Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8+ T cells which enables an effective combination immunotherapy with anti-PD-1.

Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357 mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 1015 particles (1.10 × 1010 particles/µg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8+ T cells, especially those of cancer antigen-specific CD8+ T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8+ T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents.

Transcriptomic analysis of human dermal fibroblast cells reveals potential mechanisms underlying the protective effects of visible red light against damage from ultraviolet B light.

BACKGROUND: Ultraviolet B (UVB) radiation is a major cause of skin photodamage, including the damage associated with photodermatoses, aging, and cancer. Although many studies have shown that red light has photoprotective effects on skin, the mechanisms underlying these effects are still poorly understood. OBJECTIVE: The aim of this study was to identify the photoprotective effects of visible red light against UVB-induced skin damage in normal human dermal fibroblast cells using a transcriptomic approach. METHODS: Next-generation sequencing-based transcriptomic analyses were used to profile transcriptomic alterations and identify genes that are differentially expressed by visible red light and by UVB exposure. To understand the biological networks among identified genes, a literature-based biological pathway analysis was performed. Quantitative real-time polymerase chain reaction assays were used for mRNA-level validation of selected key genes. RESULTS: We observed that visible red light contributes to skin cell protection against UVB by modulating gene expression that enhances the adaptive response to redox and inflammatory balancing and by upregulating genes involved in DNA excision repair processes. We also identified that several key genes in the red light-induced biological network were differentially regulated. CONCLUSIONS: Visible red light enhanced the UVB-protective effects in normal human skin cells via the transcriptomic modulation of genes involved in cell-protective processes. Our findings from this next-generation sequencing analysis may lead to a better understanding of the cytoprotective effects of visible red light and provide direction for further molecular or mechanistic studies.

Dehydroabietic Acid Suppresses Inflammatory Response Via Suppression of Src-, Syk-, and TAK1-Mediated Pathways.

Dehydroabietic acid (DAA) is a naturally occurring diterpene resin acid derived from coniferous plants such as Pinus and Picea. Various bioactive effects of DAA have been studied including antibacterial, antifungal, and anticancer activities. However, the anti-inflammatory mechanism of DAA remains unclear. We evaluated the anti-inflammatory effect of DAA in macrophage cell lines. Dehydroabietic acid clearly reduced nitric oxide (NO) production and inflammatory gene expression decreased according to RT-PCR results. Dehydroabietic acid displayed anti-inflammatory activity at the transcriptional level in results from NF-κB- or AP-1-mediated luciferase assays. To identify the DAA target protein, we investigated NF-κB and AP-1 pathways by Western blotting analysis. Dehydroabietic acid suppressed the activity of proto-oncogene tyrosine protein kinase (Src) and spleen tyrosine kinase (Syk) in the NF-κB cascade and transforming growth factor beta-activated kinase 1 (TAK1) in the AP-1 cascade. Using overexpression strategies, we confirmed that DAA targeted these kinases. Our findings demonstrate the anti-inflammatory effects and molecular mechanism of DAA. This suggests that DAA has potential as a drug or supplement to ameliorate inflammation.

Fine particulate matter (PM2.5) inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and skin keratinocytes.

Exposure to fine particulate matter (PM) with diameter <2.5 µm (PM2.5) causes epithelium injury and endothelial dysfunction. Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses and have roles in physiology. To date, there have been no studies investigating whether PM2.5 affects primary cilia in skin. We addressed this in the present study using normal human epidermal keratinocytes (NHEKs) and retinal pigment epithelium (RPE) cells. We found that formation of primary cilium is increased in differentiated NHEKs. However, treatment with PM2.5 blocked increased ciliogenesis in NHEKs and RPE cells. Furthermore, PM2.5 transcriptionally upregulated small proline rich protein 3 (SPRR3) expression by activating c-Jun, and ectopic expression of SPRR3 inhibits suppressed the ciliogenesis. Accordingly, treatment with c-Jun activator (anisomycin) induced SPRR3 expression, whereas the inhibitor (SP600125) recovered the ciliated cells and cilium length in PM2.5-treated cells. Moreover, c-Jun inhibitor suppressed upregulation of SPRR3 in PM2.5-treated cells. Taken together, our finding suggested that PM2.5 inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and keratinocytes.

