Early skin inflammatory biomarker is predictive of development and persistence of atopic dermatitis in infants.

BACKGROUND: The Short-Term Topical Application for Prevention of Atopic Dermatitis (STOP AD) study, a randomized, open-label trial evaluating the effect of short-term (from the first 4 postnatal days to age 8 weeks) skin barrier protection using Aveeno Dermexa Fast & Long-Lasting Balm (Johnson & Johnson, New Brunswick, NJ) in infants with a parent with allergic disease, demonstrated decreased cumulative incidence and decreased prevalence of atopic dermatitis (AD) at age 12 months. OBJECTIVE: In the STOP AD study, we aimed to identify skin biomarkers that are associated with risk of development of AD. METHODS: Skin swabs were collected from the cheek and antecubital fossa (AF) at baseline, age 8 weeks, and age 12 months from subsets of study participants from the intervention arm (n = 43 of 119) and control arm (n = 43 of 138) and were analyzed for specific cytokines (CCL27, CXCL2, human ß-defensin-1 [hBD-1], IL-18, IL-8, IL-1α, IL-1 receptor antagonist [IL-1RA], IL-1ß, S100A8/9, and IL-36γ) by ELISA. RESULTS: Higher titers of S100A8/9 at the AF at age 8 weeks in infants with the filaggrin wild-type genotype (FLGwt), but not in those with filaggrin loss-of-function mutation (FLGmut), predicted (1) development of AD in the first year of life (P = .033), (2) presence of AD at ages 6 or 12 months (P = .009 and .035, respectively), (3) persistence of AD between ages 6 and 12 months (P < .001), and (4) development of AD with the emollient intervention. CONCLUSION: Increased titers of S100A8/9 from skin swabs of the AF in high-risk infants at age 8 weeks with FLGwt were predictive of AD development in the first year of life and other AD features. These findings suggest that there are different molecular pathways leading to AD in individuals with FLGmut and in individuals with FLGwt. Early identification of infants who are likely to develop AD will allow more targeted interventions.

Skin maturation from birth to 10 years of age: Structure, function, composition and microbiome.

Infant and adult skin physiology differ in many ways; however, limited data exist for older children. To further investigate the maturation processes of healthy skin during childhood. Skin parameters were recorded in 80 participants of four age groups: babies (0-2 years), young children (3-6 years), older children (7-<10 years) and adults (25-40 years). Overall, skin barrier function continues to mature, reaching adult levels of transepidermal water loss (TEWL), lipid compactness, stratum corneum (SC) thickness and corneocyte size by the age of about 6 years. Higher levels of lactic acid and lower levels of total amino acids in the SC of babies and young children further indicate higher cell turnover rates. In all age groups, TEWL and skin surface hydration values remain higher on the face compared with the arm. Skin becomes darker and contains higher levels of melanin with increasing age. The composition of skin microbiome of the dorsal forearm in all children groups is distinct from that in adults, with Firmicutes predominating in the former and Proteobacteria in the latter. Skin physiology, along with the skin microbiome, continues to mature during early childhood in a site-specific manner.

Deciphering the Role of Skin Surface Microbiome in Skin Health: An Integrative Multiomics Approach Reveals Three Distinct Metabolite‒Microbe Clusters.

The advent of 16S RNA profiling and shotgun metagenomics has enabled a holistic approach to the study of the skin microbiome composition. Despite the interesting findings in this rapidly developing scientific area, the big question remains: What role does the microbiome play in skin physiology? To begin answering this question, we employed an integrative methodology for microbiome and metabolome analysis of skin surface samples collected from the volar forearm of healthy infants aged 3-6-months. Whereas the infant skin metabolome was dominated by amino acids, lipids, and xenobiotics, the primary phyla of the microbiome were Firmicutes, Actinobacteria, and Proteobacteria. Zooming in on the species level revealed a large contribution of commensals belonging to the Cutibacterium and Staphylococcus genera, including Cutibacterium acnes, Staphylococcus epidermidis, and S. aureus. This heterogeneity was further highlighted when combining the microbiome with metabolome data. Integrative analyses delineated the coexistence of three distinct metabolite‒microbe clusters: one dominated by Cutibacterium linked to hydrophobic elements of the skin barrier, one associating Staphylococcus genus with amino acids relevant to the water holding capacity and pH regulation of the skin surface, and one characterized by Streptococcus and independent of any particular metabolomic profile.

An integrative multi-omic analysis reveals a major metabolic rewiring between baby foreskin keratinocytes and adult female abdominal keratinocytes.

