Figure 6

Pharmacological inhibition of V-ATPase and cathepsin D reversed the restriction of BCG growth in IL-12 and sIL-27R-treated macrophages. (A) Macrophages were treated with bafilomycin (0–1000 nM) for 4 h and then subsequently treated with green fluorescent labeled latex beads for additional 48 h. Acidified lysosomes (red) were stained with LysoTracker (100 nM). The images shown are typical results from three separate experiments. MFI was calculated as described in the Methods section. A student’s t test was used to establish statistical significance in the 95% confidence interval; * indicates P < 0.001 compared to the untreated sample. (B) Pepstatin promotes the growth of BCG in macrophages in a dose-dependent manner. Macrophages were treated with pepstatin (0–100 μM) for 4 h prior to infection and then infected with BCG for an additional 48 h. BCG were stained with Auromine O and fluorescence was measured. The mean fluorescent intensity (MFI) ± standard error for two independent experiments is shown. A student’s t test was used to establish statistical significance in the 95% confidence interval; * indicates P < 0.001 compared to the untreated sample. (C) Treatment of bafilomycin and/or pepstatin reversed growth restriction of BCG by IL-12 + sIL-27R-treated macrophages. Macrophages were either untreated or treated with IL-12 + sIL-27R, bafilomycin, and/or pepstatin for 6 h and then subsequently infected with BCG for 48 h. Data is represented as the mean CFUs recovered from infected macrophages ± standard error at 48 h for three independent experiments. A student’s t test was used to establish statistical significance in the 95% confidence interval between individual sample groups as indicated.