CD80 on skin stem cells promotes local expansion of regulatory T cells upon injury to orchestrate repair within an inflammatory environment.

Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.

Critical role of interferons in gastrointestinal injury repair.

The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.

Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds.

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell-deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.

Keratinocyte Integrin α3ß1 Promotes Secretion of IL-1α to Effect Paracrine Regulation of Fibroblast Gene Expression and Differentiation.

After cutaneous injury, keratinocytes secrete paracrine factors that regulate wound cell functions; dysregulation of this signaling can lead to wound pathologies. Previously, we established that keratinocyte integrin α3ß1 promotes wound angiogenesis through paracrine stimulation of endothelial cells. We hypothesize here that α3ß1-dependent paracrine signaling from keratinocytes regulates the differentiation state of myofibroblasts. We report that epidermal α3-knockout mice exhibit more wound myofibroblasts and fewer cyclooxygenase 2 (Cox-2)-positive dermal cells than controls. We also found that conditioned medium from α3-expressing mouse keratinocytes (MKα3+), but not from α3-null MK cells (MKα3-), induces expression of Cox-2 in fibroblasts in a time- and dose-dependent manner and that this induction is mediated by IL-1α. Compared with MKα3- cells, MKα3+ cells secrete more IL-1α and less IL-1RA, a natural IL-1 receptor antagonist. Treatment with an IL-1α neutralizing antibody, recombinant IL-1RA, or IL-1 receptor-targeting small interfering RNA suppresses MKα3+ conditioned medium-dependent induction of Cox-2 expression in fibroblasts. Finally, active recombinant IL-1α is sufficient to induce Cox-2 in fibroblasts and to inhibit transforming growth factor-ß-induced α-SMA expression. Our findings support a role for keratinocyte integrin α3ß1 in controlling the secretion of IL-1α, a paracrine factor that regulates the wound myofibroblast phenotype.

Partial thickness wound: Does mechanism of injury influence healing?

Wound healing is a complex multistep process which is temporally and spatially controlled. In partial thickness wounds, regeneration is possible from the stem cells in the edges of the wound and from the remnants of the epidermal appendages (such as hair follicles and sebaceous glands). This study examines whether the mechanism of injury influences healing of wounds of similar depth. Burn and excisional wounds were created on the back of Hampshire pigs and harvested at 7, 14, 28, 44, 57 and 70days after injury and processed for histology and immunohistochemistry. Quantitative analysis of re-epithelialisation, inflammatory response and thickness of the scar and semi-quantitative analyses of the architecture of the resultant scar were performed and subjected to statistical analysis. Results demonstrated a higher number of neutrophils, macrophages and lymphocytes present in the burn on day 7 compared to the excisional wounds. The inflammatory profile of burn wounds was higher than that of excisional wounds for the first month after injury albeit less marked than on day 7 after injury. Re-epithelialisation was markedly advanced in excisional wounds compared to burn wounds at day 7 after injury, corresponding to the higher number of hair follicles in the underlying dermis of excisional wounds at this time point. The thickness of the neo-epidermis increased with time and at day 70 after wounding, the neo-epidermis of the burn was significantly thicker than the neo-epidermis of the excisional scar. Interestingly, following partial thickness excision of skin, there was neo-dermal reformation albeit with an altered architecture, lacking the normal basket-weave pattern of collagen. The thickness of the dermis of partial thickness excisional scar was greater than that of the adjacent unwounded skin. The neo-dermis of the burn scar was even thicker, with the collagen arranged more compactly and disorganised compared to excisional scar and normal skin. This study provides evidence that the mechanism of injury does influence wound healing and the resultant scarring.

Novel Biodegradable Polymeric Microparticles Facilitate Scarless Wound Healing by Promoting Re-epithelialization and Inhibiting Fibrosis.

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content.

Time to Abandon Antimicrobial Approaches in Wound Healing: A Paradigm Shift.

