Aryl Hydrocarbon Receptor Knockout Research Articles

Sex differences in cardiac ferroptosis after acute myocardial infarction in wild type and aryl hydrocarbon receptor knockout mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): MICIN/Agencia Estatal de Investigación (DOI: 10.13039/50110001103) (NextGeneration, EU/PRTR) Background and Purpose Ferroptosis is a new regulated programmed cell death pathway, driven by iron-dependent phospholipid peroxidation, that plays an important role in the development of cardiovascular diseases. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic Helix-Loop-Helix-PER-ARNT-SIM superfamily. Upon ligand binding, AhR translocates into the nucleus and dimerizes with the AhR nuclear translocator; the heterodimer then binds to dioxin-response elements located in the promoter region of receptor-regulated genes, most of which are involved in xenobiotic metabolism. Beyond its role in metabolizing exogenous toxins as part of an adaptive chemical response, it is well known that AhR is activated by endogenous ligands and it has been postulated that AhR has important functions in physiological and/or pathological conditions. Here we studied sex differences in cardiac ferroptosis after acute myocardial infarction (AMI) in mice and the implication of defective AhR signalling on this process. Methods and Results AMI was induced in 8–10-weeks-old males and females AhR+/+ and AhR-/- mice by ligation of the left anterior descending coronary artery. Animal care and experimental procedures followed the principles of animal care (NIH publication Nº85-23 revised 1985) and the guidelines for ethical care of experimental animals of the European Union (2010/63/EU). Survival analysis showed that 16% of AhR+/+male mice and 14% of AhR+/+ female mice died 72 h after AMI surgery whereas the mortality was 100% in AhR-/-male mice and only 17% in AhR-/- female mice group. The treatment of AhR-/- AMI male mice with ferrostatin (1 mg/kg i.p) increased survival to 40%. Immunofluorescence analysis of cardiac staining levels of 8-hydroxy-2’-deoxyguanosine revealed higher oxidative stress in AhR+/+AMI male mice compared with AhR+/+AMI females, 24 h after surgery. The absence of AhR in AMI males showed an increased oxidative stress that was similar to that obtained in AMI AhR+/+ males but significantly greater than that observed in AhR-/- AMI females. Finally, analysis of differential expressions of classical genes related to ferroptosis in AhR-/- AMI male and AMI female mice, 24 h after surgery, showed, only in males, a reduced expression of ferritin heavy chain 1 and ferroportin, and an increased expression of phospholipase A2. Conclusion Our results show that loss of AhR in males but not in females favours the ferroptosis process associated with myocardial ischemia, highlighting a possible role for this nuclear receptor in cardiac remodelling and survival after AMI.

Deletion of the Aryl Hydrocarbon Receptor in Endothelial Cells Improves Ischemic Angiogenesis in Chronic Kidney Disease

Background: Chronic kidney disease (CKD) is a strong yet underappreciated risk factor for peripheral arterial disease (PAD) that exacerbates disease progression and increases risk for limb amputation and cardiovascular mortality. An estimated 25-35% of CKD patients develop PAD, but mechanistic data linking these two pathologies is limited. Several tryptophan-derived uremic metabolites are retained in the blood and tissues as a consequence of CKD and are endogenous ligands of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that has been shown to regulate angiogenesis. Hypothesis: We tested the hypothesis that endothelial cell-specific conditional deletion of the AHR would enhance ischemic limb outcomes in mice with CKD and femoral artery ligation (FAL). Methods: Twelve-week-old male and female endothelium-specific AHR knockout mice (AHRecKO) and AHR floxed mice (AHRfl/fl, Cre-negative control) were fed 0.2% adenine to induce CKD or casein control diet (n=10/group/sex) prior to undergoing FAL to induce limb ischemia. Laser Doppler flowmetry was used to measure limb perfusion. Isometric contraction of the tibialis anterior in-situ via nerve stimulus was performed to measure muscle function. Skeletal muscle mitochondrial respiratory function and H2O2 production were evaluated. Primary endothelial cells were isolated from male and female wildtype mice and treated with known AHR ligand indoxyl sulfate (IS) under hypoxic conditions to uncover sex-dependent differences in AHR activating potential. Results: In male mice with CKD, endothelium-specific ablation of the AHR resulted in significant increases in percentage of perfused capillaries within the ischemic limb (AHRecKO 73.41% ± 4.80% vs AHRfl/fl 64.75% ± 8.325%; p= 0.0074), improved laser Doppler perfusion recovery in the paw and gastrocnemius muscle, and larger myofiber cross-sectional area ( p=0.0258) compared to AHRfl/fl mice. However, these improvements did not translate to improved muscle contractile function. In contrast, deletion of the AHR in female mice with CKD had no significant impact on major outcome measures. Primary cells from male mice treated with IS had significantly higher expression of the AHR controlled gene Cyp1a1 ( p=0.026) compared to cells from female mice. Conclusion: Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. These data reveal sex-dependent differences in AHR activating potential within endothelial cells that exist in the absence of sex hormones. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Tryptophan-derived Uremic Metabolite-Ahr-Pdk4 Axis Promotes Skeletal Muscle Mitochondriopathy in Chronic Kidney Disease

