The Aryl Hydrocarbon Receptor-Dependent TGF-α/VEGF-B Ratio Correlates With Disease Subtype and Prognosis in Multiple Sclerosis.

OBJECTIVE: To evaluate the aryl hydrocarbon receptor (AHR)-dependent transforming growth factor alpha (TGF-α)/vascular endothelial growth factor B (VEGF-B) ratio, which regulates the effects of metabolic, dietary, and microbial factors on acute and chronic CNS inflammation, as a potential marker in multiple sclerosis (MS). METHODS: TGF-α, VEGF-B, and AHR agonistic activity were determined in serum of 252 patients with relapsing-remitting (RR) MS, primary and secondary progressive MS, as well as during active disease (clinically isolated syndrome [CIS] and RRMS relapse). RESULTS: The TGF-α/VEGF-B ratio and AHR agonistic activity were decreased in all MS subgroups with a stable disease course as compared to controls. During active CNS inflammation in CIS and RRMS relapse, the TGF-α/VEGF-B ratio and AHR agonistic activity were increased. Conversely, in patients with minimal clinical impairment despite long-standing disease, the TGF-α/VEGF-B ratio and AHR agonistic activity were unaltered. Finally, the TGF-α/VEGF-B ratio and AHR agonistic activity correlated with neurologic impairment and time to conversion from CIS to MS. CONCLUSIONS: The AHR-dependent TGF-α/VEGF-B ratio is altered in a subtype, severity, and disease activity-specific manner and correlates with time to conversion from CIS to MS. It may thus represent a novel marker and serve as additive guideline for immunomodulatory strategies in MS. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that serum levels of AHR, TGF-α, and VEGF-B distinguish subtypes of MS and predict the severity and disease activity of MS.

Association of aryl hydrocarbon receptor (AhR) serum level and gene rs10247158 polymorphism with anthropometric, biochemical parameters and food consumption in overweight/obese patients.

AIM: Aryl hydrocarbon receptor (AhR) plays a role in xenobiotic metabolism, which can be also activated by dietary patterns and components. AhR ligands in circulation are reported to induce weight gain, glucose intolerance and suggested to contribute to the development of obesity. In this study, we aimed to examine the relationship of the AhR gene and its polymorphisms with obesity and food consumption. METHODS: The study was conducted with 117 individuals of whom 52 had a body mass index (BMI) of <25 (normal weight) and 65 had a BMI of ≥25 (overweight/obese). The distribution of the serum level and polymorphism (rs10247158) of the participants were determined in venous blood samples using the ELISA and PCR method. Body composition and skinfold thickness of the individuals were measured and their food consumption records were analysed in the BeBiS program. RESULTS: The serum AhR, HOMA-IR, fasting blood glucose and basal insulin levels were found to be significantly higher (P < .001); however, no relationship was found between AhR polymorphisms in the overweight/obese individuals. In the overweight/obese group, the serum AhR level had a negative correlation with potassium, coffee and alcohol consumption and a positive correlation with suprailiac skinfold thickness. Dietary patterns expected to be related with increased serum AhR levels, such as fat and derivatives, were not observed in overweight/obese group; on the other hand, there was a negative correlation in normal group. CONCLUSION: In our study, the serum AhR levels of the overweight/obese individuals were found to be significantly higher. Some dietary patterns were determined to be correlated with serum AhR levels in overweight/obese group. However, the results need to be confirmed for ethnic differences and larger samples.

Aryl hydrocarbon receptor is essential for the pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by arteriopathy in the small to medium-sized distal pulmonary arteries, often accompanied by infiltration of inflammatory cells. Aryl hydrocarbon receptor (AHR), a nuclear receptor/transcription factor, detoxifies xenobiotics and regulates the differentiation and function of various immune cells. However, the role of AHR in the pathogenesis of PAH is largely unknown. Here, we explore the role of AHR in the pathogenesis of PAH. AHR agonistic activity in serum was significantly higher in PAH patients than in healthy volunteers and was associated with poor prognosis of PAH. Sprague-Dawley rats treated with the potent endogenous AHR agonist, 6-formylindolo[3,2-b]carbazole, in combination with hypoxia develop severe pulmonary hypertension (PH) with plexiform-like lesions, whereas Sprague-Dawley rats treated with the potent vascular endothelial growth factor receptor 2 inhibitors did not. Ahr-knockout (Ahr-/- ) rats generated using the CRISPR/Cas9 system did not develop PH in the SU5416/hypoxia model. A diet containing Qing-Dai, a Chinese herbal drug, in combination with hypoxia led to development of PH in Ahr+/+ rats, but not in Ahr-/- rats. RNA-seq analysis, chromatin immunoprecipitation (ChIP)-seq analysis, immunohistochemical analysis, and bone marrow transplantation experiments show that activation of several inflammatory signaling pathways was up-regulated in endothelial cells and peripheral blood mononuclear cells, which led to infiltration of CD4+ IL-21+ T cells and MRC1+ macrophages into vascular lesions in an AHR-dependent manner. Taken together, AHR plays crucial roles in the development and progression of PAH, and the AHR-signaling pathway represents a promising therapeutic target for PAH.

