217 The adenosine-generating ecto-enzyme CD73 regulates human hair growth

Abstract

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.