Aryl Hydrocarbon Receptor Deficiency Research Articles

Dietary environmental factors shape the immune defense against Cryptosporidium infection

Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. Although it is known from human cases and animal models that CD4+ Tcells play a role in curbing Cryptosporidium, the role of CD8+ T cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express and depend on the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduces CD8+ IELs, decreases their cytotoxicity, and worsens infection. Transfer of CD8+ IELs rescues severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol in newborn mice promotes resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.

Endothelial sensing of AHR ligands regulates intestinal homeostasis

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

Investigation of the aryl hydrocarbon receptor’s role in colitis as it relates to observed disease-specific sex differences

Abstract Colitis is an inflammatory bowel disease (IBD) characterized by chronic inflammation in the colon. Studies suggest that there are differences between males and females regarding colitis disease progression, severity, and response to treatment. However, there lacks clear understanding, and some inconsistencies, on what these differences are attributed to. Previously, our reports showed that activation of the aryl hydrocarbon receptor (AhR) ligand was able to reduce colitis-induced disease severity, mainly through innate lymphoid type 3 cells (ILC3), in an interleukin-22 (IL-22) dependent manner. However, noted sex differences in disease severity and treatment response were observed in the animal models of colitis. In the current study, we generated cell-specific conditional knockout (KO) mice that had AhR deficiency in Rorc-expressing immune cells (ILC3s). We induced colitis using the dextran sodium sulfate (DSS) model in wildtype (WT) control mice and conditional mouse strains with AhR deficiency in Rorc-expressing immune cells (AR mice). Weight data and serum inflammatory biomarker assays showed that male mice developed a more severe colitis compared to females, with the male AR mice even worse. In addition, while control WT DSS female mice had less severe colitis than male counterparts, AR DSS female mice had just as severe disease as males, suggesting AhR, in part, was necessary to help promote less disease severity in females. Collectively, these results show that there are sex differences in colitis induction between male and female mice, and also that these differences might in part be due to some intrinsic sex-linked expression of AhR in different cell types. The studies were supported in part by NIH grants P20GM103641.

The aryl hydrocarbon receptor regulates lipid mediator production in alveolar macrophages.

Allergic inflammation of the airways such as allergic asthma is a major health problem with growing incidence world-wide. One cardinal feature in severe type 2-dominated airway inflammation is the release of lipid mediators of the eicosanoid family that can either promote or dampen allergic inflammation. Macrophages are key producers of prostaglandins and leukotrienes which play diverse roles in allergic airway inflammation and thus require tight control. Using RNA- and ATAC-sequencing, liquid chromatography coupled to mass spectrometry (LC-MS/MS), enzyme immunoassays (EIA), gene expression analysis and in vivo models, we show that the aryl hydrocarbon receptor (AhR) contributes to this control via transcriptional regulation of lipid mediator synthesis enzymes in bone marrow-derived as well as in primary alveolar macrophages. In the absence or inhibition of AhR activity, multiple genes of both the prostaglandin and the leukotriene pathway were downregulated, resulting in lower synthesis of prostanoids, such as prostaglandin E2 (PGE2), and cysteinyl leukotrienes, e.g., Leukotriene C4 (LTC4). These AhR-dependent genes include PTGS1 encoding for the enzyme cyclooxygenase 1 (COX1) and ALOX5 encoding for the arachidonate 5-lipoxygenase (5-LO) both of which major upstream regulators of the prostanoid and leukotriene pathway, respectively. This regulation is independent of the activation stimulus and partially also detectable in unstimulated macrophages suggesting an important role of basal AhR activity for eicosanoid production in steady state macrophages. Lastly, we demonstrate that AhR deficiency in hematopoietic but not epithelial cells aggravates house dust mite induced allergic airway inflammation. These results suggest an essential role for AhR-dependent eicosanoid regulation in macrophages during homeostasis and inflammation.

