Aryl Hydrocarbon Receptor Expression Research Articles

#5502 PRO-INFLAMMATORY EFFECTS OF BISPHENOL A AND POLYETHYLENE MICROPLASTICS ON HUMAN RENAL TUBULAR CELLS

Abstract Background Growing evidence shows that microplastics (MP) and their chemical derivates contaminate the environment and accumulate in the gut, liver, lungs, and kidneys. Furthermore, MP can adsorb a wide range of toxic substances (heavy metals, polycyclic aromatic hydrocarbons, Bisphenol A [B]), causing the accumulation of multiple pollutants and so, enhancing their toxicity (the so-called “Trojan Horse” effect). An epidemic of chronic kidney disease (CKD) of uncertain etiology is emerging worldwide and understanding the involvement of environmental pollution may be critical for health policies and public health responses. Inflammation is intimately linked to renal disease, and proinflammatory cytokines and chemokines are important mediators of fibrosis in the tubulointerstitial compartments. Aims To evaluate the effects of B and Polyethylene (Pe)-MPs and their combination B+Pe-MP on human renal tubular cells (HK-2). To this end, the activation of the Aryl hydrocarbon Receptor (AHR), a transcription factor binding xenobiotics, and the expression of inflammatory molecules were explored. Method HK-2 were exposed to 0 (No Treated cells: NT), 100 nM B, 0.2 mg/ml Pe-MP (diameter 1-4 µm), and B+Pe-MP for 5-24 hrs. Cell uptake of (FL)uorescent Pe-MP was visualized by microscope; viability was assessed by MTT; AHR, HSP90, CCL2, CCR2, CCL5, and CCR5 expression was studied by rt-PCR, Western blot, and immunofluorescence Results After 5 hrs exposure, a FLPe-MP cytoplasmatic and perinuclear deposition was observed (Figure 1). After 24 hrs, Pe-MP and B+Pe-MP exposition reduced cell viability (−20% vs NT p < 0.001). The treatments increased AHR expression (1.5-3-fold vs NT, p < 0.05-0.001), which had a nuclear localization both at 5 and 24 hrs. Conversely, HSP90 expression, a chaperone that keeps AHR inactive, decreased (−20-40% vs NT, p < 0.05) (Figure 2). After 5 hrs, CCL2 levels rose (1.8-3.5 fold, p<0.05-0.0001 vs NT) as well as its receptor CCR2 (2.3-6.7 fold, p<0.05-0.01). Similarly, CCL5 and CCR5 were, respectively, 1.6-2.7 and 3.8-8.4 fold overexpressed with respect to NT (p < 0.05-0.01). Interestingly, the co-exposure significantly enhanced the expression of these inflammatory molecules Conclusion Our data suggest that B and Pe-MPs induce a pro-inflammatory response in renal tubular cells and that the combined effect of B and Pe-MPs induces a worse effect on HK2 than B or MPs alone, supporting the theory of the “trojan horse” effect mediated by MPs. Therefore, MPs could be a trigger of kidney damage, thus confirming the potential impact of the environment on the pathogenesis of the renal disease. Moreover, these data could address the importance of investigating any health implications associated with MPs.

#6827 DI(2-ETHYLHEXYL) PHTHALATE INDUCES RENAL PROXIMAL TUBULAR CELLS TO UNDERGO EPITHELIAL-MESENCHYMAL TRANSITION VIA AHR SIGNALING

