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The human cathelicidin anti-microbial protein, hCAP18 is a component of the innate immune system and has broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs upon inflammation and infection. We demonstrate here a novel role for this peptide in re-epithelialization of skin wounds. We show that high levels of hCAP18 are produced in skin in vivo upon wounding. The highest hCAP18 levels are attained at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. In chronic ulcers, however, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. Using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, we show that hCAP18 is strongly expressed in healing skin epithelium, and that treatment with antibodies raised and affinity purified against LL-37, inhibits re-epithelialization in a concentration-dependent manner. Immunoreactivity for the proliferation marker Ki67 is absent in the epithelium of such inhibited wounds, suggesting that LL-37 may play a part in epithelial cell proliferation. Thus, we suggest that, in addition to being an anti-microbial peptide, LL-37 also plays a part in wound closure and that its reduction in chronic wounds impairs re-epithelialization and may contribute to their failure to heal.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

Considering the clinical and genetic heterogeneity of psoriasis which may translate into distinct disease pathology and treatment response, correct typing of the main candidate gene HLA-C is critical but not trivial. To facilitate genotyping, we compared established techniques with our newly developed tool. Here, we propose that typing of four single nucleotide polymorphic markers within the HLA-C region correctly determines HLA-Cw*06:02 genotypes in psoriatic cases and healthy controls in a population of Caucasian origin. Typing of the SNPs presented herein proved to be precise, reliable, time and cost effective, and requiring low amount of DNA.

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein initially isolated from the specific granules of human neutrophils. It is a member of the highly heterogeneous lipocalin protein family, which shares a common tertiary structure. Its synthesis is induced in gastrointestinal epithelium in association with inflammation and malignancy. To gain insight into its potential role in other epithelia we have investigated the expression of NGAL in human skin embryonic development, in normal adult skin, and in skin associated with inflammation and neoplastic transformation. In the present study we report that the embryonic expression of NGAL appears to be regulated in a spatio-temporal pattern. It was induced in the interfollicular epidermis at 20-24 weeks of gestational age but thereafter progressively receded towards the hair follicles. In normal adult skin, NGAL was detected solely in association with hair follicles. However, strong induction of NGAL in the epidermis was seen in a variety of skin disorders characterized by dysregulated epithelial differentiation such as psoriasis, pityriasis rubra and squamous cell carcinoma. In these tissues production of NGAL was confined to spatially distinct subpopulations of keratinocytes underlying areas of parakeratosis, whereas skin samples lacking parakeratotic epithelium such as lichen ruber planus, acute contact eczema and basal cell carcinoma were negative for NGAL. Consistent with being a marker for disturbed terminal differentiation, NGAL immunoreactivity showed an inverse pattern when compared with that of the differentiation marker filaggrin. The biologic functions of NGAL in epithelia are not fully known, although an immunomodulatory role in host defense has been proposed. In addition, the transient interfollicular NGAL expression during skin embryogenesis along with the induction of NGAL in adult parakeratotic epidermis suggests it play a role in epithelial differentiation pathways.

Tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) encodes a ubiquitin-modifying protein, A20, that is a critical regulator of inflammatory responses. TNFAIP3 polymorphisms are associated with the susceptibility to multiple autoimmune diseases (AIDs) including psoriasis, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis and celiac disease. In order to refine the TNFAIP3 association signal in psoriasis and identify candidate causal variants, we performed imputation and meta-analysis of the TNFAIP3 region in five European ancestry cohorts totaling 4704 psoriasis cases and 7805 controls. We identified 49 variants whose significance exceeded a corrected Bonferroni threshold, with the top variant being rs582757 (P = 6.07 × 10(-12), odds ratio (OR) = 1.23). Conditional analysis revealed a suggestive independent association at rs6918329 (P(cond) = 7.22 × 10(-5), OR = 1.15). Functional annotation of the top variants identified several with a strong evidence of regulatory potential and several within long noncoding RNAs. Analysis of TNFAIP3 haplotypes revealed that the psoriasis risk haplotype is distinct from other AIDs. Overall, our findings identify novel candidate causal variants of TNFAIP3 in psoriasis and highlight the complex genetic architecture of this locus in autoimmune susceptibility.

