Figure 2.
Platelets differentially bind to neutrophils and T reg cells at distinct time points of an inflammatory response. Bacterial pneumonia was induced in WT mice and PF4+/+iDTR+DT (receiving DT) mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFU/mouse). Some mice received a platelet-depleting antibody (clone R300, 2 µg/g body weight) or isotype control 2 d after induction of pneumonia. (A) Platelet–neutrophil and platelet–T reg cell aggregates in the circulation were analyzed by flow cytometry. Data pooled from three independent experiments with four to six mice in each group (data pooled from at least two independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (B and C) Immunofluorescence staining and 3D reconstruction of (B) platelet–neutrophil and (C) platelet–T reg cell aggregates in peripheral blood samples. Exemplary images are shown from at least two experimental repetitions. Scale bar, 10 µm. (D) Blood counts of T reg cells were analyzed by flow cytometry (data pooled from two independent experiments with four to five samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (E) The number of T reg cells in the BAL fluid was analyzed by flow cytometry (data pooled from two independent experiments with four to six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Pneumonia was induced in WT mice by i.t. instillation of K. pneumomia (data pooled from two independent experiments with four to six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (F–K) The expression of (F) PSGL-1 and (G) Mac-1 on neutrophils, (H) P-selectin and (I) GPIb on single platelets, and (J) PSGL-1 on T reg cells and ADAM8 on neutrophils (K) was analyzed at the indicated time points (data pooled from at least two independent experiments with four to five samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (L) Isolated WT neutrophils were pretreated with a blocking PSGL-1 antibody (clone 4RA10, 5 µg/ml) or rADAM8 (1–300 pg/ml), coincubated with isolated WT platelets, and platelet–neutrophil aggregates were analyzed by flow cytometry (data pooled from two independent experiments with four to six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (M and N) After induction of bacterial pneumonia animals were treated with BK-1361 (25 µg/g) and circulating platelet–neutrophil (M) platelet–T reg cell aggregates (N) were analyzed by flow cytometry at the indicated time points (data pooled from two independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). MFI, mean fluorescence intensity; PLT, platelet.

Platelets differentially bind to neutrophils and T reg cells at distinct time points of an inflammatory response. Bacterial pneumonia was induced in WT mice and PF4+/+iDTR+DT (receiving DT) mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFU/mouse). Some mice received a platelet-depleting antibody (clone R300, 2 µg/g body weight) or isotype control 2 d after induction of pneumonia. (A) Platelet–neutrophil and platelet–T reg cell aggregates in the circulation were analyzed by flow cytometry. Data pooled from three independent experiments with four to six mice in each group (data pooled from at least two independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (B and C) Immunofluorescence staining and 3D reconstruction of (B) platelet–neutrophil and (C) platelet–T reg cell aggregates in peripheral blood samples. Exemplary images are shown from at least two experimental repetitions. Scale bar, 10 µm. (D) Blood counts of T reg cells were analyzed by flow cytometry (data pooled from two independent experiments with four to five samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (E) The number of T reg cells in the BAL fluid was analyzed by flow cytometry (data pooled from two independent experiments with four to six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Pneumonia was induced in WT mice by i.t. instillation of K. pneumomia (data pooled from two independent experiments with four to six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (F–K) The expression of (F) PSGL-1 and (G) Mac-1 on neutrophils, (H) P-selectin and (I) GPIb on single platelets, and (J) PSGL-1 on T reg cells and ADAM8 on neutrophils (K) was analyzed at the indicated time points (data pooled from at least two independent experiments with four to five samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (L) Isolated WT neutrophils were pretreated with a blocking PSGL-1 antibody (clone 4RA10, 5 µg/ml) or rADAM8 (1–300 pg/ml), coincubated with isolated WT platelets, and platelet–neutrophil aggregates were analyzed by flow cytometry (data pooled from two independent experiments with four to six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (M and N) After induction of bacterial pneumonia animals were treated with BK-1361 (25 µg/g) and circulating platelet–neutrophil (M) platelet–T reg cell aggregates (N) were analyzed by flow cytometry at the indicated time points (data pooled from two independent experiments with six samples in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). MFI, mean fluorescence intensity; PLT, platelet.

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