Ahr Knockout Mice Research Articles

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.

Abstract 703: Dioxin-induced Smooth Muscle Cell Phenotypic Modulation And Atherosclerosis Worsens In The Absence Of AHR

Background: Dioxin (TCDD, 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin) is an active constituent of tobacco smoke and air pollution that activates the aryl hydrocarbon receptor (AHR). Dioxin has been linked to an increased risk of coronary artery disease (CAD). However, the corresponded molecular mechanisms remain unclear. Smooth muscle cells (SMC) play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. In this study, we aimed to understand the role of AHR in modulating the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: SMC lineage-tracing (SMC Lin ) mice ( Tg Myh11-CreERT2 , ROSA tdT/tdT , ApoE -/- ) without any TCDD injection were used as control. TCDD were injected to both SMC Lin mice, and SMC-specific Ahr knockout mice. High fat diet (HFD) and TCDD injection started at 8 weeks of age and aortic roots were harvested at 24 weeks of age for either histology or single-cell sequencing (10X Genomics, triplicates). Histology analysis was performed at the aortic sinus of control mice (n=9), TCDD injected SMC Lin mice (n=12) and TCDD injected SMC-specific Ahr knockout mice (n=9). Immunohistochemistry was performed with anti-SM22α antibody and CD68 antibody. Results: The histological analysis of the aortic sinus showed increased lesion area after TCDD treatment in the SMC Lin control mice. In the SMC-specific knockout of Ahr , the lesion area was significantly larger compared to the SMC Lin control mice when treated with TCDD along with significantly increased presence of Tagln + cells in the lesion. These results indicated TCDD leads to adverse remodeling of atherosclerosis and that the presence of Ahr gene is protective against the athero-promoting effects of dioxin. The scRNA-seq of SMC Lin control mice treated with TCDD exposure showed that the SMC phenotypic modulation was markedly inhibited, leading to the presence of fewer fibromyocytes (FMC). Increased FMC to chondromyocytes (CMC) modulation resulted in further reduction of FMC population in the Ahr -KO mice treated with TCDD. Conclusion: Dioxin adversely remodels atherosclerotic plaque, and the lack of AHR in SMC can exacerbate the atherosclerotic process, suggesting that dioxin may exhibit its harmful effect via non-AHR dependent pathway.

Aryl Hydrocarbon Receptor (AhR) Signaling in Colonic Cells and Tumors.

The aryl hydrocarbon receptor (AhR) is overexpressed in many tumor types and exhibits tumor-specific tumor promoter and tumor suppressor-like activity. In colon cancer, most but not all studies suggest that the AhR exhibits tumor suppressor activity which is enhanced by AhR ligands acting as agonists. Our studies investigated the role of the AhR in colon tumorigenesis using wild-type and AhR-knockout mice, the inflammation model of colon tumorigenesis using mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) and APCS580/+; KrasG12D/+ mice all of which form intestinal tumors. The effects of tissue-specific AhR loss in the intestine of the tumor-forming mice on colonic stem cells, organoid-initiating capacity, colon tumor formation and mechanisms of AhR-mediated effects were investigated. Loss of AhR enhanced stem cell and tumor growth and in the AOM/DSS model AhR-dependent suppression of FOXM1 and downstream genes was important for AhR-dependent anticancer activity. Furthermore, the effectiveness of interleukin-22 (IL22) in colonic epithelial cells was also dependent on AhR expression. IL22 induced phosphorylation of STAT3, inhibited colonic organoid growth, promoted colonic cell proliferation in vivo and enhanced DNA repair in AOM/DSS-induced tumors. In this mouse model, the AhR suppressed SOCS3 expression and enhanced IL22-mediated activation of STAT3, whereas the loss of the AhR increased levels of SOCS3 which in turn inhibited IL22-induced STAT3 activation. In the APCS580/+; KrasG12D/+ mouse model, the loss of the AhR enhanced Wnt signaling and colon carcinogenesis. Results in both mouse models of colon carcinogenesis were complemented by single cell transcriptomics on colonic intestinal crypts which also showed that AhR deletion promoted expression of FOXM1-regulated genes in multiple colonic cell subtypes. These results support the role of the AhR as a tumor suppressor-like gene in the colon.

Modulation of Aryl Hydrocarbon Receptor Expression Alleviated Neuropathic Pain in a Chronic Constriction Nerve Injury Animal Model.

