Expression Levels Of Aryl Hydrocarbon Receptor Research Articles

Knock out aryl hydrocarbon receptor gene in adipose-derived mesenchymal stem cell line (PT-5006) by CRISPR/Cas9

Mesenchymal stem cells (MSCs) have garnered significant attention in biomedical studies due to their remarkable properties, such as self-renewal, differentiation into diverse cell types and immune responses. The proliferation and differentiation of MSCs are significantly influenced by the ligand-dependent transcription factor known as the aryl hydrocarbon receptor (AhR). In order to understand the roles of Ahr in adipose-derived MSCs (AD-MSCs), we disrupted the Ahr gene in this work using CRISPR/Cas9 gene editing technology. The gRNA/Cas9 dual vector and donor vector were introduced into the AD-MSC cell line (PT-5006). Green fluorescent protein (GFP) expression and puromycin resistance were used to identify the transfected cells. AhR-KO cells were cloned and confirmed by PCR and sequencing. By using RT-qPCR, the expression levels of AhR and the Ahr-related gene Cyp1B1 were investigated. The results showed that the knocked out of AhR using CRISPR/Cas9, resulting significantly decreased expression of 7.69-fold for AhR and 3.70-fold for Cyp1B1 in the cells. These cell clones and CRISPR/Cas9 vectors could be used as tools to investigate the functions of AhR in both AD-MSCs and other cell types.

#1110 The AHR antagonist CH223191 alleviates kidney injury and renal fibrosis in adenine-induced chronic kidney disease

Abstract Background and Aims The aryl hydrocarbon receptor (AhR) is a transcription factor that can be activated by a variety of endogenous and exogenous ligands, and is involved in varieties of biological processes. It has been confirmed that the expression of AhR in kidney is elevated in chronic kidney disease (CKD). However, the specific role of AhR signaling pathway in CKD and its molecular mechanism remain unclear. This study aims to investigate the role and underlying mechanism of AhR in CKD, providing potential targets for the treatment of CKD. Method Male C57/B6L mice were randomly divided into control group, model group and treatment group. The model group was fed 0.2% adenine diet for 14 days to establish CKD model, while the treatment group was treated with AhR antagonist CH223191. AhR agonist kynurenine and antagonist CH223191 was applied in mouse tubular epithelial (TCMK-1) cells to investigate the underlying mechanisms of AhR in vitro. The renal function, histological changes and renal fibrosis in mice were evaluated to explore the role of AhR in adenine-induced CKD. Histological changes were evaluated by PAS and MASSON stainings, immunohistochemistry and immunofluorescence. Western blot and qPCR were used to measure protein/gene transcription levels. Results The levels of serum creatinine (p < 0.0001) and urea nitrogen (p < 0.0001) in the model group were significantly increased, with obvious renal tubule dilation and renal fibrosis by PAS and MASSON staining. CH223191 treatment significantly reduced serum creatinine (p=0.0012) and urea nitrogen ( < 0.0001) levels in mice, and alleviated renal fibrosis histologically. Q-PCR and Western-Blot results showed that the renal expression level of AhR in the treatment group was decreased, while the expression levels of fibronectin (Fn) (p < 0.0001), α-smooth muscle actin (α-SMA) (p < 0.0001), type I collagen (p=0.0007) and transforming growth factor-β (TGF-b) (p=0.0027) were significantly decreased. In addition, the expression levels of N-GAL (p < 0.0001) and KIM-1 ( < 0.0001) molecular markers associated with kidney injury were reduced, suggesting that inhibiting AhR expression in vivo can reduce adenine-mediated kidney injury and renal fibrosis in CKD. In vitro experiments indicated that kynurenic acid could induce the expression of AhR and AhR downstream regulatory gene CYP1b1 and upregulated fibrosis-related genes in tubular epithelial cells, while CH223191 treatment downregulated the fibrosis-related genes in tubular epithelial cells. Conclusion AhR mediates renal injury and renal fibrosis in adenine-induced CKD mice. AhR antagonist CH223191 exerts a renal protective effect by reducing the expression of AhR in renal tubular epithelial cells, thereby alleviating renal injury and renal fibrosis.

