Figure 4.
The appearance of the second wave of NETs on day 14 of systemic inflammation is linked to the proinflammatory phenotype. Changes in numbers of neutrophils (Ly6G+) present in liver sinusoids (A) and quantity of NET components (B-D) between day 13 and 14 after induction of endotoxemia (LPS IP; 1 mg/kg b.w.). For NET detection, levels of extDNA (B), NE (C), and histones H2A.X (D) attached to the liver endothelium were estimated. The standard imaging time was 10 AM, but on day 13, data were also collected 9 and 17 hours later (day 13 + 9 hours and day 13 + 17 hours, respectively). On day 14 (14d), additionally, female mice were studied (♀). (E) Representative images of NETs on days 13 and 14. More time points are shown in supplemental Figure 18. The images were obtained with IVM in the liver sinusoids. The scale bar indicates 50 μm. (F) Representative images of 3D models of citrullinated histone 3 (citH3; red) attached to the endothelium on days 12, 13, and 14 and their colocalization with H2A.X (violet). More detailed images are presented in supplemental Figure 19A. The H2A.X signal was made semitransparent to visualize the overlap with citH3. The scale bar indicates 20 μm. (G-I) During day 13, the proinflammatory milieu developed in the liver manifested by increased expression of adhesion molecule PECAM-1/CD31 on endothelium (G), enhanced platelet (CD49b+) aggregation in the liver vasculature (H), and the release of IL-1β into blood (I). (J-K) Lack of additional waves of NETs before day 14 was confirmed: (J) levels of histones H2A.X and NE adhering to endothelium from day 1 through day 14 after LPS injection; and (K) localization and signal strength of H2A.X Ab K) stained for 6 to 7 days (red; Ab1) and then additionally stained (violet; Ab2) just before imaging (2 different Abs were used as explained in supplemental Figure 19B). On days 7 and 13, only H2A.X remnants (red) from the first wave were present (violet overlaid with red; percentage of signal overlap); on day 14, both the remnants (red) and new NETs (dominant violet) were visible. (Ki) Data quantification and (Kii) representative images. The scale bar indicates 50 μm. (L-O) various parameters were compared between wild-type C57BL/6J mice and PAD4-deficient mice not forming NETs 14 days after LPS. Numbers of neutrophils (L), NET formation (extDNA [M]; histones [N]), and the levels of ALT (O) were evaluated. Some mice were rechallenged with another dose of LPS or with MRSA 14 days after the initial LPS stimulation. 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). In panel Ki, the difference between H2A.X Ab1 (red) and H2A.X Ab2 (violet) on day 14 is labeled ˆ (in which ˆˆˆˆ.00001 < P ≤ .0001; t test; n = 3-10).

The appearance of the second wave of NETs on day 14 of systemic inflammation is linked to the proinflammatory phenotype. Changes in numbers of neutrophils (Ly6G+) present in liver sinusoids (A) and quantity of NET components (B-D) between day 13 and 14 after induction of endotoxemia (LPS IP; 1 mg/kg b.w.). For NET detection, levels of extDNA (B), NE (C), and histones H2A.X (D) attached to the liver endothelium were estimated. The standard imaging time was 10 AM, but on day 13, data were also collected 9 and 17 hours later (day 13 + 9 hours and day 13 + 17 hours, respectively). On day 14 (14d), additionally, female mice were studied (♀). (E) Representative images of NETs on days 13 and 14. More time points are shown in supplemental Figure 18. The images were obtained with IVM in the liver sinusoids. The scale bar indicates 50 μm. (F) Representative images of 3D models of citrullinated histone 3 (citH3; red) attached to the endothelium on days 12, 13, and 14 and their colocalization with H2A.X (violet). More detailed images are presented in supplemental Figure 19A. The H2A.X signal was made semitransparent to visualize the overlap with citH3. The scale bar indicates 20 μm. (G-I) During day 13, the proinflammatory milieu developed in the liver manifested by increased expression of adhesion molecule PECAM-1/CD31 on endothelium (G), enhanced platelet (CD49b+) aggregation in the liver vasculature (H), and the release of IL-1β into blood (I). (J-K) Lack of additional waves of NETs before day 14 was confirmed: (J) levels of histones H2A.X and NE adhering to endothelium from day 1 through day 14 after LPS injection; and (K) localization and signal strength of H2A.X Ab K) stained for 6 to 7 days (red; Ab1) and then additionally stained (violet; Ab2) just before imaging (2 different Abs were used as explained in supplemental Figure 19B). On days 7 and 13, only H2A.X remnants (red) from the first wave were present (violet overlaid with red; percentage of signal overlap); on day 14, both the remnants (red) and new NETs (dominant violet) were visible. (Ki) Data quantification and (Kii) representative images. The scale bar indicates 50 μm. (L-O) various parameters were compared between wild-type C57BL/6J mice and PAD4-deficient mice not forming NETs 14 days after LPS. Numbers of neutrophils (L), NET formation (extDNA [M]; histones [N]), and the levels of ALT (O) were evaluated. Some mice were rechallenged with another dose of LPS or with MRSA 14 days after the initial LPS stimulation. 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). In panel Ki, the difference between H2A.X Ab1 (red) and H2A.X Ab2 (violet) on day 14 is labeled ˆ (in which ˆˆˆˆ.00001 < P ≤ .0001; t test; n = 3-10).

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