Murine Malaria Infection Induces Fetal Loss Associated with Accumulation of Plasmodium chabaudi AS-Infected Erythrocytes in the Placenta
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感染与免疫杂志 2006年第5期
Center for Tropical and Emerging Global Diseases and Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602
ABSTRACT
Malarial infection in nonimmune women is a risk factor for pregnancy loss, but the role that maternal antimalarial immune responses play in fetal compromise is not clear. We conducted longitudinal and serial sacrifice studies to examine the pathogenesis of malaria during pregnancy using the Plasmodium chabaudi AS/C57BL/6 mouse model. Peak parasitemia following inoculation with 1,000 parasite-infected murine erythrocytes and survival were similar in infected pregnant and nonpregnant mice, although development of parasitemia and anemia was slightly accelerated in pregnant mice. Importantly, pregnant mice failed to maintain viable pregnancies, most aborting before day 12 of gestation. At abortion, maternal placental blood parasitemia was statistically significantly higher than peripheral parasitemia. Infected mice had similar increases in spleen size and cellularity which were statistically significantly higher than in uninfected mice. In contrast, splenocyte proliferation in response to mitogenic stimulation around peak parasitemia was statistically significantly reduced in both groups of infected mice compared to uninfected, nonpregnant mice, suggesting that lymphoproliferation is not a good indicator of the antimalarial immune responses in pregnant or nonpregnant animals. This study suggests that while pregnant and nonpregnant C57BL/6 mice are equally capable of mounting an effective immune response to and surviving P. chabaudi AS infection, pregnant mice cannot produce viable pups. Fetal loss appears to be associated with placental accumulation of infected erythrocytes. Further study is required to determine to what extent maternal antimalarial immune responses, anemia, and placental accumulation of parasites contribute to compromised pregnancy in this model.
INTRODUCTION
Malaria continues to be a major public health problem in the developing world, causing an estimated 300 to 500 million cases each year and 1 to 2 million deaths (3). In regions where malaria is endemic, the related morbidity and mortality are primarily borne by children and pregnant women. Although women living in areas of high endemicity acquire protective immunity against severe malaria, this protection is markedly compromised in the first and second pregnancies (5, 44) and is characterized by maternal anemia (33, 41, 56) and low birth weight babies (20, 60, 66) who are at increased risk to die early in life (60). In contrast, in areas with low or unstable transmission of malaria, exposure is not constant enough to result in effective immunity in the population. In these settings, pregnancy outcome is severely compromised, and pregnant women of all parities are at greater risk of developing severe disease than nonpregnant women. Pregnant women living in areas of low endemicity experience high rates of abortion, stillbirth, and low birth weight babies (47, 55, 57, 58, 79).
Most of our understanding of the biological basis for the increased susceptibility of pregnant women to malarial infection is from studies conducted with pregnant women living in high-transmission areas (4, 21, 50, 73, 74). No detailed study has been done to understand the development of the maternal antimalarial immune responses in settings of low endemicity or during early pregnancy and the resultant effects on the mother and fetus. Experimental study of malarial infection during pregnancy is particularly problematic, as ethical and logistical constraints limit the longitudinal sampling of pregnant women and the placenta is inaccessible until delivery. Finally, pregnant women in regions where malaria is endemic often do not visit antenatal clinics early during pregnancy, making it difficult to assess women who abort early in gestation. An easily manipulable rodent model for malaria in pregnancy would be of great use in overcoming these limitations and improving our understanding of the immunological basis for the poor fetal outcome in nonimmune pregnant women in areas of low endemicity.
Early studies on the interrelationship of malaria and pregnancy in mice used Plasmodium berghei. These studies reported a more severe clinical course in pregnant animals, with maternal mortality, fetal loss, and reduced litter size (67, 68). This model, however, is not suitable to study the development of early maternal antimalarial immune responses or the impact of malarial infection on early pregnancy, because the infections were initiated on day 7 of pregnancy and were lethal to the mother (29, 48, 72). Further research to characterize the immunological and molecular basis of fetal loss in murine models for malarial infection during pregnancy has not been done. Thus, the recent advances in immunology and mouse genetics have not been applied to investigate the development of immune responses in malaria during pregnancy and their effect on fetal outcome. The rodent malarial parasite Plasmodium chabaudi AS represents a very useful organism for the study of immune responses to malaria, as it shares many characteristics with the most virulent human malarial parasite, Plasmodium falciparum. Both have been shown to express variant antigens (35, 59), sequestrate in the heart, lung, and liver (23, 54), and bind to CD36 (32, 46). P. chabaudi AS infection in nonpregnant C57BL/6 (B6) mice has been well characterized and used extensively to dissect the immune response to blood-stage malaria.
In the present study the clinical consequences of experimental infection with P. chabaudi AS in pregnant B6 mice were investigated. In this model system, infected pregnant mice developed splenic immune responses comparable to infected nonpregnant mice and survived the infection but failed to maintain their pregnancies. Also, we report here for the first time the accumulation of P. chabaudi AS-infected erythrocytes in the murine placenta, a phenomenon that is associated with poor fetal outcomes.
MATERIALS AND METHODS
Mice, parasites, and experimental design. Two types of studies, longitudinal and serial sacrifice, were performed to develop and characterize a mouse model for the immunopathogenesis of malaria during pregnancy. In both studies, 8- to 9-week-old, female B6 mice were used. Original breeding pairs purchased from Jackson Laboratories, Bar Harbor, ME, were maintained and bred by brother-sister pairing for a maximum of 10 generations at the University of Georgia Animal Resources facility in accordance with the guidelines of the University of Georgia Institutional Animal Care and Use Committee. Mice were maintained on a 10-h dark and 14-h light cycle with feed and water ad libitum. To improve breeding efficiency pregnant mice were fed a breeder diet (5K67) purchased from LabDiet, Richmond, IN. To avoid bias in weight gain, nonpregnant control mice were also fed the same diet. The day on which a vaginal plug was observed in timed mated mice was considered day zero of pregnancy (gestation day [GD 0]).