Abstract Background and Aims Environmental factors account for the majority of identified risks associated with CKD of uncertain etiology (CKDu). Among the various environmental factors, plasticizer di(2-ethylhexyl) phthalate (DEHP) is widely used in modern life. The exposure to DEHP is extensive but mostly underestimated in the general population. The link between DEHP exposure and renal injuries has been scarcely reported but the underlying mechanisms are largely unknown. This study aimed to examine the mechanisms by which DEHP causes renal tubular injuries. Method Human proximal tubular cells (PTC), HK2 cells, were used as a cellular model to test the central hypothesis of this study. Cells were treated with various concentrations of DEHP at different time points. WST-1 assay was used to examine the impacts of DEHP on cell viability. Bright field microscopy and immunofluorescence were used to analyze the morphological evidence of epithelial-mesenchymal transition (EMT). Western blots were used to investigate the changes in the expression of EMT-related proteins and arylhydrocarbon receptor (AhR) signaling. Inhibitors of AhR signaling were employed to investigate the crucial roles of AhR signaling in DEHP-induced EMT in HK2 cells. Results By using WST-1 assay, we found that DEHP did not affect cell viability when the examined concentration was below 50 μM, which resembled the low-level environmental exposures. However, when the concentration was higher than 100 μM, DEHP reduced the cell viability in a dose-dependent manner. In response to DEHP, HK-2 cells changed from cuboid to spindle shape when cultured with DEHP (25 μM). This morphological evidence became evident when the treatment time was longer than 48 hours. At the protein levels, DEHP resulted in down-regulation of e-cadherin and upregulation of vimentin and α-SMA. DEHP also lead to upregulation of AhR. The inhibitor of AhR signaling, CQDP (chloroquine diphosphate) reversed the DEHP-induced EMT, which were evidenced by morphology and by the expression patterns of EMT-related proteins. Conclusion At cellular level, our results suggested that low-level environmental exposures of DEHP led to EMT in renal PTC via AhR signaling.

Ethyl Caffeate Can Inhibit Aryl Hydrocarbon Receptor (AhR) Signaling and AhR-Mediated Potentiation of Mast Cell Activation.

Ethyl caffeate (EC) is a natural phenolic compound that is present in several medicinal plants used to treat inflammatory disorders. However, its anti-inflammatory mechanisms are not fully understood. Here, we report that EC inhibits aryl hydrocarbon receptor (AhR) signaling and that this is associated with its anti-allergic activity. EC inhibited AhR activation, induced by the AhR ligands FICZ and DHNA in AhR signaling-reporter cells and mouse bone marrow-derived mast cells (BMMCs), as assessed by AhR target gene expressions such as CYP1A1. EC also inhibited the FICZ-induced downregulation of AhR expression and DHNA-induced IL-6 production in BMMCs. Furthermore, the pretreatment of mice with orally administered EC inhibited DHNA-induced CYP1A1 expression in the intestine. Notably, both EC and CH-223191, a well-established AhR antagonist, inhibited IgE-mediated degranulation in BMMCs grown in a cell culture medium containing significant amounts of AhR ligands. Furthermore, oral administration of EC or CH-223191 to mice inhibited the PCA reaction associated with the suppression of constitutive CYP1A1 expression within the skin. Collectively, EC inhibited AhR signaling and AhR-mediated potentiation of mast cell activation due to the intrinsic AhR activity in both the culture medium and normal mouse skin. Given the AhR control of inflammation, these findings suggest a novel mechanism for the anti-inflammatory activity of EC.

Fenofibrate attenuates the cytotoxic effect of cisplatin on lung cancer cells by enhancing the antioxidant defense system invitro.

Fenofibrate (FF) is a peroxisome proliferator- activated receptor (PPAR)-α agonist that is widely used for the treatment of hyperlipidemia. It has been shown to have pleiotropic actions beyond its hypolipidemic effect. FF has been shown to exert a cytotoxic effect on some cancer cells when used at higher than clinically relevant concentrations; on the other hand, its cytoprotective effect on normal cells has also been reported. The present study assessed the effect of FF on cisplatin (CDDP) cytotoxicity to lung cancer cells in vitro. The results demonstrated that the effect of FF on lung cancer cells depends on its concentration. FF at ≤50 µM, which is a clinically achievable blood concentration, attenuated CDDP cytotoxicity to lung cancer cells, whereas FF at ≥100 µM, albeit clinically unachievable, had an anticancer effect. The mechanism of FF attenuation of CDDP cytotoxicity involved PPAR-α-dependent aryl hydrocarbon receptor (AhR) expression, which in turn stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and antioxidant production, resulting in lung cancer cell protection from CDDP-evoked oxidative damage. In conclusion, the present study revealed that FF, at clinically relevant concentrations, attenuated CDDP cytotoxicity to lung cancer cells by enhancing the antioxidant defense system through activation of a pathway that involves the PPAR-α-PPAR response element-AhR xenobiotic response element-Nrf2-antioxidant response element. These findings suggested that concomitant use of FF with CDDP may compromise the efficacy of chemotherapy. Although the anticancer property of FF has recently attracted much attention, concentrations that exceed clinically relevant concentrations are required.

