Journal for ImmunoTherapy of Cancer
Sanchez-Martin, M;Wang, L;Ecsedy, J;Mcgovern, K;Zhang, M;
| DOI: 10.1136/jitc-2021-sitc2021.093
BackgroundAryl Hydrocarbon Receptor (AHR) is a ligand-activated transcription factor that regulates the activities of multiple innate and adaptive immune cell types. Multiple ligands such as kynurenine bind to AHR driving its nuclear translocation and transcriptional activation, leading to an immunosuppressive tumor microenvironment.1 2 AHR activation is implicated in tumor development in multiple cancer types. In addition, high levels of serum kynurenine are associated with resistance to checkpoint inhibitors.3 To overcome AHR-mediated immunosuppression in cancers, we developed a selective oral AHR inhibitor IK-175 and took a combined computational and tissue-based approach to select cancer indications for its clinical development.MethodsThe aim of this work is to identify tumor indications dependent on AHR signaling and design patient selection strategies based on a proprietary transcriptional signature, mRNA and protein detection assays to evaluate AHR pathway activation in tumors.ResultsGenomic profiling of solid and hematological cancers from TCGA and Project GENIE databases identified bladder and esophageal tumors among others, as frequently harboring AHR gene amplifications.A proprietary gene signature of AHR activation was developed integrating literature, pathway analysis, RNAseq and nanostring data from PBMC, T-cells and cell lines upon AHR inhibition. Transcriptional analysis of the TCGA data using this signature demonstrated bladder cancer has the highest expressions of AHR and AHR signature genes, suggesting increased pathway activity in bladder cancer relative to other cancer types. Increased AHR signature gene expression was associated with worse overall survival in the TCGA bladder cancer cohort. Furthermore, RNAscope analysis of a tissue microarray containing 10 different tumor types revealed bladder cancer had one of the highest AHR transcript expression in the tumor compartment.Finally, nuclear localization of AHR protein was assessed as an indicator of pathway activation through the development of a novel IHC method. Extensive TMA screening of AHR protein in 15 different indications demonstrated bladder cancer as the tumor type with the highest prevalence of AHR nuclear expression.ConclusionsIn summary, we demonstrated high prevalence of nuclear AHR protein expression, AHR gene amplification and target gene expression in bladder cancer, suggesting aberrant AHR activation may play an important role in the progression of this tumor type. This study provides rationale for therapeutic targeting of AHR in bladder cancer patients. Ikena is currently evaluating the anti-tumor activity of IK-175 as a single agent and in combination with nivolumab in bladder cancer in a Phase 1a/1b clinical study (NCT04200963).ReferencesQuintana FJ, Sherr DH. Aryl hydrocarbon receptor control of adaptive immunity. Pharmacol Rev 2013 Aug 1;65(4):1148-61.Murray IA, Patterson AD, Perdew GH. Aryl hydrocarbon receptor ligands in cancer: friend and foe. Nat Rev Cancer 2014 Dec;14(12):801-14.Li, Haoxin et al. ‘Metabolomic adaptations and correlates of survival to immune checkpoint blockade.’ Nature Communications 2019 Sep 25;10:1-4346.
Hidaka T, Ogawa E, Kobayashi EH, Suzuki T, Funayama R, Nagashima T, Fujimura T, Aiba S, Nakayama K, Okuyama R, Yamamoto M.
PMID: 27869817 | DOI: 10.1038/ni.3614
Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.
Kimura E, Tohyama C.
PMID: 28223923 | DOI: 10.3389/fnana.2017.00004
Aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix-Per-Arnt-Sim transcription factor family, plays a critical role in the developing nervous system of invertebrates and vertebrates. Dioxin, a ubiquitous environmental pollutant, avidly binds to this receptor, and maternal exposure to dioxin has been shown to impair higher brain functions and dendritic morphogenesis, possibly via an AhR-dependent mechanism. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study analyzed AhR mRNA expression in the brains of embryonic, juvenile, and adult mice by reverse transcription (RT)-PCR and in situhybridization. In early brain development (embryonic day 12.5), AhR transcript was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and rostral migratory stream on embryonic day 18.5, postnatal days 3, 7, and 14, and in 12-week-old (adult) mice. Hippocampal expression was abundant in the CA1 and CA3 pyramidal and dentate gyrus granule cell layers, where expression level of AhR mRNA in 12-week old is higher than that in 7-day old. These results reveal temporal and spatial patterns of AhR mRNA expression in the mouse brain, providing the information that may contribute to the elucidation of the physiologic and toxicologic significance of AhR in the developing brain.
Jaligama S, Patel VS, Wang P, Sallam A, Harding J, Kelley M, Mancuso SR, Dugas TR, Cormier SA.
PMID: 29724254 | DOI: 10.1186/s12989-018-0255-3
Abstract
BACKGROUND:
Pollutant particles containing environmentally persistent free radicals (EPFRs) are formed during many combustion processes (e.g. thermal remediation of hazardous wastes, diesel/gasoline combustion, wood smoke, cigarette smoke, etc.). Our previous studies demonstrated that acute exposure to EPFRs results in dendritic cell maturation and Th17-biased pulmonary immune responses. Further, in a mouse model of asthma, these responses were enhanced suggesting exposure to EPFRs as a risk factor for the development and/or exacerbation of asthma. The aryl hydrocarbon receptor (AHR) has been shown to play a role in the differentiation of Th17 cells. In the current study, we determined whether exposure to EPFRs results in Th17 polarization in an AHR dependent manner.
