The Ah Receptor from Toxicity to Therapeutics: Report from the 5th AHR Meeting at Penn State University, USA, June 2022.

The aryl hydrocarbon receptor (AHR) is a sensor of low-molecular-weight molecule signals that originate from environmental exposures, the microbiome, and host metabolism. Building upon initial studies examining anthropogenic chemical exposures, the list of AHR ligands of microbial, diet, and host metabolism origin continues to grow and has provided important clues as to the function of this enigmatic receptor. The AHR has now been shown to be directly involved in numerous biochemical pathways that influence host homeostasis, chronic disease development, and responses to toxic insults. As this field of study has continued to grow, it has become apparent that the AHR is an important novel target for cancer, metabolic diseases, skin conditions, and autoimmune disease. This meeting attempted to cover the scope of basic and applied research being performed to address possible applications of our basic knowledge of this receptor on therapeutic outcomes.

Borrelia burgdorferi is strong inducer of IFN-γ production by human primary NK cells.

Natural Killer (NK) cells belong to the innate lymphoid lineage and are highly present in the human skin. NK cells can produce a range of pro-inflammatory mediators, including cytokines and chemokines. The role of NK(-T) cells in the immune response towards Borrelia burgdorferi infection was studied. The production of interleukin (IL)-6, IL-1ß and interferon-gamma (IFN-γ) by human primary peripheral blood mononuclear cells (PBMCs) exposed to B. burgdorferi was assessed. Interestingly, CD56+ (NK + NK-T) cells were the only cells within the PBMC-fraction that produced IFN-γ during the first 24 h of stimulation. Within the NK(-T) cell fraction, NK cells seemed to be responsible for the IFN-γ production. Since it was previously demonstrated that both TLR2 and NOD2 receptors are involved in the recognition of B. burgdorferi, the expression of both TLR2 and NOD2 mRNA on NK cells was determined. In contrast to TLR2, NOD2 mRNA was upregulated on CD56+ (NK + NK-T) cells after Borrelia exposure. Finally, to unravel the mechanisms underlying erythema migrans (EM) development, crosstalk between CD56+ (NK + NK-T) cells and keratinocytes was investigated. CD56+ (NK + NK-T) cells activated by B. burgdorferi produced soluble mediators strongly inducing the expression of antimicrobial peptides, such as ß-defensin-2 and psoriasin in human keratinocytes. In conclusion, CD56+ (NK + NK-T) cells produced IFN-γ shortly after exposure to B. burgdorferi and released soluble mediators that were able to activate keratinocytes. These observations underscore the important role of CD56+ (NK + NK-T) cells during early host defence when Borrelia burgdorferi enters the human skin during a tick bite.

Challenges in Noninvasive Skin Biomarker Measurements in Daily Practice: A Longitudinal Study on Skin Surface Protein Detection by the Transdermal Analysis Patch in Pediatric Psoriasis.

INTRODUCTION: Skin surface proteins are potential biomarkers in psoriasis and can be measured noninvasively with the transdermal analysis patch (TAP). This study aimed to assess markers measured by TAP over time in daily clinical practice, explore their correlation with disease severity in pediatric psoriasis, and compare the TAP and tape stripping detection capability. METHODS: In this prospective observational daily clinical practice study, pediatric psoriasis patients (aged >5 to <18 years) were followed during 1 year. At each visit, TAPs were applied to lesional (n = 2), peri-lesional (n = 2), and non-lesional (n = 1) sites. Post-lesional skin was sampled if all lesions on the arms, legs, or trunk cleared. Treatment and psoriasis severity data were collected. IL-1RA, hBD-2, IL-1α, IL-8, VEGF, CXCL-1/2, CCL-27, IL-23, hBD-1, IL-22, IL-17A, KLK-5, and IL-4 levels were quantified by spot-ELISA. For the statistical analysis, Wilcoxon signed rank tests, Mann-Whitney U tests, and Spearman correlations were used. Detection capability of the TAP was compared to tape stripping in a separate cohort of adult psoriasis patients. RESULTS: 32 patients (median age 15.0 years, median Psoriasis Area and Severity Index [PASI] 5.2) were followed for a mean of 11.3 (±3.4) months with a total of 104 visits. In lesional skin (n = 197), significantly higher IL-1RA, hBD-2, IL-8, VEGF, CXCL-1/2, IL-23, hBD-1, IL-22, CCL-27, and IL-17A levels were found compared to non-lesional skin (n = 104), while IL-1α was higher in non-lesional skin. Marker levels were highly variable over time and did not correlate with disease severity measured by PASI or SUM scores. Comparison of the TAP and tape strip detection capability in adult psoriasis patients (n = 10) showed that lesional hBD-2, IL1-α, IL-8, and VEGF and non-lesional IL-1RA, hBD-2, IL-8, and VEGF were more frequently detected in tape extracts than TAPs. CONCLUSION: Due to the lack of correlation with clinical disease severity and the current detection capability of the markers measured by TAP in psoriasis, its use in regular practice is still a bridge too far.

