Contribution of CARD9‐mediated signalling to wound healing in skin

The inflammatory response after skin injury involves the secretion of a variety of cytokines and growth factors that are necessary for tissue repair. Caspase recruitment domain‐containing protein 9 (CARD9) is an essential signalling adaptor molecule for NF‐κB activation upon triggering through C‐type lectin receptors (CLRs), which are expressed in macrophages and dendritic cells. However, the role of CARD9 in inflammatory responses at the wound site has not been elucidated. In this study, we analysed the role of CARD9 in the healing process of skin wounds. Wounds were created on the backs of wild‐type (WT) C57BL/6 mice and CARD9 gene‐disrupted (knockout [KO]) mice. We analysed per cent wound closure, and the wound tissues were harvested for analysis of leucocyte accumulation and cytokine and chemokine expressions. CARD9KO mice exhibited significant attenuation of wound closure compared with WT mice on days 5, 7 and 10 postwounding, which was associated with decreased macrophage accumulation and reduced TNF‐α, IL‐1β, CCL3 and CCL4 expressions. These results suggest that CARD9 may be involved in the wound‐healing process through the regulation of macrophage‐mediated inflammatory responses.     

Noninvasive red and near‐infrared wavelength‐induced photobiomodulation: promoting impaired cutaneous wound healing

The innumerable intricacies associated with chronic wounds have made the development of new painless, noninvasive, biophysical therapeutic interventions as the focus of current biomedical research. Red and near‐infrared light‐induced photobiomodulation therapy appears to emerge as a promising drug‐free approach for promoting wound healing, reduction in inflammation, pain and restoration of function owing to penetration power in conjunction with their ability to positively modulate the biochemical and molecular responses. This review will describe the physical properties of red and near‐infrared light and their interaction with skin and highlight their efficacy of wound repair and regeneration. Near‐infrared (800–830 nm) was found to be the most effective and widely studied wavelength range followed by red (630–680 nm) and 904 nm superpulsed light exhibiting beneficial photobiomodulatory effects on impaired dermal wound healing.     

Granulocyte‐macrophage colony‐stimulating factor enhances wound healing in diabetes via upregulation of proinflammatory cytokines

Summary Background Chronic ulceration, especially in diabetes, remains a substantial clinical problem. Exogenous granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is efficacious in the treatment of chronic wound healing in both animal models and patients, but its role in diabetic wounds remains to be explored. Objectives Using a diabetic mouse model, to investigate the role of GM‐CSF in wound healing. Methods Clinical observation, histopathology, immunohistochemistry and cytokine assays. Results There was a significant reduction (50%) in GM‐CSF production in the wounds of the diabetics compared with nondiabetics. Exogenous GM‐CSF substantially enhanced the wound healing in diabetic mice, accompanied by increased interleukin‐6 and monocyte chemoattractant protein‐1 production. The elevated cytokines correlated with increased neovascularization, and infiltration of macrophages and neutrophils. GM‐CSF showed no beneficial effects in nondiabetic wound healing. Conclusions Our results provide useful guidelines for the clinical management of chronic ulceration in diabetes.     

Sirtuin‐6 deficiency exacerbates diabetes‐induced impairment of wound healing

Delayed wound healing is one of the major complications in diabetes and is characterized by chronic proinflammatory response, and abnormalities in angiogenesis and collagen deposition. Sirtuin family proteins regulate numerous pathophysiological processes, including those involved in promotion of longevity, DNA repair, glycolysis and inflammation. However, the role of sirtuin 6 (SIRT6), a NAD+‐dependent nuclear deacetylase, in wound healing specifically under diabetic condition remains unclear. To analyse the role of SIRT6 in cutaneous wound healing, paired 6‐mm stented wound was created in diabetic db/db mice and injected siRNA against SIRT6 in the wound margins (transfection agent alone and nonsense siRNA served as controls). Wound time to closure was assessed by digital planimetry, and wounds were harvested for histology, immunohistochemistry and Western blotting. SIRT6‐siRNA‐treated diabetic wound showed impaired healing, which was associated with reduced capillary density (CD31‐staining vessels) when compared to control treatment. Interestingly, SIRT6 deficiency decreased vascular endothelial growth factor expression and proliferation markers in the wounds. Furthermore, SIRT6 ablation in diabetic wound promotes nuclear factor‐κB (NF‐κB) activation resulting in increased expression of proinflammatory markers (intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, tumor necrosis factor‐α and interleukin‐1β) and increased oxidative stress. Collectively, our findings demonstrate that loss of SIRT6 in cutaneous wound aggravates proinflammatory response by increasing NF‐κB activation, oxidative stress and decrease in angiogenesis in the diabetic mice. Based on these findings, we speculate that the activation of SIRT6 signalling might be a potential therapeutic approach for promoting wound healing in diabetics.     

