The human microbiome has a fundamental role in skin homeostasis, barrier function and host immune networks. Acute and chronic skin conditions are often associated with imbalances in host-microbiome-immune interactions. Despite recent advances in skin microbiome research, there are limited models for studying these interactions in healthy and diseased skin, therefore we have developed a model to investigate how different microbiome profiles alter epidermal homeostasis and T-cell responses. Full-thickness skin models were colonized with five human microbiome species:Staphylococcus epidermidis (SE), Staphylococcus capitis (SC), Cutibacterium acnes (CA), Malassezia restricta (MR) and Malassezia globosa (MG), in singular or combined (5M) inoculations.The effect of colonization on epidermal differentiation and barrier formation was observed via the expression of K14, K10, E-cadherin and Ki67. Increased expression of K14 was observed in models colonized with SE, SC (p=0.0062) and 5M. MG colonization significantly reduced K10 expression (p=0.0180). Expression of E-cadherin significantly decreased in models colonised with MR (p=0.0489). Despite significant changes in epidermal thickness, Ki67 expression was not altered across conditions. Commensal microbes have been shown to regulate epidermal barrier function via the aryl hydrocarbon receptor(AhR). Increased nuclear expression of AhR was observed in response to SC, CA and 5M, suggesting a greater role in AhR activation. Model colonization influenced the expression of CD69, cutaneous lymphocyte antigen (CLA) and HLA-DR on T cells in response to incubating PBMCs with conditioned media from inoculated models. SE caused a significant increase in CD3+/CLA+ cells (p=0.0362) and 5M significantly reduced CD3+/HLA-DR+ cells (p=0.0304). RNA-sequencing aims to support current analysis and elucidate any more targets to be explored. Our findings suggest different microbial profiles alter epidermal barrier formation and immune responses.

Commensal microbes and humans have coevolved toward a symbiotic relationship. In healthy individuals, the human microbiome has a vital role in the development of the innate and adaptive immune system, inducing protective and regulatory immunity, and the immune system maintains an optimal balance of microbes. Several acute and chronic skin conditions are a result of imbalances in the host-microbiome relationship and are accompanied by epidermal barrier disruption. This research aims to develop three-dimensional (3D) in vitro skin models integrating microbiology and immune cells to simulate this complex relationship and the effects on epidermal health. 3D models, created using the immortalised keratinocyte cell line NTERTs seeded on collagen matrices embedded with dermal fibroblasts, were colonised for 72 hrs in singular or combination inoculums of 5 prominent microbiome species: Staphylococcus epidermidis, Staphylococcus capitis, Cutibacterium acnes, Malassezia restricta and Malassezia globosa. Colonisation of 3D models showed differences in K14, K16, K10, Ki67 and involucrin expression. Epidermal thinning was observed in models colonised with singular inoculations of S. epidermidis, C. acnes and M. restricta however, restoration of epidermal thickness was observed in models colonised with the combination inoculums compared to control models. The effects of model colonisation on T cell activity and function were investigated by measuring the expression of CD69, cutaneous lymphocyte antigen (CLA) and HLA-DR in response to incubating peripheral blood mononuclear cells (PBMCs) with conditioned medium from inoculated models. PBMCs incubated with conditioned media showed increased numbers of CLA+/CD3+ T cells and HLA-DR+/CD3+ T cells, compared to those inoculated with media from sterile controls. Increased CD69+/CD3+ T cells were seen in PBMCs incubated with media from S. epidermidis, S. capitis and M. restrict. Here we demonstrate how different microbiome profiles directly alter epidermal homeostasis, skin barrier properties and immune responses.