Carboxamide Derivatives Are Potential Therapeutic AHR Ligands for Restoring IL-4 Mediated Repression of Epidermal Differentiation Proteins.

Atopic dermatitis (AD) is a common T-helper 2 (Th2) lymphocyte-mediated chronic inflammatory skin disease characterized by disturbed epidermal differentiation (e.g., filaggrin (FLG) expression) and diminished skin barrier function. Therapeutics targeting the aryl hydrocarbon receptor (AHR), such as coal tar and tapinarof, are effective in AD, yet new receptor ligands with improved potency or bioavailability are in demand to expand the AHR-targeting therapeutic arsenal. We found that carboxamide derivatives from laquinimod, tasquinimod, and roquinimex can activate AHR signaling at low nanomolar concentrations. Tasquinimod derivative (IMA-06504) and its prodrug (IMA-07101) provided full agonist activity and were most effective to induce FLG and other epidermal differentiation proteins, and counteracted IL-4 mediated repression of terminal differentiation. Partial agonist activity by other derivatives was less efficacious. The previously reported beneficial safety profile of these novel small molecules, and the herein reported therapeutic potential of specific carboxamide derivatives, provides a solid rationale for further preclinical assertation.

The aryl hydrocarbon receptor at the forefront of host-microbe interactions in the skin: A perspective on current knowledge gaps and directions for future research and therapeutic applications.

The skin is home to a community of skin microbiota including bacteria, viruses and fungi, which are widely accepted to be of importance for skin homeostasis but also associated with skin diseases. Detailed knowledge on the skin microbiota composition and its changes in a number of skin diseases is available. Yet, specific interactions between microbes and the host skin cells or how they communicate with each other are less well understood. To identify, understand and eventually therapeutically exploit causal relationships of microbial dysbiosis with disease, studies are required that address the receptors and mediators involved in host-microbe interactions. In this perspective article, we provide an outlook on one of such receptors, namely the aryl hydrocarbon receptor (AHR). The AHR is well known for being a ligand-activated transcription factor regulating the proliferation, differentiation and function of many cell types present in the skin. Its targeting by anti-inflammatory therapeutics such as coal tar and Tapinarof is effective in atopic dermatitis and psoriasis. AHR signalling is activated upon binding of wide variety of small chemicals or ligands, including microbiota-derived metabolites. New evidence has emerged pointing towards a key role for epidermal AHR signalling through skin microbiota-derived metabolites. In response, AHR-driven expression of antimicrobial peptides and stratum corneum formation may alter the skin microbiota composition. This a self-perpetuating feedback loop calls for novel therapeutic intervention strategies for which we herein discuss the requirements in future mechanistic studies.

Terminal keratinocyte differentiation in vitro is associated with a stable DNA methylome.

The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate-whether to remain proliferative or terminally differentiate-is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome-wide MethylationEPIC beadchip array measuring approximately 850 000 probes combined with RNA sequencing of in vitro cultured non-differentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes. Moreover, only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. Although TRIM29 knock-down resulted in lower expression levels of terminal differentiation genes, these changes were minor. From these results, we conclude that-in our in vitro experimental setup-it is unlikely that changes in DNA methylation have an important regulatory role in terminal keratinocyte differentiation.

