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Severe angiopathy is a major driver for diabetes associated secondary complications. Knowledge on underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors (SPs) at the edge of non-healing diabetic wounds in a murine db/db model, closely mirroring human type II diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ SPs. This compensates for the profoundly reduced angiogenin expression in non-treated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ SPs prior to injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining SPs-based therapies of non-healing diabetic foot ulcers and other pathologies with impaired angiogenesis.

Psoriasis is one of the most common dermatoses with a heterogeneous pathogenesis which can be successfully exploited therapeutically as it is increasingly well understood. Topical therapy is the gold standard for psoriasis patients with mild disease courses and for complementary and maintenance treatment in moderate and severe forms. However, while new systemic therapies are rapidly implemented in the daily routine as our pathomechanistic understanding of psoriasis evolves, the development of topical psoriasis therapies stagnates. Modern topical treatments though would require not only new active substances but also improved galenics. Due to their unique ability to directly exert biological functions, but also to deliver drugs in optimal concentrations, enabling increased therapeutic efficacy, reduced adverse effects and improved patient compliance, nanoparticles may represent ideal drug carriers for local therapeutics in psoriasis. In recent years, a series of reports added important insights into the biology of skin‐nanoparticles interactions and on how they impact the epidermal and dermal inflammatory compartments in vitro and in psoriasis plaques. Furthermore, by targeting anti‐inflammatory substances to specific skin compartments, nanotechnological advances offer the exciting opportunity to fine‐tune skin inflammation at molecular and cellular levels, paving the road to a high‐precision, skin‐directed topical therapy in psoriasis. However, nanoparticle‐based therapies have not yet found their way into clinical routine in dermatology. We here resume the current advances in the research of nanoparticles and skin inflammation in general and psoriasis in particular and discuss how this promising technology should develop in order to fulfil the requirements of an optimal skin therapy., publishedVersion

We here investigated whether the unique capacity of mesenchymal stem cells (MSCs) to re-establish tissue homeostasis depends on their potential to sense danger associated molecular pattern (DAMP) and to mount an adaptive response in the interest of tissue repair. Unexpectedly, after injection of MSCs which had been pretreated with the calcium-binding DAMP protein S100A8/A9 into murine full-thickness wounds, we observed a significant acceleration of healing even exceeding that of non-treated MSCs. This correlates with a fundamental reprogramming of the transcriptome in S100A8/A9 treated MSCs as deduced from RNA-seq analysis and its validation. A network of genes involved in proteolysis, macrophage phagocytosis, and inflammation control profoundly contribute to the clean-up of the wound site. In parallel, miR582-5p and genes boosting energy and encoding specific extracellular matrix proteins are reminiscent of scar-reduced tissue repair. This unprecedented finding holds substantial promise to refine current MSC-based therapies for difficult-to-treat wounds and fibrotic conditions.

New biomaterials based on nanoparticles (NPs) carrying polyphenols-rich extracts (Cornus mas) recently showed promising anti-inflammatory activity in psoriasis. We aimed to understand how topically delivered silver and gold nanoparticles complexed with Cornus mas (Ag-NPs-CM, Au-NPs-CM) modulate inflammation in psoriasis at cellular and molecular level. The impact on psoriatic inflammation was assessed in vitro on pro-inflammatory macrophages, by clinical score, high-frequency ultrasonography and immunohistology of psoriasis plaques treated with Ag-NPs-CM, Au-NPs-CM or control. Incubation of pro-inflammatory macrophages with nanoparticles significantly decreased the release of NO, IL-12 and TNF-α. Immunofluorescence confirmed that nanoparticles significantly reduced CD68-positive macrophages and their IL-12 and TNF-α production in human psoriasis plaques. NPs-CM appear to repress NF-κB activation in macrophages, inhibiting the production of pro-inflammatory factors with causal role in psoriasis. Ag and Au NPs-CM represent a novel nanoparticle-based "green" technology which may provide an efficient tool for modern psoriasis therapy, circumventing immunosuppression-related side effects of biologicals.

Chronic wounds pose a stern challenge to health care systems with growing incidence especially in the aged population. In the presence of increased iron concentrations, recruitment of monocytes from the circulation and activation towards ROS and RNS releasing M1 macrophages together with the persistence of senescent fibroblasts at the wound site are significantly enhanced. This unrestrained activation of pro-inflammatory macrophages and senescent fibroblasts has increasingly been acknowledged as main driver causing non-healing wounds. In a metaphor, macrophages act like stage directors of wound healing, resident fibroblasts constitute main actors and increased iron concentrations are decisive parts of the libretto, and ��� if dysregulated ��� are responsible for the development of non-healing wounds. This review will focus on recent cellular and molecular findings from chronic venous leg ulcers and diabetic non-healing wounds both constituting the most common pathologies often resulting in limb amputations of patients. This not only causes tremendous suffering and loss of life quality, but is also associated with an increase in mortality and a major socio-economic burden. Despite recent advances, the underlying molecular mechanisms are not completely understood. Overwhelming evidence shows that reactive oxygen species and the transition metal and trace element iron at pathological concentrations are crucially involved in a complex interplay between cells of different histogenetic origin and their extracellular niche environment. This interplay depends on a variety of cellular, non-cellular biochemical and cell biological mechanisms. Here, we will highlight recent progress in the field of iron-dependent regulation of macrophages and fibroblasts and related pathologies linked to non-healing chronic wounds., publishedVersion

