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The skin microbial community is a multifunctional ecosystem aiding prevention of infections from transient pathogens, maintenance of host immune homeostasis, and skin health. A better understanding of the complex milieu of microbe-microbe and host-microbe interactions will be required to define the ecosystem’s optimal function and enable rational design of microbiome targeted interventions. Malassezia , a fungal genus currently comprising 18 species and numerous functionally distinct strains, are lipid-dependent basidiomycetous yeasts and integral components of the skin microbiome. The high proportion of Malassezia in the skin microbiome makes understanding their role in healthy and diseased skin crucial to development of functional skin health knowledge and understanding of normal, healthy skin homeostasis. Over the last decade, new tools for Malassezia culture, detection, and genetic manipulation have revealed not only the ubiquity of Malassezia on skin but new pathogenic roles in seborrheic dermatitis, psoriasis, Crohn’s disease, and pancreatic ductal carcinoma. Application of these tools continues to peel back the layers of Malassezia /skin interactions, including clear examples of pathogenicity, commensalism, and potential protective or beneficial activities creating mutualism. Our increased understanding of host- and microbe-specific interactions should lead to identification of key factors that maintain skin in a state of healthy mutualism or, in turn, initiate pathogenic changes. These approaches are leading toward development of new therapeutic targets and treatment options. This review discusses recent developments that have expanded our understanding of Malassezia ’s role in the skin microbiome, with a focus on its multiple roles in health and disease as commensal, pathogen, and protector.

A post-transcriptional switch that controls spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript is reported; these gene products—miR-198 and FSTL1—are found to have opposing functions on keratinocyte migration and wound healing. Pivotal point in wound healing Wound healing is a complicated process involving coordinated cell migration, cell proliferation and extracellular matrix remodelling in response to TGF-β and other growth factors. Prabha Sampath and colleagues report a post-transcriptional switch that controls spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript. The two have opposing effects: the secreted glycoprotein FSTL1 is necessary for wound healing and the microRNA miR-198 suppresses keratinocyte migration and is essential for epidermal homeostasis in healthy skin. In normal keratinocytes, miR-198 is predominantly expressed from the dual transcript and it in turn attenuates several keratinocyte migrating genes. After injury, TGF-β signalling switches off miR-198 expression by downregulating the RNA-binding protein KSRP, thereby promoting FSTL1 protein expression. The findings may be relevant to the pathology of chronic diabetic ulcers, in which expression of miR-198 persists. Post-transcriptional switches are flexible effectors of dynamic changes in gene expression 1 . Here we report a new post-transcriptional switch that dictates the spatiotemporal and mutually exclusive expression of two alternative gene products from a single transcript. Expression of primate-specific exonic microRNA-198 (miR-198) 2 , located in the 3′-untranslated region of follistatin-like 1 ( FSTL1 ) 3 messenger RNA, switches to expression of the linked open reading frame of FSTL1 upon wounding in a human ex vivo organ culture system. We show that binding of a KH-type splicing regulatory protein (KSRP, also known as KHSRP) to the primary transcript determines the fate of the transcript and is essential for the processing of miR-198: transforming growth factor-β signalling switches off miR-198 expression by downregulating KSRP, and promotes FSTL1 protein expression. We also show that FSTL1 expression promotes keratinocyte migration, whereas miR-198 expression has the opposite effect by targeting and inhibiting DIAPH1 , PLAU and LAMC2 . A clear inverse correlation between the expression pattern of FSTL1 (pro-migratory) and miR-198 (anti-migratory) highlights the importance of this regulatory switch in controlling context-specific gene expression to orchestrate wound re-epithelialization. The deleterious effect of failure of this switch is apparent in non-healing chronic diabetic ulcers, in which expression of miR-198 persists, FSTL1 is absent, and keratinocyte migration, re-epithelialization and wound healing all fail to occur.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Background Wound management is a critical factor when treating patients with the inherited skin fragility disease dystrophic epidermolysis bullosa (DEB). Due to genetic defects in structural proteins, skin and mucous epithelia are prone to blistering and chronic wounding upon minor trauma. Furthermore, these wounds are commonly associated with excessive pruritus and predispose to the development of life-threatening squamous cell carcinomas, underscoring the unmet need for new therapeutic options to improve wound healing in this patient cohort. Vitamin D3 is acknowledged to play an important role in wound healing by modulating different cellular processes that impact epidermal homeostasis and immune responses. In this study, we evaluate the safety and efficacy of low-dose calcipotriol, a vitamin D3 analogue, in promoting wound healing and reducing itch and pain in patients with DEB. Methods Eligible DEB patients, aged ≥ 6 years and with a known mutation in the COL7A1 gene, were recruited to a placebo-controlled, randomized, double blind, cross-over phase II monocentric clinical trial. Patients were required to have at least two wounds with a minimum size of 6 cm 2 per wound. The primary objective was to evaluate efficacy of daily topical application of a 0.05 µg/g calcipotriol ointment in reducing wound size within a 4-week treatment regimen. Secondary objectives were to assess safety, as well as the impact of treatment on pruritus, pain, and bacterial wound colonization in these patients. Results Six patients completed the clinical trial and were included into the final analysis. Topical low-dose calcipotriol treatment led to a significant reduction in wound area at day 14 compared to placebo (88.4% vs. 65.5%, P  < 0.05). Patients also reported a significant reduction of pruritus with calcipotriol ointment compared to placebo over the entire course of the treatment as shown by itch scores of 3.16 vs 4.83 ( P  <  0.05 ) and 1.83 vs 5.52 ( P  <  0.0001 ) at days 14 and 28, respectively. Treatment with low-dose calcipotriol did not affect serum calcium levels and improved the species richness of the wound microbiome, albeit with no statistical significance. Conclusions Our results show that topical treatment with low-dose calcipotriol can accelerate wound closure and significantly reduces itch, and can be considered a safe and readily-available option to improve local wound care in DEB patients. Trial Registration EudraCT: 2016–001,967-35. Registered 28 June 2016, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001967-35/AT

As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH. We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation. Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them. Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma.

