Sarcopenia is a geriatric syndrome involving progressive and generalized loss of skeletal muscle mass and strength. Aged muscles are characterized by an altered mitochondrial function, increased protein degradation, inflammation, a reduction of satellite cell (SC) number and function. It has been suggested that an altered secretory pattern involving inflammatory, growth-promoting, and remodelling factors, called senescence-associated secretory phenotype (SASP), could induce sarcopenia. Noteworthy, growing evidence highlighted the role of extracellular vesicles (EVs) as key players in the senescent cell secretome of sarcopenic muscles. This study aimed to investigate the role of senescent-like myocytes derived EVs in the fine tuning of the muscle niche. In particular, how the senescent-like myocytes EVs could affect the behaviour of myoblasts and macrophages. We focused our attention on two models of in vitro senescent-like C2C12 cells: long term cultured C2C12 (more than 40 passages, HP), or exposed to oxidative stress (OX). EVs have been purified by serial ultracentrifugation and density gradient, quantified through Nanoparticles Tracking Assay (NTA), characterized by Western Blot (WB), and administered to recipient cells. Expression analyses were performed using RT-qPCR and WB. HP and OX myocytes displayed senescence-associated characteristics such as defective myogenic differentiation process and a significantly inflammatory status compared to non-senescent C2C12 cells (cultured in standard condition at low passages, LP). The NTA analysis showed that HP myocytes released a higher amount of EVs compared to LP cells. Importantly, EVs cargo of HP and OX cells was enriched of nucleic acids, especially DNA, compared to LP. Senescent-like EVs induced a higher level of IL-6 in LP C2C12, and IL-1β and IFN-1β in RAW264.7 without affecting the DNA sensor protein STING. Interestingly, in RAW264.7 the induction of IFN-1β was significantly reduced when RAW264.7 cells were pre-exposed to chloroquine, an inhibitor of endosomal toll-like receptors (TLRs) activity. These results suggested the involvement of TLRs pathway. Therefore, the data herein reported indicated that EVs released by senescent-like myocytes are capable of induce a pro-inflammatory response in the muscle niche. This led us to hypothesize a functional relationship with the derangement of the myogenic process found in HP differentiating myocytes. Importantly, DNA associated with EVs seems to play an important role in the triggering of the inflammation process as suggested by IFN-1β expression, a mechanism that is likely to take place in endosomal compartments by stimulating TLRs.

Pro-inflammatory effect of extracellular vesicles released by senescent-like C2C12

Cioccoloni, Andrea
2021

Abstract

Sarcopenia is a geriatric syndrome involving progressive and generalized loss of skeletal muscle mass and strength. Aged muscles are characterized by an altered mitochondrial function, increased protein degradation, inflammation, a reduction of satellite cell (SC) number and function. It has been suggested that an altered secretory pattern involving inflammatory, growth-promoting, and remodelling factors, called senescence-associated secretory phenotype (SASP), could induce sarcopenia. Noteworthy, growing evidence highlighted the role of extracellular vesicles (EVs) as key players in the senescent cell secretome of sarcopenic muscles. This study aimed to investigate the role of senescent-like myocytes derived EVs in the fine tuning of the muscle niche. In particular, how the senescent-like myocytes EVs could affect the behaviour of myoblasts and macrophages. We focused our attention on two models of in vitro senescent-like C2C12 cells: long term cultured C2C12 (more than 40 passages, HP), or exposed to oxidative stress (OX). EVs have been purified by serial ultracentrifugation and density gradient, quantified through Nanoparticles Tracking Assay (NTA), characterized by Western Blot (WB), and administered to recipient cells. Expression analyses were performed using RT-qPCR and WB. HP and OX myocytes displayed senescence-associated characteristics such as defective myogenic differentiation process and a significantly inflammatory status compared to non-senescent C2C12 cells (cultured in standard condition at low passages, LP). The NTA analysis showed that HP myocytes released a higher amount of EVs compared to LP cells. Importantly, EVs cargo of HP and OX cells was enriched of nucleic acids, especially DNA, compared to LP. Senescent-like EVs induced a higher level of IL-6 in LP C2C12, and IL-1β and IFN-1β in RAW264.7 without affecting the DNA sensor protein STING. Interestingly, in RAW264.7 the induction of IFN-1β was significantly reduced when RAW264.7 cells were pre-exposed to chloroquine, an inhibitor of endosomal toll-like receptors (TLRs) activity. These results suggested the involvement of TLRs pathway. Therefore, the data herein reported indicated that EVs released by senescent-like myocytes are capable of induce a pro-inflammatory response in the muscle niche. This led us to hypothesize a functional relationship with the derangement of the myogenic process found in HP differentiating myocytes. Importantly, DNA associated with EVs seems to play an important role in the triggering of the inflammation process as suggested by IFN-1β expression, a mechanism that is likely to take place in endosomal compartments by stimulating TLRs.
2021
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2688889
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