Spermidine protects intestinal mucosal barrier function in mice colitis via the AhR/Nrf2 and AhR/STAT3 signaling pathways

Aryl hydrocarbon receptor (AhR) activation promotes intestinal barrier repair and enhances the gut mucosal barrier function in inflammatory bowel diseases (IBD). Spermidine is beneficial in several murine models of IBD and may affect AhR activity. However, the precise effects of spermidine on the intestinal barrier and AhR remain unclear. This study was designed to investigate whether spermidine affects AhR and gut barrier function in IBD models as well as, its underlying mechanism. We used dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mice, as well as, Caco2 cells incubated with TNF-α and IFN-γ to establish multiple IBD models, followed by spermidine intervention. Alcian blue/Periodic acid-Schiff (AB/PAS) staining, Fluorescein isothiocyanate (FITC)-dextran permeability assay, transepithelial electrical resistance (TER), tight junction protein (TJs) expression, and 16S rRNA scope in situ hybridization were performed to assess intestinal barrier function. AhR expression and the associated pathways were measured. AhR-targeted adeno-associated virus (AAV) and siRNA were used to explore the related molecular mechanisms. Spermidine significantly attenuated the increased intestinal permeability, decreased TER, abnormal distribution of TJs in colitis, and bacterial translocation from the gut tract. Additionally, it significantly increased AhR and Nrf2 expression and inhibited STAT3 phosphorylation. However, the protective effects of spermidine and the related alterations in pathway proteins were largely abolished by the specific inhibition of AhR. Our study demonstrated that spermidine rescues intestinal barrier defects in mice with colitis via the AhR-Nrf2 and AhR-STAT3 pathways, providing a potential therapeutic agent for IBD and other conditions associated with dysregulated gut barrier function.

Tryptophan depletion sensitizes the AHR pathway by increasing AHR expression and GCN2/LAT1-mediated kynurenine uptake, and potentiates induction of regulatory T lymphocytes

BackgroundIndoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated...

Isovitexin prevents DSS-induced colitis through inhibiting inflammation and preserving intestinal barrier integrity through activating AhR

Isovitexin (ISO) is a glycosylated flavonoid obtained from Asian rice that has been reported to have anti-inflammatory effect. However, the effects of ISO on colitis have not been reported. In the present study, we aimed to explore the protective effects of isovitexin on colitis using the dextran sodium sulfate (DSS)-induced model. In vitro, the protective mechanism was investigated in TNF-α-stimulated IEC cells. Inflammatory cytokines were measured by ELISA. The signaling pathways were measured by Western blot analysis. ISO attenuated DSS-induced colitis through reducing body weight loss and colonic histological changes. Also, the levels of TNF-α and IL-1β induced by DSS were inhibited by ISO. The MPO activity induced by DSS was attenuated by ISO. In vitro, ISO inhibited IL-6 and IL-1β production in TNF-α-stimulated cells. ISO increased the expression of tight junction proteins ZO-1 and occludin. Also, ISO inhibited TNF-α-induced NF-κB activation. In addition, ISO was found to increase the expression of aryl hydrocarbon receptor (AhR). And inhibition of AhR by its antagonist CH223191 could reverse these effects of ISO. ISO inhibited DSS-induced colitis in mice through suppressing inflammation and preserving intestinal barrier integrity through activating AhR. ISO may be useful as a potential therapeutic agent for colitis.