Although human dermal white adipose tissue (DWAT) envelopes scalp hair follicles (HFs), their underlying bidirectional communication remains unexplored. Recently, we determined that culturing human HFs with surrounding DWAT promoted hair growth and pigmentation ex vivo. The current study aimed to further expand knowledge of the human DWAT-HF axis. Intriguingly, proteomic analysis identified hepatocyte growth factor (HGF) as the most abundantly-secreted DWAT product within long-term HF+DWAT ex vivo cultures. Therefore, we chose to stimulate HFs with HGF ex vivo. Both HFs cultured with DWAT, or HGF in the absence of DWAT showed a significantly increased pigment content and number of gp100+ melanocyte cells as well as maintaining higher numbers of melanocytes with three or more dendrites. These positive effects were partially ablated by a neutralizing HGF antibody added to HF+DWAT. As expected, HGF-treated HFs displayed increased proliferation and decreased apoptosis within matrix keratinocytes, effects mirrored by culturing HFs with surrounding DWAT. Transcriptomic analysis of HFs treated with HGF (6hr) revealed that HGF up-regulated PAX3 and SOX10, as well as VEGFA and WNT5B. Gene expression analysis on 24hr cultures coupled with IPA revealed that key Wnt pathway inhibitors are down-regulated in HFs treated with HGF, whereas Wnt agonists, WNT6 and WNT10B, show an upward trend for HF+HGF and HF+DWAT. Here we provide the first evidence that human dermal adipocytes communicate with adjacent scalp HFs by secreting HGF, one of the most potent promoters of hair growth and pigmentation for which the human HF epithelium and pigmentary unit express cognate receptors (cMET). This study designates human perifollicular adipocytes and HGF-dependent DWAT-HF communication an intriguing novel target for therapeutic intervention in the future management of disorders of hair growth or pigmentation.

Although human dermal white adipose tissue (DWAT) envelopes scalp hair follicles (HFs), their underlying bidirectional communication remains unexplored. Recently, we determined that culturing human HFs with surrounding DWAT promoted hair growth and pigmentation ex vivo. The current study aimed to further expand knowledge of the human DWAT-HF axis. Intriguingly, proteomic analysis identified hepatocyte growth factor (HGF) as the most abundantly-secreted DWAT product within long-term HF+DWAT ex vivo cultures. Therefore, we chose to stimulate HFs with HGF ex vivo. Both HFs cultured with DWAT, or HGF in the absence of DWAT showed a significantly increased pigment content, number of gp100+ melanocyte cells and melanocyte dendrites. These positive effects were partially ablated by a neutralizing HGF antibody added to HF+DWAT. As expected, HGF-treated HFs displayed increased proliferation and decreased apoptosis within matrix keratinocytes, effects mirrored by culturing HFs with surrounding DWAT. Transcriptomic analysis of HFs treated with HGF (6hr) revealed that HGF up-regulated PAX3 and SOX10, as well as VEGFA and WNT5B. Gene expression analysis on 24hr cultures coupled with IPA revealed that key Wnt pathway inhibitors are down-regulated in HFs treated with HGF, whereas Wnt agonists, WNT6 and WNT10B, show an upward trend for HF+HGF and HF+DWAT. Here we provide the first evidence that human DWAT communicates with adjacent scalp HFs by secreting HGF, one of the most potent promoters of hair growth and pigmentation. This study designates human perifollicular adipocytes and HGF-dependent DWAT-HF communication an intriguing novel target for therapeutic intervention in the future management of disorders of hair growth or pigmentation.