Neutrophils are the most abundant leukocytes in the blood. In the recent decades, their crucial roles in host defense, immune regulation and tissue damage have been studied in a deeper dimension. In this protocol, we described a mouse model of immune complex-mediated vasculitis in the dorsal skin induced by Arthus reaction, and the subsequent analysis of edema, hemorrhage and tissue damage due to neutrophil activation by means of Evans blue area analysis, histology, and immunofluorescence. This protocol could facilitate the investigation of cellular therapy strategy against over-activated neutrophil-mediated tissue damage.

Objective There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT3/4 receptor–modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. Methods Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca2+ measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunohistochemical analysis. Results Tropisetron suppressed collagen synthesis induced by transforming growth factor β1 (TGFβ1). This effect was independent of 5-HT3/4 receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFβ1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. Conclusion Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.

Background: Primary cutaneous diffuse large B-cell lymphoma, leg-type (LT-DLBCL) is an extremely aggressive DLBCL subtype typically occurring at lower extremities and with very poor prognosis due to early relapses and refractory (R/R) disease. Previous studies have shown increased BCR dependence in DLBCL being observed in association with the mutation status of BCR associated genes and MYD88. Aim: Primary goal was to assess the clinical course in patients with primary cutaneous DLBCL and to elucidate the potential of alternative treatments with regard to molecular characteristics. Methods: We identified 16 patients with cutaneous DLBCL treated at our center of which 8 patients had typical localization and were histologically confirmed as LT-DLBCL. The other 8 patients showed cutaneous DLBCL at other anatomic sites (DLBCL-OS) and were classified as DLBCL/DLBCL NOS. Three R/R patients received ibrutinib as off-label individual treatment attempt (420 mg daily). We extended the clinical and molecular analysis for the ibrutinib exposed DLBCL by 1 R/R oropharyngeal DLBCL. Specimen from R/R ibrutinib exposed and 3 other patients were analyzed for CD79B, MYD88, CARD11 and BTK mutations by targeted resequencing analysis. PD-1/PD-L1 expression was assessed in 2 cases with relapse after ibrutinib. Treatment was initiated after signed informed consent. Results: The median age at diagnosis was 51 years in DLBCL-OS and 80 years in LT-DLBCL (total range 37-91). Patients received a median of 2 (0-7) treatments and response to last chemotherapy was different for DLBCL-OS (6 complete remissions (CR), 2 R/R) and LT-DLBCL (2CR, 5R/R). One LT-DLBCL patient showed stable disease (SD) without treatment. LT-DLBCL patients showed a significantly shorter median overall survival (OS) (21,5 months vs. not reached, p=0.009). After ibrutinib treatment we observed 1 ongoing CR for 10 months till date for a DLBCL-OS with CD79B p.Y197S and MYD88 L273P mutation and 1 CR for 6 months in a LT-DLBCL being WT for all sequenced genes. However, this patient relapsed with a highly proliferative disease and died shortly after. The 3rd patient with LT-DLBCL had an isolated MYD88 L265P mutation and showed a PR for 1 month. Samples from patients with indolent clinical course showed a MYD88 p.S251N and BTK p.P385S mutation (LT-DLBCL with SD) or were WT for all sequenced genes (2 patients with DLBCL-OS and ongoing CR). The patient with R/R oropharyngeal DLBCL had a MYD88 L265P and CD79B c.587A mutation, 4 prior treatment regimen and fulminant progression during the last 2 treatments. Initial response to ibrutinib was rapid with a drop of LDH levels from 2200 U/L to 620 U/L within 7 days and consecutive decrease to 323 U/L. However, this patient relapsed after 30 days of treatment. Immunomodulatory effects of ibrutinib and potential synergistic treatment with checkpoint inhibitors have previously been suggested. We specifically investigated PD-1/PD-L1 expression in tissues obtained from the two patients progressing after ibrutinib treatment. Remarkably, we observed increased expression for PD-1 (moderate) and PD-L1 (strong) in non-tumor bystander cells. Treatment with nivolumab was initiated in 1 patient with early clinical benefit. However, the patient refused the continuation of this treatment. Conclusion: Patients with LT-DLBCL are older and show a poor clinical course compared to cutaneous DLBCL at other anatomic sites. MYD88 L265P mutations were observed only in chemo-refractory cases with extranodal DLBCL. Ibrutinib can induce complete remissions and sustained responses in chemo-refractory extranodal DLBCL but relapses may be more aggressive and disseminated. Mutational patterns and ibrutinib response were in line with previous hypothetical models for sensitivity to BCR inhibition. Combining ibrutinib and checkpoint inhibitors may be considered in future trials for LT-DLBCL patients. Disclosures Weissinger: Bristol-Myers Squibb: Research Funding. Viardot:Roche: Consultancy, Honoraria; Amgen: Consultancy; Gilead Kite: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Psoriasis is a T-cell-mediated inflammatory disease. Previous studies focused on lymphocyte function-associated antigen 1 (LFA-1)-expressing T cells as a molecular target for therapeutic intervention. By contrast, information on therapeutic effects and the underlying mechanism of blocking the LFA-1 counter receptor, ICAM-1 is scarce. Here, we used the CD18 (beta2-integrin) hypomorphic (CD18hypo) mouse model of psoriasis to investigate the therapeutic role of extracellular adherence protein (Eap) of Staphylococcus aureus, which exerts antiinflammatory activities by interacting with the ICAM-1 function. We show that ICAM-1 is predominantly upregulated on endothelial cells in lesional skin of CD18hypo mice. In vitro Eap was found to disrupt cell-cell contacts between T cells and dendritic cells, and inhibit T-cell proliferation. By contrast, in vivo Eap rather blocked transmigration of T cells from vessels to inflamed skin of CD18hypo mice, but did not inhibit their proliferation and activation. Most importantly, Eap successfully suppressed the disease by blocking T-cell extravasation into the inflamed skin. Together, these data indicate that interaction between LFA-1 and ICAM-1 is causally involved in the pathogenesis of psoriasiform skin inflammation, and targeting ICAM-1 to selectively block T-cell extravasation by Eap without immune suppression may represent a potential therapeutic strategy for psoriasis.