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.

Human sperm proteome reveals the effect of environmental borne seminal polyaromatic hydrocarbons exposome in etiology of idiopathic male factor infertility.

Introduction: Polyaromatic hydrocarbons (PAHs) are considered as redox active environmental toxicants inducing oxidative stress (OS) mediated injury to cells. Oxidative predominance is reported in 30%-80% of idiopathic male infertility (IMI) patients. Hence, this work aims to unravel correlation, if any, between seminal PAH exposome and sperm function in IMI patients through a proteomic approach. Methods: Seminal PAH exposome was analyzed in 43 fertile donors and 60 IMI patients by HPLC and receiver operating characteristic (ROC) curve was applied to find out the cut-off limits. Spermatozoa proteome was analyzed by label free liquid chromatography mass spectroscopy (LC-MS/MS) followed by molecular pathway analysis using bioinformatic tools. Validation of key proteins' expression and protein oxidative modifications were analyzed by western blot. Results and discussion: Of the 16 standards toxic PAH, 13 were detected in semen. Impact of the different PAHs on fertility are Anthracene < benzo (a) pyrene < benzo [b] fluoranthene < Fluoranthene < benzo (a) anthracene <indol (123CD) pyrene < pyrene < naphthalene < dibenzo (AH) anthracene < fluorene < 2bromonaphthalene < chrysene < benzo (GH1) perylene as revealed by ROC Curve analysis (AUCROC). Benzo [a] pyrene is invariably present in all infertile patients while naphthalene is present in both groups. Of the total 773 detected proteins (Control: 631 and PAH: 717); 71 were differentially expressed (13 underexpressed, 58 overexpressed) in IMI patients. Enrichment analysis revealed them to be involved in mitochondrial dysfunction and oxidative phosphorylation, DNA damage, Aryl hydrocarbon receptor (AHR) signaling, xenobiotic metabolism and induction of NRF-2 mediated OS response. Increased 4-hydroxynonenal and nitrosylated protein adduct formation, and declined antioxidant defense validates induction of OS. Increased GSH/GSSG ratio in patients may be an adaptive response for PAH metabolism via conjugation as evidenced by over-expression of AHR and Heat shock protein 90 beta (HSP90β) in patients. Seminal PAH concentrations, particularly benzo (a) pyrene can be used as a marker to distinguish IMI from fertile ones with 66.67% sensitivity and 100% specificity (95% confidence interval) along with oxidative protein modification and expression of AHR and HSP90β.

Urolithin A Alleviates Colitis in Mice by Improving Gut Microbiota Dysbiosis, Modulating Microbial Tryptophan Metabolism, and Triggering AhR Activation

Urolithin A (UroA) is a microbial metabolite derived from ellagitannins and ellagic acid with good bioavailability. In this study, we explored the anticolitis activity of UroA and clarified the mechanism by 16S rDNA sequencing and metabonomics. UroA alleviated dextran sulfate sodium (DSS)-induced colitis in mice, characterized by a decreased disease activity index, increased colon length, and improved colonic histopathological lesions, along with inhibited phosphorylation of the mitogen-activated protein kinase signaling pathway. In addition, UroA improved gut microbiota dysbiosis and modulated the microbiota metabolome. Furthermore, targeted metabolomics focused on tryptophan catabolites showed that UroA significantly increased the production of indole-3-aldehyde (IAld) and subsequently led to increased colonic expression of aryl hydrocarbon receptor (AhR) and promoted the serum content of IL-22 in mice with colitis. Collectively, our data identified a novel anticolitis mechanism of UroA by improving gut microbiota dysbiosis, modulating microbial tryptophan metabolism, promoting IAld production, and triggering AhR/IL-22 axis activation. However, a limitation noted in this study is that these beneficial effects of UroA were found at 50 μM in vitro and 20 mg/kg in vivo, which were nonphysiological concentrations.

Metabolic profiling reveals altered tryptophan metabolism in patients with kawasaki disease.