P. chabaudi AS originally obtained from Mary M. Stevenson (McGill University and the Montreal General Hospital Research Institute, Quebec, Canada) maintained as frozen stock and by passaging through gamma interferon knockout (B6.129S7-Ifngtm1Ts; obtained from R. Tarleton, University of Georgia) or A/J mice was used for all the experiments.
The first experiment was performed to characterize the course of P. chabaudi AS infection in pregnant mice. B6 infected pregnant (IP) mice were injected intravenously on GD 0 with 1,000 P. chabaudi AS-infected murine red blood cells (iRBC) per 20 g of body weight. Control, infected, nonpregnant ([INP]) mice were similarly infected. Uninfected pregnant (UP) and uninfected nonpregnant (UNP) mice were intravenously sham injected with 200 μl of phosphate-buffered saline per 20 g of body weight as controls for pregnancy and handling. After recording clinical parameters such as body weight, hematocrit, and parasitemia on GD 0, pregnant mice were not handled until day 6 of pregnancy to avoid stress-induced blastocyst implantation failure. For consistency, other mice were also not handled on those days. Thereafter, body weights were recorded daily and hematocrit and parasitemia were recorded on alternate days and at sacrifice at GD 18 (experiment day [ED] 18). No IP mouse maintained pregnancy to GD 18 (see Results).
Because IP mice did not maintain pregnancy to term, two prospective serial sacrifice studies were conducted to assess the dynamics of malaria-induced fetal loss. Control INP, UP, and UNP mice were included. In the first study, mice were sacrificed on GD/ED 6, 9, 12, and 15 and, in the second study, on GD/ED 6, 8, 9, 10, and 11. Clinical measures such as body weight, hematocrit, and parasitemia were recorded as described above for the initial longitudinal study. In the second study, apart from recording the routine clinical measures, IP mice were observed thrice daily beginning on GD 8 to identify those in the early stages of abortion. Mice having bloody, mucoid vaginal discharge were considered to be in the early stages of abortion and were sacrificed immediately. IP mice were continuously generated until at least five mice per time point were obtained. Thus, different numbers of mice, ranging from 3 to 14, were sacrificed at different time points. At sacrifice, nonviable fetuses or resorptions were identified by their necrotic appearance and notably smaller size compared to normal, viable fetuses. Resorption scars were identified by examining the uterus under a dissection microscope. To assess development of splenomegaly, spleens were collected aseptically from mice sacrificed on GD/ED 6, 8, 9, 10, 11, and 12, and a spleen index was calculated by dividing spleen weight by body weight.
Assessment of infection. The development of parasitemia was monitored by counting at least 1,000 erythrocytes in four to five high-power fields on Giemsa-stained tail blood thin smears. The hematocrit was used as a measure of anemia. Blood collected from the tail vein into heparinized capillary tubes was centrifuged in a microhematocrit centrifuge, and percent hematocrit was calculated according to the following formula: (volume of packed erythrocytes)/(total blood volume) x 100. Body weight was recorded in grams.
Placental parasitemia. Uteri collected from mice at the time of sacrifice were fixed in 10% buffered formalin for 24 h and then paraffin embedded. Giemsa-stained 2- to 3-μm-thick placental sections were used to determine the placental parasitemia by counting at least 1,000 erythrocytes in the maternal blood spaces in the placentae of at least five embryos.
Splenocyte proliferation assay. To make a single-cell suspension, spleens collected aseptically from mice sacrificed on GD/ED 6, 9, and 12 were pressed through a sterile fine-wire mesh with 10 ml of RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, Utah), 200 mM L-glutamine (Cellgro), and penicillin (10,000 IU/ml) and streptomycin (10,000 μg/ml) (Cellgro). Cell suspensions were centrifuged at 300 x g for 10 min. Erythrocytes were lysed with Tris-buffered 0.175 M NH4Cl, and the cells were washed twice in fresh medium. The viability of the cells was determined by trypan blue exclusion. One million splenocytes were then cultured in the presence of concanavalin A (ConA; 2 μg/ml), lipopolysaccharide (LPS; 1 μg/ml), and pokeweed mitogen (PWM; 2 μg/ml) (all from Sigma) in black 96-well microtiter plates for 72 h at 37°C in a humidified CO2 incubator with an atmosphere of 5% CO2. During the last 18 h of the culture the cells were incubated with bromodeoxyuridine (BrdU) labeling solution (Roche). After removing the labeling medium, the cells were dried and the incorporation of BrdU was detected using a cell proliferation enzyme-linked immunosorbent assay with BrdU (Roche Molecular Biochemicals) using chemiluminescent detection (Lmax II Luminometer; Molecular Devices, Sunnyvale CA) following the manufacturer's instructions.
Statistical analysis. Unless otherwise noted, the SAS statistical software package (version 8.02; SAS Institute, Inc., Cary, N.C.) was used for data analysis. Proc GLM was used to analyze the significance of differences among group means for parasitemia, hematocrit, body weight, spleen index, spleen cell number, and proliferative stimulation index. Duncan's multiple range test was used to perform multiple pairwise group comparisons in cases of equal sample size; where sample sizes were unequal, Tukey's Studentized (HSD) range test was used. Comparisons of two groups with unequal variances and sample sizes were performed with Welch's analysis of variance. Resorptions evaluated over time and survival were analyzed using a two-by-two contingency table, and the significance was determined with Fisher's exact test using GraphPad Instat software (version 2.05a; San Diego, CA). Student's t test was performed to analyze the significance of differences in the number of viable fetuses between IP and UP mice and placental and peripheral parasitemias. P values of 0.05 were considered to be significant.
