Mixture Effects of Tryptophan Intestinal Microbial Metabolites on Aryl Hydrocarbon Receptor Activity

Aryl hydrocarbon receptor (AHR) plays pivotal roles in intestinal physiology and pathophysiology. Intestinal AHR is activated by numerous dietary, endogenous, and microbial ligands. Whereas the effects of individual compounds on AHR are mostly known, the effects of real physiological mixtures occurring in the intestine have not been studied. Using reporter gene assays and RT-PCR, we evaluated the combinatorial effects (3520 combinations) of 11 microbial catabolites of tryptophan (MICTs) on AHR. We robustly (n = 30) determined the potencies and relative efficacies of single MICTs. Synergistic effects of MICT binary mixtures were observed between low- or medium-efficacy agonists, in particular for combinations of indole-3-propionate and indole-3-lactate. Combinations comprising highly efficacious agonists such as indole-3-pyruvate displayed rather antagonist effects, caused by saturation of the assay response. These synergistic effects were confirmed by RT-PCR as CYP1A1 mRNA expression. We also tested mimic multicomponent and binary mixtures of MICTs, prepared based on the metabolomic analyses of human feces and colonoscopy aspirates, respectively. In this case, AHR responsiveness did not correlate with type of diet or health status, and the indole concentrations in the mixtures were determinative of gross AHR activity. Future systematic research on the synergistic activation of AHR by microbial metabolites and other ligands is needed.     

Tryptophan Metabolites and Aryl Hydrocarbon Receptor in Severe Acute Respiratory Syndrome,Coronavirus-2 (SARS-CoV-2) Pathophysiology

The metabolism of tryptophan is intimately associated with the differential regulation of diverse physiological processes, including in the regulation of responses to severe acute respiratory syndrome, coronavirus-2 (SARS-CoV-2) infection that underpins the COVID-19 pandemic. Two important products of tryptophan metabolism, viz kynurenine and interleukin (IL)4-inducible1 (IL41)-driven indole 3 pyruvate (I3P), activate the aryl hydrocarbon receptor (AhR), thereby altering the nature of immune responses to SARS-CoV-2 infection. AhR activation dysregulates the initial pro-inflammatory cytokines production driven by neutrophils, macrophages, and mast cells, whilst AhR activation suppresses the endogenous antiviral responses of natural killer cells and CD8+ T cells. Such immune responses become further dysregulated by the increased and prolonged pro-inflammatory cytokine suppression of pineal melatonin production coupled to increased gut dysbiosis and gut permeability. The suppression of pineal melatonin and gut microbiome-derived butyrate, coupled to an increase in circulating lipopolysaccharide (LPS) further dysregulates the immune response. The AhR mediates its effects via alterations in the regulation of mitochondrial function in immune cells. The increased risk of severe/fatal SARS-CoV-2 infection by high risk conditions, such as elderly age, obesity, and diabetes are mediated by these conditions having expression levels of melatonin, AhR, butyrate, and LPS that are closer to those driven by SARS-CoV-2 infection. This has a number of future research and treatment implications, including the utilization of melatonin and nutraceuticals that inhibit the AhR, including the polyphenols, epigallocatechin gallate (EGCG), and resveratrol.     

Anti-Aging Physiological Roles of Aryl Hydrocarbon Receptor and Its Dietary Regulators

The vast majority of the literature on the aryl hydrocarbon receptor is concerned with its functions in xenobiotic detoxification. However, in the course of evolution, this receptor had to have physiological (rather than toxicological) functions. Our aim was to review the aryl hydrocarbon receptor’s role in the physiological functions involved in aging. This study was performed by searching the MEDLINE and Google Academic databases. A total of 34 articles were selected that focused specifically on the aryl hydrocarbon receptor and aging, the aryl hydrocarbon receptor and physiological functions, and the combination of both. This receptor’s main physiological functions (mediated by the modulation of gene expression) were cell regeneration, the immune reaction, intestinal homeostasis, and cell proliferation. Furthermore, it was shown that the loss of this receptor led to premature aging. This process may be caused by the dysregulation of hematopoietic stem cells, loss of glucose and lipid homeostasis, increase in inflammation, and deterioration of the brain. We conclude that the aryl hydrocarbon receptor, apart from its well-established role in xenobiotic detoxication, plays an important role in physiological functions and in the aging process. Modulation of the signaling pathway of this receptor could be a therapeutic target of interest in aging.     