Microglial aryl hydrocarbon receptor enhances phagocytic function via SYK and promotes remyelination in the cuprizone mouse model of demyelination

Multiple sclerosis (MS) is an inflammatory-mediated demyelinating disease of the central nervous system (CNS). Although studies have demonstrated that microglia facilitate remyelination in demyelinating diseases, the underlying mechanisms are still not fully characterized. We found that aryl hydrocarbon receptor (AhR), an environment sensor, was upregulated within the corpus callosum in the cuprizone model of CNS demyelination, and upregulated AhR was mainly confined to microglia. Deletion of AhR in adult microglia inhibited efficient remyelination. Transcriptome analysis using RNA-seq revealed that AhR-deficient microglia displayed impaired gene expression signatures associated with lysosome and phagocytotic pathways. Furthermore, AhR-deficient microglia showed impaired clearance of myelin debris and defected phagocytic capacity. Further investigation of target genes of AhR revealed that spleen tyrosine kinase (SYK) is the downstream effector of AhR and mediated the phagocytic capacity of microglia. Additionally, AhR deficiency in microglia aggravated CNS inflammation during demyelination. Altogether, our study highlights an essential role for AhR in microglial phagocytic function and suggests the therapeutic potential of AhR in demyelinating diseases.Graphical

Akkermansia muciniphila inhibits tryptophan metabolism via the AhR/β-catenin signaling pathway to counter the progression of colorectal cancer.

Akkermansia muciniphila (A. muciniphila), a gram-negative anaerobic bacterium, is selectively decreased in the fecal microbiota of patients with colorectal cancer (CRC), but its molecular mechanism in CRC development remains inconclusive. In this study, we first confirmed the inhibitory effect of A. muciniphila on CRC formation and analyzed the metabolic role of intestinal flora in human Polyps, A-CRA (advanced colorectal adenoma) and CRC samples. To better clarify the role of A. muciniphila in CRC development, a pseudo-germ-free (GF) azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model was established, followed by infection with or without A. muciniphila. Metabolomic analysis and RNA-seq analysis showed tryptophan-mediated aryl hydrocarbon receptor (AhR) was significantly down-regulated in A. muciniphila-infected CRC mice. Then, mice with intestinal specific AhR deficiency (AhRfl/fl Cre) were generated and were used in 2 murine models: AOM/DSS treatment as a model of carcinogen-induced colon cancer and a genetically induced model using ApcMin/+ mice. Notably, AhR deficiency inhibited CRC growth in the AOM/DSS and ApcMin/+ mouse model. Moreover, AhR deficiency inhibited, rather than enhanced, tumor formation and tumor-derived organoids in Apc-deficient cells both in vivo and in vitro by activating Wnt/β-catenin signaling and TCF4/LEF1-dependent transcription. Furthermore, the antitumor effectiveness of A. muciniphila was abolished either in a human colon cancer tumor model induced by subcutaneous transplantation of AhR-silenced CRC cells, or AhR-deficienty spontaneous colorectal cancer model. In conclusion, supplementation with A. muciniphila. protected mice from CRC development by specifically inhibiting tryptophan-mediated AhR/β-catenin signaling.

Intestinal epithelial specific AhR deficient mice exhibit increased severity of experimentally induced chronic colitis

Inflammatory Bowel Diseases (IBD), including Crohn’s Disease (CD) and ulcerative colitis (UC), are associated with chronic inflammation of the gastrointestinal tract. While it is a multifactorial disease, recently the aryl hydrocarbon receptor (AhR) has been identified as a susceptibility gene for IBD. AhR is a ubiquitously expressed transcription factor and its activation results in xenobiotic detoxification by CYP1A1 and CYP1B1, as well as induction of anti‐inflammatory pathways such as IL‐10 and IL‐22. In the gut, AhR is vital in maintaining immune cell populations, proliferation, and motility. Previous studies utilizing constitutive knock out of AhR in IECs showed exacerbated inflammation in acute DSS model. How intestinal epithelial cell (IEC) AhR deficiency impacts chronic colitis resembling human IBD is not yet known. Here, we investigated the effect of removing of IEC AhR in an inducible manner utilizing DSS induced chronic colitis (resembling Th1/Th17 and Th2 infiltration as seen in CD patients) and elucidated underlying mechanisms.MethodsAhRfl/fl x Villin‐CreERT mice received intraperitoneal injections of Tamoxifen (AhRΔIECKO) to induce knockout or the vehicle (WT) for 5 days. The mice were given water or 1.0% DSS for 5 weeks, alternating weekly with water. Distal colon tissue samples were taken for myeloperoxidase (MPO) assay, immunostaining, and histology to assess severity of inflammation. Additionally, distal colon mucosa samples were collected for mRNA and protein expression. Serum was used to assess levels of LPS through ELISA.ResultsAhRΔIECKOmice given DSS had increased severity of colitis as evidenced by i) a significant decrease in the colon length when compared to the WT given DSS; ii) an increase in mRNA expression of pro‐inflammatory cytokines, IFN‐ γ and IL‐1□; iii) an increase in MPO activity in the distal colon; iv) a significant increase in the spleen to body weight ratio; and v) a significant increase in histological scores. Interestingly, DSS treatment caused an increase in lipopolysaccharide (LPS) in the serum that was significantly higher in AhRΔIECKO as compared to WT indicative of increased paracellular permeability. There were no significant differences observed in the protein expression of Occludin, Claudin‐2, and ZO‐1 between AhRΔIECKO and WT DSS groups, however AhR KO mice given DSS had significantly increased phosphorylated ERK1/2 (pERK normalized to total ERK) levels which may contribute to the increased permeability and severity of inflammation.ConclusionDeletion of AhR from intestinal epithelial cells increases severity of chronic colitis associated with increased permeability possibly through ERK1/2 dependent mechanisms.

