Cardiac progenitor cell-derived extracellular vesicles skew human macrophages towards a pro-inflammatory phenotype

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This research is support by the European Research Council (ERC) consolidator grant EVICARE (ERC-2016-COG-725229).

Repair of damaged heart tissue upon myocardial infarction remains a major challenge. Transplantation of cardiac progenitor cells (CPCs) has been studied as a potential regenerative therapy, but recent studies have shown that the cardioprotective effect of CPCs is mediated by the release of extracellular vesicles (EVs). The benefits of CPC-derived EVs have mostly been associated with stimulation of angiogenesis and inhibition of cell death. Although macrophages have been suggested to be key for cardiac repair, the effect of CPC-EVs on macrophage polarization is poorly explored.

Here, we hypothesized that CPC-derived EVs can modulate the inflammatory response after myocardial infarction by interacting with macrophages.

EVs were isolated from serum-starved CPCs by ultrafiltration followed by size exclusion chromatography. Human monocytes were isolated from the blood of healthy donors and differentiated into macrophages with M-CSF. Macrophages were subsequently stimulated with LPS + IFNy or with IL4 in order to induce an inflammatory M1 and reparative M2 phenotype, respectively. A third group of macrophages were cultured in media without LPS, IFNy or IL4 to resemble naive M0 macrophages. The obtained macrophages were exposed to CPC-EVs and analyzed by flow cytometry, bulk RNA sequencing and confocal microscopy to assess macrophage phenotype changes. Gene candidates were selected based on the p-value and fold-change, and validated by RT-PCR. Small molecule inhibitors were used to further investigate the mechanism by which CPC-EVs are taken up by macrophages and the pathways involved in macrophage response.

Stimulation of macrophages with CPC-EVs enhanced the expression of the pro-inflammatory marker CD80, while slightly decreasing the anti-inflammatory marker CD206, in M0 and M2 macrophages. CPC-EV-stimulated macrophages also adopted a morphology that reflects the inflammatory macrophage. In line with these findings, bulk RNA sequencing on M0 and M2 polarized macrophages revealed upregulation of genes involved in inflammatory response, including cytokine and both type I and II interferon signaling. EV exposure did not significantly affect gene expression in M1 polarized macrophages. Ongoing investigations will provide in-depth insight into the mechanism by which CPC-EVs induce this response in macrophages.

Our data suggests that CPC-EVs are able to induce macrophage polarization towards an inflammatory phenotype, which might have implications for CPC-EV treatment after myocardial infarction. This underlines an urgent need to understand the molecular mechanisms underlying the immunomodulatory effect of CPC-derived EVs before moving into a clinical setting.