Figure 2.
Engulfment of NE and histone H2A.X by liver macrophages (KCs) and neutrophils. (A-B) Twelve hours after endotoxemia induction, mice were administered IV with antibodies labeling NE (AF647–anti-NE antibody, violet) and H2A.X histones (AF568–anti-H2A.X antibody, red). Then the mice were imaged at 24 hours of endotoxemia by IVM. (A) The volume of histones engulfed by macrophages and neutrophils as well as the percentage of cells engaged in this process are presented in the graphs (red for H2A.X and violet for NE). (B) Representative images of 3D models of KCs (F4/80+; Alexa Fluor 488 anti-F4/80 antibody) and neutrophils (Ly6G+; Brilliant Violet 421 anti-Ly6G antibody) engulfing NET proteins, rendered from the z-stacks of mouse liver sinusoids at 24 hours of endotoxemia. The cells were made semitransparent to visualize their content. In neutrophils NE is false-labeled yellow for clarity. Scale bar indicates 20 μm. Images for neutrophils were selected in such a way that the intracellular presence of NET proteins is visible, but in this particular image, the number of involved neutrophils is higher than average. (C-E) Involvement of macrophages in NET removal was confirmed by depletion studies. To remove KCs from the liver sinusoids, mice were administered IV or IP with clodronate liposomes (clodronate; scheme in supplemental Figure 3). The former treatment depleted also monocytes. Control mice received phosphate-buffered saline (PBS)–liposomes. (C-D) After these cells were removed, in some animals endotoxemia was induced (LPS IP, 1 mg/kg b.w.; red bars) and some were left untreated (green). (C-D) NETs were visualized as the area covered by NE and the numbers of neutrophils (Ly6G+), macrophages, and monocytes (both F4/80+; elongated, branched morphology vs oval, respectively) were quantified at 4 (C) and 24 hours (D) of endotoxemia. (E) Representative images of NETs (NE, violet), KCs (red, yellow arrow head), monocytes (red, white arrow head), and neutrophils (blue) present in the livers of mice injected with clodronate or control (PBS) liposomes, visualized with IVM at 24 hours of endotoxemia; NE only (NETs; right). The scale bar indicates 50 μm. The data in the graphs are expressed as mean ± SD of at least 3 fields of view. Asterisks in the graphs indicate statistically significant differences according to the Student t test (∗.01 < P ≤ .05; ∗∗.001 < P ≤ .01; ∗∗∗.0001 < P ≤ .001; ∗∗∗∗.00001 < P ≤ .0001; n = 3-4).

Engulfment of NE and histone H2A.X by liver macrophages (KCs) and neutrophils. (A-B) Twelve hours after endotoxemia induction, mice were administered IV with antibodies labeling NE (AF647–anti-NE antibody, violet) and H2A.X histones (AF568–anti-H2A.X antibody, red). Then the mice were imaged at 24 hours of endotoxemia by IVM. (A) The volume of histones engulfed by macrophages and neutrophils as well as the percentage of cells engaged in this process are presented in the graphs (red for H2A.X and violet for NE). (B) Representative images of 3D models of KCs (F4/80+; Alexa Fluor 488 anti-F4/80 antibody) and neutrophils (Ly6G+; Brilliant Violet 421 anti-Ly6G antibody) engulfing NET proteins, rendered from the z-stacks of mouse liver sinusoids at 24 hours of endotoxemia. The cells were made semitransparent to visualize their content. In neutrophils NE is false-labeled yellow for clarity. Scale bar indicates 20 μm. Images for neutrophils were selected in such a way that the intracellular presence of NET proteins is visible, but in this particular image, the number of involved neutrophils is higher than average. (C-E) Involvement of macrophages in NET removal was confirmed by depletion studies. To remove KCs from the liver sinusoids, mice were administered IV or IP with clodronate liposomes (clodronate; scheme in supplemental Figure 3). The former treatment depleted also monocytes. Control mice received phosphate-buffered saline (PBS)–liposomes. (C-D) After these cells were removed, in some animals endotoxemia was induced (LPS IP, 1 mg/kg b.w.; red bars) and some were left untreated (green). (C-D) NETs were visualized as the area covered by NE and the numbers of neutrophils (Ly6G+), macrophages, and monocytes (both F4/80+; elongated, branched morphology vs oval, respectively) were quantified at 4 (C) and 24 hours (D) of endotoxemia. (E) Representative images of NETs (NE, violet), KCs (red, yellow arrow head), monocytes (red, white arrow head), and neutrophils (blue) present in the livers of mice injected with clodronate or control (PBS) liposomes, visualized with IVM at 24 hours of endotoxemia; NE only (NETs; right). The scale bar indicates 50 μm. The data in the graphs are expressed as mean ± SD of at least 3 fields of view. Asterisks in the graphs indicate statistically significant differences according to the Student t test (∗.01 < P ≤ .05; ∗∗.001 < P ≤ .01; ∗∗∗.0001 < P ≤ .001; ∗∗∗∗.00001 < P ≤ .0001; n = 3-4).

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