cellular senescence blog: immune clearance

Showing posts with label immune clearance. Show all posts

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

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

Why induce Cellular Senescence Rather than Apoptosis?

When cells become senescent in vitro they often become resistant to apoptotic stimuli in comparison to proliferating cells. It can be speculated that if immune cells are necessary for eliminating senescent cells, the pro-survival phenotype of senescent cells may function to favor such elimination. In conjunction with regulating immune ligands and the secretory phenotype, persistent activation of the DDR, particularly double strand breaks (DSBs), may also promote a pro-survival response to facilitate DNA repair. However, if senescent cells are not removed by the immune system, this pro-survival phenotype inadvertently promotes their persistence in tissues. Alternatively, the pro- survival phenotype of senescent cells may be an adaptive response mediated by stresses within the microenvironment to facilitate protection from further stress.

The question still arises as to why senescent cells may favor removal by the immune system rather than undergoing programmed cell death. One plausible explanation could be related to the potential function of senescent cells during cellular repair following tissue damage. During wound healing, senescent cells most likely play a positive role by (1) secreting chemo-attractants that recruit and activate immune cells to the site of injury, (2) secrete growth factors to stimulate cellular proliferation required for cellular replacement and protein synthesis and (3) the secretion of proteases to debride damaged tissue. In addition, senescent cells may help to preserve tissue integrity during wound healing, that may otherwise be lost if cells underwent apoptosis, until such time that non- resident cells from other sources, such as stem cells are present to repopulate the tissue with functional cells. In an orchestrated response, senescent cells would be subsequently eliminated by the immune system when no longer required.

Link: Physiological and Pathological Consequences of Cellular Senescence

Immune surveillance of senescent cells

Taken from: Physiological and Pathological Consequences of Cellular Senescence

The ability of senescent cells to trigger an innate immune response via the up-regulation of pro-inflammatory cytokines was first suggested to play a role in limiting tumourigenesis. This immune response was later shown to be important in the elimination of senescent stellate cells during liver damage. In natural killer (NK) cell mediated cytotoxicity, NK cells identify senescent cells by the presence of NKG2D ligands on the membrane of senescent cells. The presentation of these ligands on senescent cells might be mediated by a DDR, which was previously shown to induce their expression. In particular, it appears that the ATM-ATR pathway is important for the up-regulation of NKG2D ligands in response to stress. NK cell induced cytotoxicity of senescent cells is mediated by granule exocytosis and perforin-mediated death rather than death-receptor-induced apoptosis. The perforin mediated cytotoxicity decreases in humans with age, and might therefore contribute to accumulation of senescent cells in the organism during ageing and in age-related diseases. As discussed, senescent cells are known to accumulate with age and in disease states, suggesting that senescent cells may be evading immune surveillance or their rate of accumulation is greater than the rate of removal or both. It has been advocated that the accumulation of senescent cells with age might be the consequence of an impaired ageing immune system. In fact, immune cells can also become senescent and these changes may contribute to impaired elimination of senescent cells. Therefore, strategies to restore an ageing immune system are a compelling approach for the elimination of senescent cells and for promoting an increased health-span.

A recent study has shown that senescent HSCs can be eliminated by another component of the innate immune system, the M1-like macrophages during liver damage and tumorigenesis in the liver. Secretory factors from senescent HSCs were shown to aid the elimination of these cells by macrophages. In contrast, cells that could not become senescent due to deletion of p53 and were not targeted by macrophages. Therefore, the innate immune system appears to be an initial early barrier that regulates the presence of senescent cells in physiological conditions such as in wound healing.

The elimination of senescent cells by the adaptive immune system has also been demonstrated. OIS hepatocytes were shown to secrete cytokines to evoke an immune response leading to the elimination of senescent cells by CD4(+) T-cells, a process which required the action of macrophages. The elimination of senescent hepatocytes was required to prevent the development of liver cancer. This study mentions the attraction of T-cells by soluble factors but not the mechanism of senescent cell recognition, an area of research that still needs to be explored. However, there is some indication that RS cells may up-regulate MHC1 expression, possibly via p53. It can be speculated that MHC1 proteins in senescent cells may function to display senescence-associated antigens similar to cancer cells, allowing recognition and elimination by cytotoxic T-cells. Further research will provide multiple insights into the mechanisms and consequences of the interaction of senescent cells with the immune system.

Detecting Senescent Cells: Biomarkers

The standard SA-beta-gal staining, while indicative of the presence of senescent cells, is not an absolute marker for senescent cell and indicates increased lysosmal b-galactosidase activity. The use of several molecular markers that represent different characteristics of senescent cells is necessary (see figure). Such molecular markers can represent the cell cycle arrest machinery (e.g. p53, p21, p16), lack of cellular proliferation (e.g. lack of BrdU incorporation, Ki67), activation of the DDR (e.g. gamamH2AX or p53BP1 foci), expression of secretory factors (e.g. IL-6 and IL-8), the activation of the pathways that regulate the secretory phenotype (e.g. p-p65 or p-p38), the activation of immune surveillance-related genes and possible regulators for their pro-survival response (DCR2, p-Akt, p-Erk).