BACKGROUND: Psoriatic arthritis (PsA) and co-morbidities of psoriasis represent a significant clinical and economic burden for patients with moderate-to-severe psoriasis. Often these co-morbidities may go unrecognized or undertreated. While published data are available on the incidence and impact of some of them, practical guidance for dermatologists on detection and management of these co-morbidities is lacking.OBJECTIVE: To prepare expert recommendations to improve the detection and management of common co-morbidities in patients with moderate-to-severe psoriasis.METHODS: A systematic literature review was conducted on some common co-morbidities of psoriasis-cardiovascular (CV) diseases (including obesity, hypertension, hyperglycaemia and dyslipidaemia), psychological co-morbidities (including depression, alcohol abuse and smoking) and PsA-to establish the incidence and impact of each. Data gaps were identified and a Delphi survey was carried out to obtain consensus on the detection and management of each co-morbidity. The expert panel members for the Delphi survey comprised 10 dermatologists with substantial clinical expertise in managing moderate-to-severe psoriasis patients, as well as a cardiologist and a psychologist (see appendix) with an interest in dermatology. Agreement was defined using a Likert scale of 1-7. Consensus regarding agreement for each statement was defined as ≥75% of respondents scoring either 1 (strongly agree) or 2 (agree).RESULTS: The expert panel members addressed several topics including screening, intervention, monitoring frequency, and the effects of anti-psoriatic treatment on each co-morbidity. Consensus was achieved on 12 statements out of 22 (3 relating to PsA, 4 relating to psychological factors, 5 relating to CV factors). The panel members felt that dermatologists have an important role in screening their psoriasis patients for PsA and in assessing them for psychological and CV co-morbidities. In most cases, however, patients should be referred for specialist management if other co-morbidities are detected.CONCLUSION: This article provides useful and practical guidance for the detection and management of common co-morbidities in patients with moderate-to-severe psoriasis.

Venous leg ulcers (VLUs) are one of the most prevalent types of chronic wounds. The aim of this study was to determine the safety and dose-response efficacy of the human synthetic peptide LL-37 in the treatment of hard-to-heal VLUs. This first-in-man trial included 34 participants with VLUs and comprised a 3-week, open-label, run-in period on placebo, followed by a 4-week randomized double-blind treatment phase with twice weekly applications of LL-37 (0.5, 1.6, or 3.2 mg/mL) or placebo, and a 4-week follow-up. The healing rate constants for 0.5 and 1.6 mg/mL of LL-37 were approximately six- and threefold higher than for placebo (p = 0.003 for 0.5 mg/mL and p = 0.088 for 1.6 mg/mL). Square-root transformed wound area data showed improved healing for the 0.5 and 1.6 mg/mL dose groups compared with pretreatment values (p 0.001 and p = 0.011, respectively). Consistently, treatment with the two lower doses markedly decreased the mean ulcer area (68% for 0.5 mg/mL and 50% for 1.6 mg/mL groups). No difference in healing was observed between the groups receiving 3.2 mg/mL of LL-37 and placebo. There were no safety concerns regarding local or systemic adverse events. In conclusion, topical treatment with LL-37 for chronic leg ulcers was safe and well tolerated with the marked effect on healing predictors at the two lower doses warranting further investigations.

Psoriasis is a common and chronic inflammatory skin disease in which T cells play a key role. Effective treatment heals the skin without scarring, but typically psoriasis recurs in previously affected areas. A pathogenic memory within the skin has been proposed, but the nature of such site-specific disease memory is unknown. Tissue-resident memory T (TRM) cells have been ascribed a role in immunity after resolved viral skin infections. Because of their localization in the epidermal compartment of the skin, TRM may contribute to tissue pathology during psoriasis. In this study, we investigated whether resolved psoriasis lesions contain TRM cells with the ability to maintain and potentially drive recurrent disease. Three common and effective therapies, narrowband-UVB treatment and long-term biologic treatment systemically inhibiting TNF-α or IL-12/23 signaling were studied. Epidermal T cells were highly activated in psoriasis and a high proportion of CD8 T cells expressed TRM markers. In resolved psoriasis, a population of cutaneous lymphocyte–associated Ag, CCR6, CD103, and IL-23R expressing epidermal CD8 T cells was highly enriched. Epidermal CD8 T cells expressing the TRM marker CD103 responded to ex vivo stimulation with IL-17A production and epidermal CD4 T cells responded with IL-22 production after as long as 6 y of TNF-α inhibition. Our data suggest that epidermal TRM cells are retained in resolved psoriasis and that these cells are capable of producing cytokines with a critical role in psoriasis pathogenesis. We provide a potential mechanism for a site-specific T cell–driven disease memory in psoriasis.

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.

Non‐coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non‐coding RNA located head‐to‐head with the gene encoding the Glioma‐associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer‐associated signaling pathways. The expression of this three‐exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up‐regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFβ and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up‐regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non‐coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non‐coding RNA‐based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor., Highlights A novel negative feedback loop on Hedgehog signaling is demonstrated.The mechanism involves a non‐coding RNA antisense to the GLI1 gene, GLI1AS.GLI1AS is shown to be a target gene of the GLI1 transcription factor.GLI1AS represses gene expression at the GLI1/GLI1AS locus.GLI1AS acts as an epigenetic modifier eliciting repressive chromatin marks.

