Aryl Hydrocarbon Receptor Antagonist CH223191 Research Articles

The aryl hydrocarbon receptor differentially modulates the expression profile of antibody isotypes in a human B-cell line.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cμ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cμ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (ie activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

Indoxyl sulfate contributes to colorectal cancer cell proliferation and increased EGFR expression by activating AhR and Akt.

Although patients with chronic kidney disease (CKD) have a higher risk of colorectal cancer (CRC) aggravation, the connection between these two diseases is not well understood. Recent studies have shown that both CKD and CRC aggravation are closely related to an increased abundance of indole-producing Fusobacterium nucleatum in the gut. The indole absorbed from the gut is eventually metabolized to indoxyl sulfate in the liver. Since indoxyl sulfate is involved not only in accelerating CKD progression but also in the initiation and development of its associated complications, the present study aimed to clarify whether indoxyl sulfate induces the proliferation of CRC cells. This study found that indoxyl sulfate induced the proliferation of CRC-derived HCT-116 cells by activating the aryl hydrocarbon receptor (AhR) and the proto-oncogene Akt. The AhR antagonist CH223191 and Akt inhibitor MK2206 suppressed indoxyl sulfate-induced proliferation of HCT-116 cells. We also found that indoxyl sulfate upregulated epidermal growth factor receptor (EGFR) expression, which is associated with poor prognosis of CRC, whereas CH223191 and MK2206 repressed EGFR expression. Furthermore, indoxyl sulfate increased the sensitivity of CRC cells to EGF by upregulating EGFR expression. These findings suggest that indoxyl sulfate may be an important link between CKD and CRC aggravation.

Alhagi honey polysaccharide ameliorates ulcerative colitis by modulating gut microbiota–tryptophan metabolism via AhR activation

AbstractAlhagi honey (AH) is produced in arid and hot areas of Central Asia, and its polysaccharides (AP) are widely known for their activity in the treatment of intestinal diseases such as diarrhea. However, the therapeutic potential and mechanism of AP in ulcerative colitis (UC) remain unclear. Here, AH polysaccharide‐2 (AP2), a polysaccharide with the highest content in AP, was isolated and evaluated for its effects on dextran sulfate sodium (DSS)‐induced UC in mice. AP2 was found to alleviate UC symptoms and regulate gut microbiota dysbiosis by decreasing Helicobacter levels and increasing Lactobacillus levels. Analysis of PICRUSt2 predicted that AP2 may regulate carbohydrate and amino acid metabolism, and metabolomic analysis confirmed that AP2 promotes the metabolism of tryptophan to produce kynurenic acid (kyna). Moreover, kyna acted as an aryl hydrocarbon receptor (AhR) ligand, which activated AhR to increase the expression of the tight junction proteins claudin‐1 and occludin. Interestingly, AP2 showed similar effects in protecting the intestinal barrier and alleviating colitis as the AhR agonist 6‐formylindolo[3,2‐b]carbazole, and the AhR antagonist CH223191 partially blocked the therapeutic effect of AP2 in UC mice, indicating that the anti‐UC effect of AP2 was AhR dependent. These findings demonstrate that AP2 alleviates UC by regulating the gut microbiota and promoting tryptophan metabolism to generate kyna‐activated AhR. The insights gained from this study could help in the future development of AP2 as a drug candidate or functional food for the treatment of UC.

Wogonin improves colitis by activating the AhR pathway to regulate the plasticity of ILC3/ILC1

