Plasmodium falciparum erythrocyte membrane protein 1 variants induce cell swelling and disrupt the blood-brain barrier in cerebral malaria

Abstract

Cerebral malaria (CM) is caused by the binding of Plasmodium falciparum-infected erythrocytes (IEs) to the brain microvasculature, leading to inflammation, vessel occlusion, and cerebral swelling. We have previously linked dual intercellular adhesion molecule-1 (ICAM-1)- and endothelial protein C receptor (EPCR)-binding P. falciparum parasites to these symptoms, but the mechanism driving the pathogenesis has not been identified. Here, we used a 3D spheroid model of the blood-brain barrier (BBB) to determine unexpected new features of IEs expressing the dual-receptor binding PfEMP1 parasite proteins. Analysis of multiple parasite lines shows that IEs are taken up by brain endothelial cells in an ICAM-1-dependent manner, resulting in breakdown of the BBB and swelling of the endothelial cells. Via ex vivo analysis of postmortem tissue samples from CM patients, we confirmed the presence of parasites within brain endothelial cells. Importantly, this discovery points to parasite ingress into the brain endothelium as a contributing factor to the pathology of human CM.

Graphical abstract

Figure S1.

PfEMP1 proteins are composed of different subtypes of DBL (α, β, γ, δ, ε, ξ, x) and CIDR (α, β, γ, δ) domains with different receptor specificity. (A) Group B and C PfEMP1 predominantly have a four-domain structure, while the larger group A PfEMP1 proteins have additional DBL domains following the first or second DBL-CIDR domains. CIDR, cysteine-rich inter-domain region; ATS, acidic intracellular terminal segment. (B) CM constitutes a subset of severe disease cases and associates with group A^+CM IEs expressing dual receptor–binding PfEMP1s. ICAM-1–binding motif of such PfEMP1s here indicated by an asterisk (*) in the left circle. IEs associated with severe but noncerebral P. falciparum infection express ECPR binding group A^SM or B/A^SM PfEMP1s that do not bind ICAM-1 (left circle). IEs associated with mild P. falciparum infections express PfEMP1 that binds both ICAM-1 and CD36 or ICAM-1 only. Such group B^UM and group C^UM PfEMP1s do not carry a distinct ICAM-1–binding motif (right circle). (C) Domain structure of PfEMP1s expressed by P. falciparum parasite lines used in this study. CM associates with IEs expressing group A^+CM dual ICAM-1 and EPCR binding PfEMP1s; parasite lines expressing such PfEMP1s are HB3VAR03, 3D7 PFD1235w, and BM057. The ICAM-1–binding motif is indicated by an asterisk in their DBLβ (Lennartz et al., 2017). Non-cerebral SM associates with IEs expressing group B/A^SM (and A^SM shown in B) EPCR-binding PfEMP1s that do not bind ICAM-1. Domain cassette (DC) 8 is a chimeric gene between a group A and group B var gene. Parasite lines expressing group B/A^SM PfEMP1s are IT4VAR19 and IT4VAR20. UM associates with IEs that express group B^UM or C^UM PfEMP1 that binds both ICAM-1 and CD36 or ICAM-1. Parasite lines expressing group B^UM PfEMP1s are IT4VAR13 and HB3VAR21. The HB3VAR34 parasite line expresses a group C^UM PfEMP1. UPS, upstream promotor sequence; TM, transmembrane; NTS, N-terminal segment. Redrawn and modified from Jensen et al. (2020).

Figure 1.