Background Psoriasis is associated with a systemic inflammation and an increased frequency of the metabolic syndrome, both of which are believed to link psoriasis to an increased risk of cardiovascular disease. Objective The study aimed to investigate the systemic expression of markers of cardiovascular risk and determine their response to ultraviolet B therapy and treatment with the tumor necrosis factor-alfa inhibitor, etanercept. Methods Six markers of cardiovascular risk were measured in 28 patients with psoriasis and 28 control subjects. Results Five of the 6 investigated markers were elevated in patients with psoriasis. Four of these correlated to the body mass index and waist-hip ratio, suggesting a link to the metabolic syndrome. Total plasminogen activator inhibitor-1 remained elevated independently of these factors. The levels of the investigated risk markers decreased considerably after tumor necrosis factor-alfa inhibitor treatment but remained unaffected by ultraviolet therapy. Limitations A relatively limited study population and nonrandomization are limitations. Conclusion These findings suggest that the choice of treatment in psoriasis may influence the cardiovascular risk in patients with psoriasis and the metabolic syndrome.

MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally and play important roles in various biological processes. We previously identified miR-203 as a skin- and keratinocyte-specific microRNA. Moreover, miR-203 has been implicated in repressing 'stemness' in epidermal progenitors. Here, we investigate the expression of miR-203 and two of its targets, p63 and suppressor of cytokine signalling-3, during human skin morphogenesis. MiR-203 in situ hybridization was performed on sections of human foetal skin ranging from 14 to 22 weeks' gestation and adult skin. MiR-203 was barely detectable at 14 weeks. Its expression became prominent from week 17 and was most pronounced in the suprabasal layers of the epidermis, while p63 and SOCS-3 were preferentially expressed in the basal layer. Differentiation markers such as involucrin and filaggrin were expressed mainly in the suprabasal layers of epidermis, similar to miR-203. Our results support the involvement of miR-203 in skin morphogenesis.

Substantial epidemiological evidence indicates that psoriasis associates with a predisposition to develop metabolic dysregulation leading to obesity and insulin resistance. However, the nature of this association and the potential underlying mechanisms remain unclear. In a recent report, Gerdes et al. explored the hypothesis that wingless-type MMTV integration site, Wnt5a, which has been linked to aberrant fat cell metabolism, may be driving this process. In this study, the authors compare circulating serum levels of Wnt5a in individuals with psoriasis and compare with healthy controls matched for age, gender and BMI. The bottom-line results show higher levels of Wnt5a in psoriasis patients irrespective of BMI compared to the matched non-psoriatic controls, indicating that psoriasis per se may result in increased secretion of Wnt5a into the circulation. In addition, there was a significant difference among patients with higher levels of Wnt5a in the obese psoriasis population. The study, even though being purely descriptive, may serve to inspire a more mechanistic approach exploring not only Wnt5a, but other inflammatory pathways in between the skin and the fat.

Transforming growth factor-α (TGF-α) exerts several effects on target cells, such as neovascularization promotion and mitogenic signalling. Using immunoelectron microscopy, we show that monocytes and neutrophils, store TGF-α in cytoplasmic granules. In monocytes, TGF-α did not colocalize with components of peroxidase-positive granules or with albumin of secretory vesicles. Furthermore, no colocalization of TGF-α with components of azurophilic or specific granules or secretory vesicles was observed in neutrophils. Activated monocytes and tissue-macrophages contained much less TGF-α–positive granules, suggesting TGF-α release. Western blot analysis showed a protein of 10 kD in lysates of monocytes. TGF-α mRNA was detected in monocytoid cells from the bone marrow by in situ hybridization. This study shows for the first time that monocytes and neutrophils contain TGF-α in all stages of maturation and that TGF-α in monocytes is stored in a large population of peroxidase-negative granules suggesting a function for these granules. Monocytes and neutrophils are important effector cells in inflammatory reactions. The present finding that these cells contain TGF-α might explain complications such as fibrosis and neoplastic transformation, caused by chronic inflammation.

Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation.

Background: MicroRNAs are important posttranscriptional regulators of gene expression. MiR-203 is a miRNA preferentially expressed in the skin, and an important regulator of keratinocyte differentiation. MiR-203 has been implicated in skin diseases, in particular in psoriasis in which it is overexpressed, and in basal cell carcinoma where it acts as a tumor suppressor miRNA. Objectives: To identify novel targets for miR-203 that may be relevant in skin physiology and diseases. MaterialsMethods: Bioinformatics was used to identify putative miR-203 targets among genes expressed in keratinocytes. Interleukin-8 (IL-8) gene expression and concentration in keratinocyte medium was measured by quantitative real-time PCR and ELISA, respectively. For miRNA overexpression, resting or TNF-α-treated primary human keratinocytes were transfected with synthetic precursor of miR-203, or scramble miRNA precursors using Lipofectamine 2000. 3'UTR luciferase reporter experiments were performed to prove the direct miRNA:mRNA interaction. Site-specific mutagenesis was used to mutate the predicted miR-203 binding sites in the 3'UTR of IL-8 gene. Results: Bioinformatic analysis indentified two putative miR-203 binding sites in the 3'UTR of IL-8. MiR-203 suppressed IL-8 mRNA and protein expression in primary human keratinocytes both under resting conditions and after TNF-α treatment. Overexpression of miR-203 suppressed the luciferase activity of a reporter gene fused with the IL-8 3'UTR. The suppressive effect was abolished when the predicted binding sites of miR-203 on IL-8 3'UTR were mutated. Conclusion: We identify IL-8 as a novel target of miR-203 for posttranscriptional suppression. These findings may have relevance in diseases in which miR-203 and IL-8 expression are deregulated.

