Blood plasma contains elevated number of ENDS during sepsis.(A) Three mice each were injected i.p. with LPS (40 mg/kg) or PBS (not treated, nt). Retro-orbital blood was collected at 2 h, and plasma was prepared by centrifuging at 1,000 g for 5 min. Fluorescently labeled anti-Ly6G antibody was added, and samples were scanned with ImageStream. The images show examples of Ly6G antibody–stained ENDS. Scale bar, 10 µm. (B) Confocal images show examples of EGFP-labeled ENDS, detected in the blood plasma of Ly6G-cre-mT/mG mice harvested 2 h after i.p. LPS injection. Scale bar, 5 µm. (C and D) Count (C) and ratio (D) of neutrophil-derived (CD66b+ and CD16+) micro objects detected with ImageStream in healthy (black) and septic (gray) human plasma. Micro objects were labeled for TSs with PE-labeled CD9, CD63, and CD81 antibodies and for PS with annexin-5-eF450. Septic plasma samples contained significantly more micro objects with each marker combination (average and SEM are shown; two-tailed t test with Welch’s correction, from left to right: *, P = 0.0251; **, P = 0.029; *, P = 0.0184; **, P = 0.039). In septic samples the ratio of TS−/PS− objects was increased at the expense of the TS+/PS− objects (two-tailed t test: *, P = 0.0123; **, P = 0.003). (E) Aspect ratio (width/length ratio) of TS− and PS− neutrophil-derived micro objects in septic and healthy plasma. Objects with an aspect ratio <0.6 were considered ENDS, and objects with an aspect ratio >0.6 were considered to contain fragmented ENDS. (F) Examples of ENDS and fragmented ENDS detected with ImageStream in septic blood are shown. Each lane represents a different object, and each column represents a channel of a different marker. Scale bar is 10 µm. (G) Confocal images confirm the presence of CD16+ elongated structures in septic plasma labeled with CD16-AF647 antibody. Numbers indicate ENDS length (in micrometers). Scale bar, 5 µm. (H) ENDS content of healthy and septic blood plasma are shown. Red lines indicate average values. Statistical analysis: two-tailed T test with Welch’s correction, **, P = 0.0045. (I) ENDS count showed linear correlation with neutrophil counts. Statistical analysis was done using a correlation analysis and two-tailed test; **, P = 0.006. (J) In septic, but not healthy, samples, the plasma S100A8–S100A9 complex concentration correlated with fragmented ENDS count. Statistical analysis was done using a correlation analysis and two-tailed test; *, P = 0.011. n.s., not significant.
Figure 4.

Blood plasma contains elevated number of ENDS during sepsis. (A) Three mice each were injected i.p. with LPS (40 mg/kg) or PBS (not treated, nt). Retro-orbital blood was collected at 2 h, and plasma was prepared by centrifuging at 1,000 g for 5 min. Fluorescently labeled anti-Ly6G antibody was added, and samples were scanned with ImageStream. The images show examples of Ly6G antibody–stained ENDS. Scale bar, 10 µm. (B) Confocal images show examples of EGFP-labeled ENDS, detected in the blood plasma of Ly6G-cre-mT/mG mice harvested 2 h after i.p. LPS injection. Scale bar, 5 µm. (C and D) Count (C) and ratio (D) of neutrophil-derived (CD66b+ and CD16+) micro objects detected with ImageStream in healthy (black) and septic (gray) human plasma. Micro objects were labeled for TSs with PE-labeled CD9, CD63, and CD81 antibodies and for PS with annexin-5-eF450. Septic plasma samples contained significantly more micro objects with each marker combination (average and SEM are shown; two-tailed t test with Welch’s correction, from left to right: *, P = 0.0251; **, P = 0.029; *, P = 0.0184; **, P = 0.039). In septic samples the ratio of TS−/PS− objects was increased at the expense of the TS+/PS− objects (two-tailed t test: *, P = 0.0123; **, P = 0.003). (E) Aspect ratio (width/length ratio) of TS− and PS− neutrophil-derived micro objects in septic and healthy plasma. Objects with an aspect ratio <0.6 were considered ENDS, and objects with an aspect ratio >0.6 were considered to contain fragmented ENDS. (F) Examples of ENDS and fragmented ENDS detected with ImageStream in septic blood are shown. Each lane represents a different object, and each column represents a channel of a different marker. Scale bar is 10 µm. (G) Confocal images confirm the presence of CD16+ elongated structures in septic plasma labeled with CD16-AF647 antibody. Numbers indicate ENDS length (in micrometers). Scale bar, 5 µm. (H) ENDS content of healthy and septic blood plasma are shown. Red lines indicate average values. Statistical analysis: two-tailed T test with Welch’s correction, **, P = 0.0045. (I) ENDS count showed linear correlation with neutrophil counts. Statistical analysis was done using a correlation analysis and two-tailed test; **, P = 0.006. (J) In septic, but not healthy, samples, the plasma S100A8–S100A9 complex concentration correlated with fragmented ENDS count. Statistical analysis was done using a correlation analysis and two-tailed test; *, P = 0.011. n.s., not significant.

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