AhR Ligands Research Articles

Multi-omics insights into anti-colitis benefits of the synbiotic and postbiotic derived from wheat bran arabinoxylan and Limosilactobacillus reuteri

Exploring nutritional therapies that manipulate tryptophan metabolism to activate AhR signaling represents a promising approach for mitigating chronic colitis. Arabinoxylan is a bioactive constituent abundant in wheat bran. Here, we comprehensively investigated anti-colitis potentials of wheat bran arabinoxylan (WBAX), its synbiotic and postbiotic derived from WBAX and Limosilactobacillus reuteri WX-94 (i.e., a probiotic strain exhibiting tryptophan metabolic activity). WBAX fueled L. reuteri and promoted microbial conversion of tryptophan to AhR ligands during in vitro fermentation in the culture medium and in the fecal microbiota from type 2 diabetes. The WBAX postbiotic outperformed WBAX and its synbiotic in augmenting efficacy of tryptophan in restoring DSS-disturbed serum immune markers, colonic tight junction proteins and gene profiles involved in amino acid metabolism and FoxO signaling. The WBAX postbiotic remodeled gut microbiota and superiorly enhanced AhR ligands (i.e., indole metabolites and bile acids), alongside with elevation in colonic AhR and IL-22. Associations between genera and metabolites modified by the postbiotic and colitis in human were verified and strong binding capacities between metabolites and colitis-related targets were demonstrated by molecular docking. Our study advances the novel perspective of WBAX in manipulating tryptophan metabolism and anti-colitis potentials of WBAX postbiotic via promoting gut microbiota-dependent AhR signaling.

The Aryl Hydrocarbon Receptor Controls IFNγ-Induced Immune Checkpoints PD-L1 and IDO via the JAK/STAT Pathway in Lung Adenocarcinoma.

While immunotherapy has shown efficacy in lung adenocarcinoma (LUAD) patients, many respond only partially or not at all. One limitation in improving outcomes is the lack of a complete understanding of immune checkpoint regulation. Here, we investigated a possible link between an environmental chemical receptor implicated in lung cancer and immune regulation, (the aryl hydrocarbon receptor/AhR), a known but counterintuitive mediator of immunosuppression (IFNγ), and regulation of two immune checkpoints (PD-L1 and IDO). AhR gene-edited LUAD cell lines, a syngeneic LUAD mouse model, bulk- and scRNA sequencing of LUADs and tumor-infiltrating leukocytes were used to map out a signaling pathway leading from IFNγ through the AhR to JAK/STAT, PD-L1, IDO, and tumor-mediated immunosuppression. The data demonstrate that: 1) IFNγ activation of the JAK/STAT pathway leading to PD-L1 and IDO1 upregulation is mediated by the AhR in murine and human LUAD cells, 2) AhR-driven IDO1 induction results in the production of Kynurenine (Kyn), an AhR ligand, which likely mediates an AhR→IDO1→Kyn→AhR amplification loop, 3) transplantation of AhR-knockout LUAD cells results in long-term tumor immunity in most recipients. 4) The 23% of AhR-knockout tumors that do grow do so at a much slower pace than controls and exhibit higher densities of CD8+ T cells expressing markers of immunocompetence, increased activity, and increased cell-cell communication. The data definitively link the AhR to IFNγ-induced JAK/STAT pathway and immune checkpoint-mediated immunosuppression and support the targeting of the AhR in the context of LUAD.

