Nuclear Aryl Hydrocarbon Receptor Research Articles

Induction of Semaphorin 3A by Resveratrol and Pinostilbene via Activation of the AHR-NRF2 Axis in Human Keratinocytes.

Semaphorin 3A (SEMA3A), a nerve-repellent factor produced by keratinocytes, has an inhibitory effect on nerve extension to the epidermis. Epidermal innervation is involved in pruritus in inflammatory skin diseases such as atopic dermatitis (AD) and dry skin. We previously reported that tapinarof, a stilbene molecule, upregulates SEMA3A in human keratinocytes. We also showed that this mechanism is mediated via the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, and the nuclear factor erythroid 2-related factor 2 (NRF2) axis. Since some stilbenes activate AHR and NRF2, we attempted to identify other stilbenes that upregulate SEMA3A. We analyzed normal human epidermal keratinocytes (NHEKs) treated with 11 types of stilbenes and examined SEMA3A expression. We found that resveratrol and pinostilbene, antioxidant polyphenols, upregulated SEMA3A and increased nuclear AHR and NRF2 expression. In addition, AHR knockdown by small interfering RNA (siRNA) transfection abolished the NRF2 nuclear expression. Furthermore, AHR and NRF2 knockdown by siRNA transfection abrogated resveratrol- and pinostilbene-induced SEMA3A upregulation. Finally, we confirmed that resveratrol and pinostilbene increased SEMA3A promoter activity through NRF2 binding using ChIP-qPCR analysis. These results suggest that resveratrol and pinostilbene upregulate SEMA3A via the AHR-NRF2 axis in human keratinocytes.

Abstract 6882: Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types

Abstract Background: The aryl hydrocarbon receptor (AhR) is a transcription factor that performs various biological functions upon ligand activation. While there have been extensive studies and development of AhR-targeting anticancer therapies, including active clinical trials, research on the expression of AhR, a key biomarker, in the TME remain limited. Herein, we evaluated the nuclear and cytosolic AhR expression across five solid cancer types: head and neck squamous cell carcinoma (HNSCC), bladder cancer, colorectal cancer, esophageal cancer, and non-small cell lung cancer (NSCLC). Methods: Multiplexed immunohistochemistry (mIHC) and image cytometry were employed to investigate the AhR expression and distribution in 513 patient samples, of which 292 were from patients with one of five solid cancer types. For quantification of AhR expression levels, FCS files were generated, and the resulting data was analyzed using the R program. Results: AhR expression was predominantly high in cancer cells, followed by T cells and macrophages across all cancer types. All 513 patient samples were categorized into three clusters based on distinct expression and localization patterns of AhR. Cluster 1 exhibited high AhR expression in the cancer cell nucleus, whereas cluster 3 showed the lowest. AhR expression in the stromal macrophage nucleus was the most pronounced in cluster 2. In cluster 1, NSCLC was entirely absent, with esophageal cancer representing the largest proportion. In contrast, cluster 2 was notably different from cluster 1, with a 5% prevalence of NSCLC and a relatively higher proportion of HNSCC and colorectal cancer. The AhR expression in macrophages within cluster 2 was noticeably higher than in cluster 1. Cluster 3, which had the highest NSCLC proportion, generally displayed low AhR expression compared to other clusters, but also exhibited AhR expression in regulatory T cells (Tregs), a pattern which was not observed in other cancer types. Notably, a positive correlation was observed between the nuclear and cytosolic expressions of AhR, suggesting that AhR expression as a biomarker is independent of its subcellular localization. Furthermore, there was no clear correlation between AhR expression levels and cancer stage, grade, and metastatic status. Conclusion: We elucidated the expression profile of AhR within the TME across five cancer types, then classified the patient samples into three clusters based on their distinct AhR expression patterns. By demonstrating different expression patterns of AhR in cancer cells and immune cells, our findings are anticipated to provide a fundamental basis for clinical and immunological research on AhR-targeting therapies. Citation Format: Dong Kwon Kim, Chai Young Lee, Yu Jin Han, So Young Park, Heekyung Han, Kwangmin Na, Mi Hyun Kim, Seung Min Yang, Sujeong Baek, Youngtaek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Min Hee Hong, Sun Min Lim, Jii Bum Lee, Jae Hwan Kim, Kyoung-Ho Pyo, Byoung Chul Cho. Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6882.

