Fig. 7: MEVsCa act anti-inflammatory on endothelial cells.
From: Immune modulation by complement receptor 3-dependent human monocyte TGF-β1-transporting vesicles
a TGF-β1 vesicles align along the endothelial cells of blood vessels in liver tissue of C. albicans-infected mice as shown by immunohistochemistry as compared with uninfected mice. Liver tissue was collected 24 h after C. albicans infection of mice. Staining– red: TGF-β1 and orange: nucleic acids. Bars: 10 µm. Representative stainings of four experiments (n = 4 mice) are shown. b Tracking of purified blood cell EVs by DLSM. EVs were isolated from 5 × 107 uninfected or opsonized C. albicans-infected blood cells (BCEVs or BCEVsCa, respectively) by polymer precipitation. Representative profiles of three donors are shown. c BCEVsCa significantly increase compared with BCEVs (data are presented as mean values + /− SD, p = 0.0031, unpaired two-tailed t test, n = 3 donors). d Profiles of BCEVs or BCEVsCa as determined by DLSM using NanoSight NTA 3.2 software. Graphs were generated by overlaying the size distribution of BCEVs and BCEVsCa from three donors. e TGF-β1 is present in the membrane fraction of BCEVsCa, but not in BCEVs and the cytosol fractions of BCEVs or BCEVsCa. BCEVs or BCEVCaS were isolated from 5 × 107 blood cells, cytosol and membrane fractions separated, and TGF-β1 detected by western blot (see uncropped WB in Supplementary Fig. 7e). f TGF-β1 on BCEVsCa binds to TGF-βRII on HUVECs. HUVECs were incubated with BCEVs or BCEVsCa for 30 min, stained for TGF-β1 and TGF-βRII, and TGF-β1/TGF-βRII complexes were detected by PLA. PLA staining– red: TGF-β1/TGF-βRII complexes, blue: DNA. Bars: 10 µm. Representative data of three experiments (n = 3 donors) are shown in e, f. g HUVECs incubated with BCEVsCa show increased PLA fluorescence intensity compared with HUVECs incubated with BCEVs as measured by ZEN Black 2011 software (data are presented as mean values + /− SD, p < 0.0001, unpaired two-tailed t test, n = 32 individual cell from n = 3 donors). h HUVECs treated with BCEVsCa but not with BCEVs upregulated TGFB1 expression (data are presented as mean values + /− SD, p = 0.0106, unpaired two-tailed t test, n = 5 donors). HUVECs (3 × 106) were treated for 6 h with BCEVs or BCEVsCa each isolated from 5 × 108 blood cells. Purified RNA was evaluated by comparative qPCR. i HUVECs treated with BCEVsCa for 6 h increase intracellular TGF-β1 as observed in CLSM. Staining– green: TGF-β1 and orange: nucleic acids. Bars: 10 µm. Representative data of three independent experiments (n = 3 donors) are shown. j Intracellular TGF-β1 as measured by ZEN 2011 (data are presented as mean values + /− SD, p < 0.0001, unpaired two-tailed t test, n = 39 individual cells from n = 3 donors). k BCEVCa-treated HUVECs increase intracellular TGF-β1 as measured by ELISA (data are presented as mean values + /− SD, p = 0.0018, unpaired two-tailed t test, n = 4 donors). l HUVECs treated with recombinant TGF-β1 increase TGFB1 expression in a dose-dependent manner compared to treatment with BCEVs (data are presented as mean values + /− SD, p = 0.0470, p = 0.0213, unpaired two-tailed t test, n = 3 donors). m HUVECs incubated with C. albicans show no increase in TGFB1 expression (data are presented as mean values + /− SD, p = 0.9839, unpaired two-tailed t test, n = 3 donors).