The study was conducted to investigate which areas of the brain respond to a painful encounter of minor dermal injury (a model of clinical pain) elicited by intracutaneous injection of a minute amount of ethanol. Four healthy volunteers (27-46 years) were subjected to positron emission tomographic (PET) investigation of regional cerebral blood flow (rCBF), using [15O]butanol as tracer. The ethanol (20 microliters, 70%) and saline (20 microliters, 0.9%) were injected intracutaneously 3 times in a single-blinded, semi-randomised manner for the pain experiment. All the injections were performed, adjacent to each other, at the lateral aspect of the right upper arm. Subjective sensory intensity of pain, unpleasantness and anxiety were rated with separate 100-mm visual analogue scales together with the Spielberger's State Anxiety Inventory (Spielberger et al. 1970) and heart rate. Paired-subtraction (pixel-by-pixel) between ethanol and saline was performed. Traumatic pain significantly caused higher ratings of intensity and affect scales, i.e., pain intensity, unpleasantness and increased sympathetic activity (evidenced by tachycardia). In contrast the anxiety rating remained unchanged. Acute traumatic nociceptive pain prominently activated the hypothalamus and periaqueductal gray (PAG). In addition, activations of the prefrontal cortex (PFC), insular, anterior cingulate cortex (ACC), posterior parietal cortex (PPC), primary motor/somatosensory areas (MI/SI: face, upper arm), supplementary motor area (SMA), and cerebellum were also demonstrated. The central processing of the pain-relevant/anticipatory arousal also engaged the PAG. This study demonstrates the involvement of the human cerebral cortex in perception, arousal, cognitive evaluative processes, and, hence, affective reactions (somatic/ autonomic outflow) associated with pain. The pain stimulus of traumatic character may, by its very nature, evoke the central processing to involve both the hypothalamus and the PAG.

Thyroid tumors vary widely in biologic behavior, they range from benign adenomas to rapidly growing anaplastic carcinomas. Among thyroid neoplasms, the follicular tumor is especially suited as a model for studies of tumor cell invasion; the distinction between adenomas and carcinomas relies mainly on the presence of capsular and vascular invasion. Matrix metalloproteinases play an important role in tumor cell invasion, as they are able to degrade basement membrane and extracellular matrix components. Twenty-nine thyroid tumors of varying type and aggressiveness were selected for analysis of relative molecular weight 72,000-dalton type IV collagenase (gelatinase A) expression by mRNA in situ hybridization. Strong gelatinase A mRNA expression was seen in 10 of 14 follicular carcinomas, in none of six follicular adenomas, in all four anaplastic carcinomas, and in four of five papillary carcinomas. The expression was restricted to fibroblasts in the stroma adjacent or close to invading tumor cells. Twelve of the tumors were also investigated for expression of stromelysin 3 mRNA, no expression of which was detected in any tumor. The findings suggest that gelatinase A contributes to the invasive process and spread of aggressive thyroid tumors.

412 Characterization of the immunomodulatory effects of sebocytes on macrophages M Lovaszi, M Mattii, K Eyerich, D Kovacs, C Zouboulis, M Stahle, S Eyerich and D Torocsik 1 Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2 ZAUM e Center for Allergy and Environment, Technische Universitat and Helmholtz Center Munich, Munich, Germany, 3 Department of Dermatology and Allergy, Technische Universitat Munich, Munich, Germany, 4 Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany and 5 Unit of Dermatology and Venereology, Department of Medicine, Karolinska lnstitutet, Karolinska University Hospital, Stockholm, Sweden Macrophages surround the sebaceous glands both in the healthy and the inflamed pilosebaceous unit; however, the reason for this phenomenon and the possible role of sebocytes in it has not yet been investigated. By performing immunohistochemistry on healthy biopsies, we could demonstrate that macrophages in the close proximity of sebaceous glands are exclusively alternatively activated (CD163/FXIII-A/CD206/CD209). To investigate if sebocytes contribute to the differentiation, polarization and function of macrophages, human peripheral blood monocytes were differentiated and activated in the presence of either supernatant from the human SZ95 sebocyte cell line, or major sebum lipid components such as oleic-, linoleic-, palmitic-, stearicacids and squalene. Our results showed that with the secretion of CXCL8, sebocytes could exert a chemoattractant effect towards monocytes, while sebocytes derived lipids promoted monocyte differentiation into alternatively activated macrophages as marked by the up-regulation of CD206, CD209 and FXIII-A. Moreover, detection of the produced IL-1b, IL-6 and TNFa protein levels by Propionibacterium acnes activated macrophages revealed a selective inflammatory effect for the various sebum lipids. Our results altogether suggest a role for sebaceous glands in initiating and modulating innate immune responses via their proteins and lipids that are of possible pathologic and therapeutic relevance. 413 Epithelial resident and infiltrating dendritic cells amplify active and resolved psoriasis inflammation E Martini, M Wiken, S Cheuk, A Smed Sorensen, M Stahle and L Eidsmo Dept. of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden In psoriasis, scaly skin lesions are caused by interactions between activated keratinocytes, T cells and dendritic cells present in the skin. Here we investigate the inflammatory profiles of skin resident Langerhans cells (LCs) and infiltrating epitdermal dendritic cells (eDCs) to determine if they have the potential to steer epithelial pathology in psoriasis. A population of LangerinCD11cCD1c eDCs with variable levels of CD1a and CD14 was strictly confined to epidermis in active psoriasis lesions and could not be detected in non lesional or resolved psoriasis or healthy skin. LCs and eDCs sorted from active psoriasis displayed shared expression of genes associated with T cell activation and neutrophil attraction. Although LCs from psoriatic lesions stimulated with Toll-like receptor (TLR) ligands produced the pro-inflammatory cytokines IL-1 beta and IL-23, eDCs were the major producer of these cytokines in the epidermis, and displayed comparable cytokine production to dDCs. In resolved psoriasis eDCs are absent but LCs promptly responded to TLR stimulation with increased IL-23 production. Our results show that infiltrating eDCs together with LCs strongly contribute to the pro-inflammatory environment within the epidermal compartment of the skin. In contrast, LCs alone show the capacity to activate pathogenic tissue resident T cells in resolved psoriasis.

