Laser capture microdissection as a method for investigating the human hair follicle microbiome reveals region-specific differences in the bacteriome profile.

OBJECTIVE: Human hair follicles (HFs) are populated by a rich and diverse microbiome, traditionally evaluated by methods that inadvertently sample the skin microbiome and/or miss microbiota located in deeper HF regions. Thereby, these methods capture the human HF microbiome in a skewed and incomplete manner. This pilot study aimed to use laser-capture microdissection of human scalp HFs, coupled with 16S rRNA gene sequencing to sample the HF microbiome and overcome these methodological limitations. RESULTS: HFs were laser-capture microdissected (LCM) into three anatomically distinct regions. All main known core HF bacterial colonisers, including Cutibacterium, Corynebacterium and Staphylococcus, were identified, in all three HF regions. Interestingly, region-specific variations in α-diversity and microbial abundance of the core microbiome genera and Reyranella were identified, suggestive of variations in microbiologically relevant microenvironment characteristics. This pilot study therefore shows that LCM-coupled with metagenomics is a powerful tool for analysing the microbiome of defined biological niches. Refining and complementing this method with broader metagenomic techniques will facilitate the mapping of dysbiotic events associated with HF diseases and targeted therapeutic interventions.

Peroxisome proliferator-activated receptor-γ signalling protects hair follicle stem cells from chemotherapy-induced apoptosis and epithelial-mesenchymal transition.

BACKGROUND: Permanent chemotherapy-induced alopecia (pCIA), for which preventive interventions remain limited, can manifest with scarring. While the underlying pathomechanisms of pCIA are unclear, depletion of epithelial hair follicle (HF) stem cells (eHFSCs) is likely to play a role. OBJECTIVES: To explore the hypothesis that, besides apoptosis, eHFSCs undergo pathological epithelial-mesenchymal transition (EMT) in pCIA, thus explaining the scarring phenotype. Furthermore, we tested whether a peroxisome proliferator-activated receptor (PPAR)-γ modulator could prevent pCIA-associated pathomechanisms. METHODS: Organ-cultured human scalp HFs were treated with the cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC). Additionally, HFs were pretreated with the agonistic PPAR-γ modulator N-acetyl-GED-0507-34-Levo (NAGED), which has previously been shown to promote K15 expression and antagonize EMT in eHFSCs. RESULTS: In accordance with anticipated hair bulb cytotoxicity, dystrophy and catagen induction, 4-HC promoted apoptosis along with increased p53 expression, DNA damage and pathological EMT in keratin 15+ (K15) eHFSCs, as evidenced by decreased E-cadherin expression and the appearance of fibronectin+ and vimentin+ cells in the hair bulge. Pretreatment with NAGED protected against 4-HC-induced hair bulb cytotoxicity/dystrophy, and apoptosis, p53 upregulation and EMT in the bulge, thereby significantly preventing depletion of K15+ human eHFSCs ex vivo. CONCLUSIONS: Since a key cyclophosphamide metabolite alone suffices to damage and deplete human scalp eHFSCs by promoting apoptosis, DNA damage and EMT ex vivo, strategies to prevent pCIA need to target these pathomechanisms. Given the ability of NAGED to prevent chemotherapy-induced eHFSCs damage ex vivo, our study introduces the stimulation of PPAR-γ signalling as a novel intervention strategy for the prevention of pCIA.

New effects of caffeine on corticotropin-releasing hormone (CRH)-induced stress along the intrafollicular classical hypothalamic-pituitary-adrenal (HPA) axis (CRH-R1/2, IP3 -R, ACTH, MC-R2) and the neurogenic non-HPA axis (substance P, p75NTR and TrkA) in ex vivo human male androgenetic scalp hair follicles.