Dehydroabietic Acid Induces Regeneration of Collagen Fibers in Ultraviolet B-Irradiated Human Dermal Fibroblasts and Skin Equivalents.

BACKGROUND/AIMS: Dehydroabietic acid (DAA) is a natural phytochemical found in red pine trees and herbal plants. While DAA and its derivatives are known for improving diabetes and hyperlipidemia, the antiaging effect and its underlying mechanisms of DAA on skin have not been fully examined. Here, we assessed the antiaging effects of DAA on human dermal fibroblasts and skin equivalents. METHODS: We investigated the effect of DAA on the secretion of type I procollagen and matrix metalloproteinase-1 (MMP-1) in ultraviolet B (UVB)-irradiated neonatal normal human dermal fibroblasts (NHDFn). Using nonlinear optical imaging techniques, we visualized quantitative and qualitative changes of collagen fibers by DAA treatment in human skin equivalent models. RESULTS: DAA induces increases in type I procollagen secretion when treated on UVB-irradiated NHDFn. DAA also downregulates secretion of MMP-1 through the inhibition of the JNK signaling pathway. In human skin equivalent models, we successfully visualized the spatial distribution of collagen fibers in the dermis and found that quantity, diameter, and arrangement of collagen fibers in the dermis were significantly improved by DAA treatment. CONCLUSION: Our results suggest that DAA could be a useful agent for improving skin photoaging through the protection and regeneration of collagen fibers in skin.

Mannosylerythritol lipids ameliorate ultraviolet A-induced aquaporin-3 downregulation by suppressing c-Jun N-terminal kinase phosphorylation in cultured human keratinocytes.

Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma (PPAR-γ), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of PPAR-γ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of PPAR-γ. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen.

Coumestrol (CMS), a coumestan isoflavone, plays key roles in nodulation through communication with rhizobia, and has been used as phytoestrogens for hormone replacement therapy in humans. Because CMS content is controlled by multiple genetic factors, the genetic basis of CMS biosynthesis has remained unclear. We identified soybean genotypes with consistently high (Daewonkong) or low (SS0903-2B-21-1-2) CMS content over 2 years. We performed RNA sequencing of leaf samples from both genotypes at developmental stage R7, when CMS levels are highest. Within the phenylpropanoid biosynthetic pathway, 41 genes were tightly connected in a functional co-expression gene network; seven of these genes were differentially expressed between two genotypes. We identified 14 candidate genes involved in CMS biosynthesis. Among them, seven were annotated as encoding oxidoreductases that may catalyze the transfer of electrons from daidzein, a precursor of CMS. Two of the other genes, annotated as encoding a MYB domain protein and a MLP-like protein, may increase CMS accumulation in response to stress conditions. Our results will help to complete our understanding of the CMS biosynthetic pathway, and should facilitate development of soybean cultivars with high CMS content that could be used to promote the fitness of plants and human beings.

EGR3 Is a Late Epidermal Differentiation Regulator that Establishes the Skin-Specific Gene Network.

Late epidermal differentiation is a key step of skin barrier formation; however, the specific genetic factors that distinguish late differentiation from early differentiation remain unknown. Here, we demonstrated that EGR3 is highly expressed in the stratum granulosum, and that it contributes to late epidermal differentiation. However, its expression is lost under poorly differentiated conditions, such as parakeratosis-lesional skin. EGR3 mediated the regulation of genes located in the epidermal differentiation complex through activation of enhancers and induction of enhancer RNAs. We further identified 20 targets of EGR3 specific for late differentiation. Additionally, we discovered that EGR3- and EGR3-related genes exhibited high tissue specificity on the skin. Through weighted gene co-expression analysis, EGR3 was found to be related to the keratinocyte differentiation-related module as an important part of the skin-specific genetic network. These findings shed light on the transcriptional regulation of late epidermal differentiation, highlighting candidate targets for diseases related to disrupted differentiation.

