Christoph F.A. Vogel

Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to immune suppression associated with the induction of regulatory T cells (T(reg)) expressing the transcription factor Foxp3. The immunological mechanisms of suppression are not well understood however dendritic cells (DC) are considered a key target for AhR-mediated immune suppression. Here we show that activation of AhR by TCDD induces DC indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase-like protein (IDO2). Induction of IDO1 and IDO2 was also found in lung and spleen associated with an increase of the T(reg) marker Foxp3 in spleen of TCDD-treated C57BL/6 mice, which is suppressed by inhibition of IDO. These data indicate that AhR-activation is an important signaling pathway for IDO expression and suggest a critical role of IDO in the mechanism leading to the generation of T(reg) that mediates the immune suppression through activation of AhR.

Induction of Proinflammatory Cytokines and C-Reactive Protein in Human Macrophage Cell Line U937 Exposed to Air Pollution Particulates

Exposure to particulate matter air pollution causes inflammatory responses and is associated with the progression of atherosclerosis and increased cardiovascular mortality. Macrophages play a key role in atherogenesis by releasing proinflammatory cytokines and forming foam cells in subendothelial lesions. The present study quantified the inflammatory response in a human macrophage cell line (U937) after exposure to an ambient particulate sample from urban dust (UDP) and a diesel exhaust particulate (DEP). The effect of native UDP and DEP was compared with their corresponding organic extracts (OE-UDP/OE-DEP) and stripped particles (sUDP/sDEP) to clarify their respective roles. Exposure to OE-UDP, OE-DEP, UDP, DEP, and 2,3,7,8-tetrachlorodibenzo-p-dioxin led to a greater increase of interleukin (IL)-8, tumor necrosis factor-α, and cyclooxygenase-2 mRNA expression than did the stripped particles, whereas sUDP, sDEP, UDP, and DEP led to a greater production of C-reactive protein and IL-6 mRNA. The particles and the organic extract-induced expression of cyclooxygenase-2 and cytochrome P450 (CYP)1a1 was significantly suppressed by co-treatment with an aryl hydrocarbon receptor (AhR) antagonist, indicating that these effects are mainly mediated by the organic components, which can activate the AhR and CYP1a1. In contrast, the induction of C-reactive protein and IL-6 seems to be a particle-related effect that is AhR independent. The inflammatory response induced by particulate matter was associated with a subsequent increase of cholesterol accumulation, a hallmark of foam cells. Together, these data illustrate the interaction between particulate matter and the inflammatory response as well as the formation of cholesterol-accumulating foam cells, which are early markers of cardiovascular disease.

Cross-talk between Aryl Hydrocarbon Receptor and the Inflammatory Response A ROLE FOR NUCLEAR FACTOR-κB

Background: Aryl hydrocarbon receptor (AhR) is a protein regulating differentiation and function of immune cells. Results: NF-κB activates transcription of AhR and enhances activity of AhR-regulated genes. Conclusion: Activation of NF-κB involves RelA-mediated expression of AhR. Significance: Inflammatory stimuli and cytokines that regulate NF-κB induce AhR expression during activation and differentiation of immune cells. The aryl hydrocarbon receptor (AhR) is involved in the regulation of immune responses, T-cell differentiation, and immunity. Here, we show that inflammatory stimuli such as LPS induce the expression of AhR in human dendritic cells (DC) associated with an AhR-dependent increase of CYP1A1 (cytochrome P4501A1). In vivo data confirmed the elevated expression of AhR by LPS and the LPS-enhanced 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of CYP1A1 in thymus of B6 mice. Inhibition of nuclear factor-κB (NF-κB) repressed both normal and LPS-enhanced, TCDD-inducible, AhR-dependent gene expression and canonical pathway control of RelA-regulated AhR-responsive gene expression. LPS-mediated induction of AhR was NF-κB-dependent, as shown in mouse embryonic fibroblasts (MEFs) derived from Rel null mice. AhR expression and TCDD-mediated induction of CYP1A1 was significantly reduced in RelA-deficient MEF compared with wild type MEF cells and ectopic expression of RelA restored the expression of AhR and induction of CYP1A1 in MEF RelA null cells. Promoter analysis of the human AhR gene identified three putative NF-κB-binding elements upstream of the transcription start site. Mutation analysis of the AhR promoter identified one NF-κB site as responsible for mediating the induction of AhR expression by LPS and electrophoretic shift assays demonstrated that this NF-κB motif is recognized by the RelA/p50 heterodimer. Our results show for the first time that NF-κB RelA is a critical component regulating the expression of AhR and the induction of AhR-dependent gene expression in immune cells illustrating the interaction of AhR and NF-κB signaling.

