As humans and other mammals get older, they become less able to recover from injury or repair damage to their tissues. This happens because mammalian cells gradually lose the ability to divide to produce new cells. This process is called senescence and it helps to prevent cancer by stopping old cells that are more likely to carry harmful mutations from replicating. However the link between senescence and many age-related declines in human health has led scientists to ask whether targeting senescent cells might be one way to treat age-related conditions.

Some organisms can regenerate their tissues throughout their lives; and creatures like salamanders are even able to re-grow limbs and organs if they are lost. Scientists are eager to learn how these animals are able to do this when humans are not, and answering this and related questions might help us to develop therapies that boost our ability to recover from injury or age-related diseases.

Yun et al. took a closer look at senescence in salamanders and unexpectedly found that a large number of senescent cells appeared in a salamander limb as it regenerates. But, by the time the limb had completely regrown, these senescent cells had disappeared. Further experiments revealed that when normal and senescent cells are implanted into a salamander the senescent cells also quickly disappear.

These findings suggest that senescent cells may possibly play a role in the regeneration process, and that salamanders have a system that can efficiently destroy these cells. Previous research had suggested that parts of the immune system, in particular cells called macrophages, help to eliminate senescent cells in some tissues. Yun et al. found that macrophages did accumulate around senescent cells in the regenerating limbs of living salamanders. And when a toxin was used to destroy the macrophages in some salamanders, the senescent cells were not cleared in the way they were in salamanders with active macrophages. Hence, macrophages are an essential part of the mechanism that eliminates senescent cells from salamander tissues.

This efficient mechanism for the elimination of senescent cells could explain how salamanders are able to maintain their ability to regenerate in spite of ageing. These findings also reveal the salamander as a model system that could be used to find new ways to target senescent cells, which could be eventually used in anti-ageing therapies.

Cellular senescence was previously identified as a process that permanently halts the proliferation of normal cells in culture following replicative exhaustion (Hayflick and Moorhead, 1961). It subsequently became clear that cellular senescence is a stress response that acts both in culture and in vivo to prevent proliferation of cells exposed to oncogenic stress, such as telomere attrition, and various types of DNA damage and oncogene insertions (Serrano et al., 1997; Bodnar et al., 1998; d'Adda di Fagagna, 2008). It therefore acts as an effective anti-tumourigenic mechanism (Braig et al., 2005; Chen et al., 2005; Collado et al., 2005). However, senescent cells can also have detrimental effects on biological processes. Long-term accumulation of senescent cells leads to disruption of tissue structure and function, possibly through the acquisition of a senescence-associated secretory phenotype (Campisi, 2005; Campisi et al., 2011). This is particularly relevant in the context of ageing, as in most species there is a marked accumulation of senescent cells with time (Herbig et al., 2006; Wang et al., 2009; van Deursen, 2014). Indeed, recent studies have uncovered a causative link between cellular senescence and age-related deterioration (Baker et al., 2008, 2011, 2013), underscoring the therapeutic benefits of targeting senescent cells (Naylor et al., 2013; van Deursen, 2014), and establishing cellular senescence as a hallmark of ageing (Lopez-Otin et al., 2013).

In mammals, the ability to regenerate tissues declines with age (Sousa-Victor et al., 2014). The decline in muscle regeneration with age has recently been linked to the loss of quiescent stem cells through senescence (Sousa-Victor et al., 2014). In contrast, other vertebrates, such as zebrafish and salamanders, are able to accomplish perfect regeneration of a wide range of complex structures throughout their lifespan (Margotta et al., 2002; Azevedo et al., 2011; Eguchi et al., 2011; Itou et al., 2012). It is therefore possible that these organisms exhibit mechanisms to curtail cellular senescence, thus allowing them to undergo indefinite rounds of regeneration. However, the occurrence and regulation of cellular senescence during regeneration have not been addressed in such regeneration-competent species. Here, we analysed the process of cellular senescence in salamanders. Our results demonstrate that cellular senescence is a recurrent process during salamander limb regeneration, and is subject to a highly efficient mechanism of macrophage-dependent elimination.

