Aryl Hydrocarbon Receptor mRNA Research Articles

P05 The aryl hydrocarbon receptor pathway is dysfunctional in atopic dermatitis

Abstract Introduction and aims Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by epidermal barrier disruption and type-2 immune dysregulation. While treatment has improved with targeted biologics, clinical response varies, highlighting the need for additional therapeutic strategies. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and environmental sensor, which maintains skin barrier integrity and homeostasis by reducing inflammation. Upon activation, AHR induces the expression of CYP1A1, a biomarker of pathway activation and critical for AHR ligand metabolism. Tapinarof, a topical AHR agonist, is approved for psoriasis and is effective in phase III clinical trials in AD, highlighting the importance of this pathway in inflammatory skin disease. Nevertheless, AHR expression is increased in AD skin, leading to the hypothesis that the pathway is dysregulated, thus not exerting its anti-inflammatory effect. Methods Here we begin to perform a global investigation of the AHR pathway in AD. We measured AHR and CYP1A1 mRNA expression by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in healthy and AD skin, with spatial mRNA expression assessed by RNAscope. We also investigated AHR mRNA expression in interleukin (IL)-4 stimulated (100 ng mL−1) normal human epidermal keratinocytes (NHEKs) using qRT-PCR. Results We found that AHR mRNA is significantly upregulated in lesional AD (n = 6) compared with healthy skin (n = 9; P < 0.05), whereas CYP1A1 mRNA expression shows a downward trend in lesional AD compared with healthy skin (P = 0.063). The AHR expression pattern differs in lesional AD (n = 7) compared with healthy skin (n = 8), with AHR localizing in the epidermal basal layer. Finally, IL-4 significantly upregulated AHR mRNA (n = 4 wells; P < 0.001) expression in NHEKs compared with unstimulated cells. Conclusions Overall, our data have uncovered a dysfunctional AHR pathway in AD, with increased AHR expression, possibly driven by the AD inflammatory milieu, but reduced pathway activation. Further investigations are required to fully dissect this pathway and maximize its potential as a therapeutic target for AD.

Unbalanced Aryl Hydrocarbon Receptor Expression in Peripheral and Lesional T Cell Subsets of Atopic Dermatitis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which is involved in the pathogenesis of a variety of skin diseases such as atopic dermatitis (AD). In this study, we aimed to study the AhR-expressing cells in T helper 17 (Th17), T helper 22 (Th22), regulatory T cells (Treg) and B cells in peripheral blood and in AD skin lesions. Twenty AD patients defined according to the Chinese criteria of atopic dermatitis and eighteen healthy subjects were included in our study. The AhR-expressing Th17, Th22, Treg and total B cells in peripheral blood were measured by flow cytometry. The AhR+ Th17 cells and AhR+ Th22 cells in AD skin lesions were measured by immunofluorescence. The mRNA of AhR, interleukin (IL)-22, IL-17A, IL-10, Foxp3, RORγT and TGF-β in peripheral blood mononuclear cells (PBMCs) was measured by real-time quantitative polymerase chain reaction. The expression of AhR in peripheral CD4+ T cells, Th22 cells, Treg cells and total B cells was significantly increased in AD. AhR+IL-17A+ and AhR+IL-22+ lymphocytes were also increased in AD skin lesions. The mRNA levels of AhR, IL-22 and IL-17A in PBMCs in AD patients were significantly higher. AhR mRNA levels in PBMCs positively correlated with peripheral basophil count, peripheral eosinophil count and mRNA levels of IL-22. AhR was highly expressed in subpopulations of CD4+ T cells in peripheral blood and skin lesions of AD, suggesting that AhR might contribute to the pathogenesis of AD.

Targeting AHR Increases Pancreatic Cancer Cell Sensitivity to Gemcitabine through the ELAVL1-DCK Pathway.

The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). AHR hinders the shuttling of human antigen R (ELAVL1) from the nucleus to the cytoplasm, where it stabilises its target messenger RNAs (mRNAs) and enhances protein expression. Among these target mRNAs are those induced by gemcitabine. Increased AHR expression leads to the sequestration of ELAVL1 in the nucleus, resulting in chemoresistance. This study aimed to investigate the interaction between AHR and ELAVL1 in the pathogenesis of PDAC in vitro. AHR and ELAVL1 genes were silenced by siRNA transfection. The RNA and protein were extracted for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Direct binding between the ELAVL1 protein and AHR mRNA was examined through immunoprecipitation (IP) assay. Cell viability, clonogenicity, and migration assays were performed. Our study revealed that both AHR and ELAVL1 inter-regulate each other, while also having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, both proteins function through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-deoxycytidine kinase (DCK) molecular pathway. In conclusion, AHR and ELAVL1 interaction can form a negative feedback loop. By inhibiting AHR expression, PDAC cells become more susceptible to gemcitabine through the ELAVL1-DCK pathway.

