Fig. 2.

Uptake of R848-protocells by wound macrophages induces pro-inflammatory reprogramming and alters their behaviour. (A) Schematic for loading denatured-BSA-containing FITC-protocells with R848. (B) Spectra showing absorbance quantification of the R848 supernatant before and after protocell loading. (C) Graph showing percentage of R848 released from loaded protocells. (D) Schematic for experiments in E–J showing wound, injection site and imaging region (blue box). (E) Confocal images of wounded Tg(mpeg1:mCherry;tnfα:GFP) larvae showing TNFα-positive macrophages (white arrowheads) after each protocell treatment. (F,G) Graphs showing total macrophages (F) or percentage of TNFα-positive macrophages (G). Data in G are part of an experiment shown in Fig. S2 that also includes ‘medium’ and ‘free R848’ groups. (H) Confocal images of wounded Tg(mpeg1:nls-Clover) larvae showing macrophage tracks in the wound vicinity following each protocell treatment. Warm and cold track colours indicate faster and slower macrophage velocity, respectively. (I,J) Graphs showing velocity (I) or directionality ratio (J) of macrophages. White dashed boxes in E and H indicate wound sites; each dot in C represents the mean of all experiments analyzed, in F and G, one fish, and in I and J, each small dot represents one cell and larger dots are the mean from one fish. Error bars are mean±s.e.m. ns, not significant; **P<0.01; ***P<0.001; ****P<0.0001 (unpaired two-sided Mann–Whitney test). Mφ, macrophages; n, number of fish (F,G), number of experiments (C) or number of cells/number of fish (I,J). Scale bars: 50 μm.