Introduction: Chronic kidney disease (CKD) and the subsequent accumulation of tryptophan-derived uremic metabolites (UMs) in circulation negatively impact skeletal muscle (SkM) health and function. Specifically, indoles and kynurenines are strongly associated with SkM atrophy, weakness, and fatigue found in CKD patients. Notably, these tryptophan-derived UMs are known agonist to the ubiquitously expressed aryl hydrocarbon receptor (AHR), a ligand activated transcription factor, which has been shown to be activated in the blood of CKD patients and whose chronic activation has been proven toxic to multiple tissue types. Purpose: The purpose of this study was to determine whether chronic activation of the AHR by tryptophan-derived UMs contributes to CKD associated SkM atrophy and metabolic dysfunction. Methods: Muscle biopsies from CKD patients and adult controls with normal renal function were used to examine AHR signaling and mitochondrial function. The mechanistic role of the AHR was explored using SkM-specific AHR knockout mice (AHRmKO) and SkM-specific AHR knockdown via adeno-associated virus in mice with adenine-induced CKD, as well as ectopic expression of a constitutively active AHR (CAAHR) in mice with normal kidney function. Additional mechanistic experiments were carried out in cultured muscle cells. Outcome measures included gene and protein expression, mitochondrial bioenergetic testing, and robust muscle functional phenotyping. Results: Compared to controls with normal kidney function, AHR-dependent gene expression ( CYP1A1 and CYP1B1) was significantly upregulated in gastrocnemius SkM of CKD patients ( P=0.032) and the magnitude of AHR activation was inversely correlated with mitochondrial respiration ( P<0.001). In mice with CKD, SkM mitochondrial oxidative phosphorylation (OXPHOS) was significantly impaired and strongly correlated with both the level of tryptophan-derived UMs and AHR activation. AHRmKO significantly improved mitochondrial OXPHOS in mice with CKD (~28% increase, P=0.045) and abolished the relationship between UMs and OXPHOS. The uremic metabolite-AHR-mitochondrial axis in SkM was further confirmed using SkM-specific AHR knockdown in C57BL6J that express a high-affnity AHR allele, as well as ectopic viral expression of CAAHR in mice with normal renal function. AHR activation led to impairments in pyruvate dehydrogenase (PDH) enzyme function (~29% decrease, P<0.05), significant increases in Pdk4 expression ( P<0.05) and phosphorylation of PDH enzyme ( P<0.05), mechanistically contributing to impaired pyruvate-supported OXPHOS function. Conclusion: These findings establish a uremic metabolite-AHR-Pdk4 axis in SkM that governs mitochondrial deficits in CKD. Supported by grants from the National Institutes of Health R01-HL149704 (TER) and F31-DK128920 (TT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aryl hydrocarbon receptor knockout accelerates PanIN formation and fibro-inflammation in a mutant Kras-driven pancreatic cancer model.