Neuropathic pain is well known to occur after damage to the somatosensory system. Aryl hydrocarbon receptor (AhR) has neuroprotective effects when the central nervous system is subjected to internal and external stimulations. However, the exact mechanism by which AhR regulates neuropathic pain is poorly understood. Nerve explant culture and the chronic constrictive nerve injury (CCI) model in wild or AhR-knockout mice were used in this study. In the nerve explant culture, the ovoid number increased in the AhR−/− condition and was decreased by omeprazole (AhR agonist) in a dose-dependent manner. Increased nerve degeneration and the associated inflammation response appeared in the AhR−/− condition, and these changes were attenuated by omeprazole. High expression of AhR in the injured nerve was noted after CCI. Deletion of AhR aggravated nerve damages and this was restored by omeprazole. Deletion of AhR increased NGF expression and reduced axon number in the paw skin, but this was attenuated by omeprazole. A highly expressed inflammation reaction over the dorsal spinal cord, somatosensory cortex, and hippocampus was noted in the AhR-deleted animals. Administration of omeprazole attenuated not only the inflammatory response, but also the amplitude of somatosensory evoked potential. Deletion of AhR further aggravated the neurobehavior compared with the wild type, but such behavior was attenuated by omeprazole. Chronic constrictive nerve injury augmented AhR expression of the injured nerve, and AhR deletion worsened the damage, while AhR agonist omeprazole counteracted such changes. AhR agonists could be potential candidates for neuropathic pain treatment.

Loss of the aryl hydrocarbon receptor increases tumorigenesis in p53-deficient mice

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cell fate via activation of a diverse set of genes. There are conflicting reports describing the role of AhR in cancer. AhR-knockout mice do not develop tumors spontaneously, yet the AhR can act as a tumor suppressor in certain contexts. Loss of tumor suppression by p53 is common in human cancer. To investigate AhR function in the absence of p53, we generated mice lacking both AhR and p53. Mice deficient for AhR and p53 had shortened lifespan, increased tumorigenesis, and an altered tumor spectrum relative to control mice lacking only p53. In addition, knockout of both AhR and p53 resulted in reduced embryonic survival and neonatal fitness. We also examined the consequences of loss of AhR in p53-heterozygous mice and observed a significantly reduced lifespan and enhanced tumor burden. These findings reveal an important role for the AhR as a tumor suppressor in the absence of p53 signaling and support the development of anti-cancer therapeutics that would promote the tumor suppressive actions of the AhR.

DEHP mediates drug resistance by directly targeting AhR in human breast cancer.

Resistance to chemotherapy and hormonal therapy is a major clinical problem in breast cancer medicine, especially for cancer metastasis and recurrence. Di(2-ethylhexyl)phthalate (DEHP) affects drug resistance by an unknown mechanism of action. Here we analyzed breast cancer patients (N=457) and found that Σ4MEHP (the sum of MEHP, MEHHP, MECPP and MEOHP concentrations) in urine was significantly higher (P=0.018) in the recurrent breast cancer group compared with non-recurrent patients. Σ4MEHP-High was positively and significantly correlated with tumor stage (P=0.005), lymph node status (P=0.001), estrogen receptor status (P=0.010), Her2/Neu status (P=0.004), recurrence (P=0.000) and tumor size (P=0.002), as well as an independent prognostic marker (OR = 1.868; 95% CI = 1.424-2.451; P<0.000) associated with poor survival rates based on a positive Her2/Neu status (P=0.035). In addition, we found that DEHP inhibited paclitaxel and doxorubicin effects in breast cancer cell lines MCF-7 and MDA-MB-231 and in zebrafish and mouse tumor initiation models. DEHP induced trefoil factor 3 (TFF3) expression through the vinculin/aryl hydrocarbon receptor (AhR)/ERK signaling pathway and induced CYP2D6, CYP2C8 and CYP3A4 expression through the AhR genomic pathway to increase the epithelial-mesenchymal transition (EMT) and doxorubicin metabolism, respectably. DEHP mediated AhR-related alterations in estrogen receptor expression through the ubiquitination system, which decreased tamoxifen effects in AhR knockout mice. These findings suggest a novel therapeutic avenue by targeting AhR in drug-resistant and recurrent breast cancer.

Single-cell RNA Sequencing Reveals How the Aryl Hydrocarbon Receptor Shapes Cellular Differentiation Potency in the Mouse Colon.