RESULTS
P. chabaudi AS infection increased the incidence of resorptions and abortions in pregnant C57BL/6 mice. Our initial longitudinal experiment revealed that B6 mice infected on GD 0 with 1,000 P. chabaudi AS-infected erythrocytes were able to recover from infection (Fig. 1) with no significant differences in survival between IP and INP mice. Mortalities were observed only after GD/ED 11. Seven of 12 IP mice and 3 of 3 INP mice survived until GD/ED18 (P > 0.05 versus survival of IP mice). GD 18 was chosen as an appropriate time point for assessment of pregnancy since the duration of gestation in mice is 19 to 21 days. Although most IP mice recovered from infection, none of them went on to deliver live pups. Of the seven that progressed to GD 18, only three had any evidence of pregnancy, in the form of uterine resorption scars. This suggested that fetal loss had occurred at least several days earlier. (The pregnancy status of the other four animals could not be confirmed; thus, they were not included in any analysis.) The weight loss (Fig. 1E) and vaginal bleeding during ascending and peak parasitemia exhibited by IP mice suggested that they might have actively expelled their embryos during this time. Since fetal loss is one of the most severe and understudied consequences of malarial infection in pregnant women living in areas of low endemicity, the effects of malarial infection at different stages of fetal development were examined in greater depth.
To identify the stage of gestation at which mice start losing their pregnancies, an initial serial sacrifice study was performed by sacrificing mice on GD/ED 6, 9, 12, and 15. This study revealed that IP mice failed to maintain viable pregnancies up to GD 12 (Table 1), and the few IP mice that did proceed to GD 15 had only necrotic/resorbing embryos in their uteri (data not shown). To further characterize the events leading to fetal loss, a second, more-detailed serial sacrifice study was performed with mice being sacrificed on GD 6, 8, 9, 10, and 11, with heightened surveillance beginning on GD 8 to identify the mice in early stages of abortion. Abortions occurred at GD 10.3 ± 1.0 (mean ± standard deviation). The fetal outcome results pooled from the two serial sacrifice studies are presented in Table 1. At sacrifice, mice with vaginal bleeding were observed to have an open cervix with embryos in the cervix or vagina, demonstrating that they were actively expelling their embryos. IP mice had a significantly higher number of fetal resorptions and abortions compared to uninfected pregnant mice on GD 10 through 12. IP mice had 37.4% and 38.8% resorptions on GD 10 and 11 and 100% resorptions on GD 12 of pregnancy compared to 0, 5.1, and 3.3% in the UP group, respectively (P = 0.0001). Compared to UP mice, the number of viable fetuses was significantly reduced in IP mice on GD 12 (P = 0.0001). Thus, P. chabaudi AS infection abrogated pregnancy in B6 mice, with only nonviable fetuses and resorption scars being present in IP mice from GD 12 onwards.
Development of parasitemia and anemia is accelerated in P. chabaudi AS-infected pregnant mice. To study to what extent pregnancy impacts the course of P. chabaudi AS infection, development of parasitemia, anemia, and changes in body weight were studied in the four groups of mice. The results from the initial longitudinal study are presented in Fig. 1A, C, and E. Despite the fact that IP mice lost their pregnancies around GD 10, they were retained in this group for the purposes of analysis. Parasitemia was patent in IP mice by GD 6, was more than double that of INP mice on GD/ED 8 (P > 0.05), and peaked at 22.35% on GD 10. In contrast, peak parasitemia (19.62%) was observed on ED 12 in INP mice. However, parasitemia resolved after GD/ED 12 in all mice, decreasing to less than 2% on GD/ED 16. Since IP mice did not maintain pregnancy after GD 12, it is not possible to conclude from this study whether or not peak parasitemia and resolution of P. chabaudi AS infection differ in the context of pregnancy in B6 mice.
Hematocrit values measured on alternate days were used as an indicator of anemia. Both IP and INP mice experienced profound reductions in hematocrit during the course of infection (Fig. 1C). Development of malarial anemia was accelerated in the IP group compared to the INP group, with the percent hematocrit values being significantly lower in IP mice compared to the UP and UNP groups on GD/ED 10. However, the hematocrits of both infected groups reached their nadir on GD/ED 12 (13.67% for INP versus 14.0% for IP mice; P > 0.05) and were significantly lower than those in the UP and UNP groups (P < 0.05). These significantly lower hematocrits persisted throughout the remainder of the experiment. Interestingly, the hematocrits of the UP mice were also significantly lower than those of UNP mice on GD/ED 6 (P < 0.05). This was likely due to hemodilution associated with normal pregnancy (18).
Body weights of the mice were recorded daily and are represented in Fig. 1E. UP mice exhibited a steady increase in body weight starting on GD 10, from which time their body weights were significantly higher than all other groups (P < 0.05, GD/ED 10 to 18). IP mice also exhibited an increase in body weight during the initial stages of pregnancy, presumably due to the initial fetal development. However, both infection groups lost weight as parasitemia rose and hematocrits fell. Also, some reduction in body weight in IP mice was likely due to fetal resorption. IP and INP groups regained weight as they started to resolve the infection. As expected, UNP mice maintained their body weights throughout the experimental period.