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

Aryl hydrocarbon receptor (AhR) is a transcription factor that performs various functions upon ligand activation. Several studies have explored the role of AhR expression in tumor progression and immune surveillance. Nevertheless, investigations on the distribution of AhR expression, specifically in cancer or immune cells in the tumor microenvironment (TME), remain limited. Examining the AhR expression and distribution in the TME is crucial for gaining insights into the mechanism of action of AhR-targeting anticancer agents and their potential as biomarkers. Here, we used multiplexed immunohistochemistry (mIHC) and image cytometry to investigate the AhR expression and distribution in 513 patient samples, of which 292 are patients with one of five solid cancer types. Additionally, we analyzed the nuclear and cytosolic distribution of AhR expression. Our findings reveal that AhR expression was primarily localized in cancer cells, followed by stromal T cells and macrophages. Furthermore, we observed a positive correlation between the nuclear and cytosolic expression of AhR, indicating that the expression of AhR as a biomarker is independent of its localization. Interestingly, the expression patterns of AhR were categorized into three clusters based on the cancer type, with high AhR expression levels being found in regulatory T cells (Tregs) in non-small cell lung cancer (NSCLC). These findings are anticipated to serve as pivotal evidence for the design of clinical trials and the analysis of the anticancer mechanisms of AhR-targeting therapies.

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.

Aryl hydrocarbon receptor gene expression in ankylosing spondylitis and its correlation with interleukin-17, RAR-related orphan receptor gamma t expression, and disease activity indices.

Considering the role of T helper (Th)17 cells in the pathogenesis of ankylosing spondylitis (AS), the aim of this study was to determine the correlation between aryl hydrocarbon receptor (AHR) gene expression and the expression of Th17-related genes including interleukin (IL)-17 and RAR-related orphan receptor gamma t (RORγt) transcription factor. Thirty patients with AS (26 males, 4 females; mean age: 36.1±8.1 years) and 30 age- and sex-matched healthy individuals (26 males, 4 females; mean age: 36.2±14.6 years) were recruited for the case-control study between June 2021 and January 2022. Ribonucleic acid (RNA) was extracted from peripheral blood cells and expression levels of AHR, IL-17, RORγt, and AHR repressor (AHRR) genes were evaluated using real-time polymerase chain reaction technique. The serum level of IL-17 was evaluated with enzyme-linked immunosorbent assay. The results showed a nonsignificant elevation of AHR, IL-17, and RORγt gene expression in the patient group compared to the control. There was a direct correlation between AHR gene expression and IL-17 and RORγt genes and a negative correlation between AHR and AHRR expression. Moreover, AHR gene expression showed a weak correlation with disease activity indices, including Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, Bath Ankylosing Spondylitis Metrology Index, Bath Ankylosing Spondylitis Global Score, and Ankylosing Spondylitis Quality of Life. Moreover, the serum level of IL-17 was higher in AS patients compared to the healthy group (p=0.02). Upregulated expression of the AHR gene in ankylosing spondylitis and its correlation with IL-17 and ROR-γ t gene expression suggests that it could be a potential diagnostic and therapeutic target for AS.

Aryl hydrocarbon receptor (AhR) is regulated by hyperoxia in premature infants

Objective To investigate whether aryl hydrocarbon receptor (AhR) is involved in hyperoxia-mediated oxidative stress by observing the relationship between AhR and reactive oxygen species (ROS) in peripheral blood mononuclear cells (PBMCs) after oxygen exposure in premature infants. Methods After 48 h of oxygen inhalation at different concentrations, discarded peripheral blood was collected to separate PBMCs and plasma. ROS were labeled with MitoSOXTM Red and detected by fluorescence microscopy in PBMCs. The level of MDA in plasma was detected by thiobarbituric acid colorimetry, the level of MCP-1 in plasma was detected by enzyme-linked immunosorbent assay (ELISA), the localization of AhR was detected by immunofluorescence, and the level of AhR expression in PBMCs was detected by Western blotting. Results As the volume fraction of inspired oxygen increased, compared with those in the air control group, the levels of ROS, MDA in plasma, and MCP-1 in plasma increased gradually in the low concentration oxygen group, medium concentration oxygen group and high concentration oxygen group. The cytoplasm-nuclear translocation rate of AhR gradually increased, and the expression level of AhR gradually decreased. The levels of ROS in PBMCs, MDA in the plasma and MCP-1 in the plasma of premature infants were positively correlated with the cytoplasm-nuclear translocation rate of AhR but negatively correlated with the level of AhR expression. Conclusion Aryl hydrocarbon receptor (AhR) is regulated by hyperoxia in premature infants.

Disrupting Development: Unraveling the Interplay of Aryl Hydrocarbon Receptor (AHR) and Wnt/β-Catenin Pathways in Kidney Development Under the Influence of Environmental Pollutants.

