The transcription factor p63 plays a pivotal role in keratinocyte proliferation and differentiation in the epidermis. However, how p63 regulates epidermal genes during differentiation is not yet ...clear. Using epigenome profiling of differentiating human primary epidermal keratinocytes, we characterized a catalog of dynamically regulated genes and p63‐bound regulatory elements that are relevant for epithelial development and related diseases. p63‐bound regulatory elements occur as single or clustered enhancers, and remarkably, only a subset is active as defined by the co‐presence of the active enhancer mark histone modification H3K27ac in epidermal keratinocytes. We show that the dynamics of gene expression correlates with the activity of p63‐bound enhancers rather than with p63 binding itself. The activity of p63‐bound enhancers is likely determined by other transcription factors that cooperate with p63. Our data show that inactive p63‐bound enhancers in epidermal keratinocytes may be active during the development of other epithelial‐related structures such as limbs and suggest that p63 bookmarks genomic loci during the commitment of the epithelial lineage and regulates genes through temporal‐ and spatial‐specific active enhancers.
Synopsis
The key epithelial transcription factor p63 functions as a placeholder to bookmark genomic loci for gene regulation in epithelial development. The data suggest that different sets of co‐regulators are required to activate proliferation and differentiation genes during epidermal differentiation and developmental genes in other epithelial tissues.
We report p63‐regulated gene expression dynamics during epidermal differentiation.
Gene expression correlates with the activity of p63‐dependent enhancers rather than p63 binding itself.
Co‐regulators of p63 are required during epidermal differentiation and the development of other epithelial tissues.
The key epithelial transcription factor p63 functions as a placeholder to bookmark genomic loci for gene regulation in epithelial development. The data suggest that different sets of co‐regulators are required to activate proliferation and differentiation genes during epidermal differentiation and developmental genes in other epithelial tissues.
The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and ...diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.
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.
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BACKGROUND: Recent advances in sequencing technologies have enabled metagenomic analyses of many human body sites. Several studies have catalogued the composition of bacterial communities of the ...surface of human skin, mostly under static conditions in healthy volunteers. Skin injury will disturb the cutaneous homeostasis of the host tissue and its commensal microbiota, but the dynamics of this process have not been studied before. Here we analyzed the microbiota of the surface layer and the deeper layers of the stratum corneum of normal skin, and we investigated the dynamics of recolonization of skin microbiota following skin barrier disruption by tape stripping as a model of superficial injury. RESULTS: We observed gender differences in microbiota composition and showed that bacteria are not uniformly distributed in the stratum corneum. Phylogenetic distance analysis was employed to follow microbiota development during recolonization of injured skin. Surprisingly, the developing neo-microbiome at day 14 was more similar to that of the deeper stratum corneum layers than to the initial surface microbiome. In addition, we also observed variation in the host response towards superficial injury as assessed by the induction of antimicrobial protein expression in epidermal keratinocytes. CONCLUSIONS: We suggest that the microbiome of the deeper layers, rather than that of the superficial skin layer, may be regarded as the host indigenous microbiome. Characterization of the skin microbiome under dynamic conditions, and the ensuing response of the microbial community and host tissue, will shed further light on the complex interaction between resident bacteria and epidermis.
Schnitzler's syndrome is a chronic disabling autoinflammatory disorder, characterised by chronic urticaria, paraproteinemia and systemic inflammation. The interleukin (IL) 1 receptor antagonist ...anakinra is a very effective treatment, but requires daily injection and blocks both IL-1α and IL-1β. Canakinumab is a selective human monoclonal anti-IL-1β antibody with a long half-life. We investigated the long-term efficacy and safety of canakinumab in Schnitzler's syndrome.
In an open-label, single-treatment arm trial, eight patients with Schnitzler's syndrome received monthly injections with 150 mg canakinumab subcutaneously for 6 months, followed by a 3-month observation period. Primary outcome was complete or clinical remission at day 14. Secondary outcome measures included inflammatory markers, quality of life, time to relapse, safety and tolerability.
After stopping anakinra, patients developed moderate to severe clinical symptoms. Canakinumab induced complete or clinical remission at day 14 in all eight patients. Median C-reactive protein concentrations decreased from 169 mg/l at baseline to less than 10 mg/l on day 14 and remained low or undetectable. One patient discontinued participation on day 39 because of return of symptoms while all others remained in complete or clinical remission during the 6-month treatment period. Relapse after last canakinumab dose occurred within 3 months in four patients. For two patients, remission continued several months post-study. Five patients reported at least one adverse event, predominantly mild upper respiratory tract infections. One patient died in a traffic accident.
