Aryl Hydrocarbon Receptor Receptor Research Articles

Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways.

Indole-3-aldehyde (I3A) is an intestinal microbial metabolite that regulates inflammation in various inflammatory diseases; however, its role in chronic obstructive pulmonary disease (COPD) remains unclear. This study aimed to investigate the anti-inflammatory effects and molecular mechanisms of I3A in COPD. We constructed in vivo models using cigarette smoke (CS)-stimulated mice and in vitro models using cigarette smoke extract (CSE)-stimulated MH-S cells. The results demonstrated that I3A significantly alleviated bronchial obstruction in mice with COPD and reduced the expression of inflammatory factors such as TNF-α, IL-1β, and IL-6. Additionally, I3A decreased the levels of matrix metalloproteinases MMP2, MMP12, and inhibited the NF-κB p65/NLRP3 pathways. Further investigation revealed that I3A inhibited NF-κB activity by suppressing p65 phosphorylation and nuclear translocation in CSE-stimulated MH-S cells. The activation of the NF-κB and NLRP3 signaling pathways is mediated by histone deacetylase 5 (HDAC5) and HDAC6, both of which are inhibited by I3A. Subsequent experiments indicated that aryl hydrocarbon receptor (AHR) knockdown attenuated the inhibitory effect of I3A on pro-inflammatory cytokines and the HDACs/NF-κB/NLRP3 signaling pathways, highlighting the dependence of I3A's anti-inflammatory effects on the AHR receptor. KEY MESSAGES: I3A effectively reduced lung inflammation in COPD mice by inhibiting the NF-κB pathway. In CSE-stimulated MH-S cells, I3A suppressed p65 phosphorylation and nuclear translocation, thereby inhibiting NF-κB activity. The activation of the NF-κB/NLRP3 pathways by HDAC5 and HDAC6 was diminished by I3A. Through the activation of the AHR receptor, I3A suppressed the activities of HDAC5/6, leading to a decrease in inflammatory factor levels.

Screening study of hydroxytyrosol metabolites from in vitro fecal fermentation and their interaction with intestinal barrier repair receptor AhR.

Olive oil polyphenol hydroxytyrosol (HT) significantly repairs intestinal barrier function, but its absorption in the stomach and small intestine is limited. The metabolites of unabsorbed HT that reach the colon are crucial, yet their effects on colonic microbiota and intestinal barrier repair remain unclear. This study utilized in vitro simulated digestion and colonic fecal fermentation to investigate HT's digestion and fermentation. Results indicated that 79.25% of HT potentially reached the colon intact. Further 16S rDNA, targeted, and untargeted metabolomics analyses showed that HT can be decomposed by colonic microbiota, producing aromatic hydrocarbon metabolites and regulating gut microbiota structure. It promotes the growth of gut microbiota, such as Bacteroides, Faecalibacterium, Klebsiella, and Lachnospira, which degrade HT. Additionally, HT's intervention conversely affected the production of tryptophan-derived metabolites and short-chain fatty acids (SCFAs). Subsequently, computer-simulated molecular docking technology was used to simulate the binding affinity between HT metabolites and derived metabolites and the intestinal barrier repair-related receptor aryl hydrocarbon receptor (AhR). Indole-3-acetic acid, indole-3-acetaldehyde, skatole, kynurenine, and homovanillic acid could tightly bind to the amino acid residues of the AhR receptor, with binding energies all ˂-6.0kcal/mol, suggesting that these metabolites may enhance the intestinal barrier function through the AhR signaling pathway.

AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection.

In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationships between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3s) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3s and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor influences various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, to promote their growth and development, suggesting that AhR has a broad impact on intestinal health. In conclusion, our study demonstrated the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.IMPORTANCEWe observed high expression of the AhR receptor on pig and human ILC3s, although its expression was negligible in mouse ILC3s. ILC3s are closely related to the gut microbiota, particularly the secretion of IL-22 stimulated by microbial signals, which plays a crucial regulatory role in intestinal immunity. In our study, we found that metabolites produced by beneficial gut bacteria interact with ILC3s through AhR, thereby maintaining intestinal immune homeostasis in pigs. Moreover, LGG feeding can enhance the activation of ILC3s and promote IL-22 secretion in the intestines of piglets, ultimately preventing PEDV infection.