In the initial longitudinal study, the clinical parameters such as development of parasitemia and anemia were monitored only on alternate days. To further characterize the course of infection, the development of parasitemia and anemia were monitored daily in the serial sacrifice study starting from GD/ED 8, and the experiments were terminated on GD/ED 12, the time point at which no viable fetuses were evident in IP mice (Table 1). Similar to the longitudinal study, the development of parasitemia was accelerated in IP mice and was significantly different from INP mice on GD 8 (P = 0.0004) (Fig. 1B). However, peak parasitemia was observed on GD/ED 11 in both IP (27.17%) and INP (25.04%) mice (P > 0.05). The discrepancy in the results for peak parasitemia between this and the longitudinal study is likely due to our failure to prepare blood smears on GD/ED 11. Anemia also developed earlier in IP mice and was significantly different from all the groups on GD 8, 9, and 10 (P < 0.05). However, hematocrit values for all infected mice were statistically significantly lower than that of UP and UNP mice on GD/ED 10 postinfection and reached their nadir on day 12 postinfection (INP, 11.88%; IP, 11.33%; P < 0.05 versus uninfected mice).
As in the longitudinal study, IP mice exhibited an initial increase in body weight which was significantly different from INP mice on GD/ED 6 through 9 (P < 0.05). By GD/ED 8, IP mice gained, on average (± standard deviation), 1.2 ± 1 g (6.8% ± 6.3% of average GD 0 body weight). However, as the infection progressed, both infected groups lost weight compared to UP mice on GD 9 through 12 (P < 0.05). The lowest body weights for IP and INP mice were recorded on GD 12 (8.8% ± 15.3% and 13.4% ± 5.4% loss in IP and INP mice, respectively, compared to average GD 0 weight). Compared to peak body weight on GD 8, IP mice lost 13.1% ± 9.8% by GD 12. This loss of body weight in IP mice could be due to a combined effect of malaria-induced cachexia and fetal loss. In contrast to IP mice, UP mice exhibited a steady increase in body weight starting at GD 6, increasing 9.1% ± 5.7% and 31.1% ± 13.1% at GD 8 and 12, respectively, relative to starting weight.
Accumulation of P. chabaudi AS-infected erythrocytes in the placentae of pregnant mice. Sequestration of P. chabaudi AS-infected erythrocytes has been reported in the heart, liver, lungs, and spleen of infected mice (13, 23), and placental sequestration of P. falciparum is associated with poor fetal outcome in human pregnancy (49). Thus, it was of interest to determine whether the observed pregnancy loss in P. chabaudi AS-infected pregnant mice is associated with an accumulation of iRBCs in placentae. To investigate this possibility, placental parasitemias scored on Giemsa-stained, 2-μm-thick placental sections were compared with the corresponding peripheral parasitemias. Indeed, there was massive accumulation of P. chabaudi AS-infected erythrocytes in the maternal sinusoids of the placentae (Fig. 2) with placental parasitemias in GD 10 and 11 mice being statistically significantly higher than in the peripheral blood (41.9% ± 12.7% versus 22.4% ± 8.0%; P = 0.003). To characterize the dynamics of placental accumulation of iRBCs, parasitemias were determined in mice on GD 9 (a day before abortions were observed), GD 10 (both aborting and nonaborting), and GD 11 (all aborting) of pregnancy (Fig. 3). Whereas GD 9 parasitemias were low in both placental and peripheral blood, parasitemias in placental sections from mice undergoing abortions on GD 10 and 11 were >40% higher than in the peripheral blood (47.1% and 42.4% versus 27.1% and 24.4%, respectively). A trend toward higher placental parasitemia in GD 10 nonaborting mice was not statistically significant (P > 0.05). These data demonstrate that there is an accumulation of P. chabaudi AS-infected RBCs in the placentae of infected mice, with statistically significantly higher levels than in the peripheral blood being found only in mice undergoing abortion. Contrary to what has been reported in the placentae of malaria-infected pregnant women (22, 45, 74), preliminary histopathological analysis revealed little accumulation of monocyte/macrophages in the placentae of IP mice (J. Poovassery et al., unpublished data).
Splenic function in P. chabaudi AS-infected pregnant mice. Pregnancy-associated immunomodulation is thought to play an important role in the increased susceptibility of pregnant women to malarial infection. To assess the immunocompetence of IP mice, changes in spleen size and cellularity as well as the proliferative response of splenocytes to mitogens at different stages of infection were evaluated. To compensate for significant differences in the body weights of the pregnant and nonpregnant mice, splenomegaly was evaluated using a spleen index (see Materials and Methods). For UNP mice, spleen parameters were recorded only on ED 6, 9, and 12. A comparison of the spleen indexes among the four groups is presented in Fig. 4A. Although both pregnant groups showed some level of persistent splenomegaly beginning on GD/ED 6 (both versus UNP, P < 0.05 for days 6, 9, and 12), P. chabaudi AS infection, regardless of pregnancy, resulted in large, progressive increases in the splenic index beginning on GD/ED 9 (compared to uninfected mice, days 9 and 12 [P < 0.05]).
Spleen cell number was also high in infected mice (Fig. 4B), increasing dramatically from GD/ED 8 compared to uninfected mice. Whereas splenocyte count in INP mice persisted at an elevated but constant level from days 8 to 11, spleen cellularity peaked on GD 9 in IP mice ([9.46 ± 1.3] x 107); this value was significantly different from UP ([2.93 ± 0.4] x 107), UNP ([1.53 ± 0.3] x 107), and INP ([4.93 ± 0.8] x 107) mice (P < 0.05). This enhancement of spleen cell number in IP mice was at least in part pregnancy related, since UP mice also had a higher splenocyte count compared to UNP mice, beginning at GD/ED 6 (GD/ED 6 and 12, P < 0.05). Overall, with the exception of a transient, accelerated expansion of spleen cellularity at GD/ED 9 in IP mice, increases in both spleen weight and cellularity were comparable in IP and INP mice.
Splenocyte function was assessed by the proliferative response to mitogenic stimulation (Fig. 5). At day 6 of infection, responses were comparable among the four experimental groups. However, at time points corresponding to ascending and peak parasitemia, responses in infected mice decreased relative to uninfected mice. Proliferation in response to LPS stimulation was significantly higher in UNP mice compared to INP mice on ED 9 and 12 (P < 0.05) and to IP mice on GD/ED 9 (P < 0.05). Proliferation of splenocytes from the IP and INP groups in response to ConA and PWM was also statistically significantly lower than that of the UNP group on GD/ED 12 (P < 0.05). A tendency for IP mice to have a higher LPS response compared to INP mice on GD 12 was not statistically significant (P > 0.05).
