Figure 4

Figure 4

(A and B) Sema3f^fl/flMrp8Cre^–/– (WT) and Sema3f^fl/flMrp8Cre^+/– (KO) mice were challenged with nebulized LPS and sacrificed at 0, 2, and 6 hours following LPS challenge. Blood and lung tissues were harvested with lung digest for Ly6G staining (neutrophil number). In a parallel series of experiments, lungs were instilled with agarose gel at 6 hours, then fixed and stained with the endothelial marker CD31 (green) and the neutrophil marker S100A9 (red). Lungs were imaged by confocal microscopy (Zeiss LSM 880 with Airyscan) with 3D reconstruction and neutrophil position relative to the blood vessels assigned, using Imaris software version 9.1 (neutrophil, white arrows) (C). Percentage of total neutrophils per 10⁶ μm³ lung tissue is shown, with a minimum of 190 neutrophils quantified per mouse (D). Data are mean ± SEM from 3 individual experiments (n = 3–5). (E and F) WT and KO mice were challenged with intratracheal instillation of S. pneumoniae, and lung bacterial counts (E) and BAL and lung neutrophil counts (F) were undertaken 14 hours after challenge. Data are mean ± SEM from 2 individual experiments (n = 6–10). Statistical analysis was by 2-way ANOVA with Sidak’s post hoc test (A, B, and D) and Mann-Whitney (E) and 1-tailed unpaired t test (F). *P < 0.05.