Baitouweng decoction alleviates ulcerative colitis by regulating tryptophan metabolism through DOPA decarboxylase promotion.

Baitouweng decoction (BTW) is a classic botanical drugs formula that has been widely used clinically for the treatment of gut-related disorders in China. However, its role in ameliorating ulcerative colitis (UC) remains to be explored. The study aimed to determine the therapeutic efficacy and potential mechanism of action of BTW on dextran sodium sulfate (DSS)-induced colitis mice. In vivo: 3.5% DSS-induced experimental colitis mice were treated with BTW (Pulsatilla chinensis (Bunge) Regel, Phellodendron chinense C. K. Schneid, Coptis chinensis Franch and Fraxinus chinensis Roxb), kynurenine or DOPA decarboxylase (DDC) inhibitor (carbidopa). In vitro: Caco-2 cells were stimulated with TNF-α to activate inflammation and later treated with various concentrations of BTW and carbidopa. Model evaluation included body weight, disease activity index (DAI) score, colon length and histopathology. Cytokine levels were measured by flow cytometry. Protein levels were analyzed by proteomics and functionally annotated. The levels of tryptophan metabolites in mouse serum and colon were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Alcian Blue/Phosphate Acid Schiff (AB/PAS) staining, immunohistochemistry and western blot were used to assess the intestinal barrier function and detect the protein expression levels. BTW significantly reduced the DAI, ameliorated colonic injury and regulated inflammatory cytokines in DSS-induced colitis mice. The botanical drugs formula also promoted intestinal epithelial barrier repair by enhancing the expression of the tight junction (TJ) proteins. Tryptophan metabolic signaling pathway was significantly enriched in DSS-induced UC mice, and BTW decreased the level of kynurenine, increased indole metabolites. The therapeutic effect of BTW was evidently reduced when kynurenine was given to mice. Also, BTW promoted DDC protein expression and activated the aryl hydrocarbon receptor (AHR)/IL-22 signaling pathway. BTW improves ulcerative colitis by promoting DDC expression, regulating the conversion of tryptophan metabolism from the kynurenine pathway to the indole metabolism pathway, thereby modulating tryptophan metabolism to increase indole metabolites, and activating AHR receptors to restore intestinal barrier function.

1,4-Diol Hq (TBHQ) vs 1,4-dithiol (TBDT); simulation of safe antioxidant with a lower carcinogenic activity.

tert-Butylhydroquinone (TBHQ) is an antioxidant and preservative used in unsaturated vegetable oils and processed foods. However, when consumed in higher doses daily, it may pose a threat to public health by potentially increasing the risk of cancer, as it has an affinity with both the aryl hydrocarbon receptor (AhR) and the estrogen receptor alpha (ERα). This study aimed to examine the impact of substituting the 1,4-diol of TBHQ with 1,4-dithiol, referred to as TBDT, on the carcinogenic and antioxidant systems using computational methods. The binding affinity of TBHQ and TBDT to the two carcinogenic receptors, AhR and ERα, as well as to the antioxidant receptor Keap1 alone and in connection with Nrf2 (Nrf2-Keap1) was investigated through docking analysis. The results indicated a decrease in TBDT's binding strength to ERα and AhR when assessed using Molegro Virtual Docker (P-value: 0.0001 and 0.00001, respectively), AutoDock Vina (P-value: 0.0001 and 0.0001), and the online server Fast DRH (P-value: 0.0001 and 0.0001). However, TBDT's binding affinity to Keap1 was predicted to be significantly stronger than TBHQ's by both MVD and AutoDock Vina (P-value: 0.0001 and 0.04), while its binding to Nrf2-Keap1 assessed to be stronger only by MVD (P-value: 0.0001). These findings suggest that TBDT not only exhibits higher antioxidant activity as a better ligand for the antioxidant system but also shows lower affinity with the AhR and ERα receptors. Therefore, TBDT can be considered a safer compound than TBHQ.