The pathogenesis of pancreas cancer (PDAC) remains poorly understood, hindering efforts to develop a more effective therapy for PDAC. Recent discoveries show the aryl hydrocarbon receptor (AHR) plays a crucial role in the development of several cancers, and can be targeted for therapeutic effect. However, its involvement in the pathogenesis of PDAC remains unclear. To address this gap, we evaluated the role of AHR in the development of PDAC pre-cancerous lesions in vivo. We created a global AHR-null, mutant Kras-driven PDAC mouse model (A-/-KC) and evaluated the changes in PDAC precursor lesion formation (Pan-IN 1, 2, and 3) and associated fibro-inflammation between KC and A-/-KC at 5 months of age. We then examined the changes in the immune microenvironment followed by single-cell RNA-sequencing analysis to evaluate concomitant transcriptomic changes. We identified a significant increase in PanIN-1 lesion formation and PanIN-1 associated fibro-inflammatory infiltrate in A-/-KC vs KC mice. This was associated with significant changes in the adaptive immune system, particularly a decrease in the CD4+/CD8+ T-cell ratio, as well as a decrease in the T-regulatory/Th17 T-cell ratio suggesting unregulated inflammation. These findings show the loss of AHR results in heightened Kras-induced PanIN formation, through modulation of immune cells within the pancreatic tumor microenvironment.

Abstract 2649: A new role for the aryl hydrocarbon receptor (AhR) as a proximal mediator of immune checkpoints (PD-L1/2 and IDO1) in non-small cell lung cancer: Insights into an interferon-induced, LncRNA-and JAK/STAT-mediated signaling pathway

Abstract Non-small cell lung cancer (NSCLC) is the second most common cancer and accounts for ~127,000 deaths/year in the U.S. Its current ~30% five-year survival rate is an improvement from a decade ago thanks in part to the introduction of immune checkpoint inhibitors including PD1- and PD-L1-specific monoclonal antibodies. While PD-1/PD-L1-targeted immunotherapy is effective in many cases, not all patients respond and complete remissions still only occur in a minority of patients. Therefore, a greater understanding of immune checkpoint regulation in human NSCLC is needed. Here, we investigated a novel role for the aryl hydrocarbon receptor (AhR), an environmental carcinogen receptor previously associated with smoking-induced lung cancers, in the regulation of immune checkpoints PD-L1, PD-L2, and IDO1. Computational analyses of existing databases for protein-protein interactions linked to the AhR signaling pathway suggested crosstalk between the AhR, type I and type II Interferon (IFN), and JAK/STAT signaling pathways. AhR deletion from human lung A549 adenocarcinoma cells significantly reduced expression of JAK2, STAT1, STAT2, and the JAK/STAT signaling targets IRF1 and IRF9. Type II IFNγ significantly increased expression of those genes as well as the immune checkpoints IDO1, PD-L1, and PD-L2 in an AhR-dependent fashion. Interestingly, AhR deletion also eliminated IFNγ-mediated induction of a recently described long non-coding RNA, INCR1, which is encoded across the CD274/CD273 locus, and which facilitates JAK2, CD274/PD-L1, and IDO1 translation. Concomitantly, AhR knockout increased baseline expression of an RNA-binding protein, HNRNPH1, known to sequester JAK2 and CD274 mRNA thereby reducing IDO, PD-L1 and PD-L2 expression. Similarly, the AhR was shown to regulate Type I IFNα-mediated induction of STAT1, STAT2, CD274/PD-L1, and CD273/PD-L2. The data indicate that IFN induction of key immune checkpoints in lung adenocarcinoma cells is dependent on the AhR through its control of a conventional JAK/STAT pathway and an unconventional lncRNA (INCR1) and RNA-binding protein (HNRNPH1) pathway. Citation Format: Brian Lara, Megan Snyder, Zhongyan Wang, Jocelyn Fimbres, Eric Yang, David H. Sherr. A new role for the aryl hydrocarbon receptor (AhR) as a proximal mediator of immune checkpoints (PD-L1/2 and IDO1) in non-small cell lung cancer: Insights into an interferon-induced, LncRNA-and JAK/STAT-mediated signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2649.

Aryl Hydrocarbon Receptor Regulates Muc2 Production Independently of IL-22 during Colitis.