DISCUSSION
In nonimmune pregnant women, malarial infection during the first or second trimester has been reported to be associated with high rates of abortion (42). No detailed study has been done, however, to investigate antimalarial immune responses during early pregnancy in the absence of preexisting immunity or the effect of these responses on fetal outcome. Some progress toward this end has been made using nonimmune, pregnant rhesus monkeys infected with Plasmodium coatneyi (16). In this model, infected monkeys experienced increased rates of abortion and intrauterine growth retardation associated with placental pathology (15, 16). Also, the leukocyte profiles were altered in infected pregnant monkeys, with lower CD4+ and CD8+ T-cell and B-cell counts that were suggestive of pregnancy-associated immunomodulation (17). As an alternative approach, we have developed a mouse model for the study of adverse fetal outcomes associated with malaria. The short gestation time, availability of inbred, congenic, and gene knockout mice, and the well-characterized immune system of the laboratory mouse allow us to address questions and undertake experiments that, due to ethical, financial, and biological constraints, cannot easily be performed in humans or nonhuman primates.
In this model system, both IP and INP B6 mice survived P. chabaudi AS infection. Although the development of parasitemia and anemia was accelerated in IP mice, there was no significant increase in peak parasitemia or reduction in hematocrit as a function of pregnancy. Regardless of whether or not mice were pregnant at the beginning of the experiment, all were able to ultimately control parasitemia, reducing it to less than 2% by ED 16. The 2- to 3-day delay observed in development of patent and peak parasitemia in this study compared to most published literature on P. chabaudi AS infection in B6 mice is likely due to the difference in the size of the inoculum (103 versus 106) and the difference in the route of infection (intravenous versus intraperitoneal) (10).
The clinical outcome observed in IP mice was different from previous observations made in other rodent model systems for malaria during pregnancy, which showed a more severe course for pregnant animals (19, 29, 48, 72). Differences in rodent strain and parasite species, inoculum size, and the gestation day on which infection was initiated may be responsible for the observed differences between those studies and the current results. P. chabaudi AS infection has been shown to result in nonlethal infections in BALB/c, C57BL/6, and C57BL/10 mice and lethal infections in A/J and DBA/2 mice (14, 38, 61). B6 mice develop moderate levels of acute primary parasitemia and resolve the infection by 4 to 5 weeks postinfection by mounting a Th1 cytokine-biased immune response early during the course of infection (14, 63, 82). In contrast, susceptible A/J mice mount an early Th2 cytokine-biased immune response and succumb to infection by 10 to 12 days postinfection (53, 63).
It has been well demonstrated that successful pregnancy requires a bias against Th1-type and toward Th2-type cytokines (24, 52, 75). Since the survival rate of IP mice was comparable to INP mice, it is likely that the former also developed a proinflammatory/Th1 cytokine-biased immune response early during the infection to control the parasitemia, but at the expense of their pregnancies. It is noteworthy that malaria-induced abortion in Plasmodium vinckei-infected mice was shown to be dependent on proinflammatory tumor necrosis factor alpha (11). The results of the present study are consistent with that finding in that viable pregnancy in IP mice was completely abolished. Most failed to carry their fetuses beyond mid-gestation, with higher rates of fetal resorption and abortion compared to UP mice, and none delivered live, term pups. Spontaneous abortions during the first trimester have been reported in P. coatneyi-infected, nonimmune, pregnant rhesus monkeys (16) and in P. falciparum-infected, nonimmune pregnant women (42, 47, 58, 79). However, the immunologic mechanisms that lead to fetal loss during such nonimmune malarial infections remain to be fully elucidated.
To begin to address this issue, we assessed the development of splenomegaly as well as the splenocyte proliferative response in the presence of mitogens in infected and uninfected mice. In murine models it has been shown that during acute malarial infections the spleen plays important roles in parasite clearance (77), development of pathogen-specific T- and B-cell responses (36, 81), and provision of strong hematopoietic support (71, 78). Spleen cellularity and architecture also change dramatically during malarial infection (27, 62). Depending on the mouse strain and parasite species, these changes have been shown to be associated with either resistance or susceptibility to infection. Thus, development of massive splenomegaly has been found to correlate with resistance to P. chabaudi AS infection in resistant B6 mice but not in susceptible A/J mice (62). In agreement with this, both IP and INP B6 mice developed massive splenomegaly and, in a longitudinal study, survived the infection, albeit, for IP mice, in the absence of viable pregnancy. Also, contrary to expectation, IP mice had the highest splenocyte counts at one time point corresponding to ascending parasitemia. However, after the initial peak on day 9, both IP and INP mice exhibited a decrease in splenocyte count after peak parasitemia, as reported in the case of P. chabaudi AS-infected BALB/c mice (27).
In vitro proliferation of splenocytes isolated from both IP and INP mice during the acute phase of the infection was significantly reduced compared to uninfected mice. Malaria patients frequently show reduced immune responses not only to the malaria parasite but also to unrelated antigens (31, 70, 80), suggesting that an active immunosuppressive mechanism may be operating during the course of malarial infection. One possibility is nitric oxide-mediated suppression of splenocyte proliferation, as was reported for responses to ConA in P. chabaudi AS-infected B6 mice (34). This has also been demonstrated in other protozoan infections (28, 76). Furthermore, it is noteworthy that NO has been linked to pregnancy loss in mice (26). Recently, it was suggested that CD4+ CD25+ regulatory T cells may be involved in the immunosuppression observed with Plasmodium yoelii strain 17XL infection in BALB/c mice (30). Apart from the mechanisms proposed in nonpregnant mice, pregnancy-specific immunosuppression may be also operating in IP mice prior to pregnancy loss (69).