Activation of AHR by ITE improves cardiac remodelling and function in rats after myocardial infarction.

Left ventricular remodelling subsequent to myocardial infarction (MI) constitutes a pivotal underlying cause of heart failure. Intervention with the nontoxic endogenous aryl hydrocarbon receptor (AHR) agonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) in the acute phase of MI has been shown to ameliorate cardiac function, but its role in the chronic phase remains obscured. This study explores the beneficial role of ITE in delaying the progression of heart failure in the chronic phase of MI. MI rats established by ligating the left anterior descending coronary artery were treated with the indicated concentration of the AHR agonist ITE or vehicle alone. Echocardiography was performed to determine cardiac structure and function; myocardial morphology and fibrosis were observed by haematoxylin and eosin and Masson's trichrome staining; serum biochemical indices, BNP, and inflammatory cytokine levels were detected by enzyme-linked immunosorbent assay; F4/80+ iNOS+ M1 macrophages and F4/80+ CD206+ M2 macrophages were detected by immunofluorescence; the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay was used to detect the apoptosis of cardiomyocytes; ultrastructural changes in myocardial tissue were observed by transmission electron microscopy; and Cyp1a1, Akt, P-Akt, p70S6K, P-p70S6K, Bcl-2, Bax, caspase-3, and cleaved caspase-3 protein levels were determined via Western blotting. We found that therapy with the AHR agonist ITE rescued cardiac remodelling and dysfunction in rats with MI and attenuated myocardial fibrosis, inflammation, and mitochondrial damage. Further studies confirmed that ITE dose-dependently improved myocardial cell apoptosis after MI, as demonstrated by reduced levels of the apoptosis-related proteins cleaved caspase-3 and Bax but increased expression levels of Bcl-2. These effects were attributed to ITE-induced activation of AHR receptors, leading to the down-regulation of Akt and p70S6K phosphorylation. The AHR agonist ITE alleviates cardiomyocyte apoptosis through the Akt/p70S6K signalling pathway, thereby rescuing left ventricular adverse remodelling and cardiac dysfunction after MI.

The molecular effects of Asperuloside against thermogenesis and anti-inflammatory process through multiple recent obesity pathways: An anti-obesity drug discovery by in-silico analysis

ObjectivesAdenosine receptor signaling and suppressing potential pathways such as the aryl hydrocarbon receptor (AHR) and takeda G-protein-coupled receptor-5(TGR5) have been identified as potential targets for enhancing metabolic health. Certain adenosine receptor (AR) ligands have been suggested to reduce inflammation and improve thermogenesis in adipose tissue. MethodsThis study employed in-silico biomolecular fractions of adenosine receptors and other potential targets to understand the mechanism of action of Asperuloside. Additionally, the anti-obesity potential of Asperuloside, a dual-acting ligand with A2A adenosine receptor (A2AAR) agonist and A3 adenosine receptor (A3AR) agonist activities, were examined using computational analysis in the obesity model. The impact of Asperuloside on inflammation and thermogenesis was studied through diverse protein structures such as the A2AAR complex with agonist/A2AAR complex with antagonist, the rhodopsin mutant with bound galphact peptide (as A3 adenosine receptor), The Human TGR5 complex with synthetic agonist 23H, and AHR receptors antagonism. ResultsThe study found that Asperuloside has therapeutic affinity for the binding site of adenosine receptors and revealed a novel binding interaction that helps reduce inflammation and improve thermogenesis-mediated obesity. ConclusionAsperuloside may have anti-obesity effects through its dual-acting ligand with A2AAR and A3AR agonist activities. This study provides a major step towards understanding the mechanism of action of Asperuloside and its potential use as an anti-obesity drug. In vivo tests will help ascertain its pharmacokinetic characteristics, metabolite production in animals, and the effects of chronic daily absorption.

