Study on the effects of the adipose tissue-derived mesenchymal stem cell secretome on the innate inflammatory response

Resumen

Adipose tissue-derived mesenchymal stem cells (MSC) have been shown to exert beneficial effects on some inflammatory conditions, which have been mainly related to the secretion of paracrine factors. Therefore, MSC have been investigated as potential therapeutic tools for the treatment of inflammatory and autoimmune diseases. The MSC secretome contains soluble factors and vesicular components known as extracellular vesicles (EV) which play an important role in different physiological processes. In this work, we have investigated the potential of mouse MSC conditioned medium (CM) to modulate the innate inflammatory response as well as the contribution of its components. Our study has analyzed the effects of MSC and their CM on the in vivo innate inflammatory response induced by zymosan in the mouse air pouch model. These results indicate that MSC control the early response and this effect is mainly mediated by the secretome (CM) of these cells. MSC and CM significantly decreased cell migration, degranulation and the production of relevant inflammatory mediators. The effect on cell migration was related to the downregulation of chemoattractants such as LTB4, MCP-1 and KC. In addition, MSC and CM significantly reduced the levels of pro-inflammatory mediators IL-1β, IL-6 and TNF-α at the early stage of the inflammatory response, which can be the consequence of the inhibition of nuclear factor-κB activation. MSC and CM also decreased the production of PGE2, which may be dependent on the reduction in mPGES-1 expression. We have studied and characterised the composition of the CM from MSC which contains soluble factors and EV (exosomes and microvesicles). Proteomic analysis indicated that apyrase is the most abundant protein in CM followed by phosphate-binding protein PstS and phosphate-import protein PhnD. In addition, the presence of chemokines such as KC, MCP-1 and MCP-5 was detected by a chemokine array. Macrophages form the first line of defence as a component of the innate immune response. They are also the link between the innate and the adaptive response. The regulation of macrophage activation by lipopolysaccharide (LPS) is critical for controlling the initial phases of the immune response. Therefore, we have investigated the in vitro effects of CM and its components (soluble fraction and EV) in mouse peritoneal macrophages stimulated with LPS. CM regulates phagocytosis, cell migration and production of relevant inflammatory mediators. CM and its EV-free fraction significantly reduced the production of IL-1β, TNF-α and NO in macrophages whereas the EV fraction was effective to a lower extent. We have also shown that apyrase may partly contribute to the anti-inflammatory effects of the soluble fraction while chemokines such as KC, MCP-1 and MCP-5 may be involved in the migration of neutrophils and monocytes observed in co-culture experiments with macrophages. Our data indicate that the soluble fraction plays a more important role than EV in mediating the anti-inflammatory effects of CM in mouse peritoneal macrophages. Activation of macrophages by LPS involves the TLR4 which drives pro-inflammatory signalling. We have shown that the anti-inflammatory activity of CM in this in vitro model may be related, at least in part, to the downregulation of TLR4 expression leading to the reduction of TLR4 signalling.