Group A^+CM IEs induce clustering of ICAM-1 on brain endothelial cells. (A1–A4) IEs expressing group A^+CM ICAM-1 and EPCR dual-binding PfEMP1s with a specific ICAM-1–binding motif associated with CM. IE lines in this category include HB3VAR03-IEs, 3D7 PFD1235w-IEs, and BM057-IEs. (B1–B4) IEs expressing EPCR-binding group B/A^SM PfEMPs associated with SM that do not bind ICAM-1. IE lines in this category include IT4VAR19-IEs and IT4VAR20-IEs. (C1–C4) IEs expressing dual group B^UM ICAM-1– and CD36-binding PfEMP1s associated with UM lacking the motif found in group A^+CM PfEMP1s. IE line in this category is IT4VAR13-IEs. (D1–D4) IEs expressing group B^UM or C^UM PfEMP1s associated with UM that do bind ICAM-1, but lack the motif found in group A^+CM PfEMP1s. IE lines in this category include HB3VAR21-IEs and HB3VAR34-IEs. See also Fig. S1 and Table 1. (A1–D1) Schematic of the four different receptor binding IE phenotypes (group A^+CM, group B/A^SM, group B^UM, and group C^UM) used in this study. (A2 and B2–D2) Wide-field (A2) and confocal images (B2–D2) of hCMEC/D3 brain microvascular cells incubated with IEs. ICAM-1 is green (FITC), and DAPI nuclei stain is blue. The bottom insets of each panel show brightfield images of IEs (black arrows) present in the framed box to the left. Top insets are an enlargement of the framed box. Blue staining in the top insets shows the presence of parasite nuclei. The images are representative of at least three independent experiments. Scale bars, 10 µm. (A2) hCMEC/D3 cells incubated with dual ICAM-1– and EPCR-binding group A^+CM (HB3VAR03) IEs show ICAM-1 (green) clustered around base of the IEs. hCMEC/D3 cells incubated with (B2) EPCR-binding group B/A^SM (IT4VAR19), (C2) dual ICAM-1– and CD36-binding group B^UM (IT4VAR13), and (D2) ICAM-1–binding group C^UM (HB3VAR34) IEs do not induce clustering of ICAM-1. (A3–D3) Pseudo-coloring of the images in A2–D2. As indicated by the color bar, areas of high ICAM-1 levels are white and yellow, while areas with low ICAM-1 levels are dark blue. Scale bars, 10 µm. The graph to the right of each pseudo-colored image shows quantification of the ICAM-1 (RFU) clustering around bound IEs as compared with control (i.e., hCMEC/D3 cells incubated with noninfected erythrocytes). Calculations were based on a minimum of three independent experiments with at least 50 IEs counted per experiment. Shown are mean values ± SD; statistical significance was calculated using an unpaired t test (*, P ≤ 0.001; ns, not significant). (A3) Group A^+CM IEs (HB3VAR03) recruit or induce ICAM-1 clustering (evidenced by white, yellow, and red colors) by contrast to (B3) group B/A^SM (IT4VAR19), (C3) group B^UM (IT4VAR13), and (D3) group C^UM (HB3VAR34) IEs. n, nuclei. Images were processed with Fiji. (A4–D4) Binding (adhesion) of IEs to hCMEC/D3 cells at 1 h (lighter color) and 6 h (darker color). Shown are mean values ± SD of a minimum of three independent experiments conducted in duplicate. Statistical analysis was performed using an unpaired t test (**, P = 0.02; ***, P = 0.009; ****, P < 0.001; and ns, not significant). ICAM-1– and ECPR-binding group A^+CM IEs are HB3VAR03, 3D7 PFD1235w, and BM057 (A4); EPCR-binding group B/A^SM IEs are IT4VAR19 and IT4VAR20 (B4); ICAM-1– and CD36-binding group B^UM IE is IT4VAR13 (C4); and ICAM-1–binding group B^UM and C^UM IEs are HB3VAR21 and HB3VAR34 (D4), respectively. ctrl, control.

Figure S2.

var gene and PfEMP1 expression profiles of HB3VAR21 and HB3VAR34 parasite lines. (A) Ethidium bromide–stained group B^UM HB3VAR21-IE with (gray histogram) and without (black histogram) rat anti-HB3VAR21_DBLβ_D4 antiserum. (B) Transcript level of each var gene of HB3VAR21-IE relative to control gene (seryl-tRNA synthetase). The domain architecture of the dominant expressed PfEMP1 (HB3VAR21 or KOB63129.1) is shown. (C) Ethidium bromide–stained group C^UM HB3VAR34-IE with (gray histogram) and without (black histogram) rat HB3VAR34_DBLβ_D4 antiserum. (D) Transcript level of each var gene of HB3VAR34-IE relative to control gene (seryl-tRNA synthetase). The domain architecture of the dominant expressed PfEMP1 (HB3VAR34 or KOB58843) is shown. The data represent a minimum of three independent experiments conducted in duplicate. Abs, antibodies.