Activation of aryl hydrocarbon receptor stimulates GLUT‐mediated glucose transport in human renal proximal tubular cells

Aryl hydrocarbon receptor (AhR), a ligand‐activated transcription factor, plays a major role in detoxification of xenobiotics. AhR deficiency has been shown to attenuate renal injury induced by diabetes. It is interesting whether the beneficial effect of AhR deficiency on renal injury in diabetes involve in alteration of intracellular glucose in renal tubular cells. The aim of this study was to investigate the effect and mechanism of AhR on glucose uptake into human renal proximal tubular cells. Exposure HK‐2 cells, human renal proximal tubular cell line, with omeprazole, an AhR agonist, increased glucose transporter (GLUT)‐mediated [3H]‐2‐Deoxy‐D glucose uptake into the cells in concentration‐dependent manner. The stimulatory effect of omeprazole was reduced by co‐incubation with CH223191, an AhR antagonist. These results indicate that the stimulatory effect of omeprazole on GLUT transport function regards activation of AhR. The kinetic study showed that omeprazole significantly decreased half‐maximal transport (Kt) but not maximal rate of transport (Jmax) of GLUT, suggesting that AhR might increase affinity of GLUT‐mediated glucose uptake into the cells. In addition, activation of AhR significantly induced cellular level of ROS. Inhibition of ROS using N‐acetyl‐L‐cysteine (NAC) inhibited the stimulatory effect of AhR on GLUT‐mediated glucose uptake. These results support that activation of AhR increases GLUT‐mediated glucose accumulation via increase in ROS production in human renal proximal tubular cells. Inhibition of AhR might be a target for treatment of high glucose‐induced renal injury from diabetes.

I3C-MEDIATED PROTECTION AGAINST COLITIS DEPENDENT ON AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS

Abstract Many current treatments for ulcerative colitis (UC) often causes side effects warranting safe therapeutic strategies to control the pathogenesis of colitis. Current study was aimed to ascertain the possible role of aryl hydrocarbon receptor (AhR) in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. We investigated how intestinal regulatory mediators were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis and had a higher disease score with increased inflammation in the colon compared to the controls. Also, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Transcriptome analysis of RNA isolated from enriched CECs of experimental mice showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice compared to controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids validated these results. In summary, AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

SUMMARYExternal and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.

AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS IS ESSENTIAL FOR I3C-MEDIATED PROTECTION AGAINST COLITIS

Abstract Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease that affects the colon and is characterized by localized tissue injury and disruption of the intestinal epithelial barrier. Pathophysiology of UC includes genetic predisposition, epithelial barrier defects, dysregulated immune responses, microbial dysbiosis, and environmental factors. Several therapeutic interventions for colitis have been identified, but many current treatments have various negative side effects, which is alarming given the incidence of UC is increasing around the world. Thus a search for safe therapeutic strategies to control the pathogenesis of colitis is still greatly needed. Previous studies from our laboratory reported that indole-3-carbinol (I3C), a natural plant product and ligand for the aryl hydrocarbon receptor (AhR), exhibited protective effects against colitis through innate lymphoid type 3 (ILC3) cells in an interleukin-22 (IL-22) dependent manner. The aim of this study was to ascertain the possible role of AhR in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. Using in vivo mouse studies and in vitro primary CEC organoid cultures, we investigated how key intestinal regulatory mediators (e.g. mucins and anti-microbial peptides, or AMPs) were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis. Results showed that AV mice had a higher disease score and increased inflammation in the colon when compared to wild type (WT) or littermate (LM) controls. In addition, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. This suggested that the IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice when compared to wild type (WT) or littermate (LM) controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids from WT and AV mice validated these results. Collectively, these data showed that AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