Even though its development starts early in utero, neonatal skin is still immature at birth relative to adult and undergoes a maturation process extending to the first years of life. It is now established that the stratum corneum is thinner and dryer and that skin contains less natural moisturizing factors and lipids in newborns compared to children and adults. Moreover, it has been shown that skin surface area expansion is not linear throughout life and is peaking perinatally, suggesting that baby skin has a higher epidermal cellular turnover. Despite growing resources showing differences between adult and infant skin physiology, molecular and metabolic specificities of baby skin are still poorly understood. To address this critical knowledge gap, we performed an integrative transcriptomic and metabolomic study comparing human primary foreskin and abdominal keratinocytes from male babies and female adults, respectively. Based on state-of-the-art integrative frameworks, our analyses revealed a major shift in the global energetic metabolism in baby foreskin keratinocytes compared to adult abdominal keratinocytes, highlighting increased amino acid metabolism and mitochondrial oxidative phosphorylation in baby cells to fuel the citric acid cycle, while showing glycolysis as the major cell energy source in adult cells.

A Predictive Self-Organizing Multicellular Computational Model of Infant Skin Permeability to Topically Applied Substances.

Computational models of skin permeability are typically based on assumptions of fixed geometry and homogeneity of the whole epidermis or of epidermal strata and are often limited to adult skin. Infant skin differs quantitatively from that of the adult in its structure and its functional properties, including its barrier function to permeation. To address this problem, we developed a self-organizing multicellular epidermis model of barrier formation with realistic cell morphology. By modulating the parameters relating to cell turnover reflecting those in adult or infant epidermis, we were able to generate accordingly two distinct models. Emerging properties of these models reflect the corresponding experimentally measured values of epidermal and stratum corneum thickness. Diffusion of an externally applied substance (e.g., caffeine) was simulated by a molecular exchange between the model agents, defined by the individual cells and their surrounding extracellular space. By adjusting the surface concentration and the intercellular exchange rate, the model can recapitulate experimental permeability data after topical exposure. By applying these parameters to an infant model, we were able to predict the caffeine concentration profile in infant skin, closely matching experimental results. This work paves the way for a better understanding of skin physiology and function during the first years of life.

In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like.

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin.

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative-senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age-related phenotypic and functional alteration of fibroblasts from 'young' (<35 years) and 'old' (>50 years) donors. Our results brought evidence of the senescent status of 'old' fibroblasts by senescence associated ß-galactosidase (SA-ßgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in 'old' fibroblasts as well as a subset of upregulated genes involved in senescence features. In 'old' fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFß. Old fibroblasts were also more sensitive to oxidative stress than 'young' ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of 'old' fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.

Surfactants have multi-fold effects on skin barrier function.

BACKGROUND: The stratum corneum (SC) is responsible for the barrier properties of the skin and the role of intercorneocyte skin lipids, particularly their structural organization, in controlling SC permeability is acknowledged. Upon contacting the skin, surfactants interact with the SC components leading to barrier damage. OBJECTIVE: To improve knowledge of the effect of several classes of surfactant on skin barrier function at three different levels. METHODS: The influence of treatments of human skin explants with six non-ionic and four ionic surfactant solutions on the physicochemical properties of skin was investigated. Skin surface wettability and polarity were assessed through contact angle measurements. Infrared spectroscopy allowed monitoring the SC lipid organization. The lipid extraction potency of surfactants was evaluated thanks to HPLC-ELSD assays. RESULTS: One anionic and one cationic surfactant increased the skin polarity by removing the sebaceous and epidermal lipids and by disturbing the organization of the lipid matrix. Another cationic surfactant displayed a detergency effect without disturbing the skin barrier. Several non-ionic surfactants disturbed the lipid matrix organization and modified the skin wettability without any extraction of the skin lipids. Finally two non-ionic surfactants did not show any effect on the investigated parameters or on the skin barrier. CONCLUSIONS: The polarity, the organization of the lipid matrix and the lipid composition of the skin allowed describing finely how surfactants can interact with the skin and disturb the skin barrier function.

T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and ß6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results indicate that T-plastin might be considered as a major actor in the mechanisms underlying calcineurin/NFAT-dependent keratinocyte migration and may explain wound-healing defects observed in patients under calcineurin inhibitor long-term treatment.

ORAI1 calcium channel orchestrates skin homeostasis.

To achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca(2+) channel that is a major molecular counterpart for Ca(2+) influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCß-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis.

The role of the skin barrier in modulating the effects of common skin microbial species on the inflammation, differentiation and proliferation status of epidermal keratinocytes.

