Figure 8.

Staphylocoagulases prevent NETs (neutrophil extracellular traps) from constraining bacterial infection and promoting tissue destruction. A, Brightfield images of a Brown-Hopps Gram stain (n=10, 10, 4) with bacteria represented in purple. B, Click-iT Plus TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay stained images (n=10,10, 4), depicting apoptotic cells in red. C and D, Fluorescence microscopy images (n=10, 10, 4) of Staphylococcus aureus in green and neutrophil-specific marker Ly6G in red (C), and MPO (myeloperoxidase) in red and H3Cit (citrullinated histone H3) in green (D). DNA is stained with Hoechst 33342 in blue. Scale bars represent 200 μm. E–L, Quantifications of extraluminal leukocyte infiltration (E), TUNEL (F), bacteria (H), Ly6G (K), and MPO (L)-positive area situated outside infected thrombi and H3Cit inside the thrombus (I) in control (C57Bl/6J) mice with infective endocarditis induced by wild-type (WT; black, n=10) S. aureus, and in control (C57Bl/6J and PAD4 (peptidylarginine deiminase 4)^fl/fl, dark gray, n=10) and neutrophil-selective PAD4 knockout mice (MRP8Cre⁺×PAD4^fl/fl, light gray, n=4) with infective endocarditis induced by S. aureus coaΔvwb (coagulase and von Willebrand factor binding protein). G, Spearman correlation between the H3Cit-positive area and TUNEL-positive area, both located outside the infected vegetations caused by WT (n=10, C57Bl/6J) and S. aureus ΔcoaΔvwb (n=10, C57Bl/6J and PAD4^fl/fl). J, Spearman correlation between bacteria located outside the vegetation and H3Cit inside the thrombus infected with WT (n=10, C57Bl/6J) and S. aureus ΔcoaΔvwb (n=10, C57Bl/6J and PAD4^fl/fl). Median (interquartile range) are represented in E and F, H and I, K and L. Significance was determined by Kruskal-Wallis test with Dunn post test (E and F, H and I, K and L). MRP8Cre+ indicates myeloid related protein 8, Cre recombinase expressing.