Kawasaki disease (KD) is a childhood vasculitis disease that is difficult to diagnose, and there is an urgent need for the identification of accurate and specific biomarkers. Here, we aimed to investigate metabolic alterations in patients with KD to determine novel diagnostic and prognostic biomarkers for KD. To this end, we performed untargeted metabolomics and found that several metabolic pathways were significantly enriched, including amino acid, lipid, and tryptophan metabolism, the latter of which we focused on particularly. Tryptophan-targeted metabolomics was conducted to explore the role of tryptophan metabolism in KD. The results showed that Trp and indole acetic acid (IAA) levels markedly decreased, and that l-kynurenine (Kyn) and kynurenic acid (Kyna) levels were considerably higher in patients with KD than in healthy controls. Changes in Trp, IAA, Kyn, and Kyna levels in a KD coronary arteritis mouse model were consistent with those in patients with KD. We further analyzed public single-cell RNA sequencing data of patients with KD and revealed that their peripheral blood mononuclear cells showed Aryl hydrocarbon receptor expression that was remarkably higher than that of healthy children. These results suggest that the Trp metabolic pathway is significantly altered in KD and that metabolic indicators may serve as novel diagnostic and therapeutic biomarkers for KD.

Activated intestinal microbiome-associated tryptophan metabolism upregulates aryl hydrocarbon receptor to promote osteoarthritis in a rat model

To evaluate the role of aryl hydrocarbon receptor in the pathogenesis of osteoarthritis (OA) and its association with intestinal microbiome-related tryptophan metabolism. Cartilage was isolated from OA patients undergoing total knee arthroplasty and analyzed for expression of aryl hydrocarbon receptor (AhR) and cytochrome P450 of family 1, subfamily A, and polypeptide 1 (CyP1A1). To gain mechanistic insights, OA model was induced in Sprague Dawley rats after antibiotic pretreatment combined with a tryptophan-rich diet (or not). The severity of OA was assessed eight weeks after surgery according to the Osteoarthritis Research Society International grading system. Expression of AhR, CyP1A1 as well as markers of bone and cartilage metabolism, inflammation, and intestinal microbiome-related tryptophan metabolism was assessed. Severity of OA in cartilage from patients positively correlated with expression of AhR and CyP1A1 in chondrocytes. In the rat model of OA, antibiotic pretreatment led to lower expression of AhR and CyP1A1 and lower serum levels of lipopolysaccharide (LPS). Conversely, antibiotics upregulated Col2A1 and SOX9 in cartilage, which mitigated the cartilage damage and synovitis, reduced the relative abundance of Lactobacillus. Additional tryptophan supplementation activated intestinal microbiome-related tryptophan metabolism, antagonizing the effects of antibiotics, exacerbating OA synovitis. Our study established an underlying intestinal microbiome associated tryptophan metabolism-OA connection which sets a new target for exploring OA pathogenesis. The alteration of tryptophan metabolism might prompt the activation and synthesis of AhR, accelerating the development of OA.

Indol-3-Carbinol ameliorates colitis in mice by AhR-mediated production of antimicrobial peptides and mucins

Abstract Indole-3-carbinol (I3C) is a dietary compound that acts as a ligand for the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals and a regulator of the immune response. The pathogenesis of colitis involves multiple interactions between microbial dysbiosis, dysregulation of the host immune response, environmental factors, and host genetic profile. Indeed, studies have shown defective expression of AhR and antimicrobial peptides (AMPs) particularly defensins in inflammatory bowel disease (IBD) patients. Here, we investigated how I3C influences the production of AMPs, particularly b-defensins, through AhR and colonic microbiota composition in Anti-CD40 or dextran sulfate sodium (DSS)-induced colitis. Our analysis found that I3C attenuates colitis through AhR activation leading to increased expression of murine b-defensins (mBDs) by Colonic Epithelial Cells, resulting in the restoration of healthy gut microbiota and prevention of colonic inflammation. We analyzed the colonic expression of AMPs and mucins. Our analysis found that I3C significantly increased the mRNA expression of AhR, AMPs such as mBDs, Reg4, and mucins, when compared to vehicle-treated mice with colitis. Similarly, the mRNA levels of AhR, AMPs, and mucins were significantly increased in DSS+I3C-treated colon adenocarcinoma cells MC-38 (murine) and Caco2 (human) when compared to DSS-treated cells. To conclude, our findings demonstrate that I3C inhibits colitis primarily through AhR-mediated induction of AMPs and protective mucins, resulting in the enrichment of healthy gut microbiota (This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896). This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896

