Transcription Factor Aryl Hydrocarbon Receptor Research Articles

Transcription Factors AhR and ARNT Regulate the Expression of CYP6SX1 and CYP3828A1 Involved in Insecticide Detoxification in Bradysia odoriphaga.

Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to β-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.

Sustained AhR activity programs memory fate of early effector CD8+ T cells

Identification of mechanisms that program early effector T cells to either terminal effector T (Teff) or memory T (Tm) cells has important implications for protective immunity against infections and cancers. Here, we show that the cytosolic transcription factor aryl hydrocarbon receptor (AhR) is used by early Teff cells to program memory fate. Upon antigen engagement, AhR is rapidly up-regulated via reactive oxygen species signaling in early CD8+ Teff cells, which does not affect the effector response, but is required for memory formation. Mechanistically, activated CD8+ T cells up-regulate HIF-1α to compete with AhR for HIF-1β, leading to the loss of AhR activity in HIF-1αhigh short-lived effector cells, but sustained in HIF-1αlow memory precursor effector cells (MPECs) with the help of autocrine IL-2. AhR then licenses CD8+ MPECs in a quiescent state for memory formation. These findings partially resolve the long-standing issue of how Teff cells are regulated to differentiate into memory cells.

Dietary environmental factors shape the immune defense against Cryptosporidium infection

Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. Although it is known from human cases and animal models that CD4+ Tcells play a role in curbing Cryptosporidium, the role of CD8+ T cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express and depend on the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduces CD8+ IELs, decreases their cytotoxicity, and worsens infection. Transfer of CD8+ IELs rescues severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol in newborn mice promotes resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.

Type 3 Innate Lymphoid Cells Promote Acute Myeloid Leukemia Immune Evasion and Expansion

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy originated in the bone marrow with poor overall survival rates of less than 30%. The immune system has been shown to play an important role in detecting and eliminating leukemic cells. Recent studies have described a population of immune cells termed Innate lymphoid cells (ILCs). ILCs are known to regulate both innate and adaptive immunity in an antigen-independent manner and are believed to be among the first immune cells to interact with malignantly transformed cells. Natural killer (NK) cells are a subset of ILCs and are known to inhibit AML progression, however the impact of other ILCs is not yet fully understood. Type 3 ILCs (ILC3s) have key roles in maintaining homeostasis in mucosal immunity through their ability to secrete cytokines including IL-17, IL-22, TNFα and GM-CSF (pro-myeloid). An expansion of ILC3s has recently been observed in solid tumors, however the overall functional impact of ILC3s in the setting of AML remains poorly understood and warrants further investigation. We first utilized an MLL-partial tandem duplication and FLT3-internal tandem duplication Flt3-ITD double knock-in mutation murine model of AML that recapitulates two clinically relevant mutations in human AML. Using this AML model, we discovered an expansion of ILC3s in the bone marrow of leukemic mice compared to wild-type control mice (2.364% +/- 0.40% AML vs 0.045% +/- 0.40% WT, n=5, p<0.0001). We also observed an increase in ILC3s in the bone marrow of untreated AML patients relative to age matched donor controls (7.013% +/- 2.6% vs. 2.328% +/- 2.6%). Our published data have previously shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. AHR is an important transcription factor in ILC3 development. Therefore, we hypothesized that the ILC3 increase was due at least in part to AHR-mediated increased ILC3 differentiation. To address this hypothesis, we isolated the ILC precursor (ILCP) from normal human donors and co-cultured with AML cells. By regulating AHR activity in this system, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (28.02% +/- 3.89% without AML, 60% +/- 5.5% with AML and 27% +/- 5% AML+AHR inhibitor, n=15, p<0.001). Next, we sought to determine the direct impact of ILC3s on AML proliferation and differentiation that could contribute to AML relapse. We utilized a colony forming assay (CFU) assay in the presence or absence of ILC3s. Bone marrow from primary untreated AML patients was cultured alone or with ILC3s (Lin-CD94-NKp44+). AML samples co-cultured with ILC3s had significantly increased colony formation relative to AML alone (69.4 CFU AML+ILC3 vs 42.7 CFU AML alone, n=8, p<0.05). Post-culture, we observed significant increases in the transcript of GM-CSF and TNFα in ILC3s co-cultured with AML marrow as compared to ILC3s alone. Furthermore, in our immunocompetent murine AML model, we observed significantly higher production of GM-CSF and TNFα by ILC3s in the bone marrow of leukemic mice relative to nonleukemic controls (GM-CSF: 11.08% +/- 1.6 AML vs 1.128% +/- 1.6 WT, n=10, p<0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p<0.05), suggesting a potential mechanism of ILC3-mediated increased colony formation. Lastly, to determine if ILC3 expansion impacted NK cell immune surveillance, we co-cultured normal donor human ILC3s with AML cells, separated by transwell, for 48 hours before subjecting the AML cells to an NK cell cytotoxicity assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect AML cells from NK cell-mediated killing (24.3% killing +/- 3.81% AML alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p<0.01). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion to both support AML proliferation and protect AML blasts from immune surveillance. Future studies will focus in exploring how to target these interactions to design effective therapeutic approaches and improve patient clinical outcomes.