Despite recent progress recognizing the importance of aryl hydrocarbon receptor (Ahr)-dependent signaling in suppressing colon tumorigenesis, its role in regulating colonic crypt homeostasis remains unclear. To assess the effects of Ahr on intestinal epithelial cell heterogeneity and functional phenotypes, we utilized single-cell transcriptomics and advanced analytic strategies to generate a high-quality atlas for colonic intestinal crypts from wild-type and intestinal-specific Ahr knockout mice. Here we observed the promotive effects of Ahr deletion on Foxm1-regulated genes in crypt-associated canonical epithelial cell types and subtypes of goblet cells and deep crypt-secretory cells. We also show that intestinal Ahr deletion elevated single-cell entropy (a measure of differentiation potency or cell stemness) and RNA velocity length (a measure of the rate of cell differentiation) in noncycling and cycling Lgr5+ stem cells. In general, intercellular signaling cross-talk via soluble and membrane-bound factors was perturbed in Ahr-null colonocytes. Taken together, our single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating putative stem cell driver genes, cell potency lineage decisions, and cell-cell communication in vivo. PREVENTION RELEVANCE: Our mouse single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating colonic stemness and cell-cell communication in vivo. From a cancer prevention perspective, Ahr should be considered a therapeutic target to recalibrate remodeling of the intestinal stem cell niche.

Behavioural adaptations after antibiotic treatment in male mice are reversed by activation of the aryl hydrocarbon receptor

The intestinal microbiota plays an important role in regulating brain functions and behaviour. Microbiota-dependent changes in host physiology have been suggested to be key contributors to psychiatric conditions. However, specific host pathways modulated by the microbiota involved in behavioural control are lacking. Here, we assessed the role of the aryl hydrocarbon receptor (Ahr) in modulating microbiota-related alterations in behaviour in male and female mice after antibiotic (Abx) treatment. Mice of both sexes were treated with Abx to induce bacterial depletion. Mice were then tested in a battery of behavioural tests, including the elevated plus maze and open field tests (anxiety-like behaviour), 3 chamber test (social preference), and the tail suspension and forced swim tests (despair behaviour). Behavioural measurements in the tail suspension test were also performed after microbiota reconstitution and after administration of an Ahr agonist, β-naphthoflavone. Gene expression analyses were performed in the brain, liver, and colon by qPCR. Abx-induced bacterial depletion did not alter anxiety-like behaviour, locomotion, or social preference in either sex. A sex-dependent effect was observed in despair behaviour. Male mice had a reduction in despair behaviour after Abx treatment in both the tail suspension and forced swim tests. A similar alteration in despair behaviour was observed in Ahr knockout mice. Despair behaviour was normalized by either microbiota recolonization or Ahr activation in Abx-treated mice. Ahr activation by β-naphthoflavone was confirmed by increased expression of the Ahr-target genes Cyp1a1, Cyp1b1, and Ahrr. Our results demonstrate a role for Ahr in mediating the behaviours that are regulated by the crosstalk between the intestinal microbiota and the host. Ahr represents a novel potential modulator of behavioural conditions influenced by the intestinal microbiota.

Protective Effect of Saffron in Mouse Colitis Models Through Immune Modulation.

People with inflammatory bowel disease (IBD) including ulcerative colitis are at risk for colorectal cancer. Despite available effective drugs used to treat IBD, many patients fail or lose response over time with some displaying drug-induced adverse events. Saffron (Crocus sativus) has been reported to have anti-inflammatory properties. Its protective role in IBD has not been explored extensively. To establish whether saffron treatment alleviates inflammation in experimental colitis. Colitis was induced in C57BL/6 mice with 3% DSS and treated with either saffron doses (7.5, 15, 20, 25mg/kg body weight) or vehicle through daily gavage. On day 11, mice were euthanized and analyzed for gross and microscopic inflammation. Distal colon segments were collected for mRNA and protein expression of HO-1 protein and GPX2, (the downstream targets of NRF-2). Nrf-2 translocation from cytosol to nucleus was confirmed by immunofluorescence, and further Nrf-2 protein expression in nuclear and cytosolic fraction of colon was analyzed by immunoblot. Immune cells were isolated from the lamina propria of mouse colon for flow cytometry-based immunophenotyping. Colitis was also induced in C57BL/6 Ahr knockout and wild type mice to explore the involvement of Ahr-dependent pathways in saffron's protective effect(s). The therapeutic effect of saffron was further validated in another TNBS model of colitis. Saffron 20mg/kg body weightshowed improved colon gross and histology features and led to better body weight, colon length, histology score, and reduced disease activity index (DAI). Saffron significantly decreased pro-inflammatory macrophages (M1), while increasing anti-inflammatory macrophages (M2) and IL10 + dendritic cells. Saffron treatment also enhanced CD3 + T and CD3 + CD8 + T cells followed by increase in different CD3 + CD4 + T cells subsets like CD25 + T cells, FoxP3 + CD25 + regulatory T cells, and CD4 + FOXP3 + CD25-regulatory T cells. Immunoblot analysis showed a significant increase in HO-1/GPX2 protein expression. With saffron treatment, Nrf-2 translocation into nucleus from cytosol also supports the involvement of Nrf-2 and its downstream targets in the protective effect of saffron. Further, we demonstrated that saffron in part exert anti-inflammatory effect through activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways. These data demonstrate saffron's therapeutic potential and its protective role in part via Ahr/Nrf-2 pathways and regulatory innate and adaptive immune cells.

