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.

Impact of cigarette smoke on physical-chemical and molecular proprieties of human skin in an ex vivo model.

BACKGROUND: This is a study about the skin ageing exposome, focusing on the effect of cigarette smoke. Human living skin explants (HSE) were exposed to cigarette smoke (CS) of two cigarettes for 2 hours using a custom-made exposure chamber, the Pollubox® . Effects on the surface physico-chemistry and molecular properties of the skin were analyzed and reported for the first time. BASIC PROCEDURES: To this end, transcriptomic study followed by immunohistochemistry, MDA (Malondialdehyde Dosage), and surface physio-chemistry data: surface free energy determination, TEWL (Trans Epidermal Water Loss), skin pH and FT-IR (Fourier Transform-Infrared) spectroscopy of the explant were collected from untreated and treated HSE. MAIN FINDINGS: Results showed a decrease of the total surface free energy of the treated HSE. This decrease reflected higher interactions with polar compounds from the environment and consequently a decrease of the surface hydrophobicity. Additionally, an increase of TEWL and skin pH was observed after treatment. The transcriptomic analysis showed downregulation of mitochondrial genes (PON2-NDUFA4L2-ATP1A1-ALDH2-PRODH) combined with an increase of MDA in CS-treated HSE. CONCLUSIONS: CS-induced oxidation of lipids at HSE surface alters the skin barrier: interactions with polar products are enhanced and the lipid chain packing at the surface is modified. Consequently, skin permeability could increase which correlated with repression of CA9 and AQP1 genes. Beside activation of AHR-NRF2 pathway in CS-exposed HSE, our results suggested that mitochondrial functions were strongly impacted and oxidized lipids failed to be eliminated promoting skin barrier alteration. A mitophagy activity was suggested through the confirmation of PINK1 accumulation in the epidermis by immunostaining.

Pilot Comparative Study of the Topical Action of a Novel, Crosslinked Resilient Hyaluronic Acid on Skin Hydration and Barrier Function in a Dynamic, Three-Dimensional Human Explant Model.

BACKGROUND: Hyaluronic acid (HA) is a popular ingredient in topical formulations for cosmetic improvement of the skin. Most formulations contain linear, non-crosslinked HA oligomers, low molecular weight (LMW) HA, and/or high molecular weight (HMW) HA. Crosslinking of HA enhances its clinical longevity and mechanical characteristics. The objective of this study was to characterize the topical effects of a new, crosslinked resilient HA (RHA) that is also available as a cohesive, tissue-integrating injectable filler, compared with non-crosslinked HMW HA and LMW HA. Living human skin explants that preserve the 3-dimensional structure of in vivo skin were used to maximize clinical relevance. METHODS: Standardized doses of each HA product were applied daily for 9 days to human skin explant surfaces. Untreated explants served as controls. Water content of the stratum corneum and entire epidermis was analyzed by Raman spectroscopy. Transepidermal water loss (TEWL) was measured to assess skin barrier function. Explant morphology and microrelief were evaluated by optical and scanning electron microscopy. RESULTS: Crosslinked RHA achieved a significant increase in epidermal water content (7.6%) over the control. Spectral cartography confirmed a higher epidermal water content with RHA than with HMW HA or LMW HA. TEWL was reduced by 27.8% with RHA, and by 15.6% with HMW HA, but increased by 55.5% with LMW HA. Cutaneous microrelief improved with RHA. Corneocyte cohesion improved with RHA and HMW HA. CONCLUSIONS: This comparative, multimodal study demonstrated greater benefits of topical crosslinked RHA over linear HMW HA or LMW HA in reducing TEWL, retaining and redistributing water within the epidermis, maintaining skin integrity, and improving skin barrier structure and function. RHA was a more efficacious humectant than LMW HA, and a more efficacious occlusive moisturizer than HMW HA. These integrative epidermal repair activities are of significant value for addressing primary deficits of aging skin, improving tolerance to retinoids and other topical agents, and optimizing procedural outcomes. A combination of topical and injectable HA provides an elegant model of synergistic, multi-level skin restoration.

The effect of oral collagen peptide supplementation on skin moisture and the dermal collagen network: evidence from an ex vivo model and randomized, placebo-controlled clinical trials.