RESULTS:
Exposure to EPFRs resulted in Th17 and IL17A dependent pulmonary immune responses including airway neutrophilia. EPFR exposure caused a significant increase in pulmonary Th17 cytokines such as IL6, IL17A, IL22, IL1β, KC, MCP-1, IL31 and IL33. To understand the role of AHR activation in EPFR-induced Th17 inflammation, A549 epithelial cells and mouse bone marrow-derived dendritic cells (BMDCs) were exposed to EPFRs and expression of Cyp1a1 and Cyp1b1, markers for AHR activation, was measured. A significant increase in Cyp1a1 and Cyp1b1 gene expression was observed in pulmonary epithelial cells and BMDCs in an oxidative stress and AHR dependent manner. Further, in vivo exposure of mice to EPFRs resulted in oxidative stress and increased Cyp1a1 and Cyp1b1 pulmonary gene expression. To further confirm the role of AHR activation in pulmonary Th17 immune responses, mice were exposed to EPFRs in the presence or absence of AHR antagonist. EPFR exposure resulted in a significant increase in pulmonary Th17 cells and neutrophilic inflammation, whereas a significant decrease in the percentage of Th17 cells and neutrophilic inflammation was observed in mice treated with AHR antagonist.
CONCLUSION:
Exposure to EPFRs results in AHR activation and induction of Cyp1a1 and in vitro this is dependent on oxidative stress. Further, our in vivo studies demonstrated a role for AHR in EPFR-induced pulmonary Th17 responses including neutrophilic inflammation.
International immunopharmacology
Yan, B;Mao, X;Hu, S;Wang, S;Liu, X;Sun, J;
PMID: 37104918 | DOI: 10.1016/j.intimp.2023.110166
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.
Bensreti, H;Yu, K;Alhamad, D;Shaver, J;Kaiser, H;Zhong, R;Whichard, W;Parker, E;Grater, L;Faith, H;Johnson, M;Cooley, M;Fulzele, S;Hill, W;Isales, C;Hamrick, M;McGee-Lawrence, M;
| DOI: 10.2139/ssrn.4409572
Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10mg/kg) or vehicle via intraperitoneal injection, once daily, 5x/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn’s effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.
Journal of the Canadian Association of Gastroenterology
Abdullah, N;Defaye, M;Hassan, A;Cumenal, M;Iftinca, M;Young, D;Ohland, C;Dufour, A;McCoy, K;Altier, C;
| DOI: 10.1093/jcag/gwab049.229
Background Pain is the most common cause of disability in IBD. What causes inter-individual variability in chronic pain after successful treatment of inflammation remains elusive. We have shown that activation of TRPV1+ colonic nociceptors is essential for the establishment of persistent pain in DSS colitis. Nociceptor development coincides with microbial colonization, while early life dysbiosis can lead to visceral hypersensitivity in adulthood. Whether the microbiota dictates nociceptor development and pain susceptibility remains unknown. Here we test the hypothesis that the microbiota programs nociceptor specification during early development, rendering them more susceptible to sensitization later in life. We have identified the aryl hydrocarbon receptor (AHR) that senses bacterial-derived metabolites as a candidate target that orchestrates transcriptional regulation in nociceptors. Aims We investigated the developmental regulation of nociceptors by the microbiome and how it influences pain sensitivity. We will determine the effects of AHR activation on nociceptor lineage and function as well as the long term impact of AHR signaling on pain sensitivity. Methods We have developed a germ-free (GF) TRPV1-GFP reporter mouse that was used to phenotype and visualise TRPV1+ nociceptors in the absence of a microbiota. We will isolate TRPV1+ neurons by FACS to identify genes that are under the control of the microbiota and to characterise the phosphoproteome of TRPV1+ nociceptors in GF conditions. Finally, we will investigate the role of AHR signaling in nociceptors both acutely and during development. Results We showed a reduction in thermal pain threshold and a reduction in capsaicin test responses in GF mice. The number and size of DRG neurons was unchanged in GF mice. Examination of molecular markers for peptidergic (CGRP) and non-peptidergic (IB4) neurons did not show a difference. Finally, there was no difference in the expression of TRPV1, suggesting post-translational modification of the channel. In cultured DRG neurons, we found a decrease in capsaicin induced action potentials and a decrease in the amplitude of the capsaicin response in GF mice. Using RNAscope, we showed that TRPV1+ neurons express AHR. Conclusions Our results highlight the importance of bacterial composition in regulating the development of nociceptors and pain sensitivity in adulthood. Furthermore, we are the first to demonstrate the expression of AHR in sensory neurons. These findings point to a role of the microbiota in programming nociceptors during development. My work will advance our understanding of the role of commensal bacteria in regulating pain and could lead to recommendations for the treatment of neonates in early life to reduce their risk of developing chronic pain later in life. Funding Agencies CAG, CIHR
RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma
Lamb, D;De Sousa, D;Quast, K;Fundel-Clemens, K;Erjefält, JS;Sandén, C;Hoffmann, HJ;Kästle, M;Schmid, R;Menden, K;Delic, D;
PMID: 34022896 | DOI: 10.1186/s12931-021-01743-7
RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.
Obata, Y;Castaño, Á;Fallesen, TL;Bon-Frauches, AC;Boeing, S;Huseynova, A;McCallum, S;Lasrado, R;Heanue, TA;Pachnis, V;
PMID: 35676375 | DOI: 10.1038/s41596-022-00697-4
The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.
ACE2 expression is regulated by AhR in SARS-CoV-2-infected macaques
Cellular & molecular immunology
Lv, J;Yu, P;Wang, Z;Deng, W;Bao, L;Liu, J;Li, F;Zhu, Q;Zhou, N;Lv, Q;Wang, G;Wang, S;Zhou, Y;Song, J;Tong, WM;Liu, Y;Qin, C;Huang, B;
PMID: 33795851 | DOI: 10.1038/s41423-021-00672-1