Nuclear localization of STING1 competes with canonical signaling to activate AHR for commensal and intestinal homeostasis

Extensive studies demonstrate the importance of the STING1 (also known as STING) protein as a signaling hub that coordinates immune and autophagic responses to ectopic DNA in the cytoplasm. Here, we report a nuclear function of STING1 in driving the activation of the transcription factor aryl hydrocarbon receptor (AHR) to control gut microbiota composition and homeostasis. This function was independent of DNA sensing and autophagy and showed competitive inhibition with cytoplasmic cyclic guanosine monophosphate (GMP)-AMP synthase (CGAS)-STING1 signaling. Structurally, the cyclic dinucleotide binding domain of STING1 interacted with the AHR N-terminal domain. Proteomic analyses revealed that STING1-mediated transcriptional activation of AHR required additional nuclear partners, including positive and negative regulatory proteins. Although AHR ligands could rescue colitis pathology and dysbiosis in wild-type mice, this protection was abrogated by mutational inactivation of STING1. These findings establish a key framework for understanding the nuclear molecular crosstalk between the microbiota and the immune system.

Polyphenol and glucosinolate-derived AhR modulators regulate GPR15 expression on human CD4+ T cells

Diets high in fruit and vegetables are perceived to be beneficial for intestinal homeostasis, in health as well as in the context of inflammatory bowel diseases (IBDs). Recent breakthroughs in the field of immunology have highlighted the importance of the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) as a critical regulator of mucosal immunity, including the intestinal trafficking of CD4+ helper T cells, an immune cell subset implicated in a wide range of homeostatic and pathogenic processes. Specifically, the AhR has been shown to directly regulate the expression of the chemoattractant receptor G Protein-Coupled Receptor 15 (GPR15) on CD4+ T cells. GPR15 is an important gut homing marker whose expression on CD4+ T cells in the peripheral circulation is elevated in patients suffering from ulcerative colitis, raising the possibility that, in this setting, the beneficial effect of a diet rich in fruits and vegetables may be mediated through the modulation of GPR15 expression. To address this, we screened physiologically-relevant polyphenol and glucosinolate metabolites for their ability to affect both AhR activity and GPR15 expression. Our complementary approach and associated findings suggest that polyphenol and glucosinolate metabolites can regulate GPR15 expression on human CD4+ T cells in an AhR-dependent manner.

Tyrosol: Repercussion of the Lack of a Hydroxyl-Group in the Response of MCF-7 Cells to Hypoxia.

In solid tumors, such as breast cancer, hypoxic microenvironment worsens patient prognoses. We have previously reported in MCF-7 breast cancer cells that, under hypoxic conditions, hydroxytyrosol (HT) downregulates the level of reactive oxygen species, reduces the expression of hypoxia inducible factor-1α (HIF-1α), and, at high concentrations, can bind to the aryl hydrocarbon receptor (AhR). With this background, the present study investigated whether the most abundant extra virgin olive oil (EVOO) phenolic compound tyrosol (TYR), with a chemical structure similar to HT but with only one hydroxyl group, exerts comparable effects. Our results revealed that, although TYR did not show any antioxidant activity in hypoxic MCF-7 cells, it inhibited the PI3K/Akt/mTOR/S6 kinase (S6K) pathway and reduced the expression of HIF-1α and some of its target genes. Besides, TYR showed a lower binding affinity with the cytosolic transcription factor AhR, and even reduced its transcriptional activity. Some of these results are positive to control tumor progression in a hypoxic environment; however, they are observed at doses unachievable with diet intake or nutraceutical presentations. Considering that EVOO phenols can have synergistic effects, a mixture of low doses of TYR and other phenols could be useful to achieve these beneficial effects.