Association of aryl hydrocarbon receptor transactivating activity, a potential biomarker for persistent organic pollutants, with the risk of gestational diabetes mellitus.

Persistent organic pollutants(POPs) are suggested to be potential risk factors for gestational diabetes mellitus(GDM). We examined the hypothesis that the aryl hydrocarbon receptor trans-activating(AhRT) activity, a potential biomarker for the presence of POPs, could be a GDM risk factor in pregnant women. A total of 390 GDM and 100 normal pregnant(non-GDM) subjects in the Korea National Diabetes Program cohort voluntarily participated. We measured AhRT activity and concentrations of ATP and reactive oxygen in the serum collected at the screening of the participants for GDM using recombinant Hepa1c1c7 cells. Odds ratios(ORs) and 95% confidence intervals(CIs) were estimated using multivariable logistic regression models. The sensitivity and specificity of AhRT activity for GDM diagnostics were measured by receiver operating characteristic(ROC) analysis. Body mass index at pre-pregnancy and delivery and systolic blood pressure were significantly higher in the GDM group. AhRT activity was higher, and ATP concentrations were lower in the GDM group than the non-GDM group(P < 0.0001). AhRT activity was significantly higher in the GDM group(OR 29.3, 95% CI 10.9-79.1) compared with non-GDM(P < 0.0001). Serum glucose concentration at 1 h after a 50 g glucose challenge(glucose-50) was moderately correlated with AhRT activity(r2 = 0.387) and negatively correlated with ATP production(r2 = -0.650). In the ROC curve, AhRT activity had 70.9% sensitivity and 90.0% specificity for glucose-50, a GDM screening method. In conclusion, this study suggests that serum AhRT activity is positively associated with the risk of GDM.

Aryl Hydrocarbon Receptor Plasma Agonist Activity Correlates With Disease Activity in Progressive MS.

OBJECTIVE: The relationship between serum aryl hydrocarbon receptor (AHR) agonistic activity levels with disease severity, its modulation over the course of relapsing-remitting MS (RRMS), and its regulation in progressive MS (PMS) are unknown. Here, we report the analysis of AHR agonistic activity levels in cross-sectional and longitudinal serum samples of patients with RRMS and PMS. METHODS: In a cross-sectional investigation, a total of 36 control patients diagnosed with noninflammatory diseases, 84 patients with RRMS, 35 patients with secondary progressive MS (SPMS), and 41 patients with primary progressive MS (PPMS) were included in this study. AHR activity was measured in a cell-based luciferase assay and correlated with age, sex, the presence of disease-modifying therapies, Expanded Disability Status Scale scores, and disease duration. In a second longitudinal investigation, we analyzed AHR activity in 13 patients diagnosed with RRMS over a period from 4 to 10 years and correlated AHR agonistic activity with white matter atrophy and lesion load volume changes. RESULTS: In RRMS, AHR ligand levels were globally decreased and associated with disease duration and neurologic disability. In SPMS and PPMS, serum AHR agonistic activity was decreased and correlated with disease severity. Finally, in longitudinal serum samples of patients with RRMS, decreased AHR agonistic activity was linked to progressive CNS atrophy and increased lesion load. CONCLUSIONS: These findings suggest that serum AHR agonist levels negatively correlate with disability in RRMS and PMS and decrease longitudinally in correlation with MRI markers of disease progression. Thus, serum AHR agonistic activity may serve as novel biomarker for disability progression in MS.

1-Methyl-D-tryptophan activates aryl hydrocarbon receptor, a pathway associated with bladder cancer progression.

