Lipid droplet formation in Mycobacterium tuberculosis infected macrophages requires IFN-γ/HIF-1α signaling and supports host defense
Fig 2
M. tuberculosis acquisition of host lipids does not correlate with LD formation in infected macrophages.
(A-D) Fluorescently labeled fatty acid BODIPY FL C16 was added to media of M. tuberculosis 635-Turbo infected BMDM 3 days post-infection. Bacterial lipid accumulation was analyzed by confocal microscopy at 2 hours and 8 hours after BODIPY FL C16 addition in the absence of IFN-γ activation (A,B), and with IFN-γ activation (C,D). (E) Bacterial accumulation of BODIPY FL C16 during M. tuberculosis infection of BMDM without IFN-γ activation [TB] and with IFN-γ activation [TB/G] was quantified using CellProfiler from images as in (A-D). Each data point is quantified from bacteria contained within ~100 infected BMDM. (F) BMDM were infected with M. tuberculosis 635-Turbo in the absence of IFN-γ. 3 days post-infection, BMDM were fixed, BODIPY 493/503 stained, and analyzed by SIM. Punctate BODIPY 493/503 staining co-localizing with M. tuberculosis was observed. TEM of infected BMDM at the same time point shows that these BODIPY 493/503 puncta are bacterial lipid inclusions (inset). (G) IFN-γ activated BMDM were infected and analyzed as in (F). SIM and TEM imaging indicates that in IFN-γ activated BMDM, BODIPY 493/503 signal does not localize with M. tuberculosis and bacterial lipid inclusions are not present (inset). All figures are representative of three experiments with the exception of TEM, which was performed once in triplicate. Error bars are standard deviation, **p<0.01, ***p<0.001 by unpaired t-test.