Selective Depletion of Staphylococcus aureus Restores the Skin Microbiome and Accelerates Tissue Repair after Injury.

Our skin is home to a diverse community of commensal microorganisms integral to cutaneous function. However, microbial dysbiosis and barrier perturbation increase the risk of local and systemic infection. Staphylococcus aureus is a particularly problematic bacterial pathogen, with high levels of antimicrobial resistance and direct association with poor healing outcome. Innovative approaches are needed to selectively kill skin pathogens, such as S aureus, without harming the resident microbiota. In this study, we provide important data on the selectivity and efficacy of an S aureus-targeted endolysin (XZ.700) within the complex living skin/wound microbiome. Initial cross-species comparison using Nanopore long-read sequencing identified the translational potential of porcine rather than murine skin for human-relevant microbiome studies. We therefore performed an interventional study in pigs to assess the impact of endolysin administration on the microbiome. XZ.700 selectively inhibited endogenous porcine S aureus in vivo, restoring microbial diversity and promoting multiple aspects of wound repair. Subsequent mechanistic studies confirmed the importance of this microbiome modulation for effective healing in human skin. Taken together, these findings strongly support further development of S aureus-targeted endolysins for future clinical management of skin and wound infections.

The Skin Microbiome: Current Landscape and Future Opportunities.

Our skin is the largest organ of the body, serving as an important barrier against the harsh extrinsic environment. Alongside preventing desiccation, chemical damage and hypothermia, this barrier protects the body from invading pathogens through a sophisticated innate immune response and co-adapted consortium of commensal microorganisms, collectively termed the microbiota. These microorganisms inhabit distinct biogeographical regions dictated by skin physiology. Thus, it follows that perturbations to normal skin homeostasis, as occurs with ageing, diabetes and skin disease, can cause microbial dysbiosis and increase infection risk. In this review, we discuss emerging concepts in skin microbiome research, highlighting pertinent links between skin ageing, the microbiome and cutaneous repair. Moreover, we address gaps in current knowledge and highlight key areas requiring further exploration. Future advances in this field could revolutionise the way we treat microbial dysbiosis associated with skin ageing and other pathologies.

Epithelial arginase-1 is a key mediator of age-associated delayed healing in vaginal injury.

Pelvic organ prolapse is a disorder that substantially affects the quality of life of millions of women worldwide. The greatest risk factors for prolapse are increased parity and older age, with the largest group requiring surgical intervention being post-menopausal women over 65. Due to ineffective healing in the elderly, prolapse recurrence rates following surgery remain high. Therefore, there is an urgent need to elucidate the cellular and molecular drivers of poor healing in pelvic floor dysfunction to allow effective management and even prevention. Recent studies have uncovered the importance of Arginase 1 for modulating effective healing in the skin. We thus employed novel in vitro and in vivo vaginal injury models to determine the specific role of Arginase 1 in age-related vaginal repair. Here we show, for the first time, that aged rat vaginal wounds have reduced Arginase 1 expression and delayed healing. Moreover, direct inhibition of Arginase 1 in human vaginal epithelial cells also led to delayed scratch-wound closure. By contrast, activation of Arginase 1 significantly accelerated healing in aged vaginal wounds in vivo, to rates comparable to those in young animals. Collectively, these findings reveal a new and important role for Arginase 1 in mediating effective vaginal repair. Targeting age-related Arginase 1 deficiency is a potential viable therapeutic strategy to promote vaginal healing and reduce recurrence rate after surgical repair of pelvic organ prolapse.

Cellular Senescence in Acute and Chronic Wound Repair.

Cellular senescence, once thought an artifact of in vitro culture or passive outcome of aging, has emerged as fundamental to tissue development and function. The senescence mechanism importantly halts cell cycle progression to protect against tumor formation, while transiently present senescent cells produce a complex secretome (or SASP) of inflammatory mediators, proteases, and growth factors that guide developmental remodeling and tissue regeneration. Transiently present senescence is important for skin repair, where it accelerates extracellular matrix formation, limits fibrosis, promotes reepithelialization, and modulates inflammation. Unfortunately, advanced age and diabetes drive pathological accumulation of senescent cells in chronic wounds, which is perpetuated by a proinflammatory SASP, advanced glycation end-products, and oxidative damage. Although the biology of wound senescence remains incompletely understood, drugs that selectively target senescent cells are showing promise in clinical trials for diverse pathological conditions. It may not be long before senescence-targeted therapies will be available for the management, or perhaps even prevention, of chronic wounds.