Abstract 644: Gut microbiota-derived metabolites as regulators of immunity in pre-cancer

Abstract Introduction: Tryptophan (Trp) as an essential amino acid that can only be obtained through diet and is a precursor of serotonin and melatonin and required for protein and niacin biosynthesis. In addition, Trp is appreciated for its influence on both host and microbial metabolism. Trp is shunted into the Kyn pathway (mostly) and indolic pathway (bacterial degradation), especially in the immune cells. Both Kyn and Indole/indole derivatives, in part by activating the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR), can become dysfunctional in cancer. Most individuals with cancer such as colon cancer, exhibit increased L-kynurenine levels, which in part is explained by progression of immunosuppressive phenotype and excessive inflammatory signaling molecules. To date, studies in mice and humans have linked Kyn and AHR as potent modulators of host immunity to colon cancer (CRC). Nothing is yet known about their role in pre-cancer, during the development of adenomatous polyps. We hypothesized that increases in plasma Kyn and I3A and their receptor AHR on immune cells would be detected in patients with pre-malignant polyps, suggesting dysbiosis and increased colon cancer risk due to immune modulation. Methods: We measured with mass spectrometry Kyn and I3A in plasma samples of 88 polyp patients and compared to 22 colon cancer patients. We analyzed by flow cytometry PBMCs from 64 newly diagnosed advanced polyp patients (40-70 years old) and 9 age-matched colon cancer patients in order to evaluate AHR expression in different subsets of T cells, NKT cells and NK cells, and their possible association with exhaustion markers Tim-3, LAG-3 and PD-1, as well as Granzyme K, and the presence of immunosuppressive Treg and MDSC. Results: We showed high level of expression of gut microbiota-derived metabolites in plasma of colon pre-cancer compared to cancer patients. We identified CD8+ T effector memory cells (TEM) as primary T cell population expressing their receptor AHR in polyp patients, which had a statistically significant association with Granzyme K expression, which is a hallmark of inflammaging. Conclusions: Our results suggest gut microbiota-derived metabolites as potential mediators of immunosuppression in the setting of premalignancy, which could increase cancer risk and be targets of cancer-preventing strategies. Citation Format: Amir Hossein Mohseni, Sedigheh Taghinezhad-S, Pamela L. Beatty, Olivera J. Finn. Gut microbiota-derived metabolites as regulators of immunity in pre-cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 644.

ARNTL2 upregulation of ACOT7 promotes NSCLC cell proliferation through inhibition of apoptosis and ferroptosis

BackgroundRecent studies have reported that the circadian transcription factor aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) promotes the metastatic progression of lung adenocarcinoma. However, the molecular mechanisms of ARNTL2 in non-small cell lung cancer (NSCLC) cell growth and proliferation remain to be explored.MethodsThe expression of ARNTL2 and acyl-CoA thioesterase 7 (ACOT7) in lung cancer patients was analyzed based on TCGA database. Gain-of-function of ARNTL2 and ACOT7 was conducted by transfecting the cells with plasmids or lentivirus. Knockdown assay was carried out by siRNAs. Western blot and qRT-PCR were performed to check the protein and mRNA expression. Dual luciferase and ChIP-qPCR assay was applied to check the interaction of ARNTL2 on ACOT7’s promoter sequence. Triglyceride level, MDA production, the activity of casapase 3 to caspase 7, and lipid ROS were measured by indicated assay kit. Cellular function was detected by CCK8, colony formation and flow cytometry analysis of cell death and cell cycle.ResultsWe demonstrated that ARNTL2 upregulation of ACOT7 was critical for NSCLC cell growth and proliferation. Firstly, overexpression of ARNTL2 conferred the poor prognosis of LUAD patients and supported the proliferation of NSCLC cells. Based on molecular experiments, we showed that ARNTL2 potentiated the transcription activity of ACOT7 gene via direct binding to ACOT7’s promoter sequence. ACOT7 high expression was correlated with the worse prognosis of LUAD patients. Gain-of-function and loss-of-function experiments revealed that AOCT7 contributed to NSCLC cell growth and proliferation. ACOT7 regulated the apoptosis and ferroptosis of NSCLC cells, while exhibited no effect on cell cycle progression. ACOT7 overexpression also potentiated fatty acid synthesis and suppressed lipid peroxidation. Lastly, we showed that ARNTL2 knockdown and overexpression inhibited and promoted the cellular triglyceride production and subsequent cell proliferation, which could be reversed by ACOT7 overexpression and knockdown.ConclusionOur study illustrated the oncogenic function of ARNTL2/ACOT7 axis in the development of NSCLC. Targeting ARNTL2 or ACOT7 might be promising therapeutic strategies for NSCLC patients with highly expressed ARNTL2.

