Photobiomodulation Controls Keratinocytes Inflammatory Response through Nrf2 and Reduces Langerhans Cells Activation.

Photobiomodulation (PBM) is rapidly gaining traction as a valuable tool in dermatology for treating many inflammatory skin conditions using low levels of visible light or near-infrared radiation. However, the physiological regulatory pathways responsible for the anti-inflammatory effect of PBM have not been well defined. Since previous studies showed that nuclear factor-erythroid 2 like 2 (Nrf2) is a master regulator of the skin inflammatory response, we have addressed its role in controlling inflammation by PBM. Primary human keratinocytes (KCs) stimulated with 2,4-dinitrochlorobenzene (DNCB) to mimic pro-inflammatory stress were illuminated with two wavelengths: 660 nm or 520 nm. Both lights significantly reduced the mRNA expression of the DNCB-triggered TNF-α, IL-6, and IL-8 cytokines in KCs, while they enhanced Nrf2 pathway activation. PBM-induced Nrf2 is a key regulator of the inflammatory response in KCs since its absence abolished the regulatory effect of light on cytokines production. Further investigations of the mechanisms contributing to the immunoregulatory effect of PBM in inflamed human skin explants showed that 660 nm light prevented Langerhans cells migration into the dermis, preserving their dendricity, and decreased pro-inflammatory cytokine production compared to the DNCB-treated group. This study is the first to report that the PBM-mediated anti-inflammatory response in KCs is Nrf2-dependent and further support the role of PBM in skin immunomodulation. Therefore, PBM should be considered a promising alternative or complementary therapeutic approach for treating skin-related inflammatory diseases.

S-Palmitoylation of Tyrosinase at Cysteine500 Regulates Melanogenesis.

Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine500 in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine500 by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.

Safety and efficacy of a novel home-use device for light-potentiated (LED) skin treatment.

Skin structure and function results from a dynamic interplay between dermal and epidermal cell types. Optimizing skin health through an effective and long-lasting skin care regime therefore requires a global approach, encompassing various mechanisms to stimulate this interplay beyond the action scope of a classical topical solution. This study evaluates the impact of a novel home-use device combining a topical serum, light-emitting diodes and massage on the clinical signs of extrinsic skin aging. The innovative principle relies on potentiating the effect of active ingredients contained in the topical serum with visible and near infra-red photons to prevent extracellular matrix degradation and promote its reconstruction. After in vitro and ex vivo investigations, a clinical study assessed the safety and efficacy of a daily treatment with the home-use device for 28 days. A significant increases in skin density and radiance while reducing the wrinkles was obtained with no side effects.

Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes.

To gain insight for the role of mast cell-produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin-treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2 ) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti-inflammation role in epidermis of human skin.

Protection against ultraviolet A-induced oxidative damage in normal human epidermal keratinocytes under post-menopausal conditions by an ultraviolet A-activated caged-iron chelator: a pilot study.

BACKGROUND/PURPOSE: Human skin is constantly exposed to ultraviolet A (UVA), which can generate reactive oxygen species and cause iron release from ferritin, leading to oxidative damage in biomolecules. This is particularly true in post-menopausal skin due to an increase in iron as a result of menopause. As iron is generally released through desquamation, the skin becomes a main portal for the release of excess iron in this age group. In the present study, we examined a strategy for controlling UVA- and iron-induced oxidative stress in skin using a keratinocyte post-menopausal cellular model system. METHODS: Keratinocytes that had been cultured under normal or high-iron, low-estrogen conditions were treated with (2-nitrophenyl) ethyl pyridoxal isonicotinoyl hydrazone (2-PNE-PIH). 2-PNE-PIH is a caged-iron chelator that does not normally bind iron but can be activated by UVA radiation to bind iron. Following incubation with 2-PNE-PIH, the cells were exposed to 5 J/cm² UVA and then measured for changes in lipid peroxidation and ferritin levels. RESULTS: 2-PNE-PIH protected keratinocytes against UVA-induced lipid peroxidation and ferritin depletion. Further, 2-PNE-PIH was neither cytotoxic nor did it alter iron metabolism. CONCLUSION: 2-PNE-PIH may be a useful deterrent against UVA-induced oxidative stress in post-menopausal women.