BACKGROUND: Indoleamine 2, 3-dioxygenase-1 (IDO1) is a promising target for immunotherapy in bladder cancer (BC). IDO1 breaks-down tryptophan to generate kynurenine derivatives, which may activate the aryl hydrocarbon receptor (AHR). AHR is an important target for carcinogens, but its association with BC progression was unknown. Two IDO1 inhibitors used in clinical trials are 1-methyl-D-tryptophan (MT) and INCB240360. Because MT is an aromatic hydrocarbon, it may be a ligand for AHR. We hypothesized that AHR could be associated with BC progression and that MT could activate AHR in BC. METHODS: BC patients (n = 165) were selected from the Gene Expression Omnibus database. A cut-off point for relative expression of AHR and cytochrome 450 enzymes (CYP1A1, CYP1A2, and CYP1B1; markers of AHR activation) was determined to compare with the grade, stage, and tumor progression. For in vitro experiments, RT4 (grade 1) and T24 (grade 3) BC cells were incubated with MT and INCB240360 to evaluate the expression of AHR and CYP1A1. RESULTS: AHR activation was associated with grade, stage, and progression of BC. T24 cells express more CYP1A1 than RT4 cells. Although IDO1 expression and kynurenine production are elevated in T24 cells concomitantly to CYP1A1 expression, IDO1 inhibitors were not able to decrease CYP1A1 expression, in contrast, MT significantly increased it in both cell lines. CONCLUSION: In conclusion, it is rational to inhibit IDO1 in BC, among other factors because it contributes to AHR activation. However, MT needs to be carefully evaluated for BC because it is an AHR pathway agonist independently of its effects on IDO1.

Increased AHR Transcripts Correlate With Pro-inflammatory T-Helper Lymphocytes Polarization in Both Metabolically Healthy Obesity and Type 2 Diabetic Patients.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor whose transcription activity is regulated by small compounds provided by diet, xenobiotics, and metabolism. It has been proven to be involved in energy homeostasis and inflammation in most recent years. Epidemiologically, exposure to xenobiotic AHR ligands contributes to obesity and type 2 diabetes (T2D). AHR is also the critical transcription factor determining the lineage commitment of pro-inflammatory Th17 and Th22 cells from naïve CD4+ T lymphocytes. It has been well-illustrated in animal models that IL-22, the major effector cytokine of Th17 and Th22 cells, played a major role in the interaction of metabolism and gut microbiota. But there were still missing links between gut microbiota, IL-22, and metabolism in humans. Our previous findings indicated that elevated circulating levels of IL-22 and frequencies of Th22 cells were associated with insulin resistance in both patients with obesity and T2D. Additionally, the hyperactive Th17 and Th22 cells phenotype also correlate with islets ß-cell dysfunction in T2D. In this study, we made efforts to determine AHR expressions in peripheral blood mononuclear cells (PBMCs) from patients with T2D and metabolically healthy obesity (MHO). Correlation analyses were conducted to assess the possible link between AHR and the metabolic and inflammatory context. We revealed that mRNA expression of AHR was up-regulated and correlated with the percentage of Th17, Th22 as well as Th1 cells. Elevated plasma levels of IL-22 and IL-17 also correlated with increased AHR transcripts in PBMCs from both MHO and T2D patients. The transcription factor AHR may thus have a plausible role in the interaction between metabolism and pro-inflammatory status of patients in the development of obesity and T2D.

Serum biomarkers from cell-based assays for AhRL and MIS strongly predicted the future development of diabetes in a large community-based prospective study in Korea.

Exposure to environment-polluting chemicals (EPC) is associated with the development of diabetes. Many EPCs exert toxic effects via aryl hydrocarbon receptor (AhR) and/or mitochondrial inhibition. Here we investigated if the levels of human exposure to a mixture of EPC and/or mitochondrial inhibitors could predict the development of diabetes in a prospective study, the Korean Genome and Epidemiological Study (KoGES). We analysed AhR ligands (AhRL) and mitochondria-inhibiting substances (MIS) in serum samples (n = 1,537), collected during the 2008 Ansung KoGES survey with a 4-year-follow-up. Serum AhRL, determined by the AhR-dependent luciferase reporter assay, represents the contamination level of AhR ligand mixture in serum. Serum levels of MIS, analysed indirectly by MIS-ATP or MIS-ROS, are the serum MIS-induced mitochondria inhibiting effects on ATP content or reactive oxygen species (ROS) production in the cultured cells. Among 919 normal subjects at baseline, 7.1% developed impaired glucose tolerance (IGT) and 1.6% diabetes after 4 years. At the baseline, diabetic and IGT sera displayed higher AhRL and MIS than normal sera, which correlated with indices of insulin resistance. When the subjects were classified according to ROC cut-off values, fully adjusted relative risks of diabetes development within 4 years were 7.60 (95% CI, 4.23-13.64), 4.27 (95% CI, 2.38-7.64), and 21.11 (95% CI, 8.46-52.67) for AhRL ≥ 2.70 pM, MIS-ATP ≤ 88.1%, and both, respectively. Gender analysis revealed that male subjects with AhRL ≥ 2.70 pM or MIS-ATP ≤ 88.1% showed higher risk than female subjects. High serum levels of AhRL and/or MIS strongly predict the future development of diabetes, suggesting that the accumulation of AhR ligands and/or mitochondrial inhibitors in body may play an important role in the pathogenesis of diabetes.