Figure 2.

ICAM-1–enriched microvilli and transmigratory ring/docking structures on brain hCMEC/D3 endothelial cells. (A–C) hCMEC/D3 brain endothelial cells were incubated with parasites representative of (A) group A^+CM IEs (HB3VAR03), (B) group B^UM IEs (IT4VAR13), or (C) noninfected erythrocytes (RBC indicates RBC controls). ICAM-1 expression at the apical, middle, and basal surfaces of hCMEC/D3 cells were imaged using confocal microscopy. ICAM-1 is green (FITC), and DAPI nuceli stain is blue. n denotes the nuclei and is annotated in apical images. (A) The group A^+CM IEs (HB3VAR03) induce ICAM-1–enriched circular ring/docking structures of different sizes ranging from 1.6 to 3.7 µm. White arrows in A2 and A3 point to HB3VAR03-IEs. (A–C) The images are representative of a minimum of three independent experiments. Scale bars, 20 µm. (D) Percentage of hCMEC/D3 endothelial cells covered with ICAM-1–enriched microvilli following incubation with HBVAR03-IEs, IT4VAR13-IEs, or RBC controls. ON, overnight co-culture. All graphs show mean values ± SD for 200 cells per condition, for a minimum of three independent experiments conducted in duplicate. See also Fig. S3.

Figure 3.

Group A^+CM IEs induced altered morphology of endothelial cells. (A–C) Electron microscopy images of brain microvascular cells (hCMEC/D3) co-cultured with (A) group A^+CM HB3VAR03-IEs, (B) group B^UM IT4VAR13-IEs, and (C) RBC controls (RBC). The images are representative of four independent experiments. Scale bars, A1–A5, 2 µm; B and C scale bars, 10 µm. (A1) Microvilli on hCMEC/D3 co-cultured with HB3VAR03-IE. (A2 and A3) Adherent HB3VAR03-IE on surface of hCMEC/D3. (A3) HB3VAR03-IE interacting with endothelial surface. (A4) Adherent HB3VAR03-IE showing invaginations of cell membrane, “pinching” microvilli (black arrowhead) protruding from endothelial membrane. (A5) Microvilli extending around an HB3VAR03-IE (black arrowhead) with electron-dense knobs seen around the IE membrane. Inset shows knobs (white arrowhead) at larger magnification. n, nuclei.

Figure S3.

ICAM-1–enriched microvilli on brain hCMEC/D3 endothelial cells. Percentage of hCMEC/D3 endothelial cells covered with ICAM-1–enriched microvilli following incubation with group A^+CM (3D7 PFD1235w and BM057), group B^UM (HB3VAR21), or group C^UM (HBVAR34) IEs. Shown are mean values ± SD for a minimum of three independent experiments conducted in duplicate.

Figure 4.

Brain microvascular endothelial cells internalize P. falciparum–IEs. (A–C) Electron microscopy images of brain microvascular cells (hCMEC/D3) co-cultured with (A) group A^+CM HB3VAR03-IEs, (B) group B^UM IT4VAR13-IEs, or (C) RBC controls (RBC). The images are representative of four independent experiments. Scale bars, all images, 2 µm. (A1–A6) hCMEC/D3 with internalized HB3VAR03 P. falciparum–IE at 8 and 12 h. n, nucleus of endothelial cell. Parasite hemozoin is indicated by a white asterisk (*) in A1–A4 and A6. (A3) Black arrowheads point to shrunken and ruffled membrane. (A5) Black arrows point to secretory pods associated with release of von Willebrand factor. White arrow points to one of several mitochondria. (A6) Inset shows enlargement of hCMEC/D3 showing aggregated cytoplasm indicative of actin stress fibers.

Figure 5.