We previously reported that an aryl hydrocarbon receptor (AhR) ligand, indole-3-carbinol (I3C), was effective at reducing colitis severity through immune cell-mediated interleukin-22 (IL-22) production. Intestinal epithelial cells (IECs) are also involved in regulating colitis, so we investigated their AhR-mediated mechanisms in the current report. A transcriptome analysis of IECs in wildtype (WT) mice revealed that during colitis, I3C regulated select mucin proteins, which could be attributed to goblet cell development. To address this, experiments under in vivo colitis (mice) or in vitro colon organoid conditions were undertaken to determine how select mucin proteins were altered in the absence or presence of AhR in IECs during I3C treatment. Comparing WT to IEC-specific AhR knockout mice (AhRΔIEC), the results showed that AhR expression was essential in IECs for I3C-mediated protection during colitis. AhR-deficiency also impaired mucin protein expression, particularly mucin 2 (Muc2), independently of IL-22. Collectively, this report highlights the important role of AhR in direct regulation of Muc2. These results provide justification for future studies aimed at determining how AhR might regulate select mucins through mechanisms such as direct transcription binding to enhance production.

Chronic aryl hydrocarbon receptor activity impairs muscle mitochondrial function with tobacco smoking.

Accumulating evidence has demonstrated that chronic tobacco smoking directly contributes to skeletal muscle dysfunction independent of its pathological impact to the cardiorespiratory systems. The mechanisms underlying tobacco smoke toxicity in skeletal muscle are not fully resolved. In this study, the role of the aryl hydrocarbon receptor (AHR), a transcription factor known to be activated with tobacco smoke, was investigated. AHR related gene (mRNA) expression was quantified in skeletal muscle from adult controls and patients with chronic obstructive pulmonary disease (COPD), as well as mice with and without cigarette smoke exposure. Utilizing both skeletal muscle-specific AHR knockout mice exposed to chronic repeated (5days per week for 16weeks) cigarette smoke and skeletal muscle-specific expression of a constitutively active mutant AHR in healthy mice, a battery of assessments interrogating muscle size, contractile function, mitochondrial energetics, and RNA sequencing were employed. Skeletal muscle from COPD patients (N=79, age=67.0±8.4years) had higher levels of AHR (P=0.0451) and CYP1B1 (P<0.0001) compared to healthy adult controls (N=16, age=66.5±6.5years). Mice exposed to cigarette smoke displayed higher expression of Ahr (P=0.008), Cyp1b1 (P<0.0001), and Cyp1a1 (P<0.0001) in skeletal muscle compared to air controls. Cigarette smoke exposure was found to impair skeletal muscle mitochondrial oxidative phosphorylation by ~50% in littermate controls (Treatment effect, P<0.001), which was attenuated by deletion of the AHR in muscle in male (P=0.001), but not female, mice (P=0.37), indicating there are sex-dependent pathological effects of smoking-induced AHR activation in skeletal muscle. Viral mediated expression of a constitutively active mutant AHR in the muscle of healthy mice recapitulated the effects of cigarette smoking by decreasing muscle mitochondrial oxidative phosphorylation by ~40% (P=0.003). These findings provide evidence linking chronic AHR activation secondary to cigarette smoke exposure to skeletal muscle bioenergetic deficits in male, but not female, mice. AHR activation is a likely contributor to the decline in muscle oxidative capacity observed in smokers and AHR antagonism may provide a therapeutic avenue aimed to improve muscle function in COPD.

The aryl hydrocarbon receptor in β-cells mediates the effects of TCDD on glucose homeostasis in mice

