-dioxygenase-1 (IDO1) is a promising antitumor target and predictive biomarker in a variety of cancers. Hence, we performed this meta-analysis to evaluate the clinicopathological and prognostic significance of IDO1 in head and neck squamous cell carcinoma (HNSCC). MethodsWe searched PubMed, Embase, Web of Science and Scopus databases from inception to May 2024, to identify studies measuring the clinicopathological and prognostic significance of IDO1 in HNSCC. The role of IDO1 in HNSCC was evaluated by pooled hazard ratios (HR), odd ratios (OR) and 95% confidence intervals (CI). The meta-analysis was performed using the meta package in R. Omics analyses on IDO1 were also performed. ResultsTen studies (1,119 participants) were included in the review. The analysis showed an insignificant relationship between IDO1 expression and poor overall survival, and progression free survival as indicated by the pooled HR (HR: 1.65, 95% CI: 0.68–4.02), (HR: 1.73, 95% CI: 0.63–4.77), respectively. Additionally, elevated expression of IDO1 was significantly associated with tumor T stage (OR: 1.44, 95% CI: 1.06–1.94). However, it was insignificantly correlated with positive lymph node metastasis (N status) (OR: 1.11; 95% CI: 0.82–1.50) and tumor-node-metastasis (TNM) stage (OR: 1.14; 95% CI: 0.79–1.64). ConclusionWhile higher IDO1 expression is associated with the risk of advanced tumor stage in HNSCC, its impact on overall and progression-free survival remains inconclusive. Further research is needed to elucidate its prognostic significance and therapeutic potential.

Evaluation of TNF-α and IFN-γ primed conditioned medium of mesenchymal stem cell in acetic acid-induced mouse model of acute colitis

IBD, an autoimmune-inflammatory disorder that affects people who are genetically prone to inflammation. There is a lot of interest in MSC-CM therapy, especially when primed with TNF-α + IFN-γ. Throughout the study, data were collected on the percentage of apoptotic cells, gene expression of ZO-1, Foxp3, GATA3, IDO-1, Muc2, T-bet, Notch1, TNFR2, and ROR-γt, colon weight and length, histopathological analysis, and DAI. TNF-α and IL-10 levels were assessed in addition to the NO level. The results suggest that primed MSC-CM improved DAI, mucosal deterioration, intestinal inflammation and NO concentration. The amount of TNF-α was decreased, but IL-10 and the colon’s percentage of apoptotic cells was increased. The mRNA expression of ZO-1, Foxp3, GATA3, IDO-1, and Muc2 genes increased greatly in the treatment groups, while the expression of T-bet, Notch1, TNFR2, and ROR-γt genes has decreased. These studies suggest that primed MSC-CM may combine with common treatments to improve responsiveness.

Rifaximin ameliorates influenza A virus infection-induced lung barrier damage by regulating gut microbiota

Prior research has indicated that the gut-lung-axis can be influenced by the intestinal microbiota, thereby impacting lung immunity. Rifaximin is a broad-spectrum antibacterial drug that can maintain the homeostasis of intestinal microflora. In this study, we established an influenza A virus (IAV)-infected mice model with or without rifaximin supplementation to investigate whether rifaximin could ameliorate lung injury induced by IAV and explore the molecular mechanism involved. Our results showed that IAV caused significant weight loss and disrupted the structure of the lung and intestine. The analysis results of 16S rRNA and metabolomics indicated a notable reduction in the levels of probiotics Lachnoclostridium, Ruminococcaceae_UCG-013, and tryptophan metabolites in the fecal samples of mice infected with IAV. In contrast, supplementation with 50 mg/kg rifaximin reversed these changes, including promoting the repair of the lung barrier and increasing the abundance of Muribaculum, Papillibacter and tryptophan-related metabolites content in the feces. Additionally, rifaximin treatment increased ILC3 cell numbers, IL-22 level, and the expression of RORγ and STAT-3 protein in the lung. Furthermore, our findings demonstrated that the administration of rifaximin can mitigate damage to the intestinal barrier while enhancing the expression of AHR, IDO-1, and tight junction proteins in the small intestine. Overall, our results provided that rifaximin alleviated the imbalance in gut microbiota homeostasis induced by IAV infection and promoted the production of tryptophan-related metabolites. Tryptophan functions as a signal to facilitate the activation and movement of ILC3 cells from the intestine to the lung through the AHR/STAT3/IL-22 pathway, thereby aiding in the restoration of the barrier.Key points• Rifaximin ameliorated IAV infection-caused lung barrier injury and induced ILC3 cell activation.• Rifaximin alleviated IAV-induced gut dysbiosis and recovered tryptophan metabolism.• Tryptophan mediates rifaximin-induced ILC3 cell activation via the AHR/STAT3/IL-22 pathway.