Staphylococcus aureus-derived extracellular vesicles induce monocyte recruitment by activating human dermal microvascular endothelial cells in vitro.

BACKGROUND: Atopic dermatitis (AD) represents the most common inflammatory skin disorder in children showing massive infiltration of immune cells. The colonization of AD-afflicted skin by Staphylococcus aureus and S. aureus-derived extracellular vesicles (SEVs) has been associated with AD pathogenesis; however, the molecular mechanism underlying SEV-mediated inflammatory responses remains unclear. OBJECTIVE: We investigated how SEVs can mediate inflammatory responses in AD pathogenesis by examining the effect of SEVs on human dermal microvascular endothelia cells (HDMECs). METHODS: HDMECs were treated with SEVs, and the expression of cell adhesion molecules or cytokines was assessed using RT-qPCR, Western blot or cytokine array analyses. The receptor for SEVs and related signalling molecules in HDMECs were addressed and verified via gene knockdown or inhibitor experiments. The recruitment assay of human THP-1 monocytic cells on HDMECs was performed after SEV treatment in the presence or absence of the verified receptor or signalling molecule. RESULTS: SEVs, but not other gram-positive bacteria-derived extracellular vesicles, directly activated HDMECs by increasing the expression of cell adhesion molecules (E-selectin, VCAM1 and ICAM1) and that of IL-6, the inflammatory cytokine; consequently, they enhanced the recruitment of THP-1 monocytic cells to HDMECs. The SEV-induced HDMEC activation was dependent on Toll-like receptor 4 and the NF-κB signalling pathway, which was rapidly activated within 1 hour post-treatment and followed by an upregulation of cell adhesion molecules and IL-6 at later time-points. Moreover, SEV-mediated HDMEC responses were more rapid and intense than those induced by the same protein concentrations of S. aureus extracts. CONCLUSIONS & CLINICAL RELEVANCE: SEVs as proinflammatory factors could mediate immune cell infiltration in AD by efficiently inducing endothelial cell activation and monocyte recruitment, which may provide insights into alleviating the S. aureus-mediated onset or progression of AD and its phenotypes.

Mannosylerythritol lipids inhibit melanogenesis via suppressing ERK-CREB-MiTF-tyrosinase signalling in normal human melanocytes and a three-dimensional human skin equivalent.

Hyperpigmentation is caused by excessive production of melanin in melanocytes. Mannosylerythritol lipids (MELs) are glycolipid biosurfactants that are abundantly produced by yeasts and used commercially in cosmetics. However, the potential depigmenting efficacy of MELs has not been evaluated. In this study, the depigmentary effect of MELs was tested in primary normal human melanocytes (NHMs), α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells (murine melanoma cells) and a human skin equivalent (MelanoDerm) using photography, Fontana-Masson (F&M) staining and two-photon microscopy. Mannosylerythritol lipids significantly decreased the melanin contents in NHMs and α-MSH-stimulated B16 cells. Consistent with these findings, MELs treatment had a clear whitening effect in a human skin equivalent, brightening the tissue colour and reducing the melanin content. The molecular mechanism underlying the anti-melanogenic effect of MELs treatment was examined by real-time PCR and Western blotting. Mechanistically, MELs clearly suppressed the gene expression levels of representative melanogenic enzymes, including tyrosinase, Tyrp-1 and Tyrp-2, by inhibiting the ERK/CREB/MiTF signalling pathway in NHMs. This work demonstrates for the first time that MELs exert whitening effects on human melanocytes and skin equivalent. Thus, we suggest that MELs could be developed as a potent anti-melanogenic agent for effective whitening, beyond their use as a biosurfactant in cosmetics.