ObjectiveChronic exposure to persistent organic pollutants (POPs) is associated with increased incidence of type 2 diabetes, hyperglycemia, and poor insulin secretion in humans. Dioxins and dioxin-like compounds are a broad class of POPs that exert cellular toxicity through activation of the aryl hydrocarbon receptor (AhR). We previously showed that a single high-dose injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, aka dioxin; 20 μg/kg) in vivo reduced fasted and glucose-stimulated plasma insulin levels for up to 6 weeks in male and female mice. TCDD-exposed male mice were also modestly hypoglycemic and had increased insulin sensitivity, whereas TCDD-exposed females were transiently glucose intolerant. Whether these effects are driven by AhR activation in β-cells requires investigation. MethodsWe exposed female and male β-cell specific Ahr knockout (βAhrKO) mice and littermate Ins1-Cre genotype controls (βAhrWT) to a single high dose of 20 μg/kg TCDD and tracked the mice for 6 weeks. ResultsUnder baseline conditions, deleting AhR from β-cells caused hypoglycemia in female mice, increased insulin secretion ex vivo in female mouse islets, and promoted modest weight gain in male mice. Importantly, high-dose TCDD exposure impaired glucose homeostasis and β-cell function in βAhrWT mice, but these phenotypes were largely abolished in TCDD-exposed βAhrKO mice. ConclusionOur study demonstrates that AhR signaling in β-cells is important for regulating baseline β-cell function in female mice and energy homeostasis in male mice. We also show that β-cell AhR signaling largely mediates the effects of TCDD on glucose homeostasis in both sexes, suggesting that the effects of TCDD on β-cell function and health are driving metabolic phenotypes in peripheral tissues.

The Role of Aryl Hydrocarbon Receptor in Bone Biology.

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is crucial in maintaining the skeletal system. Our study focuses on encapsulating the role of AhR in bone biology and identifying novel signaling pathways in musculoskeletal pathologies using the GEO dataset. The GEO2R analysis identified 8 genes (CYP1C1, SULT6B1, CYB5A, EDN1, CXCR4B, CTGFA, TIPARP, and CXXC5A) involved in the AhR pathway, which play a pivotal role in bone remodeling. The AhR knockout in hematopoietic stem cells showed alteration in several novel bone-related transcriptomes (eg, Defb14, ZNF 51, and Chrm5). Gene Ontology Enrichment Analysis demonstrated 54 different biological processes associated with bone homeostasis. Mainly, these processes include bone morphogenesis, bone development, bone trabeculae formation, bone resorption, bone maturation, bone mineralization, and bone marrow development. Employing Functional Annotation and Clustering through DAVID, we further uncovered the involvement of the xenobiotic metabolic process, p450 pathway, oxidation-reduction, and nitric oxide biosynthesis process in the AhR signaling pathway. The conflicting evidence of current research of AhR signaling on bone (positive and negative effects) homeostasis may be due to variations in ligand binding affinity, binding sites, half-life, chemical structure, and other unknown factors. In summary, our study provides a comprehensive understanding of the underlying mechanisms of the AhR pathway in bone biology.

Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration

Improving regeneration of damaged thymus is important for reconstituting T-cell immunity. Interleukin-22 (IL-22) was proved to improve thymus regeneration through recovering thymic epithelial cells (TECs). The IL-22 receptor IL-22RA1 is crucial for mediating IL-22 functions. Mechanism that regulates IL-22RA1 expression is unknown. Through using TECs-conditional knockout mice, we found aryl hydrocarbon receptor (AHR) is important for thymus regeneration, because Foxn1-cre-mediated AHR knockout (AhrKO) significantly blocks recovery of thymus cells. Giving mice the AHR inhibitor CH-223191 or the AHR agonist FICZ blocks or accelerates thymus regeneration, respectively. AhrKO-mediated blockade of thymus regeneration could not be rescued by giving exogenous IL-22. Mechanistically, AhrKO mice shows decreased IL-22RA1 expression. In the murine TECs cell line mTEC1 cells, targeting AHR shows an impact on IL-22RA1 mRNA levels. Using chromatin immunoprecipitation and luciferase reporter assays, we find AHR co-operates with STAT3, binds the promotor region of IL-22RA1 gene and transcriptionally increases IL-22RA1 expression in mTEC1 cells. Foxn1-cre-mediated IL-22RA1 knockout (Il22ra1KO) blocks thymus regeneration after irradiation. Furthermore, targeting AHR or IL-22RA1 has significant impacts on severity of murine chronic graft-versus-host disease (cGVHD), which is an autoimmune-like complication following allogeneic hematopoietic cell transplantation. Giving FICZ decreases cGVHD, whereas Il22ra1KO exacerbates cGVHD. The impacts on cGVHD are associated with thymus regeneration and T-cell immune reconstitution. In conclusion, we report an unrecognized function of TECs-expressed AHR in thymus regeneration and AHR transcriptionally regulates IL-22RA1 expression, which have implications for improving thymus regeneration and controlling cGVHD.

