AHR Signaling Interacting with Nutritional Factors Regulating the Expression of Markers in Vascular Inflammation and Atherogenesis.

There is strong evidence that exposure to fine particulate matter (PM2.5) and a high-fat diet (HFD) increase the risk of mortality from atherosclerotic cardiovascular diseases. Recent studies indicate that PM2.5 generated by combustion activates the Aryl Hydrocarbon Receptor (AHR) and inflammatory cytokines contributing to PM2.5-mediated atherogenesis. Here we investigate the effects of components of a HFD on PM-mediated activation of AHR in macrophages. Cells were treated with components of a HFD and AHR-activating PM and the expression of biomarkers of vascular inflammation was analyzed. The results show that glucose and triglyceride increase AHR-activity and PM2.5-mediated induction of cytochrome P450 (CYP)1A1 mRNA in macrophages. Cholesterol, fructose, and palmitic acid increased the PM- and AHR-mediated induction of proinflammatory cytokines in macrophages. Treatment with palmitic acid significantly increased the expression of inflammatory cytokines and markers of vascular injury in human aortic endothelial cells (HAEC) after treatment with PM2.5. The PM2.5-mediated activation of the atherogenic markers C-reactive protein (CRP) and S100A9, a damage-associated molecular pattern molecule, was found to be AHR-dependent and involved protein kinase A (PKA) and a CCAAT/enhancer-binding protein (C/EBP) binding element. This study identified nutritional factors interacting with AHR signaling and contributing to PM2.5-induced markers of atherogenesis and future cardiovascular risk.

Role of NF-kB RelB in Aryl Hydrocarbon Receptor-Mediated Ligand Specific Effects.

Here, we investigate the role of RelB in the regulation of genes which were identified to be induced in an aryl hydrocarbon receptor (AhR)-dependent manner and critically involved in regulation of immune responses. We analyzed the expression of genes of the AhR gene battery, cytokines, and immune regulatory enzymes in bone marrow-derived macrophages (BMM) and thymus of B6 wildtype (wt) mice and RelB knockout (RelB-/-) mice after treatment with various AhR ligands. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced expression of indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2 was significantly repressed in thymus of RelB-/- mice but not in BMM derived from RelB-/- mice. Interestingly, the induced and basal expression of the cytokines interleukin (IL)-17A, IL-22, and CCL20 required the functional expression of RelB. The RelB-dependent expression of CCL20 was induced by the AhR ligands TCDD and 6-formylindolo[3,2-b]carbazole (FICZ), whereas indole-3-carbinol (I3C) suppressed CCL20 in lipopolysaccharide (LPS)-activated wt BMM. The LPS-induced expression of IL-6 and IL-10 was enhanced by TCDD and FICZ, whereas I3C significantly suppressed these cytokines in BMM. The exposure to FICZ led to higher increases of IL-17A and IL-22 mRNA compared to the effect of TCDD or I3C in thymus of wt mice. On the other hand, TCDD was the strongest inducer of CYP1A1, AhR Repressor (AhRR), and IDO2. In summary, these findings provide evidence for the important role of RelB in the transcriptional regulation of cytokines and enzymes induced by AhR ligands.

Aryl hydrocarbon receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells.

Currently, it is not well understood how ligands of the aryl hydrocarbon receptor (AhR) modify inflammatory responses triggered by Toll-like receptor (TLR) agonists in human dendritic cells (DCs). Here, we show that AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the tryptophan derivatives 6-formylindolo[3,2-b] carbazole (FICZ), kynurenine (kyn), and the natural dietary compound indole-3-carbinol (I3C) differentially modify cytokine expression in human monocyte-derived DCs (MoDCs). The results show that TLR-activated MoDCs express higher levels of AhR and are more sensitive toward the effects of AhR ligands. Depending on the cytokine, treatment with AhR ligands led to a synergistic or antagonistic effect of the TLR-triggered response in MoDCs. Thus, activation of AhR increased the expression of interleukin (IL)-1ß, but decreased the expression of IL-12A in TLR-activated MoDCs. Furthermore, TCDD and FICZ may have opposite effects on the expression of cytochrome P4501A1 (CYP1A1) in TLR8-activated MoDCs indicating that the effect of the specific AhR ligand may depend on the presence of the specific TLR agonist. Gene silencing showed that synergistic effects of AhR ligands on TLR-induced expression of IL-1ß require a functional AhR and the expression of NF-κB RelB. On the other hand, repression of IL-12A by TCDD and FICZ involved the induction of the caudal type homeobox 2 (CDX2) transcription factor. Additionally, the levels of DC surface markers were decreased in MoDCs by TCDD, FICZ and I3C, but not by kyn. Overall, these data demonstrate that AhR modulates TLR-induced expression of cytokines and DC-specific surface markers in MoDCs involving NFκB RelB and the immune regulatory factor CDX2.