The healing effect of over‐the‐counter wound healing agents applied under semiocclusive film dressing

Basal cell carcinoma (BCC), the most common cancer in the U.S.A., is treated primarily with local excision. In some cases, lesion size, location or extent prevent complete resection. Locally advanced BCC responds to systemic therapy with the Hedgehog pathway inhibitor vismodegib, but withdrawal of treatment may result in disease relapse. Here we present a case of locally advanced auricular BCC treated with induction vismodegib and radiation, resulting in durable local control and an acceptable level of acute toxicity.     

Fibroblast‐specific upregulation of Flightless I impairs wound healing

The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell‐specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue‐specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast‐specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue‐specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non‐inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non‐tissue‐specific upregulation of Flii suggesting that fibroblast‐derived Flii may have an important role in the wound healing response.     

IL‐17A synergistically enhances TLR3‐mediated IL‐36γ production by keratinocytes: A potential role in injury‐amplified psoriatic inflammation

Skin injury can trigger formation of new lesions in psoriasis (Koebner phenomenon). The mechanisms through which injury exacerbates psoriasis are unclear. During wound repair, epidermal keratinocytes are activated and produce abundant IL‐36γ, further promoting the skin inflammation. IL‐17A is the cornerstone cytokine in the pathogenesis of psoriasis. We sought to investigate the effects of IL‐17A on injury‐induced keratinocyte activation and IL‐36γ production. Here, we demonstrated that dsRNA released from necrotic keratinocytes induced the expression of IL‐36γ. Silencing of TLR3 by siRNA decreased the IL‐36γ induction by necrotic keratinocyte supernatant. Co‐stimulation with dsRNA and IL‐17A synergistically increased the expression of IL‐36γ and other proinflammatory mediators (CCL20, CXCL8, DEFB4 and LCN2) in keratinocytes. The synergistic effects were not dependent on TLR3 upregulation, TNF receptor signalling and mRNA stabilization. Co‐stimulation with dsRNA and IL‐17A resulted in an accumulation of IκBζ. The synergistic upregulation of IL‐36γ and proinflammatory mediators were inhibited by IκBζ siRNA. Co‐stimulation with IL‐17A and poly(I:C) markedly activated the p38 MAPK and NF‐κB pathway, compared with poly(I:C). Blockade of p38 MAPK and NF‐κB suppressed dsRNA/IL‐17A–mediated IκBζ and IL‐36γ induction. These findings demonstrated that IL‐17A synergistically enhanced the dsRNA‐mediated IL‐36γ production through a p38 MAPK‐, NF‐κB–, and IκBζ‐dependent mechanism.     

Improved cutaneous wound healing after intraperitoneal injection of alpha‐melanocyte‐stimulating hormone