Effects of enhancing the expression of aryl hydrocarbon receptor in post-traumatic mice macrophages on the inflammatory cytokine level and bactericidal ability

Objective: To explore the expression pattern of aryl hydrocarbon receptor (AhR) in mice peritoneal macrophages (PMs) after major trauma and analyze the effects of enhanced AhR expression on the inflammatory cytokine level and bactericidal ability after trauma. Methods: The experimental study method was used. Forty 6-8-week-old male C57BL/6J mice (the same mouse age, sex, and strain below) were divided into control group, post trauma hour (PTH) 2 group, PTH 6 group, and PTH 12 group according to the random number table (the same grouping method below), with 10 mice in each group. Mice in the latter 3 groups were constructed as severe trauma model with fracture+blood loss, while mice in control group were left untreated. The primary PMs (the same cells below) were extracted from the mice in control group, PTH 2 group, PTH 6 group, and PTH 12 group when uninjured or at PTH 2, 6, and 12, respectively. Then the protein and mRNA expressions of AhR were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction, respectively, and the gene expressions of AhR signaling pathway related molecules were analyzed by transcriptome sequencing. Twenty mice were divided into control group and PTH 6 group, with 10 mice in each group, and the PMs were extracted. The level of ubiquitin of AhR was detected by immunoprecipitation. Twelve mice were divided into dimethyl sulfoxide (DMSO) alone group, PTH 6+DMSO group, MG-132 alone group, and PTH 6+MG-132 group, with 3 mice in each group. After the corresponding treatment, PMs were extracted, and the protein expression of AhR was detected by Western blotting. Twenty mice were constructed as PTH 6 model. Then, the PMs were extracted and divided into empty negative control adenovirus (Ad-NC) group and AhR overexpression adenovirus (Ad-AhR) group. The protein expression of AhR was detected by Western blotting at 36 h after some PMs were transfected with the corresponding adenovirus. The rest cells in Ad-NC group were divided into Ad-NC alone group and Ad-NC+endotoxin/lipopolysaccharide (LPS) group, and the rest cells in Ad-AhR group were divided into Ad-AhR alone group and Ad-AhR+LPS group. The expressions of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in the cell supernatant were detected by enzyme-linked immunosorbent assay at 12 h after the corresponding treatment (n=6). Twenty mice were obtained to extract PMs. The cells were divided into control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group, and the intracellular bacterial load was detected by plate spread method after the corresponding treatment (n=6). Data were statistically analyzed with one-way analysis of variance, least significant difference test, analysis of variance for factorial design, and independent sample t test. Results: Compared with 1.16±0.28 of control group, the protein expressions of AhR in PMs in PTH 2 group (0.59±0.14), PTH 6 group (0.72±0.16), and PTH 12 group (0.71±0.17) were all significantly decreased (P<0.05). The overall comparison of the difference of AhR mRNA expression in PMs among control group, PTH 2 group, PTH 6 group, and PTH 12 group showed no statistical significance (P>0.05). The AhR signaling pathway related molecules included AhR, AhR inhibitor, cytochrome P450 family member 1b1, cytochrome P450 family member 11a1, heat shock protein 90, aryl hydrocarbon receptor-interaction protein, and heat shock protein 70 interaction protein. The heat shock protein 90 expression of PMs in PTH 2 group was higher than that in control group, while the expressions of other molecules did not change significantly after trauma. Compared with that in control group, the level of ubiquitin of AhR in PMs in PTH 6 group was increased. Compared with that in DMSO alone group, the protein expression of AhR in PMs in PTH 6+DMSO group was decreased, while that in PMs in MG-132 alone group had no significant change. Compared with that in PTH 6+DMSO group, the protein expression of AhR in PMs in PTH 6+MG-132 group was up-regulated. At transfection hour 36, compared with that in Ad-NC group, the protein expression of AhR in PMs in Ad-AhR group was increased. At treatment hour 12, compared with those in Ad-NC+LPS group, the expressions of IL-6 and TNF-α in PM supernatant of Ad-AhR+LPS group were significantly decreased (with t values of 4.80 and 3.82, respectively, P<0.05). The number of intracellular bacteria of 1×106 PMs in control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group was (3.0±1.8), (41.8±10.2), (1.8±1.2), and (24.2±6.3) colony forming unit, respectively. Compared with that in PTH 6+Ad-NC group, the number of intracellular bacteria of PMs in PTH 6+Ad-AhR group was significantly decreased (t=3.61, P<0.05). Conclusions: Ubiquitin degradation of AhR in PMs of mice after major trauma results in decreased protein expression of AhR. Increasing the expression of AhR in post-traumatic macrophages can reduce the expressions of LPS-induced inflammatory cytokines IL-6 and TNF-α, and improve the bactericidal ability of macrophages after trauma.

