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.

Novel methodologies for host-microbe interactions and microbiome-targeted therapeutics in 3D organotypic skin models.

BACKGROUND: Following descriptive studies on skin microbiota in health and disease, mechanistic studies on the interplay between skin and microbes are on the rise, for which experimental models are in great demand. Here, we present a novel methodology for microbial colonization of organotypic skin and analysis thereof. RESULTS: An inoculation device ensured a standardized application area on the stratum corneum and a homogenous distribution of bacteria, while preventing infection of the basolateral culture medium even during prolonged culture periods for up to 2 weeks at a specific culture temperature and humidity. Hereby, host-microbe interactions and antibiotic interventions could be studied, revealing diverse host responses to various skin-related bacteria and pathogens. CONCLUSIONS: Our methodology is easily transferable to a wide variety of organotypic skin or mucosal models and different microbes at every cell culture facility at low costs. We envision that this study will kick-start skin microbiome studies using human organotypic skin cultures, providing a powerful alternative to experimental animal models in pre-clinical research. Video Abstract.

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 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. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin 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 novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases.

Gram-positive anaerobic cocci guard skin homeostasis by regulating host-defense mechanisms.

In atopic dermatitis (AD), chronic skin inflammation is associated with skin barrier defects and skin microbiome dysbiosis including a lower abundance of Gram-positive anaerobic cocci (GPACs). We here report that, through secreted soluble factors, GPAC rapidly and directly induced epidermal host-defense molecules in cultured human keratinocytes and indirectly via immune-cell activation and cytokines derived thereof. Host-derived antimicrobial peptides known to limit the growth of Staphylococcus aureus-a skin pathogen involved in AD pathology-were strongly upregulated by GPAC-induced signaling through aryl hydrocarbon receptor (AHR)-independent mechanisms, with a concomitant AHR-dependent induction of epidermal differentiation genes and control of pro-inflammatory gene expression in organotypic human epidermis. By these modes of operandi, GPAC may act as an "alarm signal" and protect the skin from pathogenic colonization and infection in the event of skin barrier disruption. Fostering growth or survival of GPAC may be starting point for microbiome-targeted therapeutics in AD.

Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes.

Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease.

CYSRT1: An Antimicrobial Epidermal Protein that Can Interact with Late Cornified Envelope Proteins.

Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced in vivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire.

Influence of FLG loss-of-function mutations in host-microbe interactions during atopic skin inflammation.

BACKGROUND: Loss-of-function mutations in the filaggrin (FLG) gene directly alter skin barrier function and critically influence atopic inflammation. While skin barrier dysfunction, Th2-associated inflammation and bacterial dysbiosis are well-known characteristics of atopic dermatitis (AD), the mechanisms interconnecting genotype, transcriptome and microbiome remain largely elusive. OBJECTIVE: In-depth analysis of FLG genotype-associated skin gene expression alterations and host-microbe interactions in AD. METHODS: Multi-omics characterization of a cohort of AD patients carrying heterozygous loss-of-function mutations in the FLG gene (ADMut) (n = 15), along with matched wild-type (ADWt) patients and healthy controls. Detailed clinical characterization, microarray gene expression and 16 S rRNA-based microbial marker gene data were generated and analyzed. RESULTS: In the context of filaggrin dysfunction, the transcriptome was characterized by dysregulation of barrier function and water homeostasis, while the lesional skin of ADWt demonstrated the specific upregulation of pro-inflammatory cytokines and T-cell proliferation. S. aureus dominated the microbiome in both patient groups, however, shifting microbial communities could be observed when comparing healthy with non-lesional ADWt or ADMut skin, offering the opportunity to identify microbe-associated transcriptomic signatures. Moreover, an AD core signature with 28 genes, including CCL13, CCL18, BTC, SCIN, RAB31 and PCLO was identified. CONCLUSIONS: Our integrative approach provides molecular insights for the concept that FLG loss-of-function mutations are a genetic shortcut to atopic inflammation and unravels the complex interplay between genotype, transcriptome and microbiome in the human holobiont.

Identification of Keratinocyte Mitogens: Implications for Hyperproliferation in Psoriasis and Atopic Dermatitis.

