39th Annual Meeting of the European Society for ...

39th Annual Meeting of the European Society for Dermatological Research 09-12 September 2009, Budapest, Hungary

Abstract Table of Contents Square brackets beside poster numbers denote [ORAL] Presentations: [001-024] are Plenary, [025-096] Concurrent

ESDR 2009 Immunology Abstracts 001-089 Clinical Investigation and Epidemiology Abstracts 090-185 Photobiology, Pigmentation and Melanoma Abstracts 186-261 Genetic Disease, Gene Expression and Gene Therapy Abstracts 262-330 Epidermis and Hair Structure and Function Abstracts 331-419 Carcinogenesis and Angiogenesis Abstracts 420-468 Cell Adhesion, Matrix Biology and Growth Factors Abstracts 469-520 Auto-Immunity, Inflammation and Infectious Diseases Abstracts 521-615 Satellite Meeting at ESDR 2009 Abstracts from Neurobiology of the Skin Symposium 10 September 2009, Budapest, Hungary

ABSTRACTS

2 Journal of Investigative Dermatology (2009), Volume 129

ABSTRACTS 001 [Oral 009]

Generation of myeloid derived suppressor cells during the progression of murine RET melanoma Taku F1, Mahnke K1, Ring S1, Umansky V2, Aiba S3, Enk AH1 1University of Heidelberg, Heidelberg, Germany 2German Cancer Research Center, Heidelberg, Germany 3 Tohoku University, Graduate School of Medicine, Sendai, Japan Myeloid derived suppressor cells (MDSC) comprise a phenotypically heterogeneous population of cells (CD11b+, Gr-1+, Ly6G+, Ly6C+), which can be found in tumor bearing mice and in patients with cancer. Though there are several reports, which suggest that MDSC suppress the activity of T cells during tumor growth, the exact mechanism of their suppressive function is still unclear. To investigate the expression of suppressive molecules (B7H1, B7H3, B7H4) by MDSC during melanoma growth, we injected RET melanoma cells into C57BL/6 mice. After tumors had reached defined sizes, mice were sacrificed and single cell suspensions of tumors and spleens were prepared. FACS analysis revealed significant numbers of Ly6C+CD124+ MDSC within the CD11b+ cells in the tumor. Interestingly, there were almost no Ly6G+ cells within the CD11b+ population in the tumor. Moreover, the CD11b+Ly6C+ cells initially upregulated the expression of B7H1, B7H3 and B7H4, whereas these molecules were gradually downregulated during tumor growth. When CD11b+ cells were isolated by MACS and co-cultivated with syngeneic CD4+ T cells and anti-CD3 antibodies, tumor-derived CD11b+ cells suppressed the proliferation of CD4+ T cells by NO production. In contrast, splenic CD11b+ cells from tumor bearing mice or control mice did not upregulate suppressive molecules in both Ly6G+ or Ly6C+ populations. As expected from FACS data, splenic CD11b did not suppress the proliferation of syngeneic CD4+ T cells. Our results suggest that the main population of tumor infiltrating CD11b+ cells were Ly6C+Ly6G- cells, which suppress T cell proliferation by upregulation of suppressive molecules.

002 [Oral 012]