Aryl Hydrocarbon Receptor in Post-Mortem Hippocampus and in Serum from Young, Elder, and Alzheimer's Patients.

One of the characteristics of the cerebral aging process is the presence of chronic inflammation through glial cells, which is particularly significant in neurodegeneration. On the other hand, it has been demonstrated that the aryl hydrocarbon receptor (AHR) participates in the inflammatory response. Currently, evidence in animal models shows that the hallmarks of aging are associated with changes in the AHR levels. However, there is no information concerning the behavior and participation of AHR in the human aging brain or in Alzheimer's disease (AD). We evaluated the expression of AHR in human hippocampal post-mortem tissue and its association with reactive astrocytes by immunohistochemistry. Besides this, we analyzed through ELISA the AHR levels in blood serum from young and elder participants, and from AD patients. The levels of AHR and glial fibrillar acid protein were higher in elder than in young post-mortem brain samples. AHR was localized mainly in the cytosol of astrocytes and displayed a pattern that resembles extracellular vesicles; this latter feature was more conspicuous in AD subjects. We found higher serum levels of AHR in AD patients than in the other participants. These results suggest that AHR participates in the aging process, and probably in the development of neurodegenerative diseases like AD.

Aryl hydrocarbon receptor expression in serum, peripheral blood mononuclear cells, and skin lesions of patients with atopic dermatitis and its correlation with disease severity.

BACKGROUND: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which is critically involved in the pathogenesis of a variety of skin diseases. The aim of this study was to detect AhR and its downstream regulators including cytochrome P450 (CYP1A1), AhR nuclear translocation (ARNT), and aryl hydrocarbon receptor repressor (AhRR) in serum, peripheral blood mononuclear cells (PBMCs), and skin lesions in patients with atopic dermatitis (AD). METHODS: Twenty-nine AD patients defined according to the criteria of Hanifin and Rajka and Chinese criteria of AD were included. Subjects without allergic and chronic diseases were recruited as controls. Patients and controls were selected from the dermatology outpatient clinic of Peking University People's Hospital from August 1 to December 31 in 2018. Enzyme-linked immunosorbent assay was performed to detect serum AhR level. The mRNA of AhR, AhRR, ARNT, and CYP1A1 in PBMCs were measured by real-time quantitative polymerase chain reaction. AhR expression in skin lesions was measured by immunohistochemistry. RESULTS: AhR was significantly higher expressed in serum (41.26 ±â€Š4.52 vs. 33.73 ±â€Š2.49 pmol/L, t = 6.507, P < 0.001) and skin lesions (0.191 ±â€Š0.041 vs. 0.087 ±â€Š0.017, t = 10.036, P < 0.001) of AD patients compared with those of controls. The mRNA levels of AhR (1.572 ±â€Š0.392 vs. 1.000 ±â€Š0.173, t = 6.819, P < 0.001), AhRR (2.402 ±â€Š1.716 vs. 1.000 ±â€Š0.788, t = 3.722, P < 0.001), CYP1A1 (2.258 ±â€Š1.598 vs. 1.000 ±â€Š0.796, t = 3.400, P = 0.002) in PBMCs of AD patients were higher compared with those of controls. The difference in mRNA levels of ARNT was not statistically significant between the patients and controls (1.383 ±â€Š0.842 vs. 1.000 ±â€Š0.586, t = 1.653, P = 0.105). AhR mRNA levels in PBMCs positively correlated with eczema area and severity index score and serum interleukin-6 levels. CONCLUSION: AhR and its downstream regulators were highly expressed in serum, PBMCs, and skin of AD patients, which might contribute to the pathogenesis of AD.

