Development of severe pathology in immunized pregnant mice challenged with lethal malaria parasites

Abstract

Pregnant women are highly susceptible to malaria infection because of their low immunity and are at increased risk of maternal illness or death, in addition to spontaneous abortion, stillbirth, premature delivery, and low birth weight. However, the detailed pathogenesis of maternal malaria remains unclear. In this study, we evaluated a mouse model that shows similar severe pathological features of pregnant women during Plasmodium falciparum infection and investigated the pathogenesis of maternal malaria. Pregnant mice immunized by infection with an attenuated parasite, Plasmodium berghei XAT, were more susceptible to virulent P. berghei NK65 challenge/infection than were nonpregnant mice and showed high levels of parasitemia and a poor pregnancy outcome associated with placental pathology, such as accumulation of parasitized red blood cells, in the late phase of pregnancy. Notably, the pregnant immune mice challenged/infected with P. berghei NK65 developed liver injury associated with microvesicular fatty infiltration in late pregnancy. The pathological features were similar to acute fatty liver of pregnancy. Higher levels of gamma interferon and nitric oxide (NO) were found in plasma from pregnant immune mice infected with P. berghei NK65 than in plasma from nonpregnant mice. These findings suggest that development of liver injury and placental pathology in pregnant immune mice challenged/infected with P. berghei NK65 is accompanied by enhanced production of proinflammatory cytokines.

Fig 1

Mice immunized with P. berghei XAT are highly susceptible to lethal malaria infection during pregnancy. Female C57BL/6 mice were immunized with P. berghei XAT. On day 30 after infection with P. berghei XAT, female mice were mated with male mice for 1 day. Mice with or without a vaginal plug were not challenged (A) or were challenged with 1 × 10⁴ pRBCs of P. berghei NK65 (B). (A) Course of parasitemia in mice infected with P. berghei XAT. Bar indicates pregnancy period. (B) Course of parasitemia in P. berghei XAT-immunized mice challenged/infected with P. berghei NK65. Asterisks indicate a statistically significant difference (P < 0.005). Experiments were performed in triplicate, and representative data are shown. Results are expressed as means ± standard deviations of 3 to 5 mice. (C) Expression of GFP on P. berghei XAT. Fifty thousand cells were counted and analyzed by a FACSCalibur cytometer. (Left) Blood from P. berghei XAT-infected mice on day 14. GFP-expressing P. berghei XAT was detected by a FACSCalibur cytometer. (Middle) Blood from nonpregnant IM+NK mice on day 12 after P. berghei NK65 challenge infection. (Right) Blood from pregnant IM+NK mice on day 12 after P. berghei NK65 challenge infection. Results are expressed as means ± standard deviations of 5 to 8 mice, and representative data are shown.

Fig 2

Low levels of hematocrit and P. berghei-specific IgG in P. berghei XAT-immunized mice challenged with P. berghei NK65. (A) Hematocrit on day 16 postmating. (B) P. berghei-specific IgG levels in plasma on day 16 postmating. Data using plasma diluted 1:16 for detection of IgG are shown. Asterisks indicate a statistically significant difference (P < 0.005). Experiments were performed in triplicate, and representative data are shown. Results are expressed as means ± standard deviations of 3 to 5 mice.

Fig 3

Poor pregnancy outcome and placental pathology in P. berghei XAT-immunized mice challenged with P. berghei NK65. Fetuses and placentas were obtained from mice on day 18 postmating. (A) Fetus weight. (B) Number of fetuses. Asterisks indicate a statistically significant difference (P < 0.005). Experiments were performed in triplicate, and representative data are shown. Results are expressed as means ± standard deviations of 3 to 5 mice. (C to H) H&E staining of placental tissue. Arrowheads indicate pRBCs. (C and D) Placentas from pregnant uninfected mice. Bars, 200 μm (C) and 100 μm (D). (E and F) Placentas from pregnant immune mice. Bars, 200 μm (E) and 100 μm (F). (G and H) Placentas from pregnant immune mice challenged with P. berghei NK65. Bars, 200 μm (G) and 100 μm (H). Experiments using 3 to 5 mice were performed in triplicate, and representative data are shown.

Fig 4

Severe liver dysfunction in pregnant immune mice challenged with P. berghei NK65. Plasma AST (A), ALT (B), and glucose (C) levels on day 16 postinfection. Asterisks indicate a statistically significant difference (P < 0.05 compared with pregnant uninfected mice). Experiments were performed in triplicate, and representative data are shown. Results are expressed as means ± standard deviations of 3 to 5 mice.

Fig 5

Extensive necrotic regions in immune mice challenged with P. berghei NK65. Liver tissue was stained with H&E. Arrowheads indicate focal necrosis of liver cells. Bars, 1 mm. (A) Pregnant uninfected mice. (B) Pregnant immune mice. (C) Pregnant immune mice challenged with P. berghei NK65. (D) Cumulative liver injury incidence of pregnant mice. Values on the y axes represent the overall percentages of the total number of pregnant mice. Categories were pregnant uninfected mice (uninfected, n = 10), pregnant immune mice (IM, n = 10), and pregnant immune mice challenged with P. berghei NK65 (IM+NK, n = 21).

Fig 6

Accumulation of fat in hepatocytes of pregnant immune mice challenged with P. berghei NK65. Liver tissue staining with H&E (A to D) and with Sudan IV (E to H). Arrowheads indicate cytoplasmic microvesiculation of liver cells. Arrows indicate microvesicular fat. Bars, 100 μm. (A and E) Pregnant uninfected mice on day 16 postmating. (B and F) Pregnant immune mice on day 16 postmating. (C, D, G, and H) Pregnant immune mice challenged with P. berghei NK65 on day 12 postmating (C and G) and on day 16 postmating (D and H). (I) Quantification of fat accumulation in hepatocytes. Fat droplets in the photographs were enumerated using the BZ-II Analyzer software. Asterisks indicate a statistically significant difference compared with uninfected mice (*, P < 0.05; **, P < 0.005). Experiments were performed in triplicate, and representative data are shown. Results are expressed as means ± standard deviations of 3 to 5 mice.

Fig 7

Upregulation of proinflammatory cytokines in pregnant immune mice challenged with P. berghei NK65. Plasma was collected from uninfected and infected mice on day 16 postmating. (A) Plasma IFN-γ levels. (B) Plasma NO levels. (C) Plasma IL-10 levels. Asterisks indicate a statistically significant difference compared with uninfected mice (*, P < 0.05; **, P < 0.005). Experiments were performed in triplicate, and representative data are shown. Results are expressed as means ± standard deviations of 3 to 5 mice.