A chronic pro-inflammatory environment contributes to the physiopathology of actinic lentigines.

Actinic lentigines (AL) or age spots, are skin hyperpigmented lesions associated with age and chronic sun exposure. To better understand the physiopathology of AL, we have characterized the inflammation response in AL of European and Japanese volunteers. Gene expression profile showed that in both populations, 10% of the modulated genes in AL versus adjacent non lesional skin (NL), i.e. 31 genes, are associated with inflammation/immune process. A pro-inflammatory environment in AL is strongly suggested by the activation of the arachidonic acid cascade and the plasmin pathway leading to prostaglandin production, along with the decrease of anti-inflammatory cytokines and the identification of inflammatory upstream regulators. Furthermore, in line with the over-expression of genes associated with the recruitment and activation of immune cells, immunostaining on skin sections revealed a significant infiltration of CD68+ macrophages and CD4+ T-cells in the dermis of AL. Strikingly, investigation of infiltrated macrophage subsets evidenced a significant increase of pro-inflammatory CD80+/CD68+ M1 macrophages in AL compared to NL. In conclusion, a chronic inflammation, sustained by pro-inflammatory mediators and infiltration of immune cells, particularly pro-inflammatory M1 macrophages, takes place in AL. This pro-inflammatory loop should be thus broken to normalize skin and improve the efficacy of age spot treatment.

Actinic lentigines from Japanese and European volunteers share similar impaired biological functions.

BACKGROUND: Hyperpigmented spots develop earlier and with a higher incidence in Asian individuals compared with Europeans. Although actinic lentigines (AL) are very common, the biological events underlying their formation remain ill-defined. OBJECTIVE: AL from Japanese volunteers were characterized through morphological and gene expression analyses. Data were then compared with published data on European volunteers. METHODS: AL on hands were selected through dermoscopic imaging and pattern scoring in Japanese women. Skin biopsies of AL and adjacent non-lesional (NL) skin were processed for histology and gene expression profiling. Japanese and European studies were compared after harmonizing the data using the same mathematical and statistical methods. RESULTS: Histologically, AL from Japanese individuals revealed deep epidermal invaginations with melanin accumulation in the depth of epidermal rete ridges. Transcriptomic data identified 245 genes differentially expressed in AL versus NL skin samples, associated with the different skin compartments and multiple functional families and biological processes, such as epidermal homeostasis, extracellular matrix organization and ion binding/transmembrane transport. Strikingly, melanogenesis-related genes were not significantly modulated in AL compared with NL skin. Comparison of the molecular profiles of Japanese and European AL showed that a huge majority of genes were modulated in the same way, recapitulating the overall biological alterations. CONCLUSION: AL from Japanese volunteers exhibited morphological and molecular alterations of the whole skin structure with impairment of multiple biological functions similar to that found in European women. These findings will contribute to the development of efficient treatments of AL lesions.

Melanosome Distribution in Keratinocytes in Different Skin Types: Melanosome Clusters Are Not Degradative Organelles.

The melanosome pattern was characterized systematically in keratinocytes in situ in highly, moderately, and lightly pigmented human skin, classified according to the individual typological angle, a colorimetric measure of skin color phenotype. Electron microscopy of skin samples showed qualitatively and quantitatively that in highly pigmented skin, although melanosomes are mostly isolated and distributed throughout the entire epidermis, clusters are also observed in the basal layer. In moderately and lightly pigmented skin, melanosomes are concentrated in the first layer of the epidermis, isolated-but for most of them, grouped as clusters of melanocores delimited by a single membrane. Electron tomography resolving intracellular three-dimensional organization of organelles showed that clustered melanocores depict contacts with other cellular compartments, such as endoplasmic reticulum and mitochondria. Additionally, immunogold labelling showed that clusters of melanocores do not correspond to autophagosomes or melanophagosomes but that they present, similarly to melanosomes in melanocytes, features of nonacidic, nondegradative organelles. Overall, these observations suggest that melanocore clusters do not correspond to autophagic organelles but represent reservoirs or protective structures for melanosome integrity and function. These results open avenues for understanding the basis of skin pigmentation in different skin color phenotypes.

Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells.

UNLABELLED: Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. SIGNIFICANCE: This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach.

Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging.

To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is associated with photo-aging.

Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity.

The regenerative capacity of human interfollicular epidermis is closely linked to the potential of immature keratinocytes present within its basal layer. The availability of selection methods and culture systems allowing precise assessment of basal keratinocyte characteristics is critical for increasing our knowledge of this cellular compartment. This report presents a multi-parametric comparative study of basal keratinocytes selected according to two different principles: 1) high adhesion capacity on a type-I collagen-coated substrate [Adh⁺⁺⁺], 2) high cell-surface expression of α6-integrin [Itg-α6 (high)]. Importantly, analysis performed at the single-cell level revealed similar primary clone-forming efficiency values of 45.5% ±â€Š6.7% [Itg-α6(high)] and 43.7% ±â€Š7.4% [Adh⁺⁺⁺], which were markedly higher than those previously reported. In addition, both methods selected keratinocytes exhibiting an extensive long-term growth potential exceeding 100 cell doublings and the capacity for generating a pluristratified epidermis. Our study also included a global transcriptome comparison. Genome-wide profiling indicated a strong similarity between [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocytes, and revealed a common basal-associated transcriptional signature. In summary, cross-analysis of [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocyte characteristics showed that these criteria identified highly equivalent cellular populations, both characterized by unexpectedly high growth capacities. These results may have broad impacts in the tissue engineering and cell therapy fields.