Despite low splenocyte proliferative responses, both IP and INP mice survived P. chabaudi AS infection, suggesting that the in vitro proliferative response of splenocytes is not a good indicator of the ability of infected mice to mount an effective immune response to malarial infection. As demonstrated in the case of nonpregnant B6 mice, an early proinflammatory/Th1 cytokine-biased immune response may be relatively more important for protection in IP and INP mice (64). This, however, may not translate to immune responses that can clear parasites from the placenta. Ultimately, it will be necessary to investigate in detail the immunological events occurring in the placental environment and in the spleen of P. chabaudi AS-infected pregnant mice to fully elucidate the protective and pathogenic immune mechanisms at play in pregnant mice. Furthermore, development of an experimental system that allows IP mice to progress to term pregnancy will be necessary for investigation of pregnancy-associated alterations in disease course and immune patterns throughout gestation. In this context, it will also be of value to assess the course of infection and outcome of pregnancy in previously malaria-exposed mice; all of these issues are currently being addressed in our laboratory.
Although the peak peripheral parasitemia was not significantly different between IP and INP mice, placental parasitemia was >40% higher than peripheral parasitemia in IP mice at the time of abortion, which effectively translates to a higher total parasite load in these animals. Sequestration of infected erythrocytes in several organs has been reported in P. chabaudi AS-infected nonpregnant mice (13, 23). Furthermore, the in vitro binding of the P. chabaudi AS-infected RBCs to endothelial cells has been shown to be mediated through CD36 (a well-characterized receptor utilized by P. falciparum-infected erythrocytes to bind to endothelial cells) (32), but other receptors are likely to be involved as well (46). Sequestration of P. falciparum-infected erythrocytes in the placental intervillous blood space is a key feature of malarial pathogenesis in pregnant women (49, 74) and is thought to be mediated largely through interaction with chondroitin sulfate A (21). The accumulation of P. chabaudi AS-infected erythrocytes in the placentae of infected mice, which has not been described before, may be a manifestation of specific placental sequestration. It is noteworthy that, like human trophoblasts, murine trophoblasts express a low-sulfated chondroitin sulfate (C. Gowda, personal communication). Clearly, further detailed studies of the interactions between P. chabaudi AS iRBCs and fetal trophoblast cells are required to define the biological mechanisms of placental parasite accumulation in mice and the pathogenic implications thereof. Our laboratory is currently investigating the impact of iRBC binding to trophoblasts on immunopathological events at the human (39) and murine maternofetal interfaces.
As reported in the case of P. chabaudi AS-infected nonpregnant B6 mice, both IP and INP mice experienced profound anemia (14). Although the lowest hematocrit levels and the day on which these levels were reached were not different between IP and INP mice, the development of anemia was faster in IP mice. Because UP mice also developed some anemia, it is likely that the faster rate in IP mice was not entirely malaria specific. Pregnancy-associated hemodilution has been reported in rats (18, 37) and pregnant women (7). In general, several factors may contribute to the complex process of anemia during malarial infection. Sequestration of infected RBCs (2), rupture of iRBCs during schizogony, development of autoantibodies (1, 25, 43), and ineffective erythropoiesis (12, 51, 83) may all contribute. Furthermore, it was recently suggested that the proinflammatory cytokine macrophage migration inhibitory factor (MIF) may play an important role in malarial anemia (40). It is of interest that MIF expression is massively upregulated in the placentae of malaria-infected pregnant women (9) and is specifically secreted by trophoblasts bound by P. falciparum-infected RBCs (8). Further study will be required to determine the relative roles of all of these factors, particularly that of MIF, in anemia and other protective and pathogenic immune mechanisms during pregnancy in B6 mice.
In addition to immune responses and placental accumulation of parasites, anemia may play a role in the observed pregnancy loss in this model. However, most of the abortions occurred between days 10 and 11, which is 1 to 2 days before peak anemia. Additionally, while anemia has been shown to be associated with low birth weight (6) and preterm labor (65), it is not associated with fetal loss in rodents or in humans. Furthermore, for cases of low birth weight among women with severe malarial anemia (<7 g/dl; 36% below normal level), Brabin and Piper (6) calculated that anemia alone can account for only about 10% of infant low birth weight cases, whereas malaria (with all associated immunopathogenic effects) can account for 40%. Thus, factors other than anemia likely play dominant roles in inducing pregnancy loss in P. chabaudi AS-infected B6 mice. We are actively pursuing this line of investigation.
In conclusion, this study shows that P. chabaudi AS infection leads to poor pregnancy outcomes in B6 mice. Although the splenocytes from both IP and INP mice exhibited reduced proliferation in response to mitogens compared to UNP mice, both IP and INP mice exhibited a comparable increase in spleen size and cell number during the course of infection and all survived the infection, albeit in the absence of viable postmidgestational pregnancy for IP. This suggests that IP mice develop peripheral antimalarial immune responses that are sufficient to control parasitemia during primary infection. Despite effective control of peak parasitemia, IP mice experienced massive accumulation of iRBCs in their placentae and failed to maintain their pregnancies beyond GD 12. This suggests that while peripheral parasitemia is controlled immunologically, this response is not sufficient to control localized placental parasitemia but may, paradoxically, contribute to fetal loss. Continued characterization of this model will contribute significantly to our understanding of the molecular and cellular immunological mechanisms involved in fetal loss during malarial infection.
ACKNOWLEDGMENTS
This work was supported by a Faculty Grant from the University of Georgia Research Foundation and State of Georgia funds (Veterinary Medical Experiment Station) as well as National Institutes of Health grant no. AI50240 to J.M.M. Jayakumar Poovassery is a recipient of a University of Georgia Department of Infectious Diseases Doctoral Assistantship.
