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The interaction and clearance of senescent vascular cells by the innate immune system

Periodic Reporting for period 1 - ICSIS (The interaction and clearance of senescent vascular cells by the innate immune system)

Reporting period: 2015-06-01 to 2017-05-31

Very little is known about the mechanisms by which senescent cells interact with innate immune cells. Understanding how these processes work and how they may fail will be valuable for developing therapeutic interventions for boosting immune clearance of senescent cells. Such treatments could be used to treat or prevent a variety of different diseases. In doing so, such research will lead to improvements in healthcare interventions which will enable everyone to live healthier, longer lives.

The overall objective of this study was to evaluate the impact of an early (acute) versus a late (chronic) senescent cell secretome on monocyte/macrophage function. It was suspected that if senescent cells were not killed by immune cells and persisted, their secretory phenotype would become altered (chronic) which could promote disease. Specific objectives included the assessment of (1) immunogenic factors on senescent cells, (2) the senescent secretome on markers of monocyte/macrophage activation/differentiation, (3) the impact of the senescent secretome on physiological responses by monocytes/macrophages including migration, adhesion and phagocytosis, and (4) the impact of a chronic senescent secretome on pathological responses by monocytes/macrophages.

This research demonstrated that an acute senescent secretome promotes the differentiation of monocytes into anti-inflammatory M2-like macrophages associated with increased macrophage migration, adhesion and phagocytosis. However, a chronic senescent secretome promoted pathological features in monocytes/macrophages that has the potential to promote disease development.
RT-PCR and ELISA was initially undertaken to assess the expression of immunogenic factors in senescent cells (fibroblasts), with results showing a general increase in chemokine/cytokine signalling. Next we assessed the impact of an acute senescent secretome on the ability of monocytes to differentiate and polarise into macrophage subsets. We assessed various markers that are normally associated with macrophage differentiation by RT-PCR and flow cytometry. We found that factors secreted by senescent cells do indeed promote the differentiation of monocytes into macrophages. The inflammatory profile of these macrophages was also evaluated by RT-PCR and ELISA. Inflammatory molecule expression was lower in macrophages exposed to an acute senescent secretome, suggesting they develop an anti-inflammatory response important for tissue repair. Next we assessed the physiological responses accompanied by macrophage differentiation and found that migration, adhesion and phagocytosis were all increased. Co-culture experiments of monocytes and senescent cells showed that direct killing of senescent cells did not occur under these conditions.

Following on from these findings which focused specifically on an acute senescent secretome, we evaluated the response of monocytes/macrophages to a chronic senescent secretome. Reduced cell migration was observed and associated with a reduction in cell surface receptor expression (CXCR1 and CXCR2) in monocytes/macrophages. These receptors are important for sensing certain secreted compounds. No significant difference in monocyte/macrophage adhesion or phagocytosis in response to a chronic senescent secretome was observed when compared to an acute senescent secretome.

We also observed a further elevation in the expression of CD36, a lipid transporter involved in pathological foam cell formation. Foam cells are macrophages loaded with fats such as oxidised low-density lipoprotein (ox-LDL). Such cells are known to play a role in the development of atherosclerosis-thickening of the arteries. Our data suggests that senescent cells may promote elevated extracellular ox-LDL which leads to increased CD36 expression and ox-LDL uptake in monocytes/macrophages.

Monocytes treated with a chronic senescent secretome also reduced T-cell proliferation. L-arginine is required for T-cell proliferation. However, a chronic senescent secretome promotes the upregulation of arginase 1 expression, an enzyme that breaks down L-arginine. This suggests that a reduction in T-cell proliferation may be due to lack of L-arginine. This immunosuppressive response is also linked to an elevation in TGFβ expression and upregulation of PPARγ in monocytes/macrophages.

Overall, these findings demonstrate that the senescent secretome regulates monocyte/macrophage function and that a chronic senescent secretome can promote a pathological, immunosuppressive environment. These findings clearly demonstrate that the senescent secretome can change with time, becoming detrimental. By further understanding the specific changes and the mechanisms involved, future therapeutic interventions could be targeted to reverse this process. In doing so, it may be possible to improve immune clearance of senescent cells with age. This data will be used to apply for further funding to investigate just that. In addition, a manuscript is currently in progress for submission to a scientific journal. Following publication, these findings will be made available in an easy to read format suitable for the public in the form of a blog (cellsen.wixsite.com/senescentcell). It is anticipated that this data will also be presented at conferences such as the annual International Cell Senescence Association (www.cellsenescence.info) meetings.
Scientific Impact: This project demonstrated that the impact of the senescent secretome can become altered with time, leading to detrimental changes that can potentially promote disease development. Research to date that has focused on the senescent secretome tend not to take into consideration the time-frame over which cells are senescent. Thus, it is anticipated that these findings will influence other researchers to take into consideration the possibility of different effects of an acute and chronic senescent secretome in their models. In doing so, a clearer understanding of the role of cell senescence in physiological and pathological contexts will become more evident. This will inevitably accelerate scientific progress focused on therapeutic interventions aimed at improving health through the targeting of senescent cells.

Fellowship Impact: Previous to this research fellowship, the Fellows primary scientific experience was concerned with molecular cell biology, with few skills involving immune biology. Through the undertaking of this project, the Fellow received training in the field of immunology that places him in a unique position to undertake further study in an emerging field of research focused on immunosurviellance of senescent cells. Specific scientific training, acquired data, student mentoring, lab management, conference attendance, networking, scientific writing, grant reviewing and grant writing undertaken during this fellowship places the Fellow in a strong position for the successful application of further funding and appointment as a principle investigator. A scientific manuscript is currently in preparation.
biological outcome of acute and chronic senescent cells