Transcriptional responses of brain endothelium to Plasmodium falciparum patient-derived isolates in vitro

Abstract

A hallmark of cerebral malaria (CM) is sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature. Binding of IE to endothelium reduces microvascular flow and, combined with an inflammatory response, perturbs endothelial barrier function, resulting in breakdown of the blood-brain barrier (BBB). Cytoadherence leads to activation of the endothelium and alters a range of cell processes affecting signaling pathways, receptor expression, coagulation, and disruption of BBB integrity. Here, we investigated whether CM-derived parasites elicit differential effects on human brain microvascular endothelial cells (HBMECs), as compared to uncomplicated malaria (UM)-derived parasites. Patient-derived IE from UM and CM clinical cases, as well as non-binding skeleton-binding protein 1 knockout parasites, were overlaid onto tumour necrosis factor (TNF)-activated HBMECs. Gene expression analysis of endothelial responses was performed using probe-based assays of a panel of genes involved in inflammation, apoptosis, endothelial barrier function, and prostacyclin synthesis pathway. We observed a significant effect on endothelial transcriptional responses in the presence of IE, yet there was no significant correlation between HBMEC responses and type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and level of IE binding to HBMECs, as we detected the same change in endothelial responses when employing both binding and non-binding parasites. Our results suggest that interaction of IE with endothelial cells in this co-culture model induces some endothelial responses that are independent of clinical origin and independent of the expression of the major variant antigen Plasmodium falciparum erythrocyte membrane protein 1 on the IE surface.

Importance: Cerebral malaria (CM) is the most prevalent and deadly complication of severe Plasmodium falciparum infection. A hallmark of this disease is sequestration of P. falciparum-infected erythrocytes (IE) in brain microvasculature that ultimately results in breakdown of the blood-brain barrier. Here, we compared the effect of P. falciparum parasites derived from uncomplicated malaria (UM) and CM cases on the relative gene expression of human brain microvascular endothelial cells (HBMECs) for a panel of genes. We observed a significant effect on the endothelial transcriptional response in the presence of IE, yet there is no significant correlation between HBMEC responses and the type of clinical syndrome (UM or CM). Furthermore, there was no correlation between HBMEC gene expression and both binding itself and the level of IE binding to HBMECs. Our results suggest that interaction of IE with endothelial cells induces endothelial responses that are independent of clinical origin and not entirely driven by surface Plasmodium falciparum erythrocyte membrane protein 1 expression.

Keywords: HBMEC; Plasmodium falciparum; cerebral malaria; cytoadherence; endothelium; gene expression.

Fig 1

IE of both UM and CM clinical isolates affect HBMEC transcriptional responses as compared to RBC. Differential gene expression of HBMECs after 6-hour co-culture with patient-derived parasites compared to their corresponding RBCs for BCL2A1 (A), CYP1A1 (B), KLF2 (C), KLF4 (D), PTGIS (E), and PTGS2 (F), calculated as relative to the corresponding 6-hour HBMEC-media control. Mean HBMEC gene expression (fold change) was compared in two ways for the selected genes: (left) Welch’s t-test comparing HBMEC-RBC and HBMEC-IE for UM or CM isolates and (right) paired t-test comparing all RBC controls and the combined HBMEC-IE of all isolates. The Welch’s t-test results shown in the graphs are from Fig. S1. Each point on the plot represents one sample FC, calculated as a mean of the technical triplicates, and the mean and 95% confidence intervals are indicated for each group. For the paired analysis, the mean FC was calculated from technical triplicates for each IE and corresponding RBC sample and are colored red for UM isolates and blue for CM isolates. A paired t-test was performed comparing the mean FC of HBMEC-IE with the corresponding HBMEC-RBC. Lines between the groups indicate that the RBC sample on the left was the control for the corresponding isolate sample on the right. Black lines represent samples from experiment 1, and gray lines represent samples from experiment 2. Two-tailed tests were performed for both analyses, and the dotted line in the plots marks the baseline of FC = 1. Significance is depicted by the P-value: *,0.01–0.05; **, 0.001–0.01; ***, 0.0001–0.001; ****, <0.0001.