Pre- and Postoperative Antioxidant Use, Aryl Hydrocarbon Receptor (AhR) Activation and Clinical Outcome in Different Treatment Groups of Breast Cancer Patients

BackgroundCancer patients often use antioxidants that may interact with adjuvant treatments. The purpose was to investigate pre- and postoperative antioxidant use in relation to clinicopathological characteristics and prognosis in different breast cancer treatment groups. Methods and patientsPre- and postoperative antioxidant (vitamin A, C, E, carotenoids, or Q10) or multivitamin use was self-reported by patients from Lund (n = 1855) and Helsingborg (n=478), Sweden. Patients were followed for up to 15 years. Clinical data were obtained from patient charts. The aryl hydrocarbon receptor (AhR) was evaluated in tumor tissue arrays from 915 patients from Lund and with Western blot in MCF-7 and MDA-MB-231 cells. ResultsAbout 10% of patients used antioxidants. Nuclear AhR (AhRnuc) positivity was twice as common in preoperative antioxidant users compared to non-users. In mechanistic studies vitamin C increased AhR levels and its downstream target CYP1B1, indicating AhR activation. There were significant interactions between tumor AhRnuc status and preoperative antioxidant use in relation to clinical outcome. In all patients, antioxidant use (other than multivitamins) at both visits was associated with poorer prognosis, while use only at the follow-up visit was associated with better prognosis, compared with no use at either visit. ConclusionThe clinical impact of antioxidants depended on antioxidant type, timing of use, and tumor AhR activation. Antioxidants may influence clinical outcome by activation of the master regulator AhR in addition to interference with free radicals. Further studies are needed to identify breast patients that might improve or worsen their prognosis when using antioxidants postoperatively.

Danshen ligustrazine injection ameliorates MCAO/R rats through the AhR-SOCS2 axis in association with gut microbiota-derived tryptophan metabolism

BackgroundDanshen Ligustrazine Injection (DSLI), composed of the extract of Salvia miltiorrhiza Bunge and Conioselinum anthriscoides 'Chuanxiong', is commonly used in the clinical treatment of ischemic stroke, which is the most common type of cerebrovascular disease, affected by multiple factors. The brain-gut axis is a new type of influencing factor for ischemic stroke, however, there is a lack of systematic research on the relationship between microbiota and host metabolic phenotype. This study aimed to investigate the host metabolic phenotype-microbiome interface in ischemic stroke and the immunomodulatory effects of DSLI. MethodsIn the study, liquid chromatography-mass spectrometry was used to primarily analyze the quality markers (Q-markers) and the polyphenols of DSLI. Based on 16S rRNA gene sequencing and metabolomics, the variations of microbial composition in different groups were characterized. In addition, a network diagram was constructed to reveal the interrelationship between DSLI-regulated gut microbiota changes and DSLI-regulated brain metabolites in MCAO/R rats. The expression of the aryl hydrocarbon receptor (AhR) in the colon and brain by immunofluorescence and suppressors of cytokine signaling 2 (SOCS2) by western blot were further detected. ResultsDiminished metabolic diversity and alterations in the tryptophan metabolism pathway were a feature of IS in the study. The results revealed that Clostri_UCG-014 was positively correlated with indoleacrylic acid, indole-3-methylacetate, and Ruminococcaceae was strongly related with L-tryptophan, 3-methylindole, and indole. The results of immunofluorescence showed that the expression of AhR in the colon and brain tissues of rats in the model group was greatly reduced compared with that in the sham-operated group, and was heavily activated with the treatment of DSLI. The levels of aryl hydrocarbon nuclear receptor ARNT2 and SOCS2 were down-regulated in the brains of Model rats, whereas increased by DSLI, further confirming that the brain-gut microbiota-Trp metabolism exists in the pathological changes of stroke and the intervention mechanism of DSLI. ConclusionsThe present study findings suggest Clostri_UCG-014, Ruminococcaceae, and Firmicute_RF39 play an important role in improving the regulation of immune function in stroke through tryptophan metabolites by acting on the AhR-SOCS2 axis.

