Unveiling the skeletal muscle secretome: characterization and functional analysis of cytokines and extracellular vesicles during muscle regeneration

The adult skeletal muscle is daily subjected to physical and mechanical stress, leading to tissue micro- and macro- injuries. In order to repair the damage, muscles activate a finely orchestrated regeneration process. During this regenerative process, immune cells, in coordination with mononuclear resident cells, actuate an inflammatory response that contributes to remove apoptotic debris and to repair the tissue. Although the adult skeletal muscle has a high regeneration potential, during the life span of an individual, in some pathological conditions, muscle regeneration fails, compromising tissue functionality. This is often caused by aberrant cell-cell communication that results in the deposition of fibrotic and adipose infiltrates. In my work I investigated in vivo the changes in the profile of the muscle secretome during tissue regeneration aiming at providing system-level information about cell cross-talks in order to find new targetable regulatory circuits to prevent tissue degeneration in pathological conditions. To this end, I analysed the expression patterns of 77 cytokines modulated during the regeneration process. Next, I focused on the third day of muscle regeneration, performing an RNAseq experiment on the four main muscle mononuclear cell populations: immune cells, endothelial cells, satellite cells (SCs) and fibroadipogenic progenitors (FAPs). This approach allowed me to annotate each cell-type with the cytokines and receptors they have the potential to synthetize. I used these expression data to build a cell-cell interaction network by connecting cells that synthetize the cytokines with those that synthetize the corresponding receptors. I next selected 12 cytokines whose receptors were exposed on FAP cells and tested their ability to modulate FAP adipogenic potential. I found that IL1α and IL1β were able to significantly and potently inhibit FAPs adipogenesis, while EGF and BTC promoted FAPs proliferation. In addition to the analysis of the cytokine expression profile, I also characterized the extracellular vesicles (EVs) secreted during the muscle regeneration process following acute damage. To this end I performed ex vivo cultures of both uninjured and injured muscles. For vesicle characterization I also developed a flow-cytometry method to perform single-vesicles analysis of my samples. I studied the EVs heterogeneity by analysing the modulation of their content during muscle regeneration. I observed that EVs differentially modulate the uptake of RNA and proteins into their lumen. I also investigated the EVs capability to interact with the main muscle-resident cell populations, by monitoring whether and how the EVs content could be released into the recipient cells. Moreover, I studied how EVs would perturb proliferation and differentiation trajectories of SCs and FAPs. I concluded that both cytokines and EVs secreted during muscle regeneration can inhibit the in vitro adipogenic differentiation of FAPs. Moreover, I found that EVs stimulate both proliferation and differentiation of SCs. Overall, this thesis provides a first system-level characterization of molecules secreted in vivo during muscle regeneration. Lastly, my in silico analysis offers an inferred framework of potential cell-cell interactions that need to be further explored experimentally and whose functional impact on muscle regeneration in vivo needs to be validated.