Understanding the intricate molecular mechanisms governing aryl hydrocarbon receptor (AHR) and Wnt/β-Catenin pathways crosstalk is of paramount importance for elucidating normal development. We investigated the repercussions of aberrant activation of these signaling pathways on kidney development. HEK-293 cells were subjected to AHR and Wnt activators and inhibitors for 3 and 24h. Subsequently, pregnant adult female BALB/c mice were administered treatments at gestation day 9 (GD-9), and embryos were analyzed at GD-18 using a combination of cellular, molecular, stereological, and histopathological techniques. Our results demonstrated a noteworthy escalation in oxidative stress and gene expression endpoints associated with apoptosis. Moreover, stereological analyses exhibited alterations in cortex, proximal tubule, and kidney tissue vessels volumes. Remarkably, co-treatment with 6-formylindolo [3,2-b] carbazole (FICZ) and cadmium (Cd) resulted in a significant reduction in glomerulus volume, while elevating the volumes of distal tubule, Henle loop, and connective tissue, compared to the control group. Histopathological investigations further confirmed structural changes in the loop of Henle and proximal tubule, alongside a decline in glomerular volume. Additionally, the expression levels of AHR and Ctnnb1 genes significantly increased in the Cd-treated group compared to the control group. Enhanced expression of apoptosis-related genes, including Bcl-x, Bax, and Caspase3, along with alterations in mitochondrial membrane potential and cytochrome C release, was observed. In contrast, Gsk3 gene expression was significantly decreased. Our findings robustly establish that chemical pollutants, such as Cd, disrupt the AHR and Wnt/β-Catenin physiological roles during developmental stages by inhibiting the metabolic degradation of FICZ.

Omeprazole activates aryl hydrocarbon receptor to reduce hyperoxia-induced oxidative stress in the peripheral blood mononuclear cells from premature infants

Objective To investigate the correlation between the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) in peripheral blood mononuclear cells (PBMCs) of premature infants, to demonstrate the protective role of AhR against hyperoxia-induced oxidative stress in premature infants and to provide a rational basis for the use of omeprazole (OM) as a new treatment for bronchopulmonary dysplasia (BPD). Methods From January 2021 to June 2021, 1–3 ml of discarded peripheral blood was collected from premature infants of gestational age less than 32 weeks who were not taking inhaled oxygen and were admitted to the Department of Neonatology of the Affiliated Hospital of Southwest Medical University. Using a random number table, the PBMCs were randomly assigned to each of the following groups: the control group, air + OM group, hyperoxia group, and hyperoxia + OM group. After 48 h of in vitro modeling and culture, PBMCs and the culture medium of each group were collected. Immunofluorescence analysis was used to examine ROS levels in PBMCs. A full-spectrum spectrophotometer was used to examine malondialdehyde (MDA) levels in the culture medium. Enzyme-linked immunosorbent assay (ELISA) was used to examine monocyte chemotactic protein 1 (MCP-1) levels in culture medium. Immunofluorescence analysis was used to examine the intracellular localization of AhR. Western blotting was used to examine the expression level of AhR in PBMCs. Results Compared with those in the control group, the levels of ROS, MDA, and MCP-1 and the cytoplasm-nuclear translocation rate of AhR in the air + OM group did not change significantly (p > 0.05), but the expression level of AhR increased significantly (p < 0.05). The levels of ROS, MDA, and MCP-1 and the cytoplasm-nuclear translocation rate of AhR significantly increased in the hyperoxia group (p < 0.05), and the expression level of AhR was significantly reduced (p < 0.05). Compared with those in the hyperoxia group, the levels of ROS, MDA, and MCP-1 in the hyperoxia + OM group were significantly reduced (p < 0.05), and the cytoplasm-nuclear translocation rate of AhR and the expression level of AhR were significantly increased (p < 0.05), but did not reach the level of the control group (p < 0.05). Conclusion OM can activate AhR to inhibit hyperoxia-induced oxidative stress in the PBMCs from premature infants.

The aryl hydrocarbon receptor (AhR) activation mediates benzo(a)pyrene-induced overexpression of AQP3 and Notch1 in HaCaT cells.

The aim of this study was twofold: (1) evaluate the effect of benzo[a]pyrene (BaP) on expression levels of AQP3 and Notch1 genes in HaCaT cells exposed "in vitro" and (2) investigate the possible biological role of assessed genes by bioinformatics methods. Cells were exposed to increasing concentrations of BaP (0.0-4.0 μM) for 1-4 days. After treatments, cell viability and expression levels of AhR, CYP1A1, AQP3, and Notch1 genes were evaluated. The possible biological role of assessed genes was evaluated using bioinformatics tools. Low cytotoxicity in HaCaT cells dosed with BaP was detected. A significant overexpression (p < .05) of CYP1A1, AQP3, and Notch1 was found in exposed HaCaT cells. The gene expression upregulation was dependent on AhR activation. The bioinformatics analysis showed that these genes were enriched in related cancer signaling pathways. The findings suggest that AQP3 and Notch1 are upregulated by AhR activation in HaCaT cells exposed to BaP.

