High susceptibility of human dendritic cells to invasion by the intracellular pathogens Brucella suis, B. abortus, and B. melitensis

Abstract

Bacteria from the Brucella genus are able to survive and proliferate within macrophages. Because they are phylogenetically closely related to macrophages, myeloid dendritic cells (DCs) constitute potential targets for Brucella bacteria. Here we report that DCs display a great susceptibility to Brucella infection. Therefore, DCs might serve as a reservoir and be important for the development of Brucella bacteria within their host.

FIG. 1.

Intracellular proliferation of Brucella spp. (A) A total of 10⁵ macrophages or DCs infected with B. suis GFP, B. abortus GFP, or B. melitensis GFP were cultured on eight-chamber Labtek slides in gentamicin-containing medium. At 48 h after the onset of infection, the green fluorescence of the Brucella GFP was visualized by videomicroscopy. Results are representative of five different experiments. (B) A total of 10⁵ DCs and macrophages were infected with B. suis GFP in 48-well plates. The intracellular development of bacteria was then followed by measuring the number of CFU/well at different times p.i. Data are means ± standard errors of the means (SEM) of 18 different experiments. (C) DCs were infected with the B. suis WT or the indicated mutants. The intracellular development of bacteria was then followed by measuring the number of CFU/well at different times p.i. Data are means ± SEM of three independent experiments. (D) DCs were infected with B. suis bvrR or control strain B. suis bcsp31::GFP. The intracellular development of bacteria was then followed by measuring the number of CFU/well at different times p.i. Data are means ± SEM of four independent experiments. *, differences between DCs and macrophages (B) or mutants and controls (C and D) were statistically significant (i.e., P < 0.01, as calculated using a paired t test).

FIG. 2.

Comparison of Brucella phagocytosis results for dendritic cells and macrophages. Macrophages (open circles) or DCs (closed circles) were cultured in the presence of different bacterium/cell or bead/cell ratios for 1 h. Afterward, cells were washed and resuspended in PBS-EDTA buffer. Percentages of cells associated with GFP-B. suis WT (A), GFP-B. suis manB (B), or FITC-stained beads (C) were determined by flow cytometry analysis. Data are means ± SEM of six independent experiments. *, differences between DCs and macrophages were statistically significant (i.e., P < 0.01, as calculated using a paired t test after required data transformations).

FIG. 3.

Implication of lipidic microdomains in Brucella phagocytosis. Macrophages (open circles) or DCs (closed circles) were left untreated or were pretreated with filipin (A, B, and C) or β-methyl-cyclodextrin (D and E) for 30 min. They were then infected for 1 h with B. suis (MOI = 50) (A and D), B. suis manB (MOI = 50) (B and E), or FITC-stained beads (MOI = 10) (C) in the presence of the drugs. The percentages of labeled cells were determined by flow cytometry (A, B, and C) and fluorescence microscopy (D and E). The ratio (percentage of infection in treated cells)/(percentage of infection in untreated cells) was calculated. Data are ratio means ± SEM of three independent experiments. *, differences between treated and untreated cells were statistically significant (i.e., P < 0,01, as calculated using a paired t test after required data transformations).

FIG. 4.

Visualization of bacterium-cell interaction by scanning electron microscopy. DCs (A to H) or macrophages (I to P) were cultured in the presence of B. suis (A, B, E, F, I, J, M, and N), B. suis manB (C, G, K, and O), or latex beads (D, H, L, and P) for 15 or 30 min as indicated. Magnifications are indicated on each individual picture. The figure panels are representative of five independent experiments. The percentages of bacteria associated with villosities were evaluated by analyzing more than 200 cells presenting bacteria on their surfaces.