Dendritic Cell - Fibroblast Crosstalk via TLR9 and AHR Signaling Drives Lung Fibrogenesis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring and loss of lung function. With limited treatment options, patients succumb to the disease within 2-5 years. The molecular pathogenesis of IPF regarding the immunologic changes that occur is poorly understood. We characterize a role for non-canonical aryl-hydrocarbon receptor signaling (ncAHR) in dendritic cells (DCs) that leads to production of IL-6 and IL-17, promoting fibrosis. TLR9 signaling in myofibroblasts is shown to regulate production of TDO2 which converts tryptophan into the endogenous AHR ligand kynurenine. Mice with augmented ncAHR signaling were created by crossing floxed AHR exon-2 deletion mice (AHR Δex2 ) with mice harboring a CD11c-Cre. Bleomycin was used to study fibrotic pathogenesis. Isolated CD11c+ cells and primary fibroblasts were treated ex-vivo with relevant TLR agonists and AHR modulating compounds to study how AHR signaling influenced inflammatory cytokine production. Human datasets were also interrogated. Inhibition of all AHR signaling rescued fibrosis, however, AHR Δex2 mice treated with bleomycin developed more fibrosis and DCs from these mice were hyperinflammatory and profibrotic upon adoptive transfer. Treatment of fibrotic fibroblasts with TLR9 agonist increased expression of TDO2. Study of human samples corroborate the relevance of these findings in IPF patients. We also, for the first time, identify that AHR exon-2 floxed mice retain capacity for ncAHR signaling.

Short-chain fatty acids suppresses astrocyte activation by amplifying Trp-AhR-AQP4 signaling in experimental autoimmune encephalomyelitis mice.

The function of astrocytes in response to gut microbiota-derived signals has an important role in the pathophysiological processes of central nervous system (CNS) diseases. However, the specific effects of microbiota-derived metabolites on astrocyte activation have not been elucidated yet. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice as a classical MS model. The alterations of gut microbiota and the levels of short-chain fatty acids (SCFAs) were assessed after EAE induction. We observed that EAE mice exhibit low levels of Allobaculum, Clostridium_IV, Clostridium_XlVb, Lactobacillus genera, and microbial-derived SCFAs metabolites. SCFAs supplementation suppressed astrocyte activation by increasing the level of tryptophan (Trp)-derived AhR ligands that activating the AhR. The beneficial effects of SCFAs supplementation on the clinical scores, histopathological alterations, and the blood brain barrier (BBB)-glymphatic function were abolished by intracisterna magna injection of AAV-GFAP-shAhR. Moreover, SCFAs supplementation suppressed the loss of AQP4 polarity within astrocytes in an AhR-dependent manner. Together, SCFAs potentially suppresses astrocyte activation by amplifying Trp-AhR-AQP4 signaling in EAE mice. Our study demonstrates that SCFAs supplementation may serve as a viable therapy for inflammatory disorders of the CNS.

Dysregulated tryptophan metabolism and AhR pathway contributed to CXCL10 upregulation in stable non-segmental vitiligo

BackgroundTryptophan metabolism dysregulation has been observed in vitiligo. However, drawing a mechanistic linkage between this metabolic disturbance and vitiligo pathogenesis remains challenging. ObjectiveAim to reveal the characterization of tryptophan metabolism in vitiligo and investigate the role of tryptophan metabolites in vitiligo pathophysiology. MethodsLC-MS/MS, dual-luciferase reporter assay, ELISA, qRT-PCR, small interfering RNA, western blotting, and immunohistochemistry were employed. ResultsKynurenine pathway activation and KYAT enzyme-associated deviation to kynurenic acid (KYNA) in the plasma of stable non-segmental vitiligo were determined. Using a public microarray dataset, we next validated the activation of kynurenine pathway was related with inflammatory-related genes expression in skin of vitiligo patients. Furthermore, we found that KYNA induced CXCL10 upregulation in keratinocytes via AhR activation. Moreover, the total activity of AhR agonist was increased while the AhR concentration per se was decreased in the plasma of vitiligo patients. Finally, higher KYAT, CXCL10, CYP1A1 and lower AhR expression in vitiligo lesional skin were observed by immunohistochemistry staining. ConclusionThis study depicts the metabolic and genetic characterizations of tryptophan metabolism in vitiligo and proposes that KYNA, a tryptophan-derived AhR ligand, can enhance CXCL10 expression in keratinocytes.

