Optimization of BeWo model to investigate placental responses to Plasmodium falciparum infected erythrocytes

Abstract

Background: Establishment of an in vitro model to study placental malaria is essential for understanding the biology and pathogenesis of placental malaria. We defined experimental variables for obtaining responses of BeWo cells to placental binding Plasmodium falciparum infected erythrocytes (IE, CS2 parasites).

Materials and methods: Experimental variables included i) concentration of forskolin, a cyclic adenosine monophosphate inducer important in the induction of syncytialisation of BeWo, ii) suitable period of incubating BeWo with forskolin, and iii) ratio of IE to BeWo cells and length of incubation to induce physiological changes in BeWo cells, including the vasculogenic factors vascular endothelial growth factor A (VEGFA), endoglin, and angiopoietin-2; an anti-angiogenic factor (inhibin A); a regulator of cell growth, mammalian target of rapamycin (mTOR); a chemokine (IL-8); and the cytokine macrophage inhibition factor. The human hormone, chorionic gonadotrophin was used as a marker for syncytialisation.

Results: We showed that 72 hrs incubation of BeWo with 10 μm forskolin resulted in higher levels of syncytialisation and hCG secretion. Overall, the best condition was to co-culture syncytialised BeWo with a 10:1 ratio of IE for 48 hours. Under these conditions, when co-cultured with IE, BeWo produced increased amounts of IL-8 (p=0.0001), VEGF (p=0.001) and endoglin (p=0.001).

Conclusion: The model can be used to evaluate the impact of IE, inflammatory cytokines and other factors associated with placental malaria on syncytiotrophoblast function.

Figure 1.

Effect of forskolin on syncytial formation of BeWo, as indicated by hCG secretion, and cell viability. A: Concentration of hCG secreted by BeWo treated with 10, 25 or 50 μm of for-skolin in DMSO. Experiment done in triplicate and pooled for analysis. B: Viability of BeWo treated with different concentrations of forskolin. 1 x 10⁴ BeWo were treated with different concentrations of forskolin in DMSO or DMSO alone for different periods of time. Data represent the mean ± SE (n=6). Two ways ANOVA-Bonferroni post test was used. No significant difference between viability of BeWo treated with 10 μm forskolin at 48, 72 and 96 hrs was observed (p=0.62). The increase in cell viability in the media (control) is due to proliferation of BeWo cells; this proliferation does not happen once syncytial formation occurs.

Figure 2.

BeWo cell monolayers. A: before and, B: after treatment with 10 μm forskolin for 72 hrs. Red asterisks indicate monolayer of syncytialised BeWo.

Figure 3.

IL-8 secretions by BeWo cells treated with different concentrations of intact CS2 infected erythrocytes (IE) using normal red blood cells (nRBC) as controls. BeWo cells were treated with 10 μm forskolin, and incubated with different concentrations of IE overnight or 48 hrs. Data represent the mean ± SE (n=6). Two-way ANOVA-Bonferroni post test was used. Three asterisk indicate a significant (P<0.0001) difference between the amount of IL-8 secreted by BeWo treated with 10 IE and 10 nRBC at 48 hrs incubation.

Figure 4.

Relative RNA expression of selected markers by BeWo treated with different concentrations of intact CS2 infected erythrocytes (IE) and normal RBC (nRBC) (mean RNA expression relative to the internal control, β-actin gene). Forskolin-treated BeWo cells were cultured with different concentrations of IE and nRBC overnight or for 48 hrs. Data represent Mean ± SE (n=6). A: VEGFA, and B: mTOR (two-way ANOVA-Bonferroni post test was used. Asterisks indicate a significant difference between the mean RNA expression of VEGFA (p<0.001) and mTOR (p <0.05) by BeWo treated with 10 IE and 10 nRBC respectively, at 48 hrs post incubation.

Figure 5.

Time course expression of selected markers in BeWo cells. 2 x 10⁵ forskolin-treated BeWo were incubated with 2 x10⁶ CS2 IE using nRBC as control and incubated for 6, 12, 24 or 48 hrs. Figure shows relative levels of RNA expression of Inhibin A (top), ANGPT 2 (middle) and endoglin (bottom) relative to β-actin. Data represent mean ± SE (n=6). Two asterisks indicate a significant difference between the mean RNA expression of en-doglin (p<0.001) between BeWo treated with 10 IE and 10 nRBC respectively at 48 hrs post incubation (two-way ANOVA with Bonferroni post test).

Figure 6.

Time course expression of MIF (ng/ml) in BeWo cells. 2 x 10⁵ forskolin-treated BeWo were incubated with 2 x10⁶ CS2 IE using nRBC as control and incubated for 6, 12, 24 or 48 hrs. Supernatants were pooled from triplicates experiments and so only single assay of pooled samples were measured.