Ameliorating effect of dipotassium glycyrrhizinate on an IL-4- and IL-13-induced atopic dermatitis-like skin-equivalent model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is not fully understood. Defects in skin barrier function and dysregulation of the Th2 immune response are thought to be pivotal in AD pathogenesis. In this study, we used keratinocytes and AD-like skin equivalent models using Th2 cytokines IL-4 and IL-13. The keratinocytes and AD-like skin model were used to investigate the effect of dipotassium glycyrrhizinate (KG), which is widely used as an anti-inflammatory agent for AD treatment. KG decreased AD-related gene expression in keratinocytes stimulated with Th2 cytokines. KG alleviated AD-like phenotypes and gene expression patterns and inhibited release of AD-related cytokines in the AD-like skin equivalent models. These findings indicate KG has potential effectiveness in AD treatment and AD-like skin equivalent models may be useful for understanding AD pathogenesis.

Irritating effects of sodium lauryl sulfate on human primary keratinocytes at subtoxic levels of exposure.

Chemical agents that can potentially cause skin irritation are typically tested in animal models or in vitro assays of cell viability or cytokine expression. However, these methods do not always provide translatable results and are not sufficiently sensitive for subtoxicity detection. Here, we introduce the mechanical properties of keratinocytes as novel endpoints for the safety assessment of chemical agents at the subtoxicity level. Human primary keratinocytes were treated with various concentrations of sodium lauryl sulfate (SLS) and their biological properties (proliferation, membrane integrity, inflammatory response, and morphology) were observed. Their biomechanical and geometrical parameters (stiffness and surface roughness) were also investigated by atomic force microscopy. Keratinocyte morphophysiological changes and inflammatory responses were significant at ≥25 µM SLS. The keratinocytes became less stiff due to changes in the distribution of F-actin filaments and α-tubulin; these changes were significant even at lower doses of SLS (≤10 µM). The morphophysiological changes of keratinocytes were clearly seen at a relatively high dose of SLS, while the mechanical properties of keratinocytes responded linearly to SLS at lower doses. Therefore, changes in mechanical properties can be used as new endpoints for in vitro toxicity testing with keratinocytes.

HIF-1α-mediated BMP6 down-regulation leads to hyperproliferation and abnormal differentiation of keratinocytes in vitro.

Hypoxia-inducible factor-1α (HIF-1α) has been reported to be up-regulated in psoriatic epidermis, resulting in increased proliferation and abnormal differentiation of human keratinocytes (KCs). However, the role of HIF-1α in psoriatic epidermis, which is mainly composed of KCs, is poorly understood. Here, we show that morphogenic protein 6 (BMP6) is down-regulated when HIF-1α is upregulated in patients with psoriasis skin lesions. HIF-1α overexpression in primary human KCs promoted proliferation and inhibited terminal differentiation. Furthermore, HIF1-α repressed the expression of BMP6 by binding directly to the hypoxia-response element (HRE) in the BMP6 promotor region, which shows that BMP6 is a novel target gene of HIF-1α. We also found that HIF-1α-mediated BMP6 suppression could alter the proliferation status by modulating the expression levels of cell cycle regulatory proteins and also affect the early differentiation of KCs. Therefore, we suggest that HIF-1α-dependent BMP6 suppression has a critical role in the induction of hyper-proliferation and abnormal differentiation in psoriatic KCs.

RIP4 upregulates CCL20 expression through STAT3 signalling in cultured keratinocytes.

