Figure 5. TTP promotes neutrophil apoptosis in a cell-intrinsic manner.

(A) Apoptosis was analyzed in peritoneal neutrophils elicited using 2 × 10⁸ CFU HK S. pyogenes 16 hours prior to flushing of the peritoneal cavity. Representative flow plots show annexin V and live/dead staining of Ly6G⁺Ly6C⁺ neutrophils. The threshold for annexin V⁺ signal was based on fluorescence minus one (FMO) staining without annexin V. Numbers represent the percentage of cells in the respective quadrants. Dot plot for 3 pooled experiments (n = 13 TTP^fl/fl; n = 17 TTP^ΔM) depicts the percentages of early apoptotic neutrophils (living annexin V⁺). (B) Neutrophils, elicited as in A, were analyzed for mitochondrial superoxide production (n = 9 TTP^fl/fl; n = 8 TTP^ΔM), polarization of mitochondrial membrane (n = 7 TTP^fl/fl; n = 8 TTP^ΔM), and active caspase-3 (n = 8 TTP^fl/fl; n = 8 TTP^ΔM). (C) TTP^fl/fl and TTP^ΔM BMDMs were incubated with pHrodo-stained nonapoptotic or apoptotic TTP^fl/fl and TTP^ΔM neutrophils at a BMDM/neutrophil ratio of 1:2. CytD-pretreated BMDMs served as a control for adherent neutrophils. Bar graph shows the percentage of pHrodo⁺F4/80⁺ BMDMs (n = 3). Error bars indicate the mean ± SD. (D) Casein-elicited neutrophils from TTP^fl/fl and TTP^ΔM mice were differently labeled, mixed at a 1:1 ratio, and injected i.p. into 4 recipient TTP^fl/fl animals, in which peritonitis was induced 3 hours earlier using HK S. pyogenes. Representative flow plots 18 hours after transplantation and dot plots show retrieved transplanted neutrophils and the percentage of annexin V⁺ cells therein. (E) Neutrophil migration through a collagen matrix was followed by live imaging. Box plots show the average speed and displacement length of TTP^fl/fl and TTP^ΔM neutrophils in untreated conditions or in a CXCL1 gradient. Error bars in A, B, and D represent the mean. Statistical analysis was determined by unpaired (A–D) or paired (E) Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.