Persistent Stimulation of Human Mesenchymal Stem/Stromal Cells with TNF-α and IFN-γ Affects the Release of Large Extracellular Vesicles with Immunoregulatory Phenotype.

Abstract

Mesenchymal stem/stromal cells (MSCs) possess immunoregulatory capacity, which is enhanced in an inflammatory environment. Participation of extracellular vesicles (EVs) in this function is proposed, as they can transport various immunoregulatory molecules. However, the impact of the inflammatory microenvironment on the load of the different types of EVs released by these cells is not fully known. Therefore, this work analyzes in detail the temporal effect of IFN-γ, alone or in combination with TNF-α (TNF-α + IFN-γ), on the cargo of immunoregulatory molecules (programmed cell death ligand 1 [PD-L1], CD73, and intercellular adhesion molecule 1 [ICAM-1]) in large extracellular vesicles (L-EVs) released by human bone marrow mesenchymal stem cells (BM-MSCs). The presence of these molecules on the surface of L-EVs was determined by flow cytometry. Our results demonstrate that exposing BM-MSCs to TNF-α + IFN-γ for 24 h increases the percentage of PD-L1+ and CD73+ L-EVs. However, if this stimulus persists, the release of L-EVs with an immunoregulatory phenotype (PD-L1+, CD73+, and PD-L1+CD73+) decreases. The impact of pro-inflammatory cytokines on the transport of ICAM-1 by L-EVs is late, since up to 72 h of treatment with IFN-γ or TNF-α + IFN-γ, the percentage of ICAM-1+ L-EVs increases. In contrast, stimulation with IFN-γ for 72 h favors the release of CD73^high and ICAM-1^high L-EVs, but this effect also decreases in the presence of TNF-α. Our study generates novel knowledge about the impact of the inflammatory microenvironment on the cargo composition of L-EVs released by BM-MSCs and demonstrates, for the first time, that the prolonged presence of TNF-α reduces the cargo of immunoregulatory molecules in these structures.