WITHDRAWN: Interleukin-35 dampens T helper 22 phenotype shift in CD4+CD25+CD127dim/- regulatory T cells in primary biliary cholangitis

The phenotype shift in regulatory T cells (Tregs) contributes to immunopathogenesis of autoimmune diseases. The current study was aimed to investigate the regulatory function of interleukin-35 (IL-35) to T helper 22 (Th22) cell phenotype shift in Tregs in primary biliary cholangitis (PBC). Fifty-five PBC patients and twenty-four controls were enrolled. CD4+CD25+CD127dim/- Tregs and Th22 cells were investigated by flow cytometry. Forkhead box P3 (FoxP3) and aryl hydrocarbon receptor (AhR) mRNA levels were assessed by real-time polymerase chain reaction. Plasma IL-10 and IL-22 levels were measured by ELISA. Purified Tregs were stimulated with exogenous IL-35, and were co-cultured with autologous CD4+CD25- T cells. Cellular proliferation and cytokine production was measured. Purified Tregs were also cultured into Th22 condition in the presence or absence of exogenous IL-35, and Th22 phenotype were assessed. PBC patients had lower levels of Treg percentage, FoxP3 mRNA, and plasma IL-10, while had higher levels of Th22 proportion, AhR mRNA, and plasma IL-22. Tregs from PBC patients showed reduced immunosuppressive activity, which presented as increased cellular proliferation, interferon-γ production and decreased IL-35/IL-10 secretion in co-culture system. Tregs shifted into Th22 phenotype in PBC patients with elevated CCR4, CCR6, and CCR10 expression as well as increased IL-22 production. IL-35 not only enhanced inhibitory function of Tregs but also suppressed phenotype shift of Tregs into Th22 phenotype in PBC patients. This process was accompanied by elevation of IL-10 and transforming growth factor-β1 secretion by Tregs from PBC patients. The present data suggested that reduced IL-35 might be insufficient to maintain Tregs function and phenotype shift from Tregs into Th22 phenotype in PBC patients.

Crosstalk between the aryl hydrocarbon receptor (AhR) and the peroxisome proliferator-activated receptor gamma (PPARγ) as a key factor in the metabolism of silver nanoparticles in neuroblastoma (SH-SY5Y) cells in vitro

The potential usefulness of silver nanoparticles (AgNPs) in anticancer therapy has been postulated for many years. However, little is known to date about the exact impact of such NPs on intracellular detoxication pathways. Therefore, the aim of this study was to determine the impact of AgNPs on the AhR-PPARγ-CYP1A1 pathway in neuroblastoma (SH-SY5Y) cells. The obtained results showed a decrease in the metabolic activity of the SH-SY5Y cells at the 50 and 100 μg/mL concentrations with an increase in caspase-3 activity. An increase in the intercellular ROS production was observed at the 1 and 10 μg/mL concentrations. The co-treatment of the AgNP-treated cells with the AhR and PPARγ inhibitors abolished the effect of the tested AgNPs in the SH-SY5Y cells. In turn, the CYP1A1 activity assay showed a decrease in this parameter in the AgNP-treated cells. Moreover, the gene expression analysis demonstrated that AgNPs were able to increase the AhR and CYP1A1 mRNA expression and decrease the PPARγ gene expression after the 6-h treatment. In turn, an increase in the AhR and PPARγ protein expression was observed after 24 h. Summarizing, the study shows for the first time that AgNPs with a 5-nm diameter size are able to exert a cytotoxic effect on SH-SH5Y cells in a ROS-dependent manner affect the AhR-PPARγ-CYP1A1 pathway inter alia by inhibiting the activity of CYP1A1. This is important due to given present research approaches using such NPs as enhancer agents in the modern PPARγ inhibitor-based anticancer therapy.