Skin Aging in Long-Lived Naked Mole-Rats Is Accompanied by Increased Expression of Longevity-Associated and Tumor Suppressor Genes.

Naked mole-rats (NMRs) (Heterocephalus glaber) are long-lived mammals that possess a natural resistance to cancer and other age-related pathologies, maintaining a healthy life span >30 years. In this study, using immunohistochemical and RNA-sequencing analyses, we compare skin morphology, cellular composition, and global transcriptome signatures between young and aged (aged 3‒4 vs. 19‒23 years, respectively) NMRs. We show that similar to aging in human skin, aging in NMRs is accompanied by a decrease in epidermal thickness; keratinocyte proliferation; and a decline in the number of Merkel cells, T cells, antigen-presenting cells, and melanocytes. Similar to that in human skin aging, expression levels of dermal collagens are decreased, whereas matrix metalloproteinase 9 and matrix metalloproteinase 11 levels increased in aged versus in young NMR skin. RNA-sequencing analyses reveal that in contrast to human or mouse skin aging, the transcript levels of several longevity-associated (Igfbp3, Igf2bp3, Ing2) and tumor-suppressor (Btg2, Cdkn1a, Cdkn2c, Dnmt3a, Hic1, Socs3, Sfrp1, Sfrp5, Thbs1, Tsc1, Zfp36) genes are increased in aged NMR skin. Overall, these data suggest that specific features in the NMR skin aging transcriptome might contribute to the resistance of NMRs to spontaneous skin carcinogenesis and provide a platform for further investigations of NMRs as a model organism for studying the biology and disease resistance of human skin.

Combined Metallomics/Transcriptomics Profiling Reveals a Major Role for Metals in Wound Repair.

Endogenous metals are required for all life, orchestrating the action of diverse cellular processes that are crucial for tissue function. The dynamic wound healing response is underpinned by a plethora of such cellular behaviours, occurring in a time-dependent manner. However, the importance of endogenous metals for cutaneous repair remains largely unexplored. Here we combine ICP-MS with tissue-level RNA-sequencing to reveal profound changes in a number of metals, and corresponding metal-regulated genes, across temporal healing in mice. Wound calcium, magnesium, iron, copper and manganese are elevated at 7 days post-wounding, while magnesium, iron, aluminium, manganese and cobalt increase at 14 days post-wounding. At the level of transcription, wound-induced pathways are independently highly enriched for metal-regulated genes, and vice versa. Moreover, specific metals are linked to distinct wound-induced biological processes and converge on key transcriptional regulators in mice and humans. Finally, we reveal a potential role for one newly identified transcriptional regulator, TNF, in calcium-induced epidermal differentiation. Together, these data highlight potential new and diverse roles for metals in cutaneous wound repair, paving the way for further studies to elucidate the contribution of metals to cellular processes in the repair of skin and other tissues.

A role for estrogen in skin ageing and dermal biomechanics.

The skin is the body's primary defence against the external environment, preventing infection and desiccation. Therefore, alterations to skin homeostasis, for example with skin ageing, increase susceptibility to skin disease and injury. Skin biological ageing is uniquely influenced by a combination of intrinsic and extrinsic (primarily photoageing) factors, with differential effects on skin structure and function. Interestingly, skin architecture rapidly changes following the menopause, as a direct result of reduced circulating 17ß-estradiol. The traditional clinical benefit of estrogens are supported by recent experimental data, where 17ß-estradiol supplementation prevents age-related decline in the skin's structural and mechanical properties. However, the off-target effects of 17ß-estradiol continue to challenge therapeutic application. Here we discuss how ageing alters the physiological and structural properties of the dermal extracellular matrix, and explore how estrogen receptor-targeted therapies may restore the mechanical defects associated with skin ageing.

Wound senescence: A functional link between diabetes and ageing?

Arguably, the two most important causes of pathological healing in the skin are diabetes and ageing. While these factors have historically been considered independent modifiers of the healing process, recent studies suggest that they may be mechanistically linked. The primary contributor to diabetic pathology is hyperglycaemia, which accelerates the production of advanced glycation end products, a characteristic of ageing tissue. Indeed, advanced age also leads to mild hyperglycaemia. Here, we discuss emerging literature that reveals a hitherto unappreciated link between cellular senescence, diabetes and wound repair. Senescent cells cause widespread destruction of normal tissue architecture in ageing and have been shown to be increased in chronic wounds. However, the role of senescence remains controversial, with several studies reporting beneficial effects for transiently induced senescence in wound healing. We recently highlighted a direct role for senescence in diabetic healing pathology, mediated by the senescence receptor, CXCR2. These findings suggest that targeting local tissue senescence may provide a therapeutic strategy applicable to a broad range of chronic wound types.

