Recrudescent Plasmodium berghei from pregnant mice displays enhanced binding to the placenta and induces protection in multigravida

Abstract

Pregnancy-associated malaria (PAM) is associated with placenta pathology and poor pregnancy outcome but the mechanisms that control the malaria parasite expansion in pregnancy are still poorly understood and not amenable for study in human subjects. Here, we used a set of new tools to re-visit an experimental mouse model of pregnancy-induced malaria recrudescence, BALB/c with chronic Plasmodium berghei infection. During pregnancy 60% of the pre-exposed primiparous females showed pregnancy-induced malaria recrudescence and we demonstrated that the recrudescent P. berghei show an unexpected enhancement of the adherence to placenta tissue sections with a marked specificity for CSA. Furthermore, we showed that the intensity of parasitemia in primigravida was quantitatively correlated with the degree of thickening of the placental tissue and up-regulation of inflammation-related genes such as IL10. We also confirmed that the incidence of pregnancy-induced recrudescence, the intensity of the parasitemia peak and the impact on the pregnancy outcome decreased gradually from the first to the third pregnancy. Interestingly, placenta pathology and fetal impairment were also observed at low frequency among non-recrudescent females. Together, the data raise the hypothesis that recrudescent P. berghei displays selected specificity for the placenta tissue enabling on one hand, the triggering of the pathological process underlying PAM and on the other hand, the induction of PAM protection mechanisms that are revealed in subsequent pregnancies. Thus, by exploiting P. berghei pregnancy-induced recrudescence, this experimental system offers a mouse model to study the susceptibility to PAM and the mechanisms of disease protection in multigravida.

Figure 1. Malaria susceptibility is increased during pregnancy with detrimental effects for the progeny.

Representative parasitemia curves of BALB/c females infected with P. berghei - GFP (day 0) and treated with chloroquine for 3 days starting at day 7 (Panel A). Parasitemias of females maintained without male (non-pregnants) are represented in the upper plot. The lower plot shows 5 typical parasitemia curves of recrudescent primigravida. In Panel B average litter size and birth weight of 20 litters from non-infected primiparous females are compared with the average litter size of 29 litters and the birth weight average of 6 litters, from recrudescent primiparous females. Error bars represent standard error (***, p<0.001).

Figure 2. Peripheral parasitemia correlates with the reduction of placental blood sinusoids area.

(A) The blood sinusoidal area is plotted against the peripheral parasitemia peak observed in the pregnancy of primiparous females. The area of placental blood sinusoids, expressed as a fraction of the total placental area, was obtained using an automated morphometric procedure as described in Materials and Methods section. In recrudescent females, the degree of parasitemia was correlated with sinusoidal area reduction (correlation coefficient for recrudescent females is 0.45, P-value = 0.0012). Representative photomicrograph of placental sections HE stained from non-infected (B) and recrudescent (C–D) mothers. Accumulation of inflammatory cells (C), trophoblast thickening (arrows) and presence of iRBC (D) in blood sinusoids (arrowheads) are evidenced in placenta tissue from recrudescent mothers. Scale bars represent 15 µm in (B–D).

Figure 3. Placenta pathology is associated with altered gene expression of inflammation markers.

qRT-PCR of placenta tissue was used to detect the expression of cell type–specific genes indicating infiltration of inflammatory cells: Klrd1 gene for Natural Killer cells, Cd3e gene for T cells, Ncf2 gene for neutrophils and Mgl2 for macrophages (A). Placental gene expression was quantified for relevant markers of monocyte/macrophage chemotaxy (B), inflammation mediators (C) and vascular stress (D). RNA expression was quantified in 15 placentas from recrudescent primiparous BALB/c females and in 8 uninfected placentas, collected on G19. In (E) placental IL10 mRNA expression was separately analyzed in 5 placentas showing moderate pathology (+) and 4 placentas showing severe (++) pathology. Relative quantification was obtained with normalization by β-actin for (A), (C), (D) and (E) and by GAPDH for (B). Results are plotted as fold change over the respective non-infected controls and each bar represents the mean±s.e.m. (*, P-value<0.05).

Figure 4. P. berghei iRBC from recrudescent females show enhanced adhesion to placenta.

iRBC from males, non-pregnant females and recrudescent primiparous females were incubated on uninfected placental sections and the adherent parasitized cells were counted as described in Materials and Methods section (A). Adhesion assays were also performed after pre-treatment of placental sections with chondroitinase ABC or heparinase (negative control) (B, upper plot). Adhesion inhibition assays were carried out by pre-incubating iRBC from recrudescent females with 1 mg/ml concentrations of CSA or CA (negative control) (B, lower plot). In panel B the proportion of bound iRBC is expressed as a percentage of the control (non-treated placentas or non-preincubated iRBC, in upper and lower plots, respectively). Error bars represent the mean±s.e.m. of three independent experiments. (***, P-value<0.001).

Figure 5. Recrudescence incidence and peripheral parasitemia are decreased in multigravida.

(A) Frequency of females with high recrudescence (above 5% parasitemia), patency (parasitemia between 1% and 5%) and no recrudescence (parasitemia <1%) according to parity. Recrudescence incidence is significantly associated with parity (P-value = 0.001, Chi-square test). (B) Box-plots illustrate the range of the peripheral parasitemia peak according to parity. The parasitemia peak in the first pregnancy was significantly different from the second (P-value = 0.004) and third pregnancies (P-value = 0.006). * and ^○ represent extremes and outliers, respectively.

Figure 6. Reduced adverse pregnancy outcomes in multigravida.

Box-plots of the average litter size (A) and average newborn birth weight (B) according to parity (first, second and third pregnancy). Pregnancy outcome was significantly different in primigravida as compared to multigravida and non-infected pregnant females (***, P-value<0.001; **, P-value<0.01; *, P-value<0.05).

Figure 7. Occasional placenta pathology in non-recrudescent pregnant females.

Photomicrographs of HE-stained placental sections of sporadic cases of placental pathology in non-recrudescent females. The figure shows presence of iRBC adhered to the syncytiotrophoblast layer (A, insert) and in blood sinusoids (arrowheads) as well as trophoblast thickening (arrow). Scale bar represents 15 µm.