Fig 1: ATP preferentially packages membrane TNF within shed MVs. A) Scanning (left; scale bar, 1 µm) and transmission (right; scale bar, 0.2 µm) EM of RAW cells illustrates membrane blebbing and MV formation in response to ATP. B) RAW cell–derived MVs, identified as CD45+ or CD11b+ particles by flow cytometry (left and middle), significantly increased in response to ATP (right; n = 6–10). C) These MVs contained TNF, accounting for the above missing TNF from the cells but in the form of 26 kDa transmembrane pro-TNF isoform rather than 17 kDa soluble mature TNF (left and middle; n = 4–8). ATP packaged substantial amounts of pro-TNF within MVs (right; n = 4–8). D) To confirm these results in primary cells, we exposed mouse BMDMs to LPS (1 µg/ml, 1 h) followed by ATP (3 mM, 15 min). Similarly, ATP induced significant release of MVs (left; n = 6), which contained substantive amounts of pro-TNF (right; n = 6). E) Confocal microscopy illustrates that these BMDM-derived MVs, identified as CD11b+ (green, top left) particles negative for nuclear materials (DAPI-, top right), actually contained TNF (red, bottom left, colocalization shown in the bottom right combined image). Scale bars, 1 µm. F) Immune EM demonstrates transfer of pro-TNF (black dots, red arrows) from BMDMs to MVs during their formation, and these pro-TNF molecules gradually localized to MV membrane surface (hence, membrane TNF) (right). Scale bars, 0.2 µm. Parametric or nonparametric data are displayed as means ± sd or box-whisker plots showing the median, IQR, and minimum or maximum values, respectively. **P < 0.01, ***P < 0.001.
Fig 2: sEV-uptake and -mediated effects in vitro. Primary murine astroglia cells were incubated with PKH67-labeled sEVs (pseudo-green) from young (sEVyoung) and old mice (sEVold) for 24 h. Representative images of immunofluorescence staining for (A) GFAP (pseudo-red), (B) Iba1 (pseudo-red), and (C) CD11b (pseudo-red). Nuclei (blue) were counter-stained with DAPI Scale bar 50 µm. (D) Summary bar graphs of the relative mean fluorescence intensity (MFI) activation genes. NTC: Non-treated cells. Bars represent the mean ± SEM of MFI. * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA with Dunn’s multiple comparisons test.
Fig 3: a-Syn binds to CD11b. (A) Purified recombinant human a-Syn aggregates were mixed with microglial lysates to allow a-Syn to react with CD11b, and then the mixtures were immunoprecipitated with antibody against a-Syn. Binding was detected via immunoblot for CD11b and a-Syn. (B) Confocal microscopy analysis further delineated the interaction between a-Syn and CD11b on the cell surface. This experiment was performed 4 independent times with duplication. Bar = 10 µm.
Fig 4: Mindin binds with integrin Mac-1. (A) HEK293T cells stably expressing Mac-1 were transfected with mindin-Myc and stained with anti-Myc or anti-HA antibodies (left panels). RAW264.7 macrophages were pre-treated with mindin for 12 h and then stained with anti-mindin, anti-CD11b and anti-CD18 antibodies (right panels). B-D, Immunoprecipitation assays were performed using mouse anti-HA or anti-Myc antibodies, and the results were analysed by Western blotting with rabbit anti-HA or anti-Myc antibodies
Fig 5: CD11b deficiency attenuates a-Syn-induced NOX2 activation and microglial activation in vivo. (A) a-Syn or vehicle was injected into the SN of WT and CD11b-/- mice. (B) The midbrain tissues of mice were dissected and the membrane translocation of p47phox was measured by using western blot. The density of blot was quantified. (C) Microglia in the SN were stained with anti-Iba-1 antibody and the representative images were shown. Activated microglia displayed enlarged cell body size and high staining density. (D) The density of Iba-1 staining in the SN was quantified (6 sections each mouse). Results are expressed as mean ± SEM. n = 3; N.S, not significant; **p < 0.01.
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