Figure 1.
P. falciparum parasite invasion of A, B and O erythrocytes.
Levels of parasite invasion and maturation during two cycles of growth of P. falciparum clone ITG in A, B and O erythrocytes. Data are presented as the combined results of 4 independent experiments using erythrocytes from healthy human volunteers with blood groups A (n = 7 donors), B (n = 4 donors) and O (n = 6 donors). Data are shown as box-and-whiskers plots, representing interquartile and complete ranges, with the horizontal line in each box indicating the median. Statistical significance was determined by a one way ANOVA. Invasion is defined as the percentage of ring parasitemia, as measured 24 hours and 72 hours after inoculation. Maturation is defined as the percentage of trophozoite parasitemia, as measured 48 hours and 96 hours after inoculation. There were no significant differences observed in the invasion and maturation of P. falciparum parasites in A, B or O erythrocytes.
Figure 2.
Infected O erythrocytes are phagocytosed more avidly by human macrophages.
P. falciparum (ITG and 3D7)-infected and uninfected A, B and O erythrocytes were incubated with A and O blood group macrophages for 90 minutes (ring-stage) or for 120 minutes (mature-stage). The phagocytic index was then calculated by determining the number of internalized parasites in ≥250 macrophages and the data were normalized to the average phagocytic index of infected A erythrocytes. (A) Data for ring-stage parasitized erythrocytes are presented as the combined results of two independent experiments. Data for each blood group (n = 3 donors/group) are shown as box-and-whiskers plot, representing interquartile and complete ranges, with the horizontal line in each box indicating the median. There was a significant increase in the phagocytic uptake of ring-stage infected O erythrocytes compared to A and B infected erythrocytes (*p = 0.022, and **p = 0.007, respectively; Student's t-test for paired samples with Bonferroni correction for multiple comparisons). (B) Data for mature-stage parasitized erythrocytes represent the combined results of six independent experiments using multiple donors per group (A: n = 6; B: n = 3; O: n = 4) and are shown as box-and-whiskers plots, representing interquartile and complete ranges, with the horizontal line in each box indicating the median. There was significantly enhanced phagocytic uptake of infected O erythrocytes observed compared to A and B infected erythrocytes (*p<0.01 and **p<0.05, respectively; Student's t-test with Bonferroni correction for multiple comparisons). (C) Macrophages were isolated from donors of blood groups A and O and phagocytosis assays were run in parallel. The blood group of the infected erythrocytes was found to influence phagocytic uptake, with infected O erythrocytes being preferentially phagocytosed (*p<0.01, two-way ANOVA), whereas the blood group of the macrophage had no effect on the uptake of infected A and O erythrocytes (p>0.05). Data represent three independent experiments normalized to the average phagocytic uptake of infected A erythrocytes by macrophages isolated from blood group A donors.
Figure 3.
Murine monocytes phagocytose infected O erythrocytes more efficiently in vivo than infected A or B erythrocytes.
C57BL/6 mice were injected intraperitoneally with 50×106 purified mature-stage P. falciparum parasites cultivated in human A, B or O erythrocytes. Three hours after injection, resident monocytes were collected, washed, and plated on glass coverslips. (A) The phagocytic index was calculated by counting the number of internalized parasites within 250 monocytes and the data were normalized to the average phagocytic index of infected A erythrocytes. Data represent three independent experiments using the P. falciparum strain ITG and each blood group is represented by at least 3 different donors, A (n = 5), B (n = 3) and O (n = 5). Bar graphs represent the mean±SEM. There was a significant increase in the phagocytic uptake of infected O erythrocytes compared to the phagocytic uptake of infected A or B erythrocytes (*p<0.05; Student's t-test with Bonferroni correction for multiple comparisons). (B) Photomicrographs show representative examples of increased uptake of mature-stage infected (i) O erythrocytes, versus mature-stage infected (ii) B erythrocytes. Images were acquired with an Olympus BX41 microscope and an Infinity capture camera at 1000× magnification.
Figure 4.
Increasing phagocytosis of erythrocytes correlates with decreasing A antigen.
