Activated Lymphocytes Secretome Inhibits Differentiation and Induces Proliferation of C2C12 Myoblasts

Abstract

Background/Aims: ageing is associated with a marked decline in immune function which may contribute to the local environment that can influence the regenerative process of skeletal muscle cells. Methods: Herein, we focused on determining the effect of an activated immune system secretome on myoblast differentiation and proliferation as possible means to attenuate adverse effects of muscle aging. C2C12 myoblasts were used as model to assess the impact of lymphocyte conditioned media (CM) following anti-CD3/IL-2 activation. Results: Myoblasts cultured with activated lymphocytes CM exhibited reduced morphological and biochemical differentiation (98±20, p<0.005) and increased entry to the S Phase of the cell cycle (61%±7, p<0.001), when compared with myoblasts cultured with non-activated lymphocytes CM. Associated with increased proliferation and reduced differentiation, muscle specific transcription factors MyoD and myogenin were significantly reduced in C2C12 treated with activated lymphocytes CM vs control CM, respectively (myoD: 0.5±0.12 fold reduction P<0.005); myogenin: 0.38±0.08 fold reduction; p<0.005). Moreover, key protein of proliferation pERK1/2 increased (46±11U/ml, p<0.05) whereas mediator of differentiation pAkt decreased (21±12U/ml, p<0.05) in C2C12 treated with activated vs. non-activated CM. Conclusion: our data demonstrate that, following activation, secretome of the immune system cells elicit marked regulatory effects on skeletal muscle growth and differentiation; enhancing the former with the loss of the latter.

Plasma from exercised rats administered to sedentary rats induces systemic and tissue inflammation

“…, ; Al‐Shanti et al. ; Stanford et al. ), no study has investigated the possible contribution of circulating factors in an exercise setting.…”

The lymphocyte secretome from young adults enhances skeletal muscle proliferation and migration, but effects are attenuated in the secretome of older adults

“…Cells were cultured at 37°C, 5% CO 2 for 4 days in RPMI‐1640 containing 10% (v/v) AB human serum, 1% Penicillin/Streptomycin, and 50 U/mL human recombinant interleukin‐2 (rhIL‐2, R & D System, Abingdon, UK) in the absence or presence of 5 μ g/mL anti‐CD28 (BD Pharmingen ™ , San Diego, CA) (Al‐Shanti et al. ).…”

“…Three control conditions were also prepared: young and old CM2, containing secretomes obtained from young and old nonactivated lymphocytes (negative control); young and old CM3, containing the secretome from young and old activated lymphocytes, but the secretomes were boiled prior to preparing the conditioned media; CM4, containing the RPMI‐1640 with IL‐2 and anti‐CD3, but without the lymphocytes (Al‐Shanti et al. ).…”

“…Recently, we reported that the proteins secreted by activated lymphocytes, collected from young adults, elicit marked regulatory effects on skeletal muscle proliferation and differentiation; enhancing the former and postponing the latter (Al‐Shanti et al. ). Thus, we hypothesized that a conditioned media prepared from lymphocytes isolated from young adults would improve skeletal muscle satellite cell proliferation and migration, while effects of lymphocytes from older adults would be attenuated.…”

From Innate to Adaptive Immune Response in Muscular Dystrophies and Skeletal Muscle Regeneration: The Role of Lymphocytes

“…Persistence of T cells in dystrophic muscle may actually modulate inflammatory milieu and immune cells activity but may also directly interfere with muscle cell function through lymphocyte-released cytokines and chemokines [63, 64]. Indeed, it was recently shown that conditioned medium from activated T-cells, previously cultured in anti-CD3 coated dishes in the presence of IL2 [65], induced proliferation of C2C12 muscle cell, a mouse derived muscle cell line widely used as a model of myogenesis in vitro , and prevents myogenic differentiation, as compared to conditioned medium derived from resting T cells [66]. …”