Possible Involvement of the Upregulation of ΔNp63 Expression Mediated by HER2-Activated Aryl Hydrocarbon Receptor in Mammosphere Maintenance

Cancer stem cells (CSCs) contribute to the drug resistance, recurrence, and metastasis of breast cancers. Recently, we demonstrated that HER2 overexpression increases mammosphere formation via the activation of aryl hydrocarbon receptor (AHR). In this study, the objective was to identify the mechanism underlying mammosphere maintenance mediated by HER2 signaling-activated AHR. We compared the chromatin structure of AHR-knockout (AHRKO) HER2-overexpressing MCF-7 (HER2-5) cells with that of wild-type HER2-5 cells; subsequently, we identified TP63, a stemness factor, as a potential target gene of AHR. ΔNp63 mRNA and protein levels were higher in HER2-5 cells than in HER2-5/AHRKO cells. Activation of HER2/HER3 signaling by heregulin treatment increased ΔNp63 mRNA levels, and its induction was decreased by AHR knockdown in HER2-5 cells. The results of the chromatin immunoprecipitation assay revealed an interaction between AHR and the intronic region of TP63, which encodes ΔNp63. A luciferase reporter gene assay with the intronic region of TP63 showed that AHR expression increased reporter activity. Collectively, our findings suggest that HER2-activated AHR upregulates ΔNp63 expression and that this signaling cascade is involved in CSC maintenance in HER2-expressing breast cancers.     

Amino Alcohol Acrylonitriles as Activators of the Aryl Hydrocarbon Receptor Pathway: An Unexpected MTT Phenotypic Screening Outcome

Lead (Z)-N-(4-(2-cyano-2-(3,4-dichlorophenyl)vinyl)phenyl)acetamide, 1 showed MCF-7 GI₅₀=30 nM and 400-fold selective c.f. MCF10A (normal breast tissue). Acetamide moiety modification ( 13 a - g ) to introduce additional hydrophobicity was favoured with MCF-7 breast cancer cell activity enhanced at 1.3 nM. Other analogues were potent against the HT29 colon cancer cell line at 23 nM. Textbook SAR data was observed in the MCF-7 cell line, in an MTT assay, via the ortho ( 17 a ), meta ( 17 b ) and para ( 13 f ). The amino alcohol -OH moiety was pivotal, but no stereochemical preference noted. But, these data did not fit our homology modelling expectations. Aberrant MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) screening results and metabolic interference confirmed by sulforhodamine B (SRB) screening. Interfering analogues resulted in 120 and 80-fold CYP1A1 and CYP1A2 amplification, with no upregulation of SULT1A1. This is consistent with activation of the AhR pathway. Piperidine per-deuteration reduced metabolic inactivation. 3-OH / 4-OH piperidine analogues showed differential MTT and SRB activity supporting MTT assay metabolic inactivation. Data supports piperidine 3-OH, but not the 4-OH, as a CYP substrate. This family of β-amino alcohol substituted 3,4-dichlorophenylacetonitriles show broad activity modulated via the AhR pathway. By SRB analysis the most potent analogue was 23 b , (Z)-3-(4-(3-(4-phenylpiperidin-1-yl)-2-hydroxypropoxy)phenyl)-2-(3,4-dichlorophenyl)-acrylonitrile.     

Aryl Hydrocarbon Receptor Repressor Is Hypomethylated in Psoriasis and Promotes Psoriasis-like Inflammation in HaCaT Cells

It is known that DNA hypomethylation of aryl hydrocarbon receptor repressor (AhRR), one of the epigenetic markers of environmental pollutants, causes skin diseases. However, the function and mechanisms are still unknown. We aimed to determine whether AhRR is hypomethylated in PBMC of psoriasis patients, as well as to examine the expression of psoriasis-related inflammatory cytokines and antimicrobial peptides after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in HaCaT cells overexpressing or silencing AhRR. AhRR was determined by qPCR, Western blot, immunohistochemistry, and immunocytochemistry in skin tissue and HaCaT cells. DNA methylation of AhRR was performed by Infinium Human Methylation450 BeadChip in PBMC of psoriasis patients and methylation-specific PCR (MSP) in HaCaT cells. NF-κB pp50 translocation and activity were performed by immunocytochemistry and luciferase reporter assay, respectively. We verified AhRR gene expression in the epidermis from psoriasis patients and healthy controls. AhRR hypomethylation in PBMC of psoriasis patients and pAhRR-HaCaT cells was confirmed. The expression level of AhRR was increased in both TCDD-treated HaCaT cells and pAhRR-HaCaT cells. NF-κB pp50 translocation and activity increased with TCDD. Our results showed that AhRR was hypomethylated and overexpressed in the lesional skin of patients with psoriasis, thereby increasing AhRR gene expression and regulating pro-inflammatory cytokines through the NF-κB signaling pathway in TCDD-treated HaCaT cells.     

