Figure 6
Figure 6. Exosomes isolated from THP-1 cells infected with intracellular pathogens but not with heat-killed BCG or LPS-treated cells contain proinflammatory activity. (A) Exosomes were isolated from THP-1 cells activated for 72 hours with LPS (200 ng/mL) or infected for 72 hours with heat-killed M bovis BCG (HI-BCG), live M bovis BCG, or S typhimurium SL1344. Exosomes were added at 1 or 5 μg to uninfected THP-1 cells, and 24 hours after treatment, cell-conditioned media were assayed for TNF-α by ELISA. Data are represented as means plus or minus standard deviation for duplicate wells. (B) S typhimurium–infected THP-1 cells were incubated with N-rhodamine-phosphoatidylethanolamine (Rh-PE) to label the MVBs. Trafficking of LPS in the infected cells was measured over time using permeablized cells stained with a primary antibody to LPS and FITC-labeled secondary antibody. Representative confocal images are shown for the different time points; coincident staining between LPS and Rh-PE appear yellow in the merged images. Nuclei were stained with the bisalkylaminoanthraquinone fluorphore Draq5 (scale bar = 5 μm). (C) Flow cytometry of exosomes isolated from uninfected or Salmonella-infected THP-1 cells after coating on sulfate/aldehyde beads and probing with a monoclonal antibody against LPS (closed peaks). Data are shown as MFI. Open peaks represent background MFI of exosome-coated beads probed with isotype control monoclonal antibody. Data shown are representative of 2 independent experiments. (D) TLR4−/− and WT C57BL/6 BMMs were treated with 10 μg of exosomes isolated from uninfected or S typhimurium–infected THP-1 cells. TNF-α levels were measured 24 hours after exosome treatment by ELISA. (E) THP-1 cells were left uninfected or infected with T gondii or M bovis BCG for 72 hours. Exosomes were isolated from the cell-conditioned media. Uninfected C57BL/6 BMMs were treated for 24 hours with the different exosome preps, and the amount of secreted TNF-α was measured by ELISA. The ELISA data are representative of 2 separate experiments and are expressed as the means plus or minus standard deviation of duplicate wells.

Exosomes isolated from THP-1 cells infected with intracellular pathogens but not with heat-killed BCG or LPS-treated cells contain proinflammatory activity. (A) Exosomes were isolated from THP-1 cells activated for 72 hours with LPS (200 ng/mL) or infected for 72 hours with heat-killed M bovis BCG (HI-BCG), live M bovis BCG, or S typhimurium SL1344. Exosomes were added at 1 or 5 μg to uninfected THP-1 cells, and 24 hours after treatment, cell-conditioned media were assayed for TNF-α by ELISA. Data are represented as means plus or minus standard deviation for duplicate wells. (B) S typhimurium–infected THP-1 cells were incubated with N-rhodamine-phosphoatidylethanolamine (Rh-PE) to label the MVBs. Trafficking of LPS in the infected cells was measured over time using permeablized cells stained with a primary antibody to LPS and FITC-labeled secondary antibody. Representative confocal images are shown for the different time points; coincident staining between LPS and Rh-PE appear yellow in the merged images. Nuclei were stained with the bisalkylaminoanthraquinone fluorphore Draq5 (scale bar = 5 μm). (C) Flow cytometry of exosomes isolated from uninfected or Salmonella-infected THP-1 cells after coating on sulfate/aldehyde beads and probing with a monoclonal antibody against LPS (closed peaks). Data are shown as MFI. Open peaks represent background MFI of exosome-coated beads probed with isotype control monoclonal antibody. Data shown are representative of 2 independent experiments. (D) TLR4−/− and WT C57BL/6 BMMs were treated with 10 μg of exosomes isolated from uninfected or S typhimurium–infected THP-1 cells. TNF-α levels were measured 24 hours after exosome treatment by ELISA. (E) THP-1 cells were left uninfected or infected with T gondii or M bovis BCG for 72 hours. Exosomes were isolated from the cell-conditioned media. Uninfected C57BL/6 BMMs were treated for 24 hours with the different exosome preps, and the amount of secreted TNF-α was measured by ELISA. The ELISA data are representative of 2 separate experiments and are expressed as the means plus or minus standard deviation of duplicate wells.

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