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cei12157-sup-0001-fig_s1.tif3982K

Fig. S1. F-actin purse string quantification by Photoshop software. Microphotographs were taken with a fluorescence microscope (Olympus IX81). The fluorescence intensity measured in square sections (arrowhead) defined along the leading edge of the migration front of epithelial cells was measured using Photoshop software. The quantified intensity of phalloidin staining reflects the F-actin purse string formation. At least five wounds per experimental condition were analysed.

cei12157-sup-0002-fig_s2.tif2028K

Fig. S2. Neutralizing anti-transforming growth factor (TGF)-β antibodies did not reduce the properties of bone marrow-derived macrophages (BMM) significantly to assist epithelial repair. Epithelial repair was performed in the presence of hepatocyte growth factor (HGF) (0·25 ng/ml), TGF-β (1 ng/ml; R&D Systems), BMM (250 000) and/or anti-TGF-β neutralizing monoclonal antibody [20 μg/ml (1D11 clone)]. The data presented show the cumulated results of three independent experiments; bars are means ± standard error of the mean. P-values were calculated by the Mann–Whitney U-test.

cei12157-sup-0003-fig_s3.tif2953K

Fig. S3. Impact of inflammatory stimuli on macrophage-assisted epithelial repair. One million heat-inactivated Escherichia coli or 10 ng/ml interleukin (IL)-1β were added to 250 000 bone marrow-derived macrophages (BMM) co-incubated with wounded epithelial cell monolayer for 18 h without insert. At the end of the incubation, we measured wound repair and HGF concentrations. The data presented show the cumulated results of three independent experiments; bars are means ± standard error of the mean. P-values were calculated by the Mann–Whitney U-test; n.d.: not done.

cei12157-sup-0004-fig_s4.tif1296K

Fig. S4. Impact of inflammatory stimuli on human macrophage-assisted epithelial repair. Human monocyte-derived macrophages (MDM) were activated for 24 h with heat-inactivated Escherichia coli at 50 multiplicity of infection (MOI) and 105 were incubated with wounded epithelial cell monolayer. No statistical differences were found between the repair activities of activated or quiescent monocyte-derived macrophages.

cei12157-sup-0005-fig_s5.tif3120K

Fig. S5. Viability and phenotypical characterization of Crohn's disease (CD) and healthy donor (HD) macrophages. (a) CD14-positive cells were selected by magnetic sorting from peripheral blood mononuclear cells and plated at density of 500 000–1 000 000 cells/ml for 5 days. On days 1 and 4, non-adherent cells were discarded and at day 5 adherent macrophages were recovered by scraping and numerated. The data show the percentage of recovered living macrophages compared to the initial number of plated CD14-positive cells. The data presented show the cumulated results of nine HD and nine CD patients; bars are means ± standard error of the mean. *No statistical difference (P-values were calculated by the Mann–Whitney U-test). (b) Macrophages were surface-stained with monoclonal antibodies to CD14 (clone M5E2; BD Biosciences), CD11b (clone ICRF44; BD Biosciences), CD11c (clone B-ly6; BD Biosciences), human leucocyte antigen D-related (HLA-DR) (clone G46-6; BD Biosciences), CD33 (clone WM53; BD Biosciences), CD68 (clone Y1/82A; BD Biosciences), CD116 (clone hGMCSFR-M1; BD Biosciences), CD163 (clone GHI/61; Biolegend), CD206 (clone 15.2; Biolegend), CX3CR1 (clone 2A9-1, MBL Nagoya, Japan). Data were acquired using a fluorescence activated cell sorter (FACS)scan flow cytometer (BD Biosciences) and analysed with CellQuest software (BD Biosciences). Dead cells were excluded by propidium iodide staining.

cei12157-sup-0006-fig_s6.tif2667K

Fig. S6. Active transforming growth factor (TGF)-β and epidermal growth factor (EGF) levels in human co-cultures. Active TGF-β and EGF protein concentrations measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) in the culture supernatant of monocytes derived-macrophages co-cultivated for 72 h with wounded epithelial cells in Dulbecco's modified Eagle's medium (DMEM) 0·1% fetal calf serum (FCS). No differences between TGF-β and EGF levels for healthy donors (HD) (eight individuals), Crohn's disease (CD) (11 individuals) and ulcerative colitis (UC patients) (four individuals) can be observed. Comparison between several groups to identify a trend was performed by a one-way analysis of variance (anova) test with Trend test as post-test.

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