Pre-Clinical Assessment of Single-Use Negative Pressure Wound Therapy During In Vivo Porcine Wound Healing.

Objective: Traditional negative pressure wound therapy (tNPWT) systems can be large and cumbersome, limiting patient mobility and adversely affecting quality of life. PICO™, a no canister single-use system, offers a lightweight, portable alternative to tNPWT, with improved clinical performance. The aim of this study was to determine the potential mechanism(s) of action of single-use NPWT (sNPWT) versus tNPWT. Approach: sNPWT and tNPWT were applied to an in vivo porcine excisional wound model, following product use guidelines. Macroscopic, histological, and biochemical analyses were performed at defined healing time points to assess multiple aspects of the healing response. Results: Wounds treated with single-use negative pressure displayed greater wound closure and increased reepithelialization versus those treated with traditional negative pressure. The resulting granulation tissue was more advanced with fewer neutrophils, reduced inflammatory markers, more mature collagen, and no wound filler-associated foreign body reactions. Of note, single-use negative pressure therapy failed to induce wound edge epithelial hyperproliferation, while traditional negative pressure therapy compromised periwound skin, which remained inflamed with high transepidermal water loss; features not observed following single-use treatment. Innovation: Single-use negative pressure was identified to improve multiple aspects of healing versus traditional negative pressure treatment. Conclusion: This study provides important new insight into the differing mode of action of single-use versus traditional negative pressure and may go some way to explaining the improved clinical outcomes observed with single-use negative pressure therapy.

Cellular benefits of single-use negative pressure wound therapy demonstrated in a novel ex vivo human skin wound model.

Negative pressure wound therapy is a widely used treatment for chronic, nonhealing wounds. Surprisingly, few studies have systematically evaluated the cellular and molecular effects of negative pressure treatment on human skin. In addition, no study to date has directly compared recently available single-use negative pressure modalities to traditional negative pressure devices in a controlled setting. Here we developed a novel large-scale ex vivo human skin culture system to effectively evaluate the efficacy of two different negative pressure wound therapy modalities. Single-use and traditional negative pressure devices were applied to human ex vivo wounded skin sheets cultured over a period of 48 hours. Cellular tissue response to therapy was evaluated via a combination of histological analysis and transcriptional profiling, in samples collected from the wound edge, skin adjacent to the wound, and an extended skin region. Single-use negative pressure wound therapy caused less damage to wound edge tissue than traditional application, demonstrated by improved skin barrier, reduced dermal-epidermal junction disruption and a dampened damage response. Transcriptional profiling confirmed significantly less activation of multiple pro-inflammatory markers in wound edge skin treated with single-use vs traditional negative pressure therapy. These findings may help to explain the greater efficacy of sNPWT in the clinic, while offering a noninvasive system to develop improved NPWT-based therapies.

Wound healing: cellular mechanisms and pathological outcomes.

Wound healing is a complex, dynamic process supported by a myriad of cellular events that must be tightly coordinated to efficiently repair damaged tissue. Derangement in wound-linked cellular behaviours, as occurs with diabetes and ageing, can lead to healing impairment and the formation of chronic, non-healing wounds. These wounds are a significant socioeconomic burden due to their high prevalence and recurrence. Thus, there is an urgent requirement for the improved biological and clinical understanding of the mechanisms that underpin wound repair. Here, we review the cellular basis of tissue repair and discuss how current and emerging understanding of wound pathology could inform future development of efficacious wound therapies.

Senescence in Wound Repair: Emerging Strategies to Target Chronic Healing Wounds.

Cellular senescence is a fundamental stress response that restrains tumour formation. Yet, senescence cells are also present in non-cancerous states, accumulating exponentially with chronological age and contributing to age- and diabetes-related cellular dysfunction. The identification of hypersecretory and phagocytic behaviours in cells that were once believed to be non-functional has led to a recent explosion of senescence research. Here we discuss the profound, and often opposing, roles identified for short-lived vs. chronic tissue senescence. Transiently induced senescence is required for development, regeneration and acute wound repair, while chronic senescence is widely implicated in tissue pathology. We recently demonstrated that sustained senescence contributes to impaired diabetic healing via the CXCR2 receptor, which when blocked promotes repair. Further studies have highlighted the beneficial effects of targeting a range of senescence-linked processes to fight disease. Collectively, these findings hold promise for developing clinically viable strategies to tackle senescence in chronic wounds and other cutaneous pathologies.