Switching on/off aryl hydrocarbon receptor and pregnane X receptor activities by chemically modified tryptamines

Microbial indoles have been demonstrated as selective or dual agonists and ligands of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). However, structural determinants of microbial indoles selectivity towards both receptors remain elusive. Here, we studied the effects of existing and newly synthesized derivatives of indole microbial metabolite tryptamine on the activity of AhR and PXR receptors. We show that the elongation of indolyl-3-alkaneamine chain, indole N-methylation and conversion of indolyl-3-alkaneamines to oleamides resulted in a major increase of PXR activity and in parallel loss of AhR activity. Using reporter gene assays, RT-PCR and TR-FRET techniques, we have characterized in detail the activation of PXR by novel indolyl-3-alkanyl-oleamides, 1-methyltryptamine and 1-methyltryptamine-acetamide. As a proof of concept, we demonstrated anti-inflammatory and epithelial barrier-protective activity of lead derivatives in intestinal Caco-2 cells, employing the measurement of expression of pro-inflammatory chemokines, tight junction genes, trans-epithelial electric resistance TEER, and dextran-FITC permeability assay. In conclusion, we show that a subtle chemical modifications of simple microbial indole metabolite tryptamine, leads to substantial changes in AhR and PXR agonist activities.

Effect-based evaluation of water quality in a system of indirect reuse of wastewater for drinking water production

Indirect potable reuse of wastewater is a practice that is gaining attention, aiming to increase freshwater supplies to meet water scarcity. However, reusing effluent wastewater for drinking water production comes with a paired risk of adverse health effects, due to the potential presence of pathogenic microorganisms and hazardous micropollutants. Disinfection is an established method to reduce microbial hazards in drinking water, but it has been associated with formation of disinfection by-products (DBPs). In this study, we performed an effect-based assessment of chemical hazards in a system wherein a full-scale trial of disinfection by chlorination, of the treated wastewater was performed prior discharge to the reciepient river. The presence of bioactive pollutants was assessed along the entire treatment system, starting from incoming wastewater to finished drinking water at seven sites in and around the Llobregat River in Barcelona, Spain. Samples were collected in two campaigns, with and without applied chlorination treatment (13 mg Cl2/L) to the effluent wastewater. The water samples were analysed for cell viability, oxidative stress response (Nrf2 activity), estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity and activation of NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling using stably transfected mammalian cell lines. Nrf2 activity, estrogen receptor activation and AhR activation was detected in all investigated samples. Overall, removal efficiencies were high in both wastewater treatment and drinking water treatment samples for most of the studied endpoints. No increase in oxidative stress (Nrf2 activity) could be attributed to the additional chlorination treatment of the effluent wastewater. However, we found an increase in AhR activity and a reduction of ER agonistic activity after chlorination treatment of effluent wastewater. The bioactivity detected in finished drinking water was considerably lower compared to what was found in effluent wastewater. We could thus conclude that indirect reuse of treated wastewater for drinking water production can be possible without compromising drinking water quality. This study contributed important knowledge in efforts to increase the reuse of treated wastewater as a source for drinking water production.

Abstract 644: Gut microbiota-derived metabolites as regulators of immunity in pre-cancer