BACKGROUND: Human hair is highly responsive to stress, and human scalp hair follicles (HFs) contain a peripheral neuroendocrine equivalent of the systemic hypothalamic-pituitary-adrenal (HPA) stress axis. Androgenetic alopecia (AGA) is supposed to be aggravated by stress. We used corticotropin-releasing hormone (CRH), which triggers the HPA axis, to induce a stress response in human ex vivo male AGA HFs. Caffeine is known to reverse testosterone-mediated hair growth inhibition in the same hair organ culture model. OBJECTIVES: To investigate whether caffeine would antagonize CRH-mediated stress in these HFs. METHODS: HFs from balding vertex area scalp biopsies of men affected by AGA were incubated with CRH (10-7 mol L-1 ) with or without caffeine (0·001% or 0·005%). RESULTS: Compared to controls, CRH significantly enhanced the expression of catagen-inducing transforming growth factor-ß2 (TGF-ß2) (P < 0·001), CRH receptors 1 and 2 (CRH-R1/2) (P < 0·01), adrenocorticotropic hormone (ACTH) (P < 0·001) and melanocortin receptor 2 (MC-R2) (P < 0·001), and additional stress-associated parameters, substance P and p75 neurotrophin receptor (p75NTR ). CRH inhibited matrix keratinocyte proliferation and expression of anagen-promoting insulin-like growth factor-1 (IGF-1) and the pro-proliferative nerve growth factor receptor NGF-tyrosine kinase receptor A (TrkA). Caffeine significantly counteracted all described stress effects and additionally enhanced inositol trisphosphate receptor (IP3 -R), for the first time detected in human HFs. CONCLUSIONS: These findings provide the first evidence in ex vivo human AGA HFs that the stress mediator CRH induces not only a complex intrafollicular HPA response, but also a non-HPA-related stress response. Moreover, we show that these effects can be effectively antagonized by caffeine. Thus, these data strongly support the hypothesis that stress can impair human hair physiology and induce hair loss, and that caffeine may effectively counteract stress-induced hair damage and possibly prevent stress-induced hair loss.

Thyroxine restores severely impaired cutaneous re-epithelialisation and angiogenesis in a novel preclinical assay for studying human skin wound healing under "pathological" conditions ex vivo.

Impaired cutaneous wound healing remains a major healthcare challenge. The enormity of this challenge is compounded by the lack of preclinical human skin wound healing models that recapitulate selected key factors underlying impaired healing, namely hypoxia/poor tissue perfusion, oxidative damage, defective innervation, and hyperglycaemia. Since organ-cultured human skin already represents a denervated and impaired perfusion state, we sought to further mimic "pathological" wound healing conditions by culturing experimentally wounded, healthy full-thickness frontotemporal skin from three healthy female subjects for three days in either serum-free supplemented Williams' E medium or in unsupplemented medium under "pathological" conditions (i.e. hypoxia [5% O2], oxidative damage [10 mM H2O2], absence of insulin, excess glucose). Under these "pathological" conditions, dermal-epidermal split formation and dyskeratosis were prominent in organ-cultured human skin, and epidermal reepithelialisation was significantly impaired (p < 0.001), associated with reduced keratinocyte proliferation (p < 0.001), cytokeratin 6 expression (p < 0.001) and increased apoptosis (p < 0.001). Moreover, markers of intracutaneous angiogenesis (CD31 immunoreactivity and the number of of CD31 positive cells and CD31 positive vessel lumina) were significantly reduced. Since we had previously shown that thyroxine promotes wound healing in healthy human skin ex vivo, we tested whether this in principle also occurs under "pathological" wound healing conditions. Indeed, thyroxine administration sufficed to rescue re-epithelialisation (p < 0.001) and promoted both epidermal keratinocyte proliferation (p < 0.01) and angiogenesis in terms of CD31 immunoreactivity and CD31 positive cells under "pathological" conditions (p < 0.001) ex vivo. This demonstrates the utility of this pragmatic short-term ex vivo model, which recapitulates some key parameters of impaired human skin wound healing, for the preclinical identification of promising wound healing promoters.

Exploring the human hair follicle microbiome.

Human hair follicles (HFs) carry complex microbial communities that differ from the skin surface microbiota. This likely reflects that the HF epithelium differs from the epidermal barrier in that it provides a moist, less acidic, and relatively ultraviolet light-protected environment, part of which is immune-privileged, thus facilitating microbial survival. Here we review the current understanding of the human HF microbiome and its potential physiological and pathological functions, including in folliculitis, acne vulgaris, hidradenitis suppurativa, alopecia areata and cicatricial alopecias. While reviewing the main human HF bacteria (such as Propionibacteria, Corynebacteria, Staphylococci and Streptococci), viruses, fungi and parasites as human HF microbiome constituents, we advocate a broad view of the HF as an integral part of the human holobiont. Specifically, we explore how the human HF may manage its microbiome via the regulated production of antimicrobial peptides (such as cathelicidin, psoriasin, RNAse7 and dermcidin) by HF keratinocytes, how the microbiome may impact on cytokine and chemokine release from the HF, and examine hair growth-modulatory effects of antibiotics, and ask whether the microbiome affects hair growth in turn. We highlight major open questions and potential novel approaches to the management of hair diseases by targeting the HF microbiome.

Frontal fibrosing alopecia shows robust T helper 1 and Janus kinase 3 skewing.