PfEMP1– and ICAM-1–dependent internalization of P. falciparum–IE. (A) Brain microvascular endothelial cells (hCMEC/D3) with internalized group A^+CM (HB3VAR03, 3D7 PFD12352, and BM057), group B/A^SM (IT4VAR19 and IT4VAR20), group B^UM (IT4VAR13 and HB3VAR34), and group C^UM (HB3VAR34) IEs (Table 1 and Fig. S1) after 4–12 h co-culture. All graphs show mean values ± SD for 200 cells per condition for a minimum of three independent experiments conducted in duplicate. (B) Internalization of IEs depends on the PfEMP1 sub-type expressed by the parasite isolate and on ICAM-1. Internalization of HB3VAR03-IEs and IT4VAR13-IEs was assessed with homologous αPfEMP1 (αHB3VAR03_D4, αIT4VAR13_D4 at 1:100), αICAM-1 (clone 15.2, 10 µg/ml), αEPCR (10 µg/ml), αCD31 (10 µg/ml), αCD36 (10 µg/ml), or control (ctrl) IgG (10 µg/ml) antibody. Graphs show mean values ± SD of a minimum of three independent experiments conducted in duplicate. Statistical analysis was done using one-way ANOVA with Tukey’s multiple comparison test (*, P < 0.001; **, P = 0.005). Only significant findings are shown. (C) Internalization of HB3VAR03-IEs into BOECs in monolayers was visualized using confocal microscopy. The image shows a representative orthogonal view of HB3VAR03-IEs (white arrowheads) surrounded by ICAM-1. ICAM-1 is green (FITC), and DAPI nuceli stain is blue. n denotes the nuclei of the BOEC. Scale bar, 20 µm. (D) Human BOECs and (E) human brain microvascular endothelial cells (HBMECs) with internalized group A^+CM (HB3VAR03) and group B^UM (IT4VAR13) IEs. The percentages of cells (hCMEC/D3, BOEC, and HBMEC) with internalized IEs (A, B, D, and E) were quantified by wide-field microscopy. Data in D show the mean of three independent experiments, while data in E show the mean values ± SD of two independent experiments conducted in duplicate.

Figure S4.

3D BBB spheroids are composed of three different cell types. (A) FACS histogram showing pericytes (NG2, neural/glial antigen 2), astrocytes (GFAP, glial fibrillary acidic protein), and human cerebral microvascular endothelial cells (hCMEC/D3 and CD31). The data are a representative FACS plot from two independent experiments conducted in duplicate. (B) Representative brightfield image of BBB spheroid. Scale bar, 100 µm. (C) Schematic of spheroid with a core comprised of astrocytes (AC), bound by pericytes (PC), and surrounded by endothelial cells (EC) at the outer surface.

Figure 6.

