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. 2022 May 2;13:878194. doi: 10.3389/fimmu.2022.878194

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Copyright © 2022 Kang, Huang, Gajendrareddy, Lu, Shirazi, Ravindran and Cooper

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effect of TNFα EVs on macrophage polarization. (A) Representative confocal images of fluorescently labeled control and TNFα EVs (green) endocytosed by macrophages. Scale bar=20μm in all images. The red staining in the images indicate actin counter stain and blue staining shows the nuclei (DAPI). (B) Dose-dependent endocytosis of fluorescently labeled control and TNFα EVs by primary macrophages. (C) Fold change in gene expression of pro-inflammatory markers in M1 polarized macrophages in the presence/absence of EVs. Note that TNFα EVs significantly reduce the expression levels of IL-1β, iNOS and TNFα indicating a reduction in M1 polarization. (D) Fold change in the expression levels of M2 marker genes in M2 polarized macrophages in the presence/absence of EVs. Note the significant increase in the expression levels of M2 markers CD206, IL-10 and Arg1 indicating an increase in M2 polarization. (E) Multiplex ELISA determined quantitative expression of pro-inflammatory cytokines in M1 polarized macrophages in the presence/absence of EVs. Note the significant reduction in cytokine expression in the presence of TNFα preconditioned MSC EVs. (F) Multiplex ELISA determined quantitative expression of IL-10 and VEGF in M2 polarized macrophages. *: statistical significance (P < 0.05) with respect to untreated control, #: represents statistical significance (P < 0.05) between control and TNFα EV groups as calculated by Tukey’s test post ANOVA.