To investigate the occurrence of cellular senescence in the salamander system, we first adapted and validated reagents that allow the identification of senescent cells in cell culture and tissue sections. Cellular senescence is a mechanism that irreversibly arrests the proliferation of damaged or dysfunctional cells (Shay and Roninson, 2004; Campisi, 2005), and is triggered upon DNA insults such as UV irradiation (Campisi, 2011). We therefore exposed A1 salamander limb cells to UV, while stabilising the level of p53, which is required for the induction of senescence in certain mammalian contexts (Figure 1A). Up to 80% of the irradiated cells entered a state of cell cycle withdrawal within 12 days that, in contrast to quiescence, was accompanied by high levels of Gadd45 expression, permanent γH2AX foci, and high levels of senescence-associated β-galactosidase (SA-βgal), a hallmark of cellular senescence (Dimri et al., 1995; Campisi, 2011) (Figure 1B–F). The timing of SA-βgal induction in salamander cells is similar to that reported in mammalian fibroblasts undergoing stress-induced senescence (Parrinello et al., 2003).

Figure 1

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Senescent cells are characterised by an irreversible cell cycle arrest. While proliferating or quiescent A1 cells re-enter the cell cycle upon serum stimulation (indicated by the incorporation of the nucleotide analogue EdU), SA-βgal positive A1 cells do not, as demonstrated by double SA-βgal/Edu staining (Figure 1G). Furthermore, the cells identified by the SA-βgal staining exhibit other markers of senescence (Campisi, 2005) such as sustained production of reactive oxygen species (ROS), and extended mitochondrial and lysosomal networks (Figure 1F,H) relative to non-senescent parallel cultures. In addition, these cells acquire a secretory phenotype comprising a number of cytokines, signalling and matrix remodelling factors (Figure 1I). It is nothworthy that many of these factors, such as the interleukins IL-1a, IL-1b, IL-13, IL-7, IFNγ (Interferon gamma), TNF-α (Tumor Necrosis Factor alpha), M-CSF (Macrophage Colony-Stimulating Factor), the matrix metelloproteases MMP1, 3 and 9, uPA (urokinase-type Plasminogen Activator) and Cathepsin D, have been previously reported to integrate the senescence-associated phenotype (SASP) in various contexts of mammalian senescence (Coppé et al., 2010). Together, these data suggest that the regulation and properties of the senescent state are comparable in salamanders and mammals.

Induction of cellular senescence during salamander limb regeneration

In order to determine whether cellular senescence occurs in vivo within normal and regenerating salamander tissues, we adapted the SA-βgal staining to detect senescent cells within salamander tissues and addressed whether senescence occurs during limb regeneration in an adult salamander, the newt (Notophthalmus viridescens). We found that there is a significant induction of cellular senescence during the intermediate stages of regeneration (mid-blastema and palette), but that the number of senescent cells diminishes subsequently (Figure 2A,B). Within the regenerating limb, senescent cells are found at the level of the amputation plane, including cartilage and muscle, and within the blastema, including fibroblast, monocyte-like cells, and epidermal glands (Figure 2—figure supplement 1). To corroborate that SA-βgal⁺ cells exhibit other hallmarks of cellular senescence in vivo, we performed SA-βgal/EdU stainings in regenerating or normal tissues following systemic EdU injections. We were unable to detect SA-βgal⁺/EdU⁺ cells under any condition examined (Figure 2C), suggesting that SA-βgal⁺ cells are withdrawn from the cell cycle, a characteristic of senescent cells.

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Similar observations were made in another salamander species, the axolotl (Ambystoma mexicanum), both in mature animals as well as at early stages of development (Figure 2D,E). Interestingly, the induction and subsequent disappearance of senescent cells appear to be characteristic of regeneration, as no significant induction of senescence takes place during normal limb development (Figure 2E, Figure 2—figure supplement 2), in contrast to previous findings in amniotic limb development (Banito and Lowe, 2013; Munoz-Espin et al., 2013; Storer et al., 2013). These observations suggest that cellular senescence is a normal process during salamander limb regeneration, and is subject to dynamic regulation.

