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.