Utility of PD-1, PD-L1, and IDO-1 Stains in Ocular Extranodal Marginal Zone Lymphoma (MZL) and Diffuse Large B-cell Lymphoma (DLBCL).

Extranodal marginal zone lymphoma (EMZL) is the most common subtype of ocular lymphomas. Diffuse large B-cell lymphoma (DLBCL) and EMZL with large-cell transformation present diagnostic challenges. Radiotherapy is the standard treatment for ocular lymphomas, but complications and relapse are common. Diagnostic utility in challenging cases, as well as treatment options using immune checkpoint inhibitors, are unclear in ocular lymphomas. We herein investigated the PD-1, PD-L1, and IDO1 staining patterns in 20 cases of ocular lymphomas, including EMZL (n=14), EMZL with increased large cells (n=2), and DLBCL (n=4). PD-1, PD-L1, and IDO1 staining was not detected in lymphoma cells in any cases but was observed within the tumor microenvironment in all cases. Positivity for PD-1, PD-L1, and IDO1 in inflammatory cells was seen either intratumorally or peritumorally. In all 6 cases with significantly more large B cells, the density of PD-1, PD-L1, and IDO1 expression in the tumor microenvironment was higher than that of the remaining 14 cases without large B cells ( P -value<0.0001), whereas other clinicopathologic features showed no statistical correlation. Increased expression of PD-1, PD-L1, and IDO1 in the inflammatory milieu in cases with large cells may provide diagnostic utility in small biopsies as well as therapeutic potential.

Hyperactivation of mTOR/eIF4E Signaling Pathway Promotes the Production of Tryptophan-To-Phenylalanine Substitutants in EBV-Positive Gastric Cancer.

Although messenger RNA translation is tightly regulated to preserve protein synthesis and cellular homeostasis, chronic exposure to interferon-γ (IFN-γ) in several cancers can lead to tryptophan (Trp) shortage via the indoleamine-2,3-dioxygenase (IDO)- kynurenine pathway and therefore promotes the production of aberrant peptides by ribosomal frameshifting and tryptophan-to-phenylalanine (W>F) codon reassignment events (substitutants) specifically at Trp codons. However, the effect of Trp depletion on the generation of aberrant peptides by ribosomal mistranslation in gastric cancer (GC) is still obscure. Here, it is shows that the abundant infiltrating lymphocytes in EBV-positive GC continuously secreted IFN-γ, upregulated IDO1 expression, leading to Trp shortage and the induction of W>F substitutants. Intriguingly, the production of W>F substitutants in EBV-positive GC is linked to antigen presentation and the activation of the mTOR/eIF4E signaling pathway. Inhibiting either the mTOR/eIF4E pathway or EIF4E expression counteracted the production and antigen presentation of W>F substitutants. Thus, the mTOR/eIF4E pathway exposed the vulnerability of gastric cancer by accelerating the production of aberrant peptides and boosting immune activation through W>F substitutant events. This work proposes that EBV-positive GC patients with mTOR/eIF4E hyperactivation may benefit from anti-tumor immunotherapy.

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, non-lethal Plasmodium yoelii strain 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.

Exploring the Surface: Sampling of Potential Skin Cancer Biomarkers Kynurenine and Tryptophan, Studied on 3D Melanocyte and Melanoma Models.

Early detection of cancer via biomarkers is vital for improving patient survival rates. In the case of skin cancers, low-molecular-weight biomarkers can penetrate the skin barrier, enabling non-invasive sampling at an early stage. This study focuses on detecting tryptophan (Trp) and kynurenine (Kyn) on the surface of reconstructed 3D melanoma and melanocyte models. This is examined in connection with IDO-1 and IL-6 expression in response to IFN-γ or UVB stimulation, both crucial factors of the melanoma tumor microenvironment (TME). Using a polystyrene scaffold, full-thickness human skin equivalents containing fibroblasts, keratinocytes, and melanocytes or melanoma cells were developed. The samples were stimulated with IFN-γ or UVB, and Trp and Kyn secretion was measured using HPLC-PDA and HPLC-MS. The expression of IDO-1 and IL-6 was measured using RT-qPCR. Increased Trp catabolism to Kyn was observed in IFN-γ-stimulated melanoma and melanocyte models, along with higher IDO-1 expression. UVB exposure led to significant changes in Kyn levels but only in the melanoma model. This study demonstrates the potential of skin surface Trp and Kyn monitoring to capture TME metabolic changes. It also lays the groundwork for future in vivo studies, aiding in understanding and monitoring skin cancer progression.

Metabolic profiling of tryptophan pathways: Implications for obesity and metabolic dysfunction-associated steatotic liver disease.

