Fig. 8 | Communications Biology

Fig. 8

From: The sickle cell trait affects contact dynamics and endothelial cell activation in Plasmodium falciparum-infected erythrocytes

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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