Iron overload in Plasmodium berghei-infected placenta as a pathogenesis mechanism of fetal death

Abstract

Plasmodium infection during gestation may lead to severe clinical manifestations including abortion, stillbirth, intrauterine growth retardation, and low birth weight. Mechanisms underlying such poor pregnancy outcomes are still unclear. In the animal model of severe placental malaria (PM), in utero fetal death frequently occurs and mothers often succumb to infection before or immediately after delivery. Plasmodium berghei-infected erythrocytes (IEs) continuously accumulate in the placenta, where they are then phagocytosed by fetal-derived placental cells, namely trophoblasts. Inside the phagosomes, disruption of IEs leads to the release of non-hemoglobin bound heme, which is subsequently catabolized by heme oxygenase-1 into carbon monoxide, biliverdin, and labile iron. Fine-tuned regulatory mechanisms operate to maintain iron homeostasis, preventing the deleterious effect of iron-induced oxidative stress. Our preliminary results demonstrate that iron overload in trophoblasts of P. berghei-infected placenta is associated with fetal death. Placentas which supported normally developing embryos showed no iron accumulation within the trophoblasts. Placentas from dead fetuses showed massive iron accumulation, which was associated with parasitic burden. Here we present preliminary data suggesting that disruption of iron homeostasis in trophoblasts during the course of PM is a consequence of heme accumulation after intense IE engulfment. We propose that iron overload in placenta is a pathogenic component of PM, contributing to fetal death. The mechanism through which it operates still needs to be elucidated.

Keywords: fetal death; heme; iron; pregnancy malaria; trophoblast.

FIGURE 1

Phagocytosis by trophoblasts of infected mouse placentas at end-stage gestation. (A) placental histology showing trophoblasts [cytotrophoblasts (CTB) and syncythiotrophoblasts (STB)], maternal blood space (MBS), endothelial cell (EC), and fetal capillary (FC) corresponding to the scheme on the upper right-hand panel. (B,C) placental sections showing engulfed IEs (arrows in B,C) in trophoblasts. Arrowheads point to IEs. Scale bar 25 μm.

FIGURE 2

Trophoblast response to IE binding. (A) Cytoadhesion of IE to trophoblast (TB) via VAR2CSA-CSA interaction leads to chemokine production by these activated cells (B) and recruitment of inflammatory cells to the site (C). Sequence of events compiled based on studies from (Fried and Duffy, 1996; Salanti et al., 2004; Lucchi et al., 2008; Srivastava et al., 2010).

FIGURE 3

Iron accumulation in placenta is dependent on dose of infection. Pregnant mice were infected with either 10⁴ or 10⁶ Plasmodium berghei-infected erythrocytes (IEs). Placentas from Plasmodium-infected pregnant mice were collected at G19/20 and processed for iron measurements as previously described (Gozzelino et al., 2012). Briefly placentas were dehydrated overnight, dissolved in 3 M HCl/trichloroacetic acid (TCA) 10% for 24 h. Labile iron was detected by colorimetric reagent (bathophenanthroline-disulfonic acid; BPTS) and absorbance was measured by spectrophotometer (SmartSpec 3000, Bio-Rad); **p < 0.01.

FIGURE 4

Iron accumulation in trophoblasts. Placentas from Plasmodium-infected pregnant females were evaluated at G19 for iron accumulation by Prussian blue staining in placental sections. (A,B) Non-infected placenta showing maternal blood space (MBS) and fetal capillary (FC) separated by trophoblast. (C,D) Infected placenta from a live fetus that shows no iron accumulation in trophoblasts and fairly preserved tissue structure; (E,F) Infected placenta from a dead fetus showing significant iron accumulation (blue) in trophoblast and loss of integrity of the tissue structure. Iron seems to be accumulated in phagocytic trophoblasts (surrounded area in F and box in G) and at the interface between trophoblast and fetal capillary (arrows in F) but not in MBS (E–G). Areas delimitated in A,C,E are magnified (crop and zoom) in B,D,F, respectively. Scale bars: 25 μm (A,C,E) and 10 μm (G). Arrowheads point to infected erythrocytes.

FIGURE 5

Hypothesis explaining heme-iron overload in trophoblast according to IE load. Left panel: (A) increased phagocytosis of IEs and possibly non-IEs by cytotrophoblast (CTB) and syncythiotrophoblasts (STB) in low blood flow regions in maternal blood space (MBS) where IE and non-IE are sequestered; (B) disruption of erythrocytes in the phagocytic vacuole leads to free heme exposure; downregulation of FLVCR1a on trophoblasts might cause free heme accumulation due to decreased heme export; (C) heme catabolism by HO-1 generates large amounts of iron which may exceed ferritin binding capacity; (D) free iron (Fe²⁺) induces cell death by ROS. Right panel: (A) lower placental infection might not affect FLVCR1a expression and phagocytosis is decreased; (B) excessive free heme is exported from the cell; (C) iron from heme catabolism is neutralized by ferritin; and (D) trophoblasts are protected from programmed cell death.