BACKGROUND: Skin dryness and an accelerated fragmentation of the collagen network in the dermis are hallmarks of skin aging. Nutrition is a key factor influencing skin health and consequently its appearance. A wide range of dietary supplements is offered to improve skin health. Collagen peptides are used as a bioactive ingredient in nutricosmetic products and have been shown in preclinical studies to improve skin barrier function, to induce the synthesis of collagen and hyaluronic acid, and to promote fibroblast growth and migration. Our aim was to investigate the effect of oral supplementation with specific collagen peptides on skin hydration and the dermal collagen network in a clinical setting. METHODS: Two placebo-controlled clinical trials were run to assess the effect of a daily oral supplementation with collagen peptides on skin hydration by corneometry, on collagen density by high-resolution ultrasound and on collagen fragmentation by reflectance confocal microscopy. Human skin explants were used to study extracellular matrix components in the presence of collagen peptides ex vivo. RESULTS: Oral collagen peptide supplementation significantly increased skin hydration after 8 weeks of intake. The collagen density in the dermis significantly increased and the fragmentation of the dermal collagen network significantly decreased already after 4 weeks of supplementation. Both effects persisted after 12 weeks. Ex vivo experiments demonstrated that collagen peptides induce collagen as well as glycosaminoglycan production, offering a mechanistic explanation for the observed clinical effects. CONCLUSION: The oral supplementation with collagen peptides is efficacious to improve hallmarks of skin aging.

Anti-inflammaging and antiglycation activity of a novel botanical ingredient from African biodiversity (Centevita™).

PURPOSE: The aim of this study was to investigate the topical efficacy of a new purified extract from Madagascar, Gotu Kola (Centella asiatica [L.] Urban), both on human explants and on human volunteers, in relation to skin wrinkling and skin protection against ultraviolet light exposure. The extract, with a peculiar content of biologically active molecules, was investigated as a novel anti-inflammaging and antiglycation agent. Its typical terpenes, known as collagen synthesis promoters, represent at least 45% of the extract. It also contains a polyphenolic fraction cooperating to the observed properties. METHODS: C. asiatica purified extract was assayed on human skin explants maintained alive, and several parameters were evaluated. Among the most relevant, the thymine dimerization was evaluated by immunostaining. Malondialdehyde formation was evaluated as free-radical scavenging marker by enzyme-linked immunosorbent assay. The expression of interleukin-1α was observed by enzyme-linked immunosorbent assay as well. The product was further evaluated as an antiglycation agent, being glycation quantified by the advanced glycation product carboxymethyl lysine. C. asiatica purified extract was also evaluated as an antiwrinkling agent in a single-blind, placebo-controlled study. Formulated in a simple oil-in-water emulsion, the extent of wrinkling was assessed by skin replicas, skin firmness, skin elasticity, and collagen density measurements. RESULTS: C. asiatica purified extract could protect DNA from ultraviolet light-induced damage, decreasing the thymine photodimerization by over 28% (P<0.05). A reduced (26%, P<0.01) expression of interleukin-1α was also observed, supporting its anti-inflammatory potential. C. asiatica purified extract showed in vitro a total inhibition of carboxymethyl lysine formation induced by the glycating agent methylglyoxal. A clear epidermal densification of collagen network in the papillary dermis was observed. These in vitro data have been confirmed by clinical results. CONCLUSION: These results qualify C. asiatica purified extract as an antiaging ingredient, addressing skin damage caused by inflammaging and glycation by relying on the synergy of triterpens and polyphenolics.

Effects of a skin neuropeptide (substance p) on cutaneous microflora.

BACKGROUND: Skin is the largest human neuroendocrine organ and hosts the second most numerous microbial population but the interaction of skin neuropeptides with the microflora has never been investigated. We studied the effect of Substance P (SP), a peptide released by nerve endings in the skin on bacterial virulence. METHODOLOGY/PRINCIPAL FINDINGS: Bacillus cereus, a member of the skin transient microflora, was used as a model. Exposure to SP strongly stimulated the cytotoxicity of B. cereus (+553±3% with SP 10(-6) M) and this effect was rapid (<5 min). Infection of keratinocytes with SP treated B. cereus led to a rise in caspase1 and morphological alterations of the actin cytoskeleton. Secretome analysis revealed that SP stimulated the release of collagenase and superoxide dismutase. Moreover, we also noted a shift in the surface polarity of the bacteria linked to a peel-off of the S-layer and the release of S-layer proteins. Meanwhile, the biofilm formation activity of B. cereus was increased. The Thermo unstable ribosomal Elongation factor (Ef-Tu) was identified as the SP binding site in B. cereus. Other Gram positive skin bacteria, namely Staphylococcus aureus and Staphylococcus epidermidis also reacted to SP by an increase of virulence. Thermal water from Uriage-les-Bains and an artificial polysaccharide (Teflose®) were capable to antagonize the effect of SP on bacterial virulence. CONCLUSIONS/SIGNIFICANCE: SP is released in sweat during stress and is known to be involved in the pathogenesis of numerous skin diseases through neurogenic inflammation. Our study suggests that a direct effect of SP on the skin microbiote should be another mechanism.

