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Background It is widely accepted that NKG2D+cells are critically involved in alopecia areata (AA) pathogenesis. However, besides being expressed in CD8+T-cells and NK cells, NKG2D is also found in human γδT-cells. AA lesional hair follicles (HFs) overexpress NKG2D and γδTCR activating ligands, e.g. MICA and CD1d, and chemoattractants for γδT-cells, such as CXCL10. Objective To investigate whether abnormal activities of γδT-cells may be involved in AA pathogenesis. Methods We analyzed the number and activation status of γδT-cells in human healthy, lesional and non-lesional AA scalp biopsies by FACS and/or quantitative (immuno-)histomorphometry. Results In healthy human scalp skin, the few skin-resident γδT-cells were found to be mostly Vδ1+, non-activated (CD69−NKG2Ddim) and positive for CXCL10, and CXCL12 receptors. These Vδ1+T-cells predominantly localized in/around the HF infundibulum. In striking contrast, the number of Vδ1+T-cells was significantly higher around and even inside the proximal (suprabulbar and bulbar) epithelium of lesional AA HFs. These cells also showed a pro-inflammatory phenotype, i.e. higher NKG2D, and IFN-γ and lower CD200R expression. Importantly, more pro-inflammatory Vδ1+T-cells were seen also around non-lesional AA HFs. Lesional AA HFs also showed significantly higher expression of CXCL12. Conclusion Our pilot study introduces skin-resident γδT-cells as a previously overlooked, but potentially important, mostly (auto-)antigen-independent, new innate immunity protagonist in AA pathobiology. The HF infiltration of these activated, IFN-γ-releasing cells already around non-lesional AA HFs suggest that Vδ1+T-cells are involved in the early stages of human AA pathobiology, and may thus deserve therapeutic targeting for optimal AA management.

Anagen stage hair follicles (HFs) exhibit "immune privilege (IP)" from the level of the bulge downwards to the bulb. Both passive and active IP mechanisms protect HFs from physiologically undesired immune responses and limit immune surveillance. IP is relative, not absolute, and is primarily based on absent, or greatly reduced, intra-follicular antigen presentation via MHC class I and II molecules, along with prominent expression of "no danger" signals like CD200 and the creation of an immunoinhibitory signalling milieu generated by the secretory activities of HFs. Perifollicular mast cells, Tregs and other immunocytes may also contribute to HF IP maintenance in healthy human skin. Collapse of anagen hair bulb IP is an essential prerequisite for the development of alopecia areata (AA). In AA, lesional HFs are rapidly infiltrated by NKG2D + T cells and natural killer (NK) cells, while perifollicular mast cells acquire a profoundly pro-inflammatory phenotype and interact with autoreactive CD8+ T cells. Using animal models, significant functional evidence has accumulated that demonstrates the dominance of the immune system in AA pathogenesis. Purified CD8+T-cell and NK cell populations alone, which secrete fγ, suffice to induce the AA phenotype, while CD4+T-cells aggravate it, and Tregs and iNKT cells may provide relative protection against AA development. While IP collapse may be induced by exogenous agents, inherent IP deficiencies might confer increased susceptibility to AA for some individuals. Thus, a key goal for effective AA management is the re-establishment of a functional HF IP, which will also provide superior protection from disease relapse.

