Aryl Hydrocarbon Receptor Ligands Research Articles

Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increasedwhenendothelialAHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Endothelial sensing of AHR ligands regulates intestinal homeostasis

Endothelial cells line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance and coordinate angiogenesis and lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric endothelial cells actively sense and integrate such signals is currently unknown. Here we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for endothelial cell sensing of dietary metabolites in adult mice and human primary endothelial cells. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic endothelial cells. Analyses of AHR-mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR deficiency in adult mice resulted in dysregulated inflammatory responses and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human endothelial cells, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the effect of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting endothelial cell quiescence and vascular normalcy.

Bojungikgi-tang improves skin barrier function and immune response in atopic dermatitis mice fed a low aryl hydrocarbon receptor ligand diet

BackgroundThe aryl hydrocarbon receptor (AhR) is a transcription factor that plays a crucial role in regulating the immune system and maintaining skin barrier function. AhR signaling is pivotal in the pathogenesis of inflammatory diseases such as atopic dermatitis (AD), and the absence of AhR ligands further contributes to the progression or worsening of AD symptoms.MethodsAD was induced with 2,4-dinitrochlorobenzene (DNCB), and Bojungikgi-tang (BJIKT) was administered orally daily for 10 weeks. Serum IgE, splenocyte IL-4, and IFN-γ levels, skin barrier genes, and AhR target gene expressions were analyzed using RNA-sequencing analysis. Spleen tissues were extracted for fluorescence-activated cell sorting (FACS) analysis to analyze the effect of BJIKT on immune responses. A correlation analysis was conducted to analyze the correlation between immune markers and skin barrier genes and AhR target genes.ResultsBJIKT effectively improved AD symptoms in AD mice fed a low AhR ligand diet by reducing neutrophil and eosinophil counts, lowering IgE levels in the blood, and decreasing IL-4 and IFN-γ levels in the splenocytes. Additionally, BJIKT significantly reduced epithelial skin thickness and transepidermal water loss (TEWL) values and reversed the decreased expression of skin barrier genes. BJIKT also considerably altered the expression of AhR target genes, including Ahr, Ahrr, cytochrome P450 1A1 (CYP1A1), and CYP1B1. Furthermore, AhR target pathway genes were negatively correlated with immune cell subtypes, including CD4 + and CD8 + T cells and macrophages (CD11b + F4/80 +) at the systemic level.ConclusionsBJIKT can regulate AhR activation and may help reduce inflammation in AD by regulating the expression of skin barrier genes and immune responses.

L-Kynurenine participates in cancer immune evasion by downregulating hypoxic signaling in T lymphocytes

ABSTRACT Malignant tumors often escape anticancer immune surveillance by suppressing the cytotoxic functions of T lymphocytes. While many of these immune evasion networks include checkpoint proteins, small molecular weight compounds, such as the amino acid L-kynurenine (LKU), could also substantially contribute to the suppression of anti-cancer immunity. However, the biochemical mechanisms underlying the suppressive effects of LKU on T-cells remain unclear. Here, we report for the first time that LKU suppresses T cell function as an aryl hydrocarbon receptor (AhR) ligand. The presence of LKU in T cells is associated with AhR activation, which results in competition between AhR and hypoxia-inducible factor 1 alpha (HIF-1α) for the AhR nuclear translocator, ARNT, leading to T cell exhaustion. The expression of indoleamine 2,3-dioxygenase 1 (IDO1, the enzyme that leads to LKU generation) is induced by the TGF-β-Smad-3 pathway. We also show that IDO-negative cancers utilize an alternative route for LKU production via the endogenous inflammatory mediator, the high mobility group box 1 (HMGB-1)-interferon-gamma (IFN-γ) axis. In addition, other IDO-negative tumors (like T-cell lymphomas) trigger IDO1 activation in eosinophils present in the tumor microenvironment (TME). These mechanisms suppress cytotoxic T cell function, and thus support the tumor immune evasion machinery.

Effect of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor agonists in growing pigs.

