Tianling Wei

MiR-155 is overexpressed in patients with atopic dermatitis and modulates T-cell proliferative responses by targeting cytotoxic T lymphocyte–associated antigen 4

BACKGROUND MicroRNAs (miRNAs) are short noncoding RNAs that suppress gene expression at the posttranscriptional level. Atopic dermatitis is a common chronic inflammatory skin disease characterized by the presence of activated T cells within the skin. OBJECTIVE We sought to explore the role of miRNAs in the pathogenesis of atopic dermatitis. METHODS Global miRNA expression in healthy and lesional skin of patients with atopic dermatitis was compared by using TaqMan MicroRNA Low Density Arrays. miR-155 expression in tissues and cells was quantified by means of quantitative real-time PCR. The cellular localization of miR-155 was analyzed by means of in situ hybridization. The regulation of cytotoxic T lymphocyte-associated antigen (CTLA-4) by miR-155 was investigated by using luciferase reporter assays and flow cytometry. CTLA-4 expression and functional assays were performed on T(H) cells overexpressing miR-155. RESULTS miR-155 was one of the highest-ranked upregulated miRNAs in patients with atopic dermatitis. In the skin miR-155 was predominantly expressed in infiltrating immune cells. miR-155 was upregulated during T-cell differentiation/activation and was markedly induced by T-cell activators in PBMCs in vitro and by superantigens and allergens in the skin in vivo. CTLA-4, an important negative regulator of T-cell activation, was identified as a direct target of miR-155. Overexpression of miR-155 in T(H) cells resulted in decreased CTLA-4 levels accompanied by an increased proliferative response. CONCLUSION miR-155 is significantly overexpressed in patients with atopic dermatitis and might contribute to chronic skin inflammation by increasing the proliferative response of T(H) cells through the downregulation of CTLA-4.

MiR-125b, a MicroRNA Downregulated in Psoriasis, Modulates Keratinocyte Proliferation by Targeting FGFR2

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes.

Protein Kinase C-Dependent Upregulation of miR-203 Induces the Differentiation of Human Keratinocytes

Terminal differentiation of keratinocytes is a multistep process that requires a coordinated program of gene expression. We aimed to explore the possible involvement of a previously unreported class of non-coding RNA genes, microRNAs (miRNAs) in keratinocyte differentiation by using miRNA expression profiling. Out of 365 miRNAs tested, 7 showed significant change between keratinocytes cultured in low or high calcium concentration. The highest-ranked upregulated gene was miR-203, whose expression was significantly upregulated in response to calcium and other inducers of keratinocyte differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and vitamin D(3). Differentiation-induced upregulation of miR-203 expression was blocked by treatment with specific inhibitors of protein kinase C (PKC), GF109203X, and Ro31-8220. Moreover, our results showed that the activator protein-1 (AP-1) proteins c-Jun and JunB regulate miR-203 expression in keratinocytes. In contrast to inducers of keratinocyte differentiation, epidermal growth factor and keratinocyte growth factor suppressed miR-203 expression in keratinocytes below the basal level. Overexpression of miR-203 in keratinocytes resulted in enhanced differentiation, whereas inhibition of miR-203 suppressed calcium-induced terminal differentiation as judged by involucrin expression. These results suggest that upregulation of miR-203 in human keratinocytes is required for their differentiation and is dependent on the activation of the PKC/AP-1 pathway.

MicroRNA-203 functions as a tumor suppressor in basal cell carcinoma

Basal cell carcinoma (BCC) of the skin represents the most common malignancy in humans. MicroRNAs (miRNAs), small regulatory RNAs with pleiotropic function, are commonly misregulated in cancer. Here we identify miR-203, a miRNA abundantly and preferentially expressed in skin, to be downregulated in BCCs. We show that activation of the Hedgehog (HH) pathway, critically involved in the pathogenesis of BCCs, as well as the EGFR/MEK/ERK/c-JUN signaling pathway suppresses miR-203. We identify c-JUN, a key effector of the HH pathway, as a novel direct target for miR-203 in vivo. Further supporting the role of miR-203 as a tumor suppressor, in vivo delivery of miR-203 mimics in a BCC mouse model results in the reduction of tumor growth. Our results identify a regulatory circuit involving miR-203 and c-JUN, which provides functional control over basal cell proliferation and differentiation. We propose that miR-203 functions as a ‘bona fide’ tumor suppressor in BCC, whose suppressed expression contributes to oncogenic transformation via derepression of multiple stemness- and proliferation-related genes, and its overexpression could be of therapeutic value.

The expression of microRNA‐203 during human skin morphogenesis

Please cite this paper as: The expression of microRNA‐203 during human skin morphogenesis. Experimental Dermatology 2010; 19: 854–856.

Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis

Please cite this paper as: Injury downregulates the expression of the human cathelicidin protein hCAP18/LL‐37 in atopic dermatitis. Experimental Dermatology 2009; 19: 442–449.

MiR-155 is overexpressed in atopic dermatitis and modulates T cell proliferative responses by targeting CTLA-4

S2 Journal of Investigative Dermatology (2010), Volume 130 007 Hypothyroidism improves random-pattern skin fl ap survival in rats Sina Rahimpour1, Behtash G. Nezami1, Negin Karimian1, Maryam SotoudehAnvari2, Sara Khalaj1, Laleh Montaser-Kouhsari1, Ahmadreza Dehpour1 1Pharmacology department, school of medicine,Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic of, 2Department of surgical and clinical pathology,Terhan Heart Center,Terhan University of Medical Sciences,Tehran, Iran, Islamic Republic of The protective effect of hypothyroidism against ischemic or toxic conditions is shown in various tissues. We investigated the effect of hypothyroidism and acute local effect of propylthiouracil (PTU) and methimazole (MMI) on the outcome of lethal ischemia in this fl ap model. Forty-two Sprauge-Dawley rats were randomly divided into 7 groups. In all groups, dorsal fl aps with caudal pedicles were elevated at midline and fl ap survival was measured at the seventh day after surgery. The fi rst group served as control and received 1 ml of 0.9% saline solution into their fl ap before fl ap elevation. In groups 2 and 3, hypothyroidism was induced by administration of either PTU 0.05% or MMI 0.04% in their drinking water for four weeks. Next four groups received local injections of MMI (10, 20, 50 or 100 μg/fl ap) before fl ap elevation. Local PTU injection was ignored due to insolubility of the agent. Hypothyroidism was induced in chronic PTU and MMI treated groups, and animals in these groups showed signifi cant increase in their fl ap survival compared to control euthyroid rats (79.47 ± 10.49% and 75.48 ± 12.93% vs. 52.26 ± 5.75%, respectively, P< 0.01). Acute local treatment of skin fl aps with MMI failed to signifi cantly affect the fl ap survival. This study demonstrates for the fi rst time that hypothyroidism survives random-pattern skin fl aps in rats. 008 VEGF plays a key role enhancing epidermal and blood vessel protection against stress Ludivine Mur1, Cedric Pouzet1, Catherine Serre1, Catherine Gondran1, Eric Bauza1, Jean Marie Botto1, Claude Dal Farra2, Nouha Domloge1 1Vincience ISP Global Skin Research Center, Sophia Antipolis, France, 2ISP Corporate Research Center, Wayne, United States Vascular endothelial growth factor (VEGF) is a crucial element of endothelial cells and angiogenesis and plays diverse roles in skin photoaging, hypoxia, and wound healing. We investigated the expression of VEGF-A and VEGFR-2 (Flk-1), its major receptor, in different cell lines by RT-PCR. VEGF-A and Flk-1 immunofl uorescence studies showed that IV09.006, a compound designed to target VEGF pathway, increased the expression of these two proteins in normal human keratinocytes (NHK) and endothelial cells. Ex vivo studies showed that VEGF-A expression in the epidermis is mainly located in the suprabasal layers. UVB irradiation and H2O2 stresses increased VEGF-A expression in the epidermis. In parallel, pre-treatment with IV09.006 was shown to protect skin from stress damage. For in vivo evaluation, we used chicken chorio-allantoïc membrane (CAM). IV09.006 active was applied directly on the CAM for 24h, and then a stress induced by UVB irradiation (60 mJ/cm2) or H2O2 (10 mM) was applied. A time course observation of the blood vessel network was performed after each stress condition. Our study showed that pre-induction of VEGF-A and Flk-1 enabled a better maintenance of the blood vessel network, preventing vasodilatation and coagulation induced by stress. Taken together, these results indicate that the positive modulation of VEGF-A and Flk-1 expression could be linked to a better preservation of the epidermis from UVB and oxidative stress-induced damage, as well as a protection of the blood vessel network from these stresses. 009 [Oral 109] A unique population of infl ammatory macrophages induced by iron impairs cutaneous wound healing Anca Sindrilaru1, Thorsten Peters1, Corina Baican2, Johannes Weiss1, Meinhard Wlaschek1, Cord Sunderkötter3, Karin Scharffetter-Kochanek1 1University of Ulm, Dept of Dermatology & Allergic Diseases, Germany, 2University of Cluj-Napoca, Dept of Dermatology & Venerology, Cluj-Napoca, Romania, 3University of Münster, Dept of Dermatology, Germany Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic infl ammatory disorders like arteriosclerosis, multiple sclerosis and chronic venous leg ulcers (CVU). However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic infl ammation and affect resident fi broblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with CVU, a model disease for macrophage-driven chronic infl ammation, while establishing a murine model closely refl ecting its pathogenesis. Here