NKG2D ligands mediate immunosurveillance of senescent cells

Abstract

Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis.  Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis.

Link: http://impactaging.com/papers/v8/n2/full/100897.html

Atypical senescent states: Metabolic stress-induced senescence

Metabolic stress, defined here as a combination of aerobic glycolysis and mitochondria dysfunction can potentially trigger a senescent state.  All organisms that use aerobic glycolysis form reactive acyclic α-oxoaldehydes (e.g. methylglyoxal and glyoxal) spontaneously from triosephosphates and by a wide variety of other routes (Thornalley, 2009).  These dicarbonyl compounds are highly reactive and damage proteins through non-enzymatic modification producing a wide variety of covalent adducts (AGEs).  Elevated levels of methylglyoxal and glyoxal are known to be cytotoxic and although the mechanism of action remains imprecisely defined, it can be blocked by ROS scavengers, suggesting that oxidative stress mediates at least some of the deleterious effects (Shangari and O’Brian, 2004).

Cytosolic and mitochondrial protection from dicarbonly damage is primarily mediated through the action of the glyoxalase system that consists of two enzymes, glyoxalase I and II.  However, in cultures of WI38 fibroblasts a significant reduction in the activity of glyoxalase-I occurs with serial passage (Ahmed et al. 2010).  Treatment of cultures of ASF2 human adult dermal fibroblasts with micro or millimolar concentrations of glyoxal or methylglyoxal renders them senescent within 72 hours.  This was defined by the presence of typical senescent morphology, irreversible growth arrest and increased SA-β-Gal activity (Sejersen & Rattan, 2009).  Further studies (Larsen et al. 2012) extended these observations to immortalized human mesenchymal stem cells (MSCs) and demonstrated that treatment with physiologically reflective (Han et al. 2007) concentrations of glyoxal for 72 hours led to senescence without significant cell death (although massive cell death occurred at higher glyoxal concentrations).  Elevated levels of SA-β-Gal, p16 and DNA damage (as measured by COMET) accompanied the growth arrest.  Interestingly, a profound reduction in the ability of these senescent MSCs to differentiate into functional osteoblasts (as determined by alkaline phosphatase and mineralization assays) was also observed.   Given the imbalances in glucose metabolism that accompany mammalian ageing (and diabetes), the authors proposed that this type of metabolic stress might underlie age-related changes in bone function.   Unfortunately, no markers of immunogenic conversion have yet been measured in this system and whilst the presence of DNA damage could indicate the likelihood of a secretory response, this cannot be assumed.  Thus, the propensity of senescence human MSCs to be cleared by the immune system remains unknown and is of considerable physiological significance.

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion

Atypical senescent states: TGFβ-induced senescence and Developmentally programmed senescence

TGFβ-induced senescence: A growing body of evidence suggests that the members of the transforming growth factor beta (TGF- β) family can induce a senescence-like state. Experimentally, senescence has been predominantly, but not exclusively, characterized by the presence of senescence-associated beta galactosidase (SA-β-Gal) staining and the up-regulation of cyclin dependent kinase inhibitors (CDKi) (see below). Human prostate basal cells treated with TGF-β1/2/3 show increased SA-β-Gal activity, which is associated with the flattened, and enlarged cell morphology typical of adherent senescent cells in vitro (Untergasser et al. 2003). Similarly TGF-β1 has been reported to induce a senescent state in bone marrow mesenchymal stem cells as a result of increased mitochondria ROS production (Wu et al. 2014). These cells also showed SA-β-Gal staining and an increased expression of p16. Yu et al. (2010) demonstrated that TGF-β2 could induce a senescent-like state in human trabecular meshwork cells. Again, this was associated with SA-β-Gal staining, increased levels of p16 at both the message and protein level and a reduction in the level of pRB protein. No impact on p21 mRNA or protein expression was observed in response to TGF-β2 exposure. Other groups have also reported a role for TGF-β signaling in inducing a senescent state (Senturk et al. 2010, Minagawa et al. 2011, Acosta et al. 2013). 

It is generally accepted that SA-β-Gal staining should be used in conjunction with several other senescent markers, as it does not appear to detect senescent cells specifically (Severino et al, 2000). However, other than the expression of CDKi, it appears that the phenotypes of cells induced to enter senescence by exposure to TGF-βs have been poorly characterized, especially in regard to immunogenic conversion. Some cell types that become senescent via this route may be cleared by the immune system in a manner analogous to those undergoing developmentally programmed senescence. Others may not and this area represents a fruitful field for further investigation.