Activation of the aryl hydrocarbon receptor by the widely used Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2).

Small molecular weight protein kinase inhibitors are frequently used tools to unravel the complex network of cellular signal transduction under certain physiological and pathophysiological conditions. 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) is a widely used compound to block the activity of Src family kinases, the major group of non-receptor tyrosine kinases, which trigger multiple cellular signaling pathways. Here, we show that PP2 induces cytochrome P450 1A1 mRNA expression and enzyme activity in a dose-dependent manner in human HepG2 hepatoma cells and NCTC 2544 keratinocytes. By means of reporter gene assays, RNA interference, electrophoretic mobility shift assay, and competitive ligand-binding assay, we further demonstrate that PP2 is a ligand for the aryl hydrocarbon receptor (AHR), an intracellular chemosensor that regulates xenobiotic metabolism, environmental stress responses, and immune functions. Upon ligand-dependent activation, the AHR translocates into the nucleus and dimerizes with the AHR nuclear translocator (ARNT) to modulate the expression of its target genes. In addition, AHR activation is frequently accompanied by an activation of the tyrosine kinase c-Src, resulting in stimulation of cell-surface receptors and downstream signal transduction. As PP2 activates the AHR/ARNT pathway by simultaneously blocking c-Src-mediated alternative signaling routes, this compound may be a suitable tool to study the contribution of the different AHR-dependent signaling pathways to biological processes and adverse outcomes. On the other hand, the unexpected property of PP2 to stimulate AHR/ARNT signaling should be carefully taken into account in future investigations in order to avoid a false interpretation of experimental results and molecular interrelations.

Combustion-derived flame generated ultrafine soot generates reactive oxygen species and activates Nrf2 antioxidants differently in neonatal and adult rat lungs.

BACKGROUND: Urban particulate matter (PM) has been epidemiologically correlated with multiple cardiopulmonary morbidities and mortalities, in sensitive populations. Children exposed to PM are more likely to develop respiratory infections and asthma. Although PM originates from natural and anthropogenic sources, vehicle exhaust rich in polycyclic aromatic hydrocarbons (PAH) can be a dominant contributor to the PM2.5 and PM0.1 fractions and has been implicated in the generation of reactive oxygen species (ROS). OBJECTIVES: Current studies of ambient PM are confounded by the variable nature of PM, so we utilized a previously characterized ethylene-combusted premixed flame particles (PFP) with consistent and reproducible physiochemical properties and 1) measured the oxidative potential of PFP compared to ambient PM, 2) determined the ability of PFPs to generate oxidative stress and activate the transcription factor using in vitro and ex vivo models, and 3) we correlated these responses with antioxidant enzyme expression in vivo. METHODS: We compared oxidative stress response (HMOX1) and antioxidant enzyme (SOD1, SOD2, CAT, and PRDX6) expression in vivo by performing a time-course study in 7-day old neonatal and young adult rats exposed to a single 6-hour exposure to 22.4 µg/m3 PFPs. RESULTS: We showed that PFP is a potent ROS generator that induces oxidative stress and activates Nrf2. Induction of the oxidative stress responsive enzyme HMOX1 in vitro was mediated through Nrf2 activation and was variably upregulated in both ages. Furthermore, antioxidant enzyme expression had age and lung compartment variations post exposure. Of particular interest was SOD1, which had mRNA and protein upregulation in adult parenchyma, but lacked a similar response in neonates. CONCLUSIONS: We conclude that PFPs are effective ROS generators, comparable to urban ambient PM2.5, that induce oxidative stress in neonatal and adult rat lungs. PFPs upregulate a select set of antioxidant enzymes in young adult animals, that are unaffected in neonates. We conclude that the inability of neonatal animals to upregulate the antioxidant response may, in part, explain enhanced their susceptibility to ultrafine particles, such as PFP.