Tryptophan diet promotes myeloid-derived suppressor cell response via aryl hydrocarbon receptor in Sjogren’s syndrome

Abstract Myeloid-derived suppressor cells (MDSCs) are heterogeneous myeloid cell populations with immunosuppressive capacity, which contribute to immune regulation and tolerance induction. We previously reported impaired MDSC function in patients with primary Sjögren’s syndrome (pSS) and mice with experimental SS (ESS). In this study, we first found that aryl hydrocarbon receptor (AhR) was highly expressed by human and murine polymorphonuclear MDSCs (PMN-MDSCs). Indole-3-propionic acid (IPA), a natural AhR ligand produced from dietary tryptophan, significantly promoted PMN-MDSC differentiation and their suppressive function on CD4 +T cells. In contrast, feeding tryptophan-free diet resulted in decreased PMN-MDSC response, a phenotype that could be reversed by IPA supplementation. Notably, AhR expression was reduced in PMN-MDSCs during ESS development, while AhR antagonism resulted in exacerbated ESS pathology and dysregulated T effector cells, which could be phenocopied by tryptophan-free diet. Interferon regulatory factor 4 (IRF4), a repressive transcription factor, was upregulated in PMN-MDSCs during ESS progression. ChIP analysis revealed the binding capacity of IRF4 to the promoter region of AhR, while IRF4 deficiency markedly enhanced AhR-mediated PMN-MDSC responses. Furthermore, tryptophan-enriched diet or dietary supplementation of IPA markedly ameliorated salivary glandular pathology in ESS mice with restored MDSC immunosuppressive function. Together, our results identify a novel function of AhR in modulating PMN-MDSC response and demonstrate the therapeutic potential of targeting AhR for the treatment of pSS.

Single cell transcriptomics identify AHR and CD9 as markers of CD14 +atypical B cells

Abstract B lymphocytes are adaptive immune cells responsible for the production of antigen specific antibodies and the release of pro- and anti-inflammatory cytokines. Pro-inflammatory B cells, often termed atypical B cells (at B cell) are a heterogenous population of B lymphocytes associated with aging, infection, and autoimmunity. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor important for xenobiotic metabolism. However, AHR is heavily implicated in immune cell differentiation as a regulator of cell fate decisions. at B cells are believed to represent an alternative B cell lineage but the role of AHR in at B cells is not known. We have reported that human, primary CD5 +innate-like B cells (ILB) are preferentially sensitive to modulation by AHR-activation compared to CD5 −B cells. CD5 +ILB are a heterogenous population of B cells enriched in IgM memory B cells, B1 B cells, marginal zone B cells, regulatory B cells, and at B cells. We used Single cell transcriptomics to elucidate AHR expression in human naïve, circulating CD19 +B cells isolated directly from PBMC and enriched for CD5 protein. We found that AHR expression correlated with expression of CD14and were enriched in a myeloid gene signature. These results were validated in isolated CD9 +B cells and PBMC. Further, CD14 expressing B cells were enriched in CD9 isolated cells and expressed higher CD11c and Tbet and lower IgD, markers of atypical B cells, compared to CD9 −B cells. These data suggest that CD9 and AHR are putative markers of CD14 +at B cells. (Supported by NIH grant P42ES004911) Supported by NIH grant P42ES004911

