Figure 5. The role of ESAT-6 in macrophage necrosis.

hMDMs were left uninfected or infected at the indicated MOI with H37Rv, an ESAT-6 mutant, a complemented ESAT-6 mutant (ESAT-6-compl.), or an RD1 mutant, and cell death was assessed by the three different staining kits after two days, and analyzed by flow cytometry, or by calcein staining. Alternatively, infected cells were lysed, dilutions were plated for viable count, and the number of CFU was enumerated. Cell culture supernatants were collected and IL-1β was assayed by ELISA. A) TUNEL analysis of DNA fragmentation after infection at MOI 10 (n = 6). B) MitoTracker analysis of mitochondrial membrane potential loss after infection at MOI 10 (n = 6). C) Plasma membrane integrity analysis after infection at MOI 10 (n = 6). D) Relative viability of hMDMs infected for two days with the different strains at MOI 40, as analyzed by calcein staining (n = 4). The values were normalized against uninfected controls from the same reading, which were set to 100%. E) The number of CFU per well after infection with the different strains at MOI 10 for two days, as determined by viable count analysis (n = 5). F) IL-1β concentration in cell culture supernatants from hMDMs infected for two days with the different strains at MOI 10 (n = 3). Bar graphs show the mean and SEM. The means were compared to that of uninfected hMDMs (A-D) or H37Rv-infected hMDMs (E-F) using ANOVA with Dunnett’s post-hoc test. A statistically significant difference is denoted by *(p<0.05), **(p<0.01), or ***(p<0.001).