The Editor’sChoice

Y. Tokura

JOURNAL OF

Dermatological Science

VOLUME 74 NUMBER 3

Page 185 June 2014 J DERMATOL SCI

Blockade of Syk ameliorates the development of murine sclerodermatous chronic graft-versus-host disease

Murine sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) is a model for human Scl-cGVHD and systemic sclerosis (SSc). Syk, a protein tyrosine kinase, is expressed in most of hematopoietic cells, fi broblasts, and endothelial cells. Huu investigated the effect of R788 (fostamatinib sodium), an oral prodrug that is rapidly converted to a potent inhibitor of Syk, R406, on Scl-cGVHD. R788 was orally administered to allogeneic recipients from day 14 to day 42 after bone marrow transplantation (BMT). In vitro, proliferation of GVHD-derived CD4+ T cells and CD11b+ cells was analyzed by R406. Allogeneic BMT increased Syk phosphorylation in T, B, and CD11b+ cells. The administration of R788 attenuated severity and fi brosis of Scl-cGVHD. The elevated expressions of CXCR4 on T cells, B cells, and CD11b+ cells were signifi cantly down-regulated by R788 treatment. R788 reduced memory CD4+ T cells. R406 inhibited proliferation of GVHD CD4+ T cells and CD11b+ cells in vitro. In addition, R788 treatment, inhibited proliferation of

CD11b+ cells in Scl-cGVHD mice. R788 treatment also reduced skin mRNA expressions of key cytokines. Blockade of Syk suppressed migration factor of immune cells and antigen-specifi c memory CD4+ T cells and proliferation and activation of GVHD CD4+ T cells and CD11b+ cells. Thus, Syk inhibitor is a potential candidate for use in treating patients with Scl-cGVHD and SSc.

Infl uence of sensory neuropeptides on human cutaneous wound healing process

Close interactions exist between primary sensory neurons of the peripheral nervous system (PNS) and skin cells. The PNS may be implicated in the modulation of different skin functions as wound healing. Chéret et al incubated injured human skin explants either with rat primary sensory neurons from dorsal root ganglia (DRG) or different neuropeptides (vasoactive intestinal peptide or VIP, calcitonin gene-related peptide or CGRP, substance P or SP) and evaluated their effects on the proliferative and extracellular matrix (ECM) remodeling phases, dermal fi broblasts adhesion and differentiation into myofi broblasts. DRG and all studied neuromediators increased fi broblasts and keratinocytes proliferation and act on the expression ratio between collagen type I and type III in favor of collagen I. Furthermore, the enzymatic activities of matrix metalloprotesases (MMP-2 and MMP-9) were increased in the fi rst days of wound healing process. The adhesion of human dermal fi broblasts and their differentiation into myofi broblasts were promoted after incubation with neuromediators. The most potent concentrations for individual tested molecules, were the lowest concentrations, corresponding to physiological concentrations. Thus, sensory neurons and their derived-neuropeptides are able to promote skin wound healing

Fig. 1. Schematic representation of the culture model used to evaluate the impact of peripheral nervous system of the wound healing process of injured human skin. Human skin wound healing assay was obtained by performing an internal injury on the 6 mm Ø human skin explants (HSE) with a smaller punch of 3 mm Ø. HSE were cocultured with DRG neurons in the same dish or incubated with neuromediators (SP, CGRP or VIP at 10–10 M, 10–8 M or 10–6 M) for 3, 7 and 10 days. Histological pictures of the injured area of HSE were realized at D3 or D10 of culture without any treatment on cryopreserved HSE, cut in 7 �m sections and stained with Trichrome stain. D = day; DRG = Dorsal root ganglia; SP = substance P; CGRP = Calcitonin Gene-related peptide; VIP = Vasoactive intestinal peptide.