Abstract Introduction: Tryptophan (Trp) as an essential amino acid that can only be obtained through diet and is a precursor of serotonin and melatonin and required for protein and niacin biosynthesis. In addition, Trp is appreciated for its influence on both host and microbial metabolism. Trp is shunted into the Kyn pathway (mostly) and indolic pathway (bacterial degradation), especially in the immune cells. Both Kyn and Indole/indole derivatives, in part by activating the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR), can become dysfunctional in cancer. Most individuals with cancer such as colon cancer, exhibit increased L-kynurenine levels, which in part is explained by progression of immunosuppressive phenotype and excessive inflammatory signaling molecules. To date, studies in mice and humans have linked Kyn and AHR as potent modulators of host immunity to colon cancer (CRC). Nothing is yet known about their role in pre-cancer, during the development of adenomatous polyps. We hypothesized that increases in plasma Kyn and I3A and their receptor AHR on immune cells would be detected in patients with pre-malignant polyps, suggesting dysbiosis and increased colon cancer risk due to immune modulation. Methods: We measured with mass spectrometry Kyn and I3A in plasma samples of 88 polyp patients and compared to 22 colon cancer patients. We analyzed by flow cytometry PBMCs from 64 newly diagnosed advanced polyp patients (40-70 years old) and 9 age-matched colon cancer patients in order to evaluate AHR expression in different subsets of T cells, NKT cells and NK cells, and their possible association with exhaustion markers Tim-3, LAG-3 and PD-1, as well as Granzyme K, and the presence of immunosuppressive Treg and MDSC. Results: We showed high level of expression of gut microbiota-derived metabolites in plasma of colon pre-cancer compared to cancer patients. We identified CD8+ T effector memory cells (TEM) as primary T cell population expressing their receptor AHR in polyp patients, which had a statistically significant association with Granzyme K expression, which is a hallmark of inflammaging. Conclusions: Our results suggest gut microbiota-derived metabolites as potential mediators of immunosuppression in the setting of premalignancy, which could increase cancer risk and be targets of cancer-preventing strategies. Citation Format: Amir Hossein Mohseni, Sedigheh Taghinezhad-S, Pamela L. Beatty, Olivera J. Finn. Gut microbiota-derived metabolites as regulators of immunity in pre-cancer [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 644.

RF15 | PMON293 Metabolic activation of tachysterol to biologically active hydroxyderivatives that act on VDR, AhR, LXRs and PPARγ receptors

Abstract Absorption of ultraviolet B (UVB) radiation by the B ring of 7-dehydrocholesterol (7-DHC) leads to the transformation of 7-DHC to previtamin D3, which after absorption of additional UVB isomerizes to tachysterol3 (T3) and lumisterol3 (L3). Previously we demonstrated that CYP11A1 can hydroxylate the side chain of vitamin D3 (D3), 7-DHC and L3. Similarly CYP27A1 can hydroxylate the side chain of L3 to biologically active hydroxyderivatives. In a continuation of these studies, we report that CYP11A1 and CYP27A1 hydroxylate T3 to 20S(OH)T3 and 25(OH)T3, respectively, plus minor unidentified hydroxyderivatives. Both 20S(OH)T3 and 25(OH)T3were detected in the human epidermis and serum. T3 was also detected in human serum and was present at a concentration of 7.3±2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 . They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR-coupled GFP (VDR-GFP) from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3 and only minimal activation by T3. The T3 hydroxyderivatives showed high affinity binding to the ligand binding domain (LBD) of the liver X receptor (LXR) α and β, and to the peroxisome proliferator-activated receptor γ (PPARg) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their previously characterized ligands. The scores for binding to the non-genomic site of the VDR were very low indicating a lack of interaction with hydroxy-T3 ligands. In conclusion, we have identified 20S(OH)T3 and 25(OH)T3 in the human epidermis and serum, identified CYP enzymes responsible for their production, demonstrated phenotypic effects on skin cells, and identified VDR, AhR, LXRs and PPARγ, and possibly RORs, as their genomic receptor targets. Thus CYP11A1, aside from its role in steroidogenesis, not only metabolizes vitamin D3, 7-DHC and L3 to biologically active metabolites, but also activates T3 to biologically active 20S(OH)T3. Similarly, CYP27A1, previously reported to act on L3, D3 and 7DHC, also activates T3 via hydroxylation at C25. We believe that these novel findings open new areas for future studies on the role of active forms of T3, not only in the skin, but also in systemic physiology and pathology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Metabolic activation of tachysterol3 to biologically active hydroxyderivatives that act on VDR, AhR, LXRs, and PPARγ receptors.

CYP11A1 and CYP27A1 hydroxylate tachysterol3 , a photoproduct of previtamin D3 , producing 20S-hydroxytachysterol3 [20S(OH)T3 ] and 25(OH)T3 , respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T3 , a smaller effect by 25(OH)T3 , and a minimal effect for their precursor, tachysterol3 . Tachysterol3 hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and β, and the peroxisome proliferator-activated receptor γ (PPARγ) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3 , comparable to their natural ligands. The scores for the non-genomic-binding site of the VDR were very low indicating a lack of interaction with tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3 .

