Fig 2: SEV-derived miR-574-5p decreases mPGES-1 and PGE2 via TLR7/8 activation in A549 cells. (a, b) Western blot analysis of A549 and 2106T cells treated with 2 µg/ml sEV, 100 ng/ml R848 (TLR7/8 ligand) or 200 mM ODN 2088 Control (ODN 2087) (TLR7/8 antagonist). Cells were treated for 24 h. MPGES-1 levels were normalized to GAPDH and folded to untreated cell samples (A549: N = 3-5, 2106T: N = 5-6). (c) PGE2-levels of A549 cells. 24 h after stimulation, cells were treated with 10 µM arachidonic acid (AA) for 15 min. PGE2-levels were measured via an enzyme-linked immunosorbent assay (Cayman Chemicals, N = 4). Results are shown as mean +SEM, unpaired t-test to other samples, *P=0.05; **P=0.01; ***P=0.001; ****P=0.0001. Unpaired t-test to untreated control § P=0.05; §§§ P=0.001; §§§§ P=0.0001

Fig 3: Increased OC differentiation by sEV-delivered miR-574-5p is mediated by TLR7/8 activation (A) Comparison of sequence homologies of miR-574-5p and miR-16-5p to RNA33. (B,C) In microscale thermophoresis (MST) affinity assay of miR-574-5p to human recombinant TLR8, 50 nM of Cy5-labeled miR-574-5p and 334 nM-10.2 pM of non-labeled TLR8 were used. As negative control, 50 nM of Cy5-labeled hsa-miR-16-5p with 209 nM-6.36 pM of TLR8 were used. After a short incubation, samples were loaded into Monolith NT.115 Standard Treated Capillaries and the MST measurement was performed (B) Dose response curve reveals a KD of 30.8 ± 5.24 nM for the interaction of miR-574-5p and TLR8. Data are shown as ± SD (N = 3). (C) MST traces shown for miR-574-5p or miR-16-5p and TLR8. An MST-on time of 1.5 s was used for analysis. (D–G) TRAP-positive OCs obtained from CD14+ monocytes cultured in presence of (D,E) 1 µg/ml ScrC- or miR-574-5p oe sEV together with 200 nM TLR7/8 inhibitor ODN 2087 or (F,G) 10–1,000 ng/ml - TLR7/8 ligand R848 added either at (D/F) monocyte stage or (E/G) M2-like macrophage stage. TRAP-positive cells with three or more nuclei were considered as OCs. The relative changes normalized to untreated control cells are given as mean + SEM (N = 4), one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.