412 Characterization of the immunomodulatory effects of sebocytes on macrophages M Lovaszi, M Mattii, K Eyerich, D Kovacs, C Zouboulis, M Stahle, S Eyerich and D Torocsik 1 Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary, 2 ZAUM e Center for Allergy and Environment, Technische Universitat and Helmholtz Center Munich, Munich, Germany, 3 Department of Dermatology and Allergy, Technische Universitat Munich, Munich, Germany, 4 Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany and 5 Unit of Dermatology and Venereology, Department of Medicine, Karolinska lnstitutet, Karolinska University Hospital, Stockholm, Sweden Macrophages surround the sebaceous glands both in the healthy and the inflamed pilosebaceous unit; however, the reason for this phenomenon and the possible role of sebocytes in it has not yet been investigated. By performing immunohistochemistry on healthy biopsies, we could demonstrate that macrophages in the close proximity of sebaceous glands are exclusively alternatively activated (CD163/FXIII-A/CD206/CD209). To investigate if sebocytes contribute to the differentiation, polarization and function of macrophages, human peripheral blood monocytes were differentiated and activated in the presence of either supernatant from the human SZ95 sebocyte cell line, or major sebum lipid components such as oleic-, linoleic-, palmitic-, stearicacids and squalene. Our results showed that with the secretion of CXCL8, sebocytes could exert a chemoattractant effect towards monocytes, while sebocytes derived lipids promoted monocyte differentiation into alternatively activated macrophages as marked by the up-regulation of CD206, CD209 and FXIII-A. Moreover, detection of the produced IL-1b, IL-6 and TNFa protein levels by Propionibacterium acnes activated macrophages revealed a selective inflammatory effect for the various sebum lipids. Our results altogether suggest a role for sebaceous glands in initiating and modulating innate immune responses via their proteins and lipids that are of possible pathologic and therapeutic relevance. 413 Epithelial resident and infiltrating dendritic cells amplify active and resolved psoriasis inflammation E Martini, M Wiken, S Cheuk, A Smed Sorensen, M Stahle and L Eidsmo Dept. of Medicine, Solna, Karolinska Institutet, Stockholm, Sweden In psoriasis, scaly skin lesions are caused by interactions between activated keratinocytes, T cells and dendritic cells present in the skin. Here we investigate the inflammatory profiles of skin resident Langerhans cells (LCs) and infiltrating epitdermal dendritic cells (eDCs) to determine if they have the potential to steer epithelial pathology in psoriasis. A population of LangerinCD11cCD1c eDCs with variable levels of CD1a and CD14 was strictly confined to epidermis in active psoriasis lesions and could not be detected in non lesional or resolved psoriasis or healthy skin. LCs and eDCs sorted from active psoriasis displayed shared expression of genes associated with T cell activation and neutrophil attraction. Although LCs from psoriatic lesions stimulated with Toll-like receptor (TLR) ligands produced the pro-inflammatory cytokines IL-1 beta and IL-23, eDCs were the major producer of these cytokines in the epidermis, and displayed comparable cytokine production to dDCs. In resolved psoriasis eDCs are absent but LCs promptly responded to TLR stimulation with increased IL-23 production. Our results show that infiltrating eDCs together with LCs strongly contribute to the pro-inflammatory environment within the epidermal compartment of the skin. In contrast, LCs alone show the capacity to activate pathogenic tissue resident T cells in resolved psoriasis.