Fig 2

When normalized to HBMEC-RBC controls, isolates within clinical categories do not induce differential responses in HBMECs. To determine the effect of the parasites, HBMEC relative gene expression when exposed to UM and CM patient-derived isolates was calculated relative to HBMEC-RBC expression, using GAPDH as the endogenous control and the 6-hour HBMEC-RBC as the normalization sample reference. Each point represents one experimental sample (mean of triplicates), and the mean FC and 95% confidence intervals for each gene were calculated using both biological replicates (n = 8). RBC-normalized HBMEC-UM is represented as “UMrn,” and RBC-normalized HBMEC-CM is represented as “CMrn.” Significance between the two clinical categories was determined by two-tailed unpaired t-tests with P-value summary defined: *,0.01–0.05. The dotted line marks the baseline of FC = 1 (red, UM isolates; blue, CM isolates).

Fig 3

Binding of patient-derived isolates to HBMECs under flow conditions. Binding of IE to TNF-stimulated HBMECs or human dermal microvascular endothelial cells (HDMECs) was determined using microfluidics. Shown is the mean ± SEM IE/mm² EC cell surface of 15 fields counted by microscopy. The level of binding for the UM- and CM-derived isolates shortly after isolation from the patients is depicted in light gray (HDMECs) and in black (HBMECs). After certain DIC binding of HBMECs is depicted in dark gray (experiment 2) and light gray with horizontal bars (experiment 1). UM1: 30 and 64 DIC, UM2: 32 and 50 DIC, UM3: 40 and 44 DIC, UM4: 25 and 38 DIC, CM1: 31 and 36 DIC, CM2: 32 and 46 DIC, CM3: 50 and 66 DIC, CM4: 42 and 46 DIC. SBP1-KO does not bind to HBMECs.

Fig 4

Observed differential effects on HBMEC transcriptional response are not due to binding to HBMECs. Relative gene expression of HBMECs exposed to patient-derived parasites that bind to HBMECs and the non-binding SBP1-KO parasites for 6 hours compared to corresponding RBCs for select genes. HBMEC-RBC samples for both binding and non-binding IE were combined for plotting. Each point on the plot represents one sample FC, calculated as a mean of the technical triplicates, and the mean and 95% confidence intervals are indicated for each group. FC was calculated using GAPDH as an endogenous control and relative to the 6-hour HBMEC-media normalization control (baseline is FC = 1). Welch’s t-tests were used to compare HBMEC-RBC and HBMEC-IE for each of the parasite groups, and the mean FC ± SEM for each gene was calculated using the two biological replicates for four UM and four CM isolates and their corresponding RBC [HBMEC-RBC (binding) n = 10, HBMEC-binding IE n = 16, HBMEC-RBC (SBP1KO) and HBMEC-SBP1KO (n = 2)]. The results of the Welch’s t-tests comparing the HBMEC-IE groups with corresponding HBMEC-RBC are summarized in Table S3. Sample grouping is represented by symbol, with triangles for HBMEC-RBC, circles for HBMEC-binding IE, and squares for HBMEC-SBP1KO. For binding IE, the red data points represent UM samples, while the blue data points represent CM samples. Samples from experiments with the non-binding parasites are in green. Significant relationships between HBMEC-IE groups and the corresponding HBMEC-RBC are indicated by straight bars over the IE groups (P < 0.05), and the P-value summaries are from the calculations performed in Table S3. For BCL2A1, KLF4, and PTGS2, the results of Welch’s t-tests comparing HBMEC-binding and HBMEC-SBP1KO are indicated by the bent lines connecting between these groups (P < 0.05), and the P-value summaries are from the calculations performed in Table S4. In all plots, the dotted line marks the baseline of FC = 1. P-value summary defined: *, 0.01–0.05; **, 0.001–0.01; ***, 0.0001–0.001; ****, <0.0001).