MiR-31 promotes Th22 differentiation through targeting Bach2 in coronary heart disease.

The aim of the present study was to investigate the role of miR-31 in Th22 differentiation in coronary heart disease (CHD). Th22 frequencies in peripheral blood of CHD patients and controls as well as in CD4+ T cells were detected by flow cytometry. The mRNA expression of Th22-associated transcription factor aryl hydrocarbon receptor (AHR) and Th22-effector cytokine interleukin (IL)-22, as well as miR-31 were examined by quantitative real-time PCR (qRT-PCR). The protein level of BTB domain and CNC homolog 2 (Bach2) was measured by Western blotting. The interaction between miR-31 and Bach2 was verified using dual luciferase reporter assay. The results showed that Th22 frequency and miR-31 expression were elevated in CHD patients. Furthermore, miR-31 mimic and Bach2 silencing significantly promoted Th22 frequency and the levels of AHR and IL-22 in CD4+ T cells from CHD patients. Further studies showed that miR-31 facilitated Th22 cell differentiation by targeting and inhibiting Bach2. Our data indicate that miR-31 promotes Th22 differentiation through targeting Bach2 in CHD.

Distinct Alterations in the Composition of Mucosal Innate Lymphoid Cells in Newly Diagnosed and Established Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Innate lymphoid cells [ILC] have been suggested to play a role in inflammatory bowel disease [IBD]. Here, we investigated the ILC compartment in intestinal biopsies and blood from distinct patient groups with Crohn's disease [CD] and ulcerative colitis [UC], either newly diagnosed or with disease established for at least 1 year. This approach allowed us to simultaneously investigate temporal, disease-specific, and tissue-specific changes in ILC composition in IBD. METHODS: ILC subset frequencies, phenotype, and transcription factor profile in blood and intestinal biopsies were investigated by multi-parameter flow cytometry analysis. Endoscopic disease severity was judged using the ulcerative colitis endoscopic index of severity and the simple endoscopic score for Crohn's disease. RESULTS: The frequency of NKp44+ILC3 was decreased in inflamed tissue, both in patients with CD and those with UC, already at the time of diagnosis, and correlated with disease severity. Simultaneously, the frequency of ILC1 was increased in patients with CD, whereas the frequency of ILC2 was increased in patients with UC. However, in patients with established UC or CD, both ILC1 and ILC2 were increased. In contrast to the ILC composition in inflamed tissue, ILC in non-inflamed tissue or blood were unchanged compared with non-IBD controls. Finally, in patients undergoing treatment with an anti-α4ß7 antibody the frequencies of ILC in peripheral blood remained unchanged. CONCLUSIONS: We report both shared and distinct changes in ILC composition depending on diagnosis and disease duration. The alterations in ILC composition in IBD occur selectively at inflamed sites in the gut.

Serum Levels of Aryl Hydrocarbon Receptor, Cytochromes P450 1A1 and 1B1 in Patients with Exacerbated Psoriasis Vulgaris.

The aryl hydrocarbon receptor (AhR) is highly expressed in psoriasis skin lesions. The aim of this study was to investigate serum concentrations of AhR, cytochromes P450 (CYP) 1A1 and 1B1 in patients with exacerbated psoriasis vulgaris treated with combined therapy of ultraviolet radiation (UVR) and crude coal tar. The analyses were performed by using enzyme-linked immunosorbent assays. Before the treatment, the patients had significantly higher serum levels of AhR and CYP1A1 than healthy controls. AhR median noticeably decreased after the therapy; nevertheless, it remained significantly higher compared to the controls. CYP1A1 levels measured before and after the therapy did not differ significantly. Serum CYP1A1 positively correlated with AhR values before and after the treatment. The serum values of CYP1B1 were very low and we did not see any differences between the study group and the control group. The study demonstrated that serum levels of AhR and CYP1A1 could indicate their immunopathological and metabolic roles in exacerbated psoriasis.

Negative association between serum aryl hydrocarbon receptor concentrations and ß-cell function in patients with no history of diabetes undergoing coronary angiography.