Therapies that reduce psoriasis symptoms may improve work productivity.To assess the effect of ixekizumab therapy on work productivity, measured by the Work Productivity and Activity Impairment-Psoriasis (WPAI-PSO).Three multicenter, randomized double-blind phase 3 trials conducted during the following periods: December 2011 through August 2014 (UNCOVER-1), May 2012 through April 2015 (UNCOVER-2), and August 2012 through July 2014 (UNCOVER-3). Adult outpatients with moderate-to-severe chronic plaque psoriasis were included.In UNCOVER-1, patients were randomized 1:1:1 to subcutaneous placebo or 80 mg ixekizumab every 2 weeks (Q2W) or every 4 weeks (Q4W) for 12 weeks; UNCOVER-2 and UNCOVER-3 also had an etanercept arm (50 mg twice weekly). Maintenance of initial ixekizumab response was evaluated in UNCOVER-1 and UNCOVER-2 during a randomized withdrawal period following week 12 through week 60. The WPAI-PSO questionnaire was administered at baseline and week 12 for all patients and at weeks 24, 36, 52, and 60 for patients in UNCOVER-1 and UNCOVER-2.Change in work productivity from baseline as measured by WPAI-PSO scores.Across trials, 5101 patients consented; 3866 were randomized (mean [SD] age, UNCOVER-1, 45.7 [12.9] y, 68.1% male; UNCOVER-2: 45.0 [13.0] y, 67.1% male; UNCOVER-3: 45.8 [13.1] y, 68.2% male). At week 12 in UNCOVER-1, the ixekizumab Q4W and ixekizumab Q2W groups showed significantly greater improvements in WPAI-PSO scores (least squares mean change from baseline [SE]) relative to placebo: absenteeism (-3.5 [0.87], P .001; -2.6 [0.84], P = .003, respectively, vs 0.2 [0.88]), presenteeism (-18.8 [1.28], P .001; -18.3 [1.24], P .001, vs 0.5 [1.30]), work productivity loss (-20.6 [1.38], P .001; -19.8 [1.33], P .001, vs -0.8 [1.40]), and activity impairment (-24.5 [1.18], P .001; -25.2 [1.15], P .001, vs 0.8 [1.18]). Similar results were obtained for UNCOVER-2 and UNCOVER-3, with the exception of absenteeism with ixekizumab Q4W in UNCOVER-2. Additionally, ixekizumab-treated patients showed significantly greater improvements in WPAI-PSO scores vs etanercept-treated patients: UNCOVER-2: presenteeism, work productivity loss, activity impairment (P .001 both doses), UNCOVER-3: activity impairment (ie, regular activities outside of work) (ixekizumab Q2W; P = .009). Improvements in WPAI-PSO scores at week 12 were sustained to at least week 60.Ixekizumab-treated patients reported short- and long-term improvements in work productivity, which could lead to reduced productivity-related cost burden in patients with psoriasis.clinicaltrials.gov Identifiers: NCT01474512, NCT01597245, NCT01646177.

Wounding of protective barriers is a major insult to the organism and immediately sets in motion a complex cascade of cellular responses in order to re-establish tissue integrity. Antimicrobial proteins, AMPs, are important components in the innate immune system and play a vital role in this process. The defensin family of proteins and the human cathelicidin, hCAP18, are the most documented AMPs in human and the focus of this review. Still, many proteins display antimicrobial activity, suggesting that the capacity to defend against microbes has been a driving force during evolution. In addition to direct killing of microbes, AMPs are involved in the inflammatory reaction through chemotaxis and control of cytokine response. Furthermore, recent data also show that AMPs have growth-factor like effects and signal via receptors promoting angiogenesis and re-epithelialization. Thus, the role of AMPs in wound healing is only beginning to be understood and may be far-reaching.

Cutaneous squamous cell carcinoma (cSCC) is the second most common human cancer. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of cancers, the role of miRNAs in cSCC is unclear. In this study, we aimed to identify tumor suppressive and oncogenic miRNAs involved in the pathogenesis of cSCC. MiRNA expression profiles in healthy skins (n = 4) and cSCCs (n = 4) were analyzed using MicroRNA Low Density Array. MiR-125b expression was analyzed by quantitative real-time PCR and in situ hybridization in skin biopsies from 40 healthy donors, 13 actinic keratosis, and 74 cSCC patients. The effect of miR-125b was analyzed in wound closure, colony formation, migration, and invasion assays in two cSCC cell lines, UT-SCC-7 and A431. The genes regulated by miR-125b in cSCC were identified by microarray analysis and its direct target was validated by luciferase reporter assay. Comparing cSCC with healthy skin, we identified four up-regulated miRNAs (miR-31, miR-135b, miR-21, and miR-223) and 54 down-regulated miRNAs, including miR-125b, whose function was further examined. We found that miR-125b suppressed proliferation, colony formation, migratory, and invasive capacity of cSCC cells. Matrix metallopeptidase 13 (MMP13) was identified as a direct target suppressed by miR-125b, and there was an inverse relationship between the expression of miR-125b and MMP13 in cSCC. Knockdown of MMP13 expression phenocopied the effects of miR-125b overexpression. These findings provide a novel molecular mechanism by which MMP13 is up-regulated in cSCCs and indicate that miR-125b plays a tumor suppressive role in cSCC.

