Fig. 5: MEVsCa downregulate IL-6 expression in human macrophages.
From: Immune modulation by complement receptor 3-dependent human monocyte TGF-β1-transporting vesicles
a MEVsCa-sβG show significantly increased protein concentrations compared with MEVs. MEVs and MEVsCa-sβG were isolated from 1 × 107 monocytes, and proteins were detected using label-free LC-MS/MS-based proteomics (p < 0.05, unpaired two-tailed t test, n = 3 different donors). b Most of the proteins in MEVsCa-sβG are extracellular exosome-related as determined by GO enrichment analysis for cellular compartments. Comparison was performed between protein contents of MEVsCa-sβG and MEVs from n = 3 donors. c MEVsCa and MEVsCa-sβG show upregulation of 68 vesicle marker proteins (ExoCarta) compared with MEVs. Protein contents of MEVsCa and MEVsCa-sβG were compared with that of MEVs from n = 3 donors. d TGF-β1 increases in MEVsAB compared with MEVs. EVs were isolated after 1 h of co-incubated monocytes (5 × 105) with opsonized apoptotic bodies (data are presented as mean values + /− SD, p = 0.0008, unpaired two-tailed t test, n = 3 donors). e TGF-β1 significantly increases in ex vivo blood cell-derived vesicles induced by apoptotic bodies (BCEVsAB) compared with control blood cell-derived vesicles (BCEVs), but not when CR3 was blocked. Vesicles were isolated from 1 h co-incubated blood cells (1 × 107) (data are presented as mean values + /− SD, p = 0.0013, p = 0.0133, unpaired two-tailed t test, n = 3 donors). f TGF-β1 significantly increases in ex vivo blood cell apoptotic body-induced vesicles (BCEVsAB) compared to control blood cell-derived vesicles (BCEVs) in wild-type mice, but not in CD11b KO mice. In all cases, vesicles were isolated from 1 h induced blood cells (1 × 107) (data are presented as mean values + /− SD, p = 0.0163, unpaired two-tailed t test, n = 3 donors). g TGF-β1 on MEVsCa but not MEVs binds to TGF-βRII on macrophages by proximity ligation assay (PLA). Cells were incubated with EVs for 30 min, and PLA assay was performed to detect TGF-β1/TGF-βRII complexes by CLSM. PLA staining– red: TGF-β1/TGF-βRII complexes, blue: DNA. Bars: 10 µm. A representative PLA of three experiments (n = 3 donors) is shown. h IL-6 is induced in LPS-stimulated macrophages in the presence of MEVs, but not MEVsCa. Blocking of TGF-β1 on MEVsCa did not reduce IL-6 production. M1 macrophages were differentiated from human blood monocytes, and IL-6 was measured by ELISA (data are presented as mean values + /− SD, p = 0.0339, p = 0.0014, p = 0.00393, unpaired two-tailed t test, n = 3 donors).