Aryl hydrocarbon receptors improve migraine-like pain behaviors in rats through the regulation of regulatory T cell/T-helper 17 cell-related homeostasis.

To investigate the effect of the aryl hydrocarbon receptor (AHR)/regulatory T cell (Treg)/T-helper 17 (Th17) cell pathway on the pathogenesis of migraine. Migraine is a disabling neurovascular disease that imposes an enormous burden on both individuals and society. The pathophysiological mechanisms of migraine remain controversial. Recent studies have suggested that immune dysfunction may be involved in the pathogenesis of migraine. The AHR, a receptor expressed on most immune cells, has been implicated in the occurrence of many autoimmune diseases; however, whether it is involved in the pathogenesis of migraine is unclear. A chronic migraine rat model was established through repeated intraperitoneal injection of nitroglycerin (NTG). The mechanical and thermal pain thresholds were assessed using von Frey filaments and radiant heat. Next, the protein expression levels of AHR in the trigeminal nucleus caudalis (TNC) region of chronic migraine (CM)-like rats were quantified and the changes in Treg/Th17-related transcription factors and inflammatory factors in the TNC were explored. To determine the role of AHR in CM, we examined the effects of the AHR agonist 2-(1'-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and AHR antagonist CH-223191 on pain behavior, c-Fos, calcitonin gene-related peptide (CGRP), AHR, and Treg/Th17-related factor expression in CM-like rats. Repeated administration of NTG significantly enhanced nociceptive hypersensitivity and increased expression of c-Fos and CGRP in rats, while AHR was significantly decreased in the TNC. In addition, the expression of the transcription factor forkhead box protein P3 and the signal transducer and activator of transcription 5 decreased significantly. In contrast, the expression of the transcription factor retinoic acid receptor-related orphan receptor γ t and signal transducer and activator of transcription 3 were significantly increased. Moreover, the mRNA level of transforming growth factor beta-1 was decreased, while that of interleukin (IL)-10 and IL-22 was increased in the TNC. The AHR agonist ITE alleviated migraine-like pain behaviors in rats, activated the AHR signaling pathway, and improved the imbalance of Treg/Th17-related transcription factors and inflammatory factors. Conversely, the AHR antagonist CH-223191 did not alleviate migraine-like pain behaviors in rats; and even exacerbated them. The AHR participates in the development of CM by regulating Treg/Th17-related homeostasis. Therefore, treatments targeting the AHR/Treg/Th17 signaling pathway could be new effective interventions for CM treatment.

Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 μmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 μmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Aryl Hydrocarbon Receptor (AHR) Gene Expression in AML Is Associated with FLT3-ITD+ AML and HLA-E Induced Immune Resistance Reversed By Ik-364

Background: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor critical for cellular metabolism, stem cell differentiation and immune control. HLA-E is a non-classical HLA molecule that is critical for NK cell activation. HLA-E is expressed on some AML blasts but genomic characterization is limited. IK-364, a tool compound generated by Ikena Oncology, is an AHR inhibitor in preclinical investigation. We set out to identify the genomic signatures associated with activation of the AHR pathway in AML samples utilizing the Beat AML dataset.Methods: Whole bone marrow or peripheral blood samples were procured from AML patients under IRB-approved protocol (eIRB# 4422). Utilizing the Beat AML database, all samples were examined based on RNA AHR expression levels (RPKM) and correlated with genomic expression. High vs. low AHR expression was based on 90 th and 10 th percentiles. Pathway enrichment from upregulated genes was identified and compared between high vs. low AHR expression. Single cell RNA sequencing by 10x genomics Chromium system by the Massively Parallel Sequencing Shared Resource at OHSU was performed with previously frozen FLT3 positive AML samples (4 high AHR and 4 low AHR). Immunophenotyping was conducted by flow cytometry. The AHR antagonist IK-364 was utilized to study the in vitro effects of AHR modulation. Cytotoxicity was measured by flow cytometric detection of caspase-3/7 and SytoxAAD activation.Results: The highest and lowest 10% expression levels of AHR in all samples in Beat AML were evaluated for mutation profiles. Ranking the top 18 mutations based on frequency we found an enrichment of FLT3 mutations in the high AHR expression group compared to the lower expression group (Figure 1A). ELN risk did not correlate with AHR expression however FAB classification M0/M1 and M4/M5 monocytic subtypes had higher AHR expression than M3 FAB subtype (Figure 2A). AHR gene expression was highly correlated with FLT3-ITD mutations (p=0.0007) (Figure 2B) and HLA-E expression (p=<0.0001) (Figure 3A). High AHR and high HLA-E joint expression in AML was associated with pathway upregulation in translocation of ZAP70 to immunological synapse, upregulation of HLA class II antigen presentation, phosphorylation of CD3 and TCR zeta chain and PD-1 signaling (p-value-1.11E-16), which are critical for innate and adaptive immune responses. AHR low and HLA-E low AML samples showed pathway enrichment for down-regulated genes including Hedgehog on state and neurexins and neuroligins (p value-0.001 and 7.61E-04 respectively). Genes upregulated in AHR high samples included CD14, CD86, HLA-DR, LILRB4, MAFB which are associated with monocytic phenotype. AHR high FLT3+ AML samples had higher protein expression of HLA-E on CD45+ blasts (p=0.03) compared to AHR low FLT3+ AML samples. Pretreatment of FLT3+ AML primary patient samples with IK-364 for 48 hours downregulates HLA-E on blasts (p=0.007) and, in a FLT3 positive AML cell line, Molm14, enhanced susceptibility to NK-cell-mediated killing (Figure 3B, 3C).Discussion: Overall, we found that high AHR gene expression in AML correlates with FLT3-ITD expression and HLA-E as well as monocytic subtype. Pathway enrichment highlight the important role of AHR in immune regulation. Furthermore, AHR antagonism by IK-364, downregulates HLA-E on AML blasts and augments NK cell mediated killing of a mutant FLT3-ITD positive AML cell line. Our results highlight a continued interest in targeting the AHR pathway to augment immune-based therapies in AML.uture studies are underway to determine the mechanism behind this critical immune regulation. [Display omitted] DisclosuresSaultz: IKENA: Research Funding. Lind: IKENA: Research Funding. Tyner: Seattle Genetics: Research Funding; Constellation: Research Funding; Agios: Research Funding; Incyte: Research Funding; Petra: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Array: Research Funding; Schrodinger: Research Funding; Astrazeneca: Research Funding. Kosaka: IKENA: Research Funding. McGovern: IKENA: Current Employment. Wang: IKENA: Current Employment. Sanchez-Martin: IKENA: Current Employment.

