Azelaic acid reduced senescence‐like phenotype in photo‐irradiated human dermal fibroblasts: possible implication of PPARγ

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long‐time treatment with AzA. We previously unrevealed that anti‐inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo‐aggravated disease, we investigated the ability of AzA to counteract stress‐induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8‐methoxypsoralen (PUVA), previously reported to activate a senescence‐like phenotype, including long‐term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence‐associated β‐galactosidase (SA‐β‐gal). We found that PUVA‐treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP‐1 release and SA‐β‐galactosidase‐positive cells. Moreover, AzA induced a reduction in ROS generation, an up‐modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA‐treated HDFs. Further evidences of AzA anti‐senescence effect were repression of p53 and p21, increase in type I pro‐collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA‐SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA‐induced senescence‐like phenotype and its ability to activate PPAR‐γ provides relevant insights into the anti‐senescence mechanism.     

Extract of Aneilema keisak inhibits transforming growth factor‐β‐dependent signalling by inducing Smad2 downregulation in keloid fibroblasts

Keloids are characterised by the excessive accumulation of extracellular matrix (ECM), especially overabundant collagen formation. In keloid fibroblasts (KFs), transforming growth factor (TGF)‐β‐dependent signalling is closely associated with a variety of keloid pathologic responses such as ECM production and fibroblast overgrowth. Thus, inhibition of TGF‐β signalling would be a potential therapeutic approach to prevent keloid scar formation. Thereby, we aimed to identify a novel TGF‐β signalling blocker among natural products using a simplified screening approach. We discovered that the extract of Aneilema keisak (A.K‐Ex) lowered TGF‐β‐dependent signalling by reducing Smad2 protein levels. Given that KFs showed altered dependency on TGF‐β for survival and proliferation, A.K‐Ex‐mediated reduction in Smad2 protein levels significantly inhibited the major characteristics of KFs such as cell growth, migration and collagen synthesis, suggesting that A.K‐Ex exhibits possible therapeutic activity on keloids.     

NADPH oxidase is a novel target of delphinidin for the inhibition of UVB‐induced MMP‐1 expression in human dermal fibroblasts

We investigated the reported antiphotoaging effects of the major anthocyanidin delphidin and sought to identify its specific molecular target during UVB‐induced MMP‐1 expression. Delphinidin treatment significantly inhibited UVB‐induced MMP‐1 expression in primary cultured human dermal fibroblasts (HDF), an effect associated with the suppression of MKK4‐JNK1/2, MKK3/6‐p38 and MEK‐ERK1/2 phosphorylation. Further investigation revealed that delphinidin significantly inhibited UVB‐induced ROS production and NOX activity. Interestingly, the inhibitory effect of delphinidin on UVB‐induced NOX activity was stronger than that of apocynin, a pharmaceutical NOX inhibitor. Fractioned cell analysis results using a Western blot assay showed that this effect occurred through the inhibition of UVB‐induced P47^phox (a NOX subunit) translocation from the cytosol to the membrane. Pull down assays demonstrated that delphinidin binds directly to P47^phox in vitro. Collectively, our results suggest that delphinidin targets NOX, resulting in the suppression of UVB‐induced MMP‐1 expression in human dermal fibroblasts.     

Overexpression of miR‐200b inhibits the cell proliferation and promotes apoptosis of human hypertrophic scar fibroblasts in vitro

Hypertrophic scarring leads to a deformed appearance and contracted neogenetic tissue, resulting in physiological and psychological problems for patients. Millions of people suffer these discomforts each year. Emerging evidence has reported that miRNA contributed to hypertrophic scarring or keloid formation. In this study, nine hypertrophic scar samples and the matched normal skin tissues were used to perform a miRNA microarray. The results of miRNA array showed that miR‐200b was downregulated by more than 2‐fold, validated by qPCR in hypertrophic scar tissues and human hypertrophic scar fibroblasts, suggesting that there was an important correlation between miR‐200b and hypertrophic scarring. We also found that miR‐200b affected hypertrophic scarring through regulating the cell proliferation and apoptosis of human hypertrophic scar fibroblasts by affecting the collagen I and III synthesis, fibronectin expression and TGF‐β1/α‐SMA signaling. Thus, our study provides evidence to support that miR‐200b may be a useful target for hypertrophic scarring management.     