Nrf2 through Aryl Hydrocarbon Receptor Regulates IL-22 Response in CD4+ T Cells.

IL-17A and IL-22 derived from Th17 cells play a significant role in mucosal immunity and inflammation. TGF-β and IL-6 promote Th17 differentiation; however, these cytokines have multiple targets. The identification and screening of additional molecules that regulate IL-17A and IL-22 responses in certain inflammatory conditions is of great clinical significance. In this study, we show that CDDO-Im, a specific Nrf2 activator, promotes IL-17A and IL-22 responses in murine Th17 cells. In contrast, CDDO-Im inhibits IL-17A response in multiple sclerosis patient-derived PBMCs. However, Nrf2 specifically regulates IL-22 response in vivo. Nrf2 acts through the regulation of antioxidant response element (ARE) binding motifs in target genes to induce or repress transcription. Promoter analysis revealed that Il17a, Rorc, and Ahr genes have several ARE motifs. We showed that Nrf2 bound to ARE repressor (ARE-R2) of Rorc and inhibited Rorc-dependent IL-17A transactivation. The luciferase reporter assay data showed that CDDO-Im regulated Ahr promoter activity. Chromatin immunoprecipitation quantitative PCR data showed that Nrf2 bound to ARE of AhR. Finally, we confirmed that the CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in CD4-specific Ahr knockout mice (AhrCD4 ). CH-223191, a specific AhR antagonist, inhibits CDDO-Im-induced IL-22 production in CD4+ T cells, which further confirmed the AhR-dependent regulation. Collectively, our data showed that Nrf2 via AhR pathways regulated IL-22 response in CD4+ T cells.

Lemon Exosome-like Nanoparticles-Manipulated Probiotics Protect Mice from C.d iff Infection.

SummaryClostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.

Aryl Hydrocarbon Receptor Activity in Hepatocytes Sensitizes to Hyperacute Acetaminophen-Induced Hepatotoxicity in Mice.

Background & AimsAcetaminophen (APAP)-induced liver injury is one of the most common causes of acute liver failure, however, a clear definition of sensitizing risk factors is lacking. Here, we investigated the role of the ligand-activated transcription factor aryl hydrocarbon receptor (Ahr) in APAP-induced liver injury. We hypothesized that Ahr, which integrates environmental, dietary, microbial and metabolic signals into complex cellular transcriptional programs, might act as a rheostat for APAP-toxicity.MethodsWildtype or conditional Ahr knockout mice lacking Ahr in hepatocytes (AlbΔ/ΔAhr) or myeloid cells (LysMΔ/ΔAhr) were treated with the specific Ahr ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) together with APAP.ResultsAhr activation by ITE, which by itself was non-toxic, exacerbated APAP-induced hepatotoxicity compared to vehicle-treated controls, causing 80% vs. 0% mortality after administration of a normally sublethal APAP overdose. Of note, Ahr activation induced hepatocyte death even at APAP doses within the therapeutic range. Aggravated liver injury was associated with significant neutrophil infiltration; however, lack of Ahr in myeloid cells did not protect LysMΔ/ΔAhr mice from exacerbated APAP hepatotoxicity. In contrast, AlbΔ/ΔAhr mice were largely protected from ITE-induced aggravated liver damage, indicating that Ahr activation in hepatocytes, but not in myeloid cells, was instrumental for disease exacerbation. Mechanistically, Ahr activation fueled hepatic accumulation of toxic APAP metabolites by up-regulating expression of the APAP-metabolizing enzyme Cyp1a2, a direct Ahr downstream target.ConclusionsAhr activation in hepatocytes potentiates APAP-induced hepatotoxicity. Thus, individual exposition to environmental Ahr ligands might explain individual sensitivity to hyperacute liver failure.

