(A) PI staining of HBMECs following exposure to pneumolysin. Cells were treated with increasing concentrations of simvastatin overnight (0.1, 1.0, and 10 μM, respectively) or with simvastatin (1 μM) supplemented with mevalonate. For each condition, 20,000 cells were analyzed by FACS for PI-positive staining. Percentages of PI-positive cells in controls were: mock, 1.5%; simvastatin, 0.1%; mevalonate, 0.2%; simvastatin plus mevalonate, 0.1%. Data represent the mean ± SD from 2 independent experiments. *P < 0.01 versus mock. (B) Western blot of pneumolysin (arrowhead) in HBMEC membranes with or without simvastatin and/or mevalonate pretreatment (molecular weight markers indicated). (C and D) Release of LDH as a measure of HBMEC lysis after 1 hour of exposure to living bacteria (C) or cholesterol-dependent toxins pneumolysin (C), tetanolysin (D), or streptolysin O (D). Pretreatment with simvastatin and reversal by mevalonate as indicated. LDH released from HBMEC monolayer completely lysed by 1% Triton X-100 was assigned a value of 100%. *P < 0.01 versus mock. Error bars represent SD. (E) SCD and WT mice with or without simvastatin treatment were challenged intratracheally with 500 ng recombinant pneumolysin in 0.5% glycerol. Lung histology was assessed at 5 hours. Effects of pneumolysin toxoid and 0.5% glycerol alone are shown as negative controls. Data are representative images from at least 3 mice per group. Scale bar: 100 μM.