Cytoskeletal regulation of inflammation and its impact on skin blistering disease epidermolysis bullosa acquisita

Actin remodelling proteins regulate cytoskeletal cell responses and are important in both innate and adaptive immunity. These responses play a major role in providing a fine balance in a cascade of biological events that results in either protective acute inflammation or chronic inflammation that leads to a host of diseases including autoimmune inflammation mediated epidermolysis bullosa acquisita (EBA). This review describes the role of the actin cytoskeleton and in particular the actin remodelling protein called Flightless I (Flii) in regulating cellular inflammatory responses and its subsequent effect on the autoimmune skin blistering disease EBA. It also outlines the potential of an antibody based therapy for decreasing Flii expression in vivo to ameliorate the symptoms associated with EBA.Zlatko Kopecki, Ralf J. Ludwig and Allison J. Cowi

Lifting the silver flakes: the pathogenesis and management of chronic plaque psoriasis

Psoriasis is a common chronic inflammatory skin condition in which patients suffer from mild to chronic plaque skin plaques. The disease manifests through an excessive inflammatory response in the skin due to complex interactions between different genetic and environmental factors. Psoriasis can affect the physical, emotional, and psychosocial well-being of patients, and currently there is no cure with treatments focusing primarily on the use of anti-inflammatory agents to control disease symptoms. Traditional anti-inflammatory agents can cause immunosuppression and adverse systemic effects. Further understanding of the disease has led to current areas of research aiming at the development of selective molecular targets to suppress the pathogenic immune responses.Heng T. Chong, Zlatko Kopecki, and Allison J. Cowi

Skin barrier and autoimmunity - mechanisms and novel therapeutic approaches for autoimmune blistering diseases of the skin

One of the most important functions of the skin besides regulating internal body temperature includes formation of the barrier between the organism and the external environment, hence protecting against pathogen invasion, chemical and physical assaults and unregulated loss of water and solutes. Disruption of the protective barrier is observed clinically in blisters and erosions of the skin that form in autoimmune blistering diseases where the body produces autoantibodies against structural proteins of the epidermis or the epidermal-dermal junction. Although there is no cure for autoimmune skin blistering diseases, immune suppressive therapies currently available offer opportunities for disease management. In cases where no treatment is sought, these disorders can lead to life threatening complications and current research efforts have focused on developing therapies that target autoantibodies which contribute to disease symptoms. This review will outline the involvement of the skin barrier in main skin-specific autoimmune blistering diseases by describing the mechanisms underpinning skin autoimmunity and review current progress in development of novel therapeutic approaches targeting the underlying causes of autoimmune skin blistering diseases.Natalie E. Stevens, Allison J. Cowin and Zlatko Kopeck

Fighting chronic wound infection – one model at a time

Wound infections are a serious medical problem for patients with non-healing chronic wounds and burn injuries. The healing of these wounds is often compromised by colonisation of many different bacteria, predisposing patients to life-threatening infections. Infected wounds continue to represent a complex problem for both health professionals and patients. Bacterial infections are a critical component of hard-to-heal wounds, often leading to biofilm formation, inhibition of innate inflammatory responses and resistance to traditional therapeutics. Over the last 20 years, tremendous progress has been made in understanding the intricacies of wound biofilm pathology and bacteria–host interactions. This has been achieved by the development of in vitro and in vivo models of wound infection. This review will discuss the challenges in the development of wound infection models and will focus on different in vivo models of cutaneous wound infection, highlighting advantages and clinical applicability of each model. It will also describe the development of novel bioluminescent models of cutaneous wound infection in vivo, which may help to revolutionise the future testing of novel antimicrobial therapeutics for the treatment of wounds.Kopecki Z, Ogunniyi AD, Trott DJ & Cowin A

Flightless I alters the inflammatory response and autoantibody profile in an OVA-induced atopic dermatitis skin-like disease

Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease characterized by excessive inflammation and disrupted skin barrier function. Although the etiology of AD is not completely understood, clinical and basic studies suggest increasing involvement of autoantibodies against intracellular proteins. An actin remodeling protein, Flightless I (Flii), has been shown to promote development of inflammatory mediated skin conditions and impairment of skin barrier development and function. Here, we sought to determine the effect of altering Flii expression on the development of AD and its contribution to autoimmune aspects of inflammatory skin conditions. Ovalbumin (OVA)-induced AD skin-like disease was induced in Flii heterozygous (Flii+/- ), wild-type (Flii+/+ ), and Flii transgenic (FliiTg/Tg ) mice by epicutaneous exposure to OVA for 3 weeks; each week was separated by 2-week resting period. Reduced Flii expression resulted in decreased disease severity and tissue inflammation as determined by histology, lymphocytic, and mast cell infiltrate and increased anti-inflammatory IL-10 cytokine levels and a marked IFN-γ Th1 response. In contrast, Flii over-expression lead to a Th2 skewed response characterized by increased pro-inflammatory TNF-α cytokine production, Th2 chemokine levels, and Th2 cell numbers. Sera from OVA-induced AD skin-like disease Flii+/- mice showed a decreased level of autoreactivity while sera from FliiTg/Tg mice counterparts showed an altered autoantibody profile with strong nuclear localization favoring development of a more severe disease. These findings demonstrate autoimmune responses in this model of OVA-induced AD-like skin disease and suggest that Flii is a novel target, whose manipulation could be a potential approach for the treatment of patients with AD.Zlatko Kopecki, Natalie E. Stevens, Heng T. Chong, Gink N. Yang and Allison J. Cowi