A novel small compound accelerates dermal wound healing by modifying infi ltration, proliferation and migration of distinct cellular components in mice

Transforming growth factor (TGF)-� is known to stimulate collagen production in dermal fi broblasts while inhibiting proliferation of epider-mal keratinocyte. A screening of small compounds that suppress type I collagen production in fi broblasts has identifi ed HSc025 that antagonizes the TGF-�/Smad signal. Yamaoka et al examined the effects of HSc025 on dermal wound healing and elucidated the underlying mechanisms. Effects of HSc025 on the wound closure process were evaluated in a murine full-thickness excisional wound healing model. Cell proliferation and migration were estimated using primary cultures of human keratinocytes and fi broblasts. Comprehensive analyses of gene expression profi les were performed using untreated and HSc025-treated fi broblasts. Oral HSc025 administration suppressed macrophage infi ltration and accelerated wound closure as early as at day 2 after the dermal excision. Treatment of cultured keratinocytes with HSc025 counteracted the inhibitory effects of TGF-� on cell proliferation and migration. On the other hand, HSc025 stimulated migration, but not proliferation, of dermal fi broblasts independently of TGF-�. Experiments using an artifi cial dermis graft revealed that HSc025 stimulated migration of collagen producing cells into the graft tissue. Pirin was identifi ed as a critical mediator accelerating fi broblast migration.

Thus, HSc025 accelerates wound healing by modifying infi ltration, prolif-eration and migration of distinct cellular components, which provides a novel insight into the therapy for intractable skin ulcer.

Fig. 5a,b. Migration of collagen-producing cells into artifi cial dermis grafts. A full-thick-ness excisional wound made on the dorsum of female COL/EGFP mice (24 to 28 weeks of age) was embedded with an artifi cial dermis graft. Mice were then treated with daily local injections of 200 �l of either saline (A) or 100 �M of HSc025 (B) into the wounds. Dermal specimens were obtained at day 7 and analyzed under a fl uorescent microscope to examine migration of EGFP-positive cells (green) into the artifi cial dermis graft. The yellow hatched line indicates the border between the autologous tissue and the embed-ded artifi cial dermis graft. The location of migrating EGFP-positive cells was divided intofour categories (red square boxes) depending on the distance from the wound edge. The cell numbers were counted and compared between untreated and HSc025-treated mice

Fig. 4a. Syk inhibitor (R406) blocks proliferation of GVHD-derived CD4+ T cells and CD11b+ cells. CD4+ T cells and CD11b+ cells were purifi ed from GVHD spleens 14 days after BMT by MACS microbead, then labeled with CFSE. (A) CFSE-CD4+ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies in presence of various concentrations of R406 for 4 days.

J DERMATOL SCI June 2014 Page 186

Distribution of IL-31 and its receptor expressing cells in skin of atopic dermatitis

Interleukin-31 (IL-31) is well known as an anti-pruritic cytokine. There is a lack of direct evidence concerning the cellular origin of IL-31 in atopic dermatitis (AD) skins, and data on the expression of IL-31RA in AD are inconsistent. Also, there is no available information regarding IL-31RA protein expression in human dorsal root ganglia (DRG), which mediates the sensation of itch and is the long-suspected source of the protein. Kato et al sought to obtain direct evidence concerning the distribution of IL-31- and IL-31RA-protein expressing cells and their characteristics in AD skin samples and in human DRG. IL-31 was detected immunohistochemically in AD skins, and representative sections were double stained with IL-31 and several immune-markers. IL-31RA was stained immunohistochemically in AD skins and normal human DRG, and representative AD skins were double stained with IL-31RA and PGP9.5 (a nerve marker). IL-31-positive cells were observed as mononuclear infi ltrating cells and as CD11b co-expressing cells in severe AD samples. As for IL-31RA, positive reactions were detected in keratinocytes and nerve fi bers in the dermis of AD and in the neurons of normal DRG. The detection of IL-31 in infi ltrating cells of severe AD skin and of IL-31RA in nerve fi bers of AD dermis and normal DRG indicates

IL-31 signaling may be a contributing factor in the persistence and exacerbation of AD skin lesions.