Effects of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor (AhR) ligands in the intestine were investigated in a pig model. Ileal digesta and faeces of pigs after feeding of eighteen different foods were analyzed. Indole, indole-3-propionic acid, indole-3-acetic acid, indole-3-lactic acid, kynurenine, tryptamine, and indole-3-aldehyde were identified in ileal digesta, which were also identified in faeces but at higher concentrations except indole-3-lactic acid, together with skatole, oxindole, serotonin, and indoleacrylic acid. The panel of tryptophan catabolites in ileal digesta and faeces varied across different foods. Eggs induced the highest overall concentration of catabolites in ileal digesta dominated by indole. Amaranth induced the highest overall concentration of catabolites in faeces dominated by skatole. Using a reporter cell line, we observed many faecal samples but not ileal samples retained AhR activity. Collectively, these findings contribute to food selection targeting AhR ligands production from dietary tryptophan in the intestine.

Six Decades of Dopamine Hypothesis: Is Aryl Hydrocarbon Receptor the New D2?

In 1957, Arvid Carlsson discovered that dopamine, at the time believed to be nothing more than a norepinephrine precursor, was a brain neurotransmitter in and of itself. By 1963, postsynaptic dopamine blockade had become the cornerstone of psychiatric treatment as it appeared to have deciphered the “chlorpromazine enigma”, a 1950s term, denoting the action mechanism of antipsychotic drugs. The same year, Carlsson and Lindqvist launched the dopamine hypothesis of schizophrenia, ushering in the era of psychopharmacology. At present, six decades later, although watered down by three consecutive revisions, the dopamine model remains in vogue. The latest emendation of this paradigm proposes that “environmental and genetic factors” converge on the dopaminergic pathways, upregulating postsynaptic transmission. Aryl hydrocarbon receptors, expressed by the gut and blood–brain barrier, respond to a variety of endogenous and exogenous ligands, including dopamine, probably participating in interoceptive awareness, a feed-back loop, conveying intestinal barrier status to the insular cortex. The conceptualization of aryl hydrocarbon receptor as a bridge, connecting vagal terminals with the microbiome, may elucidate the aspects of schizophrenia seemingly incongruous with the dopamine hypothesis, such as increased prevalence in urban areas, distance from the equator, autoantibodies, or comorbidity with inflammatory bowel disease and human immunodeficiency 1 virus. In this review article, after a short discussion of schizophrenia outcome studies and insight, we take a closer look at the action mechanism of antipsychotic drugs, attempting to answer the question: do these agents exert their beneficial effects via both dopaminergic and nondopaminergic mechanisms? Finally, we discuss potential new therapies, including transcutaneous vagal stimulation, aryl hydrocarbon receptor ligands, and restoring the homeostasis of the gut barrier.

An in vitro model of human hematopoiesis identifies a regulatory role for the aryl hydrocarbon receptor

AbstractIn vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell–free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand–activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood–derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.

An UPLC-MS/MS method for targeted analysis of microbial and host tryptophan metabolism after administration of polysaccharides from Atractylodes macrocephala Koidz. in ulcerative colitis mice

Botanic polysaccharides can be metabolized by gut microbiota into short-chain fatty acids (SCFAs) to exert extensive bioactivities, yet targeted analysis of the effect of botanic polysaccharides on other gut microbial metabolites is scarcely seen. Tryptophan metabolites such as indole and indole derivatives play import roles in health and disease development. Using polysaccharides from Atractylodes macrocephala Koidz. (AMP) in treating ulcerative colitis as the example, we checked the effects of AMP on tryptophan metabolites. After examination of pharmacological effects of AMP, we established an ultra-performance liquid chromatography coupled with mass spectrometry/mass spectrometry (UPLC-MS/MS) method to simultaneously determinate the levels of 30 tryptophan metabolites and used the method to determine the levels of these metabolites in feces and plasma. The detection results showed that 12 metabolites in feces can be detected, and 17 metabolites can be detected in plasma samples. In addition, we found out that total levels of aryl hydrocarbon receptor ligands were decreased in colitis model whereas AMP treatment can increase the levels of total ligands in both feces and plasma. The results indicated that the therapeutical effect of AMP on colitis was associated with modulation of fecal and host tryptophan metabolism. This study provides new insight into the molecular mechanisms of polysaccharides that the beneficial effects of polysaccharides can be achieved by modulating microbial tryptophan metabolism in addition to SCFAs.