Vaccination against autoimmune disease through in vivo silencing of dendritic cells with a specific IL-12/IL23p40 si-RNA Brück J1, Weigert C1, Pascolo C2, Glocova I1, Fuchs K1, Ghashghaeinia M1, Hötzenecker W1, Ghoreschi K1, Röcken M1 1Department of Dermatology, Tübingen, Germany 2 University Hospital of Zürich, Oncology, Zürich, Switzerland Experimental autoimmune encephalomyelitis (EAE) is a prototypic organ-specific autoimmune disease, mediated by inflammatory myelin-specific T helper (Th) cells. Destructive inflammation within the central nervous system -producing Th1 cells is initiated by the infiltration of IFN-g-producing Th1 cells and IL-17-producing Th17 cells. CFA stimulates dendritic cells (DC) through Toll-like receptors (TLR), and induces inflammatory mediators such as IL-12, IL-23 and IL-6 that promote the differentiation of naïve Th cells into Th1 or Th17 cells respectively. IL-12 and IL-23 are heterodimers sharing the IL-12/ IL-23p40 subunit. Experiments with knockout mice or therapies with antibodies revealed that the IL12/-23p40 subunit is crucial for the development of EAE and other autoimmune diseases. Antibodies against IL-12/IL-23p40 improve EAE and psoriasis but bear the risk of severe infections, due to the polyclonal neutralization of the IL-12/IL-23p40 molecule. In contrast to antibodies, siRNA is short lived and interferes with immune responses for very limited periods of time. We therefore asked, whether siRNA allows establishing protective immunity when applied at the time of DC activation. We synthesized various siRNA directed against IL-12/IL-23p40 and identified one candidate siRNA capable to inhibit IL-12/IL-23p40 production by DC in vitro after TLR-stimulation. Naïve SJL-mice were immunized with PLP in CFA and received either IL-12/IL-23p40 or control siRNA. In the presence of IL-12/IL-23p40 siRNA IL-12/IL-23p40 production by DC was impaired in vitro and in vivo. Vaccination of mice with IL-12/IL-23p40 siRNA during immunization significantly improved the clinical course of EAE (20% with clinical symptoms in the siRNA group vs 100% in the control). Our results may provide the basis for new vaccination strategies with IL-12/IL-23p40 siRNA establishing protective immunity against organ-specific inflammatory autoimmune diseases, including psoriasis.

003 [Oral 014]

Essential roles of actin cytoskeleton formation through mDia1 in skin dendritic cell functions Hideaki T, Gyohei E, Shuh N, Yoshiki M, Kenji K Kyoto University, Kyoto, Japan Antigen acquisition, migration, maturation, and T cell stimulation of cutaneous dendritic cells (DCs) are essential to initiate acquired cutaneous immune responses. The critical mechanism for cell machinery is formation of actin nucleation and polymerization, which are facilitated by two groups of proteins, the mDia family of formins and Wiskott-Aldrich syndrome protein (WASP). Although WASP is known to be important for immunological synapse formation, the role of mDia remains unknown. Initially, we found that mDia1 mRNA was dominantly expressed among three isotypes, mDia1-3 in bone marrow-derived dendritic cells (BMDCs) and Langerhans cells (LCs) by quantitative PCR. To evaluate its roles, we generated mDia1-deficient mice and backcrossed 10 times to C57BL/6 mice . Although the proliferation and maturation of BMDCs were comparable between wild-type mice and mDia1-deficient mice, phagocytosis of FITC-dextran and FITC-induced cutaneous DC migration were significantly impaired in mDia1-deficient mice. In addition, immunofluorescent staining with phalloidin showed incomplete actin bundle and podosome formation in mDia1-deficient mice. Moreover, two-photon microscopy for three-dimensional time-lapse imaging revealed that immunological synapse formation of mDia1-deficient DCs to OT-II CD4+ T cell were impaired. We also observed OT-II CD4 and OT-I CD8 T cell proliferation was attenuated by OVAexposed mDia-deficient BMDC. Consistently, DNFB-induced contact hypersensitivity and OVA-induced delayed-type hypersensitivity responses were impaired in mDia1deficient mice. These results suggest that actin polymerization through mDia1 is essential for cutaneous DC functions to initiate acquired cutaneous immune responses.

004 [Oral 025]