Activation of the Aryl Hydrocarbon Receptor in Muscle Exacerbates Ischemic Pathology in Chronic Kidney Disease.

Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD. AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHRmKO (skeletal muscle-specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD. PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes (Cyp1a1, Cyp1b1, and Aldh3a1) when compared with either muscle from the PAD condition with normal renal function (P<0.05 for all 3 genes) or nonischemic controls. AHRmKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions.

Aryl hydrocarbon receptor is a proviral host factor and a candidate pan-SARS-CoV-2 therapeutic target.

The emergence of a series of SARS-CoV-2 variants has necessitated the search for broad-spectrum antiviral targets. The aryl hydrocarbon receptor (AhR) senses tryptophan metabolites and is an immune regulator. However, the role of AhR in SARS-CoV-2 infection and whether AhR can be used as the target of antiviral therapy against SARS-CoV-2 and its variants are yet unclear. Here, we show that infection with SARS-CoV-2 activates AhR signaling and facilitates viral replication by interfering with IFN-I-driven antiviral immunity and up-regulating ACE2 receptor expression. The pharmacological AhR blockade or AhR knockout reduces SARS-CoV-2 and its variants' replication in vitro. Drug targeting of AhR with AhR antagonists markedly reduced SARS-CoV-2 and its variants' replication in vivo and ameliorated lung inflammation caused by SARS-CoV-2 infection in hamsters. Overall, AhR was a SARS-CoV-2 proviral host factor and a candidate host-directed broad-spectrum target for antiviral therapy against SARS-CoV-2 and its variants, including Delta and Omicron, and potentially other variants in the future.

Deletion of AhR attenuates fear memory leaving other types of memory intact

The aryl hydrocarbon receptor (AhR), a classic “environmental sensor”, has been found to play an important role in cognitive and emotional function. Recent studies showed AhR deletion led to an attenuated fear memory, providing a potential target against fear memory, whether it is the consequence of attenuated sense of fear or memory ability deficit or both is unclear. Here this study aims to work this out. The freezing time in contextual fear conditioning (CFC) reduced significantly in AhR knockout mice, indicating an attenuated fear memory. Hot plate test and acoustic startle reflex showed that AhR knockout did not change the pain threshold and hearing, excluded the possibility of sensory impairments. Results from NORT, MWM and SBT showed that deletion of AhR had little effects on other types of memory. But the anxiety-like behaviors reduced both in naïve or suffered (tested after CFC) AhR knockout mice, showing that AhR-deficient mice have a reduced basal and stressful emotional response. The basal low-frequency to high-frequency (LF/HF) ratio of the AhR knockout mice was significantly lower than that of the control group, indicating lower sympathetic excitability in the basal state, suggesting a low level of basal stress in the knockout mice. Before and after CFC, the LF/HF ratio of AhR-KO mice tended to be significantly lower than that of WT mice, and their heart rate was significantly lower; and the AhR-KO mice also has a decreased serum corticosterone level after CFC, suggesting a reduced stress response in AhR knockout mice. Altogether, the basal stress level and stress response were significant reduced in AhR knockout mice, which might contribute to the attenuated fear memory with little impairment on other types of memory, suggesting AhR as a “psychologic sensor” additional to “environmental sensor”.

A new cell line based coculture system for skin sensitisation testing in one single assay using T cells, aryl hydrocarbon receptor knockout, and co-inhibitory blockage

