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The Sickle Cell Trait Affects Contact Dynamics and Endothelial Cell Activation in Plasmodium falciparum-infected Erythrocytes

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The Sickle Cell Trait Affects Contact Dynamics and Endothelial Cell Activation in Plasmodium falciparum-infected Erythrocytes

Christine Lansche et al. Commun Biol.

Abstract

Sickle cell trait, a common hereditary blood disorder, protects carriers from severe disease in infections with the human malaria parasite Plasmodium falciparum. Protection is associated with a reduced capacity of parasitized erythrocytes to cytoadhere to the microvascular endothelium and cause vaso-occlusive events. However, the underpinning cellular and biomechanical processes are only partly understood and the impact on endothelial cell activation is unclear. Here, we show, by combining quantitative flow chamber experiments with multiscale computer simulations of deformable cells in hydrodynamic flow, that parasitized erythrocytes containing the sickle cell haemoglobin displayed altered adhesion dynamics, resulting in restricted contact footprints on the endothelium. Main determinants were cell shape, knob density and membrane bending. As a consequence, the extent of endothelial cell activation was decreased. Our findings provide a quantitative understanding of how the sickle cell trait affects the dynamic cytoadhesion behavior of parasitized erythrocytes and, in turn, endothelial cell activation.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Stage-specific and erythrocyte variant-specific adhesion dynamics. a Schematic drawing of the experimental set up. P. falciparum-infected erythrocytes labeled with SYBR Green were superfused over a confluent human dermal microvascular endothelial cell monolayer at controlled hydrodynamic conditions. The motion behavior of infected erythrocytes was recorded by fluorescence microscopy, with the focal plane positioned on top of the endothelial cell monolayer. b Motion behavior of a single infected erythrocyte as defined by the fluorescence amplitude and the translational velocity trajectories. From the trajectories, the mean translational velocity, the amplitude difference, the contact time, and the Pearson correlation coefficient between the amplitude and the velocity trajectories were obtained. cf Representative trajectories of the translational velocity, v, and fluorescence amplitude, A, of infected HbAA and HbAS erythrocytes at the trophozoite and schizont stage. The red arrows indicate velocity peaks, suggestive of a transient detachment of the parasitized HbAS erythrocyte from the substratum. The corresponding movies to the selected trajectories can be found in the supplementary information (Supplementary Movies 1-4). Wall shear stress, 0.03 Pa. a.u., arbitrary unit
Fig. 2
Fig. 2
Quantitative analysis of the adhesion dynamics. The individual velocity and amplitude trajectories were parameterized and the following parameters were obtained: a the mean translational velocity, v; b fluorescence intensity amplitude difference, ΔA, (a.u., arbitrary units); c contact time; and d Pearson correlation coefficient between fluorescence amplitude and velocity profile. Note that the data for parasitized HbAA and HbAS erythrocytes are statistically different, according to an F-test (trophozoites: F = 58, DF = 21, p < 0.001; schizonts: F = 29, DF = 21, p < 0.001). Each data point corresponds to a single cell measurement, with at least 27 cells being measured per condition. A box plot analysis is overlaid over the individual data points, with the median, 25% and 75% quartile ranges and the standard error of the mean being shown. Statistical significance was assessed, using Holm–Sidak one-way ANOVA (velocity) or Dunn’s ANOVA on ranks (amplitude and contact time). Wall shear stress, 0.03 Pa
Fig. 3
Fig. 3
Effect of cell shape on dynamic cytoadhesive behavior. a Schematic representation of the mesoscopic computer simulation, based on a deformable red blood cell model and multiparticle collision hydrodynamics, used to assess the adhesion dynamics of parasitized erythrocytes. Fluid flow lines derived from the simulation are shown around a flipping cell. Flow velocities are indicated by a color code. b Representative time series of a flipping trophozoite and a rolling schizont are shown. The upper panels show the motion behavior as recorded by phase contrast microscopy (see also Supplementary Movies 5 and 6). The lower panels show the corresponding mesoscopic simulations (see also Supplementary Movies 7 and 8). The time points at which the snapshots were recorded are indicated. Wall shear stress, 0.03 Pa; scale bar, 5 µm. c, d Simulation of the fluorescence amplitude, A, and velocity trajectory, v, of c a flipping cell with an asymmetric discoidal shape and d a rolling cell with an almost spherical shape. a.u., arbitrary units
Fig. 4
Fig. 4
Effect of membrane stiffness and knob density on dynamic cytoadhesive behavior. Simulations were performed to assess the effect of a the shear modulus, b bending modulus, and c knob density on adhesion dynamics of parasitized erythrocytes. As readouts the following parameters were obtained: translational velocity, v, contact time, fluorescence amplitude difference, ΔA, (a.u., arbitrary units), and the Pearson correlation coefficient between fluorescence amplitude and velocity trajectories. The simulations were performed for a cell with a discoidal shape (absolute volume of 79 µm3 and reduced volume of 0.79) and a wall shear stress of 0.1 Pa. The shear and bending moduli considered cover the range of values experimentally determined for infected and uninfected erythrocytes. The range of knob densities cover the values experimentally determined for HbAA and HbAS erythrocytes infected with the FCR3HDMEC strain (Supplementary Fig. 8). Statistical significance was assessed using Holm–Sidak one-way ANOVA. n.s., not significant. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 5