UV protection afforded by gel-trapped TiO2 particles.

We have developed a technology to incorporate micronized titanium dioxide (TiO(2)), together with antioxidants, in particles of a UV-visible transparent polymer gel. These particles are coated with silica to avoid clustering and the size of the micronized TiO(2) reduces the back scattering of white light. gel-trapped TiO(2) minimizes the oxidative stress exerted by UV radiation, increases the photo-stability of some accompanying ingredients, such as avobenzone. The size of the particles is in the micrometre range. This favors their permanence on the top of the stratum corneum. Gel-trapped TiO(2)-based sunscreens provide a larger SPF and two-fold larger UVA protection than equal-composition sunscreens that contain larger amounts of untrapped TiO(2).

Functional interplay between mitochondrial and proteasome activity in skin aging.

According to the mitochondrial theory of aging, reactive oxygen species (ROS) derived primarily from mitochondria cause cumulative oxidative damage to various cellular molecules and thereby contribute to the aging process. On the other hand, a pivotal role of the proteasome, as a main proteolytic system implicated in the degradation of oxidized proteins during aging, is suggested. In this study, we analyzed mitochondrial function in dermal fibroblasts derived from biopsies obtained from healthy young, middle-aged, and old donors. We also determined proteasome activity in these cells, using a degron-destabilized green fluorescent protein (GFP)-based reporter protein. We found a significant decrease in mitochondrial membrane potential in samples from aged donors, accompanied by a significant increase in ROS levels. Respiratory activity was not significantly altered with donor age, probably reflecting genetic variation. Proteasome activity was significantly decreased in fibroblasts from middle-aged donors compared with young donors; fibroblasts derived from the oldest donors displayed a high heterogeneity in this assay. We also found intraindividual coregulation of mitochondrial and proteasomal activities in all human fibroblast strains tested, suggesting that both systems are interdependent. Accordingly, pharmacological inhibition of the proteasome led to decreased mitochondrial function, whereas inhibition of mitochondrial function in turn reduced proteasome activity.

Structural and functional differences in barrier properties of African American, Caucasian and East Asian skin.

BACKGROUND: Differences in structural and functional skin characteristics have been linked with ethnical background. But racial differences in skin have not been thoroughly investigated by objective methods and the data are often contradictory. OBJECTIVES: This study was undertaken to compare skin barrier-related parameters of the stratum corneum on African American, Caucasian and East Asian skin by objective measurements. METHODS: Baseline values of trans epidermal water loss were collected on the face. Consecutive stratum corneum D-squame tape strippings were collected on the panelist's ventral forearm and face to evaluate skin barrier strength and cohesion. Stratum corneum ceramides, maturation, measured as the transglutaminase-mediated cross-linking of stratum corneum proteins, and stratum corneum trypsin like enzyme activity were measured on the D-squame tape strippings. RESULTS: East Asian and to some extent Caucasian skin was characterized by low maturation and relatively weak skin barrier. African American skin was characterized by low ceramide levels and high protein cohesion in the uppermost layers of the stratum corneum. These data can be interpreted in terms of the high prevalence of xerosis in black skin and increased skin sensitivity in East Asian skin. CONCLUSION: These results demonstrate that skin properties at the level of the stratum corneum vary considerably among these ethnic groups. This contributes to an improved understanding of physiological differences between these study populations.

Modification of skin discoloration by a topical treatment containing an extract of Dianella ensifolia: a potent antioxidant.

