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

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

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

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

Plasma-polymerized pericyte patches improve healing of murine wounds through increased angiogenesis and reduced inflammation

Pericytes have the potential to be developed as a cell therapy for the treatment of wounds; however, the efficacy of any cell therapy relies on the successful delivery of intact and functioning cells. Here, the effect of delivering pericytes on wound repair was assessed alongside the development of a surface-functionalized pericyte patch. Plasma polymerization (PP) was used to functionalize the surface of silicone patches with heptylamine (HA) or acrylic acid (AA) monomers. Human pericytes were subsequently delivered to murine excisional wounds by intradermal injection or using the pericyte-laden patches and the comparative effects on wound healing, inflammation and revascularization determined. The AA surface provided the superior transfer of the cells to de-epidermized dermis. Excisional murine wounds treated either with pericytes injected directly into the wound or with the pericyte-laden AA patches showed improved healing with decreased neutrophil infiltration and reduced numbers of macrophages in the wounds. Pericyte delivery also enhanced angiogenesis through a mechanism independent of VEGF signalling. Pericytes, when delivered to wounds, improved healing responses by dampening inflammation and promoting angiogenesis. Delivery of pericytes using PP-AA-functionalized patches was equally as effective as direct injection of pericytes into wounds. Pericyte-functionalized dressings may therefore be a clinically relevant approach for the treatment of wounds.Hannah M. Thomas, Parinaz Ahangar, Robert Fitridge, Giles T.S. Kirby, Stuart J. Mills and Allison J. Cowi

Macrophage-Mediated Inflammation in Skin Wound Healing

Published: 21 September 2022Macrophages are key immune cells that respond to infections, and modulate pathophysiological conditions such as wound healing. By possessing phagocytic activities and through the secretion of cytokines and growth factors, macrophages are pivotal orchestrators of inflammation, fibrosis, and wound repair. Macrophages orchestrate the process of wound healing through the transitioning from predominantly pro-inflammatory (M1-like phenotypes), which present early post-injury, to anti-inflammatory (M2-like phenotypes), which appear later to modulate skin repair and wound closure. In this review, different cellular and molecular aspects of macrophage-mediated skin wound healing are discussed, alongside important aspects such as macrophage subtypes, metabolism, plasticity, and epigenetics. We also highlight previous studies demonstrating interactions between macrophages and these factors for optimal wound healing. Understanding and harnessing the activity and capability of macrophages may help to advance new approaches for improving healing of the skin.Alireza Hassanshahi, Mohammad Moradzad, Saman Ghalamkari, Moosa Fadaei, Allison J. Cowin and Mohammadhossein Hassanshah

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

Human multipotent adult progenitor cell-conditioned medium improves wound healing through modulating inflammation and angiogenesis in mice

Background: Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. Methods: The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice (n = 8/group) and 100 μL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. Results: Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. Conclusion: These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.Parinaz Ahangar, Stuart J. Mills, Louise E. Smith, Xanthe L. Strudwick, Anthony E. Ting, Bart Vaes, and Allison J. Cowi

Attenuation of flightless I increases human pericyte proliferation, migration and angiogenic functions and improves healing in murine diabetic wounds

Pericytes are peri-vascular mural cells which have an important role in the homeostatic regulation of inflammatory and angiogenic processes. Flightless I (Flii) is a cytoskeletal protein involved in regulating cellular functions, but its involvement in pericyte activities during wound healing is unknown. Exacerbated inflammation and reduced angiogenesis are hallmarks of impaired diabetic healing responses, and strategies aimed at regulating these processes are vital for improving healing outcomes. To determine the effect of altering Flii expression on pericyte function, in vitro and in vivo studies were performed to assess the effect on healing, inflammation and angiogenesis in diabetic wounds. Here, we demonstrated that human diabetic wounds display upregulated expression of the Flii protein in conjunction with a depletion in the number of platelet derived growth factor receptor β (PDGFRβ) +/ neural glial antigen 2 (NG2) + pericytes present in the dermis. Human pericytes were found to be positive for Flii and attenuating its expression in vitro through siRNA knockdown led to enhanced proliferation, migration and angiogenic functions. Genetic knockdown of Flii in a streptozotocin-induced murine model of diabetes led to increased numbers of pericytes within the wound. This was associated with dampened inflammation, an increased rate of angiogenic repair and improved wound healing. Our findings show that Flii expression directly impacts pericyte functions, including proliferation, motility and angiogenic responses. This suggests that Flii regulation of pericyte function may be in part responsible for the changes in pericyte-related processes observed in diabetic wounds.Hannah M Thomas, Parinaz Ahangar, Benjamin R Hofma, Xanthe L Strudwick, Robert Fitridge, Stuart J Mills and Allison J Cowi

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research