Group A^+CM IE impact on the BBB. (A–C) Confocal images representing a minimum of three independent experiments of BBB spheroids with adherent group A^+CM IEs (total number of spheroids, n = 309). (A) 3D z-projection of a spheroid showing PKH-26–stained IE (HB3VAR03) in red and ICAM-1 in green (Alexa 488). Scale bar, 100 µm. (B) Group A^+CM IEs (HB3VAR03) binding ICAM-1 (green) and in the process of entering the endothelium. Scale bar, 20 µm. (C) Group A^+CM IEs (HB3VAR03) surrounded by ICAM-1 (green). White arrowheads point to IEs. Scale bar, 10 µm. (D) Adhesion of group A^+CM (HB3VAR03) and B^+UM (IT4VAR13) IEs to BBB spheroids in the presence of antibodies against the homologous PfEMP1 (α-PfEMP1), ICAM-1 (α-ICAM-1), EPCR (α-EPCR), PECAM-1 (α-CD31), and CD36 (α-CD36). Shown are the mean number of IEs bound per square millimeter ± SD. All data represent a minimum of three independent experiments conducted in duplicate. Statistical analysis was done using one-way ANOVA with Tukey’s multiple comparison test (*, P < 0.001). Only significant findings are shown. (E) Representative orthogonal view of confocal image of HB3VAR03 group A^+CM IEs found below the endothelial cell surface. 3D projection from confocal z-stack; IE₁ (white arrowhead) was found present in z-stack, shown is slice 23 of 57, which is 10.9 µm below the surface. White arrowhead points to the same IE (IE₁) in all three panels. Scale bar, 20 µm. (F) Group A^+CM IEs (IE₁–IE₅) found below the endothelial cell surface. 3D projection from confocal z-stack. The white arrowheads point toward internalized group A^+CM IE (HB3VAR03) at a depth of 19.8 µm (slice 44 of 60; total number of spheroids, n = 79). Scale bar, 20 µm. (G) Group A^+CM IEs (HB3VAR03) but not group B^UM IE (IT4VAR13) are internalized by BBB spheroids. Spheroids with internalized group A^+CM IEs (HB3VAR03) and group B^UM IEs (IT4VAR13). Each data point represents a single spheroid and the number of IEs or noninfected erythrocytes (RBC, red blood cell controls) visualized by confocal microscopy beyond the ICAM-1 boundary after 8 h incubation (total number of spheroids, n = 76). (H) ICAM-1–enriched ring/docking structures induced by co-culturing of endothelial cells with group A^+CM HB3VAR03-IE (left). Co-culture with IT4VAR13 group B^UM-IE does not induce such structures (right). Red arrowheads in the confocal image point to ring/docking structures; IEs are not visible in the two images (total number of spheroids, n = 309). ICAM-1 is green; nuclei stained with DAPI are blue. Scale bars, 100 µm. (I) Representative confocal image (z-projection) of spheroid with ring/docking structures induced by group A^+CM IEs (HB3VAR03). Ring/docking structures are indicated by dotted circles in top inset, and the IEs (IE₁–IE₃) found below the structures are shown in the brightfield image in the bottom inset. Black arrowheads points to IEs. Scale bar, 100 µm. (J) IE-induced alterations in barrier permeability measured as fold change ± SD in RFU-FITC dextran (70 kD) at 90 µm depth. VEGF (100 ng/ml) was used as a positive control. The data represent a minimum of three independent experiments (total number of spheroids, n = 140; 5–11 spheroids per group). Statistical analysis was done using one-way ANOVA with Tukey’s multiple comparison test (**, P = 0.001; ***, P = 0.0024). Only significant findings are shown. (K) Analysis of cell swelling. The spheroid volume (mm³) measured following coincubation with group A^+CM IE (HB3VAR03), group B^UM IE (IT4VAR13), and VEGF (100 ng/ml). The spheroids are the same as those in J. Statistical analysis was done using one-way ANOVA with Tukey’s multiple comparison test (*, P < 0.001). Only significant findings are shown. (L) Nuclei per spheroid following co-culture with group A^+CM IE (HB3VAR03), group B^UM IE (IT4VAR13), and VEGF (100 ng/ml; total number of spheroids, n = 140; 5–11 spheroids per group). Statistical analysis was done using one-way ANOVA with Tukey’s multiple comparison test; no statistical significant difference were found. Each data point in G, K, and L represents an individual spheroid. Ab, antibody; CTRL, control; n, nuclei.

Figure S5.

Group B/A^SM-IE binding EPCRs do not impact on the BBB. (A) Bar chart showing the fold change in RFU of dextran-FITC uptake after overnight incubation with group B/A^SM IT4VAR19-IEs (gray) and group B/A^SM IT4VAR20-IEs (dark gray) compared with untreated control (white). VEGF (50 ng/ml) was added as a positive control for barrier disruption (black). (B) Dot plot showing number of internalized IEs per spheroid after coincubation with IT4VAR19-IEs (gray) and IT4VAR20-IEs (dark gray). Each circle represents an individual spheroid. (C) Measurements of volume (mm³) for each individual spheroid after exposure to IT4VAR19-IEs (gray), IT4VAR20-IEs (dark gray), VEGF (50 ng/ml; black), and compared with untreated controls (white). The data in all graphs are derived from four independent experiments (total number of spheroids, n = 302), and statistical significance was determined by one-way ANOVA (*, P = 0.0338; **, P = 0.0047). Only significant findings are indicated.

Figure 7.

Brightfield image of tissue from CM patients. (A and B) May-Grünwald-Giemsa–stained postmortem brain samples from two different CM patients. Scale bars, 25 µm. Insets: Enlarged areas of interest showing IE internalized by endothelial cells. White arrows point to IEs. The assay was done blinded by two different highly experienced pathologists.