CD4+ T Cell Effector Status Is Dependent on Aryl Hydrocarbon Receptor Activation during High Salt Diet

Recent studies suggest that patients with hypertension and individuals that consume a HSD are depleted of numerous tryptophan-derived indoles and other AHR-binding metabolites that are key in intestinal CD4+ T cell homeostasis. Thus, the inappropriate immunologic response by CD4+ T cells, and their role in hypertension, may be due in part to loss of AHR activation. Therefore, we hypothesized that AHR activation contributes to maintaining effector T cell status in salt-sensitive mice. To examine the influence of a representative homeostatic or proinflammatory environment and the subsequent response to salt, we cultured naïve CD4+ T cells under homeostatic cytokine or pathogenic cytokine conditions and exposed the cultures to increased NaCl conditions with or without the AHR-activating metabolite, FICZ (6-Formylindolo(3,2-b)-carbazole). TH17 cells cultured under homeostatic high salt conditions were depressed with a concomitant increase in response to FICZ. In contrast, TH17 cells cultured under pathogenic high salt conditions had augmented IL-17A-expressing cells without a response to AHR activation. These data suggest that excess salt is disrupting the ability to induce IL-17A and increasing AHR activity overcomes this disruption to promote IL-17A, without expansion of the pathogenic TH17 pool, and this observation was specific to non-pathogenic microenvironments (homeostatic TH17: media: 11±0.6%, +NaCl: 4.9±0.3%*, +NaCl/FICZ: 9.9±0.8%, of CD4; pathogenic TH17: media: 1.0±0.2%, +NaCl: 2.2±0.2%*, +NaCl/FICZ: 2.2±0.2%*, *vs respective media, n=8/group). Given these data, we then fed salt-sensitive mice (nitric oxide synthase inhibition) a HSD or a HSD supplemented with the AHR agonist indole-3-carbinol (I3C) and examined Peyer’s patches, a lymphoid tissue in close proximity to intestinal lumen contents enriched with salt and the I3C-metabolites and noted expansion of non-pathogenic TH17 cells. In conclusion, increasing AHR ligand availability through supplementing I3C may provide a gut-centric experimental approach in targeting experimental salt-sensitivity as it relates to immunologic disturbances. F31HL151264. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dissecting the respective roles of microbiota and host genetics in the susceptibility of Card9−/− mice to colitis

BackgroundThe etiology of inflammatory bowel disease (IBD) is unclear but involves both genetics and environmental factors, including the gut microbiota. Indeed, exacerbated activation of the gastrointestinal immune system toward the gut microbiota occurs in genetically susceptible hosts and under the influence of the environment. For instance, a majority of IBD susceptibility loci lie within genes involved in immune responses, such as caspase recruitment domain member 9 (Card9). However, the relative impacts of genotype versus microbiota on colitis susceptibility in the context of CARD9 deficiency remain unknown.ResultsCard9 gene directly contributes to recovery from dextran sodium sulfate (DSS)-induced colitis by inducing the colonic expression of the cytokine IL-22 and the antimicrobial peptides Reg3β and Reg3γ independently of the microbiota. On the other hand, Card9 is required for regulating the microbiota capacity to produce AhR ligands, which leads to the production of IL-22 in the colon, promoting recovery after colitis. In addition, cross-fostering experiments showed that 5 weeks after weaning, the microbiota transmitted from the nursing mother before weaning had a stronger impact on the tryptophan metabolism of the pups than the pups’ own genotype.ConclusionsThese results show the role of CARD9 and its effector IL-22 in mediating recovery from DSS-induced colitis in both microbiota-independent and microbiota-dependent manners. Card9 genotype modulates the microbiota metabolic capacity to produce AhR ligands, but this effect can be overridden by the implantation of a WT or “healthy” microbiota before weaning. It highlights the importance of the weaning reaction occurring between the immune system and microbiota for host metabolism and immune functions throughout life. A better understanding of the impact of genetics on microbiota metabolism is key to developing efficient therapeutic strategies for patients suffering from complex inflammatory disorders.-8ZHpF322gum1G2x3b5GvyVideo

Microbiota, Tryptophan and Aryl Hydrocarbon Receptors as the Target Triad in Parkinson's Disease-A Narrative Review.