BackgroundIntestinal barrier dysfunction caused by the disrupted balance of group 3 innate lymphoid cells (ILC3)/group 1 innate lymphoid cells (ILC1) is a significant feature in the pathogenesis of inflammatory bowel disease (IBD). Activation of aryl hydrocarbon receptor (AhR) signaling contributes to the maintenance of ILC3/ILC1 balance. Wogonin, a natural flavonoid from Scutellaria baicalensis Georgi, can repair intestinal mucosal damage of IBD. However, it remains unclear if wogonin can exert a therapeutic effect by activating the AhR pathway to regulate the plasticity of ILC3/ILC1. PurposeIn this study, we investigated the immunomodulatory effects of wogonin on IBD and its potential mechanisms in vitro and in vivo. Study design and MethodsChronic colitis was induced by four cycles of 2 % DSS treatment in mice. 20 mg kg-1/day wogonin was administrated by oral gavage and mice were treated intraperitoneally with 10 mg kg-1/2 days CH223191 to block the AhR pathway. Colon tissues were processed for histopathological examination and evaluation of the epithelial barrier function by immunohistochemistry. The activation of the AhR pathway and the plasticity of ILC3/ILC1 were determined by western blot and flow cytometry. Then, we also detected the intestinal microflora and their metabolites by 16 s sequencing and non-targeted Metabolomics analysis. Furthermore, an in vitro culture system consisting of MNK3 cells and NCM460 cells, and a CETSA assay were performed to confirm the molecular mechanism. ResultsWogonin ameliorated histological severity of the colon, decreased the secretion of inflammatory factors, and increased tight junction proteins in colitis mice. These effects are associated with the tendency of conversion from ILC3 to ILC1 prevented by wogonin, which was offset by AhR antagonist CH223191. In addition, wogonin exerted the curative effect by altering gut microbiota to produce metabolites such as Kynurenic acid, and 1H-Indole-3-carboxaldehyde as AhR endogenous ligands. In vitro data further verified that wogonin as an exogenous ligand directly binds to the structural domain of AhR by CETSA. Also, the supernatant of MNK-3 cells stimulated with wogonin enhanced expression of Occludin and Claudin1 in NCM460 cells induced by LPS. ConclusionCumulatively, our study illustrated that wogonin improved the outcomes of DSS-induced chronic colitis via regulating the plasticity of ILC3/ILC1. Its specific mechanism is to binding to AhR directly, and to activate the AhR pathway indirectly by altering the tryptophan metabolisms of gut microbiota.

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.

The role of the aryl hydrocarbon receptor in mediating the effects of mono(2-ethylhexyl) phthalate in mouse ovarian antral follicles†.

Di(2-ethylhexyl) phthalate (DEHP) is a pervasive environmental toxicant used in the manufacturing of numerous consumer products, medical supplies, and building materials. DEHP is metabolized to mono(2-ethylhexyl) phthalate (MEHP). MEHP is an endocrine disruptor that adversely affects folliculogenesis and steroidogenesis in the ovary, but its mechanism of action is not fully understood. Thus, we tested the hypothesis that the aryl hydrocarbon receptor (AHR) plays a functional role in MEHP-mediated disruption of folliculogenesis and steroidogenesis. CD-1 mouse antral follicles were isolated and cultured with MEHP (0-400μM) in the presence or absence of the AHR antagonist CH223191 (1μM). MEHP treatment reduced follicle growth over a 96-h period, and this effect was partially rescued by co-culture with CH223191. MEHP exposure alone increased expression of known AHR targets, cytochrome P450 (CYP) enzymes Cyp1a1 and Cyp1b1, and this induction was blocked by CH223191. MEHP reduced media concentrations of estrone and estradiol compared to control. This effect was mitigated by co-culture with CH223191. Moreover, MEHP reduced the expression of the estrogen-sensitive genes progesterone receptor (Pgr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr) and co-treatment with CH223191 blocked this effect. Collectively, these data indicate that MEHP activates the AHR to impair follicle growth and reduce estrogen production and signaling in ovarian antral follicles.