Increased expression of flightless I in cutaneous squamous cell carcinoma affects wnt/beta-catenin signaling pathway

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/β-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/β-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased β-catenin and a decrease in the expression of the downstream effector of β-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.Gink N. Yang, Xanthe L. Strudwick, Claudine S. Bonder, Zlatko Kopecki and Allison J. Cowi

Overexpression of Flii during murine embryonic development increases symmetrical division of epidermal progenitor cells

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii(+/-), wild type: WT, transgenic Flii overexpressing: Flii(Tg/Tg)) using Western blot and immunohistochemistry. Flii(+/-) embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. Flii(Tg/Tg) led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.Gink N. Yang, Parinaz Ahangar, Xanthe L. Strudwick , Zlatko Kopecki and Allison J. Cowi

Recombinant leucine-rich repeat flightless-interacting protein-1 improves healing of acute wounds through its effects on proliferation inflammation and collagen deposition

Wound healing is an increasing clinical problem involving substantial morbidity, mortality, and rising health care costs. Leucine-rich repeat flightless-interacting protein-1 (LRRFIP-1) regulates toll-like receptor (TLR)-mediated inflammation, suggesting a potential role in the healing of wounds. We sought to determine the role of LRRFIP-1 in wound repair and whether the exogenous addition of recombinant LRRFIP-1 (rLRRFIP-1) affected healing responses. Using a model of full-thickness incisional acute wounds in BALB/c mice, we investigated the effect of wounding on LRRFIP-1 expression. The effect of rLRRFIP-1 on cellular proliferation, inflammation, and collagen deposition was also investigated. LRRFIP-1 was upregulated in response to wounding, was found to directly associate with flightless I (Flii), and significantly increased cellular proliferation both in vitro and in vivo. rLRRFIP-1 reduced Flii expression in wounds in vivo and resulted in significantly improved healing with a concurrent dampening of TLR4-mediated inflammation and improved collagen deposition. Additionally, decreased levels of TGF-β1 and increased levels of TGF-β3 were observed in rLRRFIP-1-treated wounds suggesting a possible antiscarring effect of rLRRFIP-1. Further studies are required to elucidate if the mechanisms behind LRRFIP-1 action in wound repair are independent of Flii. However, these results identify rLRRFIP-1 as a possible treatment modality for improved healing of acute wounds.Zlatko Kopecki, Natalie E. Stevens, Gink N. Yang, Elizabeth Melville and Allison J. Cowi

Chondroitin Sulfate Proteoglycan 4 as a Marker for Aggressive Squamous Cell Carcinoma

Chondroitin sulfate (CS) proteoglycan 4 (CSPG4) is a cell surface proteoglycan that is currently under investigation as a marker of cancer malignancy, and as a potential target of anticancer drug treatment. CSPG4 acts as a driver of tumourigenesis by regulating turnover of the extracellular matrix (ECM) to promote tumour cell invasion, migration as well as inflammation and angiogenesis. While CSPG4 has been widely studied in certain malignancies, such as melanoma, evidence is emerging from global gene expression studies, which suggests a role for CSPG4 in squamous cell carcinoma (SCC). While relatively treatable, lack of widely agreed upon diagnostic markers for SCCs is problematic, especially for clinicians managing certain patients, including those who are aged or infirm, as well as those with underlying conditions such as epidermolysis bullosa (EB), for which a delayed diagnosis is likely lethal. In this review, we have discussed the structure of CSPG4, and quantitatively analysed CSPG4 expression in the tissues and pathologies where it has been identified to determine the usefulness of CSPG4 expression as a diagnostic marker and therapeutic target in management of malignant SCC

Eradication of mature bacterial biofilms with concurrent improvement in chronic wound healing using silver nanoparticle hydrogel treatment

Biofilm-associated infections are a major cause of impaired wound healing. Despite the broad spectrum of anti-bacterial benefits provided by silver nanoparticles (AgNPs), these materials still cause controversy due to cytotoxicity and a lack of efficacy against mature biofilms. Herein, highly potent ultrasmall AgNPs were combined with a biocompatible hydrogel with integrated synergistic functionalities to facilitate elimination of clinically relevant mature biofilms in-vivo combined with improved wound healing capacity. The delivery platform showed a superior release mechanism, reflected by high biocompatibility, hemocompatibility, and extended antibacterial efficacy. In vivo studies using the S. aureus wound biofilm model showed that the AgNP hydrogel (200 µg/g) was highly effective in eliminating biofilm infection and promoting wound repair compared to the controls, including silver sulfadiazine (Ag SD). Treatment of infected wounds with the AgNP hydrogel resulted in faster wound closure (46% closure compared to 20% for Ag SD) and accelerated wound re-epithelization (60% for AgNP), as well as improved early collagen deposition. The AgNP hydrogel did not show any toxicity to tissue and/or organs. These findings suggest that the developed AgNP hydrogel has the potential to be a safe wound treatment capable of eliminating infection and providing a safe yet effective strategy for the treatment of infected wounds.Hanif Haidari, Richard Bright, Sanjay Garg, Krasimir Vasilev, Allison J. Cowin and Zlatko Kopeck