Antimicrobial peptides and pro-inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli.

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human ß defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-ß, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology.

Comparative study of normal and sensitive skin aerobic bacterial populations.

The purpose of this study was to investigate if the sensitive skin syndrome, a frequent skin disorder characterized by abnormal painful reactions to environmental factors in the absence of visible inflammatory response, could be linked to a modification in the skin bacterial population. A total of 1706 bacterial isolates was collected at the levels of the forehead, cheekbone, inner elbow, and lower area of the scapula on the skin of normal and sensitive skin syndrome-suffering volunteers of both sexes and of different ages. Among these isolates, 21 strains were randomly selected to validate in a first step the Matrix-Assisted Laser Desorption/Ionization (MALDI)-Biotyper process as an efficient identification tool at the group and genus levels, by comparison to API(®) strips and 16S ribosomal RNA gene sequencing identification techniques. In a second step, identification of the skin microbiota isolates by the MALDI-Biotyper tool allowed to pinpoint some differences in terms of bacterial diversity with regard to the collection area, and the volunteer's age and gender. Finally, comparison of the skin microbiota from normal and sensitive skin syndrome-suffering volunteers pointed out gender-related variations but no detectable correlation between a phylum, a genus or a dominant bacterial species and the sensitive skin phenotype. This study reveals that there is no dysbiosis of aerobic cultivable bacteria associated with the sensitive skin syndrome and further demonstrates that the MALDI-Biotyper is a powerful technique that can be efficiently employed to the study of cultivable human skin bacteria. To our knowledge, this is the first study focusing on bacteria in the sensitive skin syndrome. These results are of potential importance for pharmaceutical and cosmetic industries, which are looking for new strategies to treat this multiparametric disorder.

Isolation of human epidermal layers by laser capture microdissection: application to the analysis of gene expression by quantitative real-time PCR.

We describe, for the first time, an efficient protocol based on laser capture microdissection (LCM) for the isolation of human epidermal layers for gene expression profiling using quantitative real-time PCR. Two areas enriched either in basal or granular layers were isolated by LCM. Skin biopsies were fixed in dry ice-cooled isopentane, cryosectioned and stained before the laser procedure. High-quality total RNA was extracted from each microdissected sample, which allowed the analysis of the spatial distribution of mRNA transcripts from 10 innate immunity-related genes within the epidermal layers. Using integrin alpha-6/integrin beta-4 and corneodesmosin/filaggrin-2 sets as gene markers for the basal and granular layers, respectively, we showed that Toll-like receptor 2, RNase 7, human beta-defensin-2 and -3, psoriasin and nucleotide-binding oligomerization domain 1 are upregulated in the suprabasal layer of normal human epidermis. Our protocol, which is based on the rapid isolation of epidermal layers, can be used to follow transcriptional processes in specific areas of the epidermis and is a very promising tool to use in the study of numerous aspects of dermatology.

Experimental 70% hydrofluoric acid burns: histological observations in an established human skin explants ex vivo model.

BACKGROUND: Hydrofluoric acid (HF) is particularly dangerous due to the potential for systemic effects and induction of severe skin necrosis through two mechanisms: corrosiveness and local tissue toxicity. In addition, because it is only partially dissociated (pK(a) 3.2), it is capable of penetrating deeply into tissues. There is a lack of experimental studies that objectively characterize the behavior of HF diffusion into human skin, specifically the kinetics of tissue penetration resulting in severe cellular lesions. METHODOLOGY/PRINCIPAL FINDINGS: We describe the cutaneous effects of HF using an established ex vivo human skin model. The diffusion of 70% HF starts within the first minute of contact at the epidermal surface and after 2 min reaches the basal layer. In the subsequent minute, the epidermis is destroyed and lesions appear in the papillary dermis after 4 min. Soon after, damage appears in the upper reticular dermis. Thus, 70% HF needs only 5 min of contact to completely penetrate human skin explants. This experiment is reproducible and corroborates previous studies and clinical effects reported in accidental HF exposures. CONCLUSION/SIGNIFICANCE: This study shows that the management of HF chemical skin exposure is a question of minutes, especially for initial decontamination. These experimental observations could be useful for objectively comparing skin decontamination methods. Further studies should help to confirm these preliminary results.