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.     

Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF‐β1

Systemic sclerosis [scleroderma (SSc)]‐associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc‐induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose‐derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor β1 (TGF‐β1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte‐conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis‐like conditions. We concluded that in the skin affected by bleomycin‐induced fibrosis, increased TGF‐β1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.     

Transforming growth factor‐β enhances matrix metalloproteinase‐2 expression and activity through AKT in fibroblasts derived from angiofibromas in patients with tuberous sclerosis complex

Background Patients with tuberous sclerosis complex (TSC) develop fibrous tumours in the brain, skin, kidney, heart and lungs due to TSC1/2 mutations. In the skin, patients develop angiofibromas that have vascular and fibrotic components in which transforming growth factor (TGF)‐β and matrix metalloproteinase (MMP)‐2 are important. Objectives To investigate if the TGF‐β axis and MMP‐2 play an important role in the pathogenesis of TSC angiofibromas. Methods Samples from TSC angiofibromas and normal skin were measured for expression of TGF‐β and MMP‐2 by immunohistochemistry and real‐time polymerase chain reaction. Fibroblasts grown from TSC angiofibromas (TSC fibroblasts) were incubated with TGF‐β. Expression of ERK, AKT and S6K was measured by Western blotting, and MMP‐2 expression and activity were determined by enzyme‐linked immunosorbent assay and gelatin zymography, respectively. Results There was an increase in the expression of TGF‐β and MMP‐2 in TSC tumours compared with those in normal skin. The baseline expression of MMP‐2 was increased in conditioned medium from TSC fibroblasts. In addition, TGF‐β enhanced MMP‐2 production and activity, which could be abrogated by pretreatment with an AKT inhibitor (LY294002) but not with rapamycin. Finally, there was a significant colocalization of TGF‐β and MMP‐2 in the TSC tumours. Conclusions There is an increase of MMP‐2 as a result of TGF‐β acting through AKT in TSC tumour cells. This regulation of the TGF‐β–AKT–MMP‐2 axis is independent of mammalian target of rapamycin (mTOR) signalling. In addition to targeting the mTOR pathway, targeting TGF‐β simultaneously could block dysregulated tissue remodelling in TSC tumours.     

Staphylococcal α‐toxin induces a functional upregulation of TLR‐2 on human peripheral blood monocytes

Resistance to bacterial skin infections, for example with Staphylococcus aureus (S. aureus), is based on the function of intact innate immune mechanisms. Toll‐like receptor (TLR)‐2 recognizes components of S. aureus and is known to be expressed on monocytes. Staphylococcal exotoxins such as staphylococcal enterotoxin B (SEB) or α‐toxin are produced by many S. aureus strains. To investigate TLR‐2 regulation and function on human monocytes upon stimulation with staphylococcal exotoxins to elucidate a putative feedback loop between different staphylococcal components. Monocytes were stimulated with α‐toxin or SEB, respectively. TLR‐2 expression and regulation as well as functional effects of TLR‐2 stimulation with Pam3Cys (TLR‐2/TLR‐1), lipoteichoic acid (LTA) (TLR‐2/TLR‐6) and peptidoglycan (PGN) (TLR‐2 and Nod) were then investigated both at the mRNA and protein level and compared to monocytes from patients with psoriasis. α‐toxin significantly upregulated TLR‐2 expression. TLR‐2 mediated IL‐1β, IL‐6 and IL‐8 secretion was significantly augmented after upregulation with staphylococcal exotoxins. CD36 expression was significantly more downregulated after TLR‐2 upregulation with SEB and consecutive LTA stimulation and TLR‐2 upregulation with α‐toxin following LTA and PGN stimulation, respectively. PGN enhanced CD54 expression after upregulation of the receptor with α‐toxin. Expression of HLA‐DR was unaffected. However, no differences were observed in monocytes from psoriasis patients compared to healthy controls. Together, our findings provide a new link between staphylococcal α‐toxin and TLR‐2 signalling in monocytes which may have implications for skin diseases where skin colonization with S. aureus and dysregulation of TLR‐2 have been described.     