The Aryl Hydrocarbon Receptor Undergoes Chaperone-Mediated Autophagy in Triple-Negative Breast Cancer Cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.     

Aryl Hydrocarbon Receptor (AhR) Activation by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) Dose-Dependently Shifts the Gut Microbiome Consistent with the Progression of Non-Alcoholic Fatty Liver Disease

Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids, including taurolithocholic acid and deoxycholic acid (microbial modified bile acids involved in host bile acid regulation signaling pathways). To investigate the effects of TCDD on the gut microbiota, the cecum contents of male C57BL/6 mice orally gavaged with sesame oil vehicle or 0.3, 3, or 30 µg/kg TCDD were examined using shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species (i.e., Lactobacillus reuteri). Increased species were also associated with dose-dependent increases in bile salt hydrolase sequences, responsible for deconjugation reactions in secondary bile acid metabolism. Increased L. reuteri levels were further associated with mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis and o-succinylbenzoate synthase, a menaquinone biosynthesis associated gene. Analysis of the gut microbiomes from cirrhosis patients identified an increased abundance of genes from the mevalonate-dependent IPP biosynthesis as well as several other menaquinone biosynthesis genes, including o-succinylbenzoate synthase. These results extend the association of lactobacilli with the AhR/intestinal axis in NAFLD progression and highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD.     

Aryl Hydrocarbon Receptor Repressor and TiPARP (ARTD14) Use Similar,but also Distinct Mechanisms to Repress Aryl Hydrocarbon Receptor Signaling

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP^−/− mouse embryonic fibroblasts (MEFs) and AHRR^−/− MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP^−/− MEFs, AHRR^−/− MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR^−/− MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP^−/− MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.     

Lipopolysaccharide-Enhanced Responses against Aryl Hydrocarbon Receptor in FcgRIIb-Deficient Macrophages,a Profound Impact of an Environmental Toxin on a Lupus-Like Mouse Model

Fc gamma receptor IIb (FcgRIIb) is the only inhibitory-FcgR in the FcgR family, and FcgRIIb-deficient (FcgRIIb^−/−) mice develop a lupus-like condition with hyper-responsiveness against several stimulations. The activation of aryl hydrocarbon receptor (Ahr), a cellular environmental sensor, might aggravate activity of the lupus-like condition. As such, 1,4-chrysenequinone (1,4-CQ), an Ahr-activator, alone did not induce supernatant cytokines from macrophages, while the 24 h pre-treatment by lipopolysaccharide (LPS), a representative inflammatory activator, prior to 1,4-CQ activation (LPS/1,4-CQ) predominantly induced macrophage pro-inflammatory responses. Additionally, the responses from FcgRIIb^−/− macrophages were more prominent than wild-type (WT) cells as determined by (i) supernatant cytokines (TNF-α, IL-6, and IL-10), (ii) expression of the inflammation associated genes (NF-κB, aryl hydrocarbon receptor, iNOS, IL-1β and activating-FcgRIV) and cell-surface CD-86 (a biomarker of M1 macrophage polarization), and (iii) cell apoptosis (Annexin V), with the lower inhibitory-FcgRIIb expression. Moreover, 8-week-administration of 1,4-CQ in 8 week old FcgRIIb^−/− mice, a genetic-prone lupus-like model, enhanced lupus characteristics as indicated by anti-dsDNA, serum creatinine, proteinuria, endotoxemia, gut-leakage (FITC-dextran), and glomerular immunoglobulin deposition. In conclusion, an Ahr activation worsened the disease severity in FcgRIIb^−/− mice possibly through the enhanced inflammatory responses. The deficiency of inhibitory-FcgRIIb in these mice, at least in part, prominently enhanced the pro-inflammatory responses. Our data suggest that patients with lupus might be more vulnerable to environmental pollutants.     