BACKGROUND: The aim of the present study was to investigate the association between serum aryl hydrocarbon receptor (AhR) levels and insulin resistance and ß-cell function in patients undergoing coronary angiography with no history of diabetes. METHODS: Patients with no history of diabetes who had undergone coronary angiography underwent an oral glucose tolerance test (OGTT) 2-4 weeks after discharge from hospital; blood samples were collected for measurements of glucose, insulin, and AhR. Patients' glucose regulation status was determined on the basis of the OGTT. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR). ß-Cell function was assessed using the insulinogenic index (IGI). RESULTS: The study included 473 patients (mean (±SD) age 61 ±12 years, 81.8% male, mean body mass index 26.1 ±3.6 kg/m2 ). Overall, mean serum AhR concentrations were 25.1 ±12.2 pg/mL. Patients with normal glucose tolerance had a lower serum AhR concentrations than patients with prediabetes or newly diagnosed diabetes (23.4 ±10.8 vs 26.2 ±13.2 and 26.9 ±12.3 pg/mL, respectively; P = 0.029). Linear regression analysis revealed that serum AhR concentrations were not associated with HOMA-IR, but were negatively associated with IGI after adjustment for several confounders, including HOMA-IR (ß = -0.162; 95% confidence interval - 0.302, -0.022; P = 0.023). CONCLUSIONS: In patients with no history of diabetes, serum AhR concentrations were negatively associated with ß-cell function, independent of several confounders, including insulin resistance.

Aryl hydrocarbon receptor is activated in patients and mice with chronic kidney disease.

Patients with chronic kidney disease (CKD) are exposed to uremic toxins and have an increased risk of cardiovascular disease. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AHR). These toxins induce a vascular procoagulant phenotype. Here we investigated AHR activation in patients with CKD and in a murine model of CKD. We performed a prospective study in 116 patients with CKD stage 3 to 5D and measured the AHR-Activating Potential of serum by bioassay. Compared to sera from healthy controls, sera from CKD patients displayed a strong AHR-Activating Potential; strongly correlated with eGFR and with the indoxyl sulfate concentration. The expression of the AHR target genes Cyp1A1 and AHRR was up-regulated in whole blood from patients with CKD. Survival analyses revealed that cardiovascular events were more frequent in CKD patients with an AHR-Activating Potential above the median. In mice with 5/6 nephrectomy, there was an increased serum AHR-Activating Potential, and an induction of Cyp1a1 mRNA in the aorta and heart, absent in AhR-/- CKD mice. After serial indoxyl sulfate injections, we observed an increase in serum AHR-AP and in expression of Cyp1a1 mRNA in aorta and heart in WT mice, but not in AhR-/- mice. Thus, the AHR pathway is activated both in patients and mice with CKD. Hence, AHR activation could be a key mechanism involved in the deleterious cardiovascular effects observed in CKD.

Uremic Solute-Aryl Hydrocarbon Receptor-Tissue Factor Axis Associates with Thrombosis after Vascular Injury in Humans.

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

Unbalanced expression of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of rheumatoid arthritis.

OBJECTIVE: The goal of this study was to analyze the role of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of patients with rheumatoid arthritis. METHODS: Flow cytometry was applied to determine the proportion of AhR positive cells in CCR6+CD4+T, CD4+CD25+T and peripheral blood peripheral mononuclear cells from each subject. AhR mRNA and CYP1A1 mRNA relative expression levels were tested by real-time PCR. RESULTS: The percentage of AhR positive cells in peripheral blood mononuclear cells was higher in RA group than that in healthy cases [(35.23±10.71)% vs. (18.83±7.32)%, p<0.01]. The expression levels of AhR and CYP1A1 were both increased in patients with RA while compared to controls [(3.71±1.63) vs. (2.00±1.27), p=0.002; (2.62±2.08) vs. (0.62±0.29), p<0.01, respectively]. In RA patients, the percentage of AhR positive cells in CD4+CD25+T cells was significantly lower than that from controls [17.90 (6.10±80.10)% vs. (52.49±19.18)%, p<0.01]; In healthy controls, the percentage of AhR positive cells in CD4+CD25+T cells was significantly higher than that in CCR6+CD4+T cells, and was also significantly higher than that in PBMCs [(52.49±19.18)% vs. (23.18±5.62)% vs. (18.06±7.80)%, X2=24.03, p<0.01]; in RA patients, the percentage of AhR positive cells in CCR6+CD4+T cells was significantly increased than that in CD4+CD25+T cells and PBMCs [(46.02±14.68)% vs. 17.90 (6.10±80.10)% vs. (34.22±10.33)%, X2=38.29, p<0.01]; Nevertheless, no statistically significant relationship was found between clinical data and AhR positive cells in CCR6+CD4+T and CD4+CD25+T cells. CONCLUSION: AhR may participate in the pathological progress of RA by controlling the differentiation of Th17 and Treg cells in peripheral blood.