Psoriasis and atopic dermatitis are chronic inflammatory skin diseases characterized by keratinocyte (KC) hyperproliferation and epidermal acanthosis (hyperplasia). The milieu of disease-associated cytokines and soluble factors is considered a mitogenic factor; however, pinpointing the exact mitogens in this complex microenvironment is challenging. We employed organotypic human epidermal equivalents, faithfully mimicking native epidermal proliferation and stratification, to evaluate the proliferative effects of a broad panel of (literature-based) potential mitogens. The KC GF molecule, the T-helper 2 cytokines IL-4 and IL-13, and the psoriasis-associated cytokine IL-17A caused acanthosis by hyperplasia through a doubling in the number of proliferating KCs. In contrast, IFN-γ lowered proliferation, whereas IL-6, IL-20, IL-22, and oncostatin M induced acanthosis not by hyperproliferation but by hypertrophy. The T-helper 2‒cytokine‒mediated hyperproliferation was Jak/signal transducer and activator of transcription 3 dependent, whereas IL-17A and KC GF induced MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase‒dependent proliferation. This discovery that key regulators in atopic dermatitis and psoriasis are direct KC mitogens not only adds evidence to their crucial role in the pathophysiological processes but also highlights an additional therapeutic pillar for the mode of action of targeting biologicals (e.g., dupilumab) or small-molecule drugs (e.g., tofacitinib) by the normalization of KC turnover within the epidermal compartment.

CRISPR-Cas9‒Based Genomic Engineering in Keratinocytes: From Technology to Application.

CRISPR-Cas9 is the most straightforward genome-editing tool to date. However, its implementation across disciplines is hampered by variable genome-editing efficiencies, reduced cell viability, and low success rates in obtaining clonal cell lines. This review aims to recognize all CRISPR-Cas9‒related work within the experimental dermatology field to identify key factors for successful strategies in the different keratinocyte (KC) cell sources available. On the basis of these findings, we conclude that most groups use immortalized KCs for generating knockout KCs. Our critical considerations for future studies using CRISPR-Cas9, both for fundamental and clinical applications, may guide implementation strategies of CRISPR-Cas9 technologies in the (experimental) dermatology field.

Antimicrobial Late Cornified Envelope Proteins: The Psoriasis Risk Factor Deletion of LCE3B/C Genes Affects Microbiota Composition.

Late cornified envelope proteins are predominantly expressed in the skin and other cornified epithelia. On the basis of sequence similarity, this 18-member homologous gene family has been subdivided into six groups. The LCE3 proteins have been the focus of dermatological research because the combined deletion of LCE3B and LCE3C genes (LCE3B/C-del) is a risk factor for psoriasis. We previously reported that LCE3B/C-del increases the expression of the LCE3A gene and that LCE3 proteins exert antibacterial activity. In this study, we analyzed the antimicrobial properties of other family members and the role of LCE3B/C-del in the modulation of microbiota composition of the skin and oral cavity. Differences in killing efficiency and specificity between the late cornified envelope proteins and their target microbes were found, and the amino acid content rather than the order of the well-conserved central domain of the LCE3A protein was found responsible for its antibacterial activity. In vivo, LCE3B/C-del correlated with a higher beta-diversity in the skin and oral microbiota. From these results, we conclude that all late cornified envelope proteins possess antimicrobial activity. Tissue-specific and genotype-dependent antimicrobial protein profiles impact skin and oral microbiota composition, which could direct toward LCE3B/C-del‒associated dysbiosis and a possible role for microbiota in the pathophysiology of psoriasis.

Vesicular hand eczema transcriptome analysis provides insights into its pathophysiology.

Hand eczema is a common inflammatory skin condition of the hands whose pathogenesis is largely unknown. More insight and knowledge of the disease on a more fundamental level might lead to a better understanding of the biological processes involved, which could provide possible new treatment strategies. We aimed to profile the transcriptome of lesional palmar epidermal skin of patients suffering from vesicular hand eczema using RNA-sequencing. RNA-sequencing was performed to identify differentially expressed genes in lesional vs. non-lesional palmar epidermal skin from a group of patients with vesicular hand eczema compared to healthy controls. Comprehensive real-time quantitative PCR analyses and immunohistochemistry were used for validation of candidate genes and protein profiles for vesicular hand eczema. Overall, a significant and high expression of genes/proteins involved in keratinocyte host defense and inflammation was found in lesional skin. Furthermore, we detected several molecules, both up or downregulated in lesional skin, which are involved in epidermal differentiation. Immune signalling genes were found to be upregulated in lesional skin, albeit with relatively low expression levels. Non-lesional patient skin showed no significant differences compared to healthy control skin. Lesional vesicular hand eczema skin shows a distinct expression profile compared to non-lesional skin and healthy control skin. Notably, the overall results indicate a large overlap between vesicular hand eczema and earlier reported atopic dermatitis lesional transcriptome profiles, which suggests that treatments for atopic dermatitis could also be effective in (vesicular) hand eczema.