Adenosine produced by CD4+CD25+ regulatory T cells suppresses contact hypersensitivity reactions by engaging the A1 adenosine receptor on vascular endothelium Ring S1, Oliver S2, Cronstein B2, Enk AH1, Mahnke K1 1University Hospital Heidelberg, Heidelberg, Germany 2New York University School of Medicine, New York, USA Injection of adenosine or its agonist NECA into TNCB sensitized mice abrogates the elicitation phase of contact hypersensitivity (CHS) reactions by blocking the adherence of leukocytes to vascular endothelium. Since Treg express the ectonucleosidases CD39/ CD73, which degrade ATP into adenosine, we set out to investigated the role of Treg derived adenosine in the suppression of the T cell - endothelial cell (EC) interaction. In initial in vitro experiments we superfused activated EC with T cells and detected substantial adherence of T cells to the EC. In contrast, pretreatment of the EC with Treg or adenosine, respectively, reduced the number of adherent T cells by 80% as compared to untreated controls. Furthermore we show that these effects are mediated by the adenosine receptor A1, since blocking of the A1 receptor restores the adherence of the T cells to the EC. Likewise in vivo, we could show that adenosine is essential for the Treg mediated anti-inflammatory effect in CHS responses, since Treg isolated from CD39 deficient mice failed to prevent adhesion of leukocytes to the endothelium as compared to wt Treg. Injection of the A1 adenosine receptor antagonist CPCPX before treatment with wt Treg abrogated their suppressive function and an ear swelling reaction was recorded. When analysing the effects of Treg on adhesion molecules involved in mediating adherence of effector cells to EC we found, that the expression of E- and P-selectin by the vascular endothelium was downregulated by Treg in vivo and in vitro. In aggregate our data indicate that CD39-driven adenosine release by Treg downregulates expression of adhesion molecules by EC via the A1 adenosine receptor providing a novel mechanism by which Treg mediate suppression of CHS responses in vivo.

005 [Oral 026]

The RhoA effector mDia1 regulate motility and function in T lymphocytes Egawa G1, Tanizaki H1, Okada T2, Narumiya S1, Miyacchi Y1, Kabashima K1 1Kyoto University, Kyoto, Japan 2RIKEN, Yokohama, Japan Lymphocytes, which are the most motile cells in whole body, exhibit dynamic change in their receptor expression, cell polarity, and constitution of cytoskeleton to reach its destination. Rho-GTPase (Rho, Cdc42, Rac) play a central role in the modulation of cytoskeleton, and it is known that genetic deletion of WASP, the effector of Cdc42, induce atopic dermatitis like skin inflammation and autoimmunity. Moreover, we recently reported that mDia1, one of the effectors of RhoA, is essential for thymocytes egress and T lymphocytes entry into the lymph nodes. These reports indicate a close relation between the actin dymamics of T lymphocytes and its immunological function.To this end, we examined T lymphocytes motility in ex vivo lymph nodes using two-photon microscope and found that mDia1-deficient T lymphocyte appeared round shape and mobility impairment. Additionally, treatment of Y-27632, a specific inhibitor of other RhoA effectors, ROCK1/2, resulted in further T lymphocytes dysmotility, suggesting adequate actin nucleation by RhoA family molecules are essential for normal T lymphocytes motility in lymph nodes. We also characterized chemotactic and homing ability of mDia1-deficient T lymphocytes and found that defect of actin regulation resulted in impairment of immunological response formation. Our findings indicate that regulation of cytoskeleton is fundamental mechanism for T lymphocytes function.

006 [Oral 027]

Local UVB-induced immunosuppression is mediated by IL-10-produing and OX40L-positive mature Langerhans cells Yoshiki R1, Kabashima K2, Sugita K1, Nakamura M1, Tokura Y1 1University of Occupational and Environmental Health, Kitakyushu, Japan 2Kyoto University, Kyoto, Japan The mechanism underlying the local UVB-induced immunosuppression is a central issue to be clarified in photoimmunology. There have been reported a considerable number of cells and factors that participate in the sensitization phase-dependent suppression, including Langerhans cells (LCs), T regulatory cells (Tregs), IL-10, and TNF-a. The recent surprising finding that LC-depleted mice rather exhibit an increased contact hypersensitivity (CHS) response urges us to re-evaluate the LC-related mechanism of UVB-induced immunosuppression. We studied the surface expression of OX40L and the intracellular expression of IL-10 in LCs and dermal dendritic cells (dDCs) from UVB (300 mJ/cm2)-irradiated mouse skin and those migrating to the regional lymph nodes from UVB-irradiated, FITC-painted mice. In suspensions of epidermal and dermal cells prepared from UVB-irradiated ears, Langerin+ MHC class II+ cutaneous DCs expressed OX40L as well as CD86 and produced IL-10 at higher levels than Langerin- MHC class II+ cells, suggesting that LCs in UVBirradiated skin elaborate the immmunoregulatory surface molecule and cytokine. When freshly isolated epidermal cells were cultured with RANKL, whose production by UVB-exposed kertinocytes was confirmed by flow cytometry, the IL-10 production by LCs was increased. In mice sensitized with FITC on the UVB-preirradiated skin, antigen-specific CD4+CD25+ Tregs capable of suppressing CHS to FITC were induced. In the UVB and FITC-treated mice, Langerin+ LCs migrating from the skin to the regional lymph nodes produced a high amount of IL-10 compared to LangerinDCs. These findings suggest that IL-10-producing mature LCs induced by RANKL are responsible for the induction of Tregs and the resultant immunosuppression.