Skin hyperpigmentation, and the reactions that precipitate it, have been linked to free radicals by the fact that free radical scavengers or antioxidants can slow that hyperpigmentation. We have screened several hundred plant extracts for antioxidants and discovered one that is both a strong antioxidant and can reduce skin hyperpigmentation. Extracts of Dianella ensifolia contain 1-(2,4-dihydrophenyl)-3-(2,4-dimethoxy-3-methylphenyl) propane (DP), which was found to inhibit the free radical 1-1-diphenyl-2-picryl-hydrazyl (DPPH) with an EC(50) value of 78 mum. DP was also found to inhibit Ultraviolet (UV)C-induced lipid oxidation with an EC(50) of about 30 mum. We next investigated the effects of this antioxidant on skin hyperpigmentation. The reduction of discoloration by different topical treatments has been assessed in human volunteers using an in vivo assay for the rate of fading of UVB-induced tan. Two pharmaceutical formulas containing 4% hydroquinone (HQ) were used as positive controls, and we tested the ability of DP, a plant-derived amphoteric antioxidant, to increase performance of non-HQ cosmetic formulations. We found that the cosmetic formula containing DP produced an increase in the rate of fading compared to the two pharmaceutical treatments containing HQ.

The flavonoid luteolin increases the resistance of normal, but not malignant keratinocytes, against UVB-induced apoptosis.

Adequate protection of skin against the carcinogenic effects of UVB irradiation is essential. Flavonoids may have a conspicuous role in cancer prevention because of their antioxidant, anti-inflammatory, and growth-inhibitory effects. Therefore, we tested the effects of the flavone luteolin (LUT) on selected parameters of the sunburn response in normal human keratinocytes, exposed to physiological doses of UVB. LUT attenuated UVB-induced cell death through delay and inhibition of intrinsic apoptotic signaling. Moreover, LUT not only predominantly affected the mitochondrial apoptosis pathway through its antioxidant capacity, but also changed the balance of Bcl2 (B-cell leukemia/lymphoma 2)-family members. Furthermore, LUT had inhibitory effects on the UVB-induced release of the inflammatory mediators, IL-1alpha and prostaglandin-E(2). Using different cell lines derived from squamous cell carcinomas, we showed that LUT did not increase the resistance of malignant keratinocytes to UVB. Our data suggest that LUT inhibits different aspects of the sunburn response, which results ultimately in an increased survival of normal keratinocytes, whereas the sensitivity of malignant cells to UVB remain unchanged. Hence, LUT might have value in new photoprotective applications or improve existing ones.

Topical application of green and white tea extracts provides protection from solar-simulated ultraviolet light in human skin.

BACKGROUND: Tea polyphenols have been found to exert beneficial effects on the skin via their antioxidant properties. AIMS: We sought to determine whether topical application of green tea or white tea extracts would prevent simulated solar radiation-induced oxidative damages to DNA and Langerhans cells that may lead to immune suppression and carcinogenesis. METHODS: Skin samples were analysed from volunteers or skin explants treated with white tea or green tea after UV irradiation. In another group of patients, the in vivo immune protective effects of green and white tea were evaluated using contact hypersensitivity to dinitrochlorobenzene. RESULTS: Topical application of green and white tea offered protection against detrimental effects of UV on cutaneous immunity. Such protection is not because of direct UV absorption or sunscreen effects as both products showed a sun protection factor of 1. There was no significant difference in the levels of protection afforded by the two agents. Hence, both green tea and white tea are potential photoprotective agents that may be used in conjunction with established methods of sun protection.

Calculation of apparent age by linear combination of facial skin parameters: a predictive tool to evaluate the efficacy of cosmetic treatments and to assess the predisposition to accelerated aging.

