Integration of transcriptomics and spatial biology analyses reveals Galactomyces ferment filtrate promotes epidermal interconnectivity via induction of keratinocyte differentiation, proliferation and cellular bioenergetics.

OBJECTIVE: Human skin is the first line of defence from environmental factors such as solar radiation and is susceptible to premature ageing, including a disruption in epidermal differentiation and homeostasis. We evaluated the impact of a Galactomyces Ferment Filtrate (GFF) on epidermal differentiation and response to oxidative stress. METHODS: We used transcriptomics, both spatial and traditional, to assess the impact of GFF on epidermal biology and homeostasis in keratinocytes (primary or immortalized) and in ex vivo skin explant tissue. The effect of GFF on cell adhesion rates, cellular ATP levels and proliferation rates were quantitated. Oxidative phosphorylation and glycolytic rates were measured under normal and stress-induced conditions. RESULTS: Transcriptomics from keratinocytes and ex vivo skin explants from multiple donors show GFF induces keratinocyte differentiation, skin barrier development and cell adhesion while simultaneously repressing cellular stress and inflammatory related processes. Spatial transcriptomics profiling of ex vivo skin indicated basal keratinocytes at the epidermal-dermal junction and cornifying keratinocytes in the top layer of the epidermis as the primary cell types influenced by GFF treatment. Additionally, GFF significantly increases crosstalk between suprabasal and basal keratinocytes. To support these findings, we show that GFF can significantly increase cell adhesion and proliferation in keratinocytes. GFF also protected overall cellular bioenergetics under metabolic or oxidative stress conditions. CONCLUSION: Our findings provide novel insights into cellular differences and epidermal spatial localization in response to GFF, supporting previous findings that this filtrate has a significant impact on epidermal biology and homeostasis, particularly on spatially defined crosstalk. We propose that GFF can help maintain epidermal health by enhancing keratinocyte crosstalk and differentiation/proliferation balance as well as promoting an enhanced response to stress.

Nicotinamide Prevents UVB- and Oxidative Stress‒Induced Photoaging in Human Primary Keratinocytes.

Nicotinamide (NAM), a NAM adenine dinucleotide precursor, is known for its benefits to skin health. Under standard culture conditions, NAM delays the differentiation and enhances the proliferation of human primary keratinocytes, leading to the maintenance of stem cells. In this study, we investigated the effects of NAM on photoaging in two-dimensional human primary keratinocyte cultures and three-dimensional organotypic epidermal models. In both models, we found that UVB irradiation and hydrogen peroxide induced human primary keratinocyte premature terminal differentiation and senescence. In three-dimensional organotypics, the phenotype was characterized by a thickening of the granular layer expressing filaggrin and loricrin, but thinning of the epidermis overall. NAM limited premature differentiation and ameliorated senescence, as evidenced by the maintenance of lamin B1 levels in both models, with decreased lipofuscin staining and reduced IL-6/IL-8 secretion in three-dimensional models, compared to those in UVB-only controls. In addition, DNA damage observed after irradiation was accompanied by a decline in energy metabolism, whereas both effects were partially prevented by NAM. Our data thus highlight the protective effects of NAM against photoaging and oxidative stress in the human epidermis and pinpoint DNA repair and energy metabolism as crucial underlying mechanisms.

Metabolic dysfunction in human skin: Restoration of mitochondrial integrity and metabolic output by nicotinamide (niacinamide) in primary dermal fibroblasts from older aged donors.

Alterations in metabolism in skin are accelerated by environmental stressors such as solar radiation, leading to premature aging. The impact of aging on mitochondria is of interest given their critical role for metabolic output and the finding that environmental stressors cause lowered energy output, particularly in fibroblasts where damage accumulates. To better understand these metabolic changes with aging, we performed an in-depth profiling of the expression patterns of dermal genes in face, forearm, and buttock biopsies from females of 20-70 years of age that encode for all subunits comprising complexes I-V of the mitochondrial electron transport chain. This complements previous preliminary analyses of these changes. "Oxidative phosphorylation" was the top canonical pathway associated with aging in the face, and genes encoding for numerous subunits had decreased expression patterns with age. Investigations on fibroblasts from older aged donors also showed decreased gene expression of numerous subunits from complexes I-V, oxidative phosphorylation rates, spare respiratory capacity, and mitochondrial number and membrane potential compared to younger cells. Treatment of older fibroblasts with nicotinamide (Nam) restored these measures to younger cell levels. Nam increased complexes I, IV, and V activity and gene expression of representative subunits. Elevated mt-Keima staining suggests a possible mechanism of action for these restorative effects via mitophagy. Nam also improved mitochondrial number and membrane potential in younger fibroblasts. These findings show there are significant changes in mitochondrial functionality with aging and that Nam treatment can restore bioenergetic efficiency and capacity in older fibroblasts with an amplifying effect in younger cells.

Nicotinamide Metabolism Modulates the Proliferation/Differentiation Balance and Senescence of Human Primary Keratinocytes.

Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NAD+), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but the underlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show that NAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In 2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers and increases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared to controls. By contrast, preventing the conversion of NAM to NAD+ using FK866 leads to premature human primary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effects are rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NAD+ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NAD+ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.