Unbalanced expression of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of rheumatoid arthritis / Expressão não equilibrada do receptor de hidrocarboneto arílico nos linfócitos T CCR6+ CD4+ e CD4+ CD25+ do sangue periférico na artrite reumatoide

ABSTRACT Objective: The goal of this study was to analyze the role of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of patients with rheumatoid arthritis. Methods: Flow cytometry was applied to determine the proportion of AhR positive cells in CCR6+CD4+T, CD4+CD25+T and peripheral blood peripheral mononuclear cells from each subject. AhR mRNA and CYP1A1 mRNA relative expression levels were tested by real-time PCR. Results: The percentage of AhR positive cells in peripheral blood mononuclear cells was higher in RA group than that in healthy cases [(35.23 ± 10.71)% vs. (18.83 ± 7.32)%, p < 0.01]. The expression levels of AhR and CYP1A1 were both increased in patients with RA while compared to controls [(3.71 ± 1.63) vs. (2.00 ± 1.27), p = 0.002; (2.62 ± 2.08) vs. (0.62 ± 0.29), p < 0.01, respectively]. In RA patients, the percentage of AhR positive cells in CD4+CD25+T cells was significantly lower than that from controls [17.90 (6.10 ± 80.10)% vs. (52.49 ± 19.18)%, p < 0.01]; In healthy controls, the percentage of AhR positive cells in CD4+CD25+T cells was significantly higher than that in CCR6+CD4+T cells, and was also significantly higher than that in PBMCs [(52.49 ± 19.18)% vs. (23.18 ± 5.62)% vs. (18.06 ± 7.80)%, X 2 = 24.03, p < 0.01]; in RA patients, the percentage of AhR positive cells in CCR6+CD4+T cells was significantly increased than that in CD4+CD25+T cells and PBMCs [(46.02 ± 14.68)% vs. 17.90 (6.10 ± 80.10)% vs. (34.22 ± 10.33)%, X 2 = 38.29, p < 0.01]; Nevertheless, no statistically significant relationship was found between clinical data and AhR positive cells in CCR6+CD4+T and CD4+CD25+T cells. Conclusion: AhR may participate in the pathological progress of RA by controlling the differentiation of Th17 and Treg cells in peripheral blood.

RESUMO Objetivo: Analisar o papel do receptor de hidrocarboneto arílico (AhR) nos linfócitos T CCR6+ CD4+ e CD4+ CD25+ no sangue periférico de pacientes com artrite reumatoide (AR). Métodos: Foi aplicada citometria de fluxo para determinar a proporção de células AhR positivas em linfócitos CCR6+ CD4+ e CD4+ CD25+ do sangue periférico e células mononucleares periféricas de cada indivíduo. Os níveis de expressão relativa de ácido ribonucleico mensageiro (do inglês ribonucleic acid, RNAm,) de AhR e RNAm de enzima de primeiro estágio essencial para o AhR (CYP1A1) foram testados por reação em cadeia de polimerase (do inglês polymerase chain reaction, PCR,) em tempo real. Resultados: A percentagem de células AhR positivas nas células mononucleares do sangue periférico foi maior no grupo com AR do que nos indivíduos saudáveis [(35,23 ± 10,71)% vs. (18,83 ± 7,32)%, (p < 0,01)]. Os níveis de expressão de AhR e CYP1A1 estavam aumentados em pacientes com AR quando comparados com os controles [(3,71 ± 1,63) vs. (2,00 ± 1,27), p = 0,002; (2,62 ± 2,08) vs. (0,62 ± 0,29), p < 0,01, respectivamente]. Em pacientes com AR, a percentagem de células AhR positivas nos linfócitos T CD4+ CD25+ foi significativamente inferior à dos controles [17,90 (6,10 ± 80,10)]% vs. (52,49 ± 19,18)%, p < 0,01]; em controles saudáveis, a percentagem de células AhR positivas nos linfócitos T CD4+ CD25+ foi significativamente mais elevada do que nos linfócitos T CCR6+ CD4+ e também foi significativamente maior do que nas células mononucleares do sangue periférico (do inglês peripheral blood mononuclear cells, PBMC,) [(52,49 ± 19,18)% vs. (23,18 ± 5,62)% vs. (18,06 ± 7,80)%, X 2 = 24,03, p < 0,01]; em pacientes com AR, a percentagem de células AHR positivas nos linfócitos T CCR6+ CD4+ era significativamente maior em comparação com os linfócitos T CD4+ CD25+ e PBMC (46,02 ± 14,68)% vs. [17,90 (6,10 ± 80.10)]% vs. (34,22 ± 10,33)%, X2 = 38,29, p < 0,01]; no entanto, não foi encontrada correlação estatisticamente significativa entre os dados clínicos e células AhR positivas em linfócitos T CCR6+ CD4+ e CD4+ CD25+. Conclusão: O Ahr pode participar do progresso patológico da AR ao controlar a diferenciação de linfócitos Th17 e Treg no sangue periférico.

