Journal: The Journal of Experimental Medicine
Article Title: Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis
Figure Lengend Snippet: NETs contribute to the pathogenesis of myocarditis. (a) Confocal immunofluorescence images of an EMB of patient 6 suffering from PVB19-positive myocarditis (see Table 1 ). Top: Images show staining of H2A-H2B-DNA, MPO, and H3Cit as well as the merged image. Bar, 20 µm. Bottom: Magnification of a selected area of the merged confocal image (box) from the top panel. Arrows indicate NETs. Bar, 10 µm. (b and c) EAM score (b) as well as evaluation of the heart/body weight ratio (c) at day 21 after induction of EAM or sham immunization (sham; mean EAM score, 0.5 ± 0.19; mean heart/body weight ratio, 0.57 ± 0.01). Mice were treated with DNase (EAM + DNase: mean EAM score, 0.8 ± 0.19; mean heart/body weight ratio, 0.62 ± 0.005), Cl-amid (EAM + Cl-amid: mean EAM score, 0.9 ± 0.20; mean heart/body weight ratio, 0.62 ± 0.01), or vehicle as control (EAM + vehicle: mean EAM score, 1.7 ± 0.25; mean heart/body weight ratio, 0.71 ± 0.04) as indicated. n = 8 for sham group; n = 26 for EAM groups. (d) Left: Representative confocal images of cardiac sections of immunized WT mice at day 21 after induction of EAM (EAM + vehicle). Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. Right: Representative confocal magnified image of NETs. Images show staining of DNA, NE, and H3Cit as well as the merged image, colocalization (coloc) of DNA/NE, and DNA/H3Cit as indicated. Bars, 10 µm. (e) Representative confocal images of cardiac sections of EAM mice at day 21 after induction of EAM and blockade of MK for 21 d. Images show staining of DNA, NE, and H3Cit as well as the merged image. Bars, 20 µm. (f–h) Histological analysis of extravasated PMN into the cardiac tissue at day 21 after induction of EAM without blocking MK (EAM + vehicle) or after application of an anti-N-MK blocking Ab (EAM + anti-N-MK). Diagrams show the number of extravasated PMNs per mm 2 (f: vehicle, 768 ± 55 PMN/mm 2 ; anti-N-MK, 158 ± 86 PMN/mm 2 ), the number of NET + PMNs per mm 2 (g: vehicle, 40 ± 6 NET + PMN; anti-N-MK = 5 ± 2 NET + PMN), and the number of NET + PMNs as a percentage of all extravasated PMNs (h: vehicle, 5.1 ± 0.5 NET + PMN [%]; anti-N-MK, 0.9 ± 0.6 NET + PMN [%]). n = 6 mice. To determine P values, ANOVA on ranks with Dunn’s multiple comparisons test was performed for c and d, and an unpaired Student's t test was used for f–h. For all panels, *, P
Article Snippet: RNA was isolated using TRIzol (15596026; Thermo Fisher) according to the manufacturer’s protocol, and genomic DNA was digested using the DNase I Amplification Grade kit (18068015; Thermo Fisher).
Techniques: Immunofluorescence, Staining, Mouse Assay, Blocking Assay