Link: Physiological and pathological consequences of cellular senescence

Physiological and pathological consequences of cellular senescence

Abstract

Cellular senescence, a permanent state of cell cycle arrest accompanied by a complex phenotype, is an essential mechanism that limits tumorigenesis and tissue damage. In physiological conditions, senescent cells can be removed by the immune system, facilitating tumor suppression and wound healing. However, as we age, senescent cells accumulate in tissues, either because an aging immune system fails to remove them, the rate of senescent cell formation is elevated, or both. If senescent cells persist in tissues, they have the potential to paradoxically promote pathological conditions. Cellular senescence is associated with an enhanced pro-survival phenotype, which most likely promotes persistence of senescent cells in vivo. This phenotype may have evolved to favor facilitation of a short-term wound healing, followed by the elimination of senescent cells by the immune system. In this review, we provide a perspective on the triggers, mechanisms and physiological as well as pathological consequences of senescent cells.

LINK: Burton and Krizhanovsky (2014) Physiological and pathological consequences of cellular senescence

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development

ABSTRACT

Tae-Won Kang, Tetyana Yevsa, Norman Woller, Lisa Hoenicke, Torsten Wuestefeld, Daniel Dauch, Anja Hohmeyer, Marcus Gereke, Ramona Rudalska, Anna Potapova, Marcus Iken, Mihael Vucur, Siegfried Weiss, Mathias Heikenwalder, Sadaf Khan, Jesus Gil, Dunja Bruder, Michael Manns, Peter Schirmacher, Frank Tacke, Michael Ott, Tom Luedde, Thomas Longerich, Stefan Kubicka & Lars Zender

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo1. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects2, 3, 4, 5. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Link: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10599.html

immune clearance of senescent and cancer cells

The age-associated increase in the incidence of disease development and cancer occurrence is often thought to be due to the gradual accumulation of damage over the lifetime of an organism. However, an alternative opinion is that damaged cells are effectively eliminated and replaced by the immune system and regenerative cells (stem cells) and only when this “remove and replace” system failures, do organisms begin to show signs of ageing.

The presence of persistent DNA damage triggers cells to enter senescence (irreversible growth arrest) to protect the cell from becoming cancerous. The presence of the persistent DNA damage in these growth-arrested cells appears to activate pathways leading to cytokine/chemokine secretion and presentation of cell surface ligands (i.e MICA, MICB, ULBP2) which can be recognized by natural killer cells (NK) and some T-cells. This may allow damaged/senescent cells to communicate with immune cells for their removal (although more evidence of this is required).

For cells to become cancerous, they need to bypass senescence (following irreparable DNA damage), often achieved by acquiring mutations in genes associated with activation and maintenance of the senescence growth arrest. When such cells bypass senescence, the persistence of DNA damage may also activate pathways leading to cytokine/chemokine secretion and presentation of NK ligands.

It is also possible that cells can become cancerous if they instead escape senescence. “Escaping” is different from “bypassing” in that these cells were once senescent. If senescent cells persist in tissues without immune clearance, it is possible that stochastic genetic/epigenetic changes may lead to activation/inactivation of genes that allow the once senescent cell to reneter the cell cycle. A consequence of this escape may be the maintenance of the pro-survival phenotype and the pro-inflammatory phenotype associated with senescence. Escaping senescence may be more pertinent in cancer cells that have become senescent in response to therapy. Escape from senescence in this instance may lead to the progression of more aggressive cancers.

I am not aware of any studies that have investigated the similarities/differences in the secretory phenotype/NK ligand activation of senescent verses cancer cells. However, if both exist due the DNA damage response activating the immune response (DDR-AIR), then they are probably very similar.

If senescent and cancer cells were always effectively being removed then the incidence of cancer and disease would greatly be reduced. However, age-associated cancer and disease does occur and this may in part be due to a failure in the immune system to effectively remove senescent/cancer cells as we age. Additionally, cancer cells can develop various strategies for evading the immune response (i.e secretion of immunosuppressive cytokines). Whether the same strategies occur in senescent cells remains to be discovered.

Although purely speculative, it is possible that some of these strategies for evading immune surveillance is a result of pro-longed exposure to the pro-inflammatory phenotype of these cells. There may be a limited biological time frame whereby the presence of the inflammatory phenotype is beneficial for cell removal. Longer exposure may lead to an adaptive response through autocrine signalling leading to changes that evade immune surveillance. For example, the secretion of immunosuppressive cytokines may be an adaptive response for preventing detrimental damage from long exposure to pro-inflammatory cytokines.

Ongoing and future investigations should aim to provide solid evidence of whether (1) the secretory phenotype of senescent cells is for the purpose of immune clearance, (2) and if so, does immune clearance fail or become impaired with age and (3) if it does fail, what are the mechanisms?