Involvement of sirtuins (Sirt1 and Sirt3) and aryl hydrocarbon receptor (AhR) in the effects of triclosan (TCS) on production of neurosteroids in primary mouse cortical neurons cultures

Epidemiological studies have shown the presence of triclosan (TCS) in the brain due to its widespread use as an antibacterial ingredient. One of the confirmed mechanisms of its action is the interaction with the aryl hydrocarbon receptor (AhR). In nerve cells, sirtuins (Sirt1 and Sirt3) act as cellular sensors detecting energy availability and modulate metabolic processes. Moreover, it has been found that Sirt1 inhibits the activation of estrogen receptors, regulates the androgen receptor, and may interact with the AhR receptor. It is also known that Sirt3 stimulates the production of estradiol (E2) via the estradiol receptor β (Erβ). Therefore, the aim of the present study was to evaluate the effect of TCS alone or in combination with synthetic flavonoids on the production of neurosteroids such as progesterone (P4), testosterone (T), and E2 in primary neural cortical neurons in vitro. The contribution of Sirt1 and Sirt3 as well as AhR to these TCS-induced effects was investigated as well. The results of the experiments showed that both short and long exposure of neurons to TCS increased the expression of the Sirt1 and Sirt3 proteins in response to AhR stimulation. After an initial increase in the production of all tested neurosteroids, TCS acting for a longer time lowered their levels in the cells. This suggests that TCS activating AhR as well as Sirt1 and Sirt3 in short time intervals stimulates the levels of P4, T, and E2 in neurons, and then the amount of neurosteroids decreases despite the activation of AhR and the increase in the expression of the Sirt1 and Sirt3 proteins. The use of both the AhR agonist and antagonist prevented changes in the expression of Sirt1, Sirt3, and AhR and the production of P4, T, and E2, which confirmed that this receptor is a key in the mechanism of the TCS action.

Perspective of Aryl Hydrocarbon Receptor Exhaustion as Cancer Causative

Over the last decade, Aryl Hydrocarbon Receptor (AHR) dragged the research community’s attention due to the associated benefits in physiology and pathology. In this study, the probable role of AHR in cancer development is discussed. The ABCG2 gene that regulates uric acid metabolism is controlled by AHR. The study noticed that the failure of AHR control over ABCG2 leads to the accumulation of uric acid, reduction of vitamin D, and hormonal imbalances that are sequentially associated with several types of cancer. Furthermore, the location of AHR in the cytoplasm and its role in the regulation of the development and differentiation of blood cells, makes it impacts on overall cells of the body, while cancer genes can be transmitted through AHR receptors in the placenta to the offspring. Overtiredness of AHR by aging, microbial, chemical, or other environmental factors escalates the risk of cancer cell growth. Consequently, immune system dysfunction is facilitated by metastasis. Meanwhile, the Tryptophan catabolism indicates the exhaustion of AHR. However, this hypothesis lacks experimental exploration for further validation.

The synergistic Reduning and cefmetazole sodium treatment of severe pneumonia is mediated by the AhR-Src-STAT3 pathway.

