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Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4+ T Lymphocytes Toward a Regulatory Profile.

ABSTRACT: Mesenchymal stromal cells (MSCs) can generate immunological tolerance due to their regulatory activity in many immune cells. Extracellular vesicles (EVs) release is a pivotal mechanism by which MSCs exert their actions. In this study, we evaluate whether mesenchymal stromal cell extracellular vesicles (MSC-EVs) can modulate T cell response. MSCs were expanded and EVs were obtained by differential ultracentrifugation of the supernatant. The incorporation of MSC-EVs by T cells was detected by confocal microscopy. Expression of surface markers was detected by flow cytometry or CytoFLEX and cytokines were detected by RT-PCR, FACS and confocal microscopy and a miRNA PCR array was performed. We demonstrated that MSC-EVs were incorporated by lymphocytes in vitro and decreased T cell proliferation and Th1 differentiation. Interestingly, in Th1 polarization, MSC-EVs increased Foxp3 expression and generated a subpopulation of IFN-?+/Foxp3+T cells with suppressive capacity. A differential expression profile of miRNAs in MSC-EVs-treated Th1 cells was seen, and also a modulation of one of their target genes, TGFbR2. MSC-EVs altered the metabolism of Th1-differentiated T cells, suggesting the involvement of the TGF-? pathway in this metabolic modulation. The addition of MSC-EVs in vivo, in an OVA immunization model, generated cells Foxp3+. Thus, our findings suggest that MSC-EVs are able to specifically modulate activated T cells at an alternative regulatory profile by miRNAs and metabolism shifting.

SUBMITTER: Franco da Cunha F

PROVIDER: S-EPMC7226573 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4<sup>+</sup> T Lymphocytes Toward a Regulatory Profile.

Franco da Cunha Flavia F Andrade-Oliveira Vinicius V Candido de Almeida Danilo D Borges da Silva Tamiris T Naffah de Souza Breda Cristiane C Costa Cruz Mario M Faquim-Mauro Eliana L EL Antonio Cenedeze Marcos M Ioshie Hiyane Meire M Pacheco-Silva Alvaro A Aparecida Cavinato Regiane R Torrecilhas Ana Claudia AC Olsen Saraiva Câmara Niels N

Cells 20200423 4

Mesenchymal stromal cells (MSCs) can generate immunological tolerance due to their regulatory activity in many immune cells. Extracellular vesicles (EVs) release is a pivotal mechanism by which MSCs exert their actions. In this study, we evaluate whether mesenchymal stromal cell extracellular vesicles (MSC-EVs) can modulate T cell response. MSCs were expanded and EVs were obtained by differential ultracentrifugation of the supernatant. The incorporation of MSC-EVs by T cells was detected by conf ...[more]

PMID: 32340348

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Project description:Cartilage and other skeletal soft tissues heal poorly after injury, in part due to their lack of vascularity and low metabolic rate. No pharmacologic approaches have proven effective in preventing chronic degenerative disease after joint injury. Mesenchymal stromal cells (MSCs) have been investigated for their ability to treat pain associated with osteoarthritis (OA) and preserve articular cartilage. Limitations of MSCs include variability in cell phenotype, low engraftment and retention rates, and inconsistent clinical outcomes. Therefore, acellular biologic therapies such as extracellular vesicles (EVs) are currently being investigated. MSC-derived EVs have been found to replicate many of the therapeutic effects of their cells of origin, but the mechanisms driving this remain unclear. Recent evidence in non-orthopedic tissues suggests MSCs can rescue injured cells by donating mitochondria, restoring mitochondrial function in recipient cells, preserving cell viability, and promoting tissue repair. Our group hypothesized that MSCs package mitochondria for export into EVs, and that these so-called "mitoEVs" could provide a delivery strategy for cell-free mitochondria-targeted therapy. Therefore, the goals of this study were to: 1) characterize the vesicle fractions of the MSCs secretome with respect to mitochondrial cargoes, 2) determine if MSC-EVs contain functional mitochondria, and 3) determine if chondrocytes can take up MSC-derived mitoEVs. We isolated exosome, microvesicle, and vesicle-free fractions from MSC-conditioned media. Using a combination of dynamic light scattering and nanoparticle tracking, we determined that MSC-EV populations fall within the three size categories typically used to classify EVs (exosomes, microvesicles, apoptotic bodies). Fluorescent nanoparticle tracking, immunoblotting, and flow cytometry revealed that mitochondrial cargoes are abundant across all EV size populations, and mitoEVs are nearly ubiquitous among the largest EVs. Polarization staining indicated a subset of mitoEVs contain functional mitochondria. Finally, flow cytometry and fluorescent imaging confirmed uptake of mitoEVs by chondrocytes undergoing rotenone/antimycin-induced mitochondrial dysfunction. These data indicate that MSCs package intact, functional mitochondria into EVs, which can be transferred to chondrocytes in the absence of direct cell-cell interactions. This work suggests intercellular transfer of healthy MT to chondrocytes could represent a new, acellular approach to augment mitochondrial content and function in poorly-healing avascular skeletal soft tissues.

Dataset Information

Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4+ T Lymphocytes Toward a Regulatory Profile.

Publications

Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4<sup>+</sup> T Lymphocytes Toward a Regulatory Profile.

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