The second wave of NETs on day 14 of systemic inflammation is induced by the prolonged presence of their first wave remnants that prime the environment for the subsequent insult/infection. Impact of the first wave of NETs on the second 1 was tested by interfering with them either on day 13 or at the beginning of endotoxemia (LPS IP; 1 mg/kg b.w.). (A-D) On day 13, mice were injected IV with ADAMTS13, DNase I, or a mixture of both. Then mice were imaged on day 14 day and the influx of neutrophils into the liver (A) as well as coverage of liver endothelium by extDNA (B), NE (C), and H2A.X histones (D) were examined. (J) Representative images of NET formation on day 14 in animals treated with ADAMTS13, DNase I, or a mixture of both on day 13 in the morning. Histones were stained with AF568-anti-H2A.X Abs (red), NE with AF647–anti-NE Abs (violet), and extDNA with SYTOX Green (bright green). The colocalization of NET components was visualized by overlaying the images from individual channels. The scale bar indicates 50 μm. (E-I) Additionally some animals were pretreated with PAD4 inhibitor before LPS or IV injected with DNase I 4 hours after LPS to prevent or remove the first wave of NETs, respectively. Then neutrophil numbers (E) and NETs were evaluated by measurement of extDNA (F), NE (G), and histones (H) on day 14. Furthermore, platelet presence/aggregation was evaluated (I). (K-O) To verify the impact of the second wave of NETs on the outcome of the subsequent insult or infection, on days 14 and 165, mice were treated with LPS (+LPS) or MRSA (+MRSA), respectively. Numbers of neutrophils (K), NET formation (extDNA [L]; histones [M]), and levels of ALT (N) were evaluated. (O) Representative images of NETs (histones) upon different treatments as in panels K-N. Histones were stained with AF568–anti-H2A.X Abs (red), NE with AF647–anti-NE Abs (violet), and extDNA with SYTOX Green (bright green). The colocalization of NET components was visualized by overlaying the images from individual channels. The data in the graphs are expressed as the mean ± SD of at least 3 fields of view. Statistically significant differences according to the 1-way ANOVA are designated by letters, in which the same letter indicates no differences between groups, and different letters indicate statistical differences (Bonferroni post hoc; n ≥ 3). Asterisks in the graphs indicate statistically significant differences according to the Student t test (∗∗∗.0001 < P ≤ .001). In panels K-N, “14 days LPS + MRSA” and “165 days LPS + MRSA” groups were compared with “24 hour MRSA” with a t test and differences are marked with ˆ (in which ˆ.01 < P ≤ .05; ˆˆ.001 < P ≤ .01; ˆˆˆˆ.00001 < P ≤ .001; n = 3).