Background Human hair follicles (HFs) express the olfactory receptor (OR)2AT4, which is selectively stimulated by the synthetic sandalwood-like odorant, Sandalore® . In organ-cultured, human scalp HFs, Sandalore® prolongs anagen and suppresses apoptosis by up-regulating intrafollicular IGF-1 mediated signaling. Aims The objective of this study is to demonstrate whether effects of Sandalore® observed ex vivo translate into a clinically relevant effect in patients with telogen effluvium. Patients/methods In a randomized, double-blinded, placebo-controlled, clinical trial, 60 female volunteers (18-65 years) affected by telogen effluvium received over a period of 24 weeks treatment with either 1% Sandalore® solution (n = 30) or placebo (identically smelling, but non-OR2AT4 activating sandalwood oil n = 30). The study read-out parameters were the degree of hair shedding, hair volume, terminal/vellus hair ratio, anagen/catagen-telogen ratio, and patient self-assessment. Results Sandalore® 1% ameliorated clinical signs of telogen effluvium, namely it reduced hair shedding, and increased hair volume and the percentage of anagen HFs, the latter two parameters significantly more than placebo when changes were calculated to baseline. Sandalore® also increased the ratio of terminal/vellus hairs at week 8. Most of the anti-hair shedding effects were seen after 8 weeks and maintained at week 24. Patient questionnaire showed that verum group patients were more satisfied than the placebo group in regard to the overall results. Conclusion This clinical trial supports previous findings of anagen-prolonging effects of Sandalore® ex vivo with similar results now reproduced in clinical practice. It also provides proof-of-principle that a topically applied cosmetic odorant acting through HF olfactory receptors can be a therapeutic alternative to treat hair loss disorders characterized by excessive hair shedding such as telogen effluvium.

Wnt signalling is one of the most fundamental molecular pathways for modulating the human hair cycle and is essential for maintaining hair growth1 . We recently identified that the Wnt antagonist, secreted frizzled related protein 1 (SFRP1), prematurely induces catagen in microdissected human scalp hair follicles (HFs) ex vivo1 . SFRP1 is inhibited by the potent hypertrichosis inducing immunosuppressant, cyclosporine A (CsA)2 , and the specific SFRP1 antagonist, WAY-316606, both of which prevent spontaneous catagen development1 .

Peroxisome proliferator-activated receptors (PPARs) are abundantly expressed in human skin, with PPAR-γ being the most intensively investigated isoform. In various ex vivo and in vivo models, PPAR-γ-mediated signalling has recently surfaced as an essential element of hair follicle (HF) development, growth and stem cell biology. Moreover, the availability of novel, topically applicable PPAR-γ modulators with a favourable toxicological profile has extended the range of potential applications in clinical dermatology. In this review, we synthesize where this field currently stands and sketch promising future research avenues, focussing on the role of PPAR-γ-mediated signalling in the biology and pathology of human scalp HFs, with special emphasis on scarring alopecias such as lichen planopilaris and frontal fibrosing alopecia as model human epithelial stem cell diseases. In particular, we discuss whether and how pharmacological modulation of PPAR-γ signalling may be employed for the management of hair growth disorders, for example, in scarring alopecia (by reducing HF inflammation as well as by promoting the survival and suppressing pathological epithelial-mesenchymal transition of keratin 15 + epithelial stem cells in the bulge) and in hirsutism/hypertrichosis (by promoting catagen development). Moreover, we explore the potential role of PPAR-γ in androgenetic alopecia, HF energy metabolism and HF ageing, and consider clinical perspectives that emanate from the limited data available on this so far. As this field of translational human hair research is still in its infancy, many open questions exist, for which we briefly delineate selected experimental approaches that promise to generate instructive answers in the near future.

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.

Murine γδT-cells have stress-surveillance functions and are implicated in autoimmunity. Yet, whether human γδT-cells are also stress sentinels and directly promote autoimmune responses in the skin is unknown. Using a novel (mini-)organ assay, we tested if human dermis resident γδT-cells can recognize stressed human scalp hair follicles (HFs) to promote an alopecia areata (AA)-like autoimmune response. Accordingly, we show that γδT-cells from healthy human scalp skin are activated (CD69+), up-regulate the expression of NKG2D and IFN-γ, and become cytotoxic when co-cultured with autologous stressed HFs ex vivo. These autologous γδT-cells induce HF immune privilege collapse, dystrophy, and premature catagen, i.e. three hallmarks of the human autoimmune HF disorder, AA. This is mediated by CXCL12, MICA, and in part by IFN-γ and CD1d. In conclusion, human dermal γδT-cells exert physiological stress-sentinel functions in human skin, where their excessive activity can promote autoimmunity towards stressed HFs that overexpress CD1d, CXCL12, and/or MICA.