In the era of a steadily increasing lifespan, neurodegenerative diseases among the elderly present a significant therapeutic and socio-economic challenge. A properly balanced diet and microbiome diversity have been receiving increasing attention as targets for therapeutic interventions in neurodegeneration. Microbiota may affect cognitive function, neuronal survival and death, and gut dysbiosis was identified in Parkinson's disease (PD). Tryptophan (Trp), an essential amino acid, is degraded by microbiota and hosts numerous compounds with immune- and neuromodulating properties. This broad narrative review presents data supporting the concept that microbiota, the Trp-kynurenine (KYN) pathway and aryl hydrocarbon receptors (AhRs) form a triad involved in PD. A disturbed gut-brain axis allows the bidirectional spread of pro-inflammatory molecules and α-synuclein, which may contribute to the development/progression of the disease. We suggest that the peripheral levels of kynurenines and AhR ligands are strongly linked to the Trp metabolism in the gut and should be studied together with the composition of the microbiota. Such an approach can clearly delineate the sub-populations of PD patients manifesting with a disturbed microbiota-Trp-KYN-brain triad, who would benefit from modifications in the Trp metabolism. Analyses of the microbiome, Trp-KYN pathway metabolites and AhR signaling may shed light on the mechanisms of intestinal distress and identify new targets for the diagnosis and treatment in early-stage PD. Therapeutic interventions based on the combination of a well-defined food regimen, Trp and probiotics seem of potential benefit and require further experimental and clinical research.

A2 UVB ON SKIN EXPOSURE MODULATES INTESTINAL HOMEOSTASIS IN MICE

Abstract Background Inflammatory diseases, like IBD, are on the rise in Western societies, in concert with changes in the environment, such as reduced sun exposure. Ultraviolet light (UV) B's effects on the skin include upregulating vitamin D and Aryl hydrocarbon receptor (Ahr) signalling, but its potential influence on distant sites, like the gut, are not well-studied. The gut and skin share a strong link, with some GI patients also experiencing skin diseases (O’Neill et al., 2016). We sought to understand how UVB exposure affects gut health, gut microbiota and its metabolic by-products. We hypothesize that UVB will have a significant impact on gut health. Aims Test the effect of UVB light using vitamin D deficient (-) and sufficient (+) diets. Elucidate Ahr activation in the small intestine and colon after UVB exposure. Explore the gut metabolome after UVB exposure. Methods Female C57BL/6 mice were fed vit D (+) or (-) diets, anaesthetized, and then 8 cm2 of their dorsal skin was shaved and exposed to UVB (250 mJ/cm2, 234 sec, 311 nm). One single dose (sample collection after 4 or 10 h) or chronic dosage (9 days every other day). Mice were euthanized to collect serum, cecal content, and stool samples (HILIC negative profile and 16S sequencing). EROD assay was used to test Ahr activation of CYP1A1. Results UVB exposure did not cause skin inflammation, but instead induced photoadaptation. When mice with vit D (-) diets were exposed to UVB, their vit D levels rose to match those on vit D (+) diets. Intestinal luminal contents induced a significant 3X increase in Ahr activation at 10 h post-exposure. After chronic UVB exposure, Ahr/CYP1A1 activity increased in Peyer’s patches (p=0.0012) and colon (p=0.01). Serum levels of AhR agonist metabolites post-UVB exposure showed significant increases in kynurenine and indole-3-acetic acid (IAA) at 10 h, while in the feces, Tryptophan levels decreased (p=0.042), but IAA significantly increased (p=0.006). IAA is a known Ahr ligand and produced by gut microbiota when they metabolize Tryptophan, indicating a microbiota response to UVB skin exposure. This was accompanied by an increase in the short chain fatty acids (butyrate, propionate, acetic acid) as well as other metabolites, despite no overt changes in microbiota composition. After 9 days of UVB, significantly increased numbers of goblet cells/crypt in both the ileum and colon. Conclusions Our study demonstrates that UVB exposure on the skin leads to increased vitamin D serum levels and Ahr activation along the gastrointestinal tract. It also induces potentially beneficial metabolic changes in the gut microbiota and differentiation of the gut epithelium, highlighting sun exposure's potential intestinal health benefits. Funding Agencies CCC, CIHR, None

AhR diminishes the efficacy of chemotherapy via suppressing STING dependent type-I interferon in bladder cancer