Skin wound healing is a complex process involving many types of cells and molecules and often results in scar tissue formation in adult mammals. However, scarless healing occurs in foetal skin and minimal scars may occur after cutaneous healing in the adult with reduced inflammation. Alpha‐melanocyte‐stimulating hormone (α‐MSH) is widely distributed within the central nervous system and in other body regions, such as the skin, and has strong anti‐inflammatory activity. The aim in the present experiments was to learn whether intraperitoneal (i.p) injection of α‐MSH just before skin wounds antagonize inflammation and improves skin wound healing in adult mice. C57BL/6 young adult mice received an i.p. injection of 1 mg/kg of α‐MSH and, 30 min later, two circular through‐and‐through holes (6.5 mm diameter) were made in their dorsal skin under anaesthesia. Control mice were wounded after vehicle injection. The wound healing process was analysed macroscopically and microscopically at 3, 7, 40 and 60 days. Skin samples were fixed in formalin, embedded in paraffin, sectioned at 5 μm, stained with H&E or toluidine blue for cell analysis or Gomori's trichrome for extracellular matrix (ECM) analysis. Other samples were fixed in DMSO+methanol, embedded in paraplast and incubated with anti‐CD45, antismooth muscle actin, anticollagen‐I and anticollagen‐III for immunofluorescence analysis. Alpha‐MSH significantly reduced the number of leucocytes, mast cells and fibroblasts at 3 and 7 days after injury. On days 40 and 60, α‐MSH reduced scar area and improved the organization of the collagen fibres indicating that it may direct the healing into a more‐regenerative/less‐scarring pathway.     

Lipocalin‐2 exacerbates psoriasiform skin inflammation by augmenting T‐helper 17 response

Lipocalin‐2 (LCN2) is an antimicrobial protein and adipokine associated with insulin resistance, obesity and atherosclerotic disease. Psoriasis is a T‐helper (Th)1/Th17‐mediated, chronic inflammatory dermatosis related to metabolic syndromes and serum LCN2 levels are elevated in psoriatic patients. We examined the in vivo effects of LCN2 on topical imiquimod (IMQ)‐induced psoriasiform skin in BALB/c mice and in vitro on human keratinocytes (KC). Clinically, i.p. injected LCN2 exacerbated erythema and scaling in IMQ‐treated murine skin compared with phosphate‐buffered saline injection alone, and it augmented interleukin (IL)‐17A, IL‐17F, IL‐22, IL‐23p19, IL‐12p40, CCL20, tumor necrosis factor‐α, chemokine (C‐X‐C motif) ligand (CXCL)1, CXCL2, DEFB4, DEFB14, LCN2 and S100A7 mRNA levels of IMQ‐treated murine skin while it did not increase the mRNA levels of interferon‐γ, IL‐12p35 or CXCL10. LCN2 in synergy with IL‐17 increased mRNA levels of CCL20, LCN2 and DEFB4A but not of CXCL10 in human KC in vitro. These results suggest that LCN2 enhances the expression of Th17 cytokines/chemokines and antimicrobial peptides in murine IMQ‐treated psoriatic skin and KC. LCN2 may potentiate the development of psoriasis via the enhancement of Th17‐ and antimicrobial peptide‐mediated inflammation.     

Vitiligo patient‐derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition

Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte‐induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial–mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF‐b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.     

Histamine suppresses regulatory T cells mediated by TGF‐β in murine chronic allergic contact dermatitis

Regulatory T cells (Tregs) suppress effector T cells and ameliorate contact hypersensitivity (CH); however, the role of Tregs in chronic allergic contact dermatitis (CACD) has not been assessed. Repeated elicitation of CH has been used to produce CACD models in mice. We previously showed that the presence of histamine facilitates the creation of eczematous lesions in this model using histidine decarboxylase (HDC) (?/?) mice. Therefore, the effects of histamine on Tregs in the CACD model were investigated in this study. CACD was developed by repeated epicutaneous application of 2, 4, 6‐trinitro‐1‐chlorobenzene (TNCB) on HDC (+/+) and HDC (?/?) murine skin to assess the effects of histamine in CACD. Histamine aggravated CACD in the murine model and suppressed the number of Tregs in the skin. Histamine also suppressed the level of TGF‐β1 in this model. Recombinant TGF‐β1 or anti‐TGF‐β1 antibody was injected into the dorsal dermis of HDC (+/+) mice daily just before TNCB challenge to determine the effects of histamine‐regulated TGF‐β on the Treg population in CACD. Recombinant TGF‐β1 injection promoted the infiltration of Tregs in the skin and the production of IL‐10; however, anti‐TGF‐β1 antibody injection suppressed the number of Tregs in the skin and the production of IL‐10. Histamine suppresses the number of Tregs in CACD, and this effect is mediated by TGF‐β.     