Impact of the Aryl Hydrocarbon Receptor on Aurora A Kinase and the G2/M Phase Pathway in Hematopoietic Stem and Progenitor Cells

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.

Tubular aryl hydratocarbon receptor upregulates EZH2 to promote cellular senescence in cisplatin-induced acute kidney injury

Acute kidney injury (AKI) is one of the serious clinical syndromes with high morbidity and mortality. Despite substantial progress in understanding the mechanism of AKI, no effective drug is available for treatment or prevention. In this study, we identified that a ligand-activated transcription factor aryl hydrocarbon receptor (AhR) was abnormally increased in the kidneys of cisplatin-induced AKI mice or tubular epithelial TCMK-1 cells. The AhR inhibition by BAY2416964 and tubular conditional deletion both alleviated cisplatin-induced kidney dysfunction and tubular injury. Notably, inhibition of AhR could improve cellular senescence of injured kidneys, which was indicated by senescence-associated β-galactosidase (SA-β-gal) activity, biomarker p53, p21, p16 expression, and secretory-associated secretory phenotype IL-1β, IL-6 and TNFα level. Mechanistically, the abnormal AhR expression was positively correlated with the increase of a methyltransferase EZH2, and AhR inhibition suppressed the EZH2 expression in cisplatin-injured kidneys. Furthermore, the result of ChIP assay displayed that EZH2 might indirectly interact with AhR promoter region by affecting H3K27me3. The direct recruitment between H3K27me3 and AhR promoter is higher in the kidneys of control than that of cisplatin-treated mice, suggesting EZH2 reversely influenced AhR expression through weakening H3K27me3 transcriptional inhibition on AhR promoter. The present study implicated that AhR and EZH2 have mutual regulation, which further accelerated tubular senescence in cisplatin-induced AKI. Notably, the crucial role of AhR is potential to become a promising target for AKI.

Identification and Functional Characterization of the Transcription Factors AhR/ARNT in Dendroctonus armandi

The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the bHLH-PAS (basic Helix-Loop-Helix-Period/ARNT/Single-minded) family of transcription factors, which participate in the sensing and transmitting stimuli of exogenous and endogenous chemical substances, and subsequently activates genes transcription involved in various detoxification and physiological functions. However, they have not been identified in Dendroctonus armandi, and their roles in the detoxification metabolism are unclear. In the present study, AhR and ARNT of D. armandi were characterized. Spatiotemporal expression profiling indicated that DaAhR and DaARNT were highly expressed in the adult and larval stages of D. armandi and mainly expressed in the midgut and Malpighian tubules of adults. Additionally, the expression of DaAhR and DaARNT significantly increased after exposure to (-)-𝛽-pinene, (+)-3-carene, and (±)-limonene. Silencing DaAhR and DaARNT increased the susceptibility of D. armandi to (-)-𝛽-pinene, (+)-3-carene, and (±)-limonene, and the activities of detoxification enzyme were also remarkably reduced. Moreover, DaCYP6DF1 and DaGSTs2 were significantly down-regulated after injections of dsAhR and dsARNT in the male and female adults, with the expression of DaCYP6DF1 decreasing by higher than 70%. The present study revealed that the transcription factors AhR and ARNT of D. armandi were induced by terpenoids and participated in the regulation of DaCYP6DF1 expression, which was associated with D. armandi's susceptibility to (-)-𝛽-pinene and (±)-limonene. These results may provide a theoretical basis for the integrated control of D. armandi and improve our comprehension of insect toxicology.