BACKGROUND: Frontal fibrosing alopecia (FFA) is a scarring alopecia with unclear pathogenesis and a progressive course. The disease has a major impact on patients' quality of life and there is a lack of effective treatment to halt disease progression. METHODS: We profiled lesional and nonlesional scalp biopsies collected in 2017 from patients with FFA (n = 12) compared with scalp biopsies from patients with alopecia areata (AA) (n = 8) and controls (n = 8) to evaluate gene and protein expression, including the primary outcome (CXCL9). We determined significant differences between biomarkers using a two-sided Student's t-test adjusting P-values by false discovery rate. RESULTS: Significant increases were seen in CD8+ cytotoxic T cells, CD11c+ dendritic cells, CD103+ and CD69+ tissue-resident memory T cells in FFA and AA vs. control scalp (P < 0·05), with corresponding significantly upregulated granzyme B mRNA, particularly in FFA (P < 0·01). In AA, cellular infiltrates were primarily concentrated at the bulb, while in FFA these were mainly localized at the bulge. FFA demonstrated significant upregulation of T helper 1/intereferon (IFN) (IFN-γ, CXCL9/CXCL10), the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway (STAT1, JAK3) and fibrosis-related products (vimentin, fibronectin; P < 0·05), with no concomitant downregulation of hair keratins and the T-regulatory marker, forkhead box P3, which were decreased in AA. The stem cell markers CD200 and K15 demonstrated significantly reduced expression only in FFA (P < 0·05). CONCLUSIONS: These data suggest that follicular damage and loss of stem cells in FFA may be mediated through immune attack in the bulge region, with secondary fibrosis and reduced but still detectable stem cells. JAK/STAT-targeting treatments may be able to prevent permanent follicular destruction and fibrosis in early disease stages.

Profiling the human hair follicle immune system in lichen planopilaris and frontal fibrosing alopecia: can macrophage polarization differentiate these two conditions microscopically?

BACKGROUND: Frontal fibrosing alopecia (FFA) is traditionally regarded as a variant of lichen planopilaris (LPP) based on histological features. Distinct clinical presentation, demographics and epidemiology suggest that differing pathogenic factors determine the final phenotype. OBJECTIVES: To map the hair follicle immune system in LPP and FFA by systematically comparing key inflammatory markers in defined hair follicle compartments. METHODS: Lesional scalp biopsies from LPP and FFA and healthy controls were stained with the following immunohistochemical markers: CD1a and CD209, CD4, CD8, CD56, CD68, CD123, CXCR3, forkhead box (FOX)P3, mast cell tryptase and cKit. Macrophage polarization was explored using CD206, CD163, CD86, receptor for advanced glycation end products (RAGE), interleukin (IL)-4 and IL-13 on paired lesional and nonlesional LPP and FFA samples. RESULTS: Increased numbers of CD8+ , CXCR3+ and FOXP3+ T cells and CD68+ macrophages were identified in the distal hair follicle epithelium and perifollicular mesenchyme in both LPP and FFA compared with controls. In both LPP and FFA, total and degranulated mast cells and CD123+ plasmacytoid dendritic cells were increased in the perifollicular mesenchyme adjacent to the bulge and infundibulum, whereas numbers of CD1a+ and CD209+ dendritic cells were significantly reduced in the infundibulum connective tissue sheath. However, only with CD68 staining was a significant difference between LPP and FFA identified, with greater numbers of CD68+ cells in LPP samples. Furthermore, the identified macrophage polarization markers downregulated CD86 and upregulated CD163 and IL-4 expression in lesional LPP compared with FFA samples. CONCLUSIONS: This comparative immunopathological analysis is the first to profile systematically the hair follicle immune system in LPP and FFA. Our analysis highlights a potential role of macrophages in disease pathobiology and suggests that macrophage polarization may differ between LPP and FFA, allowing microscopic differentiation. Linked Comment: Kinoshita-Ise. Br J Dermatol 2020; 183:419-420.

Deciphering the molecular morphology of the human hair cycle: Wnt signalling during the telogen-anagen transformation.

