Figure 7

Rapamycin enhances autophagy mediated killing of intracellular M. tuberculosis in mesenchymal stem cells. Untreated or rapamycin (µM doses indicated) treated BM-MSCs were infected with Mtb (MOI = 1). Lysates were plated on 7H11 agar for viability of intracellular Mtb, and stem cell viability evaluated using alamar blue. (a) Rapamycin caused a dose-dependent loss of viability of Mtb within MSCs. Data from one of three similar experiments are shown (**p < 0.007, 5 µM dose vs. none; ANOVA). (b) Rapamycin treated BM-MSCs retained approximately 90% viability on day 7. (c) MSC lysates on day 1 post rapamycin treatment were tested for microtubule associated with light chain (LC3) lipidation by using western blotting. (d) To confirm the specificity of rapamycin effects, MSCs were treated first with siRNA vs. beclin-1 or scrambled control followed by rapamycin (5 µM dose), and CFU counts of Mtb were determined by plating MSCs lysates. (e) MSCs were treated with rapamycin (5 µM), and after a 4 hr infection with rfpMtb, MSCs were fixed on day 1 and stained for autophagosomes using cyto-ID or an antibody to rab7 lysosomal marker. Images were analyzed using deconvolution microscopy and quantified. Images show that rapamycin induces strong colocalization of autophagosomes with rfpMtb (inset) compared with untreated cells (white bar = 5 µM). Bar graph shows data from three experiments of the percentage MSCs showing colocalization of rfpMtb with autophagic puncta. (f) Rapamycin also induces stronger staining of rab7 on rfpMtb phagosomes (inset) thus suggesting lysosomal fusion. Bar graph indicates quantification.