<i>In Vivo</i> Characterization of Neutrophil Extracellular Traps in Various Organs of a Murine Sepsis Model

<div><p>Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model <i>in vivo</i> and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.</p></div

Circulating cell-free NETs.

<p>Circulating cell-free NETs (red) were observed in the blood flow of either postcapillary venules of the cesum or hepatic sinusoids of the liver (i, ii) at 24 h after LPS (20 mg/kg) intraperitoneal administration (n = 10). NETs were anchored to the leukocyte adhering to the vascular endothelium (iii; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s010" target="_blank">Movie S5</a>). Thereafter, NETs were leaving away from leukocyte, changing shape from solid to cotton-like, and flowing down the vessel. These cotton-like NETs were arrested far away from the leukocyte (iv). They were not observed in control mice.</p

<i>In vivo</i> NETs in postcapillary venules of the cecum.

<p>The postcapillary venules of the cecum were observed at 24 h after LPS (20 mg/kg) intraperitoneal administration (n = 10). NETs (red) were detected by SYTOX Orange, Alexa Fluor 594-labeled anti-histone antibody, or Alexa Fluor 594-labeled anti-NE antibody, respectively. NETs were characterized as reticular structures anchored to leukocytes (A-i; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s006" target="_blank">Movie S1</a>), reticulolinear structures (A-ii), spot-like structures anchored to leukocytes (A-iii, iv), membranous structures on the surface of leukocytes (A-v) and linear structures anchored to leukocytes (A-vi; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s007" target="_blank">Movie S2</a>). The number of leukocyte-endothelial interactions and NETs per FOV was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#s2" target="_blank">materials and methods</a>, respectively. The number of leukocyte-endothelial interactions per FOV (B) were significantly greater in LPS treated mice than control mice (14.6±1.6 vs 0.6±0.2). The number of NETs per FOV (C) was also significantly greater in LPS treated mice than control mice (4.4±0.4 vs 0.2±0.1). Data was presented as mean+standard error. **P<0.01 versus control.</p

NETs in pulmonary capillaries of the lung.

<p>The excised lungs were investigated immediately after intravital imaging of the cecum and liver to evaluate NETs and endothelial injury. NETs (red) were observed in alveolar space (A-i; arrows) and pulmonary capillaries (A-i; arrowheads) of LPS-treated mice. The number of NETs per FOV and the score of endothelial integrity were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#s2" target="_blank">materials and methods</a>, respectively. The number of NETs per FOV was significantly greater in LPS-treated mice than control mice (B; 6.4±1.1 vs 0.3±0.1). The score of endothelial integrity (C) was lower in LPS treated mice than control mice (2.8±0.2 vs 3.7±0.2). Data was presented as mean+standard error. **P<0.01 versus control.</p

Time course of change in NETs by DNase I treatment <i>in vivo</i>.

<p>After the identification of in vivo NETs (red) in postcapillary venules of the cecum of LPS treated mice, DNase I at a dose of 1000 U was administered intravenously via a catheter (DNase I treatment group, n = 5). In control group (n = 5), the equal amount of phosphate buffered saline (PBS) was administered after NETs identification. Images were recorded by a method of 30 sec-imaging followed by 90 sec-pause for at least 30 min (at least 15 cycles) to minimize the photobleaching for NETs during the observation. Anchored NETs with optimal size in postcapillary venules of the cecum were selected to examine the time course of change in NETs by DNase I treatment. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone-0111888-g007" target="_blank">Figure 7</a> shows in vivo NETs before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone-0111888-g007" target="_blank">Figure 7-i, iii</a>) and after (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone-0111888-g007" target="_blank">Figure 7-ii, iv, v</a>) DNase I treatment.</p

<i>In vivo</i> NETs in hepatic sinusoids of the liver.

<p>The hepatic sinusoids were observed at 24 h after LPS (20 mg/kg) intraperitoneal administration (n = 10). NETs (red) were detected by SYTOX Orange, Alexa Fluor 594-labeled anti-histone antibody, or Alexa Fluor 594-labeled anti-NE antibody, respectively. NETs were characterized as spot-like structures anchored to leukocytes (A-i, ii; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s008" target="_blank">Movie S3</a>), cell-free DNA fragments (A-iii; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#pone.0111888.s009" target="_blank">Movie S4</a>), and cell-free DNA fragments within platelet aggregates (A-iv). Isolectin GS-IB4 stained hepatic sinusoidal endothelial cells were observed in LPS-treated mice (A-v) and normal mice (A-vi). The number of NETs per FOV and the score of endothelial integrity were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111888#s2" target="_blank">materials and methods</a>, respectively. The number of NETs per FOV (B) was significantly greater in LPS treated mice than normal control mice (1.7±0.2 vs 0.2±0.1). The score of endothelial integrity (C) was significantly less in LPS treated mice than control mice (.0±0.3 vs 3.6±0.2). Data was presented as mean+standard error. **P<0.01 versus control.</p