Epidermal keratinocytes sense dsRNA via the NLRP3 inflammasome,mediating interleukin (IL)‐1β and IL‐18 release

Skin epidermis, in addition to its barrier function, is able to actively sense harmful pathogens using pattern recognition receptors. In immune cells, the nucleotide‐binding oligomerization domain, leucine‐rich repeat and pyrin domain containing 3 (NLRP3) inflammasome can mediate innate immunity against viral infection via a mechanism involving viral dsRNA recognition. Epidermal keratinocytes express NLRP3 inflammasome, which can sense contact sensitizers and mite allergens, leading to pro‐interleukin (IL)‐1β and pro‐IL‐18 cleavage into their active forms. Skin often faces viral infection. However, it is unknown whether viral dsRNA can be detected by the keratinocyte NLRP3 inflammasome. We transfected polyinosinic:polycytidylic acid (poly I:C), a synthetic viral dsRNA analogue, into cultured primary human keratinocytes at the aid of Lipofectamine 2000, and found that transfected poly I:C activated caspase‐1 and induced caspase‐1‐dependent release of IL‐1β and IL‐18, which were suppressed on transfection with NLRP3 siRNA. The activation of keratinocyte NLRP3 inflammasome by transfected poly I:C was dependent on dsRNA‐induced protein kinase (PKR) activation, and priming with type I interferons upregulated NLRP3 inflammasome activation through promoting PKR activation in poly I:C‐transfected keratinocytes. In conclusion, the NLRP3 inflammasome can act as a sensor of dsRNA in epidermal keratinocytes, which may be important in both skin innate immune defense against viral infection and skin inflammation.     

CXC chemokine receptor 4 is essential for Lipo-PGE1-enhanced migration of human dermal fibroblasts

Abstract: Lipo‐PGE1 [EGLANDIN^®; a lipid microsphere‐incorporated prostaglandin E1 (PGE1)] stimulates angiogenesis and promotes the healing of skin ulcers. Because the effects of Lipo‐PGE1 on cutaneous wound healing are not completely understood, we investigated the ability of Lipo‐PGE1 to affect in vivo wound healing and regulate the migration of human dermal fibroblasts (HDFs). In a murine wound model, Lipo‐PGE1 reduced the wound size compared with control mice. Lipo‐PGE1 significantly increased HDF migration in a dose‐ and time‐dependent manner. Lipo‐PGE1 markedly increased the expression of CXC chemokine receptor 4 (CXCR4), which controls the migration of HDFs, at the mRNA and protein levels. Small interfering RNA (siRNA)‐mediated knockdown of CXCR4 inhibited Lipo‐PGE1–enhanced HDF migration. Moreover, Lipo‐PGE1 directly induced the phosphorylation of c‐Jun N‐terminal kinase (JNK), and the JNK‐specific inhibitor Sp6000125 blocked Lipo‐PGE1–enhanced migration and CXCR4 expression of HDFs. Our results demonstrate that Lipo‐PGE1 accelerates wound healing in vivo and increases the CXCR4‐mediated migration of HDFs through the JNK pathway.     

Malassezia yeasts activate the NLRP3 inflammasome in antigen‐presenting cells via Syk‐kinase signalling

Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro‐inflammatory cytokine IL‐1β. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL‐1β secretion in human antigen‐presenting cells. In contrast, keratinocytes were not able to secrete IL‐1β when exposed to Malassezia spp. Moreover, we demonstrate that IL‐1β secretion in antigen‐presenting cells was dependent on Syk‐kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro‐inflammatory responses when taken up by antigen‐presenting cells and identify C‐type lectin receptors and the NLRP3 inflammasome as crucial actors in this process.