Effects of intranasal instillation of nanoparticulate matter in the olfactory bulb

Nanoparticulate matter activates the aryl hydrocarbon receptor (AhR) pathway in the respiratory system in a process involving the AhR nuclear translocator (ARNT) and cytochrome P450 family 1, member A1 (CYP1A1). We examined changes in AhR-related pathways following intranasal instillation of nanoparticulate matter in the olfactory bulb and cerebral cortex. Twice a day for 5 days per week for 1 week or 2 weeks, 8-week-old Sprague–Dawley rats were intranasally instilled with 10 µL nanoparticulate matter (nano group; n = 36). An equal volume of saline was intranasally instilled in control rats (n = 36). One week after intranasal instillation, olfactory function and Y-maze tests were performed. The expression levels of AhR in the olfactory bulb and temporal cortex were analyzed using western blotting and immunofluorescence assays. The expression levels of AhR, CYP1A1, inducible nitric oxide synthase (iNOS), and five genes encoding cation transporters (ARNT, ATP7B, ATPB1, OCT1, and OCT2) in the olfactory bulb were analyzed using quantitative reverse transcription. The olfactory discrimination capability was reduced in the nano group compared with the control group. Proportional changes in the Y-maze test were not significantly different between the nano and control groups. AhR mRNA and protein expression in the olfactory bulb increased 1.71-fold (P < 0.001) and 1.60-fold (P = 0.008), respectively. However, no significant changes were observed in the temporal cortex. In the olfactory bulb, the expression of ARNT, ATP7B, ATPB1, and OCT2 was downregulated. CYP1A1 and iNOS expression in the olfactory bulb was upregulated compared with that in the temporal cortex. The intranasal instillation of nanoparticulate matter decreased the olfactory discrimination ability, which was accompanied by upregulation of AhR expression and downregulation of cation transporters in the olfactory bulb.

Effect of the acid suppressor omeprazole on the proliferation, migration, invasion and cell cycle of esophageal squamous cell carcinoma cells via the aryl hydrocarbon receptor pathway.