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.

Skin microbiota in health and disease: From sequencing to biology.

Microbiota live in a closely regulated interaction with their environment, and vice versa. The presence and absence of microbial entities is greatly influenced by features of the niche in which they thrive. Characteristic of this phenomenon is that different human skin sites harbor niche-specific communities of microbes. Microbial diversity is considerable, and the current challenge lies in determining which microbes and (corresponding) functionality are of importance to a given ecological niche. Furthermore, as there is increasing evidence of microbial involvement in health and disease, the need arises to fundamentally understand microbiome processes for application in health care, nutrition and personal care products (e.g. diet, cosmetics, probiotics). This review provides a current overview of state-of-the-art sequencing-based techniques and corresponding data analysis methodology for profiling of complex microbial communities. Furthermore, we also summarize the existing knowledge regarding cutaneous microbiota and their human host for a wide range of skin diseases.

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.

Targeting the Cutaneous Microbiota in Atopic Dermatitis by Coal Tar via AHR-Dependent Induction of Antimicrobial Peptides.

Skin colonization by Staphylococcus aureus and its relative abundance is associated with atopic dermatitis (AD) disease severity and treatment response. Low levels of antimicrobial peptides in AD skin may be related to the microbial dysbiosis. Therapeutic targeting of the skin microbiome and antimicrobial peptide expression can, therefore, restore skin homeostasis and combat AD. In this study, we analyzed the cutaneous microbiome composition in 7 patients with AD and 10 healthy volunteers upon topical coal tar or vehicle treatment. We implemented and validated a Staphylococcus-specific single-locus sequence typing approach combined with classic 16S ribosomal RNA marker gene sequencing to study the bacterial composition. During coal tar treatment, Staphylococcus abundance decreased, and Propionibacterium abundance increased, suggesting a shift of the microbiota composition toward that of healthy controls. We, furthermore, identified a hitherto unknown therapeutic mode of action of coal tar, namely the induction of keratinocyte-derived antimicrobial peptides via activation of the aryl hydrocarbon receptor. Restoring antimicrobial peptide levels in AD skin via aryl hydrocarbon receptor-dependent transcription regulation can be beneficial by creating a (anti)microbial milieu that is less prone to infection and inflammation. This underscores the importance of coal tar in the therapeutic aryl hydrocarbon receptor armamentarium and highlights the aryl hydrocarbon receptor as a target for drug development.

A generic workflow for Single Locus Sequence Typing (SLST) design and subspecies characterization of microbiota.

We present TaxPhlAn, a new method and bioinformatics pipeline for design and analysis of single-locus sequence typing (SLST) markers to type and profile bacteria beyond the species-level in a complex microbial community background. TaxPhlAn can be applied to any group of phylogenetically-related bacteria, provided reference genomes are available. As TaxPhlAn requires the SLST targets identified to fit the phylogenetic pattern as determined through comprehensive evolutionary reconstruction of input genomes, TaxPhlAn allows for the identification and phylogenetic inference of new biodiversity. Here, we present a clinically relevant case study of high-resolution Staphylococcus profiling on skin of atopic dermatitis (AD) patients. We demonstrate that SLST enables profiling of cutaneous Staphylococcus members at (sub)species level and provides higher resolution than current 16S-based techniques. With the higher discriminative ability provided by our approach, we further show that the presence of Staphylococcus capitis on the skin together with Staphylococcus aureus associates with AD disease.

Microbe-host interplay in atopic dermatitis and psoriasis.

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

Stable pantothenamide bioisosteres: novel antibiotics for Gram-positive bacteria.

The emergence of multidrug resistant bacteria has prioritized the development of new antibiotics. N-substituted pantothenamides, analogs of the natural compound pantetheine, were reported to target bacterial coenzyme A biosynthesis, but these compounds have never reached the clinic due to their instability in biological fluids. Plasma-stable pantothenamide analogs could overcome these issues. We first synthesized a number of bioisosteres of the prototypic pantothenamide N7-Pan. A compound with an inverted amide bond (CXP18.6-012) was found to provide plasma-stability with minimal loss of activity compared to the parent compound N7-Pan. Next, we synthesized inverted pantothenamides with a large variety of side chains. Among these we identified a number of novel stable inverted pantothenamides with selective activity against Gram-positive bacteria such as staphylococci and streptococci, at low micromolar concentrations. These data provide future direction for the development of pantothenamides with clinical potential.