Defective maintenance of pH of stratum corneum is correlated with preferential emergence and exacerbation of atopic-dermatitis-like dermatitis in fl aky-tail mice

Neutralization of stratum corneum (SC) pH, which is induced by a variety of stimuli, such as scratching, use of soap and infl ammation, can stimulate activity of serine protease (SPase). Activation of SPase induces production of thymic stromal lymphopoietin (TSLP) through protease-activated receptor-2. Both reduced expression of natural moisturizing factors, which are required for maintenance of SC pH, and the preferential development of atopic dermatitis (AD)-like dermatitis are found in fl aky-tail mice (FTM). Sakai et al examined possible correlations between disturbance of responses to an exogenous stimulus of SC neutralization and the preferential emergence of AD-like dermatitis in FTM. FTM and wild-type mice were subjected to an SC-neutralization stimulus via application of 1,1,3,3-tetramethylguanidine (TMG). TMG was applied to young mice at a time when FTM had not yet developed signifi cant dermatitis, and we examined their ability to maintain SC acidity and several parameters associated with AD-like dermatitis. The recovery of SC pH after the application of TMG was delayed in FTM. Cutaneous infl ammation with elevated SPase activity and serum levels of TSLP, thymus and activation-regulated chemokine and IgE were induced only in TMG-treated FTM. The

results suggest that defective maintenance of pH of SC is correlated with emergence and exacerbation of AD-like dermatitis in FTM.

Fig. 1. Identifying the IL-31-positive cells. (a) Microphotograph of a representative case of IL-31-positive mononuclear cells infi ltrating the AD lesion. Bar = 100 �m. (b) IL-31-positive infi ltrating cells at a higher magnifi cation. Arrow: foot process-like structure. Bar = 10 �m. (c–e) IL-31-positive cells (green) in different tissue sections of the same AD skin sample. (f–h) CD3-, CD11b- and CD11c-positive cells (red) in the same areas of the sections in c, d and e, respectively. Orange arrows: double positive cells. White arrows: single positive cells. Bar = 10 �m.

Fig. 5. SPase activity and expression of TSLP in epidermis after 2 weeks of daily application of TMG or vehicle. (a and b) Skin samples were obtained from WT and FTM after 2 weeks of daily application of TMG or vehicle. SPase activity (a) and expression of TSLP (b) in epidermis were examined by in situ zymography and immunohistochemical staining, respectively. Green staining represents SPase activity or expression of TSLP and red counterstaining represents nuclear staining with propidium iodide. (c and d) The fl uorescence intensity of SPase activity (c) or TSLP (d) in the epidermis was measured as described in the text. n = 12–13 (from three mice). SPase activity and expression of TSLP in epidermis were enhanced only in TMG-treated FTM. Scale bar = 20 �m. **p < 0.01. NS, not signifi cant.

Human in vitro skin organ culture as a model system for evaluating DNA repair

UV-exposures result in accumulation of genetic lesions that facilitate the development of skin cancer. Numerous pharmacologic agents are currently under development to both inhibit formation of DNA lesions and enhance repair. Drugs must be evaluated in vitro, currently performed in cell culture systems, before being tested on humans. Current systems do not account for the architecture and diverse cellularity of intact human skin. Liu establishd a novel, functionally viable, and reproducible in vitro skin organ culture system for studying the effects of various pharmacologic agents on DNA repair. Human skin was obtained from neonatal foreskins. Intact skin punches derived from foreskins were cultured in vitro prior to exposure to UV-irradiation, and evaluated for DNA-damage using a DNA dot blot. Serial skin biopsies were obtained from patients with actinic keratoses treated with topical imiquimod. Expression of immune-stimulating and DNA repair genes was evaluated in ex vivo and in

vitro samples. DNA dot blots revealed active repair of UV induced lesions in our in vitro skin organ culture. The photo-protective effect of sunscreen was detected, while imiquimod treatment did not enhance DNA repair in vitro. The DNA repair molecules XPA and XPF were up-regulated in the skin of imiquimod treated patients with actinic

keratoses and imiquimod treated bone marrow-derived cell lines, but not keratinocytes. This in vitro human skin organ culture model detected repair of UV-induced DNA lesions, and may be easily adapted to investigate various photo-protective drugs intended to prevent or treat skin cancer.

Fig. 3. Immunofl uorescent staining for CBPDs in tissue sections confi rmed active DNA repair. (A) At 0 h following UV irradiation, several CBPD-positive cells were detected within the epidermis (red). (B) However, at 6 h post-UV, there was a marked decrease in CBPD-positive cells, indicating active DNA repair. (For interpretation of the references to color in this fi gure legend, the reader is referred to the web version of this article.)