Impact of neutrophil extracellular traps on fluid properties, blood flow and complement activation

Abstract

Introduction: The intravascular formation of neutrophil extracellular traps (NETs) is a trigger for coagulation and blood vessel occlusion. NETs are released from neutrophils as a response to strong inflammatory signals in the course of different diseases such as COVID-19, cancer or antiphospholipid syndrome. NETs are composed of large, chromosomal DNA fibers decorated with a variety of proteins such as histones. Previous research suggested a close mechanistic crosstalk between NETs and the coagulation system involving the coagulation factor XII (FXII), von Willebrand factor (VWF) and tissue factor. However, the direct impact of NET-related DNA fibers on blood flow and blood aggregation independent of the coagulation cascade has remained elusive.

Methods: In the present study, we used different microfluidic setups in combination with fluorescence microscopy to investigate the influence of neutrophil-derived extracellular DNA fibers on blood rheology, intravascular occlusion and activation of the complement system.

Results: We found that extended DNA fiber networks decelerate blood flow and promote intravascular occlusion of blood vessels independent of the plasmatic coagulation. Associated with the DNA dependent occlusion of the flow channel was the strong activation of the complement system characterized by the production of complement component 5a (C5a). Vice versa, we detected that the local activation of the complement system at the vascular wall was a trigger for NET release.

Discussion: In conclusion, we found that DNA fibers as the principal component of NETs are sufficient to induce blood aggregation even in the absence of the coagulation system. Moreover, we discovered that complement activation at the endothelial surface promoted NET formation. Our data envisions DNA degradation and complement inhibition as potential therapeutic strategies in NET-induced coagulopathies.

Keywords: DNA; blood rheology; blood viscosity; coagulation; complement; immunothrombosis; neutrophil extracellular traps.

Figure 1

Neutrophil extracellular trap (NET) formation in hirudinated whole blood. (A) HUVECs were seeded into microfluidic channels and perfused with hirudinated blood at a shear stress of 0.5 Pa for 4 h. NETosis was induced by 15 nM PMA. Macroscopic blood aggregations were formed in PMA-treated slides but not in corresponding controls (CTL). Representative immunofluorescence staining of PMA treated slides indicates that aggregates contain DNA (DAPI), tissue factor (TF) and thrombospondin-1 (TSP1). Scale bar = 50 µm (B) Representative fluorescence microscopy images of DAPI stained slides during the experiment. CD31 staining (white) shows that the endothelium underneath the DNA-rich aggregates remained intact. Nuclei of endothelial cells are not visible because the brightness and contrast settings of the images were adjusted to the strong fluorescence signal emitted by the DNA aggregates. White boxes indicate magnified areas. Scale bar = 50 µm. (C) Quantitative analysis of the time series experiment shows that the number of DNA fibers increased time-dependently. (D) Fluorescence microscopic images of neutrophils after the flow experiment. Quantifications of images are presented in bar diagrams indicating the number of single neutrophils and neutrophil clusters in control slides in comparison to PMA-treated slides. Scatter plots indicate the number of analyzed fields of view of three independent experiments (single neutrophils) or the number of neutrophil clusters per slide of three independent experiments (neutrophil clusters). Representative fluorescence images of single neutrophils and neutrophil clusters are shown. CD15 was used as neutrophil surface marker (red). Scale bar = 100 µm. ***P<0.001, Student’s t-test.

Figure 2

PMA induced channel occlusion and effects of DNA degradation and NETosis inhibition in hirudinated whole blood. (A) Quantitative analysis of the channel occlusion is shown as bar diagram. To prove the impact of extracellular DNA and NETosis on occlusion, DNase I or Cl-A was added. Representative photographs of fluidic slides, after treatment with DNase I or CL-A are shown. (B) Plasmatic cell-free DNA was increased in PMA-treated slides in comparison to CTL slides. Amount of cell-free DNA was reduced by DNase I and Cl-A treatment. (C) CitH3 was increased in PMA-treated slides in comparison to CTL slides. Less plasmatic citH3 was measured upon DNase I or Cl-A treatment. (D) Plasmatic levels of factor XIIa were neither affected by PMA stimulation nor DNase I or Cl-A treatment. (E) C5a was significantly increased in PMA-treated slides in comparison to CTL slides. PMA-induced C5a levels were significantly decreased upon DNase I treatment. Data are shown as mean ± SD, scatter plots indicate the number of independent experiments, *p<0.05, p**<0.01, ***p<0.0005, ****p<0.0001, ns = not significant, one-way ANOVA with Tukey post hoc test.