337 Multi-level dysregulation of the AHR/CYP1A1 axis in psoriasis

The aryl hydrocarbon receptor (AHR) is an environmentally-regulated transcription factor with homeostatic and anti-inflammatory roles in skin. AHR activation leads to expression of the cytochrome P4501 (CYP1) enzyme family, which provides critical negative feedback by degrading AHR ligands. We have previously shown that excessive CYP1A1 activity impairs AHR anti-inflammatory activity in psoriasis-like skin inflammation in mice, while suppression of aberrant enzymatic activity restores AHR beneficial effect. Moreover, CYP1A1 activity is significantly enhanced in blood cells of psoriasis patients, suggesting that dysregulation of CYP1A1 activity may play a role in psoriasis. Here, we performed a multi-modal investigation of the AHR/CYP1A1 axis in the blood and skin of psoriasis patients and healthy controls, assessing availability of AHR ligands and precursors by mass spectrometry, AHR and CYP1A1 mRNA by qPCR, and CYP1A1 enzymatic activity by EROD assay. Levels of the ligand precursor tryptophan were significantly (p≤0.001) decreased in the serum of psoriasis patients (n=20) versus healthy (n=20). Expression of AHR mRNA was significantly (p≤0.001) decreased in both psoriasis blood and psoriasis lesional (PsL) skin, while CYP1A1 mRNA did not differ in blood but was significantly (p≤0.05) decreased in PsL skin. In vitro culture of skin epidermal sheets with an AHR agonist significantly (p≤0.05) upregulated CYP1A1 mRNA in healthy and psoriasis skin, but, surprisingly, CYP1A enzymatic activity was not increased by AHR ligation in PsL. We are currently studying AHR and CYP1A1 protein expression in the skin at single-cell level by imaging mass cytometry to gain further insights into the complex regulation of the pathway. Taken together, our data show that there is a multi-level dysregulation of the AHR/CYP1A1 axis in psoriasis and suggest a failure of multiple pathway checkpoints that could be targeted to restore AHR beneficial effects in skin inflammation.

A new insight into the aryl hydrocarbon receptor/cytochrome 450 signaling pathway in MG63, HOS, SAOS2, and U2OS cell lines

Osteosarcoma is the most common malignant tumor of bone, with rapid progressive growth, early distant metastases, and frequent recurrence after surgical treatment. Osteosarcoma is characterized by changes in the ratio and expression of different cytochrome P450 (CYP) isoforms that can affect the effectiveness of anticancer therapies. The inducible expression of CYP1 genes depends on the ligand-dependent functionality of the aryl hydrocarbon receptor (AHR). In this study, we examined the AHR/CYP1 signaling pathway in four osteosarcoma cell lines (MG63, HOS, SAOS2, and U2OS) induced by the known AHR ligands: indirubin, indole-3-carbinol, and beta-naphthoflavone. Using qPCR and Western blot analysis, we explored the effects of these ligands on the expression of the CYP1 genes and studied the correlation between these responses and the changes in the mRNA and protein levels of AHR and the AHR nuclear translocator (ARNT) in these osteosarcoma cell lines. The results show that the AHR/CYP1 signaling pathway retains its function only in MG63 and HOS cells, and is impaired in SAOS2 and U2OS cells. Our data should be taken into account when recommending new strategies for the treatment of osteosarcoma and when evaluating new drugs against osteosarcoma in vitro.

AHR gene expression and the polymorphism rs2066853 are associated with clinicopathological parameters in colorectal carcinoma

The relationship between red and processed meat and its risk towardcolorectal carcinoma (CRC) is not fully explored in literature. Polycyclic aromatic hydrocarbons (PAHs) are procarcinogenic molecules that are ingested with meat cooked at high temperatures. The metabolic conversion of PAHs to carcinogenic diol epoxides is in part mediated by the aryl hydrocarbon receptor (AhR)-dependent induction of CYP1A1. This study aims to examine the expression profiles and polymorphisms of the AHR (aryl hydrocarbon receptor) gene which is involved in the metabolic conversion of PAHs in patients with CRC. Genetic analysis was done in matched cancer and non-neoplastic tissues from 79 patients diagnosed with CRCs. Low AHR mRNA expression was associated with mucinous colorectal adenocarcinoma. Exon 10 of AHR showed that 27% of patients had the rs2066853 single-nucleotide polymorphism resulting in an arginine-to-lysine change at codon 554. This variant was significantly associated with a lower likelihood of perineural invasion, presence of synchronous cancer, and multiple colorectal polyps. Furthermore, rs2066853 individuals were significantly more likely to be of more advanced age and have a more favorable tumor grade and pathological stage. These results imply the pathogenic roles of AHR in PAH-associated colorectal carcinogenesis.