The induction of type-I interferons (IFN-Is) is important for the efficacy of chemotherapy. By investigating the role of amino acids in regulation of IFN-I production under chemo-drug treatment in bladder cancer (BC) cells, we find an inherent AhR-dependent negative feedback to restrain STING signaling and IFN-I production. Mechanistically, in a ligand dependent manner, AhR bridges STING and CUL4B/RBX1 E3 ligase complex, facilitating STING degradation through ubiquitin-proteasome pathway. Inhibition of AhR increases STING levels and reduces tumor growth under cisplatin or STING agonist treatment. Endogenous AhR ligands are mainly consisted of tryptophan (Trp) metabolites; dietary Trp restriction, blocking the key Trp metabolism rate-limiting enzyme IDO1 or inhibition of cellular Trp importation also show similar effect as AhR inhibition. Clinically, BC patients with higher intratumoral expression of AhR or stronger intratumoral Trp metabolism (higher IDO1 or Kyn levels) that lead to higher AhR activation show worse response rate to neoadjuvant chemotherapy (NAC).

A fatty acid-rich fraction of an endolichenic fungus Phoma sp. suppresses immune checkpoint markers via AhR/ARNT and ESR1

Lung cancer has the highest mortality rates worldwide. The disease is caused by environmental pollutants, smoking, and many other factors. Recent treatments include immunotherapeutics, which have shown some success; however, the search for new therapeutics is ongoing. Endolichenic fungi produce a whale of a lot of secondary metabolites, the therapeutic effects of which are being evaluated. Here, we used a crude extract and subfractions of the endolichenic fungus, Phoma sp. (EL006848), isolated from the Pseudevernia furfuracea. It was identified the fatty acid components, palmitic acid, stearic acid, and oleic acid, exist in subfractions E1 and E2. In addition, EL006848 and its fatty acids fractions suppressed benzo[a]pyrene (an AhR ligand)- induced expression of PD-L1 to inhibit the activity of multiple immune checkpoints. E2 subfraction, which had a higher fatty acid content than E1, downregulated expression of AhR/ARNT and several human transcription factors related to ESR1. Moreover, E2 showed a strong inhibitory effect on STAT3 expression and mild effect on NF-kB activity. These results suggest that fatty acids extracted from an endolichenic fungus can exert strong immunotherapeutic effects.

Identification of methylsulochrin as a partial agonist for aryl hydrocarbon receptors and its antiviral and anti-inflammatory activities.

Although aryl hydrocarbon receptors (AhRs) are related to the metabolic pathway of xenobiotics, recent studies have revealed that this receptor is also associated with the life cycle of viruses and inflammatory reactions. For example, flutamide, used to treat prostate cancer, inhibits hepatitis C virus proliferation by acting as an AhR antagonist, and methylated-pelargonidin, an AhR agonist, suppresses pro-inflammatory cytokine production. To discover a novel class of AhR ligands, we screened 1000 compounds derived from fungal metabolites using a reporter assay and identified methylsulochrin as a partial agonist of the aryl hydrocarbon receptor. Methylsulochrin was found to inhibit the production of hepatitis C virus (HCV) in Huh-7.5.1 cells. Methylsulochrin also suppressed the production of interleukin-6 in RAW264.7 cells. Furthermore, a preliminary structure-activity relationship study using sulochrin derivatives was performed. Our findings suggest the use of methylsulochrin derivatives as anti-HCV compounds with anti-inflammatory activity.

Emerging paradigms in exploring the interactions among diet, probiotics, and cancer immunotherapeutic response

Emerging paradigms in exploring the interactions among diet, probiotics, and cancer immunotherapeutic response

Maternal PBDE exposure disrupts gut microbiome and promotes hepatic proinflammatory signaling in humanized PXR-transgenic mouse offspring over time.

