Unravelling the role of AHR signaling in microbe-host interaction in the skin

Prof. Dr. med. Bernhard Homey, Department of Dermatology, University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf

Recent findings indicate that human health is ‘multiorganismal’. Interactions between commensal or pathogenic microbes and the hosts they colonize are central to the maintenance of homeostasis and the initiation of disease. Detailed knowledge on the skin microbiome and its alterations in a variety of skin diseases is now available. However, specific interactions between microbes and skin cells of the host, including avenues of inter-organismal cell-to-cell communication are less well understood. Recently, it became apparent that a well-known communication receptor, namely the aryl hydrocarbon receptor (AHR) plays a critical role in microbe-host interactions within the gut. The AHR acts as a key sensor of metabolites produced and released by the intestinal microbiota, where they are implicated in the regulation of the mucosal immune system and intestinal barrier function. In particular, the AHR is involved in the binding of microbial-derived metabolites of the essential amino acid L-tryptophan (L-Trp). Although various members of the gut microbiome have now been shown to be involved in pathways that yield biologically active derivatives of L-Trp activating the AHR, the situation in the skin remains largely elusive. In project 1, we aim to generate evidence supporting the role for AHR signaling as a key player in microbe-host interactions maintaining cutaneous homeostasis and when perturbed, facilitating chronic skin inflammation. We are investigating healthy skin and distinct chronic inflammatory skin diseases, such as atopic dermatitis and psoriasis, which have been shown to depend on AHR signaling. The applicant will mine datasets from the unique physical biomaterial collection of project 1 and combine these with recently available public datasets (incl. single cell RNA-Seq data) as well as newly generated transcriptome and microbiome analyses. Here, the applicant will build on these multi-omics datasets to investigate the following specific aims: (1) Unravel the microbe-driven dynamics of Trp-catabolism-dependent cutaneous AHR signaling during homeostasis and chronic skin inflammation. (2) Define functional consequences in keratinocytes, sensory neurons and Schwann cells as cellular targets for AHR ligands to mediate microbe-host interactions in vitro. (3) Validate the modulation of cutaneous homeostasis and chronic skin inflammation through microbe-induced AHR signaling in mice and man in vivo. Findings of the proposed study may shed light into the role of AHR signaling in the interaction pathways between microbes and the host. It may identify important ‘beneficial’ vs. ‘harmful’ microbes as well as their products and define novel strategies based on AHR targeting, which help to maintain cutaneous homeostasis and therapeutically modulate skin inflammation.

Cell type-specific regulation and function of the AHRR in skin inflammation and allergy

Dr. rer. nat. Heike Weighardt, LIMES Institute, Faculty of Mathematics and Natural Sciences of the University of Bonn

AHR signalling is controlled via several mechanisms to ensure proper regulation of xenobiotic metabolism and immune processes. Enzymes of the cytochrome P450 (CYP) family, namely CYP1A1, CYP1B1 and CYP1A2 and the AHR repressor (AHRR) are AHR target genes and also important feedback control mechanisms of AHR activation. To analyse the function of AHRR in vivo, we generated AHRR-reporter mice and found that AHRR expression is intense in immune cells of barrier organs, such as gut and skin, and less pronounced in epithelial cells. AHR-dependent CYP1 family members, however, might be more active in epithelial cells. Therefore, we hypothesize, that AHR signalling might be regulated differentially in distinct cell types and this might be crucial for understanding AHR function in skin. Given the importance of the crosstalk of epithelial and immune cells at barrier organs, we want to explore the function of the AHRR and the AHR-dependent CYP1 enzymes in skin inflammation and allergy. We will compare the immunopathology and disease severity of AHRR-deficient mice, mice deficient for the AHR-dependent CYP1 enzymes and mice deficient for both, CYP1 enzymes and AHRR in mouse models of contact hypersensitivity and atopic dermatitis like skin inflammation. Further, we want to address the question how AHRR and CYP1 expression are regulated on transcriptional and epigenetic levels in skin epithelial cells versus immune cells and why AHRR expression in immune cells but to a lesser degree in keratinocytes is important for AHR activation during skin homeostasis, inflammation and allergy. With this global approach we also want to identify novel differentially regulated target genes in these cell types which might contribute to AHR regulation on a single cell level. To test a putative detrimental role of forced AHRR expression in epithelial cells we will generate mice to cell type-specifically express AHRR in vivo and analyze the impact of forced AHRR expression in keratinocytes for skin homeostasis, barrier function and allergy. In summary, in this project we intend to elucidate the molecular basis for the different regulation mechanism in immune cells versus keratinocytes to broaden and intensify the knowledge of regulatory networks of AHR function in skin.

