Lysosomal cholesterol accumulation attenuates macrophage <i>Mtb</i> control.

Primary human macrophages were treated with PBS control, acLDL or oxLDL (25 μg/ml) overnight. (A) Oil Red O staining of macrophages treated overnight with 25 μg/ml acLDL or oxLDL. Pictures were taken at a 20x magnification. Macrophages were infected with Mtb H37Rv (B) (n = 32), M. bovis BCG (C) (n = 6) or Salmonella enterica serovar Typhimurium (D) (n = 6) at a MOI of 10:1. Cells were lysed at 24 h post-infection and bacterial load was determined by CFU assay. Results were normalized versus PBS control. (E) Western blot analysis of lysosomal and autophagy markers in macrophages treated with bafilomycin A1 (10 nM) or DMSO control during 24 h of H37Rv Mtb infection. Data shown is from one representative donor (n = 4). (F) Quantification of LAMP1, LAMP2, Cathepsin D, Cathepsin L (+ DMSO), p62 and LC3-II (+ bafilomycin A1) protein levels. Protein levels were first normalized to actin and subsequently versus PBS control (n = 4). (G) Macrophages were co-treated with U18666A (3 μg/ml) or DMSO control for 24 h. Cells were subsequently stained for neutral lipids (LipidTOX, green), lysosomes (Lysotracker, red) and cholesterol (filipin, blue) and analyzed by confocal microscopy. Pictures were taken at a 63x magnification. Scale bars represent 5 μM. (H) Macrophages were co-treated with U18666A (3 μg/ml) or DMSO control for 24 h pre- and post-infection with Mtb H37Rv at a MOI of 10:1. Cells were lysed at 24 h post-infection and bacterial load was determined by CFU assay. Results were normalized versus PBS control (+ DMSO). Individual donors are depicted as dots with group medians. Statistical significance was determined by Wilcoxon signed rank test. * = p p < 0.0001.