Part 2. Comparison of emergency washing solutions in 70% hydrofluoric acid-burned human skin in an established ex vivo explants model.

BACKGROUND: Hydrofluoric acid (HF) is a small and partially dissociated acid (pK(a) 3.2), able to deeply penetrate into human skin in addition to the corrosiveness of the hydrogen ion (H(+)) and the toxicity of the fluoride ion (F(-)). However, there has been a lack of experimental studies to objectively characterize the results of human HF skin exposure decontamination. METHODOLOGY/PRINCIPAL FINDINGS: A previously established experimental method using a human skin explants ex vivo model (Part 1. Experimental 70% hydrofluoric acid (HF) burns: Histological observations in an established human skin explants ex vivo model) described the lesions that appeared following 70% HF penetration. Within 5 min, 70% HF penetrates to the dermis. Using the same experimental conditions, a comparison study of two different washing protocols was performed: water + topical calcium gluconate (CaG) versus Hexafluorine(®). In these conditions, washing for 15 min with running tap water followed by topical CaG ointment only delayed burn onset, while severe tissue damage appeared later. In contrast, after washing with Hexafluorine(®) over 10 min, no histological lesions developed. These results are in accordance with the results of accidental human industrial case reports. CONCLUSION/SIGNIFICANCE: Amphoteric and hypertonic Hexafluorine(®) can deactivate H(+) and chelate F(-) ions. Based on these results, it should be considered as a promising first-aid decontamination solution to prevent or minimize significant local and systemic consequences of concentrated HF skin exposures.

Restoration of stratum corneum with nacre lipids.

To discover potential new products for the atopic dermatitis treatment, lipids extracted from nacre from the oyster Pinctada margaritifera were tested on artificially dehydrated skin explants. Expression of filaggrin and transglutaminase 1 was investigated after treatment of dehydrated skin with P. margaritifera lipid extracts according to light microscopy after labelling with specific monoclonal antibodies. The lipids were extracted from the nacre with methanol/chloroform mixture at room temperature and the extract composition was determined according to TLC and densitometry measures. Relative to the dry nacre material, a yield of extraction in lipids of 0.54% (w/w) was determined. Fatty acids, triglycerides, cholesterol and ceramides were in low abundance. Then, application of lipid formulations on skin explants previously dehydrated gave after 3 h an overexpression of filaggrin and a decrease of transglutaminase expression as shown by light microscopy. Using immunofluorescence labelling, we showed that lipids extracted from the mother of pearl of P. margaritifera induced a reconstitution of the intercellular cement of the stratum corneum. The signaling properties of the nacre lipids could be used for a development of new active product treatment against the symptoms of the dermatitis.

Human keratinocyte radiosensitivity is linked to redox modulation.

BACKGROUND: Ionising radiation-induced reactive oxygen species (ROS) overproduction induces keratinocyte alterations and constitutes one of the most common effects after therapeutic gamma-irradiation. ROS production is controlled by a complex enzymatic system. OBJECTIVE: The aim of our study is to analyse the role of radiation-induced oxidative stress in keratinocytes death by apoptosis. We hypothesized that keratinocyte capacity to hamper radiation-induced ROS generation may control their radiosensitivity. METHODS: For this purpose, an original human skin explant model was developed and two types of human epidermal cells were used: primary keratinocytes NHEK and spontaneous non-tumourigenic cell line HaCaT. RESULTS: cDNA-arrays analysis was performed 24h after a 20Gy gamma-radiation and revealed down-regulation of genes involved in oxidative stress control and the apoptosis process. This was confirmed by alterations in catalase, GPx and SOD enzymatic activities. This redox modulation was concomitant to the down-regulation of anti-apoptotic genes and up-regulation of some pro-apoptotic genes (caspase 10, ubiquitin C). Interestingly TUNEL labelling revealed an increase in the number of apoptotic cells. We also demonstrated a differential inducibility of the cell antioxidant network in two keratinocyte lines, which results in a differential cellular level of ROS, explaining their different radiosensitivities. CONCLUSION: Keratinocytes apoptosis is partly dependent on ROS production after exposure to gamma-rays. In addition, the differential radiosensitivity of keratinocytes is linked to different oxidative stress responses.