In this 9-month study, monthly 150 mg canakinumab injection was an effective and well-tolerated treatment for Schnitzler's syndrome. Our data demonstrate that IL-1β plays a pivotal role in this disease. CLINICALTRIALS.GOV: NCT01276522.
Abstract An important issue in tissue engineering is the vascularisation of the implanted construct, which often takes several weeks. In vivo, the growth factors VEGF and FGF2 show a combined effect ...on both angiogenesis and maturation of blood vessels. Therefore, we hypothesise that the addition of these growth factors to an acellular construct increases blood vessel formation and maturation. To systematically evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, five porous scaffolds were prepared and characterised, viz.: collagen, collagen with heparin, and collagen with heparin plus one or two growth factors (rrFGF2 and rrVEGF). Scaffolds were subcutaneously implanted in 3 months old Wistar rats. Of all scaffolds tested, the one with a combination of growth factors displayed the highest density of blood vessels (type IV collagen) and most mature blood vessels (smooth muscle actin). In addition, no hypoxic cells were found in this scaffold at day 7 and 21 (hypoxia inducible factor 1- α ). These results indicate that the addition of both FGF2 and VEGF to an acellular construct enhances an early mature vasculature. This opens prospects for (acellular) tissue-engineered constructs in conditions as ischaemic heart disease or diabetic ulcers.
The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this ...study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC–MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC–MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies.
Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in ...keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr−/− and Ahr+/+ murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr−/− keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr+/+ keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression, and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology.
No NLRP3 genetic variants were found in any of the analyzed cell types derived from 2 patients with Schnitzler syndrome without NLRP3 variants in whole blood (Table I). Because we have previously ...demonstrated that IL-1β is a pivotal mediator of the clinical signs of Schnitzler syndrome,2,5 we proceeded to investigate the functional consequences of the NLRP3 mosaic mutations on IL-1β and IL-6 production in PBMCs (see the Methods section in this article's Online Repository at www.jacionline.org). The identified variant is considered a benign polymorphism.E5 In patient 7 we detected a known missense mutation in the NACHT domain of the NLRP3 gene (c.1569C>G; p.F523L), which was previously reported as disease causing in 2 infants with NOMID/CINCA, a severe early-onset form of CAPS.E6Canonical SS, Variants located in a canonical splice site; Coding+SS, all variants located either in exonic regions or canonical splice sites; Indels, Insertions or deletions; Known variant (HGMD), variants described as pathogenic in the Human Gene Mutation Database (www.hgmd.org); Missense (phyloP >2.5),E4 highly conserved missense variants with a phyloP >2.5; Nonsense, nonsense variants; Nonsynonymous, variants leading to an amino acid change; Not seen before (in house), variants that have not been detected in 672 in-house sequenced whole exomes; Patient, patient who has undergone whole-exome sequencing; Total no. of variants, all variants detected by using whole-exome sequencing.
Psoriasis was until recently regarded as a T-cell-driven disease with presumed (auto)immune mechanisms as its primary cause. This view was supported by clinical data and genetic studies that ...identified risk factors functioning in adaptive and innate immunity, such as HLA-C*06, ERAP1, the IL-23 pathway, and NF-κB signaling. Candidate gene approaches and genome-wide association studies, however, have identified copy number polymorphisms of the β-defensin cluster and deletion of late cornified envelope (LCE) 3B and 3C genes (LCE3C_LCE3B-del) as psoriasis risk factors. As these genes are expressed in epithelial cells and not by the immune system, these findings may cause a change of paradigm for psoriasis, not unlike the reported filaggrin association that has profoundly changed the views on atopic dermatitis. In addition to genetic polymorphisms of the immune system, genetic variations affecting the skin barrier are likely to contribute to psoriasis. Recent studies have shown epistatic interactions involving HLA-C*06, ERAP1, and LCE3C_LCE3B-del, which makes psoriasis a unique model to investigate genetic and biological interactions of associated genes in a complex disease. We present a model for disease initiation and perpetuation, which integrates the available genetic, immunobiological, and clinical data.
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