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. 2016 Mar 31;7(3):e01023-15. doi: 10.1128/mBio.01023-15

FIG 7 .

FIG 7 

Correlation between the phagosome maturation and apoptosis phenotypes of the various M. tuberculosis mutants and their capacity to enhance macrophage TNF production. (A) For the phagosome maturation study, 1 × 105 cells of J774.16 macrophages per well were cultured in eight-well chambered slides with or without the TNF-neutralizing MP6-XT22 MAb (final concentration, 10 µg/ml) for 16 h. The macrophages, in culture medium with or without MP6-XT22, were then synchronously infected with FITC-stained WT M. tuberculosis H37Rv or the various TNF-upregulating mutants. Macrophages were washed to remove extracellular bacilli after 4 h of infection, and cultures were replenished with medium with or without MP6-XT22. Cells were fixed with 4% paraformaldehyde after incubation for an additional 4 h, permeabilized, and allowed to react with primary antibodies against LAMP1, followed subsequently by staining with fluorescently tagged secondary antibodies. Analysis performed with confocal microscopy has revealed that colocalization of M. tuberculosis mutants with LAMP1 can be significantly attenuated by TNF neutralization but that the attenuation did not attain the level of that observed in MP6-XT22-treated, WT bacillus-infected macrophages. (B) For the apoptosis study, J774.16 cells were similarly cultured, in the presence or absence of MP6-XT22, in wells of 96-well plates (MatriPlate; Brooks Life Science System) at 1 × 105 macrophages per well. Cultures were infected with the WT or with the various mutant strains of M. tuberculosis for 4 h and were then incubated for 16 h after removal of extracellular bacilli before being subjected to analysis for evidence of apoptosis using a FAM-FLICA polycaspase kit. Analysis by laser scanning cytometry (iCys; Thorlabs) has revealed that, relative to the levels seen with the MP6-XT22-treated WT H37Rv-infected cultures, the apoptosis-promoting capacity of the Δrv3087 Δrv3088 and Δrv3089 (ΔfadD13) strains can be partially and significantly attenuated upon TNF neutralization. MP6-XT22 treatment had no significant effects on the apoptosis-enhancing capacity of the Δrv3130c (Δtgs1) strain. Values are the means of the results of triplicates ± SD. *, P < 0.05; **, P < 0.005.