Hanna Niehues

Transcription factor p63 bookmarks and regulates dynamic enhancers during epidermal differentiation

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.

Gram-positive anaerobe cocci are underrepresented in the microbiome of filaggrin-deficient human skin

Mutations in the filaggrin gene, which cause the skin disease ichthyosis vulgaris and are a genetic risk factor for atopic dermatitis, alter the cutaneous microbiome thereby affecting keratinocyte host defense responses following skin barrier disruption

Epidermal equivalents of filaggrin null keratinocytes do not show impaired skin barrier function.

To the Editor: The discovery that null alleles of the filaggrin (FLG) gene are a strong genetic risk factor for atopic dermatitis (AD) has caused a paradigm shift in understanding the etiology of this disease. FLG-deficient mouse models showed barrier impairment and enhanced percutaneous allergen sensitization, exemplifying the potential functional consequences of insufficient FLG expression. Both in patients with AD carrying FLG mutations and in FLG-proficient patients, increased transepidermal water loss (TEWL) and skin permeability were noted in nonlesional skin, suggesting that skin barrier abnormalities are a general phenomenon in AD, not necessarily restricted to FLGmutations. Others reported a mild increase in TEWL in patients with ichthyosis vulgaris (IV) carrying 2 FLG null alleles, but failed to demonstrate increased TEWL in heterozygous carriers of an FLG null allele. It could be argued that in vivo there may be confounding factors such as concomitant subclinical inflammation of the apparently normal skin that would obscure the effect of FLG deficiency on barrier function per se. For this reason, 3-dimensional (3D) skin models represent excellent models to investigate skin barrier function in a well-controlled setting. In vitro studies using various keratinocyte sources, 3D models, FLG gene knockdown approaches, and barrier assays have been published (summarized in Table E1 in this article’s Online Repository at www.jacionline. org). Because of different experimental procedures, these studies appear contradictory and are difficult to interpret. None of them used genetically definedFLG null (FLG) orFLG keratinocytes, but all relied on gene knockdown approaches to lower FLG expression levels. In this study, we have used human epidermal equivalents (HEEs) generated from FLG null keratinocytes derived from patients with IV to study the effect on epidermal differentiation and barrier function, without confounding factors. We analyzed the outside-in and inside-out barrier of these equivalents using low molecular weight tracers as previously used in other studies. Remarkably, we did not observe altered skin barrier function in HEEs of completely FLG-deficient keratinocytes. All procedures for cell culture, cytokine stimulation, analysis of gene and protein expression, and epidermal barrier function were performed as described in this article’s Methods section in the Online Repository at www.jacionline.org. Experiments were performed on fully differentiated HEEs expressing all markers of normal skin (see Fig E1 in this article’s Online Repository at www.jacionline.org). For the FLG HEEs, we used keratinocytes isolated from patients with IV (N5 5) which carry the 2 most frequent mutations of the FLG gene, leading to complete absence of FLG protein as verified by immunohistochemistry (see Fig E2 in this article’s Online Repository at www.jacionline.org). Healthy volunteer keratinocytes (N 5 6) were used as control (FLG). We also considered the

Psoriasis-Associated Late Cornified Envelope (LCE) Proteins Have Antibacterial Activity

Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.

Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models

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.

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

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

Late cornified envelope (LCE) proteins: distinct expression patterns of LCE2 and LCE3 members suggest nonredundant roles in human epidermis and other epithelia

Deletion of the late cornified envelope (LCE) proteins LCE3B and LCE3C is a strong and widely replicated psoriasis risk factor. It is amenable to biological analysis because it precludes the expression of two epidermis‐specific proteins, rather than being a single‐nucleotide polymorphism of uncertain significance. The biology of the 18‐member LCE family of highly homologous proteins has remained largely unexplored so far.

Antibiotics in cell culture: friend or foe? Suppression of keratinocyte growth and differentiation in monolayer cultures and 3D skin models

keratinocyte growth and differentiation in monolayer cultures and 3D skin models Uffe H. Nygaard*, Hanna Niehues*, Gijs Rikken, Diana Rodijk-Olthuis, Joost Schalkwijk and Ellen H. van den Bogaard Department of Dermatology and Venereology, Aarhus University Hospital, Aarhus, Denmark; Department of Clinical medicine, Aarhus University, Aarhus C, Denmark; Department of Dermatology, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Nijmegen, The Netherlands Correspondence: Ellen H. van den Bogaard, Department of Dermatology, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands, Tel.: +31 (0) 24 3617245; Fax: +31 (0) 24 3541184; e-mail: ellen.vandenbogaard@radboudumc.nl *Contributed equally to this manuscript.