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR’s role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.

Age-dependent vulnerability of the ovary to AhR-mediated TCDD action before puberty: Evidence from mouse models

In female mammals, puberty and fertility are regulated by the synthesis of estradiol (E2) by the ovaries at the infantile stage and at the approach of puberty, a process which may be affected by endocrine disrupting chemicals (EDC)s acting through the Aryl hydrocarbon receptor (AhR). However, there is no information on AhR-mediated regulation of ovarian estrogenic activity during these developmental periods. Here, we assessed in mouse models, the intrinsic and exogenous ligand-induced AhR action on E2 synthesis at the infantile stage (14 days postnatal (dpn)) and at the approach of puberty (28 dpn). Intrinsic AhR pathway became activated in the ovary at the approach of puberty, as suggested by the decreased intra-ovarian expression in prototypical and steroidogenesis-related AhR targets and E2 contents in Ahr knockout (Ahr−/−) mice versus Ahr+/+ mice exclusively at 28 dpn. Accordingly, AhR nuclear localization in granulosa cells, reflecting its activity in cells responsible for E2 synthesis, was much lower at 14 dpn than at 28 dpn in C57BL/6 mice. However, AhR signaling could be activated by exogenous ligands at both ages, as revealed by FICZ- and TCDD-induced Ahrr and Cyp1a1 expression in C57BL/6 mice. Nevertheless, TCDD impacted ovarian estrogenic activity only at 28 dpn. This age-related AhR action may be ligand-dependent, since FICZ had no effect on E2 synthesis at 28 dpn. In conclusion, AhR would not regulate ovarian estrogenic activity before the approach of puberty. Its activation by EDCs may be more detrimental to reproductive health at this stage than during infancy.

A genome-wide CRISPR/Cas9 screen reveals that the aryl hydrocarbon receptor stimulates sphingolipid levels

Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.

Aryl Hydrocarbon Receptor in Cutaneous Vascular Endothelial Cells Restricts Psoriasis Development by Negatively Regulating Neutrophil Recruitment

Vascular endothelial cells (VECs) that line the interiors of blood vessels participate in physiological and inflammatory processes. All skin cell types express the aryl hydrocarbon receptor (AhR), which is involved in the pathogenesis of psoriasis. However, the role of the cutaneous VEC AhR in the pathogenesis of psoriasis remains elusive. In the present study, we found that AhR protein expression and activation were downregulated in psoriatic VECs. Furthermore, cutaneous VEC-specific AhR-knockout (AhRcVECs-KO) mice were established. Using imiquimod and IL-23-induced psoriasis models, we found that skin inflammation was exacerbated with excessive neutrophil recruitment in AhRcVECs-KO mice. Furthermore, neutrophil neutralization alleviates exacerbated inflammation in imiquimod-treated AhRcVECs-KO mice. In addition, cutaneous VECs in AhRcVECs-KO mice exhibited increased dilation and activation compared with those in control mice. Finally, AhR-deficient microvascular endothelial cells stimulated by proinflammatory cytokines showed increased ICAM-1 expression invivo and invitro, which may have facilitated neutrophil recruitment. In summary, our study demonstrates that AhR in dermal VECs restricts psoriasis development by negatively regulating neutrophil recruitment, thereby providing insight into the pathogenesis of psoriasis.

Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice

The role of aryl hydrocarbon receptor (AhR) in liver fibrosis is controversial because loss and gain of AhR activity both lead to liver fibrosis. The goal of this study was to investigate how the expression of AhR by different liver cell types, hepatic stellate cells (HSCs) in particular, affects liver fibrosis in mice. We studied the effects of AhR on primary mouse and human HSCs, measuring their activation and stimulation of fibrogenesis using RNA-sequencing analysis. C57BL/6J mice were given the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE); were given carbon tetrachloride (CCl4); or underwent bile duct ligation. We also performed studies in mice with disruption of Ahr specifically in HSCs, hepatocytes, or Kupffer cells. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and immunoblotting. AhR was expressed at high levels in quiescent HSCs, but the expression decreased with HSC activation. Activation of HSCs from AhR-knockout mice was accelerated compared with HSCs from wild-type mice. In contrast, TCDD or ITE inhibited spontaneous and transforming growth factor β-induced activation of HSCs. Mice with disruption of Ahr in HSCs, but not hepatocytes or Kupffer cells, developed more severe fibrosis after administration of CCl4 or bile duct ligation. C57BL/6J mice given ITE did not develop CCl4-induced liver fibrosis, whereas mice without HSC AhR given ITE did develop CCl4-induced liver fibrosis. In studies of mouse and humanHSCs, we found that AhR prevents transforming growth factor β-induced fibrogenesis by disrupting the interaction of Smad3 with β-catenin, which prevents the expression of genes that mediate fibrogenesis. In studies of human and mouse HSCs, we found that AhR prevents HSC activation and expression of genes required for liver fibrogenesis. Development of nontoxic AhR agonists or strategies to activate AhR signaling in HSCs might be developed to prevent or treat liver fibrosis.