A and O erythrocytes were typed using standard hemagglutinin techniques. Blood group A was further classified into A1 or A2 subgroups using Dolichos biflorus lectin. (A) Flow cytometric testing of A1, A2, O, and Bombay erythrocytes with anti-H (clone BRIC231) FITC conjugated antibody. FITC-derived fluorescence is displayed on the x axis on a logarithmic scale and the number of cells on the y axis. O (solid black line), A2 (dotted black line), A1 (dashed black line) and Bombay (filled grey). (B) A1, A2 and O erythrocytes infected with P. falciparum (ITG clone) were incubated with human monocyte-derived macrophages. The phagocytic index was calculated by determining the number of internalized parasitized erythrocytes within 250 macrophages. Data were normalized to the average phagocytic index of infected A1 cells. Data represent 3 independent experiments and each blood group is represented by multiple donors, A1 (n = 8/group), A2 (n = 3/group), and O (n = 8/group). The box plots represent the median, interquartile and complete range. The phagocytosis of infected erythrocytes increased in an H antigen dose-dependent manner (r = 0.80, *p<0.0001; Spearman's correlation test).
Figure 5.
Blood group O (H antigen) status affects the phagocytic uptake of infected erythrocytes by human macrophages.
Erythrocytes of blood group O and B were treated with B-zyme, thereby removing the terminal α-1,3-galactose from blood group B antigens. (A) Flow cytometric analysis with anti-B (clone 9621A8) and phycoerythrin (PE)-conjugated rat-anti-mouse kappa as secondary antibody. In the dot plot, the x and y axes represent FL1-derived fluorescence and PE-derived fluorescence, respectively, on logarithmic scales. Results show cleavage of the terminal galactose from B erythrocytes and enzymatic conversion from group B to erythrocytes which type as group O: (i) Native, untreated O erythrocytes, (ii) Native, untreated B erythrocytes, (iii) B-zyme-treated B erythrocytes. Group O cells mock-treated with B-zyme gave identical results to the group O untreated control (data not shown). (B) The phagocytic uptake was determined by counting the number of internalized infected erythrocytes in 250 individual macrophages and data was normalized to the average phagocytic index of infected untreated B erythrocytes. Data represent two independent experiments using the P. falciparum ITG clone. Each blood group is represented by four different donors. Bar graphs represent the mean±SEM. Significance was determined by Mann Whitney test with Bonferroni correction for multiple comparisons. There was an observed increase in the phagocytosis of untreated infected O erythrocytes when compared to untreated infected B erythrocytes (*p<0.05). The phagocytic index of the infected, B-zyme-treated B erythrocytes was significantly increased from phagocytic index of infected untreated B erythrocytes (**p<0.01). However, there was no significant difference in the uptake of treated vs. untreated infected O erythrocytes.
Figure 6.
Phosphatidylserine levels on P. falciparum-infected A, B and O erythrocytes.
Annexin staining of A, B and O uninfected and P. falciparum-infected erythrocytes was evaluated. Freshly purified erythrocytes (n = 4 for each group) from healthy donors were left uninfected or were infected with ITG, 3D7 or E8B clones and stained by annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Data are shown as box-and-whiskers plots, representing interquartile and complete ranges, with the horizontal line in each box indicating the median level of percentage of annexin-V positive cells among total cell population. There were no significant differences observed between infected A, B, or O group erythrocytes.
Figure 7.
Hemichrome deposition and band 3 aggregation are increased in infected O erythrocytes.
Hemichrome levels and band 3 aggregation were measured in uninfected, ring-stage infected and mature-stage infected A, B and O erythrocytes. (A) and (B) Hemichrome data represent the combined results of 6 independent experiments (means±SEM of ≥3 different donors per blood group) using P falciparum ITG and 3D7 clones and are expressed in nmol/ml of packed erythrocytes. Compared to infected A and B erythrocytes, hemichrome deposition was significantly increased in ring-stage infected (Figure 7A, *p = 0.005, **p = 0.038, respectively) and mature-stage infected O erythrocytes (Figure 7B, *p = 0.013, **p = 0.024, respectively; Mann Whitney for two-tailed distribution). (C) Gel filtration chromatography effluents of Tween-20 membrane extracts of P. falciparum-infected A, B and O erythrocytes (Figure 7C) were analyzed for aggregated band 3 protein as absorbance using the Bradford reagent at 595 nm and eosin-5-maleimide fluorescence indicating the location of band 3 (Figure 7C insert). Increased high molecular weight aggregates of band 3 were observed in infected group O erythrocytes. The results presented are representative of 3 independent experiments.