We thank Liliana Jaso-Friedmann and Daniel G. Colley for critical review of the manuscript.
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ABSTRACT
Malarial infection in nonimmune women is a risk factor for pregnancy loss, but the role that maternal antimalarial immune responses play in fetal compromise is not clear. We conducted longitudinal and serial sacrifice studies to examine the pathogenesis of malaria during pregnancy using the Plasmodium chabaudi AS/C57BL/6 mouse model. Peak parasitemia following inoculation with 1,000 parasite-infected murine erythrocytes and survival were similar in infected pregnant and nonpregnant mice, although development of parasitemia and anemia was slightly accelerated in pregnant mice. Importantly, pregnant mice failed to maintain viable pregnancies, most aborting before day 12 of gestation. At abortion, maternal placental blood parasitemia was statistically significantly higher than peripheral parasitemia. Infected mice had similar increases in spleen size and cellularity which were statistically significantly higher than in uninfected mice. In contrast, splenocyte proliferation in response to mitogenic stimulation around peak parasitemia was statistically significantly reduced in both groups of infected mice compared to uninfected, nonpregnant mice, suggesting that lymphoproliferation is not a good indicator of the antimalarial immune responses in pregnant or nonpregnant animals. This study suggests that while pregnant and nonpregnant C57BL/6 mice are equally capable of mounting an effective immune response to and surviving P. chabaudi AS infection, pregnant mice cannot produce viable pups. Fetal loss appears to be associated with placental accumulation of infected erythrocytes. Further study is required to determine to what extent maternal antimalarial immune responses, anemia, and placental accumulation of parasites contribute to compromised pregnancy in this model.
INTRODUCTION
Malaria continues to be a major public health problem in the developing world, causing an estimated 300 to 500 million cases each year and 1 to 2 million deaths (3). In regions where malaria is endemic, the related morbidity and mortality are primarily borne by children and pregnant women. Although women living in areas of high endemicity acquire protective immunity against severe malaria, this protection is markedly compromised in the first and second pregnancies (5, 44) and is characterized by maternal anemia (33, 41, 56) and low birth weight babies (20, 60, 66) who are at increased risk to die early in life (60). In contrast, in areas with low or unstable transmission of malaria, exposure is not constant enough to result in effective immunity in the population. In these settings, pregnancy outcome is severely compromised, and pregnant women of all parities are at greater risk of developing severe disease than nonpregnant women. Pregnant women living in areas of low endemicity experience high rates of abortion, stillbirth, and low birth weight babies (47, 55, 57, 58, 79).
Most of our understanding of the biological basis for the increased susceptibility of pregnant women to malarial infection is from studies conducted with pregnant women living in high-transmission areas (4, 21, 50, 73, 74). No detailed study has been done to understand the development of the maternal antimalarial immune responses in settings of low endemicity or during early pregnancy and the resultant effects on the mother and fetus. Experimental study of malarial infection during pregnancy is particularly problematic, as ethical and logistical constraints limit the longitudinal sampling of pregnant women and the placenta is inaccessible until delivery. Finally, pregnant women in regions where malaria is endemic often do not visit antenatal clinics early during pregnancy, making it difficult to assess women who abort early in gestation. An easily manipulable rodent model for malaria in pregnancy would be of great use in overcoming these limitations and improving our understanding of the immunological basis for the poor fetal outcome in nonimmune pregnant women in areas of low endemicity.
Early studies on the interrelationship of malaria and pregnancy in mice used Plasmodium berghei. These studies reported a more severe clinical course in pregnant animals, with maternal mortality, fetal loss, and reduced litter size (67, 68). This model, however, is not suitable to study the development of early maternal antimalarial immune responses or the impact of malarial infection on early pregnancy, because the infections were initiated on day 7 of pregnancy and were lethal to the mother (29, 48, 72). Further research to characterize the immunological and molecular basis of fetal loss in murine models for malarial infection during pregnancy has not been done. Thus, the recent advances in immunology and mouse genetics have not been applied to investigate the development of immune responses in malaria during pregnancy and their effect on fetal outcome. The rodent malarial parasite Plasmodium chabaudi AS represents a very useful organism for the study of immune responses to malaria, as it shares many characteristics with the most virulent human malarial parasite, Plasmodium falciparum. Both have been shown to express variant antigens (35, 59), sequestrate in the heart, lung, and liver (23, 54), and bind to CD36 (32, 46). P. chabaudi AS infection in nonpregnant C57BL/6 (B6) mice has been well characterized and used extensively to dissect the immune response to blood-stage malaria.
In the present study the clinical consequences of experimental infection with P. chabaudi AS in pregnant B6 mice were investigated. In this model system, infected pregnant mice developed splenic immune responses comparable to infected nonpregnant mice and survived the infection but failed to maintain their pregnancies. Also, we report here for the first time the accumulation of P. chabaudi AS-infected erythrocytes in the murine placenta, a phenomenon that is associated with poor fetal outcomes.
MATERIALS AND METHODS
Mice, parasites, and experimental design. Two types of studies, longitudinal and serial sacrifice, were performed to develop and characterize a mouse model for the immunopathogenesis of malaria during pregnancy. In both studies, 8- to 9-week-old, female B6 mice were used. Original breeding pairs purchased from Jackson Laboratories, Bar Harbor, ME, were maintained and bred by brother-sister pairing for a maximum of 10 generations at the University of Georgia Animal Resources facility in accordance with the guidelines of the University of Georgia Institutional Animal Care and Use Committee. Mice were maintained on a 10-h dark and 14-h light cycle with feed and water ad libitum. To improve breeding efficiency pregnant mice were fed a breeder diet (5K67) purchased from LabDiet, Richmond, IN. To avoid bias in weight gain, nonpregnant control mice were also fed the same diet. The day on which a vaginal plug was observed in timed mated mice was considered day zero of pregnancy (gestation day [GD 0]).
