Fig. 4
Macrophage facing S. typhimurium stimuli. A Treatments applied to S. typhimurium in order to obtain the different fractions subsequently tested. Intact bacteria consist of a gram-negative membrane (green) with superficial components such as LPS or OMP porines (yellow). Intracellular components are represented in blue, whereas extracellular secreted protein, like flagellin, PrgJ, SrfH, Sip and Sop family, are indicated in pink. Four treatments were performed to S. typhimurium: secreted protein where only extracellular molecules were collected; inactivation by PFA that generates a crosslinking (grey mesh) preserving the superficial components; a cell lysate to break the cell and release superficial and intracellular components; and an inactivation by heat that damages the membrane (green) and denatures proteins (amorphous blue balls). B Migration of macrophages in 2.5 mg/ml collagen gels towards a gradient generated at the top (+ y axis, indicated by grey dots) with S. typhimurium bacterial fractions: non-stimulated control (gray), secreted protein (pink), inactivated by PFA (yellow), cell lysate (blue) and inactivated by heat (green). Directionality of migration, where the radius is the number of cells migrating in each direction and normalized by the total number of cells per condition. C Percentage of macrophages as a function of the verticality of their migration and weighted by the total distance traveled. Those macrophages whose final position is above their initial position contribute to the bar graph exceeding the red line set at 0.5. The dots in each bar graph correspond to the percentage of macrophages whose final position is above or below their starting position, but not weighted by the total distance migrated. n (devices) = 9 (control), 6 (secreted protein), 5 (inactivated by PFA) and 5 (cell lysate) and 5 (inactivated by heat). D S. typhimurium secreted protein fraction titration assay. The y-axis shows the percentage of macrophages migrating in the y-plane weighted by the total distance moved; the x-axis shows the concentrations of serial dilutions (1:10), ranging from 10–6 to 10–2 (pink intensity), including the control without bacterial stimulus (gray). The red line set at 0.5