AHR EXPRESSION ON VIL1-EXPRESSING COLONIC EPITHELIAL CELLS IS ESSENTIAL FOR I3C-MEDIATED PROTECTION AGAINST COLITIS

Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease that affects the colon and is characterized by localized tissue injury and disruption of the intestinal epithelial barrier. Pathophysiology of UC includes genetic predisposition, epithelial barrier defects, dysregulated immune responses, microbial dysbiosis, and environmental factors. Several therapeutic interventions for colitis have been identified, but many current treatments have various negative side effects, which is alarming given the incidence of UC is increasing around the world. Thus a search for safe therapeutic strategies to control the pathogenesis of colitis is still greatly needed. Previous studies from our laboratory reported that indole-3-carbinol (I3C), a natural plant product and ligand for the aryl hydrocarbon receptor (AhR), exhibited protective effects against colitis through innate lymphoid type 3 (ILC3) cells in an interleukin-22 (IL-22) dependent manner. The aim of this study was to ascertain the possible role of AhR in vil1-expressing colonic epithelial cells (CECs) during I3C-mediated protection against colitis. Using in vivo mouse studies and in vitro primary CEC organoid cultures, we investigated how key intestinal regulatory mediators (e.g. mucins and anti-microbial peptides, or AMPs) were altered in the absence or presence of AhR in CECs during I3C treatment under colitis or colitis-like conditions. We generated conditional AhR knockout mice in vil1-expressing CECs using the cre-flox system and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the mice with AhR deficiency in CECs (AV mice) lost the protective effects of I3C treatment during colitis. Results showed that AV mice had a higher disease score and increased inflammation in the colon when compared to wild type (WT) or littermate (LM) controls. In addition, after treatment with I3C during DSS-induced colitis, AV mice were not able to prevent colitis-associated gut microbial dysbiosis even though flow cytometry analysis revealed AV mice were still capable of increasing IL-22 production by ILC3s in the colon. This suggested that the IL-22-ILC3 immune cell response was not the only major mechanism involved in I3C-mediated protection against colitis and regulation of the gut microbiome. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc3 and muc13), and tight junction proteins in AV mice when compared to wild type (WT) or littermate (LM) controls. PCR gene expression data and direct effects of I3C on CECs using colonic organoids from WT and AV mice validated these results. Collectively, these data showed that AhR expression in CECs play a critical role in I3C-mediated prevention of colitis.

The AhR ligand 2, 2′-aminophenyl indole (2AI) regulates microglia homeostasis and reduces pro-inflammatory signaling

Retinal degeneration is a leading cause of visual impairment and blindness worldwide. Microglia reactivity is a hallmark of neurodegenerative diseases and a driving force for retinal cell death and disease progression. Thus, immunomodulation emerges as a potential therapeutic option. AhR deficiency is known to trigger inflammation and previous studies revealed important roles for AhR ligands in neuroprotection without focusing on microglia. Here, we investigate the anti-inflammatory and antioxidant effects of the synthetic aryl hydrocarbon receptor (AhR) ligand 2, 2′-aminophenyl indole (2AI) on microglia reactivity. We showed that 2AI potently reduced pro-inflammatory gene expression and induced antioxidant genes in activated human and murine microglia cells, in LPS-stimulated retinal explants as well as in stressed human ARPE-19 cells. 2AI also diminished LPS-induced nitric oxide (NO) release, their neurotoxic activity on photoreceptor cells, phagocytosis, and migration in murine BV-2 cells as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory effects in BV-2 cells. Our results show for the first time, that the synthetic AhR agonist 2AI regulates microglia homeostasis, highlighting AhR as a potential drug target for immunomodulatory and antioxidant therapies.

