Fibrosis arises as part of a wound healing response that maintains organ integrity following catastrophic tissue damage, but can also contribute to a variety of human pathologies, including liver cirrhosis (Bataller and Brenner, 2005
). In this study, we identify senescent cells in fibrotic livers of CCl4
treated mice, and show that these arise from activated stellate cells – a cell type that initially proliferates in response to hepatocyte cell death and is responsible for the extracellular matrix production that is the hallmark of the fibrotic scar. Surprisingly, we see that the senescence of activated HSCs limits the accumulation of fibrotic tissue following chronic liver damage, and facilitates the resolution of fibrosis upon withdrawal of the damaging agent. Thus, we demonstrate that cellular senescence acts to limit the fibrogenic response to tissue damage, thereby establishing a role for the senescence program in pathophysiological settings beyond cancer.
Liver cirrhosis involves dramatic changes in all cellular components of the liver, being associated with hepatocyte cell death, activation of Kupffer cells and HSCs, and the invasion of inflammatory cells (Bataller and Brenner, 2005
). Previous reports have identified SA-β-gal positive cells in cirrhotic livers and suggested that these cells may arise from damaged hepatocytes. However, the immunotype of senescent cells together with their location along the fibrotic scar strongly suggests that the majority of these arise from senescent activated HSCs. Furthermore, treatments that increase or decrease the number of senescent cells in the liver have an inverse effect on activated HSC accumulation and fibrosis, and livers from mice lacking the key senescence regulators display an aberrant expansion of HSCs and enhanced fibrogenic response. However, our data does not exclude the possibility that senescent hepatocytes might be present in the liver in the later stages of liver disease.
Why activated HSCs eventually senesce remains to be determined. While telomere shortening is the driving force of replicative senescence in cultured human cells (Campisi and d’Adda di Fagagna, 2007
), mouse cells have long telomeres that probably could not shorten sufficiently to trigger senescence during our six week treatment period. By contrast, a similar phenomenon of proliferation and senescence has been described in the context of senescence induced by pro-mitogenic oncogenes in both mouse and human cells (Lin et al., 1998
). In some of these settings, senescence is mediated by hyperactive Akt signaling (Chen et al., 2005
) and, indeed, we detected phosphorylated (active) AKT in activated HSCs present in fibrotic mouse livers or that had senesced in culture (Supplementary Figure 10
). Although correlative, these results are consistent with the possibility that the senescence of activated HSCs results from the hyperproliferative signals that trigger their initial expansion.
We have shown that the senescence of activated HSCs provides a barrier that limits liver fibrosis. The hallmark of cellular senescence is its stable cell cycle arrest and, indeed, we show that this process can be triggered acutely in cultured HSCs and is associated with the downregulation of many cell-cycle regulated genes. Undoubtedly, the enforced cell cycle arrest of activated HSCs in vivo provides a brake on the fibrogenic response to damage by limiting the expansion of the cell type responsible for producing the fibrotic scar.
In addition to halting proliferation, senescent cells – including the activated HSCs studied here – can also display dramatic changes in their secretory properties. For example, senescent cells downregulate genes encoding extracellular matrix components and upregulate extracellular matrix degrading enzymes (e.g. matrix metalloproteinases), although the biological consequences of these effects have not been considered. In addition, senescent cells typically upregulate a plethora of genes known to stimulate immune surveillance. We propose that these changes contribute in a coordinated way to restrain fibrosis – on one hand by limiting the secretion of fibrogenic proteins and degrading those that are present and, on the other, signaling the immune clearance of the expanded population of activated HSCs (Suppl. Figure 11
). Thus, senescence represents a homeostatic mechanism that enables the tissue to return to its pre-damaged state. Such a scheme may be broadly relevant to other wound healing responses.
The mechanism of immune clearance of senescent activated HSCs results from the cytotoxic action of natural killer cells, although other immune components contribute as well. Hence, an antagonist of NK cell function delays the clearance of senescence cells and the resolution of fibrosis, whereas an agent that stimulates the NK cell activation has the opposite effect. Interestingly, a previous report suggested that NK cells might target fraction of activated HSCs in fibrotic livers (Radaeva et al., 2006
), though what signaled this attack was not clear. Although we can not exclude the possibility that spontaneous apoptosis or other modes of cell death contribute to the clearance of activated HSCs in vivo
, our studies suggest that, by activating immune surveillance factors, senescent cells identify themselves to the immune system enabling their efficient clearance – a process that we show can be recapitulated in vitro
. Although a detailed mechanism remains to be determined, we see that senescent activated HSCs have significantly higher expression of MICA, a ligand of NK cell receptor NKG2D. Of note, Rae family proteins, the NKG2D ligands in mice, are upregulated in response to DNA damage (Gasser et al., 2005
), which also is a trigger for cellular senescence (Bartkova et al., 2006
; Di Micco et al., 2006
; Mallette et al., 2007
We previously showed that activation of endogenous p53 in murine liver carcinomas induced senescence and tumor regression in vivo (Xue et al., 2007
). Tumor regression was associated with an upregulation of inflammatory cytokines and immune cell adhesion molecules, and several components of the innate immune system contributed to the clearance of senescent cells. The demonstration that senescent activated HSCs can be targeted through a similar mechanism further underscores the fact that, in at least some situations, senescent cells can turn over in vivo to resolve a tissue pathology. Still, not all senescent cells may be targets for the immune system. For example, in the context of benign melanocytic nevi, the accumulation of senescent cells in aged tissues may be related in part to the established decline in immune system function with age. Interestingly, as observed in the mouse model studied here, clinical data suggests that immuno-suppressed patients more rapidly progress to liver cirrhosis (Berenguer et al., 2000
), while immuno-stimulatory therapy has a protective effect (Rehermann and Nascimbeni, 2005
). Our studies suggest that immuno-stimulatory therapy to enhance senescent cell clearance should be tested for the potential treatment of patients with liver fibrosis, especially in its early stages or following short term exposure to hepatotoxic agents.
We suggest that, following tissue damage, HSCs become activated and proliferate intensely, senesce, and are eventually cleared to protect the liver from an excessive fibrogenic response to acute injury. However, in response to chronic tissue damage, for example, as produced by viral hepatitis or fatty liver disease, continual rounds of hepatocyte death and HSC proliferation allow the production of senescent cells to outpace their clearance, contributing to persistent inflammation and advancing fibrosis. Such a state, while initially beneficial, may eventually trigger the aberrant proliferation and transformation of damaged hepatocytes, leading to cancer. In fact, mixing experiments indicate that senescent fibroblasts can promote the transformation of premalignant epithelial cells in vivo
(Krtolica et al., 2001
). Such a model provides one explanation for how cirrhosis predisposes to hepatocellular carcinogenesis and may be relevant to other settings where fibrosis occurs.