Fig 2: Effect of biofilm carbohydrates on human moDCs.a Human moDCs (MR+ and DC-SIGN+) bind fucose (Fuc), Lewisx and galactose (Gal) PAA-FITC polymers but only Lewisx 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.

Fig 3: Binding of C-type DC-SIGN, MR and Dectin-2 to P. aeruginosa biofilms and planktonic cells.a PAO1, ΔwspF (Psl + /Pel+), ΔwspF Δpel (Psl + /Pel-), ΔwspF Δpsl (Psl-/Pel+) and ΔwspF Δpsl Δpel (Psl-/Pel-) were grown in 96-well plates for 24 h in X-Vivo-15 and biofilm formation was analysed using the crystal violet assay. Data analysed using One-way ANOVA and corrected for multiple comparisons using Dunnett’s multiple comparison test. N = 6-7 in triplicate. ** 0.001; **** <0.0001. b and c. PAO1, ΔwspF, ΔwspF Δpel or ΔwspF Δpsl biofilms were generated in 96-well plates for 24 h, fixed and incubated with DC-SIGN-Fc (B) or MR-CTLD4-7-Fc or Dectin-2-Fc (C) followed by anti-human Fc secondary antibody conjugated to alkaline phosphatase. Cultures of ΔwspF Δpsl Δpel were used as controls. Analysis performed using Two-way ANOVA corrected for multiple comparisons using the Dunnett’s multiple comparisons test. N = 3 in triplicate. In B * 0.0324 (for ΔwspF Δpsl); 0.0413 for ΔwspF ΔpslΔpel; ** 0.007; *** 0.0007; **** <0.0001. In C **** <0.0001. d Planktonic cultures of P. aeruginosa PAO1 and the different mutants were collected, fixed and used to coat wells of MaxiSorp plates. Wells were incubated with MR-CTLD4-7-Fc or DC-SIGN-Fc followed by anti-human Fc secondary antibody conjugated to alkaline phosphatase. DC-SIGN, but not MR, bound planktonic bacteria and binding was independent of the presence of Psl and/or Pel. Two-way ANOVA corrected for multiple comparisons using the Dunnett’s multiple comparisons test. N = 4 in triplicate. *, 0.0412 for PAO1 and 0.0148 for ΔwspF; ****, <0.0001.e. DC-SIGN binding to planktonic bacteria was dependent on the presence of CPA LPS which is absent in the Δrmd and ΔwbpL mutants. N = 3 in triplicate. *, 0.0111; ****, <0.0001. Right panel: Adherence of planktonic cells to the wells was confirmed by ELISA using an antibody against P. aeruginosa (Anti-PA). N = 3 in triplicate. *, 0.0161. Analysis performed using Two-way ANOVA corrected for multiple comparisons using the Tukey’s multiple comparisons test. Graphs show mean +/− SEM. Specificity controls for these assays are shown in Fig. S1. In all cases, reading were taken within 20 min development and all strains in each graph were tested simultaneously.

Fig 4: Inhibition of polymeric ligand uptake by DC-SIGN and MR expressing cells 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. The association of fluorescent polymeric ligands with cells was measured by flow cytometry. Representative histograms and scatter plots depicting mean ± SEM of median fluorescence intensity (MFI) are shown for each cell type. a U937-DC-SIGN cells (express DC-SIGN, but not MR) associated with fucose and Lewisx polymers and this association was reduced by HMW-1 and HMW-2, N = 3. b CHO-MR cells (express MR, but not DC-SIGN) associated with fucose and Lewisx polymers and only Lewisx association was reduced by HMW-1 and HMW-2, N = 3. One-way ANOVA corrected for multiple comparisons using the Tukey’s multiple comparisons test. *, ≤0.05. ** ≤0.01. Fuc fucose, Gal galactose.

Fig 5: Macrophages and Treg cells in the ectopic and eutopic endometrium of endometriosis patients and the normal endometrium. A The expression of CD68, CD169, CSF-1R, and DC-SIGN in macrophages, and the expression of FOXP3 in Treg cells in all tissues of the normal endometrium, eutopic endometrium of endometriosis patients, and ectopic endometrium of endometriosis patients. Representative images are shown at 400 × magnification. B The comparison between the proportion of macrophages and Treg cells in tissues of the normal endometrium, eutopic endometrium of endometriosis patients, and ectopic endometrium of endometriosis patients. The data are presented as the means ± SDs. Statistical analyses were performed by one-way analysis of variance (eutopic = 17; ectopic = 17; normal = 11), and significant differences are indicated as *P ≤ 0.05 and ***P ≤ 0.001