Therapeutic effects of in vivo administration of an inhibitor of tryptophan 2,3-dioxygenase (680c91) for the treatment of fibroids: a preclinical study

ObjectiveFibroids are characterized by marked overexpression of Tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression. DesignAnimal and ex vivo human study. SubjectsSCID mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor. InterventionDaily intraperitoneal administration of 680C91 or vehicle for two months and in vitro studies with fibroid explants. Main Outcome MeasuresTumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants. ResultsCompound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after two months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of AhR (aryl hydrocarbon receptor) in the xenografts. The expression of CYP1B1 (cytochrome P450 family 1 subfamily B member 1), TGF-β3 (transforming growth factor beta 3), FN1 (fibronectin), CDK2 (cyclin-dependent kinase 2), E2F1 (E2F transcription factor 1), IL-8 (interleukin 8) and SPARC (secreted protein acidic and cysteine rich) mRNA, were lower in the xenografts of mice treated with 680C91 as compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1 and SPARC were lower in the xenografts of 680C91 treated mice as compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-β3, FN1, CDK2, E2F1, IL8 and SPARC mRNA by tryptophan which could be blocked by co-treatment with 680C91 and the AhR antagonist CH-223191. ConclusionThe results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and ECM accumulation.

The aryl hydrocarbon receptor controls mesenchymal stromal cell-mediated immunomodulation via ubiquitination of eukaryotic elongation factor-2 kinase

Mesenchymal stem cells (MSCs) have great therapeutic advantages due to their immunosuppressive properties. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose signaling plays an important role in the immune system. AHR may be involved in the regulation of MSC-associated immunomodulatory functions. However, the mechanisms by which AHR controls the immunosuppressive functions of MSCs are not well understood. Here, we report that Ahr-deficient MSCs show decreased therapeutic efficacy against graft-versus-host disease (GVHD) compared to wild-type (WT)-MSCs. This was probably due to decreased iNOS protein expression, which is a key regulatory enzyme in MSC immunomodulation. The expression of eukaryotic elongation factor 2 kinase (eEF2K), which inhibits the elongation stage of protein synthesis, is significantly increased in the Ahr-deficient MSCs. Inhibition of eEF2K restored iNOS protein expression. AHR is known to act as an E3 ligase together with CUL4B. We observed constitutive binding of AHR to eEF2K. Consequently, ubiquitination and degradation of eEF2K were inhibited in Ahr-deficient MSCs and by the AHR antagonist CH223191 in WT-MSCs. In summary, AHR regulates the immunomodulatory functions of MSCs through ubiquitination of eEF2K, thereby controlling iNOS protein synthesis and its product, nitric oxide levels.

Quercetin ameliorates ulcerative colitis by activating aryl hydrocarbon receptor to improve intestinal barrier integrity.

Ulcerative colitis (UC) pathogenesis is largely associated with intestinal epithelial barrier dysfunction. A therapeutic approach to UC involves the repair of damaged intestinal barrier. Our study aimed to investigate whether aryl hydrocarbon receptor (AhR) mediated the intestinal barrier repair effects of quercetin to ameliorate UC. 3% dextran sulfate sodium was used to induce colitic mice, and quercetin (25, 50, and 100 mg/kg) was administered orally for 10 days to assess the therapeutic effects. In vitro, Caco-2 cells were used to explore the effect of quercetin on tight junction protein expression and AhR activation. The results showed that quercetin alleviated colitic mice by restoring tight junctions (TJs) integrity via an AhR-dependent manner (p < 0.05). In vitro, quercetin dose-dependently elevated the expressions of TJs protein ZO-1 and Claudin1, and activated AhR by enhancing the expression of CYP1A1 and facilitating AhR nuclear translocation in Caco-2 cells (p < 0.05). While AhR antagonist CH223191 reversed the therapeutic effects of quercetin (p < 0.05) and blocked quercetin-induced AhR activation and enhancement of TJs protein (p < 0.05). In conclusion, quercetin repaired intestinal barrier dysfunction by activating AhR-mediated enhancement of TJs to alleviate UC. Our research offered new perspectives on how quercetin enhanced intestinal barrier function.

Oxidative stress and inflammation are mediated via aryl hydrocarbon receptor signalling in idiopathic membranous nephropathy.

