The effect of lymphocytes secretome on skeletal muscle stem cells regeneration with ageing

Older people experience skeletal muscle wasting, in part due to impaired proliferative capacity of quiescent skeletal muscle satellite cells and. The work presented in this thesis set out to examine the hypothesis that microenvironment of skeletal muscles can be influenced by immune cell secretions, which affect satellite cell proliferation, and that beneficial immune-muscle interactions in young people are blunted in elderly. The aim of this research was to investigate the role of ageing human lymphocytes on skeletal muscle cell behaviour. For this purpose, lymphocytes were isolated from whole blood of young (aged 18-25 years) and older (aged 78-85 years) healthy volunteers and older healthy volunteers. All the participants were healthy, with no history of muscle disease and not on immunosuppressant or corticosteroids treatment that affect immune function. Lymphocytes were cultured with, or without, anti-CD3/CD28 activators for 4 days to induce release of cytokines, interleukins and growth factors into the media. The secreted proteins were used to prepare conditioned media that were used to culture C2C12 myoblasts. Secretomes were analysed and fifteen secreted Th1/Th2 cytokines and IGF-I were quantified by multiplex immunoassay. The gene expression and protein concentrations of amphiregulin were determined from T-lymphocytes lysates by real-time PCR and ELISA respectively. The levels of CD25 and FoxP3 expression in lymphocytes were examined using flow cytometry. The expression of muscle transcription factors, MyoD and Myogenin were determined by real- time PCR. Activated Mek1/Erk1/2 and Akt/mTOR(definition) were measured by multiplex immunoassay. Our results demonstrate for the first time that a decrease in the levels of amphiregulin and CD25 coincides with the increase in FoxP3 with ageing, which may be involved in suppression of lymphocytes. Seven cytokines were differentially secreted by the young- compared with the old-activated lymphocytes. The secretome from young-activated lymphocytes had 30% (P<0.005) higher IGF-I concentrations compared with old and control treatments. The conditioned media from young -activated lymphocytes increased the rate of proliferation of myoblasts by ~3-fold (P<0.005) and caused an approximate 4-fold (P<0.005) increase in migration compared with non-activated lymphocyte control media. These responses were characterised the extended proliferation of young -treated myoblasts was also associated with a decrease in MyoD and Myogenin and an increase in mediators of proliferation Mek1/Erk1/2and a decrease in the key proteins for differentiation, Akt/mTOR. In contrast, myoblasts treated with conditioned media from old-activated lymphocytes exhibited a high degree of differentiation.