Human exposure to diesel exhaust induces CYP1A1 expression and AhR activation without a coordinated antioxidant response

BackgroundDiesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE.MethodsUsing immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM10, 300 µg/m3) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo–keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed.ResultsDE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = −0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo–keto reductases, or epoxide hydrolase protein expression.ConclusionThese in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

Lactobacillus species ameliorate membranous nephropathy through inhibiting the aryl hydrocarbon receptor pathway via tryptophan-produced indole metabolites.

Membranous nephropathy (MN) is an immune-mediated glomerular disease in adults. Antibody- and antigen-bonding mechanisms have been largely clarified, but the subepithelium immune complex deposition-mediated downstream molecular mechanisms are currently unresolved. Increasing evidence has suggested that gut microbiota contribute to MN pathogenesis. In this study, we identified alterations in faecal gut microbiota and serum metabolites that mediate an aryl hydrocarbon receptor (AhR) mechanism in cationic bovine serum albumin (CBSA)-induced MN rats and in patients with idiopathic MN (IMN). Impaired renal function correlated with the relative abundance of reduced faecal probiotics, Lactobacillus and Bifidobacterium, and altered serum levels of tryptophan-produced indole derivatives (TPIDs) in MN rats. Further results showed that reduced relative abundance of five probiotics, namely Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus vaginalis, Lactobacillus reuteri and Bifidobacterium animalis, positively correlated with decreased levels of indole-3-pyruvic acid, indole-3-aldehyde and tryptamine and negatively correlated with increased levels of indole-3-lactic acid and indole-3-acetic acid in serum of MN rats. Altered five probiotics and five TPIDs also were observed in patients with IMN. Further studies showed that MN rats exhibited a significant increase in intrarenal mRNA expression of AhR and its target genes CYP1A1, CYP1A2 and CYP1B1, which were accompanied by protein expression of down-regulated cytoplasmic AhR, but up-regulated nuclear AhR, in MN rats and IMN patients. Activation of the intrarenal AhR signalling pathway may involve five TPIDs. These data suggest that gut microbiota could influence MN through TPIDs that engage host receptors.

Aryl hydrocarbon receptor is regulated via multiple mechanisms in human keratinocytes

Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polycyclic aromatic hydrocarbons of synthetic and natural origin. While a number of novel AhR ligands have been recently identified, little is known about their possible influence on AhR levels and stability. We used western blot, qRT-PCR and immunocytochemistry to determine the effects of AhR ligands on AhR expression in N-TERT (N-TERT1) immortalized human keratinocytes, and immunohistochemistry to assess patterns of AhR expression in human and mouse skin and skin appendages. While AhR was highly expressed in cultured keratinocytes and in the skin, it was found primarily in the cytoplasm, but not in the nucleus, suggesting its inactivity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and eventual inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD, FICZ, caused near-complete disappearance of AhR, and treatment with I3C resulted in substantially diminished level of AhR possibly due to ligand-induced AhR degradation. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Additionally, AhR decay was blocked by ligand-selective AhR antagonist CH223191, implying substrate-induced mechanism of degradation. Furthermore, degradation of AhR was blocked in N-TERT cells with knockdown of AhR dimerization partner ARNT (HIF1β), suggesting that ARNT is required for AhR proteolysis. However, addition of hypoxia mimetics (HIF1 pathway activators) CoCl2 and DMOG had only minor effects on degradation of AhR. Additionally, inhibition of HDACs with Trichostatin A resulted in enhanced expression of AhR in both untreated and ligand-treated cells. These results demonstrate that in immortalized epidermal keratinocytes AhR is primarily regulated post-translationally via proteasome-mediated degradation, and suggest potential means to manipulate AhR levels and signaling in the skin. Overall, the AhR is regulated via multiple mechanisms, including proteasomal ligand- and ARNT-dependent degradation, and transcriptional regulation by HDACs, implying complex system of balancing its expression and protein stability.