Membranous nephropathy (MN) patients are diagnosed by the presence of phospholipase A2 receptor (PLA2R) before they progress to renal failure. However, the subepithelium-like immunocomplex deposit-mediated downstream molecular pathways are poorly understood. The aryl hydrocarbon receptor (AHR), NF-ƙB and Nrf2 pathways play central roles in the pathogenesis and progression of chronic kidney disease. However, their mutual effects on MN require further examination. Thus, we investigated the effect of AHR signalling on the NF-ƙB and Nrf2 pathways in IMN patients, cationic bovine serum albumin (CBSA)-injected rats and zymosan activation serum (ZAS)-treated podocytes. IMN patients show significantly decreased serum total protein and albumin levels, increased urine protein levels and intrarenal IgG4 and PLA2R protein expression in glomeruli compared with controls. IMN patients exhibited increased mRNA expression of intrarenal AHR and its target genes, including CYP1A1, CYP1A2, CYP1B1 and COX-2. This increase was accompanied by significantly upregulated protein expression of CD3, NF-ƙB p65 and COX-2 and significantly downregulated Nrf2 and HO-1 expression. Similarly, CBSA-induced rats showed severe proteinuria and activated intrarenal AHR signalling. This was accompanied by significantly upregulated protein expression of intrarenal p-IκBα, NF-κB p65 and its gene products, including COX-2, MCP-1, iNOS, 12-LOX, p47phox and p67phox, and significantly downregulated protein expression of Nrf2 and its gene products, including HO-1, catalase, GCLC, GCLM, MnSOD and NQO1. These results were further verified in ZAS-induced podocytes. Treatment with the AHR antagonist CH223191 and AHRsiRNA significantly preserved podocyte-specific protein expression and improved the NF-ƙB and Nrf2 pathways in ZAS-induced podocytes. In contrast, similar results were obtained in ZAS-induced podocytes treated with the NF-ƙB inhibitor BAY 11-7082 and NF-κBp65 siRNA. However, neither method had a significant effect on AHR signalling. Collectively, these results indicate that the NF-ƙB pathway is a downstream target of AHR signalling. Our findings suggest that blocking AHR signalling inhibits oxidative stress and inflammation, thereby improving proteinuria and renal injury.

2,2',4,4'-Tetrabromodiphenyl ether and cadmium co-exposure activates aryl hydrocarbon receptor pathway to induce ROS and GSDME-dependent pyroptosis in renal tubular epithelial cells.

We have previously found that a mixture exposure of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and cadmium (Cd) causes kidney damage; however, the mechanism was not fully understood. The aryl hydrocarbon receptor (AhR) is a ligand-receptor transcription factor that plays an important role in the adaptive response or metabolic detoxification of environmental toxins. Thus, this study aimed to examine the role of AhR in kidney toxicity. BDE-47 (50 μM) or Cd (5 μM) exposure reduced cell viability in renal tubular epithelial cells (HKC), with a larger effect observed in co-treatment. The cell morphology presented pyroptotic changes, including swollen cells, large bubbles, and plasma membrane pore formation. The gene expressions of AhR, heat shock protein 90 (Hsp90), AhR nuclear translocator (ARNT), and cytochrome P450 1B1 (CYP1B1) were increased, while CYP1A1 was decreased. Reactive oxygen species (ROS) were generated, which was reduced by the AhR antagonist CH223191. The apoptosis, necrosis, and intracellular lactated hydrogenase (LDH) release was elevated, and this was attenuated by N-acetylcysteine (NAC). Furthermore, the pyroptosis pathway was activated with increased protein levels of cleaved-caspase-3 and gasdermin E N-terminal (GSDME-NT), while caspase-8, caspase-3, and GSDME were decreased. These effects were alleviated by NAC and CH223191. Our data demonstrate a combined effect of BDE-47 and Cd on nephrotoxicity by activating AhR to induce ROS contributing to GSDME-dependent pyroptosis, and retardation of the AhR pathway could reduce this toxicity.

Aryl hydrocarbon receptors improve migraine-like pain behaviors in rats through the regulation of regulatory T cell/T-helper 17 cell-related homeostasis.

