Macrophage long-term cultures as a cellular model of senescence to study proteostasis in aging immune cells

The cellular theory of aging states that human aging is the result of cellular aging, whereby an increasing proportion of cells reach senescence. During aging humans are more susceptible to infections and to develop pathologies. This could be due to immunosenescence, a condition of decline of the immune system which becomes unable to defend our body in a performing way. Mechanisms of cellular senescence in immune cells have been poorly investigated. We have developed an in vitro model which displays features of senescent cells to study macrophage senescence. This model involves long-term culturing of monocyte-derived human macrophages obtained from buffy coats. After extended culture, macrophages expressed multiple senescent markers: i.e., irreversible cell cycle arrest with increased p21 and p16 levels, lipofuscin accumulation and enlarged flattened shape. They also released pro-inflammatory mediators, as demonstrated by activation of proinflammatory signalling cascades such as NF-kB and p38 MAPK in monocytic THP-1 cells exposed to conditioned medium deriving from senescent cells. In this model we studied whether components of the proteostasis network may change during senescence. Indeed, mounting evidence indicates that the reduced ability to maintain proteostasis is intrinsic to human cell senescence. The Ub/proteasome and the autophagy/lysosomal systems are the main regulator of protein degradation. During macrophage senescence, interconversion between immunoproteasome, typically expressed in immune cells, and constitutive proteasome catalytic subunits was observed by both western immunoblotting and native gel analyses. Moreover, activation of the autophagic pathway occurred as determined by increased LC3II/LC3I and decreased p62 levels. The observed changes may contribute to age-related impairments in macrophage function. Further experiments will be directed to characterize the proteasome and autophagosome systems and their role in macrophage senescence.