Phase 1a/b open-label study of IK-175, an oral AHR inhibitor, alone and in combination with nivolumab in patients with locally advanced or metastatic solid tumors and urothelial carcinoma.

TPS3169 Background: Aryl Hydrocarbon Receptor (AHR) is a ligand-activated transcription factor that regulates the activity of multiple innate and adaptive immune cells. Kynurenine, generated from tryptophan by IDO1 and TDO2, is a ligand that binds AHR and leads to a net immunosuppressive tumor microenvironment, making AHR an attractive therapeutic target in multiple cancer types. IK-175 is a selective, small molecule inhibitor of AHR being developed as an oral agent. In preclinical mouse tumor models, IK-175 demonstrates antitumor activity as a single agent or in combination with checkpoint inhibitors. AHR immunohistochemistry (IHC) tumor microarray analysis across 15 different tumor types revealed that bladder cancer has the highest level of AHR protein expression and nuclear localization indicative of ligand-bound and active AHR signaling. Therefore, nuclear AHR in urothelial carcinoma tumors is being investigated for potential predictive clinical benefit with IK-175. Methods: This is a first-in-human, phase 1a/b, open-label, multicenter, dose-escalation and expansion study of IK-175 as a single agent and in combination with nivolumab. The primary objectives are to determine the maximum tolerated dose (MTD) and/or maximum administered dose (MAD), identify the recommended phase 2 dose (RP2D), and evaluate the safety and tolerability of IK-175 alone and in combination with nivolumab. Secondary objectives are to evaluate the pharmacokinetics (PK) of IK-175, evaluate pharmacodynamic (PD) immune effects, and assess preliminary antitumor activity. Key exploratory objectives are to evaluate the PD effects on peripheral immune cells and target gene expression, to assess candidate baseline biomarkers, and correlative analyses of tumor AHR nuclear localization with clinical response. The study is exploring tumor AHR nuclear localization by IHC as a predictive biomarker in patients with urothelial carcinoma. A minimum of 10 patients with a positive AHR nuclear localization test (cutoff for positive AHR is 65% tumor cells positive for 2+/3+ nuclear AHR by a validated IHC assay) will be enrolled in the combination arm. IK-175 is administered daily in 21 or 28 day-cycles as a single agent, and in combination with nivolumab (480 mg q4w on Day 1 of every cycle), in adult patients with advanced solid tumors (dose escalation) and urothelial carcinoma (dose expansion). Key eligibility criteria include patients with histologically confirmed solid tumors (including urothelial carcinoma) who have locally recurrent or metastatic disease that have progressed on or following all standard of care therapies deemed appropriate by the treating physician including prior checkpoint inhibitors. Estimated enrollment is 93 patients; the study began in January 2020 and is ongoing. Clinical trial information: NCT04200963.

The expression of aryl hydrocarbon receptor predicts responses to anti-PD-1 antibodies in non-small cell lung cancer.