Aryl hydrocarbon receptor acts as a tumor suppressor in a syngeneic MC38 colon carcinoma tumor model.

Background: Aryl hydrocarbon receptor (AHR), commonly known as an environmental sensor involved in the metabolism and elimination of xenobiotic substances, is also an important modulator in the development and functioning of the immune system. AHR expression is varied in the T cell subsets with the highest expression in T-helper 17 and T regulatory cells. It has been reported that AHR can act as a tumor promoter or a tumor suppressor, depending on the tumor type.Methods: In an effort to understand the role played by AHR in tumor growth, the MC38 syngeneic colon carcinoma tumor model was used on C57BL/6 or ahr knockout (KO, -/-) mice with or without AHR antagonist (CH223191) treatment. Tumor sizes were measured, and biomarkers were quantified in tumor microenvironment and draining lymph nodes using flow cytometry. Enzyme-linked immunosorbent assay was used to determine the amount of cytokines in tumors.Results: In ahr deficient mice, MC38 tumors progress more rapidly than in wild-type mice, accompanied by an increase in tumor-associated macrophages and M2 macrophages and a decrease in CD8a positive cytotoxic lymphocytes. Analysis of cytokines in the tumor microenvironment reveals a pro-inflammatory phenotype. Similar changes were observed by pharmacologic blockade of the receptor using CH223191.Conclusion: AHR acts as a tumor suppressor in mice implanted with MC38 colon carcinoma cells as evidenced by either a blockade or deficiency of AHR.

Inducible Loss of the Aryl Hydrocarbon Receptor Activates Perigonadal White Fat Respiration and Brown Fat Thermogenesis via Fibroblast Growth Factor 21.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor highly expressed in hepatocytes. Researchers have employed global and liver-specific conditional Ahr knockout mouse models to characterize the physiological roles of the AHR; however, the gestational timing of AHR loss in these models can complicate efforts to distinguish the direct and indirect effects of post-gestational AHR deficiency. Utilizing a novel tamoxifen-inducible AHR knockout mouse model, we analyzed the effects of hepatocyte-targeted AHR loss in adult mice. The data demonstrate that AHR deficiency significantly reduces weight gain and adiposity, and increases multilocular lipid droplet formation within perigonadal white adipose tissue (gWAT). Protein and mRNA expression of fibroblast growth factor 21 (FGF21), an important hepatokine that activates thermogenesis in brown adipose tissue (BAT) and gWAT, significantly increases upon AHR loss and correlates with a significant increase of BAT and gWAT respiratory capacity. Confirming the role of FGF21 in mediating these effects, this phenotype is reversed in mice concomitantly lacking AHR and FGF21 expression. Chromatin immunoprecipitation analyses suggest that the AHR may constitutively suppress Fgf21 transcription through binding to a newly identified xenobiotic response element within the Fgf21 promoter. The data demonstrate an important AHR-FGF21 regulatory axis that influences adipose biology and may represent a “druggable” therapeutic target for obesity and its related metabolic disorders.

The Aryl Hydrocarbon Receptor and the Nervous System.

The aryl hydrocarbon receptor (or AhR) is a cytoplasmic receptor of pollutants. It translocates into the nucleus upon binding to its ligands, and forms a heterodimer with ARNT (AhR nuclear translocator). The heterodimer is a transcription factor, which regulates the transcription of xenobiotic metabolizing enzymes. Expressed in many cells in vertebrates, it is mostly present in neuronal cell types in invertebrates, where it regulates dendritic morphology or feeding behavior. Surprisingly, few investigations have been conducted to unravel the function of the AhR in the central or peripheral nervous systems of vertebrates. In this review, we will present how the AhR regulates neural functions in both invertebrates and vertebrates as deduced mainly from the effects of xenobiotics. We will introduce some of the molecular mechanisms triggered by the well-known AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which impact on neuronal proliferation, differentiation, and survival. Finally, we will point out the common features found in mice that are exposed to pollutants, and in AhR knockout mice.