Alopecia areata (AA) is one of the most common forms of human hair loss. Although genetic studies have implicated autoimmune processes in AA etiology, understanding of the etiopathogenesis is incomplete. Recent research has implicated microRNAs, a class of small noncoding RNAs, in diverse autoimmune diseases. To our knowledge, no study has investigated the role of microRNAs in AA. In this study, gene-based analyses were performed for microRNAs using data of the largest genome-wide association meta-analysis of AA to date. Nominally, significant P-values were obtained for 78 of the 617 investigated microRNAs. After correction for multiple testing, three of the 78 microRNAs remained significant. Of these, miR-30b/d was the most significant microRNA for the follow-up analyses, which also showed lower expression in the hair follicle of AA patients. Target gene analyses for the three microRNAs showed 42 significantly associated target genes. These included IL2RA, TNXB, and ERBB3, which had been identified as susceptibility loci in previous genome-wide association studies. Using luciferase assay, site-specific miR-30b regulation of the AA risk genes IL2RA, STX17, and TNXB was validated. This study implicates microRNAs in the pathogenesis of AA. This finding may facilitate the development of future treatment strategies.

The collapse of the immune privilege (IP) of the anagen hair bulb is now accepted as a key element in AA pathogenesis, and hair bulb IP restoration lies at the core of AA therapy. Here, we briefly review the essentials of hair bulb IP and recent progress in understanding its complexity. We discuss open questions and why the systematic dissection of hair bulb IP and its pharmacological manipulation (including the clinical testing of FK506 and α-melanocyte-stimulating hormone analogs) promise to extend the range of future therapeutic options in AA and other IP collapse-related autoimmune diseases.

Primary cicatricial alopecia is characterized by a permanent "scarring" alopecia. This condition is characterized by the irreversible loss of hair follicles (HF) as a result of apoptosis and epithelial-mesenchymal transition (EMT) of epithelial stem cells localized in the HF bulge.We here report the procedure for experimentally induced EMT in healthy human epidermal stem cells (eSCs) using full-length HF organ culture ex vivo. The present model can be used to recapitulate the complex processes observed in scarring alopecia patient tissues, to further investigate the mechanisms involved in EMT transformation of HFeSCs, and to test substances that could prevent and/or rescue HFeSCs from EMT for the management of scarring alopecias.

Alopecia areata (AA) is a hair follicle (HF) autoimmune disease leading to hair loss. Interferon gamma (IFNγ) is regarded as the key cytokine involved in AA development, which has been shown to play a role in the collapse of HF immune privilege (IP), CD8+ T-cell-driven inflammation toward the HF bulb, and premature catagen development. We present here the procedure to induce IP collapse in human healthy HFs ex vivo. This assay can be suitable not only to investigate mechanisms involved in the HF IP collapse but also to test substances for either preventing and/or reversing the HF IP collapse for the management of AA or other autoimmune diseases sharing similar pathogenesis.

Alopecia areata (AA) is a hair loss disorder resulting from an autoimmune reaction against hair follicles. T-helper 1 cells are a major contributor to this disorder, but little is known about the role of T-regulatory cells (Tregs) in AA. Here, we analysed the distribution of circulating Treg subsets in twenty AA patients with active hair loss and fifteen healthy subjects by flow cytometry. The Treg suppressor HLA-DR+ subpopulation was significantly reduced in the patients (P