To investigate the effect of the aryl hydrocarbon receptor (AHR)/regulatory T cell (Treg)/T-helper 17 (Th17) cell pathway on the pathogenesis of migraine. Migraine is a disabling neurovascular disease that imposes an enormous burden on both individuals and society. The pathophysiological mechanisms of migraine remain controversial. Recent studies have suggested that immune dysfunction may be involved in the pathogenesis of migraine. The AHR, a receptor expressed on most immune cells, has been implicated in the occurrence of many autoimmune diseases; however, whether it is involved in the pathogenesis of migraine is unclear. A chronic migraine rat model was established through repeated intraperitoneal injection of nitroglycerin (NTG). The mechanical and thermal pain thresholds were assessed using von Frey filaments and radiant heat. Next, the protein expression levels of AHR in the trigeminal nucleus caudalis (TNC) region of chronic migraine (CM)-like rats were quantified and the changes in Treg/Th17-related transcription factors and inflammatory factors in the TNC were explored. To determine the role of AHR in CM, we examined the effects of the AHR agonist 2-(1'-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and AHR antagonist CH-223191 on pain behavior, c-Fos, calcitonin gene-related peptide (CGRP), AHR, and Treg/Th17-related factor expression in CM-like rats. Repeated administration of NTG significantly enhanced nociceptive hypersensitivity and increased expression of c-Fos and CGRP in rats, while AHR was significantly decreased in the TNC. In addition, the expression of the transcription factor forkhead box protein P3 and the signal transducer and activator of transcription 5 decreased significantly. In contrast, the expression of the transcription factor retinoic acid receptor-related orphan receptor γ t and signal transducer and activator of transcription 3 were significantly increased. Moreover, the mRNA level of transforming growth factor beta-1 was decreased, while that of interleukin (IL)-10 and IL-22 was increased in the TNC. The AHR agonist ITE alleviated migraine-like pain behaviors in rats, activated the AHR signaling pathway, and improved the imbalance of Treg/Th17-related transcription factors and inflammatory factors. Conversely, the AHR antagonist CH-223191 did not alleviate migraine-like pain behaviors in rats; and even exacerbated them. The AHR participates in the development of CM by regulating Treg/Th17-related homeostasis. Therefore, treatments targeting the AHR/Treg/Th17 signaling pathway could be new effective interventions for CM treatment.

Manuka honey activates the aryl hydrocarbon receptor: Implications for skin inflammation

Manuka honey (MH) is a complex nutritional material with antimicrobial, antioxidant and anti-inflammatory activity. We have previously shown that MH down regulates IL-4-induced CCL26 expression in immortalized keratinocytes. As MH contains potential ligands of the Aryl Hydrocarbon Receptor (AHR), a key regulator of skin homeostasis, we hypothesize that this effect is mediated via AHR activation. Here, we treated HaCaT cell lines, either stable transfected with an empty vector (EV-HaCaT) or in which AHR had been stable silenced (AHR-silenced HaCaT); or primary normal human epithelial keratinocytes (NHEK) with 2% MH for 24 h. This induced a 15.4-fold upregulation of CYP1A1 in EV-HaCaTs, which was significantly reduced in AHR-silenced cells. Pre-treatment with the AHR antagonist CH223191 completely abrogated this effect. Similar findings were observed in NHEK. In vivo treatment of the Cyp1a1Cre x R26ReYFP reporter mice strain’s skin with pure MH significantly induced CYP1A1 expression compared with Vaseline. Treatment of HaCaT with 2% MH significantly decreased baseline CYP1 enzymatic activity at 3 and 6 h but increased it after 12 h, suggesting that MH may activate the AHR both through direct and indirect means. Importantly, MH downregulation of IL-4-induced CCL26 mRNA and protein was abrogated in AHR-silenced HaCaTs and by pre-treatment with CH223191. Finally, MH significantly upregulated FLG expression in NHEK in an AHR-dependent manner. In conclusion, MH activates AHR, both in vitro and in vivo, thereby providing a mechanism of its IL4-induced CCL26 downregulation and upregulation of FLG expression. These results have potential clinical implications for atopic diseases and beyond.