Efficient surveillance of senescent cells in adult salamander tissues

To test this possibility, we implanted one thousand senescent or normal cells (labelled with nuclear GFP expressed from an integrated retrovirus) within newt limb tissues and analysed their persistence over time (Figure 3A). After an initial non-specific loss of cells following implantation, the normal cells (A1 GFP+) persist for at least 40 days and contribute to structures within the limb tissues (Figure 3A,B). In contrast, 80% of the implanted senescent cell population is cleared from the limb tissues within 2 weeks post-implantation (Figure 3A,B), consistent with a half-life of 9 days (calculated from the exponential equation corresponding to the senescent dataset, after subtraction of the initial loss observed in normal cells), comparable to the rate of clearance of senescent cells during regeneration (Figure 2). Although this experiment suggests that senescent cells are being cleared from salamander tissues, it cannot exclude the possibility that some cells may reverse their senescence state. To distinguish between these two possibilities, we performed the same experiment using retrovirally labelled nGFP senescent cells (Figure 3B and Figure 3—figure supplement 1). The rate of clearance of nGFP+ senescent cells corresponds to the rate of clearance of unlabelled senescent cells, supporting the clearance hypothesis. In contrast, the implantation of a population of nGFP cells exposed to a UV dose of 1J/m2, 10 times lower than the dose required for senescence induction (Figure 3—figure supplement 1), results in persistence of the implanted cells (Figure 3B). These data suggest that salamander tissues have a significant capacity for senescent cell clearance.

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Interestingly, the implantation of a 1:1 mixture of senescent and normal cells does not result in the persistence of the normal cells, but in the clearance of both populations (Figure 3B, Figure 4A,B), albeit with a delay when compared to the clearance of purely senescent cells. It has been recently shown that senescent cells are able to induce senescence in their neighbours, a phenomenon termed ‘senescence bystander effect’ (Nelson et al., 2012; Acosta et al., 2013). We found that this property is also present in senescent salamander cells. Cellular senescence can be induced in normal cells by co-culture with senescent cells (Figure 4C), or following incubation with purified media from senescent populations (Figure 4D), supporting the idea that senescence can be transmitted in a paracrine fashion. In addition, implantation of the 1:1 mixture of senescent + normal cells results in the induction of senescence in normal cells (Figure 4E,F), providing an explanation for their eventual clearance. Together, these results suggest that active mechanisms identify and target senescent cells for clearance within salamander tissues.

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Macrophages are a critical part of the senescent cell clearance mechanism

A series of elegant experiments have recently described a role for the immune system in the surveillance and clearance of p53 or oncogene-induced senescent hepatocytes in mice (Xue et al., 2007; Kang et al., 2011; Lujambio et al., 2013). In addition, recent evidence suggests that there is an extensive recruitment of immune cells, and in particular macrophages, to the regenerating limb in salamanders (Godwin et al., 2013). Hence, we decided to investigate if the macrophage was part of the senescence clearance mechanism. In vivo labelling using TMR-dextran, which is specifically internalised by macrophages (Figure 5A–C), as well as monocyte specific α-naphthyl acetate esterase staining (Figure 5D), reveal that macrophages and senescent cells are found in close proximity within regenerating limbs, suggesting cell-to-cell contact and engulfment in some cases. To determine whether macrophages mediate the clearance of senescent cells, we took advantage of an efficient method for macrophage depletion, based on the intravenous administration of selectively toxic, clodronate salt-filled liposomes (clodrosomes). These are internalised specifically by macrophages via phagocytosis (van Rooijen and Hendrikx, 2010) and have been shown to operate effectively in salamanders (Godwin et al., 2013). We found that treatment with clodrosomes is able to reduce the macrophage population by fivefold, compared with control DiI liposomes (Figure 6A). As with the TMR-dextran, these liposomes are specifically internalised by macrophages, as shown by their incorporation into cells expressing F4/80, a specific macrophage marker (Morris et al., 1991) (Figure 6B). We therefore used this system to induce systemic macrophage depletion in salamanders prior to the implantation of senescent or normal nGFP+ cells in contralateral regenerating limbs, and followed the fate of the implanted cells with time (Figure 6C). At 20 hr post implantation, we observed a strong accumulation of DiI-labelled macrophages in the vicinity of senescent cells (Figure 6D–F). In contrast, macrophage recruitment to areas of normal cells (control) was not significantly different from their recruitment to other areas of the limb mesenchyme (Figure 6D,F). At 2 weeks post implantation, the normal cells remained within the regenerating limbs (Figure 6G). However, the senescent cells were cleared in animals injected with control DiI liposomes, but remained in animals whose macrophages had been depleted (Figure 6G). This demonstrates that the macrophage is critical for clearance of senescent cells, and constitutes the first evidence for senescence surveillance mechanisms that operate during normal regeneration.