Whole metagenome analysis has the potential to reveal functional triggers of skin diseases, but issues of cost, robustness and sampling efficacy have limited its application. Here, we have established an alternative, clinically practical and robust metagenomic analysis protocol and applied it to 80 skin microbiome samples epidemiologically stratified for atopic dermatitis (AD). We have identified distinct non-flare, baseline skin microbiome signatures enriched for Streptococcus and Gemella but depleted for Dermacoccus in AD-prone versus normal healthy skin. Bacterial challenge assays using keratinocytes and monocyte-derived dendritic cells established distinct IL-1-mediated, innate and Th1-mediated adaptive immune responses with Staphylococcus aureus and Staphylococcus epidermidis . Bacterial differences were complemented by perturbations in the eukaryotic community and functional shifts in the microbiome-wide gene repertoire, which could exacerbate a dry and alkaline phenotype primed for pathogen growth and inflammation in AD-susceptible skin. These findings provide insights into how the skin microbial community, skin surface microenvironment and immune system cross-modulate each other, escalating the destructive feedback cycle between them that leads to AD flare. The skin microbiome in patients prone to atopic dermatitis is enriched in Streptococcus and Gemella , and is associated with an adaptive immune response, an altered eukaryotic community and a functional shift in the microbiome-wide gene repertoire.

IntroductionAtopic eczema affects 20% of UK children, and environmental factors are important in its aetiology. Several observational studies suggest an increased risk of atopic eczema in children living in hard water areas. The Softened Water for Eczema Prevention pilot trial tests the feasibility of installing domestic ion-exchange water softeners around the time of birth to reduce the risk of atopic eczema in children with a family history of atopy. A further aim is to explore the pathophysiological mechanisms for this in an embedded mechanistic study.Methods and analysisMulticentre parallel group assessor-blinded randomised controlled pilot trial. Participants are newborn babies (n=80) living in a hard water (>250 mg/L calcium carbonate) area at risk of developing atopic eczema because of a family history of atopy. Participants will be randomised prior to birth in a 1:1 ratio. The intervention group will have an ion-exchange water softener installed prior to birth. The control group will receive their usual domestic hard water supply. Follow-up will be until 6 months of age. Data will be collected at birth (baseline), 1, 3 and 6 months of age. The main outcome is the proportion of eligible families screened who are willing and able to be randomised. Several secondary feasibility and clinical endpoints will also be evaluated, alongside mechanistic outcomes. Data will be analysed on an intention-to-treat basis. There will be no hypothesis testing for the clinical outcomes. Study acceptability will be evaluated through semistructured interviews.Ethics and disseminationThis study has been reviewed and given a favourable opinion by the North West–Liverpool East Research Ethics Committee (Ref: 17/NW/0661). The results of the study will be reported at international conferences and in peer-reviewed scientific journals. We will send participating families a summary of the pilot trial results.Trial registration number NCT03270566.

Yes-associated protein (YAP) is a mechanosensor protein and a downstream effector of the Hippo kinase pathway, which controls organ growth, cell proliferation, survival, maintenance and regeneration. Unphosphorylated YAP translocates to the nucleus where it acts as a cofactor of primarily the TEAD transcription factors to activate target gene transcription and cell proliferation. Perturbed YAP activation results in tumorigenesis. The pathways downstream of activated YAP that drive cell proliferation remain relatively unexplored. In this study, we employed YAP2-5SA-∆C transgenic mice, which overexpress a mildly activated YAP mutant protein in basal keratinocytes leading to increased proliferation of the epidermal stem/progenitor cell populations. We performed massively-parallel sequencing of skin biopsy mRNA (RNA-Seq) and found dysregulation of 1491 genes in YAP2-5SA-∆C skin, including many with roles in cell activation and proliferation. Furthermore, we found that 150 of these dysregulated genes harbored YAP/TEAD binding motifs in the 3′ UTR, suggesting that these may be direct YAP/TEAD target genes in the control of epidermal regeneration. Further validation and functional characterization assays identified Plau and Tgfbr3 as prime candidate genes that may be activated by epidermal YAP activity in the mouse skin in vivo to promote keratinocyte proliferation. This study provides novel insights into the mechanisms regulated by YAP that control tissue homeostasis, and in particular in conditions where YAP is aberrantly activated such as in neoplastic and regenerative skin disease.

Objectives Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle‐like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle‐related cells. Materials and methods Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three‐dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two‐dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. Results The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core‐shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell‐cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. Conclusions Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function. Cultures comprising of dermal papilla cells, keratinocytes, and human dermal fibroblasts were prepared from sequential seeding into hydrogel microwells, in order to reproduce the spatial orientation of hair follicular cells in vivo. This work demonstrated the importance of keratinocytes in maintaining cellular compartmentalization and dermal papilla aggregation, which is necessary for hair follicle development and function.