Optimising platelet secretomes to deliver robust tissue-specific regeneration.

Promoting cell proliferation is the cornerstone of most tissue regeneration therapies. As platelet-based applications promote cell division and can be customised for tissue-specific efficacy, this makes them strong candidates for developing novel regenerative therapies. Therefore, the aim of this study was to determine if platelet releasate could be optimised to promote cellular proliferation and differentiation of specific tissues. Growth factors in platelet releasate were profiled for physiological and supraphysiological platelet concentrations. We analysed the effect of physiological and supraphysiological releasate on C2C12 skeletal myoblasts, H9C2 rat cardiomyocytes, human dermal fibroblasts (HDF), HaCaT keratinocytes, and chondrocytes. Cellular proliferation and differentiation were assessed through proliferation assays, mRNA, and protein expression. We show that supraphysiological releasate is not simply a concentrated version of physiological releasate. Physiological releasate promoted C2C12, HDF, and chondrocyte proliferation with no effect on H9C2 or HaCaT cells. Supraphysiological releasate induced stronger proliferation in C2C12 and HDF cells compared with physiological releasate. Importantly, supraphysiological releasate induced proliferation of H9C2 cells. The proliferative effects of skeletal and cardiac muscle cells were in part driven by vascular endothelial growth factor alpha. Furthermore, supraphysiological releasate induced differentiation of H9C2 and C2C12, HDF, and keratinocytes. This study provides insights into the ability of releasate to promote muscle, heart, skin, and cartilage cell proliferation and differentiation and highlights the importance of optimising releasate composition for tissue-specific regeneration.

Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy).

PURPOSE: There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. METHODS: THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. RESULTS: We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. CONCLUSIONS: The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages.

Tissue Iron Promotes Wound Repair via M2 Macrophage Polarization and the Chemokine (C-C Motif) Ligands 17 and 22.

Macrophages are important for effective iron recycling and erythropoiesis, but they also play a crucial role in wound healing, orchestrating tissue repair. Recently, we demonstrated a significant accumulation of iron in healing wounds and a requirement of iron for effective repair. Herein, we sought to determine the influence of iron on macrophage function in the context of wound healing. Interestingly, wound macrophages extensively sequestered iron throughout healing, associated with a prohealing M2 phenotype. In delayed healing diabetic mouse wounds, both macrophage polarization and iron sequestration were impaired. In vitro studies revealed that iron promotes differentiation, while skewing macrophages toward a hypersecretory M2-like polarization state. These macrophages produced high levels of chemokine (C-C motif) ligands 17 and 22, promoting wound reepithelialization and extracellular matrix deposition in a human ex vivo wound healing model. Together, these findings reveal a novel, unappreciated role for iron in modulating macrophage behavior to promote subsequent wound repair. These findings support therapeutic evaluation of iron use to promote wound healing in the clinic.

Reduced Iron in Diabetic Wounds: An Oxidative Stress-Dependent Role for STEAP3 in Extracellular Matrix Deposition and Remodeling.

Iron is crucial for maintaining normal bodily function with well-documented roles in erythropoiesis, hemostasis, and inflammation. Despite this, little is known about the temporal regulation of iron during wound healing, or how iron contributes to wound biology and pathology. In this study, we profiled tissue iron levels across a healing time-course, identifying iron accumulation during late-stage repair. Diabetic murine wounds displayed significantly reduced iron levels, delayed extracellular matrix deposition, and dysregulation of iron gene expression. In vitro studies revealed important cellular roles for iron, promoting both the deposition and remodeling of extracellular proteins. Functional studies identified oxidative stress-dependent upregulation of the iron-converting metalloreductase, STEAP3, as a key mediator of extracellular matrix production. Taken together, these data reveal a mechanistic role for iron in facilitating the remodeling stage of wound healing. Indeed, targeting tissue iron could be a promising future strategy to tackle the development and progression of chronic wounds.

Elevated Local Senescence in Diabetic Wound Healing Is Linked to Pathological Repair via CXCR2.

Cellular senescence can be broadly defined as a stable, but essentially irreversible, loss of proliferative capacity. Historically, senescence has been described as a negative outcome of advanced cellular age. It is now clear, however, that senescence represents a dynamic autonomous stress response, integral to long-term tumor suppression. Transient induction of a senescent phenotype has actually been suggested to promote regeneration in both liver and skin. Here, we explored the role of senescence in pathological aged and diabetic murine wound healing. Aged and diabetic wounds had greater numbers of senescent cells, and diabetic macrophages maintained altered retention of polarization and produced a CXCR2-enriched senescence-associated secretory phenotype (i.e., SASP). Of translational relevance, targeted expression of CXCR2 in primary human dermal fibroblasts led to paracrine induction of nuclear p21. Furthermore, a selective agonist to CXCR2 was able to reverse delayed healing in diabetic mice and accelerate ex vivo human skin wound healing. Collectively, these data suggest a hitherto unappreciated role for CXCR2 in mediating cellular senescence in pathological wound repair.