BACKGROUND: The signals that induce anagen (growth) in 'quiescent' human telogen hair follicles (HFs) are as yet unknown. Their identification promises better targeted therapeutic hair growth interventions. OBJECTIVES: Recognizing the central role of Wnt signalling in hair biology, the aim was to delineate the differential expression of key agonists, antagonists and target genes of this pathway during the telogen-to-anagen transformation of human scalp HFs. METHODS: This differential expression was studied by in situ hybridization in human telogen and early-anagen scalp HF sections. RESULTS: On anagen induction, gene expression of the Wnt ligands WNT3, WNT4 and WNT10B, the Wnt ligand secretion regulator WLS, and the Wnt target genes AXIN2 and LEF1, is significantly increased within the secondary hair germ and the dermal papilla. Conversely, expression of the secreted Wnt inhibitor SFRP1 (secreted frizzled-related protein 1) is reduced. Human epithelial HF stem cells upregulate WNT4 and WNT10A expression, suggesting that these Wnt agonists are important for stem cell activation. CONCLUSIONS: We provide the first evidence that key changes in Wnt signalling that drive murine anagen induction also occur in human scalp HFs, yet with notable differences. This provides a rational basis for Wnt-targeting therapeutic interventions to manipulate human hair growth disorders. What's already known about this topic? Upregulation of Wnt agonists and downregulation of Wnt antagonists in the secondary hair germ and/or dermal papilla drives hair growth (anagen) induction in mice. Autocrine Wnt signalling in murine epithelial hair follicle stem cells is required to maintain their stem cell function. Reduction of Wnt ligands or increased expression of Wnt antagonists induces dysregulation of the murine hair follicle cycle and causes alopecia. What does this study add? This study demonstrates for the first time that key Wnt pathway regulatory agonists, antagonists and target genes, are expressed in the human telogen-to-early-anagen transformation. On human anagen induction the Wnt ligands WNT3, WNT4 and WNT10B are increased in the regenerating epithelium, whereas the Wnt antagonist, SFRP1 (secreted frizzled-related protein 1), is reduced. Human anagen induction has fundamental differences in the expression of Wnt ligands compared with the murine system. What is the translational message? Regulation of these Wnt ligands permits targeted therapeutic interventions in human hair growth disorders and informs development of new drugs that promote or suppress anagen induction.

Theophylline exerts complex anti-ageing and anti-cytotoxicity effects in human skin ex vivo.

OBJECTIVE: Theophylline is a phosphodiesterase inhibitor that is being used clinically for asthma therapy. In addition, it is recognized as a cosmetic agent with possible anti-ageing and anti-oxidative properties. Nevertheless, how it affects human skin is still poorly examined. METHODS: Theophylline (10 or 100 µM) was administered to the culture medium of full-thickness human skin ex vivo for 24 or 72 h. RESULTS: Theophylline stimulated protein expression of the anti-oxidant metallothionein-1 and mRNA levels of collagen I and III. Assessment of fibrillin-1 immunohistology revealed enhanced structural stability of dermal microfibrils. Theophylline also exerted extracellular matrix-protective effects by decreasing MMP-2 and MMP-9 mRNA levels, partially antagonizing the effects of menadione, the potent, toxic ROS donor. In addition, it decreased menadione-stimulated epidermal keratinocytes apoptosis. Interestingly, theophylline also increased the level of intracutaneously produced melatonin, that is the most potent ROS-protective and DNA damage repair neuromediator, and tendentially increased protein expression of MT1, the melatonin receptor. Theophylline also increased the expression of keratin 15, the stem cell marker, in the epidermal basal layer but did not change mitochondrial activity or epidermal pigmentation. CONCLUSION: This ex vivo pilot study in human skin shows that theophylline possesses several interesting complex skin-protective properties. It encourages further examination of theophylline as a topical candidate for anti-ageing treatment.

OBJECTIF: la théophylline est un inhibiteur de la phosphodiestérase actuellement utilisée en clinique pour le traitement de l'asthme. En outre, elle est reconnue comme étant un agent cosmétique ayant des propriétés potentiellement anti-âge et antioxydantes. Cependant, la manière dont elle affecte la peau chez l'homme est encore très peu étudiée. MÉTHODES: de la théophylline (10 ou 100 µM) a été ajoutée dans le milieu de culture d'un échantillon de peau humaine d'épaisseur totale ex vivo pendant 24 ou 72 h. RÉSULTATS: la théophylline a stimulé l'expression de la métallothionéine-1, une protéine antioxydante, et les taux d'ARNm du collagène I et III. L'évaluation immunohistologique de la fibrilline-1 a révélé une meilleure stabilité structurale des microfibrilles du derme. La théophylline a également exercé des effets protecteurs sur la matrice extracellulaire en diminuant les taux d'ARNm des métalloprotéinases matricielles MMP-2 et MMP-9, neutralisant en partie les effets de la ménadione, puissant donneur d'espèces réactives de l'oxygène (ROS) toxiques. En outre, elle a diminué l'apoptose des kératinocytes épidermiques stimulés par la ménadione. Fait intéressant, la théophylline a également augmenté le taux de mélatonine produite de manière intra-cutanée, la mélatonine étant le plus puissant neuromédiateur protecteur contre les ROS et réparateur des lésions de l'ADN. Elle a augmenté de façon tendancielle l'expression de la protéine MT1, récepteur de la mélatonine. La théophylline a également augmenté l'expression de la kératine 15, marqueur de cellules souches, dans la couche basale épidermique, mais n'a pas modifié l'activité mitochondriale ou la pigmentation épidermique. CONCLUSION: cette étude pilote ex vivo réalisée sur de la peau humaine montre que la théophylline a plusieurs propriétés protectrices de la peau complexes et intéressantes. Ces résultats encouragent à poursuivre l'étude de la théophylline en tant que candidat à un traitement local anti-âge.