The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein β (C/EBPβ) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of β-catenin.

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated β-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of β-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of β-catenin. This article has an associated First Person interview with the first authors of the paper.

Transcription Factor AhR, Cytokines IL-6 and IL-22 in Subjects with and without Peri-Implantitis: A Case Control-Study.

Peri-implantitis is a plaque-associated condition characterized by mucosal inflammation and subsequent progressive loss of supporting bone; it is caused by bacterial biofilm, but the host response triggered by bacterial stimulation promotes the release of cells and mediators that culminate in tissue destruction. The Aryl-hydrocarbon Receptor (AhR) is associated with IL-22 production by Th22 and Th17 CD4+ Th cells. The presence of IL-6 may promote the Th22 phenotype. The present case-control study evaluated the gene expression of AhR, IL-22, and IL-6 in the peri-implant tissues of healthy and peri-implantitis patients. Tissue biopsies were collected from thirty-five volunteers (15 healthy and 20 with peri-implantitis). A real-time PCR reaction was utilized to assess the AhR, IL-22, and IL-6 gene expression levels relative to the reference gene (GAPDH). The results were analyzed using the Mann–Whitney test with a significance level of 5%. Higher levels of gene expression of AhR and IL-6 were detected in peri-implantitis tissues. The IL-22 gene expression levels did not differ between groups. In conclusion, higher gene expression levels for AhR and IL-6 were detected in the soft tissues of peri-implantitis patients. IL-22 did not vary between conditions, which may indicate the loss of the immunomodulatory role of IL-22 in periimplantitis.

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.

ILC3 Expansion in Acute Myeloid Leukemia

Abstract At homeostasis, type 3 innate lymphoid cells (ILC3s) are tissue-resident and have key roles in maintaining mucosal immunity. Recently, the expansion of ILC3s has been described in solid tumors; however, the mechanism driving this finding is not completely understood. In the setting of mouse and human acute myeloid leukemia (AML), we have observed an expansion of ILC3s. Using an AML murine model, we observed a 7-fold increase in ILC3s in the intestine (the normal ILC3 niche) of leukemic mice relative to controls and a 3-fold increase of ILC3s in the bone marrow (intestine: 0.208% +/− 0.04 AML vs 0.032% +/− 0.04 WT, n=5, p<0.001; bone marrow: 1.312% +/− 0.20 AML vs 0.067% +/− 0.20 WT, n=5, p<0.0001). We hypothesized that the ILC3 increase was due at least in part to increased ILC3 differentiation. To address this hypothesis, we isolated the innate lymphoid cell precursor (ILCP) from normal human donors and co-cultured with AML cells which resulted in increased ILC3 frequency (59% +/− 5% with AML vs 28% +/− 5% without AML, n=15, p <0.001). This observation was also recapitulated in a human PDX model in NSG mice (3.22% +/− 7.8% without AML vs 23.91% +/− 7.8% with AML, n=10; p<0.0001). Our published data have shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. By manipulating AHR activity ex vivo, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (60% +/− 5.5% with AML vs 27% +/− 5% AML+AHR inhibitor, n=15, p<0.001). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion by skewing ILCP differentiation towards ILC3. Future studies will define the role of ILC3s in AML progression. Supported by grant from NIH (1R01CA255860-01A1)

Aryl Hydrocarbon Receptor: From Homeostasis to Tumor Progression.

Transcription factor aryl hydrocarbon receptor (AHR) has emerged as one of the main regulators involved both in different homeostatic cell functions and tumor progression. Being a member of the family of basic-helix-loop-helix (bHLH) transcriptional regulators, this intracellular receptor has become a key member in differentiation, pluripotency, chromatin dynamics and cell reprogramming processes, with plenty of new targets identified in the last decade. Besides this role in tissue homeostasis, one enthralling feature of AHR is its capacity of acting as an oncogene or tumor suppressor depending on the specific organ, tissue and cell type. Together with its well-known modulation of cell adhesion and migration in a cell-type specific manner in epithelial-mesenchymal transition (EMT), this duality has also contributed to the arise of its clinical interest, highlighting a new potential as therapeutic tool, diagnosis and prognosis marker. Therefore, a deregulation of AHR-controlled pathways may have a causal role in contributing to physiological and homeostatic failures, tumor progression and dissemination. With that firmly in mind, this review will address the remarkable capability of AHR to exert a different function influenced by the phenotype of the target cell and its potential consequences.