Esophageal cancer is a malignant tumor type with one of the highest mortality rates worldwide. The aryl hydrocarbon receptor (AHR), which has been investigated in recent years, has been confirmed to be associated with the occurrence and development of esophageal cancer. AHR has a variety of different ligands, which regulate its activity following binding. The widely known acid inhibitor omeprazole (OME) also affects AHR and its downstream proteins (such as the cytochrome P450 family) by non-ligand binding; however, the mechanisms have remained to be fully elucidated. Therefore, the aim of the present study was to investigate the role of OME in esophageal squamous cell carcinoma (ESCC), whether the mechanism proceeds via the AHR pathway and how OME regulates AHR to affect the occurrence and development of esophageal carcinoma. The AHR-selective regulator OME was used to treat the ESCC cell lines TE1 and KYSE150. Western blot analysis was used to verify the effect of OME on AHR and proliferating cell nuclear antigen (PCNA) protein expression levels, while Cell Counting Kit (CCK)-8, wound-healing and Transwell assays were used to determine the proliferation, migration and invasion of the ESCCs, respectively, following treatment with OME. In addition, flow cytometry was used to investigate the cell cycle distribution of the ESCCs following incubation with OME. AHR was highly expressed in the ESCCs and following treatment with OME, the protein expression levels of AHR and PCNA were downregulated. The CCK-8 assay indicated that the proliferation of the ESCCs was also reduced following treatment with OME. Furthermore, flow cytometry revealed a notable block of the cells in G1/G0 phase, while the results of the wound-healing and Transwell assays respectively suggested that cell migration and invasion were reduced. In conclusion, OME inhibited the proliferation, migration and invasion of ESCC cells and blocked the cell cycle via the AHR pathway, which may provide a therapeutic effect on esophageal squamous cell cancer.

POS-431 ALTERED REGULATION OF TRYPTOPHAN METABOLISM AND ARYL HYDROCARBON RECEPTOR DISTRIBUTION IN RODENT POLYCYSTIC KIDNEYS

Polycystic kidney disease (PKD) is an inherited disorder characterized by stimulated cell proliferation, increased fluid secretion into cysts, and interstitial inflammation in renal tissue. Many mechanisms of disease progression have been determined in both autosomal dominant and autosomal recessive PKD, but the exact signaling pathways responsible for cystogenesis remain unclear. To explore novel mechanisms of PKD progression, we evaluated altered metabolic activity and associated receptor expression and distribution in the kidneys of 4- and 20-week-old PCK rats, an orthologous model of human autosomal recessive PKD, by mass spectrometry, quantitative analysis, real-time RT-PCR and immunofluorescence. Based on mass spectrometry and quantitative analysis results, the abundances of kynurenine and kynurenic acid were significantly higher in both 4- and 20-week-old PCK kidneys compared with age-matched wild-type kidneys. Since kynurenine and kynurenic acid are endogenous agonists of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor for activating metabolic enzymes such as those in the cytochrome P450 (CYP) superfamily, the expression of AhR was detected by real-time RT-PCR in the kidneys of wild-type and PCK rats. At 4-weeks of age with early stage PKD, the expression levels of AhR were significantly lower in PKD compared with wild-type kidneys. Intriguingly, its expression level in 20-week-old PCK kidneys with progressive stage PKD was higher compared with wild-type kidneys. Since cysts are derived from medulla in PCK kidneys, the distribution of AhR was observed by immunofluorescence mainly in renal medullary specimens. AhR is known to translocate into the nucleus upon ligand binding, and in normal kidneys at 4 weeks of age, AhR was localized to the nucleus in tubular epithelia, but not at 20 weeks of age in normal kidneys. However, in PCK kidneys at 20 weeks of age, AhR was localized in the nucleus in normal tubular epithelia and in developed cysts, while in dilated renal tubules (which develop into cysts), AhR translocated to the nucleus in PCK kidneys both at 4 and 20 weeks of age. Indolamine-2,3-dioxygenase 2 (IDO2) is an enzyme which catalyzes the conversion from tryptophan to kynurenine/kynurenic acid. In epithelial cells of dilated tubules in PCK kidneys, IDO2 was localized in the cytoplasm, suggesting that IDO2 may have a role in activating the AhR signaling pathway in cystogenesis. The current findings suggest that the progression of PKD may relate to the increased activation of tryptophan metabolism in combination with altered expression and distribution of AhR. Regulation of the kynurenine/kynurenic acid and AhR signaling pathway may have therapeutic potential against disease progression in PKD.

Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation.

Recently, a consanguineous family was identified in Israel with three children affected by Infantile Nystagmus and Foveal Hypoplasia, following an autosomal recessive mode of inheritance. A homozygous stop mutation c.1861C > T; p.Q621∗ in the aryl hydrocarbon receptor (AHR) gene (AHR; MIM 600253) was identified that co-segregated with the disease in the larger family. AHR is the first gene to be identified causing an autosomal recessive Infantile Nystagmus-related disease in humans. The goal of this study is to delineate the molecular basis of this newly discovered human genetic disorder associated with a rare AHR gene mutation. The gene and protein expression levels of AHR and selected AHR targets from leukocyte cultures of healthy subjects and the patients were analyzed. We observed significant variation between mRNA and protein expression of CYP1A1, CYP1B1, and TiPARP under rest and AHR-induced conditions. The CYP1A1 enzymatic activity in induced leukocytes also differs significantly between the patients and healthy volunteers. Intriguingly, the heterozygous subjects demonstrate CYP1A1 and TiPARP gene and protein expression similar to homozygous patients. In contrast, CYP1B1 inducibility and expression vary between hetero- and homozygous subjects. Similarity and differences in gene and protein expression between heterozygotes and homozygous patients can give us a hint as to which metabolic pathway/s might be involved in the Nystagmus etiology. Thus, we have a unique human model for AHR deficiency that will allow us the opportunity to study the biochemical basis of this rare human mutation, as well as the involvement of AHR in other physiological processes.