AHR-mediated mechanisms to overcome vascular barriers in autoimmunity

Prof. Dr. med. Dr. rer. nat. Sven Meuth, Department of Neurology, University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf

The development of Multiple sclerosis (MS), one of the most frequent immune-mediated, chronic inflammatory, and neurodegenerative diseases of the central nervous system (CNS), requires a combination of genetic predisposition and environmental triggers. Among these are cigarette smoking, air pollution, low vitamin D levels potentially caused by an insufficient response to ultraviolet (UV) light exposure, dietary intake, the intestinal microbiome, or Epstein-Barr virus infection. Many of these risk factors like UV light, components of cigarette smoke, dietary or microbiome-derived metabolites are sensed by the aryl hydrocarbon receptor (AHR). Sensing frequently takes place in the skin, thereby pointing to a link between the skin and the CNS in MS pathology (skin-CNS axis). Accordingly, in a mouse model of experimental autoimmune encephalomyelitis (EAE) we were able to show that UV irradiation reduced disease severity in wild-type but not in AHR deficient mice.

In healthy individuals the blood-brain barrier (BBB), which is established by brain microvascular endothelial cells (BMEC) prevents immune cell migration to the CNS and neuroinflammation. However, in MS the BBB is often described as “leaky” and immune cells are breaching the BBB. Processes involved in controlling BBB integrity are not completely understood and we could show that pharmacological inhibition of AHR in BMEC limited T cell transmigration, which was accompanied by a reduction in ICAM-1 expression. In line with this, mice with an endothelial cell (EC)-specific deletion of AHR developed a less severe EAE compared to wild-type controls, indicating that AHR signaling directly impacts on EC function, BBB integrity, and the infiltration of pathogenic immune cells into the CNS.

Hence, in this project we will investigate the role of AHR in the connection of inflammatory processes in the skin and the CNS with a particular focus on its function in skin and brain EC. This will help to better understand pathways mediating the infiltration of immune cells into target tissues as well as the destruction of barrier integrity. In this context we will focus on three different aspects: (1) Investigating the ligand-specific function of AHR in the blood-skin barrier (BSB) and the BBB using skin and brain EC cultures with a particular focus on tight junction proteins, barrier integrity, immune cell penetration, transcriptomic profiling, proteomics and secretomics. (2) Transferring the results obtained in vitro to in vivo settings by using autoimmune mouse models with EC-specific deletion of AHR. (3) Translating the findings from animal models into the human system using vascularized human cortical brain organoids and skin organ cultures. Together, the results from this project will increase our knowledge on how AHR signaling influences immunological barriers. Since EC integrity is implicated in the pathophysiology of autoimmunity, this could potentially result in novel therapeutic options.

AHR as a master regulator in cutaneous and systemic autoimmunity

Prof. Dr. rer. nat. Karin Loser, Institute of Immunology, Faculty VI: Medicine and Health Sciences, Carl von Ossietzky University of Oldenburg

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting many organs including the skin, the joints, the central nervous system or the kidneys. Although the pathophysiology of SLE is not completely understood yet, it has been shown that the interaction of impaired apoptotic clearance, up-regulated innate and adaptive immune responses, complement activation, autoantibodies, and imbalanced production of pro-inflammatory cytokines including type-1 interferons leads to the loss of self-tolerance. The onset of SLE requires a combination of genetic predisposition, environmental triggers, and immunological as well as hormonal factors. Among the environmental triggers of SLE, ultraviolet (UV) light is the most recognized one. UV irradiation contributes to the generation of tryptophan photoproducts, which act as endogenous ligands of the aryl hydrocarbon receptor (AHR), a transcription factor frequently suggested as sensor of external stimuli. Besides UV, dietary compounds or toxins, many of which are known as risk factors of SLE, represent AHR ligands and thus, activation of the transcription has been linked to autoimmune disorders.