Transcriptomic analysis reveals the regulatory mechanism underlying the indirubin-mediated amelioration of dextran sulfate sodium-induced colitis in mice

Context Aryl hydrocarbon receptor (AhR) agonists are potential therapeutic agents for ulcerative colitis (UC). Indirubin (IDR), which is a natural AhR ligand approved for leukemia treatment, ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. However, the therapeutic mechanisms of IDR are unknown, limiting its application. Objective This study explores the therapeutic mechanisms of IDR in DSS-induced colitis using transcriptomic analysis. Materials and methods Male BALB/c mice were categorized to six groups: normal, DSS model (2% DSS), IDR treatment (10, 20 and 40 mg/kg), and sulfasalazine (520 mg/kg) groups. The drugs were intragastrically administered for 7 consecutive days. The disease activity index (DAI) was recorded. After euthanasia, the colon length was measured, and histopathological examination, immunohistochemistry staining using F4/80, and colonic transcriptomic analysis were conducted. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting (WB) were conducted to verify our findings. Results Compared with DSS, IDR treatment decreased the DAI score by 64.9% and increased colon length by 26.2%. Moreover, it alleviated mucosal injury and reduced macrophage infiltration. Transcriptomic analysis identified several downregulated genes (Igkvs and Nlrp3), as well as Nlrp3/Il1β and hemoglobin gene networks, after IDR treatment. The abundances of NF-κB p65, NLRP3, IL-1β, and HBA decreased by 69.1, 59.4, 81.1, and 83.0% respectively, after IDR treatment. Discussion and conclusion Apart from the well-documented NF-κB signalling pathway, IL-17A, and NLRP3-IL-1β, the suppression of haemoglobin-induced lipid peroxidation could be a previously unknown mechanism of IDR. Our study can help improve its application for UC treatment.

Breast cancer progression and kynurenine pathway enzymes are induced by hexachlorobenzene exposure in a Her2-positive model.

Breast cancer is one of the leading cancers among women worldwide. Given the evidence that pesticides play an important role in breast cancer, interest has grown in pesticide impact on disease progression. Hexachlorobenzene (HCB), an aryl hydrocarbon receptor (AhR) ligand, promotes triple-negative breast cancer cell migration and invasion. Estrogen receptor β (ERβ) inhibits cancer motility, while G protein-coupled ER (GPER) modulates the neoplastic transformation. Tryptophan is metabolized through the kynurenine pathway by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), with kynurenine signaling activation often predicting worse prognosis in cancer. In this context, we examined the HCB (0.005; 0.05; 0.5 and 5μM) effect on LM3 cells, a human epidermal growth factor receptor 2 (HER2)-positive breast cancer model. Results show that HCB increases IDO and TDO mRNA levels and promotes cell viability, proliferation and migration through the AhR pathway. Moreover, HCB boosts mammosphere formation, vascular endothelial growth factor and cyclooxygenase-2 expression and reduces IL-10 levels. For some parameters, U-shaped or inverted U-shaped dose-response curves are shown. HCB alters ER levels, reducing ERβ while increasing GPER. These results demonstrate that exposure to environmentally relevant concentrations of HCB up-regulates the kynurenine pathway and dysregulates ERβ and GPER levels, collaborating in HER2-positive breast cancer progression.

Molecular networking identifies an AHR-modulating benzothiazole from white button mushrooms (Agaricus bisporus).