Antimicrobial approaches (eg, antibiotics and antiseptics) have been used for decades in the treatment of infected wounds, ulcers, and burns. However, an increasing number of meta-analyses have raised questions regarding the therapeutic value of these approaches. Newer findings show that the body actively hosts an ecosystem of bacteria, fungi, viruses, and mites on its outer surfaces, known as the microbiome, as part of its defense against pathogens. Antimicrobials would disrupt this system and thereby work against the strategy the body has chosen. Recently, a new technology, micropore particle technology (MPPT), has been identified; it is not an antimicrobial but instead acts as a passive immunotherapy that disrupts the weaponry bacteria and fungi use to inhibit the immune system, allowing the immune system to recover. Clinical findings show MPPT removes wound infections 60% quicker than antibiotics and antiseptics and promotes the healing of chronic wounds that have not responded to antimicrobials. These effects are achieved without antimicrobial action and, considering the limited therapeutic benefits of antibiotics and antiseptics for wound infections, it is valid to question the use of antimicrobial approaches in wound care and the dogma that a reduction in microbial burden will lead to a reduction in infection. Instead, it may be time to consider a paradigm shift in wound healing away from antimicrobials and towards therapies that support the immune system and the microbiome. This review covers the increasing evidence that infections on external surfaces have to be treated fundamentally differently to internal infections.

Cyclosporine for Epidermal Necrolysis: Absence of Beneficial Effect in a Retrospective Cohort of 174 Patients-Exposed/Unexposed and Propensity Score-Matched Analyses.

Cyclosporine has shown promising results for mortality in patients with Stevens-Johnson syndrome/toxic epidermal necrolysis. However, available studies included only a small number of patients and did not include a validated and homogenous control group. We present the results from a retrospective monocentric study including 174 patients with Stevens-Johnson syndrome/toxic epidermal necrolysis during 2005-2016. Among them, 95 received cyclosporine (3 mg/kg/day) plus supportive care, and 79 received supportive care only. Both a traditional exposed/unexposed method and a propensity score-matching method were used to compare the progression of skin detachment between day 0 and day 5, the proportion of patients with cutaneous re-epithelialization starting on day 5 or mucosal re-epithelialization on day 10, the duration of progression, and the number of deaths between the two groups. None of these outcomes significantly favored cyclosporine, either by the exposed/unexposed method or the propensity score method. Acute renal failure affected more patients receiving cyclosporine (P = 0.05). Overall, the results of this epidemiological study did not show a beneficial effect of cyclosporine in patients with Stevens-Johnson syndrome/toxic epidermal necrolysis. They are discordant with those previously published. The large number of patients and the use of a propensity score method provide valuable insights. The main limitation of the study is the lack of randomization.

[Hyaluronan as a key for accelerated wound healing in human 3D full thickness skin models]. / Hyaluronan als Schlüssel zur schnelleren Wundheilung in humanen 3­D-Vollhautmodellen.

BACKGROUND: Wound healing can be divided into three phases: (1) exsudation phase, (2) granulation phase, (3) regeneration phase. In particular, the epithelization phase is of great importance in order to quickly reconstitute the natural skin barrier. The aim of the present study was to determine the reepithelization kinetics of untreated and 0.5% sodium hyaluronate (NHA) treated human 3D full thickness skin models. MATERIALS AND METHODS: The test protocol consisted of topically applying 10 µl of the test substance 0.5% NHA twice a day. Evaluation of reepithelialization kinetics was carried out from days 2-6. Determination of the influence on immune response was performed based on quantification of IL-1α and IL-10. RESULTS: Application of 0.5% NHA twice a day enhanced the reepithelialization speed at all time points (p < 0.001). This observation is accompanied by a reduced expression of IL-10 paralleled by an elevated expression of IL-1α on days 2-4 (p < 0.001). DISCUSSION: The treatment of human skin models with NHA resulted in a significantly increased reepithelization velocity of wounded tissue and consequently promoted faster wound closure, compared to untreated controls. It can be assumed that the downregulation of IL-10 caused the IL1-α mediated increased immune response which finally leads to accelerated wound healing. Follow-up studies will reveal if the faster wound healing and the modulation of the immune response through the application of NHA is valid in vivo.

Dectin-1 activation induces proliferation and migration of human keratinocytes enhancing wound re-epithelialization.