Dendritic Cells and Cancer Immunotherapy: The Adjuvant Effect

Dendritic cells (DCs) are immune specialized cells playing a critical role in promoting immune response against antigens, and may represent important targets for therapeutic interventions in cancer. DCs can be stimulated ex vivo with pro-inflammatory molecules and loaded with tumor-specific antigen(s). Protocols describing the specific details of DCs vaccination manufacturing vary widely, but regardless of the employed protocol, the DCs vaccination safety and its ability to induce antitumor responses is clearly established. Many years of studies have focused on the ability of DCs to provide overall survival benefits at least for a selection of cancer patients. Lessons learned from early trials lead to the hypothesis that, to improve the efficacy of DCs-based immunotherapy, this should be combined with other treatments. Thus, the vaccine’s ultimate role may lie in the combinatorial approaches of DCs-based immunotherapy with chemotherapy and radiotherapy, more than in monotherapy. In this review, we address some key questions regarding the integration of DCs vaccination with multimodality therapy approaches for cancer treatment paradigms.     

Expression of the Aryl Hydrocarbon Receptor in Growth Plate Cartilage and the Impact of Its Local Modulation on Longitudinal Bone Growth

Although dioxin has been reported to impair bone growth in both humans and animals, the underlying mechanisms have not been clarified. We conducted this study to rule out if dioxin may directly target the growth plate, via local modulation of the aryl hydrocarbon receptor (AhR). Initial studies in rare tissue samples of the human growth plate confirmed that the AhR protein is widely expressed in growth plate cartilage. To explore the local role of the AhR, mechanistic studies were performed in a well-established model of cultured fetal rat metatarsal bones. The longitudinal growth of these bones was monitored while being exposed to AhR modulators. The AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin, did not affect bone growth at any concentrations tested (1 pM–10 nM). In contrast, the AhR antagonist, alpha-naphthoflavone, suppressed bone growth and increased chondrocyte apoptosis, although only at a high, potentially cytotoxic concentration (50 µM). We conclude that although the AhR is widely expressed in the growth plate, bone growth is not modulated when locally activated, and therefore, dioxin-induced growth failure is likely mediated through systemic rather than local actions.     

Tumour Microenvironment: Roles of the Aryl Hydrocarbon Receptor,O-GlcNAcylation,Acetyl-CoA and Melatonergic Pathway in Regulating Dynamic Metabolic Interactions across Cell Types—Tumour Microenvironment and Metabolism

This article reviews the dynamic interactions of the tumour microenvironment, highlighting the roles of acetyl-CoA and melatonergic pathway regulation in determining the interactions between oxidative phosphorylation (OXPHOS) and glycolysis across the array of cells forming the tumour microenvironment. Many of the factors associated with tumour progression and immune resistance, such as yin yang (YY)1 and glycogen synthase kinase (GSK)3β, regulate acetyl-CoA and the melatonergic pathway, thereby having significant impacts on the dynamic interactions of the different types of cells present in the tumour microenvironment. The association of the aryl hydrocarbon receptor (AhR) with immune suppression in the tumour microenvironment may be mediated by the AhR-induced cytochrome P450 (CYP)1b1-driven ‘backward’ conversion of melatonin to its immediate precursor N-acetylserotonin (NAS). NAS within tumours and released from tumour microenvironment cells activates the brain-derived neurotrophic factor (BDNF) receptor, TrkB, thereby increasing the survival and proliferation of cancer stem-like cells. Acetyl-CoA is a crucial co-substrate for initiation of the melatonergic pathway, as well as co-ordinating the interactions of OXPHOS and glycolysis in all cells of the tumour microenvironment. This provides a model of the tumour microenvironment that emphasises the roles of acetyl-CoA and the melatonergic pathway in shaping the dynamic intercellular metabolic interactions of the various cells within the tumour microenvironment. The potentiation of YY1 and GSK3β by O-GlcNAcylation will drive changes in metabolism in tumours and tumour microenvironment cells in association with their regulation of the melatonergic pathway. The emphasis on metabolic interactions across cell types in the tumour microenvironment provides novel future research and treatment directions.     

Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126)

Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1, are regulated by the aryl hydrocarbon receptor (AhR). 3,3'',4,4'',5-Pentachlorobiphenyl (PCB 126) is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA) and 5-aza-2''-deoxycytidine (5-Aza-dC), significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.