To investigate the role of AHR in systemic autoimmunity we generated a mouse model for SLE (K14-CD40L TG mice), which mimics typical characteristics of human disease including autoimmune dermatitis, nephritis, autoantibodies in the serum, and the presence of autoreactive T cells. Interestingly, AHR signaling was critical for disease development since K14-CD40L TG mice bred to an AHR-deficient background did not show any symptoms of SLE-like autoimmunity. Cell type-specific deletion of AHR revealed that AHR signaling in T cells seemed to be of minor relevance for the onset of CD40L-induced autoimmunity whereas mice deficient for AHR in Langerin⁺ dendritic cells (DC) were completely protected from disease, suggesting a central role of AHR in tissue-resident DC. Hence, we hypothesized that AHR might be critically involved in the development and progression of (systemic) autoimmune diseases by modulating the function of immune cells such as tissue-resident DC. Now we will try to better understand the ligand- and cell-specific character of AHR signaling during the onset of autoimmunity and three central questions shall be addressed: (1) Impact of AHR signaling on the development and progression of autoimmunity as well as functional relevance of the pathway in autoreactive cells; (2) Intervention in AHR signaling as potential therapeutic option for an (adjuvant) treatment of autoimmune diseases; (3) Relevance of the AHR pathway for the development of pathogenic or autoreactive human cells. Taken together, the project aims at better understanding AHR interactions in autoimmune processes, which might result in novel strategies to use this druggable ligand-activated transcription factor as a molecular target for the local or adjuvant treatment of autoimmune diseases.

The role of AHR for the gut-skin inflammatory axis

Prof. Dr. rer. nat. Charlotte Esser, IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf

Microorganisms that live in and on the body of animals and humans and the nutrients or messenger substances they produce are important for health and disease. Chemicals can be sensed through cellular receptors, and trigger adaptive reactions. One such chemical sensor, the aryl hydrocarbon receptor (AHR), is a transcription factor and becomes activated upon binding to certain small molecule chemicals (“AHR ligands”). AHR ligands include metabolites of the essential amino acid tryptophan, which are generated by certain intestinal bacteria, but are also certain indoles from food plants, e.g. from the family Brassicaceae. We had previously shown that the expression of the AHR in intestinal epithelial cells affects the composition of the intestinal microbiota. In addition, we showed using gene-deficient mice that the presence of AHR and dietary AHR ligands are important for a healthy skin barrier and may thus contribute to the gut-skin axis. For instance, addition of AHR ligands to the mouse diet attenuated pea peanut allergy in a mouse model. Furthermore, we showed that the skin barrier is weakened when the AHR is genetically lost, and could even be strengthened in wild-type mice by long-term feeding of AHR ligands. In the present project, we will investigate the role of the AHR for the gut-skin axis in mice. In particular, we want to investigate in which cells the AHR must be expressed in order to bring about a change in the gut microbiome, i.e. dysbiosis. We know from preliminary work that mice with an AHR-deficiency in intestinal epithelial cells have a different gut microbiota profile than wild-type mice. Since certain gut bacteria are known to be particularly good producers of AHR ligands, dysbiosis could lead to different levels of AHR-ligands in the serum. Central experiments of our project are feeding studies, which will allow us to investigate the role of the gut microbiota for the skin barrier using an inflammatory skin disease associated with barrier damage, atopic dermatitis, in mice. Specifically, we will orally transfer intestinal bacteria (“fecal transplants”) from AHR-deficient mice with dysbiosis into wild-type mice via gavage, and subsequently induce an AD-like inflammation using a standard method, followed by a thorough characterization of its extent. Similar experiments will be performed with high-affinity, pure AHR ligands, which are also used by other members of the consortium, and which are suspected to differentially activate the AHR pathway. This set of experiments is also about the generation of e.g. ligand- and cell-specific gene expression profiles that can be used in later meta-analyses. Finally, we will identify AHR ligands in mouse sera and attempt to associate them with the altered bacterial abundance. In the long term, we want to create a rational basis for therapeutic intervention with probiotic bacteria for the gut-skin axis.

Crosstalk between AHR signaling and retinoids in skin

Prof. Dr. med. Jean Krutmann, IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf and Medical Faculty of Heinrich Heine University Düsseldorf

Arylhydrocarbon receptor (AHR) signaling is critical for skin barrier function and photocarcinogenesis, which are also affected by retinoids. Retinoids are a family of signaling molecules which comprise vitamin A and its natural and synthetic derivatives. Under normal conditions, virtually all effects of vitamin A in the skin are mediated by all-trans retinoic acid (atRA). Formation of atRA involves as the final step the irreversible conversion of retinaldehyde to atRA by Aldehyde Dehydrogenase (ALDH) 1A enzymes, of which ALDH1A3 is the predominant isozyme in skin. Skin might be exposed to atRA as a consequence of its endogenous production by epidermal keratinocytes, but also after atRA has been topically applied for therapeutic or cosmetic indications.