Beta-glucans in temporary wound dressings have immuno-stimulatory capacities and have been shown to enhance wound healing in burn patients. Curdlan is a 1,3-linked bacterial/fungal derived beta-glucan that induces inflammatory responses via the C-type lectin receptor dectin-1 on dendritic cells (DCs). Here we investigated the effect of beta-glucan curdlan and the role of dectin-1 expressed by keratinocytes (KCs) in wound healing. Curdlan enhanced migration, proliferation and wound closure of human KCs in a dectin-1 dependent manner, both in vitro and ex vivo. Our data suggest that curdlan induces human KC proliferation and migration and could therefore be used in creams to enhance wound healing.

An in vitro wound healing model for evaluation of dermal substitutes.

Reepithelialization of skin wounds is essential to restore barrier function and prevent infection. This process requires coordination of keratinocyte proliferation, migration, and differentiation, which may be impeded by various extrinsic and host-dependent factors. Deep, full-thickness wounds, e.g., burns, are often grafted with dermal matrices before transplantation of split-skin grafts. These dermal matrices need to be integrated in the host skin and serve as a substrate for neoepidermis formation. Systematic preclinical analysis of keratinocyte migration on established and experimental matrices has been hampered by the lack of suitable in vitro model systems. Here, we developed an in vitro full-thickness wound healing model in tissue-engineered human skin that allowed analysis of the reepithelialization process across different grafted dermal substitutes. We observed strong differences between porous and nonporous matrices, the latter being superior for reepithelialization. This finding was corroborated in rodent wound healing models. The model was optimized using lentivirus-transduced keratinocytes expressing enhanced green fluorescent protein and by the addition of human blood, which accelerated keratinocyte migration underneath the clot. Our model shows great potential for preclinical evaluation of tissue-engineered dermal substitutes in a medium-throughput format, thereby obviating the use of large numbers of experimental animals.

Randomized placebo-controlled human pilot study of cold atmospheric argon plasma on skin graft donor sites.

Cold atmospheric plasma has already been shown to decrease the bacterial load in chronic wounds. However, until now it is not yet known if plasma treatment can also improve wound healing. We aimed to assess the impact of cold atmospheric argon plasma on the process of donor site healing. Forty patients with skin graft donor sites on the upper leg were enrolled in our study. The wound sites were divided into two equally sized areas that were randomly assigned to receive either plasma treatment or placebo (argon gas) for 2 minutes. Donor site healing was evaluated independently by two blinded dermatologists, who compared the wound areas with regard to reepithelialization, blood crusts, fibrin layers, and wound surroundings. From the second treatment day onwards, donor site wound areas treated with plasma (n = 34) showed significantly improved healing compared with placebo-treated areas (day 1, p = 0.25; day 2, p = 0.011; day 3, p < 0.001; day 4, p < 0.001; day 5, p = 0.004; day 6, p = 0.008; day 7, p = 0.031). Positive effects were observed in terms of improved reepithelialization and fewer fibrin layers and blood crusts, whereas wound surroundings were always normal, independent of the type of treatment. Wound infection did not occur in any of the patients, and no relevant side effects were observed. Both types of treatment were well tolerated. The mechanisms contributing to these clinically observed effects should be further investigated.

Rapamycin reduces burn wound progression by enhancing autophagy in deep second-degree burn in rats.

Burn wound progression is caused by many mechanisms including local tissue hypoperfusion, prolonged inflammation, free radical damage, apoptosis, and necrosis in burn wounds. Autophagy, a homeostatic process by which cells break down their own components, was found to protect against ischemic injury, inflammatory diseases, and apoptosis in some cases. We tested whether rapamycin, an autophagy inducer, could ameliorate burn wound progression and promote wound healing through autophagy enhancement. Using a previously described deep second-degree burn model, we first tested the effects of rapamycin on autophagic response in burn wound tissue. Autophagy levels in wound tissue of treated rats were increased as compared with controls. Furthermore, we found that laser Doppler flowmetry values and Na/K-ATPase activities were markedly higher in the treated wounds. The content of interleukin-8, methane dicarboxylic aldehyde, and myeloperoxidase activity in the wounds of treated rats were much lower than in controls. The apoptotic rates in treated wounds were much lower than controls as determined by terminal deoxynucleotidyl transferase mediated nick end labeling assay. Finally, histomorphological analysis showed that burn wound progression in the treatment group was ameliorated. The time to wound reepithelialization was shorter in the treated wounds than controls 22.5 ± 1.4 days vs. 24.8 ± 1.3 days (mean ± standard deviation, p < 0.01).