ACE increases neutrophil oxidative response. (A) ROS production in WT and Neu^ACE neutrophils under basal conditions and 48 hours after MRSA infection. ROS production by neutrophils was assessed by FCM analysis of DHR 123 oxidation. ROS production was significantly higher in Neu^ACE vs WT neutrophils. (B) ROS inhibition by DPI (3 mg/kg per day, intraperitoneal [IP]) eliminated the increased bacterial resistance of Neu^ACE mice. DPI injection was started 1 day before subcutaneous MRSA infection (1 × 10⁸ CFU/mouse flank) and continued until the end of the experiment. (i) Skin lesion area, (ii) Bacterial counts (CFU) in lesions after 3 days. (C) Superoxide production was measured by SOD-inhibitable cytochrome c reduction. Neutrophils were purified from bone marrow and challenged with MRSA in vitro (n = 5/group). (D) Upon activation, the level of NOX2 subunits was measured by western blotting of neutrophil membranes. Neutrophils were purified from bone marrow and activated with MRSA for 15 minutes, and then membranes were isolated using a kit from ThermoFisher. (E) Expression of NOX2 subunits was determined by western blotting of total neutrophil protein lysate. (F) p47-phox phosphorylation (p-Ser345) was determined in neutrophil membranes by western blot analysis. Neutrophils were purified from bone marrow and activated with MRSA (MOI ∼10): (i) groups without MRSA infection (0 minutes) were used as a control, (ii) phospho-p47-phox at 5 minutes after MRSA infection, (iii) phospho-p47-phox at 15 minutes after MRSA infection, (iv) groups with ACE inhibition by ramipril. (G) Effect of gp91 ds-tat, a specific NOX2 inhibitor, on blood killing of MRSA. Blood samples were treated with gp91 ds-tat peptide for 90 minutes (5 μM), and then infected with MRSA at ∼10⁶ CFU/mL. NOX2 inhibition eliminated the difference between WT and Neu^ACE bacterial killing. (D-G) Data are representative of at least 4 mice in each group. *P ≤ .05, **P ≤ .005, ***P ≤ .0005.