Effects of intranasal instillation of nanoparticulate matter in the olfactory bulb

Nanoparticulate matter activates the aryl hydrocarbon receptor (AhR) pathway in the respiratory system in a process involving the AhR nuclear translocator (ARNT) and cytochrome P450 family 1, member A1 (CYP1A1). We examined changes in AhR-related pathways following intranasal instillation of nanoparticulate matter in the olfactory bulb and cerebral cortex. Twice a day for 5 days per week for 1 week or 2 weeks, 8-week-old Sprague–Dawley rats were intranasally instilled with 10 µL nanoparticulate matter (nano group; n = 36). An equal volume of saline was intranasally instilled in control rats (n = 36). One week after intranasal instillation, olfactory function and Y-maze tests were performed. The expression levels of AhR in the olfactory bulb and temporal cortex were analyzed using western blotting and immunofluorescence assays. The expression levels of AhR, CYP1A1, inducible nitric oxide synthase (iNOS), and five genes encoding cation transporters (ARNT, ATP7B, ATPB1, OCT1, and OCT2) in the olfactory bulb were analyzed using quantitative reverse transcription. The olfactory discrimination capability was reduced in the nano group compared with the control group. Proportional changes in the Y-maze test were not significantly different between the nano and control groups. AhR mRNA and protein expression in the olfactory bulb increased 1.71-fold (P < 0.001) and 1.60-fold (P = 0.008), respectively. However, no significant changes were observed in the temporal cortex. In the olfactory bulb, the expression of ARNT, ATP7B, ATPB1, and OCT2 was downregulated. CYP1A1 and iNOS expression in the olfactory bulb was upregulated compared with that in the temporal cortex. The intranasal instillation of nanoparticulate matter decreased the olfactory discrimination ability, which was accompanied by upregulation of AhR expression and downregulation of cation transporters in the olfactory bulb.

AHR-dependent genes and response to MTX therapy in rheumatoid arthritis patients

Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis. Nevertheless, MTX resistance is quite a common issue in clinical practice. There are some premises that aryl hydrocarbon receptor (AhR) gene battery may take part in MTX metabolism. In the present retrospective study, we analyzed genes expression of AHR genes battery associated with MTX metabolism in whole blood of RA patients with good and poor response to MTX treatment. Additionally, sequencing, genotyping and bioinformatics analysis of AHR repressor gene (AHRR) c.565C > G (rs2292596) and c.1933G > C (rs34453673) have been performed. Theoretically, both changes may have an impact on H3K36me3 and H3K27me3. Evolutionary analysis revealed that rs2292596 may be possibly damaging. Allele G in rs2292596 and DAS28 seems to be associated with a higher risk of poor response to MTX treatment in RA. RA patients with poor response to MTX treatment revealed upregulated AhR and SLC19A1 mRNA level. Treatment with IL-6 inhibitor may be helpful to overcome the low-dose MTX resistance. Analysis of gene expression revealed possible another cause of poor response to MTX treatment which is different from that observed in the case of acute lymphoblastic leukemia.

Aryl hydrocarbon receptor modulates airway inflammation in mice with cockroach allergen-induced asthma by regulating Th17/Treg differentiation

To investigate whether aryl hydrocarbon receptor (AhR) modulates cockroach allergen (CRE)-induced asthma by regulating Th17/Treg differentiation. Mouse models of CRE-induced asthma established by sensitizing and challenging the mice with CRE were randomized into asthma model group, AhR agonist group treated with TCDD (10 μg/ kg), and AhR antagonist group treated with TCDD and CH223191 (10 mg/kg) (n=5), with 5 mice without CRE challenge as the control group. The expressions of AhR, Cyp1a1 and Cyp1b1 mRNA in the lung tissues of the mice were detected using RT-PCR, and pulmonary inflammation was evaluated with immumohistochemical staining. The expressions of inflammatory cytokines in the lungs were detected using ELISA, and the expression of Treg in the lung tissues and pulmonary lymph nodes was analyzed with flow cytometry. Both TCDD and CH223191 were capable of modulating pulmonary expressions of AhR and its downstream genes Cyp1a1 and Cyp1b1 in asthmatic mice (P < 0.002). TCDD treatment significantly decreased inflammatory cells and mucus production in the lungs of asthmatic mice, and BALFs from TCDD-treated mice with CRE challenge contained lowered levels of the proinflammatory factors including IL-4, IL-13 and IL-17A (P < 0.001) but increased anti-inflammatory factors including IL-10, IL-22 and TGF-β1 (P < 0.001). All these changes were significantly reversed by treatment with CH223191 to the levels comparable with those in the asthma model group (P>0.05). More importantly, TCDD treatment significantly increased the number of Tregs cells and FOXP3 expression and lowered RORγt mRNA expression in the lungs and pulmonary lymph nodes in asthmatic mice (P < 0.001); inhibition of AhR with CH223191, as compared with TCDD, significantly decreased the expression of CD4+CD25+Foxp3+Treg cells in the lungs and pulmonary lymph nodes and the expression of FOXP3 mRNA in lymphocytes and increased RORγt mRNA expression (P < 0.001) to the levels comparable with those in asthma model group (P>0.05). AhR activation modulates airway inflammation in mice with CRE-induced asthma by modulating the differentiation of Th17/Treg.