2628 Background: Blockade of programmed cell death receptor 1 (PD-1) by antibodies such as pembrolizumab has revolutionized treatment for patients with non-small cell lung cancer (NSCLC). However, a significant number of patients achieve only marginal benefit. The predictive abilities of the frequently used biomarkers, including programmed cell death 1 ligand (PD-L1) and tumor mutation burden (TMB), are yet limited, highlighting the urgent need for more reliable biomarkers. Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that controls the transcription of its targets including PD-L1. Whether AhR could be used as a predictive biomarker for response to PD-1 blockade immunotherapy in NSCLC warrants further investigation. Methods: We collected formalin-fixed, paraffin-embedded (FFPE) slides from 104 NSCLC patients before PD-1 blockade immunotherapy treatment, who had sufficient follow-up data for progression-free survival (PFS), and detected the expression of nuclear localized AhR (AhRN) and membrane localized PD-L1 (PD-L1M) with multiplex immunohistochemistry (mIHC). H-score was applied to characterize the specific value with the following formula [1 × (% cells intensity =1) + 2 × (% cells intensity = 2) + 3 × (% cells intensity = 3)]. Survival curve for each group was estimated by the Kaplan–Meier method and log-rank test. The diagnostic performance was assessed with the area under the receiver operating characteristic (ROC) curve (AUC), which was calculated based on the Wilcoxon rank-sum test. All statistical tests were two-sided and P values less than 0.05 were considered statistically significant. Results: Both AhRN and PD-L1M could distinguish responders from non-responders, and patients with higher H-score had longer PFS than those with lower H score. The AUC of AhRN was 0.804 (95 % CI: 0.721-0.881), higher than that of PD-L1M (0.714; 95 % CI: 0.618-0.811) and AhRN+PD-L1M (0.743; 95 % CI: 0.649-0.837), indicating AhRN might show better predictive capacity to the efficacy of PD-1 blockade immunotherapy than PD-L1M. Besides, AhRN exhibited the most significant accuracy in prognosis prediction, since the hazard ratio (HR) of AhRN was 0.1529 (95 % CI: 0.07704-0.3033, P&lt;0.0001), significantly higher than that of PD-L1M [HR:0.283 (95 % CI: 0.1525-0.525, P&lt;0.0001)] and AhRN+PD-L1M [HR: 0.2456 (95 % CI: 0.1309-0.4607, P&lt;0.0001)]. Yet, among the 54 smokers, AhRN+PD-L1M was the best classifier for the efficacy of immunotherapy, of which the AUC was 0.8937 (95 % CI: 0.816-0.971) and HR was infinitely approaching 0 ( P&lt;0.0001). Conclusions: AhR might be a reliable predictive biomarker for PD-1 blockade immunotherapy in NSCLCs.

Intrarenal 1-methoxypyrene, an aryl hydrocarbon receptor agonist, mediates progressive tubulointerstitial fibrosis in mice.

Recent studies have shown that endogenous metabolites act via aryl hydrocarbon receptor (AhR) signalling pathway in tubulointerstitial fibrosis (TIF) pathogenesis. However, the mechanisms underlying endogenous metabolite-mediated AhR activation are poorly characterised. In this study, we conducted untargeted metabolomics analysis to identify the significantly altered intrarenal metabolites in a mouse model of unilateral ureteral obstruction (UUO). We found that the levels of the metabolite 1-methoxypyrene (MP) and the mRNA expression of AhR and its target genes CYP1A1, CYP1A2, CYP1B1 and COX-2 were progressively increased in the obstructed kidney at Weeks 1, 2 and 3. Furthermore, these changes were positively correlated with progressive TIF in UUO mice. In NRK-52E, RAW 264.7 and NRK-49F cells, MP dose-dependently upregulated the mRNA expression of AhR and its four target genes and the protein expression of nuclear AhR, accompanied by the upregulated protein expression of collagen I, α-SMA and fibronectin, as well as downregulated E-cadherin expression. Consistently, oral administration of MP in mice progressively enhanced AhR activity and upregulated profibrotic protein expression in the kidneys; these effects were partially inhibited by AhR knockdown in MP-treated mice and cell lines. In addition, we screened and identified erythro-guaiacylglycerol-β-ferulic acid ether (GFA), which was isolated from Semen plantaginis, as a new AhR antagonist. GFA significantly attenuated TIF in MP-treated NRK-52E cells and mice by partially antagonising AhR activity. Our results suggest that MP activates AhR signalling, thus mediating TIF through epithelial-mesenchymal transition and macrophage-myofibroblast transition. MP is a crucial metabolite that contributes to TIF via AhR signalling pathway.

The synergistic Reduning and cefmetazole sodium treatment of severe pneumonia is mediated by the AhR-Src-STAT3 pathway.