More than 300 genetic risk loci have been identified for male pattern baldness (MPB) but little is known about the exact molecular mechanisms through which the associated variants exert their effects on MPB pathophysiology. Here, we aimed at further elucidating the regulatory architecture of the MPB risk locus on chromosome (chr.) 2q35, where we have previously reported a regulatory effect of the MPB lead variant on the expression of WNT10A. A HaploReg database research for regulatory annotations revealed that the association signal at 2q35 maps to a binding site for the transcription factor EBF1, whose gene is located at a second MPB risk locus on chr. 5q33.3. To investigate a potential interaction between EBF1 and WNT10A during MPB development, we performed in vitro luciferase reporter assays as well as expression analyses and immunofluorescence co-stainings in microdissected human hair follicles. Our experiments confirm that EBF1 activates the WNT10A promoter and that the WNT10A/EBF1 interaction is impacted by the allelic expression of the MPB risk allele at 2q35. Expression analyses across different hair cycle phases and immunhistochemical (co)stainings against WNT10A and EBF1 suggest a predominant relevance of EBF1/WNT10A interaction for hair shaft formation during anagen. Based on these findings we suggest a functional mechanism at the 2q35 risk locus for MPB, where an MPB-risk allele associated reduction in WNT10A promoter activation via EBF1 results in a decrease in WNT10A expression that eventually results in anagen shortening, that is frequently observed in MPB affected hair follicles. To our knowledge, this study is the first follow-up study on MPB that proves functional interaction between two MPB risk loci and sheds light on the underlying pathophysiological mechanism at these loci.

Olfactory receptors are expressed by different cell types throughout the body and regulate physiological cell functions beyond olfaction. In particular, the olfactory receptor OR2AT4 has been shown to stimulate keratinocyte proliferation in the skin. Here, we show that the epithelium of human hair follicles, particularly the outer root sheath, expresses OR2AT4, and that specific stimulation of OR2AT4 by a synthetic sandalwood odorant (Sandalore®) prolongs human hair growth ex vivo by decreasing apoptosis and increasing production of the anagen-prolonging growth factor IGF-1. In contrast, co-administration of the specific OR2AT4 antagonist Phenirat® and silencing of OR2AT4 inhibit hair growth. Together, our study identifies that human hair follicles can engage in olfactory receptor-dependent chemosensation and require OR2AT4-mediated signaling to sustain their growth, suggesting that olfactory receptors may serve as a target in hair loss therapy.

Alopecia areata (AA) is an organ-restricted autoimmune disease that mainly affects the hair follicle (HF). Several findings support a key primary effector role of CD8+ T cells in the disease pathogenesis. Autoreactive CD8+ T cells are not only present in the characteristic peribulbar inflammatory cell infiltrate of lesional AA HFs but are also found to be infiltrating in lesional HF epithelium where they are thought to recognize major histocompatibility complex class I-presented (auto-)antigens. However, the latter still remain unidentified. Therefore, one key aim in AA research is to identify the clonotypes of autoaggressive, intralesional CD8+ T cells. Therapeutically, this is important (a) so that these lymphocytes can be selectively eliminated or inhibited, (b) to identify the-as yet elusive-key (auto-)antigens in AA, and/or (c) to induce peripheral tolerance against the latter. Therefore, we have recently embarked on a National Alopecia Areata Foundation-supported project that attempts to isolate disease-specific, intralesional CD8+ T cells from AA skin in order to determine their TCR clonotype, using two complementary strategies. The first method is based on the enzymatic skin digestion from lesional AA skin, followed by either MACS technology and single-cell picking or FACS cell sorting, while the second method on laser microdissection. The identification of disease-specific TCRs can serve as a basis for specific AA immunotherapy along the lines sketched above and may possibly also provide prognostic biomarkers. If successful, this research strategy promises to permit, at long last, the causal therapy of AA.

Hypotrichosis simplex (HS) is a rare form of hereditary alopecia characterized by childhood onset of diffuse and progressive scalp and body hair loss. Although research has identified a number of causal genes, genetic etiology in about 50% of HS cases remains unknown. The present report describes the identification via whole-exome sequencing of five different mutations in the gene LSS in three unrelated families with unexplained, potentially autosomal-recessive HS. Affected individuals showed sparse to absent lanugo-like scalp hair, sparse and brittle eyebrows, and sparse eyelashes and body hair. LSS encodes lanosterol synthase (LSS), which is a key enzyme in the cholesterol biosynthetic pathway. This pathway plays an important role in hair follicle biology. After localizing LSS protein expression in the hair shaft and bulb of the hair follicle, the impact of the mutations on keratinocytes was analyzed using immunoblotting and immunofluorescence. Interestingly, wild-type LSS was localized in the endoplasmic reticulum (ER), whereas mutant LSS proteins were localized in part outside of the ER. A plausible hypothesis is that this mislocalization has potential deleterious implications for hair follicle cells. Immunoblotting revealed no differences in the overall level of wild-type and mutant protein. Analyses of blood cholesterol levels revealed no decrease in cholesterol or cholesterol intermediates, thus supporting the previously proposed hypothesis of an alternative cholesterol pathway. The identification of LSS as causal gene for autosomal-recessive HS highlights the importance of the cholesterol pathway in hair follicle biology and may facilitate novel therapeutic approaches for hair loss disorders in general.

