Changes of senescent cell accumulation and removal in skin tissue with ageing

Abstract

Senescent cells are known to secrete various inflammatory cytokines (known as the senescence-associated secretory phenotype factors: SASP factors) and adversely affect surrounding tissues. Senolytic drugs that selectively induce the death of senescent cells are under development.1, 2 We reported that skin inherently possesses mechanisms to remove senescent cells. In the epidermis, this is achieved by the binding of JAG1, a Notch ligand expressed on adjacent non-senescent keratinocytes, to Notch1 receptors expressed by senescent keratinocytes, which promotes the exclusion of senescent cells from the basal layer by inducing differentiation.3 Meanwhile, in the dermis, senescent cells are phagocytosed by macrophages through recognition by the phosphatidyl serine (PS) receptor STAB1.4 However, since ageing is associated with the accumulation of senescent cells in skin tissue, it is hypothesized that this accumulation is preceded by a decline in the ability to remove them. Here, we tested this hypothesis by analysing in detail the changes in the number of senescent cells and the ability of them to be removed from the skin with ageing. Marginal skin tissues at the time of surgery were collected from patients who had provided informed consent. Paraffin-embedded sections prepared from the unexposed skin tissue were subjected to immunostaining with the major senescent cell marker p16INK4A to analyse the number of senescent cells (Appendix S1). To characterize the ability for senescent cells to be removed from the skin, the expression of JAG1 in the epidermal keratinocytes and STAB1 in macrophages was analysed. The results confirmed that senescent cell accumulation was increased with age in both epidermis and dermis (Figure 1A,B). We also found that the ability to remove senescent cells decreased with age (Figure 1C,D and Figure S1A). The results also confirmed correlations between the increased rate of senescent cells and decreased function of senescent cell removal in epidermis and dermis (Figure 1E and Figure S1B). It suggested that declining in senescent cell removal ability led the accumulation of senescent cells. In addition, it was observed that while the accumulation of senescent cells was detected after 30s, the removal capacity began to decrease at 20s. Although there was a delay of the age at which senescent cell accumulation starts after the onset of declining of senescent cell removal ability, there appears to be a correlation across all ages between the removal ability and the accumulation of senescent cells (Figure 1E). This may be because the sample numbers for young ages (10s and 20s) were too small compared to those of the total to affect the correlation throughout ages. Although further investigation is necessary, because the number of samples were insufficient for accurate analysis of time series, these results suggested that the ability to remove senescent cells declined before cells started to accumulate in the skin. Moreover, it was expected the possibility of preventing the accumulation of senescent cells by maintaining the ability to remove them from a young age. We next examined whether the inability to remove senescent cells had any negative effects other than allowing these cells to accumulate. In vitro, co-culture of senescent and normal keratinocytes was found to enhance EGF gene expression in normal keratinocytes (Figure S2A). Moreover, FGF2 gene expression was enhanced in macrophages that had phagocytosed senescent fibroblasts (Figure S2B). Additionally, these improvements were reduced by inhibiting the JAG1 expression in keratinocytes or blocking macrophage phagocytosis using a substance that prevents actin polymerization (latrunculin) (Figure S2A,B). The growth factors EGF and FGF2 are crucial for maintaining skin homeostasis5 and showed decreased expression with ageing.6 In addition, cellular senescence in keratinocytes and fibroblasts did not show significant effects on their gene expressions of EGF and FGF2 (Figure S3A,B). These results suggest that the removal of senescent cells is involved in the maintenance of homeostasis not only via the removal of these harmful cells themselves, but also by stimulating the production of EGF and FGF2, which activate skin stem cells to provide new cells. Senolytic drugs only act to remove senescent cells; they do not stimulate stem cells' ability to produce new cells. Although data of this study were limited, we believe that endogenous mechanisms that eliminate senescent cells are better for maintaining skin homeostasis than senolytic drugs. The reason of the decline in the senescent cell removal function in the 20s, before senescent cells accumulate, is still a mystery. We believe that environmental factors such UV radiation, reactive oxygen species and exposure to SASP factors released by transiently senescent cells in young age may have an impact on the function of senescent cell removal. Future plans for this research include a closer look at these factors and the techniques for enhancing the efficiency of senescent cell removal. Y. Ogata and T. Yamada designed the research study. Y. Ogata and T. Yamada performed the research. Y Ogata, S. Hasegawa, K. Sugiura and H. Akamatsu carried out the data analysis and wrote the manuscript. All authors critically revised the manuscript and approved the final manuscript. We are grateful to Y. Iwata, M. Arima (Department of Dermatology, Fujita Health University School of Medicine, Aichi, Japan), Y. Ishii and M. Kawagishi-Hotta (Nippon Menard Cosmetic Co., Ltd., Aichi, Japan) for their technical support. The authors declare that there is no conflict of interest. Data sharing is not applicable to this article as no new data were created or analyzed in this study. Figure S1 Appendix S1 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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