Developmentally programmed senescence: Cells sharing features of senescence have been reported within the mesonephros and the endolymphatic sac of the inner ear in human and mouse embryos; as well as the neural roof plate and apical ectodermal ridge in rodents (Munoz-Espin et al. 2013, Storer et al. 2013). The authors hypothesize that this “developmental senescence” (DS) is a programmed part of normal embryonic development. DS was demonstrated experimentally by the presence of SA-β-Gal activity and senescence associated heterochromatin (Munoz-Espin et al. 2013). These cells seem to lack detectable DNA damage and appear to have become senescent independent of p53 and p16 and have gene expression patterns that significantly overlap with those of IMR90 fibroblasts in a state of oncogene-induced senescence. Arrest in this instance is dependent instead upon p21, regulated via the TGF-β/SMAD and PI3K/FOXO pathways (thus showing some affinity with other TGF-β induced senescent states). Interestingly, DS cells are removed during normal embryonic development by macrophages in a manner related to immune clearance of senescent cells in the mature organism (or by apoptosis should senescence fail) contributing to the formation of normal tissue architecture. Thus, the long-recognized distinction between programmed cell death in development and apoptosis in the mature organism appears to be mirrored in DS. Given that the expression of p21 in developing embryos is often attributed to ‘terminal differentiation’ (Vasey et al. 2011), it will be interesting to determine how many of these p21 positive cells are senescent cells and have undergone immunogenic conversion.

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion

Immune Ligand Expression in Senescent Cells

In addition to secreting soluble factors for the attraction of immune cells, senescent cells can also become immunogenic through the up-regulation of ligands that can specifically be recognized by immune cells.  While research into the recognition and interaction of immune cells with senescent cells is at its infancy, a number of studies have reported the up-regulation of the Natural Killer Group 2D (NKG2D) ligands in senescent cells that can be recognized by receptors on Natural Killer (NK) cells and CD8+ T-cells.  Since NKG2D ligands are not widely expressed on healthy cells, this would allow for specific recognition, interaction and elimination of senescent cells by immune cells.  As with the senescent secretome, this response is likely not exclusive to cell senescence as the same mechanism functions in immunosurveillance of tumour cells (López-Soto et al. 2014).  The human NKG2D ligands primarily consist of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6.  The transcriptional up-regulation of MICA and ULBP2 during cell senescence have been reported in senescent activated hepatic stellate cells, replicative senescent fibroblasts and HUVECs, etoposide-induced senescent fibroblasts, fusion-induced senescent fibroblasts and chemotherapy-induced senescent multiple myeloma cells (Krizhanovsky, et al. 2008, Kim et al. 2008 Chuprin et al. 2013, Soriani et al. 2014, Lackner et al, 2014).  In addition to MICA and ULBP2, microarray analysis of replicative senescent fibroblasts demonstrated an increase in the expression of ULBP1 (2.75 fold) compared to growing cells, in addition to the up-regulation of HLA-E (2 fold) (Lackner et al. 2014).  HLA-E is a non-classical MHC class I molecule that plays a role in cell recognition by NK cells. However, replicative senescent vascular smooth muscle cells do not appear to up-regulate MICA, ULBP2 or ULBP1, at least not greater than 2 fold as assessed by microarray analysis (Burton et al. 2009).  Therefore, it should not be assumed that all senescent cell types up regulate NKG2D ligands and this should be evaluated in underexplored senescent cell types. Mechanisms involved in the interaction of senescent cells with T-cells is less understood, but it appears that major histocompatibility complex class II (MHCII) expression is required for killing of pre-malignant senescent hepatocytes by T-cells (Kang et al. 2011).  Mice with liver specific MHCII deficiency resulted in impaired immunosurveillance of senescent cells.

At the mechanistic level, little is currently known about the regulation of NKG2D ligand expression in senescent cells.  Nonetheless, some extrapolation from others models is possible.  For example, MICA and MICB have been reported to be regulated by endogenous miRNAs in tumours and as a result of infection with cytomegalovirus (Stern-Ginossar et al. 2008).  Since miRNAs appear to play a role in regulating cellular senescence (Feliciano et al. 2011, Liu et al. 2012 Benhamad et al. 2012) and their expression is altered in response to DNA damage (Dolezalova et al. 2012, Wang and Taniguchi, 2013), it is possible that changes in miRNA expression also regulate the expression of immune ligands in senescent cells. 