Regulation of IDO2 by the Aryl Hydrocarbon Receptor (AhR) in Breast Cancer.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a tryptophan-catabolizing enzyme and a homolog of IDO1 with a distinct expression pattern compared with IDO1. In dendritic cells (DCs), IDO activity and the resulting changes in tryptophan level regulate T-cell differentiation and promote immune tolerance. Recent studies indicate that IDO2 exerts an additional, non-enzymatic function and pro-inflammatory activity, which may play an important role in diseases such as autoimmunity and cancer. Here, we investigated the impact of aryl hydrocarbon receptor (AhR) activation by endogenous compounds and environmental pollutants on the expression of IDO2. Treatment with AhR ligands induced IDO2 in MCF-7 wildtype cells but not in CRISPR-cas9 AhR-knockout MCF-7 cells. Promoter analysis with IDO2 reporter constructs revealed that the AhR-dependent induction of IDO2 involves a short-tandem repeat containing four core sequences of a xenobiotic response element (XRE) upstream of the start site of the human ido2 gene. The analysis of breast cancer datasets revealed that IDO2 expression increased in breast cancer compared with normal samples. Our findings suggest that the AhR-mediated expression of IDO2 in breast cancer could contribute to a pro-tumorigenic microenvironment in breast cancer.

Aryl Hydrocarbon Receptor Signaling Synergizes with TLR/NF-κB-Signaling for Induction of IL-22 Through Canonical and Non-Canonical AhR Pathways.

Interleukin 22 (IL-22) is critically involved in gut immunity and host defense and primarily produced by activated T cells. In different circumstances IL-22 may contribute to pathological conditions or act as a cancer promoting cytokine secreted by infiltrating immune cells. Here we show that bone marrow-derived macrophages (BMM) express and produce IL-22 after activation of the aryl hydrocarbon receptor (AhR) when cells are activated through the Toll-like receptor (TLR) family. The additional activation of AhR triggered a significant induction of IL-22 in TLR-activated BMM. Deletion and mutation constructs of the IL-22 promoter revealed that a consensus DRE and RelBAhRE binding element are necessary to mediate the synergistic effects of AhR and TLR ligands. Inhibitor studies and analysis of BMM derived from knockout mice confirmed that the synergistic induction of IL-22 by AhR and TLR ligands depend on the expression of AhR and Nuclear Factor-kappa B (NF-κB) member RelB. The exposure to particulate matter (PM) collected from traffic related air pollution (TRAP) and wildfires activated AhR as well as NF-κB signaling and significantly induced the expression of IL-22. In summary this study shows that simultaneous activation of the AhR and NF-κB signaling pathways leads to synergistic and prolonged induction of IL-22 by integrating signals of the canonical and non-canonical AhR pathway.

Targeting the Aryl Hydrocarbon Receptor Signaling Pathway in Breast Cancer Development.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.

Inflammatory marker and aryl hydrocarbon receptor-dependent responses in human macrophages exposed to emissions from biodiesel fuels.

Biodiesel or renewable diesel fuels are alternative fuels produced from vegetable oil and animal tallow that are being considered to help reduce the use of petroleum-based fuels and emissions of air pollutants including greenhouse gases. Here, we analyzed the gene expression of inflammatory marker responses and the cytochrome P450 1A1 (CYP1A1) enzyme after exposure to diesel and biodiesel emission samples generated from an in-use heavy-duty diesel vehicle. Particulate emission samples from petroleum-based California Air Resource Board (CARB)-certified ultralow sulfur diesel (CARB ULSD), biodiesel, and renewable hydro-treated diesel all induced inflammatory markers such as cyclooxygenase-2 (COX)-2 and interleukin (IL)-8 in human U937-derived macrophages and the expression of the xenobiotic metabolizing enzyme CYP1A1. Furthermore, the results indicate that the particle emissions from CARB ULSD and the alternative diesel fuel blends activate the aryl hydrocarbon receptor (AhR) and induce CYP1A1 in a dose- and AhR-dependent manner which was supported by the AhR luciferase reporter assay and gel shift analysis. Based on a per mile emissions with the model year 2000 heavy duty vehicle tested, the effects of the alternative diesel fuel blends emissions on the expression on inflammatory markers like IL-8 and COX-2 tend to be lower than emission samples derived from CARB ULSD fuel. The results will help to assess the potential benefits and toxicity from biofuel use as alternative fuels in modern technology diesel engines.