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ABSTRACTS 007 [Oral 028]

The role of mast cells in contact hypersensitivity using newly-generated mast cellspecific conditional depletion model Otsuka A, Miyachi Y, Kabashima K Kyoto University, Kyoto, Japan The role of mast cells in contact hypersensitivity (CHS) response is controversial because some studies have shown that mast cell-deficient mice have intact CHS responses whereas others have shown defective elicitation of CHS. In those studies, W/Wv mice containing a point mutation in stem cell factor (SCF) receptor (c-Kit), were used as mast cell-deficiency model. However, W/Wv mice were not only lack of mast cells but also anemic and melanocyte deficient. Therefore, to evaluate the role of mast cells during CHS, pure mast cell deficient mice model are required. A recent study indicated that conserved noncoding sequence 2 (CNS-2) is an essential enhancer for interleukin 4 gene transcription specific in mast cells. CNS2-deficient mice could not produce IL-4 or IL-13 in both the connective tissuetype and immature-type of mast cells but not in basophils. Taking advantage of this system, mast cell specific–diphtheria toxin receptor mediated conditional cell knock out (MaS TRECK) mice were newly generated. In this mouse, mast cells in the peritoneal cavity, skin, and stomach were completely depleted after injection of diphtheria toxin and recovered within 7 days. On the other hand, the numbers of CD4, CD8 NKT cells were not altered. We used these mice to address the roles of mast cell in the CHS response. When mast cells were depleted during both sensitization and elicitation, or only during sensitization phase or elicitation phase in CHS, the extent of ear thickness change was attenuated. Consistently, mast cell depletion suppressed edema and inflammatory cell infiltration, especially CD4+ cells, in the dermis. Therefore, MasTRECK mice are valuable system to evaluate the mast cell functions at each time point in vivo, and the above results suggest that mast cells play an essential role in CHS during both sensitization and elicitation phase.

008 [Oral 029]

Surgical denervation of skin in the KC-Tie2 mouse model of psoriasis eliminates dendritic cell infiltrate, decreases IL-23 protein expression and improves acanthosis Ostrowski SM, Belkadi AM, Matheny C, Diaconu D, Wolfram JA, Hatala DA, Ward NL Case Western Reserve University, Cleveland, USA A role for the nervous system in psoriasis is evidenced by increases in the number of nerve fibers and neuropeptides in psoriatic skin, the symmetrical distribution of plaque, and disease exacerbation related to stress. Most interestingly may be the clinical finding that following accidental denervation psoriatic plaques have been reported to clear. Although many hypotheses have been proposed, the mechanisms underlying these observations remain unclear. We recently generated the KC-Tie2 mouse, a new murine model of human psoriasis. Using this model system, we observed a ~2.5-fold increase in the number of PGP9.5+ nerve fibers in KC-Tie2 mouse back skin compared with littermate controls (p=0.0001) and a 4.1 and 9.2 fold increase in substance P (SP) and neuropeptide Y (NPY), respectively. To examine the influence of nerves and nerve-derived factors on psoriasis maintenance, we unilaterally surgically denervated the dorsal cutaneous nerves at their anatomical entry into back skin of KC-Tie2 animals. Ten days post-surgery, denervation was confirmed using PGP9.5 immunohistochemistry and measurement of protein expression of PGP9.5. We also determined that the nerve derived molecules SP and NPY were decreased in denervated compared with innervated skin. Interestingly, histological examination of the psoriasiform phenotype following cutaneous denervation revealed a a 34% decrease in acanthosis (p=0.014). CD4+ T cells and Th1 and Th17 cytokine expression remained unchanged as did the number of F4/80+ macrophages, however, the number of CD11c+ dermal DCs decreased significantly in denervated skin (50%, p=0.002), returning to control mouse levels. In addition, IL23 protein expression decreased by 33% in denervated compared with innervated skin. These data provide experimental evidence suggesting that nerves and their derived factors influence DCs and KCs and contribute to inflammation and acanthosis observed in psoriasis. Elucidation of the mechanisms underlying these findings may lead to novel therapeutic approaches for the treatment of psoriasis.

