Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , Boxplots showing quantification of MPO–DNA and dsDNA (NET structures) in the plasma of HS ( n = 53) and patients with severe sepsis (with or without septic shock) ( n = 51). The boxes indicate the 25% quantile, median and 75% quantile. b , Scatterplots displaying correlations of platelet pyroptosis (activated caspase 1 positive platelets) with the levels of MPO–DNA (HS, n = 15; severe sepsis, n = 34) complexes and dsDNA (HS, n = 20; severe sepsis, n = 29) in the plasma of patients with severe sepsis (with or without septic shock) and HS ( r , correlation coefficient; n = 49). c , Immunofluorescence analysis showing S100A8/A9-induced pyroptotic platelets induced NET formation. Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (green) and CD41 for platelets (red). Scale bars, 25 μm and 7.5 μm. d , Quantification of MPO–DNA and dsDNA in the supernatant of cells ( n = 6). e , Immunofluorescence analysis showing NET formation in PMNs treated with rmS100A8/A9-induced platelets (WT or Tlr4 −/− ) (n = 6). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (red) and CD41 for platelets (green). Scale bars, 25 μm and 7.5 μm. f , Bar graphs displaying the levels of MPO–DNA and dsDNA in the supernatant of cells treated with rmS100A8/A9-induced platelets ( Tlr4 −/− or WT) ( n = 6). g , Levels of ox-mtDNA in supernatant of S100A8/A9-induced pyroptotic platelets (for 4 h) determined using a General 8-OHdG ELISA Kit ( n = 5). h , Levels of ox-mtDNA in plasma from sepsis patients (sepsis, n = 11; severe sepsis, n = 17) and HS ( n = 13). i , Levels of ox-mtDNA in plasma of sham (n = 7) or CLP mice ( n = 9). Data were presented as mean ± s.d. Statistical analysis was conducted using two-tailed Mann–Whitney test ( a ), two-tailed Pearson’s correlation test ( b ), one-way ANOVA and Tukey’s multiple comparisons test ( d , f , g ), Kruskal–Wallis test and Dunn’s multiple comparisons test ( h ) and two-tailed unpaired t -test ( i ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , S100A8/A9 released from NETs determined by ELISA ( n = 5). b , Representative immunofluorescence images of pyroptotic platelets incubated with PMA-treated murine PMNs (WT or S100a9 −/− ). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (cyan), CD41 for platelets (red) and caspase 1 for pyroptosis (green). Scale bars, 25 μm and 5 μm. c , Bar graphs displaying mitochondrial ROS production in platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) using FACS analysis ( n = 5). d , Immunofluorescence analysis showing coexpression of CD41 (green), ASC (red) and NLRP3 (blue) in platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) ( n = 5). Purple indicates overlap. Scale bars, 5 μm and 1 μm. e , Quantified results for the NLRP3 inflammasome are shown. f , Bar graphs displaying the caspase 1 activity of platelets cocultured with PMA-treated murine PMNs (WT or S100a9 −/− ) by FACS analysis ( n = 5). Data were presented as mean ± s.d. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparisons test ( a , c , e , f ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Incubation, Staining, Activity Assay

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , PMNs ( S100a9 -/- or WT) were incubated with 50 nM PMA to induced NET formation for 4 hours, and then incubated with platelets for another 4 hours. Representative immunofluorescence of PMNs incubated with platelets. Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (cyan), CD41 for platelet (red) and activated caspase 1 for pyroptosis (green); scale bars: 25 μm and 5 μm. b , In the CLP-induced sepsis model, platelet counts in Gsdmd fl/fl PF4-Cre mice and littermate control Gsdmd fl/fl mice were assessed at 0, 2, 4, and 6 hours using a hematology analyzer (n = 5). Data was presented as mean ± SD. Two-way ANOVA and Tukey’s multiple comparisons test for b. Abbreviation is as follow: PLT, platelet; NS, not statistically significant; PMNs, polymorphonuclear neutrophils; PMA, phorbol myristate acetate; MPO, myeloperoxidase; Sham, sham-operated mice; CLP, CLP-induced sepsis mice; GSDMD, Gasdermin D.

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Incubation, Immunofluorescence, Staining

Journal: Nature Cardiovascular Research

Article Title: Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis

doi: 10.1038/s44161-022-00108-7

Figure Lengend Snippet: a , Immunofluorescence analysis showing NET formation induced by platelets from Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice treated with S100A8/A9 ( n = 5). Cells were stained with Hoechst for DNA (blue), anti-MPO for PMNs or NETs (red) and CD41 for platelets (green). Scale bars, 25 μm and 7.5 μm. b , Bar graphs displaying the levels of MPO–DNA and dsDNA in supernatant of rmS100A8/A9-induced platelets from Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice using PicoGreen fluorescent dye and MPO–DNA–ELISA, respectively ( n = 5). c – h , In the CLP-induced sepsis murine model, levels of NETs, heterodimer S100A8/A9 and proinflammatory cytokines in plasma of Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice were detected using ELISA kits after 6 h. c , d , Bar graphs displaying plasma levels of MPO–DNA complexes ( c ) and dsDNA ( d ) in mice ( n = 5). e , Bar graphs showing plasma levels of heterodimer S100A8/A9 in mice ( Gsdmd fl/fl + sham, n = 7; Gsdmd fl/fl + CLP, n = 8; Gsdmd fl/fl PF4-Cre+sham, n = 8; Gsdmd fl/fl PF4-Cre+CLP, n = 6). f – h , Bar graphs showing plasma levels of IL-1β ( f ), TNF-α ( g ) and IL-6 ( h ) in mice ( n = 5). i , Survival analysis of Gsdmd fl/fl PF4-Cre mice and Gsdmd fl/fl mice in CLP-induced sepsis model for 7 d ( n = 11). Data were presented as mean ± s.d. Statistical analysis was conducted using one-way ANOVA and Tukey’s multiple comparisons test ( b – h ) and the log-rank (Mantel–Cox) test ( i ).

Article Snippet: MPO–DNA complexes and dsDNA in plasma of human/mice and medium supernatant were detected by anti-MPO polyclonal (biotin conjugated) primary antibody (Bioss, 1:500, bs-4943R-Biotin) with a Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche, 11774425001) and Quant-iT PicoGreen dsDNA reagent (Invitrogen, catalog no. P7581) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay