Figure 3.
Platelets regulate IL-10 and TGFβ cytokine homeostasis and guide T reg cells to inflamed alveoli. (A) Bacterial pneumonia was induced in WT mice and red fluorescent platelets isolated from DsRed+ mice were injected at day 3 and detected in the BAL fluid by flow cytometry on day 5 (representative plot from two experiments with four to six mice in each group). Ratios of DsRed+:WT platelets are provided for days 3 and 5. (B) Bacterial pneumonia was induced in WT mice and platelets in the BAL fluid were analyzed by flow cytometry (data pooled from at least two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (C) Isolated DsRed+ platelets were injected into WT mice before induction of bacterial pneumonia and platelets in the BAL fluid were analyzed by flow cytometry. Ratios of DsRed+:WT platelets are provided each day (data pooled from three independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (D) Bacterial pneumonia was induced in WT and PF4+/+iDTR+DT mice (receiving DT) by i.t. instillation of viable K. pneumoniae. On day 3, platelet-depleted mice were reconstituted with isolated WT donor platelets by intravascular injection at different concentrations and recruitment of T reg cells in the BAL fluid was analyzed by flow cytometry on day 5. (E and F) Bacterial pneumonia was induced in WT, PF4+/+iDTR+DT, and DEREG mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFU/mouse) and the concentrations of the cytokines IL-10 (E) and TGFβ in the BAL (F) were analyzed by ELISA (data pooled from two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Bacterial pneumonia was induced in DEREG mice or DEREG mice that were reconstituted with isolated WT donor T reg cells or recombinant IL-10 (200 ng/mouse i.t.) 2 d after induction of pneumonia. (G and H) The number of neutrophils in the BAL fluid (G) and the percentage of apoptotic neutrophils in the BAL (H) was analyzed by flow cytometry (data pooled from two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (I and J) 14 d after induction of pneumonia, lung collagen content (I) and lung compliance (J) were analyzed (data pooled from at least two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). PLT, platelet.

Platelets regulate IL-10 and TGFβ cytokine homeostasis and guide T reg cells to inflamed alveoli. (A) Bacterial pneumonia was induced in WT mice and red fluorescent platelets isolated from DsRed+ mice were injected at day 3 and detected in the BAL fluid by flow cytometry on day 5 (representative plot from two experiments with four to six mice in each group). Ratios of DsRed+:WT platelets are provided for days 3 and 5. (B) Bacterial pneumonia was induced in WT mice and platelets in the BAL fluid were analyzed by flow cytometry (data pooled from at least two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (C) Isolated DsRed+ platelets were injected into WT mice before induction of bacterial pneumonia and platelets in the BAL fluid were analyzed by flow cytometry. Ratios of DsRed+:WT platelets are provided each day (data pooled from three independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (D) Bacterial pneumonia was induced in WT and PF4+/+iDTR+DT mice (receiving DT) by i.t. instillation of viable K. pneumoniae. On day 3, platelet-depleted mice were reconstituted with isolated WT donor platelets by intravascular injection at different concentrations and recruitment of T reg cells in the BAL fluid was analyzed by flow cytometry on day 5. (E and F) Bacterial pneumonia was induced in WT, PF4+/+iDTR+DT, and DEREG mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFU/mouse) and the concentrations of the cytokines IL-10 (E) and TGFβ in the BAL (F) were analyzed by ELISA (data pooled from two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Bacterial pneumonia was induced in DEREG mice or DEREG mice that were reconstituted with isolated WT donor T reg cells or recombinant IL-10 (200 ng/mouse i.t.) 2 d after induction of pneumonia. (G and H) The number of neutrophils in the BAL fluid (G) and the percentage of apoptotic neutrophils in the BAL (H) was analyzed by flow cytometry (data pooled from two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (I and J) 14 d after induction of pneumonia, lung collagen content (I) and lung compliance (J) were analyzed (data pooled from at least two independent experiments with four to six mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). PLT, platelet.

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