Fig 4. Role of TNFR in lipid droplet formation in M. tuberculosis-infected macrophages.

MDM were infected with mCherry-expressing M. tuberculosis and treated either with R-7050 (TNFR chemical inhibitor) or DMSO (vehicle control). When TNFR neutralizing antibodies were used, MDM were either pre-treated with the antibodies or left untreated, and then infected. Uninfected samples were included as controls. Lipid droplet content was measured as described in Fig 3. (A) Representative images of uninfected and infected macrophages obtained by imaging flow cytometry (60× magnification) and lipid droplet content determination; lipid droplets are fluorescent green signals and bacteria are orange signals. Each bar of the graph represents the average and standard deviation of median Bodipy fluorescence intensity per cell obtained from three donors. Effect of R-7050 (B) or TNFR neutralizing antibodies (C) on lipid droplet content of infected macrophages. Results are expressed as the ratio between lipid droplet content of treated and untreated cells. Graphs show the median of six (B) and ten (C) donors (each dot represents one donor). Statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) was assessed by paired (panel A) and one-sample (panels B and C) student t-tests. The comparisons in the paired tests are as indicated; the comparison in the one-sample student t-test was between treated and untreated cells. UN: uninfected, INF: infected, TNFR1 AB: TNFR1 neutralizing antibodies, TNFR2 AB: TNFR2 neutralizing antibodies.