The pathobiology of alopecia areata (AA), one of the most frequent autoimmune diseases and a major unsolved clinical problem, has intrigued dermatologists, hair biologists and immunologists for decades. Simultaneously, both affected patients and the physicians who take care of them are increasingly frustrated that there is still no fully satisfactory treatment. Much of this frustration results from the fact that the pathobiology of AA remains unclear, and no single AA pathogenesis concept can claim to be universally accepted. In fact, some investigators still harbour doubts whether this even is an autoimmune disease, and the relative importance of CD8+ T cells, CD4+ T cells and NKGD2+ NK or NKT cells and the exact role of genetic factors in AA pathogenesis remain bones of contention. Also, is AA one disease, a spectrum of distinct disease entities or only a response pattern of normal hair follicles to immunologically mediated damage? During the past decade, substantial progress has been made in basic AA-related research, in the development of new models for translationally relevant AA research and in the identification of new therapeutic agents and targets for future AA management. This calls for a re-evaluation and public debate of currently prevalent AA pathobiology concepts. The present Controversies feature takes on this challenge, hoping to attract more skin biologists, immunologists and professional autoimmunity experts to this biologically fascinating and clinically important model disease.

Background: Alopecia areata (AA) is an autoimmune disorder whose pathogenesis involves the collapse of the relative immune privilege (IP) of the hair follicle (HF). Given that vasoactive intestinal peptide (VIP) is an immunoinhibitory neuropeptide released by perifollicular sensory nerve fibres, which play a role in IP maintenance, it may modulate human HF-IP and thus be therapeutically relevant for AA. Objectives: To answer the following questions: Do human HFs express VIP receptors, and does their stimulation protect from or restore experimentally induced HF-IP collapse? Is VIP signalling defective in AA HFs?. Methods: Firstly, VIP and VIP receptor (VPAC1, VPAC2) expression in human scalp HFs and AA skin was assessed. In HF organ culture, we then explored whether VIP treatment can restore and/or protect from interferon-γ-induced HF-IP collapse, assessing the expression of the key IP markers by quantitative (immuno-)histomorphometry. Results: Here we provide the first evidence that VIP receptors are expressed in the epithelium of healthy human HFs at the gene and protein level. Furthermore, VIP receptor protein expression, but not VIP+ nerve fibres, is significantly downregulated in lesional hair bulbs of patients with AA, suggesting defects in VIP receptor-mediated signalling. Moreover, we show that VIP protects the HF from experimentally induced IP collapse in vitro, but does not fully restore it once collapsed. Conclusions: These pilot data suggest that insufficient VIP receptor-mediated signalling may contribute to impairing HF-IP in patients with AA, and that VIP is a promising candidate ‘HF-IP guardian’ that may be therapeutically exploited to inhibit the progression of AA lesions.

Alopecia areata (AA) is an organ-restricted autoimmune disease that mainly affects the hair follicle (HF). Several findings support a key primary effector role of CD8þ T cells in the disease pathogenesis. Autoreactive CD8þ T cells are not only present in the characteristic peribulbar inflammatory cell infiltrate of lesional AA HFs but are also found to be infiltrating in lesional HF epithelium where they are thought to recognize major histocompatibility complex class I-presented (auto-)antigens. However, the latter still remain unidentified. Therefore, one key aim in AA research is to identify the clonotypes of autoaggressive, intralesional CD8þ T cells. Therapeutically, this is important (a) so that these lymphocytes can be selectively eliminated or inhibited, (b) to identify the—as yet elusive—key (auto-)antigens in AA, and/or (c) to induce peripheral tolerance against the latter. Therefore, we have recently embarked on a National Alopecia Areata Foundation–supported project that attempts to isolate disease-specific, intralesional CD8þ T cells from AA skin in order to determine their TCR clonotype, using two complementary strategies. The first method is based on the enzymatic skin digestion from lesional AA skin, followed by either MACS technology and single-cell picking or FACS cell sorting, while the second method on laser microdissection. The identification of disease-specific TCRs can serve as a basis for specific AA immunotherapy along the lines sketched above and may possibly also provide prognostic biomarkers. If successful, this research strategy promises to permit, at long last, the causal therapy of AA.