Differential expression of genes in C. elegans reveals transcriptional responses to indirect-acting xenobiotic compounds and insensitivity to 2,3,7,8-tetrachlorodibenzodioxin

Caenorhabditis elegans is a well-established model organism for toxicity testing of chemical substances. We recently demonstrated its potential for bioanalysis of the toxic potency of chemical contaminants in water. While many detoxification genes are homologues to those in mammalians, C. elegans is reported to be deficient in cytochrome CYP1-like P450 metabolism and that its aryl hydrocarbon receptor (AhR) homolog encoded by ahr-1 purportedly does not interact with dioxins or any other known xenobiotic ligand. This suggests that C. elegans is insensitive for compounds that require bioactivation (indirectly acting compounds) and for dioxins or dioxin-like compounds. This study analysed genome-wide gene expression of the nematode in response to 30 μM of aflatoxin B1 (AFB1), benzo(a)pyrene (B(a)P), Aroclor 1254 (PCB1254), and 10 μM of 2,3,7,8-tetrachlorodibenzodioxin (TCDD). After 24 h of exposure in the early L4 larval stage, microarray analysis revealed 182, 86, and 321 differentially expressed genes in the nematodes treated with 30 μM of AFB1, B(a)P, and PCB1254, respectively. Among these genes, many encode xenobiotic-metabolizing enzymes, and their transcription levels were among the highest-ranked fold-changed genes. Interestingly, only one gene (F59B1.8) was upregulated in the nematodes exposed to 10 μM TCDD. Genes related to metabolic processes and catalytic activity were the most induced by exposure to 30 μM of AFB1, B(a)P, and PCB1254. Despite the genotoxic nature of AFB1 and B(a)P, no differential expression was found in the genes encoding DNA repair and cell cycle checkpoint proteins. Analysis of concentration–response curves was performed to determine the Lowest Observed Transcriptomic Effect Levels (LOTEL) of AFB1, B(a)P, and PCB1254. The obtained LOTEL values showed that gene expression changes in C. elegans are more sensitive to toxicants than reproductive effects. Overall, transcriptional responses of metabolic enzymes suggest that the nematode does metabolize AFB1, B(a)P, and PCB1254. Our findings also support the assumption that the transcription factor AhR homolog in C. elegans does not bind typical xenobiotic ligands, rendering the nematode transcriptionally insensitive to TCDD effects.

IDO1 can impair NK cells function against non-small cell lung cancer by downregulation of NKG2D Ligand via ADAM10

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the rate-limiting step in tryptophan catabolism along the kynurenine (Kyn) pathway and exerts immunosuppressive properties mainly via activation of transcription factor aryl hydrocarbon receptor (AhR) pathway. IDO1 induces NK cells dysfunction via downregulation of the activating receptor NKG2D on NK cells, but whether and how it affects the expression of NKG2D Ligand (NKG2DL) on tumor cells remains unclear. Since a disintegrin and metalloprotease 10 (ADAM10) plays a potential role in the shedding of NKG2DL and the releasing of soluble NKG2DL (sNKG2DL), we investigated how IDO1 modulates the expression of NKG2DL via ADAM10 in non-small cell lung cancer (NSCLC). We found that IDO1 expression was negatively correlated with NKG2DL expression while positively correlated with ADAM10 expression with human lung cancer brain metastasis tissue, NSCLC cells and LLC tumor-bearing mice. IDO1 could regulate ADAM10 expression via IDO1-Kyn-AhR signaling pathway and subsequently regulate NKG2DL expression. IDO1 deficiency led to retarded tumor growth and improved NK cells function in NSCLC mice. IDO1 inhibitors improved NK cells function in vitro and in vivo. The combo of IDO1 inhibitor and NK cells exhibited more therapeutic efficacy than either of the single IDO1 inhibitor or NK cells treatment.