Fig 2: Pyroptosis is induced in neutrophils in septic mice.C57 BL/6 mice were randomly assigned to sham or CLP-induced sepsis group, n = 20 per group for the survival analysis, n = 6 per group for other experiments. a Survival of sham and CLP-induced septic mice. b For pulmonary histology analysis, murine lungs were harvested. Lung injury was assessed by H&E staining. Lung injury score was recorded (Scale bar, 50 µm). c Wet/dry ratio of lung tissues were calculated. d CD11b+Ly6G+ neutrophils in the peripheral blood, spleens, and BALF of sham and CLP mice were detected by flow cytometry. e LDH activity was determined in neutrophils to indicate cell death. f Pyroptosis-related proteins were detected in the neutrophils isolated from sham or CLP-induced septic mice by western blot. g Activity of caspase 1 and caspase 4 in neutrophils of sham or CLP-induced septic mice were measured by colorimetric assay. h Concentration of IL-1ß and IL-18 in the supernatant of neutrophil cultures were assessed by ELISA. Experiments were repeated for three times independently and representative image from one of the experiments were shown. Data are presented as dot-plots of individual experiments and mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Resolving neutrophils trained by 4-PBA express elevated levels of cell surface CD86/CD200R, increased RvD1/SerpinB1, and reduced TNF-a.A Flow analyses of mature Ly6GhiCD11b+ neutrophils. WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours were subjected to flow analyses and the percentages of Ly6GhiCD11b+ neutrophils were plotted. (n = 3). B The mean fluorescent intensity (MFI) levels of Ly6G on WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours (n = 3). C The levels of CD86 and CD200R on WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours (n = 3). D The expression of RvD1 and SerpinB1 from WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours (n = 3). E The secretion of TNF-a from WT neutrophils treated with PBS or 4-PBA (1 mM) for 24 hours followed by PMA priming (100 ng/ml) for 30 min (n = 3). Data plotted as mean ± SD. ****P < 0.0001, ***P < 0.001, **P < 0.01 using two-sided Student’s t test.

Fig 4: IFN-? regulates macrophage inflammatory profile during normal tissue repair.(A) IFN-?–KO and control wound cell isolates were processed for flow cytometry using the following gating strategy selecting for single cells, live (viability dye–), CD45+, Ly6G-, CD11b+, Ly6Chi, or Ly6Clo. (B and C) Flow cytometry quantification of Ly6Chi and Ly6Clo cells in wounds (n = 4–6 per group). (D) Wound monocyte/macrophages (MF) (CD3–CD19–Ly6G–CD11b+) were isolated from WT and IFN-?–KO mice on day 3; n = 3 per group, repeated in triplicate. Gene expression of inflammatory cytokines Tnf, Il1b, and Il12 was measured via qPCR. Data were analyzed for variances, and 2-tailed Student’s t test or 1-way ANOVA was performed. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are presented as mean ± SEM.

Fig 5: Identification of conducting airway wall neutrophils using anti-Ly6G or anti-Gr-1 antibodies. (a) Three-dimensional volume-rendering z-stack images of the conducting airway wall 6 h after the mice received A. fumigatus conidia at low (left panel, grid spacing: 20 µm) and high (middle panel, grid spacing: 5 µm) magnification showing Ly6G+ (upper row, green) or Gr-1+ cells (lower row, green), the nuclei (NucBlue, blue), and the smooth muscle layer (ActinRed, light cyan). Images of indicated neutrophils were magnified (right panels) and are presented as slices (scale bar: 5 µm). (b) Quantitative analysis of Gr-1+ and Ly6G+ cells in the conducting airway of mice just before (0 h) or 6 h after A. fumigatus conidium application. The data shown represent the average of four measurements from each independent specimen (n = 4 mice). The data were analyzed using the Mann–Whitney U test. ns: not significant.