Objective Recently, we found that human dermal fibroblasts (HDFs) express melanocortin 1 receptors (MC-1R) that bind α-melanocyte–stimulating hormone (α-MSH). In search of novel therapies for scleroderma (systemic sclerosis [SSc]), we used the bleomycin (BLM) model to investigate the effects of α-MSH on collagen synthesis and fibrosis. Methods Collagen expression in HDFs was determined by real-time reverse transcription–polymerase chain reaction (RT-PCR) and Western blot analyses. Signal transduction studies included pharmacologic blockade, immunofluorescence analysis, Western blotting, and reporter–promoter assays. Oxidative stress was measured by fluorescence-activated cell sorter analysis, and anti–oxidative enzyme levels were determined by real-time RT-PCR and Western blot analyses. The effect of α-MSH in the BLM mouse model of scleroderma was assessed by histologic, immunohistochemical, real-time RT-PCR, and protein analyses. Expression of MC-1R and pro-opiomelanocortin (POMC) in skin and HDF samples from patients with SSc was determined by RT-PCR and compared with that in samples from normal controls. Results Treatment with α-MSH (and related peptides) suppressed BLM-induced expression of type I and type III collagen in HDFs, and this effect was cAMP-dependent. Neither BLM nor α-MSH altered Smad signaling, but antioxidants inhibited BLM-induced collagen expression in vitro. In addition, α-MSH suppressed BLM-induced oxidative stress and enhanced the expression of superoxide dismutase 2 (SOD2) and heme oxygenase 1 (HO-1). In the BLM mouse model, α-MSH reduced skin fibrosis and collagen content and increased tissue levels of SOD2 and HO-1. In skin and HDFs from patients with SSc, both MC-1R and POMC messenger RNAs were detected, but there were no differences compared with healthy controls. Conclusion Alpha-melanocyte–stimulating hormone and related peptides that exert their effects via MC-1R may provide a novel antifibrogenic therapeutic tool for the treatment of fibrotic diseases such as scleroderma.