S | Hair and Other Adnexal Structures 216 Human hair follicles can “smell”: OR2AT4-mediated hair growth regulation J Cheret, LM Ponce Moya, M Bertolini, T Tsai, M Alam, H Hatt and R Paus 1 Monasterium Laboratory, Muenster, Germany, 2 Ruhr-Uni-Bochum, Bochum, Germany and 3 Dermatology Research Centre, Manchester, United Kingdom Olfactory receptors (OR) are part of an evolutionarily ancient chemosensory signaling system, which regulates many cell functions beyond olfaction, incl. keratinocyte (KC) proliferation. Since hair follicle (HF) growth requires proliferation, we asked whether ORs play a role in human HF growth. We focused on OR2AT4, since OR2AT4 stimulation promotes human epidermal KC growth/migration. Human scalp HFs prominently express OR2AT4 (IF) in situ, namely in basal layer KCs of the central outer root sheath. When human scalp anagen VI HFs were treated with the specific synthetic OR2AT4 agonist, Sandalore , this significantly retarded spontaneous catagen development of HFs ex vivo and significantly decreased hair matrix KC apoptosis (TUNEL/caspase-3), possibly via down-regulating the expression of the catagen-promoting growth factor TGFb2 and up-regulating the expression of the key anagenmaintenance growth factor, IGF-1. Most of these effects were counteracted by co-administering the OR2AT4 antagonist, Phenirat . When OR2AT4 expression was silenced in human scalp HFs (the knock-down efficiency ex vivo was low), HF cycling appeared unaffected by it in short-term HF organ culture. However, OR2AT4 silencing significantly enhanced both HF KC apoptosis and TGFb2 expression. This supports that OR-mediated signalling regulates human HF KC biology in situ and suggests that OR2AT4 stimulation by (as yet unknown) endogenous ligands is required to down-regulate KC apoptosis and TGFb2 expression in human anagen HFs. These data constitute the first evidence that human HFs can “smell” in the sense that they engage in OR2AT4-mediated chemosensation to regulate their own growth. If the odorant, Sandalore , is also topically effective, it may become an adjuvant therapy for managing hair growth disorders characterized by premature catagen entry (e.g. telogen effluvium, alopecia areata). S198 Journal of Investigative Dermatology (2016), Volume 136 217 The adenosine-generating ecto-enzyme CD73 regulates human hair growth LM Ponce Moya, M Bertolini, Y Uchida, K Figlak, J Cheret, H Waldmann and R Paus 1 Dermatology, University of Munster, Munster, Germany, 2 Queen Mary University of London, London, United Kingdom, 3 Monasterium Laboratory, Munster, Germany, 4 Pathology, Oxford University, Oxford, United Kingdom and 5 Dermatology Rs Ctr, University of Manchester, Manchester, United Kingdom The immunoihibitory ecto-5’-nucleotidase CD73 metabolizes pericellular AMP to adenosine, with the latter being implicated in hair follicle (HF) growth modulation. Therefore, we hypothesized that CD73 may be expressed in human HFs and might impact on their growth. Our results revealed that CD73 is strongly expressed in HF mesenchyme, namely in endothelial and immune cells, and in the HF epithelium, in cytokeratin 74+ keratinocytes. The CD73-specific antagonist, adenosine 50-(a,b-methylene)diphosphate (APCP), strongly inhibited hair shaft production and induced catagen development (demonstrated by hair cycle staging). Interestingly, the concentration to which this effect was seen in each patient differed from 25mM to 100mM. In addition, HFs that showed reduced elongation after treatment with APCP, revealed also decreased adenosine concentration (0.1-0.2 mM) in the medium, suggesting that the APCP-dependent elongation inhibition may result from a reduction of adenosine production by CD73. This hypothesis is further confirmed by the fact that CD73-dependent elongation inhibition is reversed in pilot experiments upon treatment of HFs with APCP in combination of 0.1mM adenosine. In addition, hair shaft production and catagen transition were unaffected by silencing CD73 in the mesenchyme, suggesting the intriguing concept that only the intrafollicular CD73 activity associated with HF epithelium is functionally important for human hair growth control. Thus, our study reveals CD73 activity as a novel endogenous regulator of human growth, most likely by modulating the production of anagen-prolonging adenosine within the HF epithelium. Therefore, CD73 deserves to be explored as a novel therapeutic target in the management of human hair growth disorders, e.g. by the topical administration of CD73 inhibitors as candidate anti-hirsutism agents. 