Figure 6.
Intra-alveolar platelets drive polarization toward resolving macrophages. Bacterial pneumonia was induced in WT, PF4+/+iDTR+DT (receiving DT), and DEREG mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFUs/mouse). (A) The total number of macrophages (Mϕ) in the BAL fluid was analyzed by flow cytometry (data pooled from at least two independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Pneumonia was induced in WT mice and PF4+/+iDTR+DT (receiving DT) mice. Mice were i.t. or i.v. reconstituted with the indicated number of nonactivated or ADP-activated (10 µm ADP) isolated WT donor platelets. (B and C) The number of neutrophils in the BAL fluid (B) and the percentage of apoptotic neutrophils in the BAL (C) were 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). To conduct RNaseq analysis of macrophages in the lung during resolution of pulmonary inflammation, pro- and anti-inflammatory macrophages were isolated from BAL fluid obtained at day 5 after induction of bacterial pneumonia from WT mice and platelet-depleted WT mice. (D and E) Heat map with hierarchical tree comparing differences of gene expression values induced by platelet depletion in (D) pro-inflammatory macrophages and (E) anti-inflammatory macrophages. Each line in heat map represents the changes in a given transcript. Yellow and blue colors represent increased and decreased gene expression, respectively. (F) Venn diagram of DE genes filtered by s-value < 0.01 in either pro-inflammatory and/or anti-inflammatory macrophages after platelet depletion. (G and H) Volcano plots comparing differences of gene expression values induced by platelet depletion in (G) pro-inflammatory macrophages and (H) anti-inflammatory macrophages (data pooled from three independent experiments with three mice in each group). Pneumonia was induced in control mice, in mice in which intravascular platelets were depleted by i.v. injection of a platelet-depleting antibody (clone R300, 2 µg/g bodyweight) 2 d after induction of pneumonia, and in mice that were i.t. reconstituted with unactivated or activated WT donor platelets. (I) Macrophage polarization in the BAL fluid obtained 5 d after induction of pneumonia was analyzed by qRT-PCR (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (J) Isolated alveolar macrophages from untreated WT mice were incubated with BAL fluid obtained from control mice and platelet-depleted mice 5 d after induction of pneumonia and macrophage polarization was analyzed by qRT-PCR (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (K) Efferocytosis index of macrophages obtained from control mice and PF4+/+iDTR+DT (receiving DT) mice 5 d after induction of pneumonia (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (L) Efferocytosis index of isolated alveolar macrophages from untreated mice incubated with BAL fluid obtained from control mice and PF4+/+iDTR+DT (receiving DT) mice 5 d after induction of pneumonia (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). AI, anti-inflammatory; PI, pro-inflammatory.

Intra-alveolar platelets drive polarization toward resolving macrophages. Bacterial pneumonia was induced in WT, PF4+/+iDTR+DT (receiving DT), and DEREG mice by i.t. instillation of viable K. pneumoniae (1.5 × 107 CFUs/mouse). (A) The total number of macrophages (Mϕ) in the BAL fluid was analyzed by flow cytometry (data pooled from at least two independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). Pneumonia was induced in WT mice and PF4+/+iDTR+DT (receiving DT) mice. Mice were i.t. or i.v. reconstituted with the indicated number of nonactivated or ADP-activated (10 µm ADP) isolated WT donor platelets. (B and C) The number of neutrophils in the BAL fluid (B) and the percentage of apoptotic neutrophils in the BAL (C) were 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). To conduct RNaseq analysis of macrophages in the lung during resolution of pulmonary inflammation, pro- and anti-inflammatory macrophages were isolated from BAL fluid obtained at day 5 after induction of bacterial pneumonia from WT mice and platelet-depleted WT mice. (D and E) Heat map with hierarchical tree comparing differences of gene expression values induced by platelet depletion in (D) pro-inflammatory macrophages and (E) anti-inflammatory macrophages. Each line in heat map represents the changes in a given transcript. Yellow and blue colors represent increased and decreased gene expression, respectively. (F) Venn diagram of DE genes filtered by s-value < 0.01 in either pro-inflammatory and/or anti-inflammatory macrophages after platelet depletion. (G and H) Volcano plots comparing differences of gene expression values induced by platelet depletion in (G) pro-inflammatory macrophages and (H) anti-inflammatory macrophages (data pooled from three independent experiments with three mice in each group). Pneumonia was induced in control mice, in mice in which intravascular platelets were depleted by i.v. injection of a platelet-depleting antibody (clone R300, 2 µg/g bodyweight) 2 d after induction of pneumonia, and in mice that were i.t. reconstituted with unactivated or activated WT donor platelets. (I) Macrophage polarization in the BAL fluid obtained 5 d after induction of pneumonia was analyzed by qRT-PCR (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (J) Isolated alveolar macrophages from untreated WT mice were incubated with BAL fluid obtained from control mice and platelet-depleted mice 5 d after induction of pneumonia and macrophage polarization was analyzed by qRT-PCR (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (K) Efferocytosis index of macrophages obtained from control mice and PF4+/+iDTR+DT (receiving DT) mice 5 d after induction of pneumonia (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). (L) Efferocytosis index of isolated alveolar macrophages from untreated mice incubated with BAL fluid obtained from control mice and PF4+/+iDTR+DT (receiving DT) mice 5 d after induction of pneumonia (data pooled from three independent experiments with 5–6 mice in each group; Mann-Whitney test was used for statistical analysis, *, P < 0.05). AI, anti-inflammatory; PI, pro-inflammatory.

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