Leukocytoclastic vasculitis (LcV) is the most common form of vasculitis of the skin and usually results from deposition of immune complexes at the vessel wall. It presents in different forms and in association with different diseases. When IgA is the dominant immunoglobulin in immune complexes, systemic involvement is likely in both children and adults (Henoch-Schönlein purpura--HSP). LcV due to IgG- or IgM-containing immune complexes has less systemic involvement and a better prognosis than HSP. Other forms of LcV include cryoglobulinaemic, urticarial and ANCA-associated LcV as well as LcV associated with vasculopathy and coagulopathy in SCLE/SLE or in bacteraemia/sepsis. The aim of diagnostic guidelines is to determine the specific type and systemic involvement of LcV and to identify an underlying cause. Basic work-up should encompass history of drug intake and of preceding infections, biopsy with immunofluorescence, differential blood count, urine analysis and throat swabs. Therapy of immune complex LcV often does not require aggressive therapy due to a usually favourable course. It includes avoidance or treatment of eliciting agents and use of compression stockings to reduce purpura. There are no large prospective randomized controlled studies. Corticosteroids are indicated when there are signs of incipient skin necrosis. In chronic or relapsing LcV we suggest colchicine as a first-line and dapsone as a second-line therapy. Corticosteroids may reduce the incidence of severe renal insufficiency in children according to some studies, but there is no study showing such an effect in adults. Severe systemic vasculitis requires immunosuppressive strategies.

Allergic contact dermatitis is a T cell-mediated immune response, which in its relapsing chronic form is of high socioeconomic impact. The phosphoglycoprotein osteopontin (OPN) has chemotactic and Th1 cytokine functions and in various models is essential for robust T cell-mediated immunity. Here we demonstrate that OPN is abundantly expressed by both effector T cells and keratinocytes in allergic contact dermatitis lesions. T cells from nickel-allergic donors secrete high levels of OPN following antigen-specific stimulation. OPN may substitute for missing IFN-gamma secretion in T effector cells because low IFN-gamma-producing T cell clones secrete high levels of OPN, and OPN down-modulates their interleukin-4 expression. Furthermore, interferon-gamma from T effector cells augments OPN in allergic contact dermatitis by inducing OPN in keratinocytes, which in turn polarizes dendritic cells and attracts inflammatory cells. In the murine contact hypersensitivity (CHS) model for allergic contact dermatitis, OPN is strongly induced in antigen-specific proliferating T cells, and OPN null mice display a reduced chronic CHS inflammatory response due to a decreased influx of effector T cells. Importantly, because of its function for chronic allergic contact dermatitis, OPN may well be a therapeutic target, because anti-OPN antibody treatment in part suppresses established chronic CHS.

Psoriasis is a chronic skin disorder of unsolved pathogenesis affecting skin in 2-3% of the general population. Research into the pathogenesis of psoriasis has profited from suitable animal models. Previously, we reported on the CD18 hypomorphic (CD18(hypo)) PL/J mouse model clinically resembling human psoriasis, which is characterized by reduced expression of the common chain of beta(2)-integrins (CD11/CD18) to only 2-16% of wild-type levels. Aside from common clinical and pathophysiological features shared with human psoriasis, the psoriasiform skin disease in CD18(hypo) PL/J mice also depends on the presence of CD4(+) T-cells. This review focuses on the role of activated macrophages in the pathogenesis of CD18(hypo) T-cell-mediated mouse model of psoriasis, and extends our understanding in unrestrained pathogenic T-cells whose activation may be crucial for the recruitment and activation of macrophages within skin. The findings in the CD18(hypo) PL/J model are discussed in the context of current literatures of human and other autoimmune disorders.

Psoriasis is a complex genetic disease of unresolved pathogenesis with both heritable and environmental factors contributing to onset and severity. In addition to a disfiguring skin inflammation, approximately 10–40% of psoriasis patients suffer from destructive joint involvement. Previously, we reported that the CD18 hypomorphic PL/J mouse carrying a mutation resulting in reduced expression of the common chain of β2 integrins (CD11/CD18) spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, the same mutation on C57BL/6J background did not demonstrate this phenotype. By a genome-wide linkage analysis, two major loci were identified as contributing to the development of psoriasiform dermatitis under the condition of low CD18 expression. Using a congenic approach, we now demonstrate that the introduction of a 9-centimorgan fragment of chromosome 10 derived from the PL/J strain into the disease-resistant CD18 hypomorphic C57BL/6J was promoting the development of psoriasiform skin disease and notably also arthritis. We therefore designated this locus psoriasiform skin disease-associated locus 1 (PSD1). High numbers of CD4+ T cells and TNF-α producing macrophages were detected both in inflamed skin and joints in these congenic mice, with a complete resolution upon TNF-α inhibitor therapy or depletion of CD4+ T cells. For the first time, we have identified a distinct genetic element that contributes to the T cell-dependent development of both psoriasiform skin disease and associated arthritis. This congenic model will be suitable to further investigations of genetic and molecular pathways that cause psoriasiform dermatitis and arthritis, and it may also be relevant for other autoimmune diseases.

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-jB (NF-jB) system is activated in response to several of these stresses, we hypothesized that NF-jB activation in hepatocytes may contribute to fibrosis development. To activate the NF-jB signaling pathway in a timeand celltype-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-jB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-jB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-jB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-jB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117-1128)