OBJECTIVE:Dandruff is a very common scalp condition characterized by flaking and pruritus usually with no visible signs of inflammation, such as redness and erythema. Dandruff is considered a multifactorial condition with both microbial colonization and host factors such as sebum production thought to play a role. There is evidence of changes in epidermal morphology in the scalp skin of dandruff sufferers, with reports of an increase in mean thickness and more nucleated cell layers. The underlying mechanisms driving these morphological changes are currently unclear. The objective of this study was to fully characterize epidermal morphology in dandruff compared to healthy scalp skin and to evaluate potential mechanisms underlying any changes observed.METHODS:Scalp skin biopsies were taken from 22 healthy female subjects and 21 dandruff sufferers, from both lesional and non-lesional sites. Samples were processed, sectioned and stained using haematoxylin and eosin (H&E). To fully characterize epidermal morphology, measurements were taken of epidermal thickness, the convolution of the dermal-epidermal junction and the depth of epidermal rete ridges. To analyse changes in epidermal proliferation immunohistochemical staining was performed using Ki67, a well-established marker of cell proliferation, and quantified using image analysis.RESULTS:Histochemical analysis of skin sections revealed that in dandruff lesional samples, the epidermis was thicker, had a more convoluted dermal epidermal junction and the rete ridges were elongated, compared to healthy scalp skin. Similar directional changes in epidermal morphology, were observed in non-lesional dandruff samples, albeit to a lesser extent. Image analysis of Ki67 expression in the epidermis revealed dandruff lesional skin contained significantly more Ki67-positive proliferating keratinocytes than healthy controls samples. This suggests dandruff scalp skin epidermal keratinocytes are in a hyper-proliferative state.CONCLUSION:There were significant changes in epidermal morphology in dandruff lesional skin compared to healthy scalp skin including increased epidermal thickness, a more convoluted dermal-epidermal junction and elongation of rete ridges. Interestingly, we found there was evidence of an increase in the percentage of epidermal Ki67-positive cells, which has not been reported previously, and demonstrates dandruff is a condition displaying epidermal hyper-proliferation.

In murine skin, dermal white adipose tissue (DWAT) undergoes fluctuations in size across the hair cycle, whereas changes in size, function and metabolism of dermal adipocytes (DAs) during the human scalp hair cycle remain unexplored. Transmission electron microscopy results suggest that during anagen-catagen transition, human DAs co-opt the autophagy machinery to undergo lipophagy within their lipid droplets. Whole-mount staining of hair follicles (HFs) and surrounding DWAT for the autophagy marker LC3B confirms the increased presence of LC3B+ lipid droplets adjacent to catagen HFs; moreover, DWAT around catagen HFs engages in greater glycerol release compared to DWAT surrounding anagen HFs. Thus, we hypothesize that human DAs switch from lipogenesis during anagen to lipophagy together with lipolysis during catagen. We propose various experiments to further prove this hypothesis, whose systematic exploration should help to better characterize the functions of human DWAT and its communication with the HF.

Background: Dandruff is a multifactorial condition with both microbial colonisation and host factors such as sebum production thought to play a role. It is a common condition characterised by excess skin shedding, hyperand para-keratosis, immune cell infiltration and pruritus, often without accompanying visible signs of inflammation. Dandruff sufferers display alterations in scalp skin epidermal morphology. However, the precise biological mechanisms driving these morphological changes remain unclear.

Dermal white adipose tissue (DWAT) is a main component of human skin, composed of individual lipid-laden mesenchymal cells known as dermal adipocytes (DAs). Besides their well-known role in lipid storage and release, DAs also promote skin immunity, wound healing and hair follicle cycling and are important players in cutaneous neuroendocrinology. The ever-growing insights into DWAT functions, albeit mostly in mice, have invited speculation that it may be involved in multiple skin diseases ranging from fibrosis to alopecia and psoriasis, thus designating human DWAT a clinically relevant, but as yet insufficiently investigated skin compartment. Therefore, this practical, user-friendly guide aims to introduce the techniques available to study human DWAT in situ and ex vivo, including immunohistochemistry, immunofluorescence microscopy and analysis via quantitative immunohistomorphometry. Here, we provide information on a collection of stains comprising pre-adipocyte (Pref1) and mature adipocyte markers (Perilipin1, Caveolin1), as well as various lipid (OilRedO, BODIPY) and histochemical stains (H&E, trichrome) available for use on human DWAT. We offer the reader guidelines on fixing, processing and staining human DAs and highlight caveats and solutions to common problems that one may encounter when studying this fascinating skin compartment. We also suggest standard methods for conducting quantitative immunohistomorphometry on human DWAT and its individual adipocytes to quantify cell size, number, lipid content and fluorescence intensity of adipose-specific markers. Finally, we briefly introduce in situ hybridization, transmission electron microscopy and essentials of magnetic resonance imaging imaging as additional tools for instructively interrogating this largest, but still least-known compartment of human skin.