Established in vitro assays for regulatory testing of skin sensitisation partly suffer from only moderate sensitivity, specificity, and predictivity when testing specific groups of chemicals. This may be due to limited biomarker response in vitro in cell types that interact as crucial players of in vivo skin sensitisation pathogenesis. Here, we propose a molecular approach to overcome this limitation. In our model, we apply genome editing and blocking of immunoregulatory molecules to increase the range of biomarker modulation by sensitising chemicals. To this end, aryl hydrocarbon receptor (AhR) knockout was done by CRISPR/Cas9 technology in THP-1 cells and combined with Programmed Cell Death-Ligand (PD-L)1 blockade. AhR-knockout THP-1 in coculture with HaCaT keratinocytes showed increased CD54 expression compared to wild type cells after stimulation with 10 µmol/L dinitrochlorobenzene (DNCB) that was further enhanced by anti-PD-L1. After stimulation of AhR-knockout THP-1 with 200 µmol/L mercaptobenzothiazol or 10 µmol/L DNCB, cocultivated Jurkat T cells significantly increased expression of T cell receptor-associated CD3. No such increase was detected after prior treatment of THP-1 with 150 µmol/L of irritant sodium lauryl sulphate. Additionally, higher levels of inflammatory cytokines MIP-3α, MIP-1β, TNF-α, and IL-8 were found in supernatants of enhanced loose-fit co-culture based sensitisation assay (eLCSA) after substance treatment. Hence, eLCSA allowed to discriminate between sensitisers and non-sensitisers. Thus, inhibition of immunoinhibitory pathway signalling by combining AhR knockout and PD-L1 antibody blockage into an assay involving main acting cell types in skin sensitisation may increase sensitivity and specificity of such assays and allow potency derivation.

Abstract 296: Role Of Endothelial Cell-specific Knockout Of The Aryl Hydrocarbon Receptor In Ischemic Limb Myopathy In Mice Subjected To Hind Limb Ischemia

Introduction: Chronic kidney disease (CKD) is a risk factor for peripheral arterial disease (PAD) that accelerates disease evolution and worsens limb outcomes, however the molecular mechanisms linking the pathobiologies are ill-defined. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, has been previously shown to regulate angiogenesis. Notably, CKD results in the accumulation of tryptophan-derived metabolites that are endogenous ligands for the AHR. On this basis, we tested the hypothesis that endothelial cell-specific knockout of the AHR would enhance limb perfusion recovery and capillarity in mice with CKD subjected to hindlimb ischemia. Methods: Twelve-week-old conditional AHR knockout mice (AHR fl/fl , Cre-negative) and endothelium-specific AHR knockout mice (AHR ecKO ) were fed 0.2% adenine to induce CKD or casein control diet (n=10/group/sex). Femoral artery ligation (FAL) was performed on the left limb using the right limb as a non-ischemic control. Limb perfusion was measured using laser Doppler flowmetry. Skeletal muscle function was measured using nerve stimulus. Gastrocnemius mitochondrial respiratory function and H 2 O 2 production were assessed. Results: Regardless of genotype, mice with CKD had significantly lower renal function. Interestingly, male AHR ecKO mice with CKD displayed a significant increase in percentage of perfused capillaries within the ischemic tibialis anterior muscle (AHR ecKO 73.41% ± 4.80% vs AHR fl/fl 64.75% ± 8.325%; p= 0.0074 ) and improved laser Doppler perfusion recovery in the paw and gastrocnemius muscle compared to male AHR fl/fl mice with CKD. AHR ecKO mice had a larger mean myofiber size ( p=0.0258 ), although muscle weight and contractile function were not different. Mitochondrial respiratory capacity was significantly higher (p=0.0085) in AHR ecKO than AHR fl/fl mice. Conclusion: In mice with CKD, endothelium-specific deletion of the AHR improved blood perfusion and increased myofiber size in the ischemic limb muscle. These changes conferred improvement in muscle mitochondrial function, but not muscle strength. Interestingly, these changes were observed in male, but not female, mice.

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

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

1502 Conditional AhR knockout in fibroblasts exaggerated ITP-induced contact hypersensitivity while decreased TGF-b mediated myofibroblast differentiation

1502 Conditional AhR knockout in fibroblasts exaggerated ITP-induced contact hypersensitivity while decreased TGF-b mediated myofibroblast differentiation

Abstract 3443: Aryl hydrocarbon receptor and its ligands influence the formation of colonic tertiary lymphoid tissues