Diet-derived aryl hydrocarbon receptor (AHR) ligands have potential to maintain gut health. However, among the myriad bioactive compounds from foods, identifying novel functional ligands which would significantly impact gastrointestinal health is a challenge. In this study, a novel AHR modulator is predicted, identified, and characterized in the white button mushroom (Agaricus bisporus). Using a molecular networking approach, a methylated analog to benzothiazole was indicated in white button mushrooms, which was subsequently isolated and identified as 2-amino-4-methyl-benzothiazole(2A4). Cell-based AHR transcriptional assays revealed that 2-amino-4-methyl-benzothiazole possesses agonistic activity and upregulated CYP1A1 expression. This contrasts with previous findings that whole white button mushroom extract has overall antagonistic activity in vivo, underscoring the importance of studying the roles each chemical component plays in a whole food. The findings suggest that 2-amino-4-methyl-benzothiazole is a previously unidentified AHR modulator from white button mushroom and demonstrate that molecular networking has potential to identify novel receptor modulators from natural products.

Host-Microbiome Interactions: Tryptophan Metabolism and Aromatic Hydrocarbon Receptors after Traumatic Brain Injury.

Traumatic brain injury refers to the damage caused to intracranial tissues by an external force acting on the head, leading to both immediate and prolonged harmful effects. Neuroinflammatory responses play a critical role in exacerbating the primary injury during the acute and chronic phases of TBI. Research has demonstrated that numerous neuroinflammatory responses are mediated through the "microbiota-gut-brain axis," which signifies the functional connection between the gut microbiota and the brain. The aryl hydrocarbon receptor (AhR) plays a vital role in facilitating communication between the host and microbiota through recognizing specific ligands produced directly or indirectly by the microbiota. Tryptophan (trp), an indispensable amino acid in animals and humans, represents one of the key endogenous ligands for AhR. The metabolites of trp have significant effects on the functioning of the central nervous system (CNS) through activating AHR signalling, thereby establishing bidirectional communication between the gut microbiota and the brain. These interactions are mediated through immune, metabolic, and neural signalling mechanisms. In this review, we emphasize the co-metabolism of tryptophan in the gut microbiota and the signalling pathway mediated by AHR following TBI. Furthermore, we discuss the impact of these mechanisms on the underlying processes involved in traumatic brain injury, while also addressing potential future targets for intervention.

The opposite effect of tapinarof between IMQ and IL-23 induced psoriasis mouse models.

Tapinarof is an aryl hydrocarbon receptor (AHR) ligand which is used to treat plaque psoriasis in adults. However, the underlying mechanism is not yet fully understood. In this study, we applied two of the most studied psoriasis mouse models: topical application of imiquimod (IMQ) and subcutaneous injection of IL-23. Although both models successfully induced psoriasis-like lesions in mice, tapinarof had a completely opposite effect on the two models. Tapinarof decreased the expression of multiple essential cytokines involved in the pathological IL-23/IL-17/IL-22 axis and ameliorated IMQ-induced psoriatic dermatitis, inhibiting keratinocyte proliferation and abnormal differentiation. However, in the IL-23-injection-model, tapinarof instead aggravated the disease. Here, tapinarof increased epidermal thickness and differentiated epidermal dysplasia in mice. Our data suggest that tapinarof may have different effects on varied types of psoriasis.

Activation of the Aryl Hydrocarbon Receptor in Muscle Exacerbates Ischemic Pathology in Chronic Kidney Disease.

Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD. AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHRmKO (skeletal muscle-specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD. PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes (Cyp1a1, Cyp1b1, and Aldh3a1) when compared with either muscle from the PAD condition with normal renal function (P<0.05 for all 3 genes) or nonischemic controls. AHRmKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions.

Ethyl Caffeate Can Inhibit Aryl Hydrocarbon Receptor (AhR) Signaling and AhR-Mediated Potentiation of Mast Cell Activation.

