C5a enhances dysregulated inflammatory and angiogenic responses to malaria in vitro: potential implications for placental malaria

Abstract

Background: Placental malaria (PM) is a leading cause of maternal and infant mortality. Although the accumulation of parasitized erythrocytes (PEs) and monocytes within the placenta is thought to contribute to the pathophysiology of PM, the molecular mechanisms underlying PM remain unclear. Based on the hypothesis that excessive complement activation may contribute to PM, in particular generation of the potent inflammatory peptide C5a, we investigated the role of C5a in the pathogenesis of PM in vitro and in vivo.

Methodology and principal findings: Using primary human monocytes, the interaction between C5a and malaria in vitro was assessed. CSA- and CD36-binding PEs induced activation of C5 in the presence of human serum. Plasmodium falciparum GPI (pfGPI) enhanced C5a receptor expression (CD88) on monocytes, and the co-incubation of monocytes with C5a and pfGPI resulted in the synergistic induction of cytokines (IL-6, TNF, IL-1beta, and IL-10), chemokines (IL-8, MCP-1, MIP1alpha, MIP1beta) and the anti-angiogenic factor sFlt-1 in a time and dose-dependent manner. This dysregulated response was abrogated by C5a receptor blockade. To assess the potential role of C5a in PM, C5a plasma levels were measured in malaria-exposed primigravid women in western Kenya. Compared to pregnant women without malaria, C5a levels were significantly elevated in women with PM.

Conclusions and significance: These results suggest that C5a may contribute to the pathogenesis of PM by inducing dysregulated inflammatory and angiogenic responses that impair placental function.

Figure 1. Parasitized erythrocytes (PEs) induce C5 activation and pfGPI upregulates C5aR expression on monocytes.

(A) Serum from malaria-naïve donors was added to mature stage CS2 (CSA binding) PEs or E8B (ICAM-1 and CD36 binding), uninfected red blood cells (uRBCs) or media for 30 min and supernatants were assayed for C5a by ELISA. Increased C5a levels were observed in the supernatants of PEs compared to uRBC or control, (Student's t-test: uRBCs vs. Mature PEs ***p<0.001). Data are presented as means±SEM and are representative of four independent experiments for CS2 parasites and two independent experiments for CD36-binding parasites. (B) Human PBMCs were stimulated with pfGPI or unconjugated gold beads alone as a control for 24 h. Expression of C5a receptor was determined by flow cytometry on monocytes (CD14). The percentage of monocytes expressing C5aR was significantly higher for pfGPI-treated versus media control cells, (Student's t-test: *p = 0.0279). Data are presented as means±SEM and are representative of two independent experiments.

Figure 2. C5a potentiates pfGPI-induced inflammatory cytokines in a dose-dependent manner.

Human PBMCs were stimulated with varying concentrations of recombinant human C5a (0, 1, 5, 10, 50, 100 nM) for 24 hours, with or without pfGPI. Culture supernatants were collected and assayed for IL-6 (A) and TNF (B) by ELISA. Data were analyzed by two-way ANOVA and demonstrate an interaction effect (C5a*pfGPI) for IL-6 (***p<0.0001) and for TNF (***p<0.0001). Data are presented as means±SEM of three independent experiments.

Figure 3. C5a potentiates pfGPI-induced inflammatory cytokines and this effect is abrogated by C5a receptor blockade.

Human PBMCs were cultured with unconjugated gold beads (media control), unconjugated gold beads and C5a (50 nM), pfGPI (300 ng/mL), or a combination of C5a and pfGPI. (A–D) Supernatants were assayed for IL-6 (A), TNF (B), IL-1β (C), and IL-10 (D). A significant interaction effect between C5a and pfGPI was observed for all cytokines tested. p<0.0001 for IL-6, p = 0.0124 for TNF, p = 0.0418 for IL-1β and p<0.0001 for IL-10 (by two-way ANOVA on cumulative cytokine production over 48 h). Data are representative of three independent experiments. (E–H) Human PBMCs were treated with an α-CD88 mAb (5 µg/mL) to block the C5a receptor or an appropriate isotype control and exposed to either unconjugated gold beads (media control) or a combination of C5a (50 nM) and pfGPI (300 ng/mL) for 48 h. Supernatants were assayed for IL-6 (E), TNF (F), IL-1β (G) and IL-10 (H). Cytokine production was significantly reduced upon C5a receptor blockade; p = 0.0005 for IL-6, p = 0.0028 for TNF, p = 0.0479 for IL-1β and p = 0.0191 for IL-10 (repeated measures ANOVA comparing cytokine levels between isotype control mAb and α-CD88 mAb groups co-treated with C5a and pfGPI). Values shown are corrected for background levels observed in the media controls, and are presented as means±SEM. Data are representative of two independent experiments.

Figure 4. C5a potentiates secretion of pfGPI-induced chemokines and the anti-angiogenic factor sFlt-1.

Human PBMCs were treated with either unconjugated gold beads (media control), unconjugated gold beads and C5a (50 nM), pfGPI (300 ng/mL), or a combination of C5a and pfGPI for 48 h. Where indicated PBMCs were treated with either 5 µg/mL of α-CD88 mAb to block the C5a receptor or an appropriate isotype control and were exposed to C5a and pfGPI. Supernatants were assayed for IL-8 (A), MCP-1 (B), MIP-1α (C), MIP-1β (D) and sFlt-1 (E). A significant interaction effect between C5a and pfGPI was observed; *p = 0.0265 for IL-8, **p = 0.0010 for MCP-1, * p = 0.0177 for sFlt-1, and ***p<0.0001 for MIP-1 α, and MIP-1β (by two-way ANOVA). C5a receptor blockade significantly reduced the levels of MCP-1 (* p = 0.0388), MIP-1α (*** p = 0.0005), and MIP-1β (*** p = 0.0002) (by student's t-test). Data are presented as means±SEM and are representative of three independent experiments.

Figure 5. Primigravid women with placental malaria (PM) have increased levels of C5a in peripheral and placental plasma.

C5a levels were assayed in peripheral and placental plasma by ELISA. Samples were obtained from HIV negative primigravid women at time of delivery in Kisumu, western Kenya. C5a levels were significantly higher in (A) peripheral plasma of placenta malaria positive (PM+, n = 21) vs. placenta malaria negative (PM−, n = 45) women, *p = 0.01 by two-tailed Student's t-test on log transformed data; and (B) in placenta plasma of placenta malaria positive (PM+, n = 24) vs. placenta malaria negative (PM−, n = 47) women, *p = 0.0264 by Student's t-test two-tailed test on log transformed data.

Figure 6. Proposed mechanism for C5a-mediated dysregulation of the placental environment in PM.

Mature schizonts activate C5 and rupture releasing parasite components containing GPI that induce expression of C5aR and activate macrophages. The concomitant expression of C5aR and generation of C5a represent a potential autocrine mechanism by which C5a can amplify inflammation in P. falciparum malaria resulting in the excessive cytokine and chemokine responses that characterize PM. The dysregulated production of cytokines, chemokines and the anti-angiogenic factor sFlt-1 contribute to placental dysfunction, intrauterine growth restriction and low birth weight infants.