Activation of Aryl Hydrocarbon Receptor Induces Vascular Inflammation and Promotes Atherosclerosis in Apolipoprotein E−/− Mice

Objective—Exposure to dioxins has been shown to contribute to the development of inflammatory diseases, such as atherosclerosis. Macrophage-mediated inflammation is a critical event in the initiation of atherosclerosis. Previously, we showed that treatment of macrophages with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to aryl hydrocarbon receptor (AhR)–dependent activation of inflammatory mediators and the formation of cholesterol-laden foam cells. However, the mechanisms responsible for the formation of atherosclerotic lesions mediated through AhR have not been identified. Methods and Results—An in vitro macrophage and an apolipoprotein E (ApoE)−/− mouse model were used to determine whether chemokines and their receptors are responsible for the AhR-mediated atherogenesis. Exposure of ApoE−/− mice to TCDD caused a time-dependent progression of atherosclerosis, which was associated with induction of inflammatory genes, including interleukin-8, as well as F4/80 and matrix metalloproteinase-12. A high-fat diet enhanced the TCDD-mediated inflammatory response and aggravated the formation of complex atheromas. Treatment with a CXCR2 inhibitor and an AhR antagonist reduced the TCDD-induced progression of early atherosclerotic lesions in ApoE−/− mice. Conclusion—The results suggest that CXCR2 mediates the atherogenic activity of environmental pollutants, such as dioxins, and contributes to the development of atherosclerosis through the induction of a vascular inflammatory response by activating the AhR-signaling pathway.

Dioxin Increases C/EBPβ Transcription by Activating cAMP/Protein Kinase A

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD = dioxin) has been shown to increase the expression of C/EBPβ. The modulated expression of C/EBPβ has been suggested to be associated with toxic responses of TCDD such as wasting syndrome, diabetes, and inhibition of adipocyte differentiation. This study focused on the regulatory mechanism of TCDD-mediated transcriptional activation of C/EBPβ. Elevated C/EBPβ mRNA and protein levels in mouse embryonic fibroblasts (C3H10T½) and in mouse hepatoma cells (Hepa1c1c7) were correlated with increased binding affinity of the C/EBPβ protein. Transfection studies with different deletion constructs of the CCAAT/enhancer-binding protein promoter indicated that a small region located 60–120 bp upstream of the start site of transcription is required for activation of the C/EBPβ gene by TCDD in both cell lines tested. Further analysis using mutation constructs of the C/EBPβ promoter demonstrated that activation of the C/EBPβ promoter is mediated through incomplete cAMP-response element-binding protein (CREB) sites located close to the TATA box of the C/EBPβ gene. The protein kinase A (PKA) inhibitor H89 completely blocks the TCDD-dependent effect on C/EBPβ promoter activity, indicating that TCDD activates CREB binding via a cAMP/PKA pathway, which is supported by the increased cAMP level and PKA activity observed after TCDD treatment. Gel shift analyses demonstrated that CREB itself binds to the putative CREB motif that mediates the TCDD-dependent effect on C/EBPβ gene transcription. Cotransfection experiments with CREB and PKA expression plasmids further supported our conclusions that the TCDD-dependent effect on C/EBPβ transcription is mediated via PKA-dependent CREB activation.

Activation of inflammatory mediators and potential role of ah-receptor ligands in foam cell formation.

Epidemiological data and in vivo animal experiments have indicated that exposure to the Ah-receptor (AhR) ligand dioxin and other dioxin-like compounds can lead to cardiovascular toxicity and atherosclerosis. Here, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR ligand, on the differentiation of U937 cells into foam cells, which are considered to be early lesions of atherosclerosis. Our findings show that, like oxidized low-density lipoprotein (oxLDL), TCDD promotes the differentiation of U937 macrophages to atherogenic foam cells, verified by lipid accumulation and extensive formation of blebs on the cell surface, which are characteristics of foam cells. Through screening expression patterns of typical genes involved in atherosclerosis and foam cell generation, we could demonstrate that mRNA levels of cyclooxygenase-2, interleukin 1 β, and tumor necrosis factor-α were increased in a time- and dose-dependent manner in U937 macrophages treated with TCDD, like oxLDL, and that these changes accompanied significantly elevated levels of matrix-degrading metalloproteinases (MMP)-1, MMP-3, MMP-12, and MMP-13. Increased levels of MMPs were associated with TCDD-stimulated cell migration of U937 macrophages. These findings clearly indicate that AhR ligands, like TCDD, stimulate differentiation of U937 macrophages into potentially plaque-forming foam cells.

Interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with induced adipocyte differentiation in mouse embryonic fibroblasts (MEFs) involves tyrosine kinase c-Src