The rise in obesity highlights the need for improved therapeutic strategies, particularly in addressing metabolic dysfunction-associated steatotic liver disease (MASLD). We aim to assess the role of tryptophan metabolic pathways in the pathogenesis of obesity and in the different histological stages of MASLD. We used ultra-high performance liquid chromatography to quantify circulating levels of 15 tryptophan-related metabolites from the kynurenine, indole and serotonin pathways. A cohort of 76 subjects was analysed, comprising 18 subjects with normal weight and 58 with morbid obesity, these last being subclassified into normal liver (NL), simple steatosis (SS) and metabolic dysfunction-associated steatohepatitis (MASH). Then, we conducted gene expression analysis of hepatic IDO-1 and kynyrenine-3-monooxygenase (KMO). Key findings in obesity revealed a distinct metabolic signature characterized by a higher concentration of different kynurenine-related metabolites, a decrease in indole-3-acetic acid and indole-3-propionic acid, and an alteration in the serotonin pathway. Elevated tryptophan levels were associated with MASLD presence (37.659 (32.577-39.823) μM of tryptophan in NL subjects; 41.522 (38.803-45.276) μM in patients with MASLD). Overall, pathway fluxes demonstrated an induction of tryptophan catabolism via the serotonin pathway in SS subjects and into the kynurenine pathway in MASH. We found decreased IDO-1 and KMO hepatic expression in NL compared to SS. We identified a distinctive metabolic signature in obesity marked by changes in tryptophan catabolic pathways, discernible through altered metabolite profiles. We observed stage-specific alterations in tryptophan catabolism fluxes in MASLD, highlighting the potential utility of targeting these pathways in therapeutic interventions.

Distinct functions of wild-type and R273H mutant Δ133p53α differentially regulate glioblastoma aggressiveness and therapy-induced senescence

Despite being mutated in 92% of TP53 mutant cancers, how mutations on p53 isoforms affect their activities remain largely unknown. Therefore, exploring the effect of mutations on p53 isoforms activities is a critical, albeit unexplored area in the p53 field. In this article, we report for the first time a mutant Δ133p53α-specific pathway which increases IL4I1 and IDO1 expression and activates AHR, a tumor-promoting mechanism. Accordingly, while WT Δ133p53α reduces apoptosis to promote DNA repair, mutant R273H also reduces apoptosis but fails to maintain genomic stability, increasing the risks of accumulation of mutations and tumor’s deriving towards a more aggressive phenotype. Furthermore, using 2D and 3D spheroids culture, we show that WT Δ133p53α reduces cell proliferation, EMT, and invasion, while the mutant Δ133p53α R273H enhances all three processes, confirming its oncogenic potential and strongly suggesting a similar in vivo activity. Importantly, the effects on cell growth and invasion are independent of mutant full-length p53α, indicating that these activities are actively carried by mutant Δ133p53α R273H. Furthermore, both WT and mutant Δ133p53α reduce cellular senescence in a senescence inducer-dependent manner (temozolomide or radiation) because they regulate different senescence-associated target genes. Hence, WT Δ133p53α rescues temozolomide-induced but not radiation-induced senescence, while mutant Δ133p53α R273H rescues radiation-induced but not temozolomide-induced senescence. Lastly, we determined that IL4I1, IDO1, and AHR are significantly higher in GBMs compared to low-grade gliomas. Importantly, high expression of all three genes in LGG and IL4I1 in GBM is significantly associated with poorer patients’ survival, confirming the clinical relevance of this pathway in glioblastomas. These data show that, compared to WT Δ133p53α, R273H mutation reorientates its activities toward carcinogenesis and activates the oncogenic IL4I1/IDO1/AHR pathway, a potential prognostic marker and therapeutic target in GBM by combining drugs specifically modulating Δ133p53α expression and IDO1/Il4I1/AHR inhibitors.

Long-Term Follow-up of Levonorgestrel Intrauterine Device for Atypical Hyperplasia and Early Endometrial Cancer Reveals Relapse Characterized by Immune Exhaustion.