BackgroundReduning (RDN) is a common Chinese medicine preparation with antibacterial, anti-inflammatory, antiviral and immunomodulatory effects in respiratory infectious diseases. Clinically, it is used in combination with antibiotics, but its synergistic effect and mechanism in treating severe pneumonia remain unclear.MethodsA rat model of severe pneumonia and an in vitro coculture model consisting of A549 and THP-1 cells were used to observe the synergistic effect of RDN on severe pneumonia. The inflammatory cytokines were tested by enzyme-linked immunosorbent assay (ELISA). The localization of Aryl hydrocarbon receptor (AhR) in A549 cells was observed by immunofluorescence, and the interaction of AhR and signal transducer and activator of transcription 3 (STAT3) proteins was observed by co-immunoprecipitation. AhR-Src tyrosine kinase (Src)-STAT3 pathway in rats and A549 cells were examined by Western Blot. Histopathological changes were observed by Hematoxylin-eosin (HE) staining, X-ray and survival rates were used to evaluate the effects of paclitaxel on severe pneumonia rats.ResultsRDN regulation of Src–STAT3–interleukin 10 (IL-10) signaling pathway activation and macrophage polarization were mediated through the nuclear receptor AhR. The expression of AhR was significantly increased after RDN treatment, and this effect was accompanied by STAT3 expression increasing. Coimmunoprecipitation confirmed an interaction between AhR and STAT3 and upregulated IL-10 expression. Silencing AhR decreased Src, STAT3, and IL-10 expression. RDN activated AhR and increased Src, STAT3, and IL-10 expression. In addition, RDN regulated the polarization of macrophages RDN combined with cefmetazole sodium significantly reduced the pulmonary bacterial load, alleviated lung injury, and reduced o inflammatory factors expression, improving their survival.ConclusionsRDN can synergistically enhance the effect of cefmetazole sodium treatment in severe pneumonia, and the mechanism may involve increasing the expression level of IL-10 mediated through the AhR-Src-STAT3 pathway, driving the polarization of macrophages, and attenuating the cytokine storm to control inflammation in severe pneumonia.

1-Hydroxypyrene mediates renal fibrosis through aryl hydrocarbon receptor signalling pathway.

In chronic kidney disease (CKD), patients inevitably reach end-stage renal disease and require renal transplant. Evidence suggests that CKD is associated with metabolite disorders. However, the molecular pathways targeted by metabolites remain enigmatic. Here, we describe roles of 1-hydroxypyrene in mediating renal fibrosis. We analysed 5406 urine and serum samples from patients with Stage 1-5 CKD using metabolomics, and 1-hydroxypyrene was identified and validated using longitudinal and drug intervention cohorts as well as 5/6 nephrectomised and adenine-induced rats. We identified correlations between the urine and serum levels of 1-hydroxypyrene and the estimated GFR in patients with CKD onset and progression. Moreover, increased 1-hydroxypyrene levels in serum and kidney tissues correlated with decreased renal function in two rat models. Up-regulated mRNA expression of aryl hydrocarbon receptor and its target genes, including CYP1A1, CYP1A2 and CYP1B1, were observed in patients and rats with progressive CKD. Further we showed up-regulated mRNA expression of aryl hydrocarbon receptor and its three target genes, plus up-regulated nuclear aryl hydrocarbon receptor protein levels in mice and HK-2 cells treated with 1-hydroxypyrene, which caused accumulation of extracellular matrix components. Treatment with aryl hydrocarbon receptor short hairpin RNA or flavonoids inhibited mRNA expression of aryl hydrocarbon receptor and its target genes in 1-hydroxypyrene-induced HK-2 cells and mice. Metabolite 1-hydroxypyrene was demonstrated to mediate renal fibrosis through activation of the aryl hydrocarbon receptor signalling pathway. Targeting aryl hydrocarbon receptor may be an alternative therapeutic strategy for CKD progression.

The Prognostic Impact of Intratumoral Aryl Hydrocarbon Receptor in Primary Breast Cancer Depends on the Type of Endocrine Therapy: A Population-Based Cohort Study.