The aryl hydrocarbon receptor promotes inflammation-induced dedifferentiation and systemic metastatic spread of melanoma cells.

The aryl hydrocarbon receptor (AHR) is a ligand binding-transcription factor of the basic helix-loop-helix family regulating multiple cellular functions such as differentiation, cell cycle, apoptosis, and inflammatory reactions. In neoplastic diseases, the AHR has been described to modulate proliferation and differentiation in dichotomous ways, either inhibiting or augmenting the growth of tumors. The precise role of AHR in melanoma is mostly unknown. Here, we report a functional effect of AHR activation on inflammation-induced melanoma cell dedifferentiation and the development of lung metastases in a mouse model. Via in silico analyses of "The Cancer Genome Atlas" human melanoma cohort, we detected a correlation between AHR expression levels and a dedifferentiated melanoma cell phenotype with an invasive gene signature, which we were able to functionally recapitulate in a panel of human melanoma cell lines. Both human and mouse melanoma cell lines upregulated AHR expression after inflammatory stimulation with tumor necrosis factor-α (TNF-α). Activation of AHR in human and mouse melanoma cell lines with the endogenous ligand formylindolo(3,2-b)carbazole (FICZ) promoted inflammation-induced dedifferentiation in vitro. Importantly, mouse melanoma cells with CRISPR/Cas9-mediated disruption of the AHR gene showed impaired in vivo tumor growth after transplantation in the skin as well as decreased numbers of spontaneous lung metastases. Taken together, our results demonstrate a functional role for AHR expression in melanoma development and metastatic progression. This provides a scientific basis for future experiments that further dissect the underlying molecular mechanisms and assess the potential for AHR inhibition as part of multimodal melanoma treatment strategies.

Elevated expression of AhR and NLRP3 link polycyclic aromatic hydrocarbon exposure to cytokine storm in preschool children

BackgroundPolycyclic aromatic hydrocarbons (PAHs), as a group of persistent organic pollutants, are linked to impaired immune function and low-grade inflammation in adults and children. However, the potential of PAHs to lead to a cytokine storm associated with AhR (aryl hydrocarbon receptor) and NLRP3 (NLR family pyrin domain containing 3) in humans has been poorly studied. ObjectivesWe aimed to investigate the associations between PAH exposure, AhR and NLRP3 expression, and cytokines associated with a cytokine storm in healthy preschoolers. MethodsBasic demographic surveys and physical examinations were conducted on 248 preschoolers from an electronic waste (e-waste) recycling area (Guiyu, n = 121) and a reference area (Haojiang, n = 127). Ten urinary PAH metabolite (OH-PAH) concentrations were measured. We also measured the expression levels of AhR and NLRP3 and seventeen serum cytokine levels. ResultsThe concentrations of multiple OH-PAHs were significantly higher in the exposed group than those in the reference group, especially 1-hydroxynaphthalene (1-OH-Nap) and 2-hydroxynaphthalene (2-OH-Nap). PAH exposure was closely related to a child's living environment and hygiene habits. Expression levels of AhR and NLRP3 were significantly higher in the exposed group than in the reference group. Similarly, serum IL-1β, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-22, IL-23, and IFN-γ levels were notably higher in the e-waste-exposed children than in the reference children. After adjusting for age, gender, BMI, family income, parental education level, and second-hand smoke exposure, we found that increased PAH exposure was associated with higher AhR and NLRP3 expression and elevated IL-4, IL-10, IL-12p70, IL-18, IL-22, IL-23, TNF-α, and IFN-γ levels. The associations between PAH exposure and IL-1β, IL-18, IFN-γ, and TNF-β were mediated by NLRP3 expression, and the relationships between PAH exposure and IL-4, IL-10, IL-12p70, IL-22, IL-23, and TNF-α were mediated by AhR expression. ConclusionsOur findings suggest that the association between PAH exposure and a cytokine storm may be mediated by AhR and NLRP3 expression among preschoolers.

