Fig. 6: Effect of biofilm carbohydrates on human moDCs.

a Human moDCs (MR+ and DC-SIGN+) bind fucose (Fuc), Lewis^x and galactose (Gal) PAA-FITC polymers but only Lewis^x association is reduced by HMW-1 and HMW-2. Cells were treated with HMW-1 or HMW-2 (10 µg/ml) for 1 h, then fluorescein-labelled polymeric ligands were added for a further 1 h. Association of fluorescent polymeric ligands to moDCs was measured by flow cytometry. Representative histograms are shown as well as a graph depicting mean ± SEM of median fluorescence intensity (MFI), N = 4. One-way ANOVA corrected for multiple comparisons using the Tukey’s multiple comparisons test. ****, ≤0.0001. Fuc fucose; Gal galactose. b. Changes in human moDC morphology in the presence of biofilm-associated carbohydrate. HMW-2 (with and without endogenous LPS, 10 µg/ml, diluted in X-Vivo-15 medium) was used to coat chambers of µ-slide VI 0.4 flow slides overnight at 4 °C. MoDCs were added (5 × 10⁴ cells per channel) and incubated for 24 h. Samples were then fixed and stained for DC-SIGN (magenta) and nucleus (DAPI, blue). The figure shows representative images from unpermeabilised samples. Permeabilised samples, including secondary antibody control are shown in Supplementary Fig. S16. Cells were analysed for changes in shape (Circularity Index), size (Perimeter). Analysis of DC-SIGN labelling intensity (Raw Integrated Density and Signal per Unit Area) is shown in Supplementary Fig. S16. Data derive from 3 independent experiments, 20 cells per experiment were analysed. Statistical significance assessed using Kruskal-Wallis test corrected for multiple comparison using a Dunn’s multiple comparison test. c. LPS-free HMW-2 does not affect cytokine production by moDCs in response LPS. MoDCs (10⁵ cells per well) were added to 48 well tissue culture plates coated with different doses of LPS-free HMW-2 (10, 1, and 0.1 µg/ml) for 16 h. Cultures were incubated for 2 h and then treated for 4 h with ultrapure E. coli LPS (100 and 10 ng/ml, LPS-100 and LPS-10) and supernatants collected for cytokine quantification. N = 4. Controls: samples treated with buffer or incubated only with LPS-free HMW-2. Only samples treated with LPS-100 ng/ml in the presence and absence of LPS-free HMW-2 were analysed for IL-10.