Neutrophils maintain sex differences and provide protection during LPS challenge–induced mortality. (A) Schematic of the experimental design for neutrophil depletion during primary LPS challenge. Age-matched male and female mice received anti–Ly6G monoclonal antibody (mAb; 400 μg per mouse) to deplete neutrophils or isotype control antibody on day −1 and day 0. On day 0, mice were challenged with 5 mg/kg LPS (IP) and monitored for survival. (B) Left: representative flow cytometry plot showing depletion of neutrophils in PBLs of mice treated with anti–Ly6G Ab. Right: cumulative data of CD11b⁺Ly6G⁺ neutrophil frequencies in all isotype and anti–Ly6G-treated mice. (C) Survival curve of control and neutrophil-depleted mice following LPS challenge. (D) Data from panel C analyzed separately for male and female survival curves in IgG control and anti–Ly6G-treated groups. (E) Schematic of the experimental design for survival analysis of mice rechallenged with LPS. Age-matched male and female mice were primed with 3 mg/kg LPS (IP) on day 0. On day 7, survivors were rechallenged with a lethal dose of 35 mg/kg LPS (IP) and monitored for survival. Some LPS-primed mice were treated with anti–Ly6G Ab (400 μg/mouse) or isotype control Ab on day 6 and day 7 to deplete neutrophils before rechallenge. (F) Naïve male and female mice were treated with 35 mg/kg LPS (IP) and monitored for survival to assess baseline susceptibility. (G) Survival curve of control or neutrophil-depleted LPS-tolerized mice, rechallenged with 35 mg/kg LPS. (H) Data in panel G analyzed separately for male and female survival curves in IgG control and anti–Ly6G-treated groups. Survival curves were analyzed using the log rank (Mantel-Cox) test. P values < .05 were considered to indicate statistical significance.