Gut Dysbiosis Induced by Cardiac Pressure Overload Promotes T Cell Activation and Adverse Cardiac Remodeling Through Alterations in the Aryl Hydrocarbon Receptor Expression

Heart failure (HF), a leading cause of morbidity and mortality worldwide, has been recently associated with gut microbiota alterations (gut dysbiosis) and T cell mediated inflammation in humans, and in experimental models of HF. The gut is a major reservoir of T cells, which are in close proximity with the gut microbiota. Gut bacterial populations process nutrients and produce metabolites that signal through cellular receptors, to maintain homeostasis or induce inflammation at distal organs. The aryl hydrocarbon receptor (AhR) is one of such receptors that recognize ligands that include gut derived tryptophan metabolites to modulate immune responses in the gut and systemically. We hypothesized that cardiac pressure overload induces gut dysbiosis and contributes to adverse cardiac remodeling through T cell activation and via AhR signaling in the gut and in the heart. We performed 16S rRNA sequencing in feces from mice undergoing Sham or transverse aortic constriction (TAC) surgery to test whether cardiac pressure overload induced gut dysbiosis, and in T cell deficient (Tcra−/−) mice to evaluate the contribution of T cells in gut dysbiosis induced by TAC. In addition, we studied the role of gut microbes in T cell activation, cardiac remodeling, and AhR expression, in mice that were orally treated with an antibiotic cocktail (ABX) to deplete the microbiota, and in ABX‐treated mice that spontaneously reconstituted the microbiota upon ABX removal during the 4 weeks of TAC (TAC‐REC). TAC resulted in increased abundance of phylum Bacteroidetes, and in a decrease in genus Lactobacillus, a tryptophan derivative producer, as compared to Sham. These microbiota alterations were not observed in Tcra−/− TAC mice, which were protected from adverse cardiac remodeling and exhibited a closer gut microbiota profile to WT Sham than to WT TAC mice in principal component analysis. However, TAC did not induce leaky gut, determined by LPS levels in serum, or T cell activation in the lamina propria, but increased T cell activation in the mediastinal lymph nodes (mLNs) that drain the heart, and drastically resulted in decreased AhR expression in the gut and in the heart. Strikingly, depletion of the gut microbiota in the onset of TAC (TAC‐ABX) resulted in a reduction in the number of activated T cells in the mLNs and heart infiltrated T cells, lack of cardiac fibrosis and systolic dysfunction, and an induction of AhR expression in the gut and the heart as compared to TAC mice that had not been depleted of the microbiota. Moreover, gut microbiota reconstitution in TAC‐REC mice resulted in decreased expression of AhR in the gut and in the heart as compared to ABX treated mice, and showed similar expression levels of AhR and systolic dysfunction as TAC mice. Our data demonstrate a role of gut microbes in T cell activation and adverse cardiac remodeling induced by cardiac pressure overload that likely involves decreased AhR signaling in the gut and in the heart.Support or Funding InformationThese studies were supported by NIH HL 123658 and the American Heart Association grant 19POST34430075/2019

Benzo[a]pyrene at human blood equivalent level induces human lung epithelial cell invasion and migration via aryl hydrocarbon receptor signaling.

Benzo[a]pyrene (B[a]P), a typical carcinogenic polycyclic aromatic hydrocarbon, exists worldwide in vehicle exhaust, cigarette smoke and other polluted environments. Recent studies have demonstrated a strong association between B[a]P and lung cancer. However, whether B[a]P at human blood equivalent level can promote epithelial-mesenchymal transition (EMT), a crucial molecular event during cell malignant transformation, remains unclear. Besides, whether B[a]P facilitates this progress via aryl hydrocarbon receptor (AhR) signaling pathway also lacks scientific evidence. In our study, the transwell assay showed that 5 μg/L of B[a]P promoted BEAS-2B cell invasion and migration. In addition, the mRNA and protein expression levels of AhR and its target genes involved in B[a]P metabolism, such as AhR nuclear translocator, heat shock protein 90 and CYP1A1, were significantly increased by B[a]P exposure. Moreover, the mRNA expression levels of downstream regulatory factors related to both AhR signaling pathway and EMT, such as NRF2, K-RAS and hypoxia-inducible factor 1-alpha, were significantly increased. Furthermore, the expression level of the epithelial marker E-cadherin was significantly downregulated, while the mRNA expression of mesenchymal phenotype markers, N-cadherin, fibronectin and vimentin, were significantly upregulated. Notably, the above changes induced by B[a]P were significantly attenuated or even stopped by resveratrol (RSV), a natural phenol, also an AhR inhibitor, when the AhR signaling pathway was inhibited by RSV, demonstrating the regulatory role of AhR signaling pathway in B[a]P-induced EMT. In conclusion, B[a]P at the human blood equivalent level induces BEAS-2B cell invasion and migration through the AhR signaling pathway.