An osteopontin-derived peptide inhibits human hair growth at least in part by decreasing fibroblast growth factor-7 production in outer root sheath keratinocytes.

BACKGROUND: Given that unwanted hair growth (hirsutism, hypertrichosis) can cause major psychological distress, new pharmacological treatment strategies with safe and effective hair growth inhibitors that do not destroy the hair follicle (HF) and its stem cells need to be developed. OBJECTIVES: To establish if osteopontin-derived fragments may modulate human hair growth given that human HFs express the multifunctional, immunomodulatory glycoprotein, osteopontin. METHODS: Our hypothesis was tested ex vivo and in vivo by using a newly generated, toxicologically well-characterized, modified osteopontin-derived peptide (FOL-005), which binds to the HF. RESULTS: In organ-cultured human HFs and scalp skin, and in human scalp skin xenotransplants onto SCID mice, FOL-005 treatment (60 nmol L-1 to 3 µmol L-1 ) significantly promoted premature catagen development without reducing the number of keratin 15-positive HF stem cells or showing signs of drug toxicity. Genome-wide DNA microarray, quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry revealed decreased expression of the hair growth promoter, fibroblast growth factor-7 (FGF7) by FOL-005, while cotreatment of HFs with recombinant FGF7 partially abrogated FOL-005-induced catagen promotion. CONCLUSIONS: With caveats in mind, our study identifies this osteopontin-derived peptide as an effective, novel inhibitory principle for human hair growth ex vivo and in vivo, which deserves systematic clinical testing in hirsutism and hypertrichosis. What's already known about this topic? The treatment of unwanted hair growth (hypertrichosis, hirsutism) lacks pharmacological intervention, with only few and often unsatisfactory treatments available. Osteopontin is prominently expressed in human HFs and has been reported to be elevated during catagen in the murine hair cycle. What does this study add? We tested the effects on hair growth of a novel, osteopontin-derived fragment (FOL-005) ex vivo and in vivo. In human hair follicles, high-dose FOL-005 significantly reduces hair growth both ex vivo and in vivo. What is the translational message? High-dose FOL-005 may provide a new therapeutic opportunity as a treatment for unwanted hair growth.

Homeostasis of the sebaceous gland and mechanisms of acne pathogenesis.

BACKGROUND: Sebaceous glands (SGs) are appendages of mammalian skin that produce a mixture of lipids known as sebum. Acne vulgaris is an exceptionally common skin condition, characterized by elevated sebum production, altered sebum composition, and the formation of infundibular cysts, called comedones. Comedo-associated SGs are atrophic, suggesting that comedo formation involves abnormal differentiation of progenitor cells that generate the SG and infundibulum: the 'comedo switch'. Understanding the biological processes that govern SG homeostasis promises to highlight potential aetiological mechanisms underlying acne and other SG-associated skin disorders. RESULTS: In this review, we discuss the clinical data, genetic mouse models and in vitro research that have highlighted major hormones, paracrine factors, transcription factors and signalling pathways that control SG homeostasis. These include, but are not limited to androgens, progestogens and oestrogens; retinoids; receptor tyrosine kinases such as ErbB family receptors, fibroblast growth factor receptor 2 and insulin/insulin-like growth factor 1 receptors; peroxisome proliferator-activated receptor γ; aryl hydrocarbon receptor; and the Wnt signalling pathway. Where possible, the cellular and molecular mechanisms by which these regulatory factors control SG biology are indicated, along with considerations as to how they might contribute to acne pathogenesis. CONCLUSIONS: Future research should seek to establish the relative importance, and causative relationships, of altered sebum production, sebum composition, inflammation and abnormal differentiation of sebaceous progenitors to the process of comedo formation in acne. Such an understanding will allow for therapeutic targeting of regulatory factors that control SG homeostasis, with the aim of treating acne.