Genetic association of aromatic hydrocarbon receptor and its repressor gene polymorphisms with risk of rheumatoid arthritis in Han Chinese populations.

The goal of this study was to evaluate the potential relationship among polymorphisms of aromatic hydrocarbon receptor, aromatic hydrocarbon receptor repressor, and rheumatoid arthritis (RA) susceptibility as well as the association among the polymorphisms of aromatic hydrocarbon receptor, aromatic hydrocarbon receptor repressor, and their expression.We performed a hospital-based, case-control study of 400 patients with RA and 726 healthy controls in Han Chinese populations. Two single-nucleotide polymorphisms were selected for genotyping including aromatic hydrocarbon receptor (rs2066853) and aromatic hydrocarbon receptor repressor (rs2292596).To single-nucleotide polymorphism rs2292596, a statistically significantly increased risk of RA was found to be associated with the G allele of rs2292596; the odds ratio was 2.170 (95% confidence interval: 1.820-2.587). Unfortunately, no significant differences exhibited in the allelic and the genotype frequencies of rs2066853 between 2 groups. We failed to find any association between rs2066853, rs2292596 genotypes and their expression of patients, respectively. No statistical relationship was found between aromatic hydrocarbon receptor, aromatic hydrocarbon receptor repressor at messenger Ribonucleic acid levels and clinical data, either.This study demonstrated that the polymorphisms of rs2292596 was significant with genetic susceptibility to RA patients; furthermore, it suggests the G allele of rs2292596 might be associated with a dangerous effect on RA in Han Chinese populations.

Crowd-Sourced Verification of Computational Methods and Data in Systems Toxicology: A Case Study with a Heat-Not-Burn Candidate Modified Risk Tobacco Product.

Systems toxicology intends to quantify the effect of toxic molecules in biological systems and unravel their mechanisms of toxicity. The development of advanced computational methods is required for analyzing and integrating high throughput data generated for this purpose as well as for extrapolating predictive toxicological outcomes and risk estimates. To ensure the performance and reliability of the methods and verify conclusions from systems toxicology data analysis, it is important to conduct unbiased evaluations by independent third parties. As a case study, we report here the results of an independent verification of methods and data in systems toxicology by crowdsourcing. The sbv IMPROVER systems toxicology computational challenge aimed to evaluate computational methods for the development of blood-based gene expression signature classification models with the ability to predict smoking exposure status. Participants created/trained models on blood gene expression data sets including smokers/mice exposed to 3R4F (a reference cigarette) or noncurrent smokers/Sham (mice exposed to air). Participants applied their models on unseen data to predict whether subjects classify closer to smoke-exposed or nonsmoke exposed groups. The data sets also included data from subjects that had been exposed to potential modified risk tobacco products (MRTPs) or that had switched to a MRTP after exposure to conventional cigarette smoke. The scoring of anonymized participants' predictions was done using predefined metrics. The top 3 performers' methods predicted class labels with area under the precision recall scores above 0.9. Furthermore, although various computational approaches were used, the crowd's results confirmed our own data analysis outcomes with regards to the classification of MRTP-related samples. Mice exposed directly to a MRTP were classified closer to the Sham group. After switching to a MRTP, the confidence that subjects belonged to the smoke-exposed group decreased significantly. Smoking exposure gene signatures that contributed to the group separation included a core set of genes highly consistent across teams such as AHRR, LRRN3, SASH1, and P2RY6. In conclusion, crowdsourcing constitutes a pertinent approach, in complement to the classical peer review process, to independently and unbiasedly verify computational methods and data for risk assessment using systems toxicology.