In the past decade, the crosstalk between dermal white adipose tissue (DWAT) and hair follicles (HFs) has become of increasing interest. Murine studies have revealed that pathways involving BMP2, PDGFA, SHH and leptin are involved in the HF-DWAT axis. Despite such progress on murine models, human HF-DWAT communication remains virtually unexplored. In this study, we cultured micro-dissected HFs versus HFs surrounded by the immediate 3-4 layers of dermal adipocytes (HF+DWAT) from human scalp skin for 48hr ex vivo. Interestingly, quantitative immuno-histomorphometry of Ki67+ cells below Aubers line reveals that DWAT significantly enhances cell proliferation in the HF matrix, as well as increasing the number of DAPI+ nuclei. Furthermore, Masson Fontana staining shows a significant upregulation of melanin content within HFs cultured with the surrounding DWAT compared to HFs cultured on their own. We also carried out ex vivo cultures of a) HFs on their own b) HFs with the surrounding DWAT (HF+DWAT), and c) HFs with dissected subcutaneous scalp fat (SWAT) in the same well (HF+SWAT). Interestingly, hair shaft elongation is significantly higher in hair follicles grown together with the surrounding DWAT compared to HFs alone and HFs grown with dissected SWAT. Overall, our results suggest that DWAT-derived factors act upon human HFs via paracrine signalling to modulate key processes within the HF matrix. For the first time, we show that human DWAT influences scalp HFs ex vivo via enhancing proliferation, pigmentation and hair shaft elongation. Our results hold translational promise, and may point to the notion that deregulation of dermal adipocytes surrounding human HFs may play a more significant role in promoting hair disorders than previously imagined.

Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti‐dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays.

SynopsisFemales in South East Asia (Thailand, Indonesia and the Philippines) show concern about dark areas of skin which develop in their underarms, but little is known about the features differentiating pale and hyperpigmented axillary skin in the general population. To investigate this, a histology study was undertaken in the Philippines to define the aetiology of underarm darkening, which is postulated to be a mild form of postinflammatory hyperpigmentation (PIHP). Punch biopsies were taken from dark and light axillary skin sites of 20 female subjects, of whom seven had hyperpigmented underarms, based on an instrumental (Mexameter MX‐18, Courage and Khazaka Electronic GmbH, Cologne, Germany) measure, and 13 had not. Histological and immunohistochemical analyses were undertaken using a range of stains and antibodies, including haematoxylin‐eosin for general histopa‐thology, Masson‐Fontana for melanin, anti‐CD68 for monocytes and macrophages, Van Gieson's technique for fibrosis, anti‐proliferating cell nuclear antigen for cell mitosis, and the melanocyte‐specific immunostains, anti‐tyrosinase and anti‐tyrosinase‐related protein 1. In most cases, dark skin sites from hyperpigmented panellists had increased intensity of Masson‐Fontana, anti‐tyrosinase and/or anti‐TRP1 staining, indicative of melanocyte stimulation and increased melanin production. Furthermore, hair plucking emerged as a key stimulus to increased pigmentation. The trauma of hair plucking slightly increased the number of infiltrating mononuclear cells and macrophages that ingested melanosomes leaking from the damaged epidermis, more so in the skin of hyperpigmented panellists; this, in turn, potentially increases pigmentation. However, cell infiltration was focal, mainly near the plucked follicles, and not indicative of diffuse inflammation. The results from this study support the hypothesis that axillary darkening is mild PIHP, characterized by increased epidermal melanin, following stimulation or mild irritation of skin, with hair plucking as a key factor in this process.