Effects of a high-fat diet and global aryl hydrocarbon receptor deficiency on energy balance and liver retinoid status in male Sprague-Dawley rats

The physiological functions of the aryl hydrocarbon receptor (AHR) are only beginning to unfold. Studies in wildtype and AHR knockout (AHRKO) mice have recently disclosed that AHR activity is required for obesity and steatohepatitis to develop when mice are fed with a high-fat diet (HFD). In addition, a line of AHRKO mouse has been reported to accumulate retinoids in the liver. Whether these are universal manifestations across species related to AHR activity level is not known yet. Therefore, we here subjected wildtype and AHRKO male rats (on Sprague-Dawley background) to HFD feeding coupled with free access to 10% sucrose solution and water; controls received a standard diet and water. Although the HFD-fed rats consumed more energy throughout the 24-week feeding regimen, they did not get overweight. However, relative weights of the brown and epididymal adipose tissues were elevated in HFD-fed rats, while that of the liver was lower in AHRKO than wildtype rats. Moreover, the four groups exhibited diet- or genotype-dependent differences in biochemical variables, some of which suggested marked dissimilarities from AHRKO mice. Expression of pro- and anti-inflammatory genes was induced in livers of HFD-fed AHRKO rats, but histologically they did not differ from others. HFD reduced the hepatic concentrations of retinyl palmitate, 9-cis-4-oxo-13,14-dihydroretinoic acid and (suggestively) retinol, whereas AHR status had no effect. Hence, the background strain/line of AHRKO rat is resistant to diet-induced obesity, and AHR does not modulate this or liver retinoid concentrations. Yet, subtle AHR-dependent differences in energy balance-related factors exist despite similar weight development.

AhR expression on both Rorc-specific immune cells and Vil1-expressing colonic epithelial cells are essential for I3C-mediated protection against colitis

Abstract Colitis is an inflammatory bowel disease (IBD) characterized by dysregulation of the immune system. In our previous reports, we showed indole-3-carbinol (I3C), an aryl hydrocarbon receptor (AhR) ligand, was able to reduce colitis-induced disease severity in an interleukin-22 (IL-22) dependent manner, mainly though innate lymphoid type 3 (ILC3) cells. In the current study, we generated cell-specific conditional knockout (KO) mice that had AhR deficiency in either Rorc-expressing immune cells (ILC3s), or Vil1-expressing colonic epithelial cells (CECs). We induced colitis using the dextran sodium sulfate (DSS) model in control mice and conditional KO strains, those deficient of AhR in Rorc (AR mice) or Vil1 (AV mice) cells. Results showed that compared to control mice, both AR and AV mice lost the expected efficacy effects of I3C treatment during colitis, with higher disease score and inflammation in the colon. Interestingly, immune cell profiling by single-cell RNAseq and flow cytometry showed only AR mice lost the ability to increase IL-22 secretion by ILC3s after I3C treatment during colitis. Since AV mice were still able to increase IL-22 via ILC3s, this suggests AhR deficiency in the CECs leads to alterations in other I3C-mediated mechanisms used to prevent colitis development, independent of the IL22-ILC3 axis. Collectively these data show AhR expression in both specific immune cells and CECs play a pivotal role in I3C-mediated prevention of colitis, though their mechanisms differ.

Aryl hydrocarbon receptor deficiency causes the development of chronic obstructive pulmonary disease through the integration of multiple pathogenic mechanisms.

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease.

Activation of AhR-NQO1 Signaling Pathway Protects Against Alcohol-Induced Liver Injury by Improving Redox Balance.

Background & AimsAryl hydrocarbon receptor (AhR) is a liver-enriched xenobiotic receptor that plays important role in detoxification response in liver. This study aimed to investigate how AhR signaling may impact the pathogenesis of alcohol-related liver disease (ALD).MethodsChronic alcohol feeding animal studies were conducted with mouse models of hepatocyte-specific AhR knockout (AhRΔhep) and NAD(P)H quinone dehydrogenase 1 (NQO1) overexpression, and dietary supplementation of the AhR ligand indole-3-carbinol. Cell studies were conducted to define the causal role of AhR and NQO1 in regulation of redox balance and apoptosis.ResultsChronic alcohol consumption induced AhR activation and nuclear enrichment of NQO1 in hepatocytes of both alcoholic hepatitis patients and ALD mice. AhR deficiency exacerbated alcohol-induced liver injury, along with reduction of NQO1. Consistently, in vitro studies demonstrated that NQO1 expression was dependent on AhR. However, alcohol-induced NQO1 nuclear translocation was triggered by decreased cellular oxidized nicotinamide adenine dinucleotide (NAD+)-to-NADH ratio, rather than by AhR activation. Furthermore, both in vitro and in vivo overexpression NQO1 prevented alcohol-induced hepatic NAD+ depletion, thereby enhancing activities of NAD+-dependent enzymes and reversing alcohol-induced liver injury. In addition, therapeutic targeting of AhR in the liver with dietary indole-3-carbinol supplementation efficiently reversed alcoholic liver injury by AhR-NQO1 signaling activation.ConclusionsThis study demonstrated that AhR activation is a protective response to counteract alcohol-induced hepatic NAD+ depletion through induction of NQO1, and targeting the hepatic AhR-NQO1 pathway may serve as a novel therapeutic approach for ALD.

