Aryl Hydrocarbon Receptor Stimulation Research Articles

TCDD and CH223191 Alter T Cell Balance but Fail to Induce Anti-Inflammatory Response in Adult Lupus Mice.

Aryl hydrocarbon receptor (AhR) responds to endogenous and exogenous ligands as a cytosolic receptor, transcription factor, and E3 ubiquitin ligase. Several studies support an anti-inflammatory effect of AhR activation. However, exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early stages of development results in an autoimmune phenotype and exacerbates lupus. The effects of TCDD on lupus in adults with pre-existing autoimmunity have not been described. We present novel evidence that AhR stimulation by TCDD alters T cell responses but fails to impact lupus-like disease using an adult mouse model. Interestingly, AhR antagonist CH223191 also changed T cell balance in our model. We next developed a conceptual framework for identifying cellular and molecular factors that contribute to physiological outcomes in lupus and created models that describe cytokine dynamics that were fed into a system of differential equations to predict the kinetics of T follicular helper (Tfh) and regulatory T (Treg) cell populations. The model predicted that Tfh cells expanded to larger values following TCDD exposure compared with vehicle and CH223191. Following the initial elevation, both Tfh and Treg cell populations continuously decayed over time. A function based on the ratio of predicted Treg/Tfh cells showed that Treg cells exceed Tfh cells in all groups, with TCDD and CH223191 showing lower Treg/Tfh cell ratios than the vehicle and that the ratio is relatively constant over time. We conclude that AhR ligands did not induce an anti-inflammatory response to attenuate autoimmunity in adult lupus mice. This study challenges the dogma that TCDD supports an immunosuppressive phenotype.

Involvement of sirtuins (Sirt1 and Sirt3) and aryl hydrocarbon receptor (AhR) in the effects of triclosan (TCS) on production of neurosteroids in primary mouse cortical neurons cultures

Epidemiological studies have shown the presence of triclosan (TCS) in the brain due to its widespread use as an antibacterial ingredient. One of the confirmed mechanisms of its action is the interaction with the aryl hydrocarbon receptor (AhR). In nerve cells, sirtuins (Sirt1 and Sirt3) act as cellular sensors detecting energy availability and modulate metabolic processes. Moreover, it has been found that Sirt1 inhibits the activation of estrogen receptors, regulates the androgen receptor, and may interact with the AhR receptor. It is also known that Sirt3 stimulates the production of estradiol (E2) via the estradiol receptor β (Erβ). Therefore, the aim of the present study was to evaluate the effect of TCS alone or in combination with synthetic flavonoids on the production of neurosteroids such as progesterone (P4), testosterone (T), and E2 in primary neural cortical neurons in vitro. The contribution of Sirt1 and Sirt3 as well as AhR to these TCS-induced effects was investigated as well. The results of the experiments showed that both short and long exposure of neurons to TCS increased the expression of the Sirt1 and Sirt3 proteins in response to AhR stimulation. After an initial increase in the production of all tested neurosteroids, TCS acting for a longer time lowered their levels in the cells. This suggests that TCS activating AhR as well as Sirt1 and Sirt3 in short time intervals stimulates the levels of P4, T, and E2 in neurons, and then the amount of neurosteroids decreases despite the activation of AhR and the increase in the expression of the Sirt1 and Sirt3 proteins. The use of both the AhR agonist and antagonist prevented changes in the expression of Sirt1, Sirt3, and AhR and the production of P4, T, and E2, which confirmed that this receptor is a key in the mechanism of the TCS action.

Aryl hydrocarbon receptor (Ahr)-dependent Il-22 expression by type 3 innate lymphoid cells control of acute joint inflammation.

The aryl hydrocarbon receptor (AHR) controls several inflammatory and metabolic pathways involved in various diseases, including the development of arthritis. Here, we investigated the role of AHR activation in IL‐22‐dependent acute arthritis using the K/BxN serum transfer model. We observed an overall reduction of cytokine expression in Ahr‐deficient mice, along with decreased signs of joint inflammation. Conversely, we report worsened arthritis symptoms in Il‐22 deficient mice. Pharmacological stimulation of AHR with the agonist VAG539, as well as injection of recombinant IL‐22, given prior arthritogenic triggering, attenuated inflammation and reduced joint destruction. The protective effect of VAG539 was abrogated in Il‐22 deficient mice. Finally, conditional Ahr depletion of Rorc‐expressing cells was sufficient to attenuate arthritis, thereby uncovering a previously unsuspected role of AHR in type 3 innate lymphoid cells during acute arthritis.

