Fig. 6.

tACPA binds to (pre-)NETs, which are taken up by macrophages in vivo. a Representative images of tACPA binding in A23187-induced NET release in HV neutrophils. NETs were stained with DAPI (blue), anti-mIgG antibody (red), and ch-tACPA (green). Binding of m-tACPA and ch-tACPA to pre-NETs and extracellular NETs is indicated with yellow and white arrowheads, respectively. Scale bars: 50 µm. To induce peritoneal cell infiltration and NET formation, pristane was injected into the peritoneum of mice with 1 or 2 i.p. injections of 50 mg/kg dc-tACPA at 0 h (b) or at 0 and 12 h (c, d and e). After 24 h, inflammatory cells were harvested from the peritoneum and analyzed. b Representative images showing dc-tACPA binding to pre-NETs and NETs from the peritoneum of mice with pristane-induced peritonitis. NETs and dc-tACPA were stained with Sytox Green and anti-human IgG (red), respectively. Scale bars: 25 µm. c Representative images of tACPA-opsonized NETs, which have been phagocytosed by macrophages. NETs containing NE in combination with hIgG (tACPA) were present in macrophages from animals that had been treated with dc-tACPA only. Hoechst (blue), the macrophage marker F4/80 (magenta), anti-NE (green) and anti-hIgG (cyan). Scale bars: 10 µm. d, e Quantification of phagocytosed hIgG (% anti-hIgG per macrophage image) and neutrophil elastase (% anti-NE per macrophage image) (n = 58–176 macrophages from three mice). The results are presented as the means ± SEM. ***P < 0.001, ****P < 0.0001 using two-way ANOVA with Dunnett’s multiple comparisons test