The estimated apparent age (EAA) was estimated by a panel of trained experts, for the individuals in a cohort. Twelve independent clinical, biophysical and biochemical parameters measured on facial skin, have been identified by multiple regression analysis, which influence the EAA of a person of chronological age (CA) (under eye lines, clinically assessed crow's feet, age spots, clinically evaluated firmness, forehead lines, pores, lip lines, instrumentally evaluated firmness, instrumentally evaluated crow feet, skin texture, in vivo fluorescence related to proliferation and glycation). An algorithm has been devised to obtain the calculated age score (CAS) in a cohort of 452 female volunteers, as CAS(n) = ∑RCiPi(n) (i = 1-13, n = 1-452 and P13 = 1) where the coefficients Ci are obtained by minimizing the difference EAA - CAS, and Pi(n) are the experimental values of the i-th parameter for the n-th volunteer. The determination of CAS before and after a specific cosmetic or pharmacological anti-aging treatment can be used to objectively assess the efficacy of the treatment. The comparison of EAA(n) and of CAS(n) with CA(n) allows one to predict the susceptibility of an individual's face to undergo aging. It has been observed that the biophysical and biochemical parameters play a relevant role in the assessment of the predisposition of skin to undergo accelerated aging.

A low UVB dose, with the potential to trigger a protective p53-dependent gene program, increases the resilience of keratinocytes against future UVB insults.

One protein central in the response of human keratinocytes to ultraviolet B damage is p53. By transactivating genes involved in either cell cycle arrest or DNA repair, p53 has a leading role in the recovery from this damage. Considering this role, we wished to investigate whether the triggering of a p53-dependent gene program by repetitive ultraviolet B (UVB) exposure can induce an adaptive response in human skin cells. In particular, we examined two p53-target genes, p21/WAF1 and p53R2, with a crucial role in p53-induced cell cycle arrest and p53-induced DNA repair respectively. Exposure to a mild UVB dose was able to induce an adaptive response in human keratinocytes, leading to increased survival of cells that maintain their capacity to repair DNA damage upon exposure to apoptotic doses of UVB. Our study indicates that this adaptation response is only achieved if the interval between subsequent UVB insults allows sufficient time for the p53-induced protective gene program to be induced. Our results also demonstrate that small but quickly recurring UVB exposures are as harmful as one intense, continual exposure to UVB irradiation. Future research will be oriented toward investigating alternative ways to induce an adaptive response without pre-exposing the cells to UV.

Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences.

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.

A synthetic superoxide dismutase/catalase mimetic (EUK-134) inhibits membrane-damage-induced activation of mitogen-activated protein kinase pathways and reduces p53 accumulation in ultraviolet B-exposed primary human keratinocytes.

Salen-manganese complexes exhibit powerful superoxide dismutase and catalase activity, with pharmacologic efficacy in several oxidative-stress-associated disease models. Ultraviolet (UV) B not only induces direct DNA damage, but also generates oxidative stress. EUK-134, a salen-manganese complex, might therefore confer a direct protection against UVB-induced oxidative stress and consequently alleviate UVB-damage-induced signal transduction. We investigated the effect of EUK-134 on the UVB-induced accumulation and stabilization of the p53 protein. p53 plays a central role in the UVB response, both as sensor of UVB damage and as a mediator of a protective response. Cells treated with EUK-134 before UVB irradiation showed a significantly lower accumulation of the p53 protein in a concentration-dependent fashion. Furthermore, EUK-134 severely reduced N-terminal phosphorylation of p53. The extracellular signal-regulated kinase ERK and the stress-activated kinases JNK and p38 have been implicated in the UVB-induced N-terminal phosphorylation and accumulation of p53. Pre-treatment with EUK-134 inhibited the UVB-induced activation of these mitogen-activated protein kinase (MAPK) pathways. We hypothesize that EUK-134, by direct protection of the membrane from UVB-induced oxidative damage, reduces oxidative stress induced MAPK signaling and consequently lowers the level of p53 induction. The protection conferred by EUK-134 resulted in a significant increase in cell survival following UVB irradiation.

Antioxidant enzyme activity in human stratum corneum shows seasonal variation with an age-dependent recovery.