The receptor-interacting protein kinase 4 (RIP4), a serine/threonine kinase, is an important modulator of epidermal growth and cutaneous inflammation. We found that RIP4 expression was significantly increased in the lesional skin of psoriasis. However, the role and regulatory mechanism of RIP4 in psoriasis have not been characterized. After treatment with IL-17, RIP4 mRNA and protein levels were increased in HaCaT cells. IL-17 also activated the RIP4 promoter. To understand the functional role of RIP4 in keratinocyte and to investigate the genes regulated by RIP4, RNA-based microarray analysis was performed. Among immune response-related genes, CCL20 expression was significantly changed by RIP4. To identify RIP4-interacting protein, an immunoprecipitation assay was performed. As a result, STAT3 was identified as a new protein that interacts with RIP4. The interaction of RIP4 and STAT3 enhanced STAT3 phosphorylation. In addition, the transcriptional activity of STAT3 induced by RIP4 regulated IL-17-mediated CCL20 expression in HaCaT cells. Taken together, these findings indicate that IL-17 increased RIP4-mediated STAT3 phosphorylation by directly interacting with STAT3. Thus, transcriptional activation of STAT3 promotes the expression of CCL20. Thus, activations of these signalling pathways by RIP4 may contribute to epithermal inflammation in psoriatic keratinocytes.

Pyruvate Protects against Cellular Senescence through the Control of Mitochondrial and Lysosomal Function in Dermal Fibroblasts.

Mitochondrial dysfunction can drive cellular senescence, which is accompanied by changes in metabolism and increases in senescence-associated secretory phenotypes. Although pyruvate, a key metabolite for numerous aspects of metabolism, has been used as general supplement in synthetic media, the physiological function of pyruvate underlying its protective role against cellular senescence under normal conditions has remained unknown. Here, we show that extracellular pyruvate prevents senescence in normal human dermal fibroblasts through increasing the generation of oxidized nicotinamide adenine dinucleotide (NAD+) during the conversion to lactate. Acetylated peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), vacuolar-type H+-ATPaseV0A1 (v-ATPaseV0A1), NF-κB p65 subunit (RelA), and histone H3 accumulate under pyruvate deprivation conditions, resulting in the onset of senescence in normal human dermal fibroblasts through the accumulation of abnormal mitochondria generated by lysosomal inactivation-induced mitophagy defects, and through an increase in senescence-associated secretory phenotypes. Furthermore, pyruvate showed a protective effect against aging phenotypes in skin equivalents, which consist of a dermis and epidermis that act similarly to in vivo skin tissues. Our findings reveal a connection between pyruvate and mitochondrial dysfunction in the progression of senescence that is, to our knowledge, previously unreported. These results suggest that the pyruvate deprivation-induced senescence model can be used to study the connection between metabolism and senescence under normal conditions.

The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells.

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.

TNFSF14 inhibits melanogenesis via NF-kB signaling in melanocytes.

Melanin synthesis in melanocytes is affected by various cytokines. Here, we reported for the first time that tumor necrosis factor superfamily member 14 (TNFSF14) inhibits melanogenesis in the primary culture of human epidermal melanocytes. TNFSF14 is known to bind to its receptors herpes virus entry mediator (HVEM) and lymphotoxin ß receptor (LTßR) for signal transduction, but TNFSF14-induced hypopigmentation was independent of HVEM and LTßR in melanocytes. To explore signaling in melanocytes treated with TNFSF14, we performed RNA-seq and found that nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling is activated by TNFSF14. Further, we observed that inhibition of NF-kB effectively blocks the hypopigmentation induced by TNFSF14. We conclude that TNFSF14 inhibits melanogenesis in melanocytes via NF-κB signaling and could be applied in the treatment of cutaneous pigment disorders.

Psychological Stress Deteriorates Skin Barrier Function by Activating 11ß-Hydroxysteroid Dehydrogenase 1 and the HPA Axis.