Nonsurgical treatment options are increasingly needed for endometrial atypical hyperplasia (AH) and endometrioid endometrial cancer (EEC). Despite promising initial response rates, prospective long-term data and determinants for relapse are limited. Follow-up data from patients in our prospective phase II trial of levonorgestrel intrauterine device (LIUD) for AH/G1EEC were collected from medical records. Spatial transcriptomics (Nanostring GeoMX digital spatial profiling) with in silico cell type deconvolution and pathway analyses were employed on longitudinal biopsy samples from five patients across pre-treatment, on-treatment, and relapse. Of 43 participants exhibiting initial response to LIUD, 41 had follow-up data. Sixteen (39%) experienced relapse. Clinical factors associated with shorter response duration included younger age, initial diagnosis of G1EEC, lack of response at 6 months, premenopausal status, and Hispanic ethnicity (P < 0.05), but only 6-month response status remained a significant predictor in a multivariate model (P = 0.023). LIUD increased abundance of NK cells (ΔMCP-counter score = 46.13, FDR = 0.004) and cytotoxic lymphocytes (ΔMCP-counter score = 277.67, FDR = 0.004), as well as lymphocyte cytotoxicity markers PRF1 (log2FC = 1.62, FDR = 0.025) and GZMA (log2FC = 2.47, FDR = 0.008). NK cells were reduced at relapse (ΔMCP-counter score = -55.96, FDR = 0.02). Immune-related pathways (IFNα response and TGFβ signaling) were enriched at relapse (FDR < 0.05). IDO1 expression, reflecting immune exhaustion, was upregulated at relapse (FDR < 0.05). Upfront resistance and relapse after initial response to LIUD for AH/G1EEC impacts nearly half of patients, remaining a major hurdle for nonsurgical treatment of AH/G1EEC. Molecular studies evaluating longitudinal biopsies from a small cohort implicate immune mechanisms at relapse, including reversal of progestin-related immunomodulation and increased immune exhaustion. See related commentary by Johannet and Friedman, p. 5001.

Osteostaticytes: A novel osteoclast subset couples bone resorption and bone formation

BackgroundOsteomyelitis (OM) is an inflammatory condition of bone characterized by cortical bone devascularization and necrosis. Dysregulation of bone remodelling is triggered by OM. Bone remodelling is precisely coordinated by bone resorption and formation via a reversal phase. However, the cellular and molecular mechanisms underlying bone remodelling failure after osteomyelitis remain elusive. MethodsTo elucidate the cellular and molecular mechanism underlying bone healing after osteomyelitis, we employed single-cell RNA sequencing (scRNA-seq) to depict the atlas of human cortical bone in normal, infected and reconstructed states. Dimensionality reduction by t-stochastic neighbourhood embedding (t-SNE) and graph-based clustering were applied to analyse the detailed clusters of osteoclast lineages. After trajectory analysis of osteoclast lineages over pseudotime, real-time PCR and immunofluorescence (IF) staining were applied to identify marker gene expression of various osteoclast lineages in the osteoclast induction model and human bone sections, respectively. The potential function and communication of osteoclasts were analysed via gene set enrichment analysis (GSEA) and CellChat. The chemotactic ability of mesenchymal stem cells (MSCs) and osteoclast lineage cells in various differentiation states was determined by transwell assays and coculture assays. The effects of various osteoclast lineages on the osteogenic differentiation potential of MSCs were also determined by using this coculture system. A normal mouse tibia fracture model and an osteomyelitis-related tibia fracture model were generated via injection of luciferase-labelled Staphylococcus aureus to verify the relationships between a novel osteoclast lineage and MSCs. Then, the infection was detected by a bioluminescence imaging system. Finally, immunofluorescence staining was used to detect the expression of markers of MSCs and novel osteoclast lineages in different remodelling phases in normal and infected bone remodelling models. ResultsIn this study, we constructed a cell atlas encompassing normal, infected, and reconstructed cortical bone. Then, we identified a novel subset at the earlier stage of the osteoclast lineage that exhibited increased expression of IDO1, CCL3, and CCL4. These IDO1highCCL3highCCL4high cells, termed osteostaticytes (OSCs), were further regarded as the reservoir of osteoclasts in the reversal phase. Notably, OSCs exhibited the highest chemotactic activity, surpassing other lineage subsets. We also discovered that cells at the earlier stage of the osteoclast lineage play a significant role in recruiting mesenchymal stem cells (MSCs). Finally, the data revealed that OSCs might be positively related to the occurrence of bone MSCs and the contribution of bone remodelling. ConclusionCollectively, our findings revealed a novel stage (OSC) within the osteoclast lineage, potentially representing elusive bone reversal cells due to its increased chemotactic ability towards MSCs and potential contribution to bone remodelling. This study provides valuable insights into the intricate mechanisms of the reversal phase during bone remodelling and unveils potential therapeutic strategies for diseases associated with bone uncoupling. Translational potential of this articleThis study identified a new subset, referred to as IDO1(plus symbol) CCL3(plus symbol) CCL4(plus symbol) osteostaticytes which displayed the highest chemotactic activity among all osteoclast lineages and may serve as reversal cells in bone remodelling. These findings offer new insights and insights for understanding bone reversal-related diseases and may serve as novel therapeutic targets for conditions such as osteomyelitis and delayed bone healing.