Developmental exposure to the persistent environmental pollutant, polybrominated diphenyl ethers (PBDEs), is associated with increased diabetes prevalence. The microbial tryptophan metabolite, indole-3-propionic acid (IPA), is associated with reduced risk of type 2 diabetes and lower-grade inflammation and is a pregnane X receptor (PXR) activator. To explore the role of IPA in modifying the PBDE developmental toxicity, we orally exposed humanized PXR-transgenic (hPXR-TG) mouse dams to vehicle, 0.1 mg/kg/day DE-71 (an industrial PBDE mixture), DE-71+IPA (20 mg/kg/day), or IPA, from 4 weeks preconception to the end of lactation. Pups were weaned at 21 days of age and IPA supplementation continued in the corresponding treatment groups. Tissues were collected at various ages until 6 months of age (n = 5 per group). In general, the effect of maternal DE-71 exposure on the gut microbiome of pups was amplified over time. The regulation of hepatic cytokines and prototypical xenobiotic-sensing transcription factor target genes by DE-71 and IPA was age- and sex-dependent, where DE-71-mediated mRNA increased selected cytokines (Il10, Il12p40, Il1β [both sexes], and [males]). The hepatic mRNA of the aryl hydrocarbon receptor (AhR) target gene Cyp1a2 was increased by maternal DE-71 and DE-71+IPA exposure at postnatal day 21 but intestinal Cyp1a1 was not altered by any of the exposures and ages. Maternal DE-71 exposure persistently increased serum indole, a known AhR ligand, in age- and sex-dependent manner. In conclusion, maternal DE-71 exposure produced a proinflammatory signature along the gut-liver axis, including gut dysbiosis, dysregulated tryptophan microbial metabolism, attenuated PXR signaling, and elevated AhR signaling in postweaned hPXR-TG pups over time, which was partially corrected by IPA supplementation.

Aryl-hydrocarbon receptor agonist: A novel topical therapeutic approach for inflammatory skin diseases.

The broadening of novel therapeutic options for chronic inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis (PSO), known to be associated with dysregulation of the immune response, systemic comorbidities and a reduced quality of life for patients and caretakers, offers better chances for long-lasting disease control.1, 2 Recent advances in clarifying the mechanisms that lead to the immune imbalance in both diseases indicate a predominant IL-23/IL-17A/IL36 pathway in psoriasis, and a predominant Th2 IL-4/IL-13, with the participation of IL-17 and IL-22 axis in atopic dermatitis.1, 2 The development of targeted-oriented therapies such as biologics and small molecule inhibitors (SMIs) changed the traditional management of such inflammatory conditions, therefore optimizing the standard of care in both diseases.1, 2 One of the new foci of targeted therapies for inflammatory skin diseases is the aryl-hydrocarbon receptor (AhR)/AhR-nuclear translocator (ARNT) system, which works as a sensor for exogenous and endogenous molecules. It is expressed in the skin and has a relevant role in the skin barrier maintenance, upregulating filaggrin and loricrin and enhancing epidermal differentiation. Furthermore, the AhR complex modulates the T-helper 17/22 and T regulatory axis, and some AHR ligands also exert a role in the neutralization of oxidative stress through the activation of the nuclear factor-erythroid 2-related factor-2 (NRF2) transcription factor and a ROS-scavenging structure.3 Bissonnette et al.4 present a review article on tapinarof (3,5-dihydroxy-4-isopropylstilbene), an AhR agonist from a historical perspective. His group has been working with this component since 2007, and the initial studies focused on psoriasis.5 Tapinarof 1% cream is a topical medication applied once daily, and the review shows the main outcomes of its use in clinical trials in patients with psoriasis and AD. The key point of this review indicates the profile of tapinarof in psoriasis and AD, as follows4: Psoriasis: A phase 2, single-centre clinical trial, showed a rapid start of action in topically applied bid tapinarof 1% group than vehicle. There was a 67.5% of improvement in PGA (physician global assessment) score to clear or almost clear against 4.8% in vehicle group at week 12 in psoriasis patients. In phase 2b (multicentre trial), tapinarof 0.5% and 1%, once or twice a day compared to vehicle was tested and reached the primary endpoints (PGA0/1) with 1%. The relevant finding of phase 2b was the sustained improvement during the 4-week follow-up. Subsequentially, the studies with tapinarof 1% once a day versus placebo including 1366 psoriasis patients, demonstrated an improvement of 35.4% and 40.2% for tapinarof group, versus 6% and 6.3% for vehicle in PGA score, and a PASI (Psoriasis Area Index Severity) 75 achievement in 36.1% in tapinarof group versus 10.2% for vehicle (at week 12). In the 1-year open-label extension study, the mean duration of effect remission (PGA0/1) off-therapy was 130.1 days. Atopic dermatitis: In one single-centre study, 34 AD patients randomized (1:1:1) to tapinarof 0.5%, 1% or vehicle were treated bid for 4 weeks, showing that 50% of AD patients in both treatment groups achieved an IGA of clear or almost clear compared to 8.3% for the vehicle group (at week 5). These findings were confirmed in a phase 2 study with AD patients. A randomized phase 2b study with 247 AD patients under bid or once daily application of tapinarof 0.5%, 1% or vehicle (12 weeks), assessing safety and efficacy in adolescents 12 years and older, indicated that at week 12, 53% of the AD patients with tapinarof 1% BID and 46% with once daily application achieved the primary endpoint (IGA of clear/almost clear with a 2-point decrease proportion) versus 24% and 28% for vehicle respectively. Tapinarof is currently undergoing phase 3 trials for AD patients in >2 years of age. The present study with topical tapinarof 1% cream in both psoriasis and AD opens unlimited field for more research and expansion of its use. One advantage for such medication is the long-lasting clearing of the skin lesions despite discontinuation, which could be very beneficial to patients with chronic conditions, impacting on the need for prolonged systemic therapies and therefore impacting on the patients' safety regarding adverse events and alleviating the economic impact of the disease. There are still more points to be addressed that may be clarified after more studies are conducted, such as whether the response to treatment will be effective and homogeneous depending on: stages of the disease (acute × chronic), age and ethnic/racial immune phenotypes (folliculitis5 is a clinical finding that may be more present in patients with AD of African descent, and was one of the reported initial adverse events of tapinarof). None declared. VA has participated in clinical trials for Lilli and Sanofi and as a consultant for Abbvie, Galderma and Leopharma. RLO has participated in clinical trials for Lilli, Sanofi and Amgen and as consultant for Bayer and Abbvie. Data sharing not applicable.