BackgroundReduning (RDN) is a common Chinese medicine preparation with antibacterial, anti-inflammatory, antiviral and immunomodulatory effects in respiratory infectious diseases. Clinically, it is used in combination with antibiotics, but its synergistic effect and mechanism in treating severe pneumonia remain unclear.MethodsA rat model of severe pneumonia and an in vitro coculture model consisting of A549 and THP-1 cells were used to observe the synergistic effect of RDN on severe pneumonia. The inflammatory cytokines were tested by enzyme-linked immunosorbent assay (ELISA). The localization of Aryl hydrocarbon receptor (AhR) in A549 cells was observed by immunofluorescence, and the interaction of AhR and signal transducer and activator of transcription 3 (STAT3) proteins was observed by co-immunoprecipitation. AhR-Src tyrosine kinase (Src)-STAT3 pathway in rats and A549 cells were examined by Western Blot. Histopathological changes were observed by Hematoxylin-eosin (HE) staining, X-ray and survival rates were used to evaluate the effects of paclitaxel on severe pneumonia rats.ResultsRDN regulation of Src–STAT3–interleukin 10 (IL-10) signaling pathway activation and macrophage polarization were mediated through the nuclear receptor AhR. The expression of AhR was significantly increased after RDN treatment, and this effect was accompanied by STAT3 expression increasing. Coimmunoprecipitation confirmed an interaction between AhR and STAT3 and upregulated IL-10 expression. Silencing AhR decreased Src, STAT3, and IL-10 expression. RDN activated AhR and increased Src, STAT3, and IL-10 expression. In addition, RDN regulated the polarization of macrophages RDN combined with cefmetazole sodium significantly reduced the pulmonary bacterial load, alleviated lung injury, and reduced o inflammatory factors expression, improving their survival.ConclusionsRDN can synergistically enhance the effect of cefmetazole sodium treatment in severe pneumonia, and the mechanism may involve increasing the expression level of IL-10 mediated through the AhR-Src-STAT3 pathway, driving the polarization of macrophages, and attenuating the cytokine storm to control inflammation in severe pneumonia.

The Kynurenine Pathway Metabolites QA and KYNA induce senescence in Bone Marrow Stem Cells through the AhR Pathway

Cell senescence is emerging as a critical factor in the pathophysiology of aging bone loss. We have shown that the essential amino acid tryptophan is metabolized by IDO-1 in the periphery to generate kynurenine (KYN), and that KYN can signal though the aryl hydrocarbon receptor (AhR) transcription factor pathway to inhibit osteogenesis in bone marrow MSCs via epigenetic regulation of osteogenic genes, while also upregulating osteoclastogenic transcription factors and genes driving osteoclast activity. Further, we recently showed that KYN acting via AhR inhibits MSC autophagy while inducing senescence. Here we demonstrate that KYN metabolites downstream from KYN act via the AhR signaling pathway to inhibit autophagy and induce SASP expression and drive senescence in murine and human bone marrow MSCs. We focused on two of these metabolites, quinolinic acid (QA) and kynurenic acid (KYNA) and investigated their effects on BMSC cellular function. We demonstrated that both kynurenine pathway metabolites QA and KYNA increase biomarkers for senescence including beta-galactosidase, p21/Cdkn1 and other SASPs such as PAI-1 and TIMP-2, as well as nuclear DNA damage leading to senescent markers like H2A Ser139 phosphorylation, and the accumulation of senescence-associated hetero chromatin foci (SAHF) with H3K9-me3 labeling. Then upon treatment with the AhR inhibitor 3’4’-DMF the disruption of autophagy and induction of senescent biomarkers was blocked. Like KYN, the effects of QA and KYNA were mediated through the AhR receptor. Therefore, this presents novel therapeutic targets linked to KYN metabolite signaling via AhR to prevent senescence and bone loss.

Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut

Breastfeeding profoundly shapes the infant gut microbiota, which is critical for early life immune development, and the gut microbiota can impact host physiology in various ways, such as through the production of metabolites. However, few breastmilk-dependent microbial metabolites mediating host–microbiota interactions are currently known. Here, we demonstrate that breastmilk-promoted Bifidobacterium species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective aromatic lactic acids (indolelactic acid, phenyllactic acid and 4-hydroxyphenyllactic acid) via a previously unrecognized aromatic lactate dehydrogenase (ALDH). The ability of Bifidobacterium species to convert aromatic amino acids to their lactic acid derivatives was confirmed using monocolonized mice. Longitudinal profiling of the faecal microbiota composition and metabolome of Danish infants (n = 25), from birth until 6 months of age, showed that faecal concentrations of aromatic lactic acids are correlated positively with the abundance of human milk oligosaccharide-degrading Bifidobacterium species containing the ALDH, including Bifidobacterium longum, B. breve and B. bifidum. We further demonstrate that faecal concentrations of Bifidobacterium-derived indolelactic acid are associated with the capacity of these samples to activate in vitro the aryl hydrocarbon receptor (AhR), a receptor important for controlling intestinal homoeostasis and immune responses. Finally, we show that indolelactic acid modulates ex vivo immune responses of human CD4+ T cells and monocytes in a dose-dependent manner by acting as an agonist of both the AhR and hydroxycarboxylic acid receptor 3 (HCA3). Our findings reveal that breastmilk-promoted Bifidobacterium species produce aromatic lactic acids in the gut of infants and suggest that these microbial metabolites may impact immune function in early life.

Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of AhR receptor responsive xenobiotics

Dioxins are a group of highly toxic environmental persistent organic pollutants, which are lipophilic in nature. 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic representative of this class. TCDD causes several human health effects like endocrine disruption, carcinogenesis and reproductive toxicity mediated by aryl-hydrocarbon receptor. Current detection methods of dioxins like gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry etc. are costly and time consuming. Therefore, the present study aims to develop a relatively faster and cheaper technique called reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to detect dioxins. Cultured granulosa cells used as a model system were treated with different doses (5, 10 and 15 pg/mL) of aryl hydrocarbon receptor (AhR)responsive xenobiotic, TCDD, in accordance with maximum residue limit values. Cells were treated for 6, 12 and 24 h, respectively to study the gene expression of TCDD receptor called AhR and AhR responsive genes, CYP1A1 and CYP1B1, in a dose and time dependent manner. All targeted genes expression significantly increased after 6 and 12 h by 1.3–8 folds. For the development of RT-LAMP assay, CYP1A1 gene was used with 6 h TCDD treatment. RT-LAMP assay was standardized with optimal color change at 30 min using 50 ng of cellular RNA. In all the cases, we could distinguish RT-LAMP-positive condition from one sample to another sample due to intensity of color. The method was also validated by spectrometric method. In conclusion, the developed method will be used to screen AhR receptor responsive xenobiotics by observing the color change in RT-LAMP assay like dioxin used in the present study.

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.

Indolepropionic Acid, a Metabolite of the Microbiome, Has Cytostatic Properties in Breast Cancer by Activating AHR and PXR Receptors and Inducing Oxidative Stress.

Oncobiotic transformation of the gut microbiome may contribute to the risk of breast cancer. Recent studies have provided evidence that the microbiome secretes cytostatic metabolites that inhibit the proliferation, movement, and metastasis formation of cancer cells. In this study, we show that indolepropionic acid (IPA), a bacterial tryptophan metabolite, has cytostatic properties. IPA selectively targeted breast cancer cells, but it had no effects on non-transformed, primary fibroblasts. In cell-based and animal experiments, we showed that IPA supplementation reduced the proportions of cancer stem cells and the proliferation, movement, and metastasis formation of cancer cells. These were achieved through inhibiting epithelial-to-mesenchymal transition, inducing oxidative and nitrosative stress, and boosting antitumor immune response. Increased oxidative/nitrosative stress was due to the IPA-mediated downregulation of nuclear factor erythroid 2-related factor 2 (NRF2), upregulation of inducible nitric oxide synthase (iNOS), and enhanced mitochondrial reactive species production. Increased oxidative/nitrosative stress led to cytostasis and reductions in cancer cell stem-ness. IPA exerted its effects through aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR) receptors. A higher expression of PXR and AHR supported better survival in human breast cancer patients, highlighting the importance of IPA-elicited pathways in cytostasis in breast cancer. Furthermore, AHR activation and PXR expression related inversely to cancer cell proliferation level and to the stage and grade of the tumor. The fecal microbiome’s capacity for IPA biosynthesis was suppressed in women newly diagnosed with breast cancer, especially with stage 0. Bacterial indole biosynthesis showed correlation with lymphocyte infiltration to tumors in humans. Taken together, we found that IPA is a cytostatic bacterial metabolite, the production of which is suppressed in human breast cancer. Bacterial metabolites, among them, IPA, have a pivotal role in regulating the progression but not the initiation of the disease.