The stratum corneum, as the body's principal barrier to the environment, is continuously exposed to environmental sources of reactive oxygen species like ultraviolet light, ozone, and pollution. Reactive oxygen species are believed to be involved in cancer, aging, and inflammatory skin disorders. We have developed a method to measure catalase and superoxide dismutase activity on tape strippings from the human stratum corneum and demonstrated a gradient of antioxidant enzyme activity across the stratum corneum with decreasing levels towards the skin surface. Sun exposure resulted in a seasonal variation of the catalase activity in stratum corneum, with low activities in summer and higher activities in winter for the same person, whereas superoxide dismutase activity in stratum corneum did not seem to vary in those conditions. Exposure of human skin to broadband ultraviolet-A resulted in a dose-dependent deactivation of the catalase activity in stratum corneum within 24 h, whereas exposure to ultraviolet-B had no effect. Superoxide dismutase activity in stratum corneum was not affected by ultraviolet-A or ultraviolet-B irradiation within 24 h. After exposure to a dose of 15 J per cm2 broadband ultraviolet-A, full recovery of the catalase activity occurred in 3-4 wk at an age-dependent rate. We conclude that sun exposure results in a disturbed catalase to superoxide dismutase ratio in the stratum corneum. This may lead to an increased vulnerability to oxidative damage in stratum corneum barrier components. These results therefore stress the importance of providing efficient protection for this internal defense mechanism in sun-exposed areas of the skin.

A new dicaffeoylquinic acid butyl ester from Isertia pittieri.

A new 4,5-di-O-caffeoylquinic acid butyl ester (1) was isolated from an ethyl acetate soluble fraction of Isertia pittieri stems, along with three known compounds, 1,5-di-O-caffeoylquinic acid (2), 3,4-di-O-caffeoylquinic acid (3) and 4,5-di-O-caffeoylquinic acid (4).

Age-dependent response of energy metabolism of human skin to UVA exposure: an in vivo study by 31P nuclear magnetic resonance spectroscopy.

PURPOSE: The aim of our study was to evaluate the in vivo energy metabolism of human skin as a function of age, in conditions of rest and after a mild stress caused by a suberythemal UVA irradiation. METHODS: The kinetics of UVA-induced modifications in high-energy phosphorylated metabolites of young and old skins were non-invasively monitored over a period of 24 h using 31P nuclear magnetic resonance spectroscopy. In vivo 31P spectra were obtained on the ventral aspect of the wrist, using a NMR Imaging Spectrometer equipped with a double-tuned surface coil. Concentrations of phosphocreatine, inorganic phospate, adenosine tri-phosphate, phosphomono and phosphodiesters were calculated from the spectra and results were expressed as relative concentrations. A total of 20 subjects were enrolled in this study (n = 10 for the age group below 25 years and n = 10 for the age group above 55 years). A second experiment was then performed on 10 old subjects (mean age 60) who were treated on one wrist, twice a day for one month prior to UVA irradiation, with a product that contained active ingredients to restore barrier function and modulate the inflammatory response, the other wrist being an untreated control. RESULTS: Baseline levels of phosphorylated metabolites were similar in young and old skins. A suberythemal dose of UVA (6 J.cm-2) led to a significant decrease in the PCr/Pi ratio (index of energy status) and a significant increase in the PME/PDE ratio (index of cellular turnover rate of lipid-related metabolites) within 1 h. The observed variations were transient and the recovery was complete at T + 24 h post-UVA, although recovery was significantly slower in the older group. The disturbances were significantly reduced after treatment of the older skin with a formula that restored barrier function of the stratum corneum and modulated the inflammatory response. CONCLUSION: (i) baseline levels of energy metabolites in skin do not seem to vary with age; (ii) low dose UVA irradiation induces a rapid response in the energy metabolism of the skin; (iii) the kinetics of the response and recovery after an aggression by UVA suggest that older skin has significantly less energy rebound after a stress situation than younger skin; (iv) the energy reserve in older skin can be protected efficiently against UVA-induced stress by restoring barrier function and modulating the inflammatory response.