Figure 4

Secretion of active gMMP-9 and endothelial permeability enhancement are triggered by ANDV infection of iDCs. (A) The gMMP-9 expression in ANDV-infected iDCs was assessed by Western blotting. Uninfected iDCs (Mock), ANDV-infected iDCs (ANDV) and LPS-treated iDCs (LPS) were collected 3 h post-infection. Forty micrograms of total proteins from cell lysates were separated in an 8% SDS-PAGE gel. Following an electrical protein transfer onto a nitrocellulose membrane, an anti-gMMP-9 antibody was used to probe the presence of gMMP-9 that was revealed using an HRP-conjugated anti-mouse IgG Ab. (B) Supernatants from ANDV-infected DCs or Mock cells collected 3 h post-infection. Gelatinase activity was assayed by zymography. Gelatinolytic activity was quantified by gel densitometry using the Image J Software. Data are presented as the fold-increase of gMMP-9 activity in supernatants. Statistical significance (**, p < 0.01) was determined from five independent experiments. (C) Enhancement of the endothelial cells permeability induced by supernatant from ANDV-infected iDCs. HUVEC confluent monolayers plated onto collagen-coated transwell inserts were incubated with either ANDV supernatant, mock control (uninfected DCs supernatant) or with TNF-α (50 ng/ml) as positive control. Following 18 h at 37°C in CO2 5% after addition, within the top chamber, of 500 μg/ml FITC-conjugated Dextran, paracellular permeability was measured by reading in the bottom chamber containing the infiltrated FITC-dextran at an excitation wavelength of 485 nm and an emission of 530 nm. Data represent means of five independent experiments. **, p < 0.01.