Ethyl caffeate (EC) is a natural phenolic compound that is present in several medicinal plants used to treat inflammatory disorders. However, its anti-inflammatory mechanisms are not fully understood. Here, we report that EC inhibits aryl hydrocarbon receptor (AhR) signaling and that this is associated with its anti-allergic activity. EC inhibited AhR activation, induced by the AhR ligands FICZ and DHNA in AhR signaling-reporter cells and mouse bone marrow-derived mast cells (BMMCs), as assessed by AhR target gene expressions such as CYP1A1. EC also inhibited the FICZ-induced downregulation of AhR expression and DHNA-induced IL-6 production in BMMCs. Furthermore, the pretreatment of mice with orally administered EC inhibited DHNA-induced CYP1A1 expression in the intestine. Notably, both EC and CH-223191, a well-established AhR antagonist, inhibited IgE-mediated degranulation in BMMCs grown in a cell culture medium containing significant amounts of AhR ligands. Furthermore, oral administration of EC or CH-223191 to mice inhibited the PCA reaction associated with the suppression of constitutive CYP1A1 expression within the skin. Collectively, EC inhibited AhR signaling and AhR-mediated potentiation of mast cell activation due to the intrinsic AhR activity in both the culture medium and normal mouse skin. Given the AhR control of inflammation, these findings suggest a novel mechanism for the anti-inflammatory activity of EC.

Induction of Aryl Hydrocarbon Receptor-Mediated Cancer Cell-Selective Apoptosis in Triple-Negative Breast Cancer Cells by a High-Affinity Benzimidazoisoquinoline.

Triple-negative breast cancer (TNBC) remains a disease with a paucity of targeted treatment opportunities. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in a wide range of physiological processes, including the sensing of xenobiotics, immune function, development, and differentiation. Different small-molecule AhR ligands drive strikingly varied cellular and organismal responses. In certain cancers, AhR activation by select small molecules induces cell cycle arrest or apoptosis via activation of tumor-suppressive transcriptional programs. AhR is expressed in triple-negative breast cancers, presenting a tractable therapeutic opportunity. Here, we identify a novel ligand of the aryl hydrocarbon receptor that potently and selectively induces cell death in triple-negative breast cancer cells and TNBC stem cells via the AhR. Importantly, we found that this compound, Analog 523, exhibits minimal cytotoxicity against multiple normal human primary cells. Analog 523 represents a high-affinity AhR ligand with potential for future clinical translation as an anticancer agent.

Complex chemical signals dictate Ah receptor activation through the gut-lung axis.

The aryl hydrocarbon receptor (AHR) mediates intestinal barrier homeostasis. Many AHR ligands are also CYP1A1/1B1 substrates, which can result in rapid clearance within the intestinal tract, limiting systemic exposure and subsequent AHR activation. This led us to the hypothesis that there are dietary substrates of CYP1A1/1B1 that functionally increase the half-life of potent AHR ligands. We examined the potential of urolithin A (UroA), a gut bacterial metabolite of ellagitannins, as a CYP1A1/1B1 substrate to enhance AHR activity in vivo. UroA is a competitive substrate for CYP1A1/1B1 in an in vitro competition assay. A broccoli-containing diet promotes the gastric formation of the potent hydrophobic AHR ligand and CYP1A1/1B1 substrate, 5,11-dihydroindolo[3,2-b]carbazole (ICZ). In mice, dietary exposure to UroA in a 10% broccoli diet led to a coordinated increase in duodenal, cardiac, and pulmonary AHR activity, but no increase in activity in the liver. Thus, CYP1A1 dietary competitive substrates can lead to enhanced systemic AHR ligand distribution from the gut, likely through the lymphatic system, increasing AHR activation in key barrier tissues. Finally, this report will lead to a reassessment of the dynamics of distribution of other hydrophobic chemicals present in the diet.

TCDD-Induced Allosteric Perturbation of the AhR:ARNT Binding to DNA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals, including halogenated aromatic hydrocarbons. In this work, we investigate the effects of the binding of the AhR prototypical ligand, TCDD, on the stability of the AhR:ARNT complex, as well as the mechanisms by which ligand-induced perturbations propagate to the DNA recognition site responsible for gene transcription. To this aim, a reliable structural model of the overall quaternary structure of the AhR:ARNT:DRE complex is proposed, based on homology modelling. The model shows very good agreement with a previous one and is supported by experimental evidence. Moreover, molecular dynamics simulations are performed to compare the dynamic behaviour of the AhR:ARNT heterodimer in the presence or absence of the TCDD. Analysis of the simulations, performed by an unsupervised machine learning method, shows that TCDD binding to the AhR PASB domain influences the stability of several inter-domain interactions, in particular at the PASA-PASB interface. The inter-domain communication network suggests a mechanism by which TCDD binding allosterically stabilizes the interactions at the DNA recognition site. These findings may have implications for the comprehension of the different toxic outcomes of AhR ligands and drug design.

