Figure 3

Gingipains inhibit macrophage migration towards AC. THP-1 cell-derived MØ were exposed to putative attractants in a horizontal or vertical migration assay. For the horizontal assay (a–d), MØ were seeded to glass coverslips and treated or untreated with gingipains before loading to a Dunn horizontal chamber and exposed to putative attractants (a–d). MØ migration was monitored for 2 h at 37 °C using time-lapse video microscopy. Migration of 40 cells per assay was measured using ImageJ and Ibidi Chemotaxis and Migration Tool (V2.0). The MØ route of travel is shown by a line from the starting point (set at the cross hairs of the plots) to the final MØ position after 2 h (black dot). The relative location of AC was at the top of the plot (black diamond). (a) Representative plots showing: left panel: untreated MØ (MØ) migration with control, cell-free medium as putative attractant to reveal basal levels of MØ migration. Centre panel: untreated MØ (MØ) migration with apoptotic NØ as attractant. Right panel: Kgp-treated MØ (Kgp-MØ) migration with apoptotic NØ as attractant. (b) Forward migration index (Y FMI) to quantify MØ migration in the direction of the putative gradient. (c) Representative plots showing: left panel: untreated MØ (MØ) migration with control, cell-free medium as putative attractant to reveal basal levels of MØ migration. Centre panel: untreated MØ (MØ) migration with apoptotic B cells as attractant. Right panel: Kgp-treated MØ (Kgp-MØ) migration with apoptotic B cells as attractant. (d) Forward migration index (Y FMI) to quantify MØ migration in the direction of the putative gradient. (e) For the vertical migration assay, MØ (untreated or treated with gingipain) were seeded to a transwell above a lower well containing secretome from apoptotic NØ and MØ migration to lower chamber assessed after 10 h by cell counting. Data shown in (b and d) are representative of four distinct experiments with two replicates in each experiment. Statistical analysis was conducted using ANOVA followed by Bonferroni post hoc-test *P< 0.05, **P<0.01, ***P< 0.001