Figure 3

Activation of complement system promoted NETosis and channel occlusion in hirudinated whole blood. To induce complement activation in the fluid phase, complexes of heat aggregated immunoglobulins (c-Ig) were added to the perfusing blood. To induce complement activation at the endothelium, CD31 directed antibodies (αCD31) were deposited on the surface of HUVECs prior to the perfusion with whole blood. (A) No macroscopic channel occlusions were formed in c-Ig-treated slides, whereas channels occluded upon αCD31 treatment. (B) Plasmatic cell-free DNA was significantly increased in c-Ig- and αCD31-treated slides in comparison to the CTL group. (C) Concentration of plasmatic citH3 was significantly increased in αCD31-treated slides in comparison to the CTL group. Although c-Ig treatment increased the plasmatic concentration of citH3, the effect was statistically not significant. (D) Detection of C5a in plasma samples. In comparison to the CTL slide, C5a was significantly increased in c-Ig- and αCD31-treated slides. (E) Vascular deposition of C3d (red) was analyzed by immunofluorescence staining of flow slides after the perfusion experiment. Representative fluorescence microscopic images are shown. Scale bar = 10 µm. Quantitative analysis of microscopy images is indicated as bar diagram. (F) MAC staining (green) of single neutrophils attached to the endothelium and (G) neutrophils/NET conglomerates. CD66b (magenta) was used as a neutrophil marker. Scale bars = 50 µm. For reference, data of PMA-treated and CTL slides, which were already shown in Figure 2, are depicted again (A-D). Data are shown as mean ± SD, scatter plots indicate the number of independent experiments (A-D) or the mean fluorescence intensity (MFI) of the C3d staining per field of view of three independent experiments (E), p*<0.05, p***<0.0005, p****<0.0001, ns = not significant, one-way ANOVA with Tukey (A-E) or Dunnett’s T3 (C) post hoc test.

Figure 4

Impact of surface immobilized NETs on fluid flow. (A) NETs immobilized at the bottom (height = -0.007 cm) of the flow channel. DNA was stained with DAPI (blue) and EvaGreen (green). Scale bar = 100 µm. (B) The velocity (v) of microparticles within the flow channel was measured by microparticle velocimetry. Flow velocity profile recorded at a shear rate of 222 s^-1 and 1333 s^-1 are shown. The profile measured in control channels (red area) were superimposed with the profile measured in NET containing channels (grey area). (C) Impact of the shear rate (˙γ) on the total fluid flow. Shown is the change of the total flow in comparison to the corresponding flow within the control channels. The flow in the control channel is indicated by the dashed line. **p< 0.01, Fisher z-transformation.

Figure 5

Impact of NETs on fluid viscosity. Velocity (v) of microparticles was measured in channels containing NETs or purified DNA. Flow experiments were performed at different shear rates ranging from 150 to 1129 s^-1. (A) Representative velocity profiles at low shear rates. Image insets show the trajectories of microparticles in the center of the channel (height = 0 cm) either filled with fluid containing purified DNA (green) or NETs (red). Dashed lines indicate the standard deviation. Exposure time = 10 ms, Scale bar = 50 µm. (B) Representative velocity profiles at high shear rates. Image insets show the trajectories of microparticles in the center of the channel (height = 0 cm) either filled with fluid containing purified DNA (green) or NETs (red). Dashed lines indicate the standard deviation. Exposure time = 1 ms, Scale bar = 50 µm. (C) Shown is the viscosity (η) of the flowing fluid as a function of the applied shear rate (γ˙).