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.

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.

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.

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.

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.

Targeting Skin Barrier Function in Atopic Dermatitis.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the general population. Skin barrier dysfunction is the central abnormality leading to AD. The cause of skin barrier dysfunction is complex and rooted in genetic mutations, interactions between the immune pathway activation and epithelial cells, altered host defense mechanisms, as well as environmental influences that cause epithelial cell activation and release of alarmins (such as thymic stromal lymphopoietin) that can activate the type 2 immune pathway, including generation of interleukins 4 and 13, which induces defects in the skin barrier and increased allergic inflammation. These inflammatory pathways are further influenced by environmental factors including the microbiome (especially Staphylococcus aureus), air pollution, stress, and other factors. As such, AD is a syndrome involving multiple phenotypes, all of which have in common skin barrier dysfunction as a key contributing factor. Understanding mechanisms leading to skin barrier dysfunction in AD is pointing to the development of new topical and systemic treatments in AD that helps keep skin borders secure and effectively treat the disease.

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 integration of large-scale public data and network analysis uncovers molecular characteristics of psoriasis.

In recent years, a growing interest in the characterization of the molecular basis of psoriasis has been observed. However, despite the availability of a large amount of molecular data, many pathogenic mechanisms of psoriasis are still poorly understood. In this study, we performed an integrated analysis of 23 public transcriptomic datasets encompassing both lesional and uninvolved skin samples from psoriasis patients. We defined comprehensive gene co-expression network models of psoriatic lesions and uninvolved skin. Moreover, we curated and exploited a wide range of functional information from multiple public sources in order to systematically annotate the inferred networks. The integrated analysis of transcriptomics data and co-expression networks highlighted genes that are frequently dysregulated and show aberrant patterns of connectivity in the psoriatic lesion compared with the unaffected skin. Our approach allowed us to also identify plausible, previously unknown, actors in the expression of the psoriasis phenotype. Finally, we characterized communities of co-expressed genes associated with relevant molecular functions and expression signatures of specific immune cell types associated with the psoriasis lesion. Overall, integrating experimental driven results with curated functional information from public repositories represents an efficient approach to empower knowledge generation about psoriasis and may be applicable to other complex diseases.

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.

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.

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.

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.

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.