009 [Oral 030]

Alteration of the migratory behavior of UV-induced regulatory T cells in vivo Schwarz A, Schuller W, Schwarz T University of Kiel, Kiel, Germany UV-induced regulatory T cells (UV-Treg) inhibit the sensitization but not the elicitation of contact hypersensitivity when injected i.v, since UV-Treg express lymph node homing, but not skin homing receptors and migrate into the lymph nodes but not the skin. We demonstrated that the migration and the homing receptor expression of UV-Treg can be altered by tissue-specific antigen presenting cells (APC) in vitro. To prove whether this phenomenon can also be observed in vivo, we obtained UV-Treg from mice which were sensitized against dinitrofluorobencene (DNFB) through UVexposed skin. UV-Treg were injected i.v. into mice which were DNFB-sensitized 5 days earlier. To activate the i.v. injected UV-Treg in the lymph nodes with DNFB-bearing skin-derived APC, recipient mice were boosted with DNFB on the abdomen and ear challenged 24 hours later. Sensitized but unboosted mice were not suppressed in their challenge despite the injection of UV-Treg. When mice were boosted with DNFB 24 hours after injection of UV-Treg, the ear swelling response was suppressed, suggesting that UV-Treg upon the DNFB boost through the skin migrate into the periphery. The experiment was repeated but UV-Treg were labelled with CFSE. Ears were cut and analyzed by fluorescence microscopy. In mice which were not boosted with DNFB after injection of UV-Treg no CFSE-labelled cells were detected in the ears. In contrast, CFSE-positive cells were found in the ears of mice which were DNFB-boosted after injection. In turn, CFSE-labelled cells were detected in the lymph nodes of unboosted mice, but absent in boosted mice. This indicates a first option to alter the migratory behavior of Treg in vivo and thus may have input on strategies trying to utilize Treg not only for the prevention but also for the treatment of immune-mediated disorders.

4 Journal of Investigative Dermatology (2009), Volume 129

010 [Oral 031]

IL-4 therapy of psoriasis selectively abrogates IL-23 and TH17 immune responses in humans Guenova E1, Ghoreschi K1, Hötzenecker W1, Sauer K1, Weindl G2, Tham M1, Schäkel K3, Schaller M1, Röcken M1, Biedermann T1 1Eberhard Karls University Tübingen, Germany 2 Freie Universität Berlin, Germany 3Technische Universität Dresden, Germany Over the last decade, psoriasis was characterized as prototypic Th1 disease mediated by IFN-γ producing Th cells. More recent data suggested that also Th17 cells and the Th17 driving cytokines such as IL-23 are involved. Consequently, targeting the IL-12/23p40 subunit improved psoriasis. Systemic IL-4 therapy also significantly improved psoriasis as shown in a dose escalation study. This IL-4 treatment induced intra-lesional IL-4 expression, but did not significantly suppress IL-12 or IFN-γ. Therefore, we investigated the in vivo effects of IL-4 therapy on the IL-23/Th17 pathway in psoriasis. While IL-12p35 mRNA was rather increased following IL-4 therapy, this treatment dose dependently suppressed cutaneous IL-23p19 and IL-17 mRNA expression to