Bisphenol analogs AF, S and F: Effects on functional characteristics of porcine granulosa cells

In order to replace industrial functions of the restricted endocrine disruptor bisphenol A (BPA), its structural analogs are increasingly employed without adequate assessment of their biological actions. Our study examined effects of the bisphenols AF (BPAF), S (BPS) and F (BPF), on functions of porcine ovarian granulosa cells (GCs) with the focus on viability, steroid production (10-9–10-4M), and expression of factors (10-9–10-5M) important for the follicle development: vascular endothelial growth factor A (VEGFA), matrix metalloproteinase 9 (MMP9), forkhead box O1 (FOXO1), and aryl hydrocarbon receptor (AHR). Cell viability was not impaired by the bisphenol analogs, except for the highest BPAF concentration (10-4M). While the lower concentrations of the bisphenols were without effect, each of them reduced follicle-stimulating hormone (FSH)-induced progesterone synthesis at the highest dose. Estradiol synthesis was sensitive to BPS, inhibitory effects of which were manifested from the concentration of 10-6M. Treatment of GCs with the selected bisphenol concentrations did not result in marked alterations in steroidogenic enzyme expression. Bisphenols did not significantly modulate VEGFA mRNA expression or output either under basal or FSH-stimulated conditions. BPF at 10-5M increased MMP9 expression in FSH-stimulated cells. FSH upregulated FOXO1 expression, however, none of the bisphenols significantly affected FOXO1 levels either in basal or in FSH-stimulated conditions. AHR mRNA expression remained unchanged after bisphenol treatment. Although the significant effects of BPAF, BPS and BPF appeared only at supraphysiological doses, the results obtained indicate that BPA analogs are not inert with regard to ovarian physiology.

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

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

Aryl Hydrocarbon Receptor and Autophagy-Related Protein Microtubule-Associated Protein Light Chain 3 Expression in Psoriasis.

BackgroundThe aryl hydrocarbon receptor (AHR) and autophagy are both important to maintain skin homeostasis. However, they are also involved in skin disorders. So far, their roles in psoriasis pathogenesis are unknown.ObjectiveWe studied the immunohistochemical and gene expression of AHR, CYP1A1, and microtubule-associated protein light chain 3 (LC3) in lesional skin of psoriasis patients to determine correlations among them.MethodsWe included 24 psoriasis patients and ten healthy volunteers. Skin biopsies were collected. AHR, CYP1A1, and LC3 protein expression was examined by immunohistochemistry, immunofluorescence, and western blotting. AHR, CYP1A1, LC3, ATG5, BECN1 and Nrf2 mRNA levels were measured by quantitative polymerase chain reaction.ResultsAHR and CYP1A1 protein expression were higher in psoriasis lesional skin than in normal skin. LC3 protein expression was lower in psoriasis lesions than in normal controls. AHR and CYP1A1 protein expression in psoriasis lesions showed significant positive correlations with mean epidermal thickness and inflammatory cell density. Significant negative correlations were noted between LC3 protein expression in psoriasis lesions and the mean epidermal thickness or inflammatory cell density. A significant negative correlation was found between AHR and LC3 expression in psoriatic skin. AHR, CYP1A1 and Nrf2 mRNA expression were upregulated while LC3, ATG5, and BECN1 mRNA were down-regulated, in psoriatic lesional skin compared with normal controls.ConclusionAHR and autophagy could play a role in psoriasis pathogenesis by modifying epidermal hyperproliferation and inflammation. AHR and autophagy regulation are potential therapeutic targets in chronic inflammatory skin diseases.

Exposure to Traffic-Generated Pollutants Exacerbates the Expression of Factors Associated with the Pathophysiology of Alzheimer's Disease in Aged C57BL/6 Wild-Type Mice.

