Fig 2: SEMA3F promotes neutrophil rounding and F-actin disassembly.(A–C) sema3fa or sema3fb RNA (50 ng/µL) was injected into 1-cell-stage Tg(mpx:LifeActRuby) embryos, tail fin transection was performed at 2 dpf, and neutrophil fluorescence intensities were calculated (A). Representative images (B). White arrows indicate the direction to the wound edge. Roundness scores were calculated for neutrophils recruited to the injury (C). Data are mean ± SEM from an single experiment (n = 9). (D–G) Human blood neutrophils were pretreated with PBS or 100 nM SEMA3F before stimulating with PBS or 100 nM fMLF. Following DAPI/cell mask/phalloidin staining, neutrophil rounding was quantified by high-content widefield microscopy (1 = perfect sphere), with more than 1000 cells measured per condition (D). Pixel intensity for phalloidin was obtained using confocal microscopy (×100 objective), scale bars: 5 µm (E). F-actin cell content was quantified with more than 1000 cells measured per condition (F and G) and distribution was used to calculate a polarity index (H). Data are mean ± SEM from 4 independent experiments (n = 4–22). (I–K) Intratracheal SEMA3F (1 µM) was administered to C57BL/6 mice 24 hours after nebulized LPS or PBS, and lung tissue (I and J) or BAL fluid (K) was collected at 48 hours. Lungs were instilled with agarose gel, fixed and stained with the endothelial CD31 and the neutrophil marker S100A9, and imaged by confocal microscopy. The percentage of neutrophils of each sphericity (I) and the mean sphericity of neutrophils (J) was defined. F/G actin ratios per cell were calculated from fluorescence intensities following staining with phalloidin (F-actin) and DNAse 1 (G-actin), with a latrunculin B (LTNB) negative control (K). Data are mean ± SEM from 2 independent experiments (n = 9) (I–K). Statistical analysis was by 1-way ANOVA and Bonferroni’s post hoc test (A–D and H), with Sidak-Holm multiple comparison posttest (K) and paired t test (G) performed for time points 15 to 30 minutes during the steady state of F-actin turnover, and unpaired t test (I–J). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: Neutrophil-driven deletion of Sema3f favors a selective allocation of neutrophils in the alveolar space while retaining antimicrobial capacity.(A and B) Sema3ffl/flMrp8Cre–/– (WT) and Sema3ffl/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.