Fig. 5
Simulated trajectories of parasitized HbAA and HbAS erythrocytes. Representative translational velocity, v, and fluorescence amplitude trajectories, A, are shown for parasitized HbAA and HbAS erythrocytes at a the trophozoite and b schizont stage. The following experimentally determined parameters were used for the simulations, with the parameters given in the following order: reduced volume, knob density, bending modulus, and shear modulus. HbAA trophozoites, 0.79, 5 knobs µm−2, 84 kBT, 20 µN m−1; HbAS trophozoites, 0.66, 3 knobs µm−2, 143 kBT, 40 µN m−1; HbAA schizonts, 0.99, 8 knobs µm−2, 84 kBT, 20 µN m−1; HbAS schizonts, 0.75, 5 knobs µm−2, 143 kBT, 40 µN m−1. Wall shear stress, 0.1 Pa. c Effect of cell size on the dynamic adhesion of infected erythrocytes. Three different cell sizes were selected: 100%, 80%, and 60% of surface area. The translational velocity, amplitude difference, contact time, and Pearson correlation coefficient between fluorescence amplitude and velocity trajectories are shown for a trophozoite. The knob density was kept constant in all the cases and a bending modulus and a shear modulus of 84 kBT and 20 µN m−1, respectively, were considered. Wall shear stress, 0.1 Pa. d Effect of the knob distribution on adhesion dynamics. A homogeneous knob distribution (hom) was simulated by keeping the distance between any two consecutive knobs almost the same, whereas the heterogeneous knob distribution (heterog) represents our standard approach as described in the methods section. All other parameters were as described above. Statistical significance was assessed using Holm–Sidak one-way ANOVA (c) or Student’s two-tailed t-test (d). n.s., not significant. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 6
Fig. 6
Principal component analysis of adhesion dynamics of parasitized HbAA and HbAS erythrocytes. The score plot of the first two principal components (PC1 and PC2) is shown and the percentage of the total variance explained by each principal component is given in parenthesis. The combined principal components PC1 and PC2 explain 59.64% of the variance in the data. The plot is overlaid with the eigenvectors (gray arrows) whose length and direction indicate how influential a variable is. Each dot represents the data set obtained from a single cell
Fig. 7
Fig. 7
Contact interactions between parasitized erythrocytes and human dermal microvascular endothelial cells. a Representative examples of simulated contact footprints that parasitized HbAA and HbAS erythrocytes at the trophozoite and schizont stage leave on the substratum. For simulation parameters see Fig. 5 legend, except that, for comparative reasons, a spherical shape with a reduced volume of 0.99 was used for both parasitized HbAA and HbAS erythrocytes at the schizont stage. b Representative simulated adhesive patches are shown for parasitized HbAA and HbAS erythrocytes at the trophozoite and schizont stage. The color code indicates the relative number of productive cytoadhesive interactions between the parasitized erythrocyte and the substratum at a particular point in time. The cells are viewed from the side facing the substratum. c Experimentally determined adhesive contact maps of trophozoites and schizonts. The adhesive contact maps were obtained by projecting the amplitude profiles derived from the flow chamber experiments around the circumference of the cells, yielding an average dwell time per unit angle as an indicator of contact points between the cell and the substratum at a given spatial and temporal position
Fig. 8
Fig. 8
Reduced endothelial cell activation by infected HbAS erythrocytes. a Representative confocal microscopy images showing the subcellular localization of p65-NFĸB subunit in human dermal microvascular endothelial cells (HDMEC) upon TNF-α treatment (100 units ml−1 for 2 h) or upon co-culture with parasitized HbAA or HbAS erythrocytes (1 × 107 infected red blood cells at the trophozoite stage for 2 h) under static conditions. Medium served as a negative control. The p65-NFĸB subunit was localized using a specific polyclonal rabbit antiserum (dilution 1:200) and an Alexa-488 conjugated goat anti-rabbit IgG antiserum (1:400) as secondary antibody. The nuclei were stained using Hoechst 33342. Scale bar, 20 µm. b Percentage of HDMECs positive for nuclear p65-NFĸB staining (nuclear labeling index, NLI) as a function of the parasite load under static conditions. Infected HbAA and HbAS erythrocytes (iHbAA and iHbAS) were analyzed. TNF-α served as a positive control and uninfected HbAA and HbAS red blood cells (HbAA and HbAS) were used as negative controls. As an additional control, the effect of an isogenic knobless FCR3 line (iHbAA K−) was investigated. The mean ± SEM of at least three independent biological replicates is shown, with at least 50 endothelial cells being analyzed per condition and per biological replicate. Note the data obtained for parasitized HbAA and HbAS as well as for the HbAA erythrocytes infected with the knobless parasite line are significantly different, as determined using F-tests (F = 28; DF = 4; p < 0.001 and F = 169; DF = 4; p < 0.001, respectively). c Endothelial cell activation under flow conditions. Infected and uninfected HbAA and HbAS erythrocytes (1 × 108 cells) were superfused over a confluent monolayer of HDMECs at a wall shear stress of 0.03 Pa. A box plot analysis is overlaid over the individual data points, with the median, 25% and 75% quartile ranges and the standard error of the mean being shown. At least 50 endothelial cells were analyzed per condition and per biological replicate. Statistical significance was determined using one-way ANOVA

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