Acute stress enhances contact dermatitis by promoting nuclear factor‐κB DNA‐binding activity and interleukin‐18 expression in mice

Psychological stress adversely affects the immune system, and aggravates various skin diseases, such as psoriasis, alopecia areata and atopic dermatitis. However, the precise underlying mechanisms remain to be elucidated. The goal of this study was to use a murine restraint stress model to determine the mechanisms by which psychological stress modulates immune response in contact dermatitis. In the present study, mice were sensitized and challenged on the skin with 2,4‐dinitrofluorobenzene. Acute restraint stress was administrated to healthy or sensitized mice before challenge, and nuclear factor (NF)‐κB DNA‐binding activation of nuclear protein and expression of interleukin (IL)‐18 mRNA in murine spleen lymphocytes was detected. Chemical sympathectomy was performed using the neurotoxin 6‐hydroxy‐dopamine to determine the effect of the sympathetic nervous system. The experiment showed that restraint stress induced a series of changes which include increasing of NF‐κB DNA‐binding activity and IL‐18 mRNA expression in spleen lymphocytes and enhancement of contact hypersensitivity response, and these changes may be mediated by the sympathetic nervous system. These findings provide new insights into the roles of the nervous system in the aggravation of skin diseases.     

A dipeptidyl peptidase‐4 inhibitor promotes wound healing in normoglycemic mice by modulating keratinocyte activity

Dipeptidyl peptidase‐4 (DPP‐4) inhibitors are a well‐known and novel class of oral antihyperglycaemic drugs. DPP‐4 inhibition facilitates ulcer healing in patients with diabetes. However, the actual mechanisms, which are independent of lower blood glucose levels, are still unknown. Therefore, the aim of this study was to analyse the effect of the DPP‐4 inhibitor sitagliptin on wound healing through a glucose‐independent pathway. In this study, DPP‐4 inhibitors facilitate keratinocyte differentiation and the proliferation, increase blood flow in the cutaneous of wounds in healthy C57BL/6 mice. Additionally, the administration of the DPP‐4 inhibitor ameliorates wound healing and enhances adiponectin expression in healthy C57BL/6 mice. Taken together, our results reveal a protective role for the DPP‐4 inhibitor sitagliptin in wound healing by regulating adiponectin and phospho‐eNOS levels in keratinocytes. Based on these results, the DPP‐4 inhibitor may have therapeutic potential for healing wounds through a diabetes‐independent mechanism.     

Extracellular vesicles from human hepatic progenitor cells accelerate deep frostbite wound healing by promoting fibroblasts proliferation and inhibiting apoptosis

Frostbites are cold tissue damages frequently observed at high altitudes and under extremely cold conditions. Though their incidence rate is low, the resulting impact in affected patients can be very serious, often leading to amputations. Clinical management and the prediction of outcome can be of utmost importance to frostbite patients. A possible use of stem cell-derived extracellular vesicles (EVs) has been suggested for cutaneous wound healing and we, therefore, tested their use for the treatment of deep frostbite wound. To this end, the impacts of hHPC-derived EVs were evaluated in an in vivo animal model comprising of Kunming female mice as well as studied in vitro for the mechanism. We first characterized the EVs and these hHPC-derived EVs, when applied to treat frostbite wounds, accelerated wound healing in the in vivo animal model, as assessed by wound closure, re-epithelization thickness, collagen density and the expression of Collagen I and α-SMA. The proliferation and migration of human skin fibroblasts was also found to be increased by EVs in the in vitro experiments. The H₂O₂-induced apoptosis cell model, established to simulate the post-frostbite injury, was inhibited by EVs, with concomitant increase in the expression of Bcl-2 and decreased expression of Bax, further confirming the findings. Our novel results indicate that the application of EVs might be a promising strategy for deep frostbite wound healing.