Of note, there is compelling evidence that retinoid signaling and AHR signaling can influence each other. All of these studies, however, have focused on non-cutaneous tissues and / or have employed the difficult to degrade AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which causes long-lasting activation of AHR. It is currently not known how transient activation of AHR signaling by physiologically, environmentally or therapeutically and thus clinically relevant AHR ligands affects retinoid metabolism, how retinoids impact AHR signaling caused by such ligands, and how these interactions might be relevant for skin. In this regard, we have recently found that AHR signaling regulates synthesis of atRA in vitro in human keratinocytes and in vivo in murine skin. We also obtained evidence that AHR-induced regulation of the expression and activity of ALDH1A3 might be critically involved in this context. In addition, independent evidence suggests that a combined treatment of murine skin with atRA plus TCDD causes a proinflammatory, psoriasis-like inflammatory skin response.

We therefore hypothesize that in skin, AHR signaling and retinoid signaling influence each other, that this interaction is bidirectional and of clinical relevance, e.g. for skin barrier function. Specifically, we will systematically assess AHR ligand-mediated regulation of atRA synthesis in epidermal keratinocytes, the role of ALDH1A3 as an AHR target gene in this process, the functional relevance of endogenous retinoid synthesis as an integral part of AHR responses in keratinocytes, and we will comprehensively characterize the crosstalk of exogenously added atRA and AHR ligands in skin. In all of these studies we will use transiently activating AHR ligands of clinical relevance. We will address these topics by employing genetically engineered keratinocytes, complete as well as conditional knockout mouse models, and ex vivo human skin models. This project contributes to a better understanding of AHR signaling in skin barrier function and its importance for clinically and cosmetically used topical retinoids.

From actinic keratosis to invasive squamous cell carcinoma: Impact of AHR and p27KIP1 on malignant transformation

Dr. rer. nat. Thomas Haarmann-Stemmann, IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf

The incidence of cutaneous squamous cell carcinomas (SCC) has been increasing steadily for years. The most important risk factor for the development of this malignancy is the chronic exposure to ultraviolet (UV) radiation. The majority of SCC arises from actinic keratosis (AK), a precancerous precursor which predominantly occurs on sun-exposed skin areas. Interestingly, only a small percentage of AK develops into invasive SCC. The underlying molecular mechanisms driving this process of malignant transformation are not well understood.

In the context of photocarcinogenesis, we have shown that the aryl hydrocarbon receptor (AHR), a transcription factor, which is activated in epidermal cells by photoproducts of the amino acid tryptophan, regulates DNA damage-dependent responses in UVB-exposed keratinocytes. Specifically, AHR inhibits both DNA repair and apoptosis by modulating the intracellular localization and the protein level of the tumor suppressor p27^KIP1 (p27). Studies on extracutaneous cancers revealed that a mislocalization of p27 in the cytoplasm induced migration, invasion and other processes, which contribute to the malignant transformation. Further preliminary data indicate that proinflammatory cytokines inhibit the metabolism of AHR-activating tryptophan photoproducts, thereby amplifying the activation of the AHR-p27 axis.

In the current project, we will test the hypothesis that an inflammatory micromilieu fosters the accumulation of AHR agonists and the subsequent activation of the, in this context, oncogenic AHR-p27 axis. As a result, AK cells lose their contact inhibition, enhance their motility and grow invasive, finally leading to their transformation into malignant SCC cells. In order to test our working hypothesis, we will investigate whether 1.) an inhibition of CYP1A enzyme activity by inflammatory mediators, such as TNFα, is sufficient to induce or amplify AHR-dependent signaling pathways in AK cells, 2.) an activation of AHR in the presence of inflammatory mediators results in a cytosolic mislocalization and/or proteolysis of the p27 protein, 3.) an activation of the AHR-p27 axis induces the malignant transformation of AK cells in vitro and in vivo, and 4.) comparative immune-/histological staining and RNAseq analyses (cells, xenograft mouse model, clinical samples) enable us to identify AK-specific prognostic signatures.

The overall aim of this research project is to identify and validate the AHR-p27 axis as a molecular target for chemopreventive measures to reduce the transformation rate of premalignant AK.