Abstract 3255: A novel small molecule inhibitor of AhR suppresses the polarization and activity of M2 macrophages

Abstract Tumor associated macrophages, mainly represented by the M2-like phenotype, are a highly immunosuppressive macrophage subset that exert a pro-tumorigenic role in a variety of cancer types. Understanding the factors that promote M2 macrophage differentiation and activity is of critical importance to unlocking the potential of anti-tumor immune therapy. Thus, we investigated the role of the AhR pathway in M2 macrophages. We found the Aryl Hydrocarbon Receptor (AhR) highly expressed in in vitro polarized human M2 macrophages derived from peripheral blood mononuclear cells. Stimulation of AhR with small molecule agonists TCDD or kynurenine resulted in a significant enhancement of the suppressive activity of M2 macrophages on anti-CD3/CD28 stimulated CD4 T cell proliferation and IFNγ production. Inhibition of AhR using a novel, highly potent and selective AhR inhibitor (IDE-AhRi-1) fully inhibited the suppressive activity of M2 macrophages on T cells. Surprisingly, stimulated CD4 T cells co-cultured with IDE-AhRi-1 exposed M2 macrophages had an enhanced ability to proliferate and produce IFNγ compared with stimulated T cells in the absence of M2 macrophages. This suggests that IDE-AhRi-1 not only inhibits the suppressive mechanisms of M2 macrophages, but also boosts their ability to generate pro-inflammatory responses that enhance T-cell activity. Further studies to elucidate the AhR-driven molecular mechanisms responsible for this activity are underway. To find out whether AhR driven M2 polarization and activity is evident in human cancer, we analyzed TCGA-derived RNA sequencing data from an array of solid tumor indications. In a subset of cancer indications, high AhR activity correlated with key M2 macrophage and immuno-regulatory signature genes. Surprisingly, expression of CYP1B1 also correlated with expression of TDO2 mRNA, but not IDO1 mRNA. These finding establish AhR as a key modulator of M2 macrophages and demonstrate a correlation between AhR activity and tumor associated macrophages in human tumor samples. Furthermore, we demonstrate that IDE-AhRi-1 can potently suppress M2 macrophages and propose IDE-AhRi-1 as a novel approach for potentiating anti-tumor immunity. Citation Format: Candy Garcia, Hadia Lemar, Christina Galang, James Joseph, Marcos Gonzalez-Lopez, Jeffrey Hager, Michael P. Dillon, Fred J. Aswad. A novel small molecule inhibitor of AhR suppresses the polarization and activity of M2 macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3255.

Aryl Hydrocarbon Receptor (AHR) Antagonism As a Transformative, Dual-Mechanism Novel Therapy for Multiple Myeloma