Role of macrophage AHR/TLR4/STAT3 signaling axis in the colitis induced by non-canonical AHR ligand aflatoxin B1

Here we report that macrophage AHR/TLR/STAT signaling axis is implicated in the colon colitis induced by non-canonical AHR ligand aflatoxin B1 (AFB1). In BALB/c mice gavaged with 5, 25 and 50 µg/kg body weight/day AFB1, we observed severe colitis featured by over-recruitment of myeloid lineage immune cells such as monocytes/macrophage in colon lamina propria. Stressed and damaged colon epithelial cells were observed in low-dose group, while twisted and shortened intestinal crypts being found in middle dose group. Severe tissue damage was induced in the high-dose group. Dose-dependent increases of ROS, NO, and decrease of mitochondrial ROS-suppressor STAT3 were observed in the exposure groups. Further investigation in AFB1-treated human macrophage model found: (1) functional adaptations such as elevation of TNF-alpha and IL-6 secretion, stimulation of phagocytosis, elevation of LTE4 level; (2) overall inflammatory status confirmed by RNA-sequence analysis, in line with up-regulation of immune functional proteins such as ICAM-1, IDO-1, NF-kB-p65, NLRP3, COX-2 and iNOS; (3) mRNA disruption of mitochondrial oxidative phosphorylation complex I units and STATs; (4) perturbation of AHR/TLR/STAT3 signaling axis, including elevated AHR, TLR2, TLR4, and decreased STAT3, p-STAT3 Ser727. Mechanism investigation revealed regulatory links of ligand-dependent AHR/TLR4/STAT3. AHR-TLR4 together regulate MyD88, and STAT3 may be directly regulated by MyD88 (TLR4 downstream molecule) upon AHR/TLR4 binding with ligands. Solely protein level changes of AHR/TLR4 cannot regulate STAT3. Our study suggests that macrophage AHR/TLR4/STAT3 is involved with the colitis induced by sub-acute exposure to AFB1. Future follow-up study will focus on the intervention of the colitis using AHR-anti-inflammatory ligands.