Gene and Protein Expression in Subjects With a Nystagmus-Associated AHR Mutation.

Recently, a consanguineous family was identified in Israel with three children affected by Infantile Nystagmus and Foveal Hypoplasia, following an autosomal recessive mode of inheritance. A homozygous stop mutation c.1861C > T; p.Q621∗ in the aryl hydrocarbon receptor (AHR) gene (AHR; MIM 600253) was identified that co-segregated with the disease in the larger family. AHR is the first gene to be identified causing an autosomal recessive Infantile Nystagmus-related disease in humans. The goal of this study is to delineate the molecular basis of this newly discovered human genetic disorder associated with a rare AHR gene mutation. The gene and protein expression levels of AHR and selected AHR targets from leukocyte cultures of healthy subjects and the patients were analyzed. We observed significant variation between mRNA and protein expression of CYP1A1, CYP1B1, and TiPARP under rest and AHR-induced conditions. The CYP1A1 enzymatic activity in induced leukocytes also differs significantly between the patients and healthy volunteers. Intriguingly, the heterozygous subjects demonstrate CYP1A1 and TiPARP gene and protein expression similar to homozygous patients. In contrast, CYP1B1 inducibility and expression vary between hetero- and homozygous subjects. Similarity and differences in gene and protein expression between heterozygotes and homozygous patients can give us a hint as to which metabolic pathway/s might be involved in the Nystagmus etiology. Thus, we have a unique human model for AHR deficiency that will allow us the opportunity to study the biochemical basis of this rare human mutation, as well as the involvement of AHR in other physiological processes.

Aryl hydrocarbon receptor deficiency enhanced airway inflammation and remodeling in a murine chronic asthma model

The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Recent studies have shown that AhR is a novel master regulator of the mucosal immune system, including lungs and intestine. To elucidate the role of AhR in chronic severe asthma, AhR wild-type and knockout mice (AhR-/- ) were sensitized and challenged with ovalbumin for 4weeks. To uncover the underlying mechanisms, inflammatory cells profile and cytokines production were analyzed in bronchial lavage fluid (BALF) and lung tissue. Compared to wild-type mice, AhR-/- mice had exacerbated asthma symptoms, including airway inflammation, mucus production, airway hyperresponsiveness, and airway remodeling. BALF monocytes, neutrophils, eosinophils, and lymphocytes were all enhanced in OVA-immunized AhR-/- mice. In OVA-immunized AhR-/- mice, T helper (Th) 17 cell-specific cytokine IL-17A, as well as airway remodeling factors, including epithelial-mesenchymal transition (EMT) markers and vascular endothelial growth factor (VEGF), were all enhanced in lung tissue. Moreover, human cohort studies showed that AhR gene expression in bronchial epithelial cells decreases in severe asthma patients. Loss of AhR leads to worsening of allergic asthma symptoms, indicating its importance in maintaining normal lung function and mediating disease severity.

Lack of the aryl hydrocarbon receptor accelerates aging in mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, largely known for its role in xenobiotic metabolism and detoxification as well as its crucial role as a regulator of inflammation. Here, we have compared a cohort wild-type and AhR-null mice along aging to study the relationship between this receptor and age-associated inflammation, termed as "inflammaging," both at a systemic and the CNS level. Our results show that AhR deficiency is associated with a premature aged phenotype, characterized by early inflammaging, as shown by an increase in plasma cytokines levels. The absence of AhR also promotes the appearance of brain aging anatomic features, such as the loss of the white matter integrity. In addition, AhR-/- mice present an earlier spatial memory impairment and an enhanced astrogliosis in the hippocampus when compared with their age-matched AhR+/+ controls. Importantly, we have found that AhR protein levels decrease with age in this brain structure, strongly suggesting a link between AhR and aging.-Bravo-Ferrer, I., Cuartero, M. I., Medina, V., Ahedo-Quero, D., Peña-Martínez, C., Pérez-Ruíz, A., Fernández-Valle, M. E., Hernández-Sánchez, C., Fernández-Salguero, P. M., Lizasoain, I., Moro, M. A. Lack of the aryl hydrocarbon receptor accelerates aging in mice.