A CRISPR-Cas9 mutation in sox9b long intergenic noncoding RNA (slincR) affects zebrafish development, behavior, and regeneration.

The role of long noncoding RNAs (lncRNAs) regulators of toxicological responses to environmental chemicals is gaining prominence. Previously, our laboratory discovered an lncRNA, sox9b long intergenic noncoding RNA (slincR), that is activated by multiple ligands of aryl hydrocarbon receptor (AHR). Within this study, we designed a CRISPR-Cas9-mediated slincR zebrafish mutant line to better understand its biological function in presence or absence of a model AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slincRosu3 line contains an 18 bp insertion within the slincR sequence that changes its predicted mRNA secondary structure. Toxicological profiling showed that slincRosu3 is equally or more sensitive to TCDD for morphological and behavioral phenotypes. Embryonic mRNA-sequencing showed differential responses of 499 or 908 genes in slincRosu3 in absence or presence of TCDD Specifically, unexposed slincRosu3 embryos showed disruptions in metabolic pathways, suggesting an endogenous role for slincR. slincRosu3 embryos also had repressed mRNA levels of sox9b-a transcription factor that slincR is known to negatively regulate. Hence, we studied cartilage development and regenerative capacity-both processes partially regulated by sox9b. Cartilage development was disrupted in slincRosu3 embryos both in presence and absence of TCDD. slincRosu3 embryos also displayed a lack of regenerative capacity of amputated tail fins, accompanied by a lack of cell proliferation. In summary, using a novel slincR mutant line, we show that a mutation in slincR can have widespread impacts on gene expression and structural development endogenously and limited, but significant impacts in presence of AHR induction that further highlights its importance in the developmental process.

Transcriptomic signatures reveal a shift towards an anti-inflammatory gene expression profile but also the induction of type I and type II interferon signaling networks through aryl hydrocarbon receptor activation in murine macrophages.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a broad range of target genes involved in the xenobiotic response, cell cycle control and circadian rhythm. AhR is constitutively expressed in macrophages (Mϕ), acting as key regulator of cytokine production. While proinflammatory cytokines, i.e., IL-1β, IL-6, IL-12, are suppressed through AhR activation, anti-inflammatory IL-10 is induced. However, the underlying mechanisms of those effects and the importance of the specific ligand structure are not yet completely understood. Therefore, we have compared the global gene expression pattern in activated murine bone marrow-derived macrophages (BMMs) subsequently to exposure with either benzo[a]pyrene (BaP) or indole-3-carbinol (I3C), representing high-affinity vs. low-affinity AhR ligands, respectively, by means of mRNA sequencing. AhR dependency of observed effects was proved using BMMs from AhR-knockout (Ahr-/-) mice. In total, more than 1,000 differentially expressed genes (DEGs) could be mapped, covering a plethora of AhR-modulated effects on basal cellular processes, i.e., transcription and translation, but also immune functions, i.e., antigen presentation, cytokine production, and phagocytosis. Among DEGs were genes that are already known to be regulated by AhR, i.e., Irf1, Ido2, and Cd84. However, we identified DEGs not yet described to be AhR-regulated in Mϕ so far, i.e., Slpi, Il12rb1, and Il21r. All six genes likely contribute to shifting the Mϕ phenotype from proinflammatory to anti-inflammatory. The majority of DEGs induced through BaP were not affected through I3C exposure, probably due to higher AhR affinity of BaP in comparison to I3C. Mapping of known aryl hydrocarbon response element (AHRE) sequence motifs in identified DEGs revealed more than 200 genes not possessing any AHRE, and therefore being not eligible for canonical regulation. Bioinformatic approaches modeled a central role of type I and type II interferons in the regulation of those genes. Additionally, RT-qPCR and ELISA confirmed a AhR-dependent expressional induction and AhR-dependent secretion of IFN-γ in response to BaP exposure, suggesting an auto- or paracrine activation pathway of Mϕ.