#3048 INDOXYL SULFATE INHIBITS OSTEOGENESIS OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS THROUGH AHR/HES1/BMP2 PATHWAY

Abstract Background and Aims There is a high global burden of chronic kidney disease (CKD). The increased risk of bone fracture in patients with CKD suggests that CKD-associated factors, such as uremic toxins, might be the cause. Recent studies demonstrated that low turnover osteodystrophy and bone formation defect develop in early CKD, which indicates that uremic toxins, particularly in relatively low concentrations, can potentially be a cause. Thus far, studies about defected osteogenesis adopted high concentrations of uremic toxins (for example, indoxyl sulfate (ISx) &amp;gt;100 μM). Furthermore, the pathology is not yet fully investigated. Uremic toxin induced aryl hydrocarbon receptor (AHR) has been linked to bone formation defect, however, the mechanisms are incompletely understood. AHR inhibit osteogenesis during early processes of differentiation of early cells (stem cells), but whether uremic toxins induced AHR to affect stem cell in osteogenesis lineage has yet to be studied. The present study aims to clarify whether ISx at concentrations approximate to average in CKD patients (20 μM in free form) affect osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs), and further survey the underline signal transduction of AHR-induced defected osteogenesis. Method Mineralization of D1 cells (mouse BMSCs) was evaluated by alizarin red S staining (ARS). The mRNA expressions and protein levels of osteogenic markers including Runx2, BMP2, ALP, and osteocalcin were determined by quantitative real-time polymerase chain reaction (RT-PCR) and western blotting. AHR translocation into the nucleus was evaluated by an immunofluorescence confocal microscope. Reporter gene assays were adopted to study the transcriptional regulation of Runx2 and BMP2. Results ISx at concentrations ≤50 μM significantly downregulated mineralization of BMSCs with the most influence in the early stage of osteogenesis. ISx decreased the mRNA expressions and protein levels of osteogenic markers Runx2, BMP2, ALP, and osteocalcin. ISx of 50 μM induced AHR translocation into the nucleus and upregulated AHR target genes CYP1a1 and CYP1b1. The ISx-induced mineralization defects of BMSCs were counteracted by AHR antagonist CH223191, while downregulated gene expressions of osteogenic markers were recovered by AHR antagonist and knockdown (KD) of AHR. In mechanistic studies, ISx upregulated mRNA levels of hairy and enhancer of split (HES1), which is a primary target of NOTCH signaling. Although there were no significant differences in expressions of Notch1∼4, the upregulated Hes1 was attenuated by the antagonist and KD of AHR, which indicated that Hes1 was directly regulated by AHR. We identified putative binding sites of transcription repressor Hes1 in the promoter area of BMP2 and Runx2. Following experimental treatment of ISx, expression levels of BMP2 and Runx2 monitored by reporter assays were downregulated, which indicated that Hes1 plays as a transcription repressor to downregulated BMP2 and Runx2. Conclusion ISx under clinically relevant concentrations attenuated mineralization of BMSCs, particularly in the early stage of osteogenesis. ISx-induced AHR upregulated Hes1, which inhibited expressions of not only Runx2, but also BMP2, which has not been published before. A study of the underline signal transduction pathways relevant to the AHR genomic control system may provide potential therapy for low bone formation in early CKD.

Aryl hydrocarbon receptor is regulated via multiple mechanisms in human keratinocytes

Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polycyclic aromatic hydrocarbons of synthetic and natural origin. While a number of novel AhR ligands have been recently identified, little is known about their possible influence on AhR levels and stability. We used western blot, qRT-PCR and immunocytochemistry to determine the effects of AhR ligands on AhR expression in N-TERT (N-TERT1) immortalized human keratinocytes, and immunohistochemistry to assess patterns of AhR expression in human and mouse skin and skin appendages. While AhR was highly expressed in cultured keratinocytes and in the skin, it was found primarily in the cytoplasm, but not in the nucleus, suggesting its inactivity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and eventual inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD, FICZ, caused near-complete disappearance of AhR, and treatment with I3C resulted in substantially diminished level of AhR possibly due to ligand-induced AhR degradation. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Additionally, AhR decay was blocked by ligand-selective AhR antagonist CH223191, implying substrate-induced mechanism of degradation. Furthermore, degradation of AhR was blocked in N-TERT cells with knockdown of AhR dimerization partner ARNT (HIF1β), suggesting that ARNT is required for AhR proteolysis. However, addition of hypoxia mimetics (HIF1 pathway activators) CoCl2 and DMOG had only minor effects on degradation of AhR. Additionally, inhibition of HDACs with Trichostatin A resulted in enhanced expression of AhR in both untreated and ligand-treated cells. These results demonstrate that in immortalized epidermal keratinocytes AhR is primarily regulated post-translationally via proteasome-mediated degradation, and suggest potential means to manipulate AhR levels and signaling in the skin. Overall, the AhR is regulated via multiple mechanisms, including proteasomal ligand- and ARNT-dependent degradation, and transcriptional regulation by HDACs, implying complex system of balancing its expression and protein stability.

3, 3’-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway

Background3, 3’-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition. PurposeThis study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition. MethodsCytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo. ResultsDIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67+ F4/80+ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain. ConclusionDIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via “AhR-Nrf2/Arg-1” signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.

Astrocytic aryl hydrocarbon receptor mediates chronic kidney disease-associated mental disorders involving GLT1 hypofunction and neuronal activity enhancement in the mouse brain.

Chronic kidney disease (CKD)-associated mental disorders have been attributed to the excessive accumulation of hemodialysis-resistant indoxyl-3-sulfate (I3S) in the brain. I3S not only induces oxidative stress but is also a potent endogenous agonist of the aryl hydrocarbon receptor (AhR). Here, we investigated the role of AhR in CKD-induced brain disorders using a 5/6 nephrectomy-induced CKD mouse model, which showed increased I3S concentration in both blood and brain, anxiety and impaired novelty recognition, and AhR activation in the anterior cortex. GFAP+ reactive astrocytes were increased accompanied with the reduction of glutamate transporter 1 (GLT1) on perineuronal astrocytic processes (PAPs) in the anterior cingulate cortex (ACC) in CKD mice, and these alterations were attenuated in both neural lineage-specific and astrocyte-specific Ahr conditional knockout mice (nAhrCKO and aAhrCKO). By using chronic I3S treatment in primary astrocytes and glia-neuron (GN) mix cultures to mimic the CKD brain microenvironment, we also found significant reduction of GLT1 expression and activity in an AhR-dependent manner. Chronic I3S treatment induced AhR-dependent pro-oxidant Nox1 and AhR-independent anti-oxidant HO-1 expressions. Notably, AhR mediates chronic I3S-induced neuronal activity enhancement and synaptotoxicity in GN mix, not neuron-enriched cortical culture. In CKD mice, neuronal activity enhancement was observed in ACC and hippocampal CA1, and these responses were abrogated by both nAhrCKO and aAhrCKO. Finally, intranasal AhR antagonist CH-223191 administration significantly ameliorated the GLT1/PAPs reduction, increase in c-Fos+ neurons, and memory impairment in the CKD mice. Thus, astrocytic AhR plays a crucial role in the CKD-induced disturbance of neuron-astrocyte interaction and mental disorders.

Glutamine alleviates intestinal injury in a murine burn sepsis model by maintaining intestinal intraepithelial lymphocyte homeostasis

Intestinal intraepithelial lymphocytes (IELs) play a sentinel role in the mucosal immune system because of their unique anatomical location in the epithelial layer. The disruption of IEL homeostasis is implicated in driving the intestinal injury of many typical inflammatory disorders, such as inflammatory bowel disease (IBD) and sepsis. Therefore, it is meaningful to alleviate intestinal injury by restoring IEL homeostasis in disease conditions. This study explores the effects of glutamine on intestinal IEL homeostasis in a murine model of burn sepsis. We report that glutamine inhibits inflammatory response and reduces injury in the small intestine of burn septic mice. This effect is attributed to the maintaining of IEL homeostasis by suppressing apoptosis and restoring the disrupted subpopulation balance induced by burn sepsis. Mechanistically, we show that glutamine does not affect the IL-15 dependent mechanisms that drive the maintenance and differentiation of IELs. Instead, glutamine sustains IEL homeostasis by upregulate aryl hydrocarbon receptor (AHR) and interleukin (IL)-22 transcription and expression. Consistently, the protective roles of glutamine in burn septic mice were repressed by further supplement with an AHR antagonist CH-223191. Collectively, our study reveals a new role of glutamine to maintain IEL homeostasis by activating the AHR signaling pathway, which in turn ameliorates intestinal injury in burn sepsis.