218 Chemokine receptor CCR5 is the novel target for the treatment of alopecia areata T Ito, T Suzuki, A Funakoshi, T Fujiyama and Y Tokura 1 Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan and 2 Dermatology, Fujinomiya City General Hospital, Fujinomiya, Japan Alopecia areata (AA) is an organ-specific autoimmune disease with cell-mediated autoimmune reactions. T lymphocytes densely surround hair bulbs in the lesion of acute-phase AA, referred to as “swarm of bees”. The pathological mechanisms of “swarm of bees”, can be induced by the upregulation of Th1 chemokine expression from hair follicles that result in the resultant infiltration of CXCR3 and CCR5 Th1 or Tc1 cells into AA lesions. Here, C3H/HeJ mice with AA were treated with CCR5 inhibitor, Maraviroc, that is HIV drug by negative allosteric modulator of the CCR5 receptor. Specifically, maraviroc is a negative allosteric modulator of the CCR5 receptor. At first, AA lesions were experimentally induced in C3H/HeJ by subcutaneous injection of T cells that were derived from LNs of AA-bearing syngeneic mice and stimulated IL-2, IL-7, and IL-15. Then, maraviroc is orally administrated. Interestingly, 4/5 maraviroc-treated C3H/HeJ mice with AA showed improvement of hair loss lesions after 2 weeks. Immunohistological assessments revealed decreased number of CD4CCR5 and CD8CCR5 T cells in the lesions after maraviroc treatment. Furthermore, FACS analysis also supports revealed the reduced frequency of CD4CCR5 T cells in skin-infiltrating cells. Next, CD4+ T cells from LN cells were incubated with 0, 1 and 10 mM maraviroc for 8 hours. Then, chemotactic activity was analyzed by EZTaxiscan. Interestingly, the significant inhibition of the velocity of CD4 LN cells toward RANTES by maraviroc was found compared to PBS-treated LN cells. Mean intensity of CCR5 was slightly downregulated by maraviroc treatments in CD4 T cells. In conclusion, inhibition of chemokine receptors/chemokines can be the novel target for the treatment of AA. 219 STAT5 activation in the dermal papilla is important for hair follicle growth phase induction, hair follicle regeneration and wound healing J Legrand, R Villani, E Roy and K Khosrotehrani Centre for Clinical Research, The University of Queensland, Brisbane, QLD, Australia Hair follicles are skin appendages that undergo periods of growth (anagen), regression (catagen) and rest (telogen), regulated by their mesenchymal component, the dermal papilla (DP). Based on reports of its specific expression in the DP, we investigated STAT5 activation during hair development and cycling. STAT5 activation in the DP began in late catagen, reaching a peak in early anagen before disappearing for the rest of the cycle. This was confirmed by the expression profile of SOCS2, a STAT5 target in the DP. This pattern of expression starts after the first postnatal hair cycle. In order to conditionally ablate STAT5A and B in the DP, we used Sox18Cre/ER mice. Indeed Sox18 was expressed in developing dermal placodes for all three hair waves at E14.5, E16.5 and E18.5. Its expression post-natally was associated with zigzag hairs only. Remarkably dominant negative loss of function mutants of Sox18 did not display any Auchene or zigzag hair. Quantification of hair cycling using the Flash canonical Wnt signalling in vivo bioluminescence reporter found that conditional knockout of STAT5A/B in the DP targeted through Cre recombinase under the control of the Sox18 promoter induced at birth, resulted in delayed anagen entry compared to control. Of note, this method did not delete STAT5 in Sox2 expressing HFs. Microarray analysis of STAT5deletion versus control revealed key changes in TNFalpha, Wnt and FGF ligands, known for their role in inducing anagen entry. Pharmacological inhibition of Jak1-2 did not alter STAT5 activation in the DP. Finally, the use of DP spheroids with active STAT5 resulted in more robust hair follicle regeneration in patch assays and more robust engraftment and acceleration of wound healing in vivo. We conclude that STAT5 activation acts as a mesenchymal switch to trigger natural anagen entry in post-developmental hair follicle cycling.

Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune disease of the hair follicle (HF) in which the collapse of HF immune privilege (IP) plays a key role. Mast cells (MCs) are crucial immunomodulatory cells implicated in the regulation of T cell-dependent immunity, IP, and hair growth. Therefore, we explored the role of MCs in AA pathogenesis, focusing on MC interactions with CD8+ T-cells in vivo, in both human and mouse skin with AA lesions. Quantitative (immuno-)histomorphometry revealed that the number, degranulation and proliferation of perifollicular MCs are significantly increased in human AA lesions compared to healthy or non-lesional control skin, most prominently in subacute AA. In AA patients, perifollicular MCs showed decreased TGFβ1 and IL-10 but increased tryptase immunoreactivity, suggesting that MCs switch from an immuno-inhibitory to a pro-inflammatory phenotype. This concept was supported by a decreased number of IL-10+ and PD-L1+ MCs, while OX40L+, CD30L+, 4–1BBL+ or ICAM-1+ MCs were increased in AA. Lesional AA-HFs also displayed significantly more peri- and intrafollicular- CD8+ T-cells as well as more physical MC/CD8+ T-cell contacts than healthy or non-lesional human control skin. During the interaction with CD8+ T-cells, AA MCs prominently expressed MHC class I and OX40L, and sometimes 4–1BBL or ICAM-1, suggesting that MC may present autoantigens to CD8+ T-cells and/or co-stimulatory signals. Abnormal MC numbers, activities, and interactions with CD8+ T-cells were also seen in the grafted C3H/HeJ mouse model of AA and in a new humanized mouse model for AA. These phenomenological in vivo data suggest the novel AA pathobiology concept that perifollicular MCs are skewed towards pro-inflammatory activities that facilitate cross-talk with CD8+ T-cells in this disease, thus contributing to triggering HF-IP collapse in AA. If confirmed, MCs and their CD8+ T-cell interactions could become a promising new therapeutic target in the future management of AA.

Alopecia areata (AA) may represent a CD8+T cell-mediated, organ-specific autoimmune disease in which as yet elusive autoantigens are recognized, once they become exposed by ectopic major histocompatibility complex class I expression by anagen hair follicles (HFs) that have lost their relative immune privilege (IP). On this basis, AA research is chiefly challenged with identifying the autoreactive CD8+T cells and their cognate autoantigens as well as key inducers of HF-IP collapse and "HF-IP guardians" that prevent and/or can restore IP collapse. However, natural killer group 2D-positive (NKG2D+) cells (incl. NK, NKT, and CD8+T cells) and NKG2D-activating ligands from the MICA (MHC I-related chain A) family may also have a key role in AA pathogenesis, as a massive infiltrate of IFN-γ-secreting NKG2D+ cells alone suffices to induce the AA phenotype. Therefore, we speculate that AA may represent a stereotypic, but distinct HF response pattern to inflammatory insults associated with HF-IP collapse.