Abstract Background: In the large intestine, tertiary lymphoid tissues (TLTs) serve as localized centers of adaptive immune responses. These structures often form in response to inflammation and infection during adulthood. However, more recently TLTs have become a focal point of colon caner development and progression as their presence is often associated with positive clinical outcomes. Interestingly, several studies have demonstrated that the aryl hydrocarbon receptor (AhR) influences the development of secondary lymphoid tissues, but minimal studies have focused on its role in TLT formation. The purpose of the presented studies was to test the hypothesis that activation of AhR in intestinal epithelial cells (IECs) induces the formation of TLTs in the colon. Methods: Wild type (WT) and IEC-specific AhR knockout (CDX2PCreT2 x AhRf/f- iAhRKO) mice were used in several different experimental models. In the first study, azoxymethane (AOM) was used to induce precancerous lesions in the colon and TLT formation was evaluated in the presence and absence of AhR. Then, experiments were conducted to determine the necessity of AhR expression in IECs to influence the formation of TLTs following induction of acute inflammation induced by Dextran sodium sulfate (DSS) exposure. Finally, the ability of known, naturally occurring and microbial-derived AhR ligands (indole-3-aldehyde (I3A), 3,3ʹ-diindolylmethane (DIM), and indole-3-acetic acid (IAA)) were tested for their ability to induce TLT formation in the colon. Intestinal permeability (FITC-Dextran), expression of IEC-associated genes (real-time qPCR), and TLT formation/composition (H&amp;E staining/Immunofluorescence) were evaluated. Results: Results from the first study demonstrated that IEC-specific AhR KO mice exposed to AOM developed significantly fewer TLTs when compared to WT controls, while expression of Il-22 and other chemokines involved in TLT formation were also significantly downregulated. In the acute inflammation study, sex specific results were found. In females, loss of AhR activity in IECs reduced the formation of TLTs without significant changes in immune cell composition within their TLTs. Conversely, in males, loss of AhR lowered expression of functional-IEC genes (Ocln, Il-22), increased number of TLTs, increased T-cell density, and lower B: T cell ratio. Finally, in the other experiments, I3A induced TLT formation and DIM promoted intestinal barrier integrity through the upregulation of various tight junction genes and genes involved in the signaling associated with TLT formation. Evaluation of IAA which is a microbial-derived, AhR ligand is ongoing. Conclusion: These data indicate that AhR plays a distinct role in the formation of TLTs in the colon and these effects are likely ligand specific and may have a significant effect on colon tumor formation. Citation Format: Kimberly F. Allred, Erika L. Garcia-Villatoro, Jennifer DeLuca, Victoria Tepe, Zachary Bomstein, Arinzechukwu Ufondu, Stephen H. Safe, Robert S. Chapkin, Arul Jayaraman, Clinton D. Allred. Aryl hydrocarbon receptor and its ligands influence the formation of colonic tertiary lymphoid tissues [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 3443.

Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

ObjectiveMitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. MethodsIn this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. ResultsTranscriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. ConclusionsAhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Impact of the Aryl Hydrocarbon Receptor on Aurora A Kinase and the G2/M Phase Pathway in Hematopoietic Stem and Progenitor Cells

Recent evidence suggests that the environment-sensing transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of hematopoiesis. Yet, the mechanisms and extent of AHR-mediated regulation within the most primitive hematopoietic cells, hematopoietic stem and progenitor cells (HSPCs), are poorly understood. Through a combination of transcriptomic and flow cytometric approaches, this study provides new insight into how the AHR influences hematopoietic stem and progenitor cells. Comparative analysis of intraphenotypic transcriptomes of hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells from AHR knockout (AHR KO) and wild type mice revealed significant differences in gene expression patterns. Notable among these were differences in expression of cell cycle regulators, specifically an enrichment of G2/M checkpoint genes when Ahr was absent. This included the regulator Aurora A kinase (Aurka, AurA). Analysis of AurA protein levels in HSPC subsets using flow cytometry, in combination with inducible AHR KO or in vivo AHR antagonism, showed that attenuation of AHR increased levels of AurA in HSCs and lineage-biased MPP cells. Overall, these data highlight a potential novel mechanism by which AHR controls HSC homeostasis and HSPC differentiation. These findings advance the understanding of how AHR influences and regulates primitive hematopoiesis.