A Novel Silver Bioactive Glass Elicits Antimicrobial Efficacy Against Pseudomonas aeruginosa and Staphylococcus aureus in an ex Vivo Skin Wound Biofilm Model.

Biofilm infection is now understood to be a potent contributor to the recalcitrant nature of chronic wounds. Bacterial biofilms evade the host immune response and show increased resistance to antibiotics. Along with improvements in antibiotic stewardship, effective new anti-biofilm therapies are urgently needed for effective wound management. Previous studies have shown that bioactive glass (Bg) is able to promote healing with moderate bactericidal activity. Here we tested the antimicrobial efficacy of a novel BG incorporating silver (BgAg), against both planktonic and biofilm forms of the wound-relevant bacteria Pseudomonas aeruginosa and Staphylococcus aureus. BgAg was stable, long lasting, and potently effective against planktonic bacteria in time-kill assays (6-log reduction in bacterial viability within 2 h) and in agar diffusion assays. BgAg reduced bacterial load in a physiologically relevant ex vivo porcine wound biofilm model; P. aeruginosa (2-log reduction) and S. aureus (3-log reduction). BgAg also conferred strong effects against P. aeruginosa biofilm virulence, reducing both protease activity and virulence gene expression. Co-culture biofilms appeared more resistant to BgAg, where a selective reduction in S. aureus was observed. Finally, BgAg was shown to benefit the host response to biofilm infection, directly reducing host tissue cell death. Taken together, the findings provide evidence that BgAg elicits potent antimicrobial effects against planktonic and single-species biofilms, with beneficial effects on the host tissue response. Further investigations are required to elucidate the specific consequences of BG administration on polymicrobial biofilms, and further explore the effects on host-microbe interactions.

The role of estrogen in cutaneous ageing and repair.

Combined advances in modern medical practice and increased human longevity are driving an ever-expanding elderly population. Females are particularly at risk of age-associated pathology, spending more of their lives in a post-menopausal state. Menopause, denoted by a rapid decline in serum sex steroid levels, accelerates biological ageing across the body's tissues. Post-menopause physiological changes are particularly noticeable in the skin, which loses structural architecture and becomes prone to damage. The sex steroid most widely discussed as an intrinsic contributor to skin ageing and pathological healing is 17ß-estradiol (or estrogen), although many others are involved. Estrogen deficiency is detrimental to many wound-healing processes, notably inflammation and re-granulation, while exogenous estrogen treatment widely reverses these effects. Over recent decades, many of the molecular and cellular correlates to estrogen's beneficial effect on normal skin homeostasis and wound healing have been reported. However, disparities still exist, particularly in the context of mechanistic studies investigating estrogen receptor signalling and its potential cellular effects. New molecular techniques, coupled with increased understanding of estrogen in skin biology, will provide further opportunities to develop estrogen receptor-targeted therapeutics.

Comparing the Effectiveness of Polymer Debriding Devices Using a Porcine Wound Biofilm Model.

Objective: Debridement to remove necrotic and/or infected tissue and promote active healing remains a cornerstone of contemporary chronic wound management. While there has been a recent shift toward less invasive polymer-based debriding devices, their efficacy requires rigorous evaluation. Approach: This study was designed to directly compare monofilament debriding devices to traditional gauze using a wounded porcine skin biofilm model with standardized application parameters. Biofilm removal was determined using a surface viability assay, bacterial counts, histological assessment, and scanning electron microscopy (SEM). Results: Quantitative analysis revealed that monofilament debriding devices outperformed the standard gauze, resulting in up to 100-fold greater reduction in bacterial counts. Interestingly, histological and morphological analyses suggested that debridement not only removed bacteria, but also differentially disrupted the bacterially-derived extracellular polymeric substance. Finally, SEM of post-debridement monofilaments showed structural changes in attached bacteria, implying a negative impact on viability. Innovation: This is the first study to combine controlled and defined debridement application with a biologically relevant ex vivo biofilm model to directly compare monofilament debriding devices. Conclusion: These data support the use of monofilament debriding devices for the removal of established wound biofilms and suggest variable efficacy towards biofilms composed of different species of bacteria.