ARYL HYDROCARBON RECEPTOR REGULATES GENES IN COLONIC EPITHELIAL CELLS TO MITIGATE COLITIS SEVERITY IN THE DSS-INDUCED MURINE MODEL

Abstract Current treatment of ulcerative colitis (UC) leaves a clear unmet need to target the colonic epithelial cells (CECs). In our previous study we found that aryl hydrocarbon receptor (AhR) expression on CECs has a vital role during indole-3-carbinol (I3C)-mediated protection against colitis. However, the precise mechanism behind this remains unclear. We investigated how intestinal regulatory components were altered in the absence or presence of AhR in CECs during I3C treatment under colitis (mouse model) or colitis-like conditions (colonic organoid). We generated conditional AhR-CEC knockout mice (AV) and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the AV mice lost the protective effects of I3C against colitis. AV mice had higher disease scores and increased inflammation compared to controls. I3C treated AV mice that had DSS induced colitis were not able to regulate the colitis-associated parameters, even though flow cytometry analysis revealed AV mice were still capable of increasing protective interleukin-22 (IL-22) production in the colon. These results prompted us to study the CEC-specific mechanism involved in I3C-mediated protection against colitis. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc2 and muc13), and tight junction proteins (Cld3 and Cld4) in AV mice when compared to controls. RT-PCR data and direct effects of I3C on CECs (immunostaining) using organoids validated these results. This data concluded that AhR-dependent signaling orchestrates I3C-mediated protection of colitis. NIH 5P20GM103641

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.

Astragaloside IV attenuates indoxyl sulfate-induced injury of renal tubular epithelial cells by inhibiting the aryl hydrocarbon receptor pathway.

Astragalus membranaceus Fisch. ex Bunge has long been used to treat chronic kidney disease (CKD) in China. However, the mechanism of action requires further study. Indoxyl sulfate accumulation is the key cause of CKD progression. The aryl hydrocarbon receptor (AhR) plays an essential role in the renal tubular injury induced by indoxyl sulfate (IS). We explored the effects of Astragaloside IV (AS-IV), a minor component of the flowering perennial Astragalus membranaceus Fisch. ex Bunge, on AhR activity during IS-induced injury of renal tubular epithelial cells. C57BL/6 mice fed a 0.2% adenine diet (adenine+IS) and intraperitoneally injected with IS were used to study the protective effects of AS-IV, and specifically the effect on the AhR. In addition, apoptosis (annexin/PI), oxidative stress and the AhR pathway were investigated in IS-stimulated HK-2cells treated with AS-IV. The binding of AS-IV to the AhR was assessed in a molecular docking analysis. AhR knockdown using AhR siRNA allowed determination of the effects of AS-IV in IS-stimulated HK-2cells. AS-IV inhibited tubulointerstitial injury in adenine+IS mice. While AS-IV did not reduce serum IS levels, it did inhibit AhR expression in the kidney. In IS-stimulated HK-2cells, AS-IV also dramatically reduced apoptosis, decreased oxidative stress responses and inhibited the expression of the AhR pathway. The molecular docking analysis showed surface binding of AS-IV to the AhR. Following AhR knockdown in HK-2cells, IS-induced apoptosis was reduced and could not be further reduced by AS-IV. By targeting the AhR, AS-IV may alleviate IS-induced renal tubular injury, thus offering a novel therapeutic approach to the treatment of chronic renal failure.

Uremic Toxin Indoxyl Sulfate Promotes Macrophage-Associated Low-Grade Inflammation and Epithelial Cell Senescence.

