CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Blockade of IL-13 signaling improves skin barrier function and biology in patients with moderate to severe atopic dermatitis.

BACKGROUND: Interleukin (IL)-13 is a key driver of inflammation and barrier dysfunction in atopic dermatitis (AD). While there is robust evidence that tralokinumab, a monoclonal antibody neutralizing IL-13, reduces inflammation and clinical disease activity, less is known about its effects on barrier function. OBJECTIVES: To characterize effects of tralokinumab treatment on skin barrier function. METHODS: Transepidermal water loss (TEWL), stratum corneum hydration (SCH), natural moisturizing factor (NMF) content, histopathological characteristics, biomarker expression and microbiome composition were evaluated in lesional, non-lesional, and sodium lauryl sulfate (SLS)-irritated skin of 16 AD patients over the course of 16 weeks of tralokinumab treatment. RESULTS: All clinical severity scores decreased significantly over time. At week 16, mean TEWL in target lesions decreased by 32.66% (p = 0.01), and SCH increased by 58.44% (p = 0.004), along with histological reduction in spongiosis (p = 0.003), keratin 16 expression and epidermal thickness (p = 0.001). In parallel, there was a significant decrease in several barrier dysfunction-associated and pro-inflammatory proteins such as fibronectin (p = 0.006), CCL17/TARC (p = 0.025) and IL-8 (p = 0.014), with significant changes already at week 8. Total bacterial load and Staphylococcus aureus abundance were significantly reduced from week 2. CONCLUSION: Tralokinumab treatment improves skin physiology, epidermal pathology, and dysbiosis, further highlighting the pleiotropic role of IL-13 in AD pathogenesis.

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.

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.

Duplicated Enhancer Region Increases Expression of CTSB and Segregates with Keratolytic Winter Erythema in South African and Norwegian Families.

Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals.

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.

Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.

Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of ichq (Cst6-deficient; Cst6-/-) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-Cst6)Cst6ichq/ichq mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to Cst6-/-Ctsl+/- mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-Cst6)Cst6ichq/ichq mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.

Analysis of protein-protein interaction between late cornified envelope proteins and corneodesmosin.

Deletion of two members of the late cornified envelope (LCE) family, LCE3B and LCE3C (LCE3C_LCE3B-del), has been identified as risk factor for psoriasis with a possible role in skin barrier function. Moreover, genetic interaction between LCE3C_LCE3B-del and HLA-C*06, located in the psoriasis susceptibility regions 4 and 1 (PSORS4 and 1), has been reported in several populations. Because of high linkage disequilibrium between the PSORS1 genes HLA-C*06 and corneodesmosin (CDSN), both genes are potentially involved in psoriasis. As corneodesmosin and LCE proteins are both constituents of the stratum corneum, we investigated potential direct protein-protein interactions between six LCE proteins and two corneodesmosin sequence variants. Partial colocalization of LCE2 and CDSN was observed in normal and psoriasis skin using immunofluorescence microscopy. Co-expression of eCFP-LCE and mRFP-CDSN proteins in COS-1 cells and human adult keratinocytes, and GST pull-down results did not provide evidence for direct interactions between LCE proteins and CDSN variants.

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.

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.

Lead optimization of aryl hydrocarbon receptor ligands for treatment of inflammatory skin disorders.

Therapeutic aryl hydrocarbon receptor (AHR) modulating agents gained attention in dermatology as non-steroidal anti-inflammatory drugs that improve skin barrier properties. By exploiting AHR's known ligand promiscuity, we generated novel AHR modulating agents by lead optimization of a selective AHR modulator (SAhRM; SGA360). Twenty-two newly synthesized compounds were screened yielding two novel derivatives, SGA360f and SGA388, in which agonist activity led to enhanced keratinocyte terminal differentiation. SGA388 showed the highest agonist activity with potent normalization of keratinocyte hyperproliferation, restored expression of skin barrier proteins and dampening of chemokine expression by keratinocytes upon Th2-mediated inflammation in vitro. The topical application of SGA360f and SGA388 reduced acute skin inflammation in vivo by reducing cyclooxygenase levels, resulting in less neutrophilic dermal infiltrates. The minimal induction of cytochrome P450 enzyme activity, lack of cellular toxicity and mutagenicity classifies SGA360f and SGA388 as novel potential therapeutic AHR ligands and illustrates the potential of medicinal chemistry to fine-tune AHR signaling for the development of targeted therapies in dermatology and beyond.

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.

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.

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.

Psoriasis risk genes of the late cornified envelope-3 group are distinctly expressed compared with genes of other LCE groups.

Deletion of the late cornified envelope (LCE) genes LCE3B and LCE3C has recently been identified as a risk factor for psoriasis. Expression of 16 LCE genes of LCE groups 1, 2, 3, 5, and 6 was examined in vivo and in vitro. Quantitative PCR demonstrated that moderate to high LCE expression was largely confined to skin and a few oropharyngeal tissues. Genes of the LCE3 group demonstrated increased expression in lesional psoriatic epidermis and were induced after superficial injury of normal skin, whereas expression of members of other LCE groups was down-regulated under these conditions. Immunohistochemistry and immunoelectron microscopy demonstrated that LCE2 protein expression was restricted to the uppermost granular layer and the stratum corneum. Stimulation of in vitro reconstructed skin by several psoriasis-associated cytokines resulted in induction of LCE3 members. The data suggest that LCE proteins of groups 1, 2, 5, and 6 are involved in normal skin barrier function, whereas LCE3 genes encode proteins involved in barrier repair after injury or inflammation. These findings may provide clues to the mechanistic role of LCE3B/C deletion in psoriasis.