The aryl hydrocarbon receptor (AhR) is a master regulator of multiple pathways involved in breast cancer, and influences the estrogen receptor alpha (ER) and aromatase/CYP19A1. The purpose of this study was to elucidate the interplay between intratumoral levels of AhR and aromatase, patient characteristics (including AhR and CYP19A1 genotypes), clinicopathological features, and prognosis in breast cancer patients receiving adjuvant treatments. A prospective cohort of 1116 patients with primary breast cancer in Sweden, included 2002–2012, was followed until June 30th 2019 (median 8.7 years). Tumor‐specific AhR (n=920) and aromatase levels (n=816) were evaluated on tissue microarrays using immunohistochemistry. Associations between cytoplasmatic (AhRcyt) and nuclear (AhRnuc) AhR levels, intratumoral aromatase, clinicopathological features, and prognosis in different treatment groups were analyzed. Low AhRcyt levels (n=183) and positive intratumoral aromatase (n=69) were associated with estrogen receptor (ER)– status and more aggressive tumors. Genotypes were not associated with their respective protein levels. The functional AhR Arg554Lys GG genotype was associated with recurrence-free survival in switch-therapy (sequential tamoxifen/aromatase inhibitors (AI) or AI/tamoxifen) treated patients (HRadj 0.42; 95% CI 0.22–0.83). High AhRcyt levels were associated with longer recurrence-free survival during the first 10 years of follow-up among tamoxifen-only treated patients (HRadj 0.40; 95% CI 0.23–0.71) compared to low AhRcyt levels, whereas an almost inverse association was seen in patients with switch-therapy (P interaction=0.023). Intratumoral aromatase had little prognostic impact. These findings warrant confirmation in an independent cohort, preferably in a randomized clinical trial comparing different endocrine regimens. They might also guide the selection of breast cancer patients for clinical trials with selective AhR modulators.

Neurons expressing the aryl hydrocarbon receptor in the locus coeruleus and island of Calleja major are novel targets of dioxin in the mouse brain

The aryl hydrocarbon receptor (AhR) acts as a receptor that responds to ligands, including dioxin. The AhR–ligand complex translocates from the cytoplasm into the nucleus to induce gene expression. Because dioxin exposure impairs cognitive and neurobehavioral functions, AhR-expressing neurons need to be identified for elucidation of the dioxin neurotoxicity mechanism. Immunohistochemistry was performed to detect AhR-expressing neurons in the mouse brain and confirm the specificity of the anti-AhR antibody using Ahr−/− mice. Intracellular distribution of AhR and expression level of AhR-target genes, Cyp1a1, Cyp1b1, and Ahr repressor (Ahrr), were analyzed by immunohistochemistry and quantitative RT-PCR, respectively, using mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The mouse brains were shown to harbor AhR in neurons of the locus coeruleus (LC) and island of Calleja major (ICjM) during developmental period in Ahr+/+ mice but not in Ahr−/− mice. A significant increase in nuclear AhR of ICjM neurons but not LC neurons was found in 14-day-old mice compared to 5- and 7-day-old mice. AhR was significantly translocated into the nucleus in LC and ICjM neurons of TCDD-exposed adult mice. Additionally, the expression levels of Cyp1a1, Cyp1b1, and Ahrr genes in the brain, a surrogate of TCDD in the tissue, were significantly increased by dioxin exposure, suggesting that dioxin-activated AhR induces gene expression in LC and ICjM neurons. This histochemical study shows the ligand-induced nuclear translocation of AhR at the single-neuron level in vivo. Thus, the neurotoxicological significance of the dioxin-activated AhR in the LC and ICjM warrants further studies.

The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity.

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed.

Metabolites in a mouse cancer model enhance venous thrombogenicity through the aryl hydrocarbon receptor–tissue factor axis

AbstractPatients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer–specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

The Prognostic Impact of the Aryl Hydrocarbon Receptor (AhR) in Primary Breast Cancer Depends on the Lymph Node Status.

Increasing evidence implicates the aryl hydrocarbon receptor (AhR) as a possible regulator of mammary carcinogenesis. This study aims to clarify its prognostic impact in breast cancer (BC). Meta-analyses performed at the mRNA level demonstrated that the predictive value of AhR expression in BC depends on the lymph node (LN) status. AhR expression and sub-cellular location were then analyzed by immunohistochemistry in 302 primary BC samples. AhR was expressed in almost 90% of cases with a predominant nuclear location. Nuclear and cytoplasmic AhR levels were significantly correlated and associated with the expression of RIP140 (receptor-interacting protein of 140 kDa), an AhR transcriptional coregulator and target gene. Interestingly, total and nuclear AhR levels were only significantly correlated with short overall survival in node-negative patients. In this sub-group, total and nuclear AhR expression had an even stronger prognostic impact in patients with low RIP140-expressing tumors. Very interestingly, the total AhR prognostic value was also significant in luminal-like BCs and was an independent prognostic marker for LN-negative patients. Altogether, this study suggests that AhR is a marker of poor prognosis for patients with LN-negative luminal-like BCs, which warrants further evaluation.

Newly Identified Metabolites Connect Colon Cancer to Thrombosis

Patients with cancer are at 4-to-7-fold higher risk of venous thromboembolism (VTE). The risk of cancer-associated VTE varies with the type of cancer, underscoring the presence of cancer type-specific drivers and a need for appropriate animal models, both of which have been understudied. Here, we generated a colon cancer-specific mouse model and probed for the development of VTE and factors that might induce it. To this end, a group of athymic animals injected with human colon adenocarcinoma cell line (HT-29) underwent inferior vena cava (IVC) ligation, a validated model of venous thrombosis. Compared to corresponding controls (age- and gender-matched), mice on regular chow diet with xenografts exhibited significantly higher IVC clot weights. Plasma analysis of metabolites led us to identify the tryptophan metabolites, Kynurenine and Indoxyl Sulfate, which are known activator of the Aryl Hydrocarbon Receptor (AHR), as significantly elevated in the cancer model (2-3 fold; p<0.05). Plasma from these animals activated the AHR pathway and augmented both tissue factor (TF) and Plasminogen Activator Inhibitor 1 (PAI-1) levels in venous endothelial cells. PAI-1 is a known target of AHR pathway in other cells. Using an AHR inhibitor, we showed that the above effects of the plasma depended on AHR activation. Consistent with these findings, immunostaining of the endothelial layer of IVC in animals with xenografts revealed predominantly nuclear AHR (sign of activation) and clear increases in TF and PAI-1 expression- telltale signs of an activated AHR-TF/PAI-1 axis. As TF augments thrombogenesis and PAI-1 inhibits thrombolysis, these results may explain the enhanced thrombogenicity in this model. While this model represents a novel tool to mechanistically probe mediators of cancer-associated VTE, it links for the first-time cancer-associated VTE to a set of metabolites. Our data raise a possibility of AHR-TF/PAI-1 axis as a mediator of VTE in other tumors, worthy of exploration given its potential as a biomarker and a therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibromatosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its endogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and trimethoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr-/- mice during myelin development. AHR ablation leads to locomotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr-/- mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased β-catenin levels and stimulated the binding of β-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myelination and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors.

Aryl hydrocarbon receptor expression by macrophages and lymphocytes within infiltrates in BK polyomavirus associated nephropathy

BackgroundBK virus nephropathy (BKPyVN) is a major complication after renal transplantation. Little is known about the intra renal immune response during BKPyVN. The role of macrophages remains elusive. The activation of aryl hydrocarbon receptor (AHR) - a transcription factor involved in drug metabolism - plays a key role in inflammation and viral tolerance through modulation of macrophages polarization. Since AHR has not been studied in kidney transplantation, our aim was to compare the AHR expression within renal grafts in BKPyVN with T-cell mediated rejection (TCMR) as a control. MethodsWe evaluated AHR expression in kidney grafts from BKPyVN (n=8) with TCMR as control (n=6) among cases with available frozen material for AHR gene intragraft transcription measurement and stainings for AHR, CD68 and CD45. ResultsAHR transcription was higher in BKPyVN grafts versus TCMR (p=0.03). While CD68+ or CD45+ cell expression did not differ within infiltrates (median score=3 in both groups; p=1.0 and 0.69, respectively), a higher proportion of nuclear AHR expression was found in BKPyVN for CD68+ and CD45+ cells when compared with TCMR (score median 2 vs 0; p=0.007 and 1 vs 0; p=0.013, respectively). ConclusionsWe describe for the first time a higher expression of AHR in inflammatory cell infiltrates from BKPyVN versus TCMR renal biopsies. Further studies are required to explore AHR as a potential target in the modulation of inflammatory response in BKPyVN with known modulating ligands.