BACKGROUNDThe aryl hydrocarbon receptor (AHR) is a transcription factor that regulates xenobiotic-metabolizing enzymes in response to exogenous polycyclic aromatic hydrocarbons. However, we have described a critical role for AHR in normal lymphocyte development and identified the functional expression of AHR in multiple myeloma (MM). Interest is growing in AHR as a novel anti-cancer therapeutic target in solid tumors, but very little comparatively is known about AHR in hematologic malignancies. Epidemiologic data suggest that exposure to the AHR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzene and dioxin may be linked to increased rates of MGUS and MM in Vietnam-era veterans as well as firefighters at the World Trade Center attacks in September, 2001. Whereas we have shown that AHR antagonism facilitates normal, healthy natural killer (NK) cell lymphocyte development and acquisition of cytolytic potential, herein, we show that AHR antagonism impairs MM cell viability. In addition, we characterize the direct effects of AHR antagonism on MM cells as well as complementary mechanisms by which the NK cell versus MM effect is augmented, further strengthening ongoing interest in targeting AHR as a novel anti-MM therapy. METHODSThe MM cell lines MM.1S, MM.1R, OPM-2, and U266 were obtained from ATCC. Primary cells from healthy donors and patients with MM were procured under IRB-approved protocols. AHR expression was studied by Western blot, flow cytometry and real time PCR. AHR function was assessed by expression of the downstream target CYP1A1 by real time PCR. Clinical MM outcomes were determined with data obtained from a publically available database. Cell proliferation and viability were studied by light microscopy (trypan blue exclusion) and Sytox. Immunophenotyping was conducted by flow cytometry. Cytotoxicity was measured by 51Cr release assay. The AHR antagonists CH233191 and CB7993113 and the high affinity AHR agonist 5,11-dihydroindolol[3,2-b]carbazole-6-carboxaldehyde (FICZ) were utilized to study the in vitro effects of AHR modulation. RESULTSAHR transcript and protein are expressed in human NK cells, plasma cells and B cells, as well as MM cell lines and primary MM samples. AHR agonism increases transcription of CYP1A1 in MM cells whereas AHR antagonism suppresses CYP1A1; these observations confirm that AHR is functionally active in MM. AHR is abundantly expressed from the CD38brightCD27(+) MM stem cell compartment to primary plasma cell leukemia samples. MM cases with high AHR expression are associated with inferior survival as compared to MM cases with low AHR expression (median OS = 50 months versus not reached at 70 months, p = 0.00002). AHR stimulation reduces expression of CD38, CD56, and CD138 and upregulates CD10, CD11a, CD13, CD19, CD20, CD27, CD40, CD45, and CD117 consistent with MM clonogenic progenitor cells previously described as “stem-like” and drug resistant. AHR antagonism suppresses MM cell line and primary MM cell viability, including cytogenetically high-risk, plasma cell leukemia primary samples. These effects are preserved in 7- and 14-day washout experiments. AHR antagonism increases expression of CD38 (2.1-fold) and SLAMF7 (CS1, 1.54-fold) among other antigens. AHR antagonism enhances NK cell cytolytic potential by promoting expression of CD94, TRAIL, perforin and granzyme B, whereas in MM cells, AHR antagonism upregulates surface expression of NK cell activating ligands: CD112, CD54, ULBP1, ULBP2, ULBP3, ULBP4, CD155, MICA/B, and DR4 and DR5. Pretreatment of MM cell targets with an AHR antagonist enhances susceptibility to NK-cell cytotoxicity (p < 0.005) and augments NK-cell mediated ADCC in combination with daratumumab (p = 0.001) and elotuzumab (p = 0.001) versus controls. CONCLUSIONSThese data confirm functional expression of AHR in MM and may provide insight to growing epidemiologic data indicting a link between environmental exposure to aromatic hydrocarbons and subsequent risk of MM. In addition, as opposed to the direct, deleterious effects of AHR antagonism on MM cell viability, AHR antagonism promotes NK cell development, surveillance of MM, and direct as well as ADCC-mediated cytotoxicity against MM targets. Taken together, these data strongly support the concept of targeting AHR as a novel direct anti-MM therapy and anti-MM immunotherapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

TGF\u03b21 signaling sustains aryl hydrocarbon receptor (AHR) expression and restrains the pathogenic potential of TH17 cells by an AHR-independent mechanism