Figure 5

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Figure 6

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It is clear that salamanders possess a rapid and efficient mechanism to recognise and clear senescent cells that either arise endogenously, or are introduced from culture. Our study demonstrates that a robust macrophage-dependent surveillance mechanism operates in normal and regenerating tissues of adult salamanders, and this allows them to circumvent the negative effects associated with the long-term accumulation of senescent cells, such as the disruption of tissue structure and function (Campisi, 2005; Rodier and Campisi, 2011). These surveillance mechanisms are particularly significant for limb regeneration because there is a notable induction of cellular senescence during this process. Consistent with this, recent reports show that systemic macrophage depletion during salamander limb regeneration leads to defects in this process (Godwin et al., 2013). This has also been observed during zebrafish fin regeneration (Petrie et al., 2014). Furthermore, cellular senescence has been recently shown to contribute to the decline in muscle regenerative ability upon ageing in mammals (Wang et al., 2009; Baker et al., 2011; Sousa-Victor et al., 2014). In light of these observations, we propose that the effective immune clearance of senescent cells in salamanders supports their ability to undergo multiple rounds of regeneration through their lifespan (Figure 7).

Figure 7

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The mechanisms underlying the induction of senescent cells and their clearance, for example the involvement of other immune cell types in their surveillance, constitute an important outstanding issue. The analysis of these problems has been limited by lack of reagents, as well as cellular and molecular tools for in vitro modelling of senescent-immune cell interactions as well as gain or loss-of-function approaches to evaluate induction and clearance of endogenous senescent cells. Further progress in this area will enable the exploration of these important matters.

Nevertheless, it is interesting that we find a consistent induction of cellular senescence during regeneration, suggesting that this could be integral to the mechanism. Senescent cells could play a positive role during regeneration, a proposal that is consistent with recent findings that senescence contributes to tissue remodelling during mouse development (Munoz-Espin et al., 2013; Storer et al., 2013; Campisi, 2014) and wound healing (Krizhanovsky et al., 2008; Jun and Lau, 2010; Demaria et al., 2014). Alternatively, senescent cells could contribute to regeneration indirectly, by promoting the efficient recruitment of macrophages, since the latter have been shown to promote tissue remodelling and patterning in various contexts, including regeneration (Lobov et al., 2005; Qian and Pollard, 2012; Petrie et al., 2014). In either case, the senescence-associated secretory phenotype is likely to play an important role. These are important issues that merit further examination.

It has recently been suggested that targeting senescent cells could lead to therapeutic strategies for age-related pathologies (Naylor et al., 2013; van Deursen, 2014). Here, we identify an animal with an efficient mechanism for surveillance of senescent cells operating through adulthood. Analysis of this mechanism could lead to the identification of novel therapeutic targets for the amelioration of age-related disorders and extension of healthspan.

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Author details

1. Maximina H Yun

   Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom

   Contribution

   MHY, Conceived the project, Designed and performed experiments, Analysed/interpreted data, Wrote and revised the manuscript

   For correspondence

   maximina.yun@ucl.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

2. Hongorzul Davaapil

   Institute of Ophthalmology, University College London, London, United Kingdom

   Contribution

   HD, Performed experiments and analysed/interpreted data

   Competing interests

   The authors declare that no competing interests exist.

3. Jeremy P Brockes

   Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom

   Contribution

   JPB, Designed experiments, Analysed/interpreted data and revised the manuscript

   Competing interests

   The authors declare that no competing interests exist.

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