MicroRNAs are short non-coding RNAs that regulate gene expression. Previously, in a genome-wide screen, we found deregulation of microRNA expression in psoriasis skin. MicroRNA-21 (miR-21) is one of the microRNAs significantly up-regulated in psoriasis skin lesions. To identify the cell type responsible for the increased miR-21 level, we compared expression of miR-21 in epidermal cells and dermal T cells between psoriasis and healthy skin and found elevated levels of miR-21 in psoriasis in both cell types. In cultured T cells, expression of miR-21 increased markedly upon activation. To explore the function of miR-21 in primary human T helper cells, we inhibited miR-21 using a tiny seed-targeting LNA-anti-miR. Specific inhibition of miR-21 increased the apoptosis rate of activated T cells. Our results suggest that miR-21 suppresses apoptosis in activated T cells, and thus, overexpression of miR-21 may contribute to T cell-derived psoriatic skin inflammation.

Item does not contain fulltext BACKGROUND: Clinical trials to test investigational drugs for the treatment of chronic plaque psoriasis currently lack standards for comparison of efficacy and safety data.The majority of studies do not address the important need for long-term treatment. METHODS: The International Psoriasis Council (IPC) conducted two surveys of its members to assess the need for gold standards, active comparators, and long-term therapy in clinical trials. In Survey 1, 30 participants delivered viewpoints on active comparators for topical therapy (six questions), systemic therapy (nine questions), and continuous versus intermittent therapy (six questions). In Survey 2, 31 participants provided input on gold standards for treatment (five questions), appropriate comparators (four questions), and continuous versus intermittent therapy (six questions). The IPC leadership interpreted the results after each survey. RESULTS: The majority of participants (77% in Survey 1 and 89% in Survey 2) agreed that studies of investigative treatments should include an active comparator. Participants described the most important feature of a gold standard as a treatment that: is widely used and generally accepted (45%); has the best efficacy (42%); and is well tolerated (13%). The majority agreed that gold standards should be dependent on: patient subgroup; location/extent of psoriasis; and psoriasis subtype/morphology. It was also agreed that continuous therapy for more than 3 years is needed for patients with moderate-to-severe plaque psoriasis. We have provided an expert opinion regarding the definition of a gold standard in psoriasis and have also established that no single treatment can be the gold standard across all subgroups and types of the disease. CONCLUSIONS: A single gold standard for the treatment of psoriasis does not exist. The complexity and heterogeneity of psoriasis requires different gold standards for the various manifestations of psoriasis and for subgroups of patients reconciling comorbidities. Of note, 17 experts out of 30 selected methotrexate as the most nominated gold standard amongst systemic agents. The experts support the election of an active comparator as one that is most efficacious over just the best in a particular class. In concordance, 87% of respondents agreed that good tolerability is therefore not the lead criterion for selection of an active comparator in favor of effectiveness and broad use. Patients with moderate-to-severe plaque psoriasis require continuous therapy; this statement was supported by 93% of the experts. Reasons for considering long-term therapy included appearance of comorbidities, impairment of quality of life, possibility of relapse, and subjective complaints such as itch, pain, and joint disease. 01 augustus 2011

Contains fulltext : 87874.pdf (Publisher’s version ) (Closed access) BACKGROUND: Psoriasis, an inflammatory disorder of the skin, can significantly impact on a patient's quality of life, affecting their daily activities and families. The onset of psoriasis in childhood is quite common; however, the treatment of moderate-to-severe disease in this population is challenging, with a paucity of data reported and few licensed agents available. METHODS: A Delphi survey was conducted among a panel of European expert dermatologists and physicians with a particular interest in pediatric inflammatory disorders. The survey covered the aspects of psoriasis types, psoriatic arthritis, diagnosis and treatment options in childhood. RESULTS: A series of consensus opinions were reached, detailing the current practice in Europe for the diagnosis and treatment of psoriasis in childhood. These opinions are presented in the context of evidence from the literature and the current licensure status and indications of therapies for psoriasis in childhood. CONCLUSIONS: These data provide detailed information on the current practices in Europe for treating psoriasis in childhood. 01 oktober 2010

MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally and play important roles in various biological processes. We previously identified miR-203 as a skin- and keratinocyte-specific microRNA. Moreover, miR-203 has been implicated in repressing 'stemness' in epidermal progenitors. Here, we investigate the expression of miR-203 and two of its targets, p63 and suppressor of cytokine signalling-3, during human skin morphogenesis. MiR-203 in situ hybridization was performed on sections of human foetal skin ranging from 14 to 22 weeks' gestation and adult skin. MiR-203 was barely detectable at 14 weeks. Its expression became prominent from week 17 and was most pronounced in the suprabasal layers of the epidermis, while p63 and SOCS-3 were preferentially expressed in the basal layer. Differentiation markers such as involucrin and filaggrin were expressed mainly in the suprabasal layers of epidermis, similar to miR-203. Our results support the involvement of miR-203 in skin morphogenesis.