Multiple studies report a strong correlation between traffic-generated air pollution-exposure and detrimental outcomes in the central nervous system (CNS), including Alzheimer's disease (AD). Incidence of AD is rapidly increasing and, worldwide, many live in regions where pollutants exceed regulatory standards. Thus, it is imperative to identify environmental pollutants that contribute to AD, and the mechanisms involved. We investigated the effects of mixed gasoline and diesel engine emissions (MVE) on the expression of factors involved in progression of AD in the hippocampus and cerebrum in a young versus aged mouse model. Young (2 months old) and aged (18 months old) male C57BL/6 mice were exposed to either MVE (300μg/m3 PM) or filtered air (FA) for 6 h/d, 7 d/wk, for 50 d. Immunofluorescence and RT-qPCR were used to quantify oxidative stress (8-OHdG) and expression of amyloid-β protein precursor (AβPP), β secretase (BACE1), amyloid-β (Aβ), aryl hydrocarbon receptor (AhR), cytochrome P450 (CYP) 1B1, angiotensin-converting enzyme (ACE1), and angiotensin II type 1 (AT1) receptor in the cerebrum and hippocampus, in addition to cerebral microvascular tight junction (TJ) protein expression. We observed age-related increases in oxidative stress, AhR, CYP1B1, Aβ, BACE1, and AT1 receptor in the CA1 region of the hippocampus, and elevation of cerebral AβPP, AhR, and CYP1B1 mRNA, associated with decreased cerebral microvascular TJ protein claudin-5. MVE-exposure resulted in further promotion of oxidative stress, and significant increases in AhR, CYP1B1, BACE1, ACE1, and Aβ, compared to the young and aged FA-exposed mice. Such findings suggest that MVE-exposure exacerbates the expression of factors in the CNS associated with AD pathogenesis in aged populations.

Indoleamine 2,3-Dioxygenase Mediates Survival of Chronic Lymphocytic Leukemia B Cells through Aryl Hydrocarbon Receptor By Inducing Mcl1

Introduction: Chronic lymphocytic leukemia (CLL) is a dynamic disease in which monoclonal B cells proliferate within the pseudo-follicular centers in lymphoid organs and then they accumulate due to an intrinsic defect of apoptosis. Leukemic cells are considered as "addicted to the host" since extrinsic signals from the microenvironment strongly influence the establishment of a progressive immunosuppression for malignant cell growth and survival. The cytoplasmic enzyme indoleamine 2,3-dioxygenase (IDO) mediates the conversion of the essential amino acid tryptophan (Trp) into metabolic byproducts such as kynurenine (Kyn). Kyn and other secondary metabolites are endogenous activators of the aryl hydrocarbon receptor (AHR), a ligand-controlled transcription factor that mediates cellular responses to toxins or endogenous ligands. The IDO-Kyn-AHR axis plays important roles in carcinogenesis and cancer progression. The mechanisms that promote inflammation around tumor tissues and determine immune tolerance consist in Trp depletion, which induces T cell apoptosis, and in Kyn-mediated AHR activation that inhibits effector T cells and promotes regulatory T cells differentiation. IDO protein is expressed in human hematologic malignancies and its level is correlated with a poor prognosis and chemoresistance. The IDO activity, measured as the Kyn/Trp ratio, was reported to be increased in CLL cases comparing to normal controls. Aim: We wondered to characterize the expression of IDO and AHR in CLL patients and to dissect the biological function of the IDO-Kyn-AHR axis. Methods: Gene transcription and protein expression were evaluated by real time PCR and western blot. Enzymatic activity was assessed through ELISA. Survival was measured with PI/annexin V assay. Overexpression and silencing of target genes was obtained by nucleofection. Results: Firstly, we observed that CLL cells expressed both IDO and AHR at variable levels. Moreover, we found that several microenvironmental signals such as IFNγ, LPS, anti-IgM, CpG oligo DNA, CD40L and TNFα were able to up-regulate IDO and AHR mRNA and protein. To characterize the pathways able to mediate IDO expression, we stimulated CLL cells with IFNγ and CD40L. Using ruxolitinib, an inhibitor of JAK-STAT pathway, we found that IFNγ induced IDO through STAT1 signaling. Again, CD40L stimulation determined IDO overexpression through the non-canonical NF-kB pathway, as assessed by treating cells with NF-κB inducing kinase inhibitor, NIK SMI1. We also confirmed that IFNγ-treated CLL cells were able to produce a functional IDO enzyme by measuring Kyn production and Trp consumption by ELISA. The strong increase in the Kyn/Trp ratio induced by IFNγ was significantly reduced by ruxolitinib treatment. To verify if Kyn produced by CLL cells could act through an autocrine loop on AHR, leukemic cells were treated with Kyn. We observed that Kyn mediated AHR translocation from the cytoplasm to the nuclei, inducing its activation as assessed by up-regulation of CYP1A1, a known AHR target gene. Of interest, we found that Kyn treatment improved CLL cells survival. Analyzing the anti-apoptotic proteins of the Bcl2 family after Kyn treatment, we found the induction of Mcl1, that was affected by adding CH-223191, an antagonist of AHR. Moreover, we transfected CLL cells with an IDO vector. The up-regulation of IDO increased CLL cells survival through the induction of Mcl1. Accordingly, when CLL cells were silenced for AHR, we observed a reduction of their survival. Conclusion: Our data demonstrate the constitutive expression of IDO and AHR in CLL cells. Furthermore, the tumor microenvironment promotes the induction of IDO and AHR through a complex signaling crosstalk with leukemic cells. Our findings underline that IDO-Kyn-AHR axis is active in CLL cells and promotes Mcl1 expression, sustaining the survival of CLL cells. Disclosures Luppi: Gilead Sci: Consultancy, Speakers Bureau; MSD: Consultancy; Sanofi: Consultancy; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy; Novartis: Consultancy, Speakers Bureau. Marasca:Gilead Sci: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria.