Psychological stress (PS) increases endogenous glucocorticoids (GC) by activating the hypothalamic-pituitary-adrenal axis. The negative effects of GC on skin barrier function under PS have been well-established. However, endogenous GC can also be active when cortisone (inactive form) is converted to cortisol (active form) by 11ß-hydroxysteroid dehydrogenase type I (11ß-HSD1) in the peripheral tissue. Here, we evaluated the changes in 11ß-HSD1 and barrier function under PS. Elevated 11ß-HSD1 in oral mucosa correlated with increased cortisol in the stratum corneum and deteriorated barrier function. Expression of 11ß-HSD1 in the oral mucosa correlated with that in the epidermal keratinocytes. We further investigated whether barrier function improved when PS was relieved using a selective serotonin reuptake inhibitor (SSRI) in patients with anxiety. Decreased 11ß-HSD1 and improved barrier function were observed after SSRI treatment. The collective findings suggest that elevated 11ß-HSD1 under PS increases the level of cutaneous GC and eventually impairs barrier function. PS-alleviating drugs, such as SSRI, may help to treat PS-aggravated skin diseases.

Exposure of human melanocytes to UVB twice and subsequent incubation leads to cellular senescence and senescence-associated pigmentation through the prolonged p53 expression.

BACKGROUND: Ultraviolet radiation (UVR) is a well-known factor in skin aging and pigmentation, and daily exposure to subcytotoxic doses of UVR might accelerate senescence and senescence-associated phenomena in human melanocytes. OBJECTIVE: To establish an in vitro melanocyte model to mimic the conditions of repeated exposure to subcytotoxic doses of UVB irradiation and to investigate key factor(s) for melanocyte senescence and senescence-associated phenomena. METHODS: Human epidermal melanocytes were exposed twice with 20 mJ/cm2 UVB over a 24-h interval and subsequently cultivated for 2 weeks. Senescent phenotypes were addressed morphologically, and by measuring the senescence-associated ß-galactosidase (SA-ß-Gal) activity, cell proliferation capacity with cell cycle analysis, and melanin content. RESULTS: The established protocol successfully induced melanocyte senescence, and senescent melanocytes accompanied hyperpigmentation. Prolonged expression of p53 was responsible for melanocyte senescence and hyperpigmentation, and treatment with the p53-inhibitor pifithrin-α at 2-weeks post-UVB irradiation, but not at 48 h, significantly reduced melanin content along with decreases in tyrosinase levels. CONCLUSION: Melanocyte senescence model will be useful for studying the long-term effects of UVB irradiation and pigmentation relevant to physiological photoaging, and screening compounds effective for senescence-associated p53-mediated pigmentation.

Propionibacterium acnes-Derived Extracellular Vesicles Promote Acne-Like Phenotypes in Human Epidermis.

Acne vulgaris is an inflammatory disease occurring in the pilosebaceous unit and is the most common skin condition in young people. A gram-positive bacterium, Propionibacterium acnes, has been suspected to contribute to the development of acne. Here, we report that P. acnes constitutively releases extracellular vesicles (EVs) exhibiting typical EV morphology and size. Moreover, the P. acnes-derived EVs (PEVs) can induce acne-like phenotypes in human epidermal keratinocytes and a reconstituted human skin model. PEVs significantly induced inflammatory cytokines IL-8 and GM-CSF and dysregulated epidermal differentiation by increasing proliferating keratinocytes and decreasing epidermal keratin 10 and desmocollin 1 levels. PEVs showed strong effects, evoking these responses at earlier time points compared with P. acnes extract at the same protein concentration. We verified that PEVs were internalized via clathrin-dependent endocytosis into keratinocytes and that PEV-induced cellular responses occurred via Toll-like receptor 2-dependent signal cascades. Furthermore, PEVs showed a stronger effect than keratinocytes in inducing inflammatory cytokines in myeloid cells. Collectively, our study suggests that PEVs induce acne-like phenotypes in a unique way; therefore, inhibiting the release of EVs from P. acnes or targeting PEV-mediated signaling pathways could represent an alternative method for alleviating acne occurrence and phenotypes.