Role of the AHR pathway in UV-induced initiation and progression of melanoma and in resistance to therapy

Prof. Dr. med. Thomas Tüting, Department of Dermatology, University Hospital Magdeburg, Medical Faculty of the Otto von Guericke University Magdeburg

Melanoma, the most lethal type of skin cancer, accounts for almost 2% of cancer cases worldwide, with an increasing incidence due to the unbroken trend of recreational UV exposure. The aryl hydrocarbon receptor (AHR), a ligand binding transcription factor of the basic helix-loop-helix Per-ARNT-Sim family, was initially described as the receptor for the dioxin TCDD and has been shown to mediate the detoxification of environmental pollutants. More recently, a multitude of physiological and endogenous ligands have been discovered, along with increasing evidence of physiological roles of AHR in cell proliferation, differentiation and inflammatory responses. UV light has been shown to generate the high affinity AHR agonist FICZ that promotes skin tanning as a protective response against UV. However, in cancer cells AHR activation promotes tumor progression. The current project is based on the hypothesis that AHR signaling promotes protective melanocyte responses to UV irradiation in physiological settings, whereas in pathologic settings, AHR signaling enhances malignant transformation as well as metastatic progression and impairs the efficacy of T cell directed immunotherapies. In the first work package of the project, we will use melanocyte-specific, conditional AHR knockout mice to characterize the impact of AHR signaling on the response of melanocytes to UV light. Additionally, we will also analyze the role of AHR signaling on UV-induced tumor development and progression by utilizing the Hgf-Cdk4 melanoma model crossed with the melanocyte-specific AHR knockout mice. In the second work package, we will assess the role of AHR signaling on immunotherapy. For this, we will use our transplantable melanoma cell line HCmel12 with and without a CRISPR mediated AHR knockout and perform adoptive T-cell transfer. Furthermore, we will also combine adoptive cell transfer therapies with pharmacological AHR inhibition in mice transplanted with HCmel12 melanoma cells to assess the future prospect of therapeutic AHR inhibition in man. In the third work package, we will characterize the AHR signaling dynamics in melanocytes and melanoma cells in vitro. Subsequently, we attempt to analyze the impact of AHR signaling on inflammation-induced melanoma cell plasticity and unravel the underlying molecular mechanism. In the final work package, we will analyze the impact of AHR signaling on the dynamics of melanoma cell transcriptional plasticity in response to inflammatory and therapeutic stimuli via single cell RNA sequencing. Additionally, we will also characterize AHR signaling on the differentiation trajectories of cutaneous melanocytes and melanoma cells following UVB irradiation. We expect that the insights gained from this project will provide a rationale for the future therapeutic modulation of AHR signaling in the treatment of melanoma.

Role of the AHR in cutaneous adverse drug reactions

Dr. med. Stephan Meller, Department of Dermatology, University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf

Adverse drug reactions (ADR) are common and often represent a serious threat to patients’ health and that may compromise their treatment continuation. ADR are mostly classified in on-target and off-target reactions. On-target reactions are very common and are caused by an augmentation of the known therapeutic and pharmacological action of a medication. In contrast, off-target reactions consist of unpredictable immune and non-immune reactions. Off-target non-immune ADR may involve innate immune responses but are not based on a specific hypersensitivity due to an immunological memory. Recently, the applicant and his collaborators demonstrated that vemurafenib antagonizes the canonical aryl hydrocarbon receptor (AHR) signaling pathway resulting in the development of an off-target non-immune reaction presenting as inflammatory skin rashes or phototoxic reactions. The AHR is a transcriptional factor that orchestrates multiple functions following its activation by a variety of ligands including xenobiotics, natural products, microbiome metabolites, and (pseudo-) endogenous molecules. Apart from skin homeostasis, the AHR pathway controls immune-mediated skin responses and is also involved in cutaneous pathological processes such as atopic dermatitis and plaque psoriasis. Moreover, the AHR pathway regulates the metabolism of common drugs. Activation of AHR stimulates canonical and non-canonical pathways. The canonical and the non-canonical AHR signaling pathways are tightly balanced in healthy skin thereby contributing to skin homeostasis. Preliminary unpublished data demonstrate that not only vemurafenib but also several other drugs present as AHR ligands and display antagonistic activity against the AHR in vitro. In the proposed project the applicant aims to characterize the role of the AHR in ADR and put forward the concept of modulating of AHR activity as a new mechanism of an off-target non-immune ADR. For this purpose, the applicant will first use empirical data as well as in silico-prediction models to identify drugs interfering with AHR-signaling (aim #1), second confirm the biologic effects of AHR-antagonistic drugs (aim #2), third validate AHR antagonism signatures in biomaterial of ADR patients ex vivo (aim #3), and fourth establish a diagnostic cell-based tool to identify AHR-mediated drug eruptions in vitro (aim #4). The experimental methods are established in the laboratories of the applicant, his collaborators or at the core facilities of the University of Düsseldorf. Significance of the project: The proposed project will identify clinically relevant drugs that interfere with AHR signaling. Findings of this project will increase our understanding of molecular and cellular pathways inducing ADR based on an off-target non-immune ADR. The results will provide new translational concepts to improve the clinical management of ADR.