BACKGROUND: Skin resident microbial species are often thought of either as pathogenic or commensal. However, little is known about the role of the skin barrier in modulating their potential for causing disease. To investigate this question we measured the effects of three microbial species commonly found on the skin (Staphylococcus epidermidis, Staphylococcus aureus, and Propionibacterium acnes) on a reconstructed human epidermal model by either applying the bacteria on the model surface (intact barrier) or adding them to the culture medium (simulating barrier breach). RESULTS: When added to the medium, all of the tested species induced inflammatory responses and keratinocyte cell death with species-specific potency. P. acnes and S. epidermidis induced specific alterations in the expression of keratinocyte differentiation and proliferation markers, suggesting a barrier reparation response. S. aureus induced complete keratinocyte cell death. On the contrary, topically applied S. epidermidis and P. acnes caused no inflammatory response even when tested at high concentrations, while topical S. aureus induced a weak reaction. None of the tested species were able to alter the expression of keratinocyte differentiation or expression markers, when applied topically. CONCLUSIONS: We show that the skin barrier prevents the effects of common skin bacteria on epidermal keratinocyte inflammation, differentiation and proliferation and highlight the importance of skin barrier in defending against the pathogenic effects of common skin bacteria.

A purified feverfew extract protects from oxidative damage by inducing DNA repair in skin cells via a PI3-kinase-dependent Nrf2/ARE pathway.

BACKGROUND: Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) - antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms. OBJECTIVE: To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells. METHODS: These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR. RESULTS: PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner. CONCLUSIONS: Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors.

NF-κB accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a -112/-61-bp region of the COL1A1 promoter in human skin fibroblasts.

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

In the shadow of the wrinkle: theories.

As time passes, wrinkles typically appear. These skin depressions that become deeper and deeper draw more and more coarser lines on almost all the visible parts of aging individual's skin. They are indeed the most obvious and maybe disliked signs of skin aging, and thus, preventing and treating them are a major topic for dermo-cosmetic laboratories. However, the cause and occurrence mechanism of these simplistic looking lines are not yet fully understood. Wrinkling is thought to be a complex biophysical process resulting from repeated strains on a progressively, structurally and biochemistry altered aging skin with impaired mechanical properties. Focus is made on the specific histological features of the wrinkle compared to the surrounding aging skin. The numerous age-related changes in human skin that are supposed to be involved in wrinkling are briefly reviewed, and the current theories on wrinkle formation linked to these changes are also discussed.

The p65 subunit of NF-κB inhibits COL1A1 gene transcription in human dermal and scleroderma fibroblasts through its recruitment on promoter by protein interaction with transcriptional activators (c-Krox, Sp1, and Sp3).

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

Age-associated modifications of Base Excision Repair activities in human skin fibroblast extracts.

Base Excision Repair (BER) is the predominant repair pathway responsible for removal of so-called small DNA lesions such as abasic sites (AP site), uracil (U), 8-oxo-7,8-dihydroguanine (8oxoG), thymine glycol (Tg). In this study, we investigated effect of aging on excision efficacy of several endogenous base lesions and AP sites using an in vitro multiplexed fluorescent approach on support (parallelized oligonucleotide cleavage assay). Human fibroblasts nuclear extracts from 29 donors of different ages were characterized in their ability to simultaneously excise the different lesions. Clearly, three different groups of lesions emerged according to the efficiency of their cleavage: one exhibited very high cleavage efficiency (AP sites and U paired with G), one showed intermediate cleavage efficiency (U paired with A and Tg). The third group included 8oxoG, A paired with 8oxoG, T at CpG site and hypoxanthine (Hx) and displayed poor repair. Aging was significantly associated with modification of excision efficiency for AP sites, uracil, Tg and 8oxoG. Repair rate decreased for the first three lesions and the most drastic effects were observed for repair of U:A. Surprisingly, excision of 8oxoG increased with aging suggesting a completely different regulation or adaptation for the initiation step of this related specific repair pathway.

Parthenolide-depleted Feverfew (Tanacetum parthenium) protects skin from UV irradiation and external aggression.

The skin is under continual assault from a variety of damaging environmental factors such as ultraviolet irradiation and atmospheric pollutants, and as organisms age the cumulative damage exceeds the capacity of endogenous antioxidant defenses resulting in chronic inflammation and premature aging. Botanical extracts such as Feverfew containing naturally occurring antioxidants could replenish the depleted cutaneous stores and perhaps forestall these degenerative changes. A parthenolide-depleted extract of Feverfew (PD-Feverfew), which was free of sensitization potential, was found to possess free radical scavenging activity against a wide range of reactive oxygen species and with greater activity than Vitamin C. In vitro, PD-Feverfew restored cigarette smoke-mediated depletion of cellular thiols, attenuated the formation of UV-induced hydrogen peroxide and reduced pro-inflammatory cytokine release. In vivo, topical PD-Feverfew reduced UV-induced epidermal hyperplasia, DNA damage and apoptosis. In a clinical study PD-Feverfew treatment significantly reduced erythema versus placebo 24 h post-UV exposure. Through the ability to scavenge free radicals, preserve endogenous antioxidant levels, reduce DNA damage and induce DNA repair enzymes, which can help repair damaged DNA, parthenolide-depleted extract of Feverfew may protect skin from the numerous external aggressions encountered daily by the skin and reduce the damage to oxidatively challenged skin.