SCD mice were pretreated daily for 5 days with vehicle control (A–C) or simvastatin (D–F) and then challenged intranasally with pneumococci. Lungs were harvested at 18 hours for histopathology (H&E staining: A, B, D, and E) or apoptosis (TUNEL: C and F). See text for full description; sections are representative of at least 6 mice per group. Scale bars: 100 μM.

(A) WT and SCD mice fitted with cranial windows were injected intravenously with fluorescent beads coated with the pneumococcal adhesin CbpA to assess activation-dependent adhesion to cerebral vessels (representative images at ×4 magnification). Negative control beads coated with BSA were also utilized in both WT and SCD mice as indicated. (B) Quantitation of bead adherence is presented as average ± SD of beads/×4 field for 10 fields per animal from at least 3 independent experiments. *P < 0.01 versus SCD mock; **P < 0.01 versus WT mock. (C) Ptafr^–/– mice were transplanted with WT (squares; n = 11) or SCD (circles; n = 11) bone marrow. Survival following intranasal pneumococcal challenge was monitored after mice received mock (open symbols; n = 11) or simvastatin treatment (filled symbols; n = 11). Survival of Ptafr^+/+ SCD mice (triangles; from Figure 1) is shown for comparison.

(A) Survival of WT versus SCD mice receiving mock treatment (n = 26 and n = 24, respectively) or simvastatin (n = 27 and n = 32, respectively) following intranasal challenge with pneumococci; *P = 0.023 versus SCD, **P = 0.0001 versus SCD. (B) Blood and lung bacterial titers at 24 hours after challenge of SCD mice receiving mock or simvastatin treatment. (C) Blood and lung bacterial titers at 24 hours after challenge of WT mice receiving mock or simvastatin treatment. Each symbol represents an individual mouse; horizontal lines indicate the mean; dashed lines indicate limit of detection. *P < 0.01, mock versus simvastatin; Mann-Whitney U test.

(A) Immunohistochemical detection (purple) of PAFr expression on HBMECs activated with TNF-α with or without simvastatin. Original magnification, ×200. (B) Relative bacterial adhesion to activated HBMECs with or without pretreatment with simvastatin, and reversal by mevalonate. *P < 0.01 versus no treatment; ^†P < 0.01 versus statin; ^‡P < 0.01 versus TNF plus statin. Error bars represent SD. (C) Detection of PAFr by Western blot analysis in activated HBMEC lysates and infected lung homogenates of SCD mice with or without simvastatin treatment. β-Actin served as the loading control. (D) Uninfected lung sections of SCD (with or without simvastatin), WT, and SCD Ptafr^–/– mice showing bronchial epithelium stained for PAFr (purple). Arrowheads indicate staining in sections showing patchy expression. Original magnification, ×100.

(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.