Blocking the Aryl Hydrocarbon Receptor Alleviates Myocardial Ischemia/Reperfusion Injury in Rats.

Restoring the blood perfusion of ischemic heart tissues is the main treatment for myocardial ischemia. However, the accompanying myocardial ischemia reperfusion injury (IRI) would aggravate myocardial damage. Previous studies have confirmed that aryl hydrocarbon receptor (AhR) is closely correlated to kidney and intestinal IRI. The present study aimed to explore the relationship between AhR and myocardial IRI. An oxygen glucose deprivation/reoxygenation (OGD/R) model of H9c2 cells and an ischemia/reperfusion (I/R) model of Sprague-Dawley rat myocardium were established. OGD/R cells and myocardial IRI rats were treated with different concentrations of the AhR antagonist CH-223191 or agonist 6-formylindolo[3,2-b] carbazole (FICZ). Under the conditions of normoxia and hypoxia/reoxygenation, the activity of cardiomyocytes, lactate dehydrogenase (LDH) and cell reactive oxygen species (ROS) were detected. In rats, myocardial pathological damage and markers of myocardial injury were detected. According to the results of the cell viability, LDH and ROS tests in vitro, both CH-223191 and FICZ showed no myocardial protection under OGD/R conditions. However, the histological staining and analysis of myocardial injury marker LDH in vitro revealed that CH-223191 could significantly reduce the myocardial IRI. AhR exhibited a different effect on myocardial IRI in vitro and in vivo. In vivo, CH-223191 could significantly alleviate the myocardial IRI, suggesting that inhibition of AhR may play a role in myocardial protection, and AhR may serve as a potential treatment target for myocardial IRI.

Bifidobacterium longum mediated tryptophan metabolism to improve atopic dermatitis via the gut-skin axis

ABSTRACT Gut microbial disturbance affects allergic diseases including asthma, atopic dermatitis (AD) via the aberrant immune response. Some Bifidobacterial species and strains have been reported to improve AD via modulating immune-microbe interactions in patients. However, the effective metabolites and mechanism of alleviating AD in bifidobacteria remain to be elucidated. This study aimed to explore the microbial metabolite and mechanism of Bifidobacterium longum to improve AD. Based on shotgun metagenomic sequencing and UHPLC Q-Exactive-MS targeted metabolic experiments in vitro and in vivo, we focused on tryptophan metabolism and indole derivatives, which are endogenous ligands for aryl hydrocarbon receptor (AHR). Indole-3-carbaldehyde (I3C), a tryptophan metabolite of B. longum CCFM1029 activated AHR-mediated immune signaling pathway to improve AD symptoms in animal and clinical experiments. B. longum CCFM1029 upregulated tryptophan metabolism and increased I3C to suppress aberrant T helper 2 type immune responses, but these benefits were eliminated by AHR antagonist CH223191. Furthermore, B. longum CCFM1029 reshaped gut microbial composition in AD patients, increased fecal and serum I3C, and maintained the abundance of Lachnospiraceae related to tryptophan metabolism of gut microbiota. The results suggested that based on the interactions of the gut-skin axis, B. longum CCFM1029 upregulated tryptophan metabolism and produced I3C to activate AHR-mediated immune response, alleviating AD symptoms. Indole derivates, microbial metabolites of tryptophan, may be the potential metabolites of bifidobacteria to alleviate AD via the AHR signaling pathway.