The power and potential of BIOMAP to elucidate host-microbiome interplay in skin inflammatory diseases.

The two most common chronic inflammatory skin diseases are atopic dermatitis (AD) and psoriasis. The underpinnings of the remarkable degree of clinical heterogeneity of AD and psoriasis are poorly understood and, as a consequence, disease onset and progression are unpredictable and the optimal type and time point for intervention are as yet unknown. The BIOMAP project is the first IMI (Innovative Medicines Initiative) project dedicated to investigating the causes and mechanisms of AD and psoriasis and to identify potential biomarkers responsible for the variation in disease outcome. The consortium includes 7 large pharmaceutical companies and 25 non-industry partners including academia. Since there is mounting evidence supporting an important role for microbial exposures and our microbiota as factors mediating immune polarization and AD and psoriasis pathogenesis, an entire work package is dedicated to the investigation of skin and gut microbiome linked to AD or psoriasis. The large collaborative BIOMAP project will enable the integration of patient cohorts, data and knowledge in unprecedented proportions. The project has a unique opportunity with a potential to bridge and fill the gaps between current problems and solutions. This review highlights the power and potential of the BIOMAP project in the investigation of microbe-host interplay in AD and psoriasis.

Skin Surface Protein Detection by Transdermal Analysis Patches in Pediatric Psoriasis.

INTRODUCTION: Transdermal analysis patches (TAPs) noninvasively measure soluble proteins in the stratum corneum. Ultimately, such local protein profiles could benefit the search for biomarkers to improve personalized treatment in psoriasis. This study aimed to explore the patient friendliness and protein detection by TAP in pediatric psoriasis in daily clinical practice. METHODS: In this observational study, TAPs measuring CXC chemokine ligand (CXCL)-1/2, CC chemokine ligand (CCL)-27, interleukin (IL)-1RA, IL-23, IL-1α, IL-8, IL-4, IL-22, IL-17A, vascular endothelial growth factor (VEGF), human beta-defensin (hBD)-2, hBD-1, and kallikrein-related peptidase (KLK)-5 were applied on lesional, peri-lesional, and non-lesional skin sites of psoriasis patients aged >5 to <18 years. Discomfort during TAP removal as an indicator for patient friendliness was assessed by visual analogue scale (VAS; range 0-10). RESULTS: Thirty-two patients (median age 14.0 years) were included, of which 19 were treated with solely topical agents and 13 with systemic treatment. The median VAS of discomfort during TAP removal was 1.0 (interquartile range 1.0). Significantly higher levels in lesional versus non-lesional skin were found for IL-1RA, VEGF, CXCL-1/2, hBD-2, and IL-8, whereas lower levels were found for IL-1α. Skin surface proteins were measured in both treatment groups, with significant higher lesional levels of KLK-5, IL-1RA, hBD-2, IL-1α, IL-23, and CCL-27 in the systemic treatment group. CONCLUSION: The TAP platform holds the potential for patient-friendly and noninvasive monitoring of skin-derived proteins in pediatric psoriasis patients in daily clinical practice.

Know your enemy: Unexpected, pervasive and persistent viral and bacterial contamination of primary cell cultures.

In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination.

Deficiency of the human cysteine protease inhibitor cystatin M/E causes hypotrichosis and dry skin.

PURPOSE: We aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. METHODS: Exome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. RESULTS: We identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. CONCLUSION: The herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.

Pharmacologic inhibition of hypoxia-inducible factor (HIF)-hydroxylases ameliorates allergic contact dermatitis.

BACKGROUND: When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway. METHODS: Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development. RESULTS: Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway. CONCLUSION: These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.

Past, present and future of in vitro 3D reconstructed inflammatory skin models to study psoriasis.