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by ligand highly expressed on TH17 cells, and AHR-deficient CD4+ T cells have impaired production of IL-17A and IL-22. Although AHR activation can exacerbate in vivo TH17 cell-mediated autoimmunity, accumulating data indicate that AHR is a nonpathogenic TH17 marker. Thus it remains unclear how AHR activation is regulated and impacts on the generation of TH17 subsets. Here we demonstrated that AHR pathway is activated during in vitro pathogenic TH17 polarization, but it is quickly downregulated. Under these conditions, additional AHR activation promoted IL-22 but not IL-17A. Interestingly, AHR high sustained expression and IL-17A promotion were only achieved when TGFβ1 was present in the culture. In addition to the effect on AHR regulation, TGFβ1 presented a dual role by simultaneously suppressing the TH17 pathogenic phenotype acquisition. This latter effect was independent of AHR stimulation, since its activation did not confer a TH17 anti-inflammatory profile and Ahr−/− cells did not upregulate any TH17 pathogenic marker. Through the use of EAE model, we demonstrated that AHR is still functional in encephalitogenic CD4+ T cells and the adoptive transfer of Ahr−/− TH17 cells to recipient mice resulted in milder EAE development when compared to their WT counterparts. Altogether, our data demonstrated that although AHR is highly expressed on in vitro-generated nonpathogenic TH17 cells, its ligation does not shift TH17 cells to an anti-inflammatory phenotype. Further studies investigating the role of AHR beyond TH17 differentiation may provide a useful understanding of the physiopathology of autoimmune diseases.

HIF-1α-induced xenobiotic transporters promote Th17 responses in Crohn's disease

In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis.Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.

Aryl hydrocarbon receptor induced intratumoral aromatase in breast cancer.

Aryl hydrocarbon receptor (AhR) inhibits estrogen receptor (ER) pathway, which may suppress estrogen-dependent cell proliferation. However, the correlation between AhR stimulation and intratumoral estrogen synthesis, especially through aromatase, has not been reported to date. In the present study, we examined this correlation in breast cancer cells. We examined AhR and aromatase immunoreactivity in 29 patients with invasive ductal carcinoma. We performed in vitro studies using three breast carcinoma cell lines, MCF-7, T47D, and MDA-MB-231. AhR stimulation induced the mRNA expression of the aromatase gene in vitro in three breast carcinoma cell lines, and increased estrogen synthesis in MCF-7 cell line. Results of microarray analysis showed that AhR-induced aromatase expression was associated with BRCA1 induction. Analysis of patients with breast cancer showed a significant positive correlation between intratumoral AhR and aromatase status. We also compared the effects of AhR stimulation on the induction of intratumoral estrogen synthesis and inhibition of the ER signaling pathway, because AhR exerts contradictory effects on estrogen action in breast carcinoma cells. AhR-induced aromatase expression persisted for a significantly longer duration than AhR-induced ER pathway inhibition. Moreover, breast carcinoma cells treated with an AhR agonist tended to show earlier cell proliferation after removing the agonist than cells not treated with the AhR agonist. The results of the present study suggest that AhR stimulates estrogen-dependent progression of breast carcinoma by inducing aromatase expression under some conditions. These results provide new insights on the possible roles of environmental toxins in breast cancer development.

Signaling network map of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a multi-domain cytosolic protein that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. Ligand binding induces a conformational change in AHR and promotes nuclear translocation of the receptor (Kewley et al. 2004). AHR can either bind to exogenous (polycyclic aromatic hydrocarbons, dioxins, cigarette smoke) or endogenous ligands (arachidonic acid and leukotrienes, heme metabolites, UV photoproducts of tryptophan) within the cytoplasm. Exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene are known to activate AHR and mediate cellular toxic response. AHR is also activated by dietary compounds including indole-3-carbinol and flavonoids that mediate various physiological activities in the body (Nguyen and Bradfield 2008; Marconett et al. 2010). AHR ligands are classified as agonists and antagonists depending on the ability of ligands to activate or inhibit AHR induced activity. Previous reports indicate that ligands such as kaempferol, resveratrol, galangin, chrysin and quercetin act either as agonists or antagonists based on the ligand concentration and type of cells induced (Zhang et al. 2003). Thus, diversity of ligands makes AHR signaling a very dynamic and complex. AHR in its inactive state is located in the cytoplasm and forms a complex with molecular chaperones, such as heat shock protein 90 (HSP90) and co-chaperons such as p23 and AHR-interacting protein (AIP) (Trivellin and Korbonits 2011). In presence of ligand, AHR undergoes nuclear translocation where it interacts with AHR nuclear transporter (ARNT) or AHR repressor (AHRR) through the PAS domain. It has been reported that nucleocytoplasmic transport mechanism of AHR varies between humans and mice. In humans, AHR both in stimulated or unstimulated state can undergo nuclear translocation complexed with AIP. In contrast, association of AIP prevents nucleocytoplasmic shuttling of AHR in mice in both stimulated and unstimulated states (Ramadoss et al. 2004). Once AHR is translocated to the nucleus it forms a heterodimer complex with ARNT and binds to xenobiotic response elements located in the promoter region of the target genes. This complex induces coordinated transcription of detoxifying enzymes for efficient absorption, distribution and elimination of xenobiotics from the body (Abel and Haarmann-Stemmann 2010). Apart from this, AHR is known to exhibit endogenous functions such as cell proliferation, cell differentiation and apoptosis. It also acts as an endogenous regulator in several developmental and physiological processes including neurogenesis, hematopoietic stem cell regulation, cellular stress response, immunoregulation and reproductive health (Lindsey and Papoutsakis 2012; Kadow et al. 2011; Hansen et al. 2014). AHR is associated with various pathological and physiological disorders in the body including autoimmune diseases (Veldhoen et al. 2008), inflammation (Podechard et al. 2008; Ovrevik et al. 2014), cardiovascular diseases (Kerley-Hamilton et al. 2012; Savouret et al. 2003) and cancer. Activation of AHR in presence of cigarette smoke has been well documented in lung cancer (Martey et al. 2005; Tsay et al. 2013). Cigarette smoke induced AHR is also known to mediate immune signaling mechanism in chronic obstructive pulmonary disease (COPD) (Chen et al. 2011). Apart from playing an essential role in COPD and lung cancer, AHR expression has been reported in other cancers and adenomas. AHR is reported to be downregulated in growth hormone secreting pituitary adenomas (Jaffrain-Rea et al. 2009), while, increased expression levels of AHR are associated with tumorigenesis in medulloblastoma (Dever and Opanashuk 2012). The enhancement of AHR levels under ligand stimulation induced cell cycle arrest has been reported in pancreatic and gastric cancer (Koliopanos et al. 2002; Peng et al. 2009). Depending upon the type of ligand stimulation, AHR is either known to promote or inhibit tumor progression. Stimulation of AHR with TCDD, 2,3,7,8-tetrachlorodibenzofuran and 3,3′-diindolylmethane inhibits invasiveness and cell growth in breast cancer (Hall et al. 2010). In contrast, stimulation of AHR with n-butyl benzyl phthalate and dibutyl phthalate ligands enhances tumorigenic properties in breast cancer cells (Hsieh et al. 2012). Therefore, AHR could serve as a potential therapeutic target in several cancers (Murray et al. 2014) and hence it is important to develop AHR signaling pathway to understand the mechanism of AHR mediated tumor progression and regression. Although, the diverse role of AHR is documented in literature however a detailed network of AHR signaling is lacking. In this study, we have curated literature information pertaining to AHR induced signaling and developed a pathway map to facilitate better understanding of this receptor.

Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon receptor

Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.

Aryl hydrocarbon receptor agonist, leflunomide, protects the ischemic-reperfused kidney: role of Tregs and stem cells

The aryl hydrocarbon receptor (AHR) has emerged as a major modulator of inflammatory processes. We tested the hypothesis that AHR activation protects the ischemic-reperfused kidney in association with the suppression of the inflammatory response. Accordingly, male mice were treated with the nondioxin AHR agonist, leflunomide (40 mg/kg ip); vehicle-treated animals served as controls. Thereafter, the right kidney was subjected to an ischemia (45 min)-reperfusion (4 h) insult, while the left kidney served as a sham control. Renal cells prepared from ischemic-reperfused kidneys of leflunomide-treated mice displayed preservation of mitochondrial membrane potential (Ψ(m)) and decreased apoptosis and necrosis compared with vehicle-treated ischemic-reperfused kidneys. Leflunomide treatment increased regulatory T cells (Tregs; forkhead box P3+) and IL-10-positive cells but reduced IL-17- and IL-23-expressing cells in both the peripheral blood and kidney cells, indicative of down-regulation of inflammatory responses. Leflunomide treatment also increased mobilization of stems cells subsets (i.e., mesenchymal and hematopoietic stem cells and endothelial progenitor cells) in the peripheral blood and promoted their recruitment into the ischemic-reperfused kidney. Collectively, the results indicate that AHR stimulation may represent a novel renoprotective mechanism likely involving mobilization and recruitment of Tregs and stem cells into the damaged kidney.

Abstract 452: Impact of Aryl Hydrocarbon Receptor Agonist, Leflunomide, on Components of Stem Cells in the Setting of Renal Ischemia-Reperfusion Injury

The aryl hydrocarbon receptor (AHR) has emerged as a regulator of bone marrow-derived stem cells (BMDSCs). Further, mobilization and homing of BMDSCs into the ischemic-reperfused kidney contribute importantly to the repair and recovery of the injured tissue. We tested the hypothesis that the recently described non-dioxin AHR agonist, leflunomide, mobilizes BMDSCs into the ischemic-reperfused kidney in association with reduction in cell death. Accordingly, male mice were treated with leflunomide (40 mg/kg; i.p.). Thereafter, the right kidney of each animal was subjected to ischemia (45 min) followed by reperfusion (4 hr) while the left kidney served as sham control; vehicle-treated animals served as controls. Peripheral blood and renal cells prepared from kidneys of experimental groups were subjected to flow cytometry-based assays using markers for endothelial progenitor cells (EPCs; Sca1 + /CD31 + ), mesenchymal stem cells (MSCs; Sca1 + /CD11b - ) and hematopoietic stem cells (HSCs; Sca1 + /CD11b + ). Compared to the vehicle-treated mice, peripheral blood of leflunomide-treated mice showed significantly higher levels of MSCs and HSCs with the effect more marked for the latter. On the other hand, the ischemic-reperfused, but not sham, kidneys of leflunomide-treated animals displayed significant and similar increase in the level of each component of BMDSCs compared to their vehicle-treated counterparts. Further, leflunomide-treated mice displayed preservation of mitochondrial membrane potential as well as decreased apoptosis and necrosis compared to vehicle-treated ischemic-reperfused or sham kidneys. Collectively, the results indicate that AHR stimulation may represent a novel renoprotective mechanism likely involving mobilization and recruitment of components of Sca1+ stem cells into the damaged kidney.

Notch signaling drives IL-22 secretion in CD4+T cells by stimulating the aryl hydrocarbon receptor

CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses.

Inflammatory Signaling and Aryl Hydrocarbon Receptor Mediate Synergistic Induction of Interleukin 6 in MCF-7 Cells

The pleiotropic cytokine interleukin 6 (IL-6) is involved in immune cell homeostasis. Additionally, IL-6 expression and signaling in tumor cells have been shown to elicit both protumor and antitumor properties. There is a plethora of mechanistic knowledge regarding how IL-6 signal transduction translates to biological responses. However, there is little understanding as to what factors control IL-6 expression within a tumor cell environment. The studies presented herein show that, in MCF-7 breast and ECC-1 endocervical cancer cells, the stimulation of aryl hydrocarbon receptor (AHR) activity, in combination with IL-1beta or phorbol 12-myristate 13-acetate (PMA) treatment, results in a marked synergistic induction of IL-6 levels over what is seen without AHR activation. Chromatin immunoprecipitation experiments suggest that the regulation of IL-6 mRNA expression occurs at the chromatin level, as AHR presence on the IL-6 promoter was observed in response to treatment with AHR ligand. Synergistic induction of IL-6 expression was sustained for 72 hours, with accumulation of IL-6 protein reaching levels 4.8-fold above IL-1beta treatment alone. In addition, transcriptional regulation of the prototypic AHR responsive gene Cyp1a1 was negatively regulated by PMA and IL-1beta treatment. Silencing of RELA expression alleviated IL-1beta-mediated repression of AHR transcriptional activity, whereas PMA-mediated repression was maintained. Additionally, small interfering RNA studies reveal that AHR and RELA are necessary for synergistic induction of IL-6. The findings presented here reveal the AHR as a potential therapeutic target for selective modulation of IL-6 expression in some tumor cell types. The data also suggest a possible previously unrecognized mechanism of AHR-mediated tumor promotion.