Soriani et al demonstrated that the up-regulation of MICA in senescent multiple myeloma cells was dependent upon the DDR (Soriani et al. 2014).   In other systems, NKG2D ligands have also been shown to be up-regulated in response to DNA damage and Ras activation via ATM and ATR (Gasser et al. 2005, Cerboni et al. 2014).  Inhibition of the ATM or ATR pathways prevented the up-regulation of immune ligands. 

It is also possible that the up-regulation of immune ligands on senescent cells is mediated via the secretory response.  In addition to activating and attracting immune cells, the senescent secretome may serve to up-regulate immune ligands in an autocrine or paracrine manner.  It has been shown for example, that TNFα can up-regulate MICA on human endothelial cells and that the addition of exogenous MICA seems to induce senescence in HUVECs (Lin et al. 2011), but the extent to which this occurs under more physiologically reflective situations remains unclear. 

Immune ligands can also be up-regulated in response to various other forms of cell stress such as heat shock, metabolic stress and endoplasmic reticulum (ER) stress (Cerwenka, 2009, Valés-Gómez et al. 2008).  Thus, as with the secretory response, mechanisms exists that can up-regulate immune ligands independent of DNA damage.  Given that this is an important aspect of senescent cell clearance and the number of cell types in which the up-regulation of immune ligands has been shown is limited, a more detailed study of this aspect of immunogenic conversion seems warranted.

While senescent cells are likely eliminated by the immune system during normal physiological processes, it has been speculated that the accumulation of senescent cells with age could be due to inefficient elimination by an ageing immune system (Burton, 2009).  In fact, immune cells may themselves undergo cellular senescence, a process that requires further investigations (Effros et al. 2005, Rajagopalan et al. 2012). As such, induction of cell senescence in immune cells may represent one aspect of immunosenescence, the gradual deterioration of the immune system, which consequently leads to impaired immunosurveillance of non-immune senescent cells.  It can be speculated that impaired immunosurveillance may result from altered expression of surface receptors on immune cells that impair recognition and interaction with target senescent cells (and cancer cells).  In addition, it is possible that aged or senescent immune cells do not respond as efficiently to chemoattractants secreted by senescent cells.  In order to understand the mechanisms associated with age-related changes resulting in impaired immunosurveillance of senescent cells, we must first fully understand the normal processes governing immune clearance of senescent cells.  However, evaluating the hypothesis that aged or senescent immune cells display a reduced capacity to target senescent cells and the physiological impact of this decline can still be assessed.  If this were indeed found to be the case, the rejuvenation of an ageing immune system would represent an attractive approach for promoting health span.

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion

Cellular senescence: from growth arrest to immunogenic conversion

Abstract

Cellular senescence was first reported in human fibroblasts as a state of stable in vitro growth arrest following extended culture. Since that initial observation, a variety of other phenotypic characteristics have been shown to co-associate with irreversible cell cycle exit in senescent fibroblasts. These include (1) a pro-inflammatory secretory response, (2) the up-regulation of immune ligands, (3) altered responses to apoptotic stimuli and (4) promiscuous gene expression (stochastic activation of genes possibly as a result of chromatin remodeling). Many features associated with senescent fibroblasts appear to promote conversion to an immunogenic phenotype that facilitates self-elimination by the immune system. Pro-inflammatory cytokines can attract and activate immune cells, the presentation of membrane bound immune ligands allows for specific recognition and promiscuous gene expression may function to generate an array of tissue restricted proteins that could subsequently be processed into peptides for presentation via MHC molecules. However, the phenotypes of senescent cells from different tissues and species are often assumed to be broadly similar to those seen in senescent human fibroblasts, but the data show a more complex picture in which the growth arrest mechanism, tissue of origin and species can all radically modulate this basic pattern. Furthermore, well-established triggers of cell senescence are often associated with a DNA damage response (DDR), but this may not be a universal feature of senescent cells. As such, we discuss the role of DNA damage in regulating an immunogenic response in senescent cells, in addition to discussing less established “atypical” senescent states that may occur independent of DNA damage.

Link: Burton and Faragher (2015) Cellular senescence: from growth arrest to immunogenic conversion. AGE. DOI 10.1007/s11357-015-9764-2