Adipocyte differentiation of mouse embryonic fibroblasts (MEFs) derived from c-Src wild-type or c-Src-deficient (abbreviated as MEF+/+ and MEF-/- hereafter) C57BL/6 mice was induced by ascorbic acid (A) and beta-glycerophosphate (G). TCDD clearly suppressed differentiation of MEF+/+, but not MEF-/-, as measured by increased accumulation of triglycerides associated with increased expression of adipocyte differentiation-specific genes such as peroxisome proliferators activated receptor (PPAR)gamma, stearoyl-CoA desaturase (SCD-1). Studies on inducibility of TCDD-activated genes such as cytochrome P450 (CYP)1A1 and CYP1B1 revealed a comparable dose response in both MEF+/+ and MEF-/-. Furthermore, the binding activity of AhR complexes to xenobiotic response elements (XREs) was similar in both cell lines. We further studied the effect of TCDD on CCAAT/enhancer binding proteins (C/EBP), which are known to be important regulators of cell differentiation. TCDD induced C/EBPbeta and C/EBPdelta mRNA expression and DNA binding activity in a time- and dose-dependent manner in MEF+/+ but not in MEF-/-. The levels of C/EBPbeta and C/EBPdelta were still elevated in differentiated MEF+/+ after 10 days of treatment with TCDD. In MEF-/-, C/EBPbeta and C/EBPdelta are highly expressed constitutively. In contrast to MEF+/+, TCDD does not cause any significant change of these transcription factors in MEF-/-. These data indicate that suppression of differentiation by TCDD in MEF requires a functional c-Src activity and induced levels of C/EBPbeta and C/EBPdelta, including their maintenance at high levels by TCDD, rather than ultimate high levels of these C/EBP isoforms.

Aryl hydrocarbon receptor signaling regulates NF-κB RelB activation during dendritic-cell differentiation.

How the aryl hydrocarbon receptor (AhR) regulates dendritic‐cell (DC) differentiation is unknown. We show that activation of AhR by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) caused enhanced differentiation from immature DCs (IDCs) to mature DCs (MDCs) in the bone‐marrow‐derived DCs (BMDC) from B6 wild‐type mice but not in the BMDCs from AhR‐null mice as indicated by the expression of CD11c and class II major histocompatibility complex (MHC). Enhanced maturation of BMDCs was associated with elevated levels of CD86 and an increased AhR‐dependent nuclear accumulation of nuclear factor‐kappa‐light‐chain enhancer of activated B cell (NF‐κB) member RelB in BMDCs. The expression of interleukin (IL) 10 and chemokine DC‐CK1 was suppressed, whereas that of CXCL2, CXCL3 and IL‐22 was significantly increased in AhR‐activated BMDCs. Furthermore, TCDD induced expression of the regulatory enzymes indoleamine 2,3‐dioxygenase (IDO1) and indoleamine 2,3‐dioxygenase‐like 1 (IDO2). Increased expression of IDO2 was associated with coexpression of the cell‐surface marker CCR6. Interestingly, mRNA expression of the chemokine receptor CCR6 was drastically decreased in AhR‐null IDCs and MDCs. Overall, these data demonstrate that AhR modifies the maturation of BMDCs associated with the induction of the regulatory enzyme IDO and altered expression of cytokine, chemokines and DC‐specific surface markers and receptors.

AhR deficiency impairs expression of LPS-induced inflammatory genes in mice

Recent reports suggest the participation of the aryl hydrocarbon receptor (AhR) in the induction mechanism of the NF-κB signaling pathway. In the current study we challenged C57BL/6 wild-type (WT) and AhR deficient (AhR(-/-)) mice with bacterial lipopolysaccharide (LPS) to investigate the role of the AhR in expression profiles of LPS and NF-κB target genes. Further, we analyzed the effect of LPS on the DNA binding activity of NF-κB, C/EBP and AP-1 transcription factors in liver and lung from WT and AhR(-/-) mice. The results show that the LPS-induced expression of several target genes was impaired in AhR(-/-) mice compared to WT mice. Depending on the target gene, the target tissue as well as the time of treatment, the deficiency of AhR may cause an inhibition or increase of the LPS-induced gene expression. The binding activity of NF-κB, C/EBP and AP-1 transcription factors was also affected in a time- and tissue-dependent manner. The current study shows that the AhR is implemented in LPS-induced inflammatory gene expression in vivo even in the absence of exogenous ligands of the AhR. The main implication of this finding is that the AhR functions in Toll-like receptor (TLR) and NF-κB signaling after activation by a classical stimulus, such as LPS.

Effects of src-deficiency on the expression of in vivo toxicity of TCDD in a strain of c-src knockout mice procured through six generations of backcrossings to C57BL/6 mice.

The effect of TCDD was studied in c-src-deficient C57BL6-src(tm1sor) (N6 src -/- and -/+) mice, and their wild-type littermate mice (N6 src +/+). The former was created from the original strain of B6, 129-src(tm1sor) mice through six generations of backcrossings with C57BL6 mice. The results of a high dose TCDD toxicity tests in male mice indicated that N6 src-/+ mice were significantly less responsive to the toxic action of TCDD (115 microg/kg single i.p. injection) than N6 src+/+ mice in terms of reduced % body weight gain, the increase in the liver to body weight ratio, and the decrease in the adipose tissue to liver weight ratio and in the weight of pancreas. To understand the cause for these differential effects of TCDD we studied TCDD-induced changes in several biochemical parameters at day 10 and found that most drastically affected ones were glycogen depletion and phosphoenolpyruvate carboxykinase (PEPCK) downregulation. In addition, the degree of triglyceride accumulation in liver was less pronounced in N6-/+ than in N6+/+ mice. These findings suggest that the absence of c-src expression indeed affects the development of selected, TCDD-induced toxic endpoints that are related to wasting syndrome.