Psoriasis is a common chronic inflammatory skin disease with a significant socio-economic impact that can greatly affect the patients' quality of life. The prevailing dogma in the aetiology and pathophysiology of this complex disease is that skin cells, immune cells and environmental factors contribute to psoriatic skin inflammation. For a better understanding of the disease pathogenesis, models are required that mimic the disease and which can be used to develop therapeutics. Over the last decades, in vitro human reconstructed skin models have been widely used in dermatological research and have also been developed to mimic psoriatic skin. This viewpoint summarizes the most commonly used in vitro models and the latest accomplishments for the combination of the dermal and epidermal compartments with other cell types and factors that are important players in the psoriatic skin environment. We aim to critically list the most complete and best-validated models that include major psoriasis hallmarks with regard to gene and protein expression profile and epidermal morphology, but also discuss the shortcoming of the current models. This viewpoint intends to guide the development of in vitro 3D skin models that faithfully mimic all features of psoriatic skin. Such model will enable fundamental biological studies for a better understanding of the aetiology and pathophysiology of psoriasis and aid in novel therapeutic target identification and drug development studies.

3D skin models for 3R research: The potential of 3D reconstructed skin models to study skin barrier function.

The skin barrier is an important shield regulating the outside-in as well as inside-out penetration of water, nutrients, ions and environmental stimuli. We can distinguish four different barrier compartments: the physical, chemical, immunological and microbial skin barrier. Well-functioning of those is needed to protect our body from the environment. To better understand the function and the contribution of barrier dysfunction in skin diseases, 3D skin or epidermal models are a valuable tool for in vitro studies. In this review, we summarize the development and application of different skin models in skin barrier research. During the last years, enormous effort was made on optimizing these models to better mimic the in vivo composition of the skin, by fine-tuning cell culture media, culture conditions and including additional cells and tissue components. Thereby, in vitro barrier formation and function has been improved significantly. Moreover, in this review we point towards changes and chances for in vitro 3D skin models to be used for skin barrier research in the nearby future.

The Effects of Human Beta-Defensins on Skin Cells in vitro.

BACKGROUND: Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing. OBJECTIVE: We aimed to investigate the effects of human ß-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration. METHODS: Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation. RESULTS: We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction. CONCLUSION: Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining.

The "Alarmins" HMBG1 and IL-33 Downregulate Structural Skin Barrier Proteins and Impair Epidermal Growth.

The epidermal-derived "alarmins" high-mobility group box 1 (HMGB1) protein and interleukin-33 (IL-33) are upregulated in patients with atopic dermatitis. How-ever, their capacity as pro-inflammatory cytokines and their derived effects on skin barrier regulation are poorly elucidated. We investigated the impact of HMGB1 and IL-33 on gene transcription, protein expression and epidermal differentiation across 3 distinct keratinocyte in vitro models. Primary keratinocytes from healthy donors were used in submerged monolayer cultures, 3D human epidermis equivalents and 3D human skin equivalents. All keratinocyte models underwent 96-h stimulation with HMGB1 (100 µM) or IL-33 (100 ng/ml) using IL-4 (50 ng/ml) as positive control of regulation and vehicle as negative control. We found that HMGB1 and IL-33 downregulated transcription of several genes from members of the epidermal differentiation complex, including filaggrin. Furthermore, HMGB1 downregulated the expression of the encoded proteins in the upper epidermis. Finally, IL-33 and HMGB1 ultimately led to impaired epidermal growth and maturation. In conclusion, HMGB1 and IL-33 could play a significant role in the atopic dermatitis pathophysiology due to negative regulation of structural proteins, stratum corneum formation and epidermal growth.

APR-246/PRIMA-1(MET) rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations.

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models.

Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair.

The Aryl Hydrocarbon Receptor Regulates Epidermal Differentiation through Transient Activation of TFAP2A.

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as an indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multiomics analyses using human skin keratinocytes revealed that upon ligand activation, AHR binds open chromatin to induce expression of transcription factors, for example, TFAP2A, as a swift response to environmental stimuli. The terminal differentiation program, including upregulation of barrier genes, FLG and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases.