Research ArticleCELL BIOLOGY

Extracellular vesicles derived from ODN-stimulated macrophages transfer and activate Cdc42 in recipient cells and thereby increase cellular permissiveness to EV uptake

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Science Advances  24 Jul 2019:
Vol. 5, no. 7, eaav1564
DOI: 10.1126/sciadv.aav1564
  • Fig. 1 EVs transport ODN into recipient cells.

    (A) EVs released from FITC-ODN–treated DsRed+ cells (marked by arrows). DsRed+ cells were treated with 0.5 μM FITC-ODN for 12 hours before real-time monitoring by confocal microscopy for 4 hours. Snapshot images taken at 0, 15, and 30 min were shown for the visualization of EV release. Scale bar, 5 μm. (B) Nanoparticle tracking analysis (NTA) displaying the size distribution of particles around the expected size of EVs isolated from the cell culture. EVs were diluted 50 times for NTA analysis. The images are representative of three independent experiments. (C) Immunofluorescence images of EVs isolated from untreated cells (CTL-EV), treated with 0.5 μM ODN (ODN-EV), or treated with 0.5 μM FITC-ODN (FITC-ODN-EV). After 12 hours of the treatment, EVs were isolated from the TLR9-overexpressing human embryonic kidney (HEK) 293 cell culture medium by ultracentrifugation. Then, the isolated EVs were mounted with a fluoromount aqueous mounting medium onto glass slides and observed by the Nikon N-SIM microscopy system. Scale bar, 1 μm. (D) Confocal images showing that FITC-ODN-EV entered DsRed+ cells and colocalized with TLR9 in the endosome. RAW 264.7 macrophage cells were incubated with 0.5 μM FITC-ODN for 12 hours. After the isolated EVs were then added into untreated cells, TLR9 and EEA1 were stained with their respective antibodies, followed by confocal microscopy analysis. Scale bar, 5 μm.

  • Fig. 2 ODN enhances EV transfer between cells expressing TLR9.

    (A) Schematic diagram showing the Cre-LoxP reporter system used to visualize the transfer of Cre+ recombinase [cyan fluorescent protein (CFP), cyan] activity. A red-to-green color switch is induced in reporter+ cells (red) upon the transfer of Cre activity from CFP+ Cre+ cells. (B) Dose-dependent activation of TLR9 by ODN. HEK-Blue hTLR9 cells were treated by different concentrations of ODN (0.2, 0.4, 0.6, 0.8, and 1.0 μM) for 24 hours, and activation was determined by luminescence assay. SEAP, secreted embryonic alkaline phosphatase; OD620, optical density at 620 nm. (C) Images of HEK-293 cultures consisting of a mixture of TLR9-Cre+ (CFP+) and reporter+ (DsRed+) cells. Cells were treated with 0.5 or 1 μM ODN for 24 hours. Graph represents the percentage of reporter+ cells with green fluorescent protein (GFP) signals. Scale bars, 50 μm. ns, not significant. (D) Images of HEK-293 reporter+ cells at the bottom well of a Transwell system and TLR9-Cre+ (cyan) cells in the upper well with or without ODN treatment. TLR9-Cre+ cells were treated with 0.2 or 1 μM ODN for 24 hours and then subcultured in Transwell. Cells were washed twice by centrifugation before the subculture. Graph represents the percentage of reporter+ cells with GFP. Scale bars, 100 μm. (E) NTA displaying the size distribution of EVs isolated from RAW 264.7 cell cultures with (ODN-EV) or without (CTL-EV) the treatment of ODN. The images are representative of three independent experiments. Graph represents the fold change of the numbers between CTL-EV and ODN-EV. (F) Images of RAW 264.7 cells taking up CTL-EV or ODN-EV. CTL-EV and ODN-EV were stained with PKH67 (green), and the plasma membrane of recipient cells was labeled by anti-Glut1 (red). Graph represents the percentage of reporter+ cells or RAW 264.7 cells with green fluorescence signals. Scale bars, 20 μm. Graphs show means ± SEM. [*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant using analysis of variance (ANOVA) one-way test (C and D) or unpaired Student’s t test (E and F)]. Results are representative of at least three independent experiments.

  • Fig. 3 Proteomic analysis and Western blot analysis of protein cargos of various EVs.

    (A) Proteomics analysis of EVs isolated from RAW 264.7 cell cultures in the absence (CTL-EV) or presence of 0.5 μM ODN (ODN-EV) for 12 hours. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was used to identify the proteins. (B) The protein interaction network was generated using the STRING database. Proteins with levels 1.5-fold higher in ODN-EV compared with CTL-EV were first analyzed by KEGG analysis. Then, proteins enriched in “endocytosis” signaling were chosen for the network analysis. (C) Comparison of the level of Cdc42 in CTL-EV and ODN-EV. The relative protein level was quantified by LC-MS analysis. (D) Western blot analysis of Cdc42 protein in cells treated with CTL-EV and ODN-EV. RAW 264.7 cells were treated with CTL-EV or ODN-EV for 12 hours before lysis of cells for Western blot analysis. The images are representative of three repeated experiments. (E) Quantitative reverse transcription polymerase chain reaction analysis of Cdc42 mRNA levels in cells treated with CTL-EV and ODN-EV. Graphs in (C) and (E) show means ± SEM. (*P < 0.05; ns, not significant using unpaired Student’s t test). Results are representative of at least three independent experiments.

  • Fig. 4 Cdc42 enhances the cellular uptake of EVs.

    (A) Western blot analysis of the level of Cdc42 in cells expressing Cdc42Q61L. (B) Confocal images of reporter+ cells expressing Cdc42. In the Cre-LoxP reporter system (see Fig. 1A), Cre+ cells were cocultured with reporter+ cells for 48 hours after the transfection of Cdc42Q61L plasmid. Scale bars, 100 μm. (C) Western blot analysis of the level of Cdc42 in reporter+ cells, which were treated with 0, 50, or 100 nM Cdc42 small interfering RNA (siRNA) for 48 hours. (D) Confocal microscopy images of reporter+ cells. Both Cre+ cells and reporter+ cells were treated with 100 nM Cdc42 siRNA. After 6 hours, the medium was replaced with fresh medium. Then, Cre+ cells were treated with 0.5 μM ODN or 20 μM GW4869 for 24 hours before being mixed with reporter+ cells. After 24 hours, cells were observed by confocal microscopy. Scale bars, 50 μm. Graphs in (B) and (D) represent the percentage of reporter+ cells with GFP signals and show means ± SEM. [**P < 0.01 using unpaired Student’s t test (B); *P < 0.05 using ANOVA one-way test (D)]. The percentage of cells with GFP signals was calculated in three different fields of each condition. Images are representative of at least three independent experiments.

  • Fig. 5 The effect of EV and ODN-EV on Cdc42 activity.

    (A) RAW 264.7 cells were serum-starved for 24 hours before actin filament staining with FITC-phalloidin. Cells were treated for 10 min with TNF-α (20 ng/ml) and 24 hours with 0.5 μM CpG ODN after serum starvation. Scale bars, 20 μm. The asterisks (*) highlight the locations of filopodia. Graph represents the percentage of RAW 264.7 cells with filopodia. (B) Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of TNF-α in THP-1 cells, which were treated with 0.5 μM ODN. (C) RAW 264.7 cells were serum-starved for 24 hours before actin filament staining with FITC-phalloidin. Cells were treated with CTL-EV or ODN-EV for the indicated time period after serum starvation. Cells treated with serum for 10 min served as a positive control. Scale bars, 20 μm. The asterisks (*) highlight the locations of filopodia. Graph represents the percentage of RAW 264.7 cells with filopodia. (D) Cdc42 pull-down activation assay showed that the Cdc42 activity was blocked by TNF-α inhibition. QNZ (EVP4593) is a known TNF-α inhibitor. ML141 is a known Cdc42 inhibitor. PAK, human p21 activated kinase; PBD, p21-binding domain. Graphs show means ± SEM. [**P < 0.01; ***P < 0.001; ns, not significant using ANOVA one-way test (A and C) or unpaired Student’s t test (B)]. Results are representative of at least three independent experiments.

  • Fig. 6 Schematic representation of the proposed mechanism for ODN-stimulated EV uptake via modulation of Cdc42.

    EVs secreted from ODN-activated macrophages carry both Cdc42 and ODN. When EVs internalize into the recipient cells, Cdc42 is transferred from EVs to cells, further increasing the level of Cdc42 in the cells. ODN carried by EVs also induces the release of TNF-α from recipient macrophages, which, in turn, activates Cdc42, forming a positive feedback loop to promote further endocytosis of EVs.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaav1564/DC1

    General Methods

    Fig. S1 Characterization of EVs isolated from cell culture.

    Fig. S2. Effect of ODN on EV transfer and cell viability.

    Fig. S3. GO bioinformatics analyses of proteins that are more enriched in ODN-EV compared with CTL-EV.

    Fig. S4. The quantification of secreted EVs from the Cdc42-overexpressing cells.

    Fig. S5. ODN or ODN-EV increases the level of TNF-α in macrophages.

    Movie S1. Movie showing real-time EV secretion from FITC-ODN–treated DsRed-marked cells.

  • Supplementary Materials

    The PDF file includes:

    • General Methods
    • Fig. S1 Characterization of EVs isolated from cell culture.
    • Fig. S2. Effect of ODN on EV transfer and cell viability.
    • Fig. S3. GO bioinformatics analyses of proteins that are more enriched in ODN-EV compared with CTL-EV.
    • Fig. S4. The quantification of secreted EVs from the Cdc42-overexpressing cells.
    • Fig. S5. ODN or ODN-EV increases the level of TNF-α in macrophages.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Movie showing real-time EV secretion from FITC-ODN–treated DsRed-marked cells.

    Files in this Data Supplement:

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