Figure 3.

ACE overexpression in neutrophils enhances antibacterial resistance in mice. (A) Resistance to MRSA infection in WT and Neu^ACE mice. Mice were infected subcutaneously with MRSA (2 × 10⁸ CFU/mouse flank) under basal and neutrophil-depleted conditions. For neutrophil depletion, mice were administered anti-mouse polymorphonuclear neutrophil antibody (250 µg/day/mouse) beginning 1 day before MRSA infection and continuing until the end of the experiment. (i) Representative image of skin lesions at day 3 postinfection, (ii) skin lesion area, and (iii) bacterial burden (CFU) per lesion. (B) Similar experiments were conducted with mice treated with ramipril (36.3 mg/L in drinking water) for 1 week before MRSA infection. (C) Blood from Neu^ACE mice cleared MRSA more effectively than blood from WT mice at both 2 and 5 hours. Blood samples were infected with MRSA at ∼10⁶ CFU/mL. However, when this assay was performed with blood from mice pretreated with ramipril for 7 days, no difference in bacterial clearance was observed between the 2 groups. (D) Bacterial phagocytosis (internalization) was determined using GFP-Staph. Bone marrow neutrophils were purified and infected with GFP-Staph. (MOI ∼20). The time-dependent percentage of GFP⁺ neutrophils was determined by FCM (n = 4/group). (E) Measurement of neutrophil phagocytic killing. Following 20 minutes of phagocytosis (MOI ∼10, considered 0 time) and killing extracellular bacteria with gentamycin (400 µg/mL), the loss of GFP from neutrophils was determined by FCM. This parallels the killing of ingested bacteria (n = 4/group). (F) Intracellular killing of MRSA in bone marrow neutrophils. After 20 minutes of phagocytosis (MOI ∼10, considered 0 time), extracellular bacteria were killed with gentamycin and then bacterial survival was determined by CFU counting at 0, 2, and 5 hours (n = 4/group). *P ≤ .05, **P ≤ .005, ***P ≤ .0005. NS, nonsignificant.