Dietary tryptophan deficiency promotes gut RORγt+ Treg cells at the expense of Gata3+ Treg cells and alters commensal microbiota metabolism

Micronutrient deficiency is a major cause of disease throughout the world. Yet, how perturbations influence the immune-microbiome interface remains poorly understood. Here, we report that loss of dietary tryptophan (Trp) reshapes intestinal microbial communities, including the depletion of probiotic L.reuteri, drives transcriptional changes to immune response genes in the intestinal ileum, and reshapes the regulatory Tcell (Treg) compartment. Dietary Trp deficiency promotes expansion of RORγt+ Treg cells and the loss of Gata3+ Tregs in a microbiota-dependent manner. In the absence of dietary Trp, provision of the AhR ligand indole-3-carbinol is sufficient to restore the Treg compartment. Together, these data show that dietary Trp deficiency perturbs the interaction between the host and its bacterial symbionts to regulate Treg homeostasis via the deprivation of bacterially derived Trp metabolites. Our findings highlight an essential role for immune-microbiome crosstalk as a key homeostatic regulator during nutrient deficiency.

Insights into the toxicities of UV-328, UV-329, UV-P in HepG2 cells and their roles in AHR-mediated pathway

The widespread high concentrations of benzotriazole ultraviolet stabilizers (BUVSs) in many biotic and abiotic samples have raised urgent concerns of their adverse effects on environmental and human health. In this study, we investigated the toxicity of three typical BUVSs (UV-328, UV-329, UV-P) with HepG2 cells in vitro. Results indicated that the three BUVSs showed weak cytotoxicity in HepG2 cells at concentrations lower than 50 μM. Transcriptional analysis indicated that the toxic effects of the three chemicals followed the order of UV-P > UV-329 > UV-328. UV-P and UV-329 may act as potential environmental diabetogens by significantly enriching several diabetic related items in both GO and KEGG analysis. Moreover, UV-P and UV-329 significantly upregulated the expression of AHR target genes (CYP1A1, CYP1A2, UGT1A1, etc.), and increased the ethoxyresorufin-O-deethylase (EROD) activity and exhibited agonistic activity toward AHR in the XRE-mediated luciferase reporter gene assay. Molecular docking assay also indicated that UV-329 and UV-P had higher binding affinities to AHR-LBD than UV-328. In brief, our findings indicated that UV-P and UV-329 were potential agonist of AHR ligand, and may exert more toxicity than UV-328 in inducing liver toxicity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces multigenerational alterations in the expression of microRNA in the thymus through epigenetic modifications.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand, is an environmental contaminant that is known for mediating toxicity across generations. However, whether TCDD can induce multigenerational changes in the expression of microRNAs (miRs) has not been previously studied. In the current study, we investigated the effect of administration of TCDD in pregnant mice (F0) on gestational day 14, on the expression of miRs in the thymus of F0 and subsequent generations (F1 and F2). Of the 3200 miRs screened, 160 miRs were dysregulated similarly in F0, F1, and F2 generations, while 46 miRs were differentially altered in F0 to F2 generations. Pathway analysis revealed that the changes in miR signature profile mediated by TCDD affected the genes that regulate cell signaling, apoptosis, thymic atrophy, cancer, immunosuppression, and other physiological pathways. A significant number of miRs that showed altered expression exhibited dioxin response elements (DRE) on their promoters. Focusing on one such miR, namely miR-203 that expressed DREs and was induced across F0 to F2 by TCDD, promoter analysis showed that one of the DREs expressed by miR-203 was functional to TCDD-mediated upregulation. Also, the histone methylation status of H3K4me3 in the miR-203 promoter was significantly increased near the transcriptional start site in TCDD-treated thymocytes across F0 to F2 generations. Genome-wide chromatin immunoprecipitation sequencing study suggested that TCDD may cause alterations in histone methylation in certain genes across the three generations. Together, the current study demonstrates that gestational exposure to TCDD can alter the expression of miRs in F0 through direct activation of DREs as well as across F0, F1, and F2 generations through epigenetic pathways.

SOC-IV-11 Comparison of the abundance of AhR ligands in fecal water between IBD patients and healthy controls

SOC-IV-11 Comparison of the abundance of AhR ligands in fecal water between IBD patients and healthy controls