In this study, we investigated the impact of the uremic toxin indoxyl sulfate on macrophages and tubular epithelial cells and its role in modulating the response to lipopolysaccharide (LPS). Indoxyl sulfate accumulates in the blood of patients with chronic kidney disease (CKD) and is a predictor of overall and cardiovascular morbidity/mortality. To simulate the uremic condition, primary macrophages and tubular epithelial cells were incubated with indoxyl sulfate at low concentrations as well as concentrations found in uremic patients, both alone and upon LPS challenge. The results showed that indoxyl sulfate alone induced the release of reactive oxygen species and low-grade inflammation in macrophages. Moreover, combined with LPS (proinflammatory conditions), indoxyl sulfate significantly increased TNF-α, CCL2, and IL-10 release but did not significantly affect the polarization of macrophages. Pre-treatment with indoxyl sulfate following LPS challenge induced the expression of aryl hydrocarbon receptor (Ahr) and NADPH oxidase 4 (Nox4) which generate reactive oxygen species (ROS). Further, experiments with tubular epithelial cells revealed that indoxyl sulfate might induce senescence in parenchymal cells and therefore participate in the progression of inflammaging. In conclusion, this study provides evidence that indoxyl sulfate provokes low-grade inflammation, modulates macrophage function, and enhances the inflammatory response associated with LPS. Finally, indoxyl sulfate signaling contributes to the senescence of tubular epithelial cells during injury.

Abstract 2890: Investigating dynamic IDO-Kyn-Ahr pathway induced tumor immunosuppression using imaging modality to optimize therapeutic Intervention

Abstract Despite great clinical responses in several cancers to immune checkpoint blockade (ICB) intervention, there persist many mechanisms of tumor evasion. Tryptophan catabolism by enzymes Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) into metabolite L-Kynurenine (Kyn) has been shown to drive an immunosuppressive tumor microenvironment (TME). We have shown tumors overexpressing IDO1 recruit and activate the Treg/macrophage suppressive axis and blockade of aryl hydrocarbon receptor (AHR) activation abrogates this. Preclinical models demonstrate inhibition of IDO1 delayed tumor growth and increased T cell function. However, a phase III clinical trial in melanoma patients combining IDO1 inhibition with anti-PD-1 blockade showed no improved efficacy when compared to anti-PD-1 blockade. Kyn can act as a key signaling molecule through activation of AHR. We hypothesize that AHR engagement by kyn within the TME is highly dynamic and elevated AHR engagement must coincide with intervention to maximize therapeutic response. Using an AHR biosensor, we designed a construct with a dioxin response element (DRE) promoter that is engaged upon AHR-ligand complex, translocated to the nucleus, and drives a luciferase gene with a luminescence readout. This allows us to use an image-guided approach to track AHR bioactivity in real-time. In-vitro, we were able to validate our construct using transduced murine B16 melanoma and 4T1 breast cancer models. We treated transduced cells with endogenous AHR ligands, kynurenine, and AHR agonist (FICZ), and observed a significant dose-dependent increase in AHR reporter readout. Additionally, the AHR inhibitor (CH-223191) abrogated AHR activity and subsequent luminescence below control threshold. We implanted both DRE transduced B16 (B16-DRE) and 4T1 (4T1-DRE) intradermally and intramammary fat pad respectively. Tumors were imaged and we observed total luminescence flux corresponding to increased tumor growth. After three weeks, we assessed IDO, AHR, and HIF1a expression which all converge on the IDO-Kyn-AhR pathway using a broad panel of lymphocyte and myeloid markers from tumors and spleen. We identified high expression of IDO, AHR, and HIF1a markers in myeloid cells with an increased frequency of dendritic cells and macrophages. Future experiments will focus on using our reporter construct in the context of ICB treatment and assessing AHR activity overtime as well as sampling peripheral blood to assess the magnitude of tryptophan, kyn, and other polycyclic hydrocarbons including gut microbiome-derived indoles. The characterization of the IDO-Kyn-AhR modulation after therapeutic intervention will inform the rational design of treatment regimens to abrogate immune suppression associated with AHR activity in combination with ICB treatment. Citation Format: Mamadou Alpha Bah, Inna Serganova, Sadna Budhu, Tanya Schild, Jonathan F. Khan, Kayvan R. Keshari, Jedd D. Wolchok, Taha Merghoub. Investigating dynamic IDO-Kyn-Ahr pathway induced tumor immunosuppression using imaging modality to optimize therapeutic Intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2890.