Figure 2.
Both DC-SIGN and MMR contribute to the recognition strength of C. albicans by immature dendritic cells (imDC). (A) Schematic set-up showing a single C. albicans cell immobilized on a tip-less AFM cantilever interacting with an imDC expressing different C-type lectins such as DC-SIGN and MMR. (B) Example brightfield image that shows how a C. albicans cell on the tip (indicating by dashed white ellipse) interacts with different imDCs. The positions at which the C. albicans is brought into contact for 10 seconds with three imDCs are indicated by asterisks. (C) Example FD-curves of C. albicans - imDC interactions are shown of without (medium control) and with specifically blocking DC-SIGN and MMR (with 30 µg/ml anti-DC-SIGN and 30 mM mannose for 30 minutes). In FD-curves the work and Fmax are measured such as indicated by the shaded area and depth of the curve, respectively. The zoom shows single membrane tether rupture steps (arrows). (D,E) The relative detachment force (D) and work (E) needed to detach an C. albicans cell from the imDC before and after blocking by CA-mannan, anti-DC-SIGN, mannose, or a combination of mannose and the antibody (N ≥ 3 independent experiments; N ≥ 20 cells; N≥ 20 FD-curves per condition). Significance levels by Mann-Whitney (n.s. = not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (F) imDC cells were incubated with FITC-labeled C. albicans in the presence or absence of blocking agents; anti-DC-SIGN antibody, mannose and EGTA. The percentage of cells that bound C. albicans was calculated by flow cytometry. Data are normalized to medium conditions. (G) Loading probability distribution of all rupture steps detected in the FD-curves prior to and after blocking show different distributions that peak at 112 pN (medium), 102 pN (MMR block), 122 pN (DC-SIGN block), and 76 pN (both blocked) (N > 450 rupture steps). The normalized probability plots are shown and the ‘dashed’ curves are scaled relative to the number of rupture steps per FD-curve (arbitrary units).