we show that iron overloading of macrophages in CVU and in irondextran-treated murine full-thickness excisional wounds induce a novel macrophage population in situ which up-regulate CD163, the hemoglobin-haptoglobin scavenger receptor for iron. CD163hi wound macrophages mount a persistent pro-infl ammatory activation state with high expression of M1 markers (TNFahighIL-12highCD11bhighCCR2high) and the concomitant intermediate expression of anti-infl ammatory M2 markers (IL-4RamedDectin-1medCD36medCD206med), suggestive for an incomplete switch towards the tissue repair-promoting M2 activation phenotype. We show that ‘hybrid’ M1/M2 activated macrophages via enhanced TNFa and hydroxyl radicals release – perpetuate infl ammation and install a p16INK4a-dependent senescence program in resident fi broblasts eventually leading to tissue breakdown and impaired wound healing. Understanding the role of macrophage activation for persistency or resolution of infl ammation in chronic venous ulcers and other chronic infl ammatory diseases holds substantial promise for the development of novel therapies for these diffi cult-to-treat conditions. 010 [Oral 110] Toll-like receptor signaling in dendritic cells is suffi cient to mediate Imiquimodinduced psoriasis-like skin infl ammation in mice Christian Wohn1, Errol Prens2, Sabina Onderwater1, Edwin Florencia2, Heike Weighardt3, Björn Clausen1 1Dept of Immunology, ErasmusMC, University Medical Center, Rotterdam, Netherlands, 2Dept of Dermatology, ErasmusMC, University Medical Center, Rotterdam, Netherlands, 3Institut für Umweltmedizinische Forschung, Heinrich-Heine-Universität Düsseldorf, Germany Psoriasis is a common infl ammatory skin disease characterized by sharply demarcated chronic red plaques covered by white scales. Based on the observation that Imiquimod (IMQ) treatment leads to de novo development or relapse of psoriasis in patients, we established a new mouse model for human psoriasis. Daily application of IMQ onto mouse back skin induces infl amed scaly lesions resembling plaque type psoriasis. These lesions show increased epidermal proliferation, abnormal cell differentiation, neoangiogenesis and infi ltrates consisting of neutrophils, CD4+ T cells, conventional and plasmacytoid dendritic cells (DC). We previously demonstrated that lesion development is critically dependent on IL-23 and IL-17. However, the cell types triggering the infl ammatory process remain elusive. IMQ activates diverse cells of the immune system after binding to toll-like receptors (TLR)-7/8. To investigate the role of different (skin) DC in initiating IMQ-induced psoriasis, we generated MyD88-defi cient mice in which TLR-signaling can be conditionally switched on by Cre-mediated excision of a stop cassette (MyD88stp/stp mice). As expected, MyD88stp/stp animals are resistant to IMQ-induced psoriasis. In contrast, mice with selective reconstitution of TLR signaling in all CD11c+ DC acquire full-blown psoriasiform skin disease following IMQ painting. Intriguingly, mice with TLR signaling restricted to Langerin+ DC subsets, including epidermal Langerhans cells and Langerin+ dermal DC, develop attenuated disease. These data imply that DC are suffi cient to mediate IMQ-induced psoriasis. In ongoing experiments we are further dissecting the requirement of different DC subsets for the induction of disease. 011 [Oral 063] Human IL-10 producing regulatory B cells control CD4+ T cell proliferation Jean-David Bouaziz1, Sébastien Calbo2,3, Maud Maho-Vaillant2, Anne Saussine1, Martine Bagot1, Armand Bensussan1, Philippe Musette2 1INSERM U976, Saint Louis Hospital, Paris, France, 2INSERM U905, Charles Nicolle Hospital, Rouen, France, 3Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore The existence of interleukin 10 (IL-10) producing regulatory B cells that downmodulate infl ammation has already been validated in mice. Especially, a potent subset of regulatory B cells with a CD1dhighCD5+CD19high phenotype was found to decrease contact hypersensitivity in an IL-10-dependent manner. In humans, a transitional B cell subset has recently been shown to exhibit regulatory capacities in vitro.We investigated the existence of IL-10 producing B cells in human blood and spleen and evaluated their phenotype. We also tested the optimal in vitro conditions to trigger IL-10 production by B cells and tested their capacities to regulate CD4+CD25T cell proliferation.We were able to detect after ex vivo short time stimulation, IL-10 producing B cells in human blood and spleen (1.8% and 1.1% of blood and spleen human B cells produced IL-10 as detected by intracellular cytokine staining). Blood IL-10 producing B cells were relatively enriched within the memory (CD27+) and transitional (CD38highCD24high) B cell subsets but IL-10 producing B cells were detected within the whole B cell lineage. Combined CpG-B and anti-immunoglobulin stimulation, rather than CD40L-CD40 pathway, was the most potent stimulus for inducing IL-10 secretion (ELISA assay). Under thes