A Protective Role of Aryl Hydrocarbon Receptor Repressor in Inflammation and Tumor Growth.

The aryl hydrocarbon receptor (AhR) is known for mediating the toxicity of environmental pollutants such as dioxins and numerous dioxin-like compounds, and is associated with the promotion of various malignancies, including lymphoma. The aryl hydrocarbon receptor repressor (AhRR), a ligand-independent, transcriptionally inactive AhR-like protein is known to repress AhR signaling through its ability to compete with the AhR for dimerization with the AhR nuclear translocator (ARNT). While AhRR effectively blocks AhR signaling, several aspects of the mechanism of AhRR's functions are poorly understood, including suppression of inflammatory responses and its putative role as a tumor suppressor. In a transgenic mouse that overexpresses AhRR (AhRR Tg) we discovered that these mice suppress 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)- and inflammation-induced tumor growth after subcutaneous challenge of EL4 lymphoma cells. Using mouse embryonic fibroblasts (MEF) we found that AhRR overexpression suppresses the AhR-mediated anti-apoptotic response. The AhRR-mediated inhibition of apoptotic resistance was associated with a suppressed expression of interleukin (IL)-1ß and cyclooxygenase (COX)-2, which was dependent on activation of protein kinase A (PKA) and the CAAT-enhancer-binding protein beta (C/EBPß). These results provide mechanistic insights into the role of the AhRR to suppress inflammation and highlight the AhRR as a potential therapeutic target to suppress tumor growth.

Transgenic Overexpression of Aryl Hydrocarbon Receptor Repressor (AhRR) and AhR-Mediated Induction of CYP1A1, Cytokines, and Acute Toxicity.

BACKGROUND: The aryl hydrocarbon receptor repressor (AhRR) is known to repress aryl hydrocarbon receptor (AhR) signaling, but very little is known regarding the role of the AhRR in vivo. OBJECTIVE: This study tested the role of AhRR in vivo in AhRR overexpressing mice on molecular and toxic end points mediated through a prototypical AhR ligand. METHODS: We generated AhRR-transgenic mice (AhRR Tg) based on the genetic background of C57BL/6J wild type (wt) mice. We tested the effect of the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of cytochrome P450 (CYP)1A1 and cytokines in various tissues of mice. We next analyzed the infiltration of immune cells in adipose tissue of mice after treatment with TCDD using flow cytometry. RESULTS: AhRR Tg mice express significantly higher levels of AhRR compared to wt mice. Activation of AhR by TCDD caused a significant increase of the inflammatory cytokines Interleukin (IL)-1ß, IL-6 and IL-10, and CXCL chemokines in white epididymal adipose tissue from both wt and AhRR Tg mice. However, the expression of IL-1ß, CXCL2 and CXCL3 were significantly lower in AhRR Tg versus wt mice following TCDD treatment. Exposure to TCDD caused a rapid accumulation of neutrophils and macrophages in white adipose tissue of wt and AhRR Tg mice. Furthermore we found that male AhRR Tg mice were protected from high-dose TCDD-induced lethality associated with a reduced inflammatory response and liver damage as indicated by lower levels of TCDD-induced alanine aminotransferase and hepatic triglycerides. Females from both wt and AhRR Tg mice were less sensitive than male mice to acute toxicity induced by TCDD. CONCLUSION: In conclusion, the current study identifies AhRR as a previously uncharacterized regulator of specific inflammatory cytokines, which may protect from acute toxicity induced by TCDD. CITATION: Vogel CF, Chang WL, Kado S, McCulloh K, Vogel H, Wu D, Haarmann-Stemmann T, Yang GX, Leung PS, Matsumura F, Gershwin ME. 2016. Transgenic overexpression of aryl hydrocarbon receptor repressor (AhRR) and AhR-mediated induction of CYP1A1, cytokines, and acute toxicity. Environ Health Perspect 124:1071-1083; http://dx.doi.org/10.1289/ehp.1510194.