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Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA–induced metabolic stress in reconstructed human skin

ABSTRACT: Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

SUBMITTER: Ung T

PROVIDER: S-EPMC8589997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Despite their preponderance amongst the ultraviolet (UV) range received on Earth, the biological impacts of longwave UVA1 rays (340-400 nm) upon human skin have not been investigated so thoroughly. Nevertheless, recent studies have proven their harmful effects and involvement in carcinogenesis and immunosuppression. In this work, an in vitro reconstructed human skin model was used for exploring the effects of UVA1 at molecular, cellular and tissue levels. A biological impact of UVA1 throughout the whole reconstructed skin structure could be evidenced, from morphology to gene expression analysis. UVA1 induced immediate injuries such as generation of reactive oxygen species and thymine dimers DNA damage, accumulating preferentially in dermal fibroblasts and basal keratinocytes, followed by significant cellular alterations, such as fibroblast apoptosis and lipid peroxidation. The full genome transcriptomic study showed a clear UVA1 molecular signature with the modulation of expression of 461 and 480 genes in epidermal keratinocytes and dermal fibroblasts, respectively (fold change> = 1.5 and adjusted p value<0.001). Functional enrichment analysis using GO, KEGG pathways and bibliographic analysis revealed a real stress with up-regulation of genes encoding heat shock proteins or involved in oxidative stress response. UVA1 also affected a wide panel of pathways and functions including cancer, proliferation, apoptosis and development, extracellular matrix and metabolism of lipids and glucose. Strikingly, one quarter of modulated genes was related to innate immunity: genes involved in inflammation were strongly up-regulated while genes involved in antiviral defense were severely down-regulated. These transcriptomic data were confirmed in dose-response and time course experiments using quantitative PCR and protein quantification. Links between the evidenced UVA1-induced impacts and clinical consequences of UVA1 exposure such as photo-aging, photo-immunosuppression and cancer are discussed. These early molecular events support the contribution of UVA1 to long term harmful consequences of UV exposure and underline the need of an adequate UVA1 photoprotection.

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Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA–induced metabolic stress in reconstructed human skin

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