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging.We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p.Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3′,4′-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.

Benzo[a]pyrene at human blood equivalent level induces human lung epithelial cell invasion and migration via aryl hydrocarbon receptor signaling.

Benzo[a]pyrene (B[a]P), a typical carcinogenic polycyclic aromatic hydrocarbon, exists worldwide in vehicle exhaust, cigarette smoke and other polluted environments. Recent studies have demonstrated a strong association between B[a]P and lung cancer. However, whether B[a]P at human blood equivalent level can promote epithelial-mesenchymal transition (EMT), a crucial molecular event during cell malignant transformation, remains unclear. Besides, whether B[a]P facilitates this progress via aryl hydrocarbon receptor (AhR) signaling pathway also lacks scientific evidence. In our study, the transwell assay showed that 5 μg/L of B[a]P promoted BEAS-2B cell invasion and migration. In addition, the mRNA and protein expression levels of AhR and its target genes involved in B[a]P metabolism, such as AhR nuclear translocator, heat shock protein 90 and CYP1A1, were significantly increased by B[a]P exposure. Moreover, the mRNA expression levels of downstream regulatory factors related to both AhR signaling pathway and EMT, such as NRF2, K-RAS and hypoxia-inducible factor 1-alpha, were significantly increased. Furthermore, the expression level of the epithelial marker E-cadherin was significantly downregulated, while the mRNA expression of mesenchymal phenotype markers, N-cadherin, fibronectin and vimentin, were significantly upregulated. Notably, the above changes induced by B[a]P were significantly attenuated or even stopped by resveratrol (RSV), a natural phenol, also an AhR inhibitor, when the AhR signaling pathway was inhibited by RSV, demonstrating the regulatory role of AhR signaling pathway in B[a]P-induced EMT. In conclusion, B[a]P at the human blood equivalent level induces BEAS-2B cell invasion and migration through the AhR signaling pathway.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis.

To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). Murine models of A. fumigatus keratitis were established by scraping the central epithelium of mouse cornea, daubing A. fumigatus on the cornea and covering with a contact lens. The mice were randomly divided into the control group and the A. fumigatus-infected (A.F.) group for 1, 3 and 5d respectively, which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection. In this study, immunofluorescence staining was used to detect the expression and localization of AhR in mouse corneas, and the mRNA and protein of AhR were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. In addition, mouse peritoneal macrophages were stimulated by A. fumigatus with or without the pretreatment of AhR antagonist CH223191 and AhR agonist FICZ, and the tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and Arg-1 mRNA were detected by RT-PCR. According to the results of the slit light photography, it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3rd day after the infection of A. fumigatus. Contrasted with the control group, the expression of AhR in the corneal epithelial cells infected with A. fumigatus was significantly increased detected by immunofluorescence staining. AhR mainly expressed in the nucleus and cytoplasm of corneal epithelial cells. Consistent with the transcriptional level of AhR mRNA, the expression level of AhR protein reached the peak on the 3rd day after infection which was detected by Western blot. Furthermore, RT-PCR showed that CH223191 up-regulated the expression of TNF-α and iNOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages; inversely, FICZ reduced the expression of TNF-α and iNOS while elevated the expression of IL-10 and Arg-1. AhR is involved in the pathogenesis of A. fumigatus keratitis and induced immune protection in anti-A. fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.