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 TH cells through the downregulation of CTLA-4. (J Allergy Clin Immunol 2010; 126: 581-9.) 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.

Contains fulltext : 89179.pdf (Publisher’s version ) (Closed access) To identify new susceptibility loci for psoriasis, we undertook a genome-wide association study of 594,224 SNPs in 2,622 individuals with psoriasis and 5,667 controls. We identified associations at eight previously unreported genomic loci. Seven loci harbored genes with recognized immune functions (IL28RA, REL, IFIH1, ERAP1, TRAF3IP2, NFKBIA and TYK2). These associations were replicated in 9,079 European samples (six loci with a combined P < 5 x 10 and two loci with a combined P < 5 x 10). We also report compelling evidence for an interaction between the HLA-C and ERAP1 loci (combined P = 6.95 x 10). ERAP1 plays an important role in MHC class I peptide processing. ERAP1 variants only influenced psoriasis susceptibility in individuals carrying the HLA-C risk allele. Our findings implicate pathways that integrate epidermal barrier dysfunction with innate and adaptive immune dysregulation in psoriasis pathogenesis. 01 november 2010

Contains fulltext : 89593.pdf (Publisher’s version ) (Closed access) Psoriasis is now classified as an immune-mediated inflammatory disease (IMID) of the skin. It is being recognized that patients with various IMIDs, including psoriasis, are at higher risk of developing "systemic" co-morbidities, e.g., cardiovascular disease (CVD), metabolic syndrome, and overt diabetes. In non-psoriatic individuals, the pathophysiology of obesity, aberrant adipocyte metabolism, diabetes, and CVDs involves immune-mediated or inflammatory pathways. IMIDs may impact these co-morbid conditions through shared genetic risks, common environmental factors, or common inflammatory pathways that are co-expressed in IMIDs and target organs. Given that pathogenic immune pathways in psoriasis are now well worked out and a large number of inflammatory mediators have been identified in skin lesions, in this review we will consider possible mechanistic links between skin inflammation and increased risks of (1) obesity or metabolic alterations and (2) CVD. In particular, we will discuss how well-established risk factors for CVD can originate from inflammation in other tissues. 01 juli 2010

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

Metalloproteinases are thought to be important for tumor invasion and metastasis. We used in situ hybridization with 35S-labeled cRNA probes to localize sites of expression for 92-kDa type IV collagenase mRNA in sections of nodular basal cell carcinoma. Positive signal for 92-kDa type IV collagenase mRNA was detected in eosinophilic granulocytes within inflammatory infiltrates surrounding the tumor nodules. Eosinophils, however, were not adjacent to tumor cells, suggesting that metalloenzyme production by these granulocytes in this disease may be targeted more to stromal components than to remodeling or destruction of the basement lamina. The identity of the eosinophils was confirmed by cell morphology and specific histochemical staining. No resident or other migratory cells were positive for enzyme mRNA in these samples. Signal specificity for in situ hybridization was shown by a duplication of the results with complementary oligomeric probes and by a lack of signal in sections hybridized with a sense RNA probe or nonspecific oligomer. No signal for 92-kDa type IV collagenase mRNA was detected in circulating eosinophils or in eosinophils associated with Hodgkin's lymphoma. These data suggest that eosinophils migrate into the dermis and express type IV collagenase in response to basal cell carcinoma and that this process may have a role in tumor growth.

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.

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. Abstract: Reduced production of antimicrobial peptides was proposed to contribute to susceptibility for skin infections in atopic dermatitis (AD). Focusing on the human cathelicidin protein, hCAP18, the aim of the present study was to explore whether reduced hCAP18 expression is a constitutive trait in AD and if established inducers affect the expression of hCAP18 in the skin of AD. First, we compared levels of hCAP18 mRNA between lesional skin in AD and psoriasis and verified significantly lower expression of hCAP18 mRNA in AD. In non-lesional skin, however, there was no difference between AD, psoriasis and healthy, indicating that there is no constitutive defect in the production of hCAP18 in AD patients. In healthy skin, hCAP18 was reported to be rapidly induced following wounding and here we verified this pattern in healthy controls and in psoriasis. In AD lesions, however, the expression of hCAP18 mRNA was markedly suppressed following wounding. Obviously, the inflammation in AD lesions neutralizes the expected induction of hCAP18 and even induces suppression. Notably, the mechanism to upregulate hCAP18 following vitamin D treatment was functional in lesional as well as in non-lesional AD indicating that the CAMP gene is normally regulated in this respect. In addition, cultured primary keratinocytes from non-lesional skin of psoriasis, AD and healthy skin, upregulated hCAP18mRNA following treatment with vitamin D. Itching is a hallmark of AD and scratching inevitably injures the skin. Failure to upregulate hCAP18 in eczema following injury is likely to affect antimicrobial protection and tissue repair in AD.