P. chabaudi AS originally obtained from Mary M. Stevenson (McGill University and the Montreal General Hospital Research Institute, Quebec, Canada) maintained as frozen stock and by passaging through gamma interferon knockout (B6.129S7-Ifngtm1Ts; obtained from R. Tarleton, University of Georgia) or A/J mice was used for all the experiments.
The first experiment was performed to characterize the course of P. chabaudi AS infection in pregnant mice. B6 infected pregnant (IP) mice were injected intravenously on GD 0 with 1,000 P. chabaudi AS-infected murine red blood cells (iRBC) per 20 g of body weight. Control, infected, nonpregnant ([INP]) mice were similarly infected. Uninfected pregnant (UP) and uninfected nonpregnant (UNP) mice were intravenously sham injected with 200 μl of phosphate-buffered saline per 20 g of body weight as controls for pregnancy and handling. After recording clinical parameters such as body weight, hematocrit, and parasitemia on GD 0, pregnant mice were not handled until day 6 of pregnancy to avoid stress-induced blastocyst implantation failure. For consistency, other mice were also not handled on those days. Thereafter, body weights were recorded daily and hematocrit and parasitemia were recorded on alternate days and at sacrifice at GD 18 (experiment day [ED] 18). No IP mouse maintained pregnancy to GD 18 (see Results).
Because IP mice did not maintain pregnancy to term, two prospective serial sacrifice studies were conducted to assess the dynamics of malaria-induced fetal loss. Control INP, UP, and UNP mice were included. In the first study, mice were sacrificed on GD/ED 6, 9, 12, and 15 and, in the second study, on GD/ED 6, 8, 9, 10, and 11. Clinical measures such as body weight, hematocrit, and parasitemia were recorded as described above for the initial longitudinal study. In the second study, apart from recording the routine clinical measures, IP mice were observed thrice daily beginning on GD 8 to identify those in the early stages of abortion. Mice having bloody, mucoid vaginal discharge were considered to be in the early stages of abortion and were sacrificed immediately. IP mice were continuously generated until at least five mice per time point were obtained. Thus, different numbers of mice, ranging from 3 to 14, were sacrificed at different time points. At sacrifice, nonviable fetuses or resorptions were identified by their necrotic appearance and notably smaller size compared to normal, viable fetuses. Resorption scars were identified by examining the uterus under a dissection microscope. To assess development of splenomegaly, spleens were collected aseptically from mice sacrificed on GD/ED 6, 8, 9, 10, 11, and 12, and a spleen index was calculated by dividing spleen weight by body weight.
Assessment of infection. The development of parasitemia was monitored by counting at least 1,000 erythrocytes in four to five high-power fields on Giemsa-stained tail blood thin smears. The hematocrit was used as a measure of anemia. Blood collected from the tail vein into heparinized capillary tubes was centrifuged in a microhematocrit centrifuge, and percent hematocrit was calculated according to the following formula: (volume of packed erythrocytes)/(total blood volume) x 100. Body weight was recorded in grams.
Placental parasitemia. Uteri collected from mice at the time of sacrifice were fixed in 10% buffered formalin for 24 h and then paraffin embedded. Giemsa-stained 2- to 3-μm-thick placental sections were used to determine the placental parasitemia by counting at least 1,000 erythrocytes in the maternal blood spaces in the placentae of at least five embryos.
Splenocyte proliferation assay. To make a single-cell suspension, spleens collected aseptically from mice sacrificed on GD/ED 6, 9, and 12 were pressed through a sterile fine-wire mesh with 10 ml of RPMI 1640 (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, Utah), 200 mM L-glutamine (Cellgro), and penicillin (10,000 IU/ml) and streptomycin (10,000 μg/ml) (Cellgro). Cell suspensions were centrifuged at 300 x g for 10 min. Erythrocytes were lysed with Tris-buffered 0.175 M NH4Cl, and the cells were washed twice in fresh medium. The viability of the cells was determined by trypan blue exclusion. One million splenocytes were then cultured in the presence of concanavalin A (ConA; 2 μg/ml), lipopolysaccharide (LPS; 1 μg/ml), and pokeweed mitogen (PWM; 2 μg/ml) (all from Sigma) in black 96-well microtiter plates for 72 h at 37°C in a humidified CO2 incubator with an atmosphere of 5% CO2. During the last 18 h of the culture the cells were incubated with bromodeoxyuridine (BrdU) labeling solution (Roche). After removing the labeling medium, the cells were dried and the incorporation of BrdU was detected using a cell proliferation enzyme-linked immunosorbent assay with BrdU (Roche Molecular Biochemicals) using chemiluminescent detection (Lmax II Luminometer; Molecular Devices, Sunnyvale CA) following the manufacturer's instructions.
Statistical analysis. Unless otherwise noted, the SAS statistical software package (version 8.02; SAS Institute, Inc., Cary, N.C.) was used for data analysis. Proc GLM was used to analyze the significance of differences among group means for parasitemia, hematocrit, body weight, spleen index, spleen cell number, and proliferative stimulation index. Duncan's multiple range test was used to perform multiple pairwise group comparisons in cases of equal sample size; where sample sizes were unequal, Tukey's Studentized (HSD) range test was used. Comparisons of two groups with unequal variances and sample sizes were performed with Welch's analysis of variance. Resorptions evaluated over time and survival were analyzed using a two-by-two contingency table, and the significance was determined with Fisher's exact test using GraphPad Instat software (version 2.05a; San Diego, CA). Student's t test was performed to analyze the significance of differences in the number of viable fetuses between IP and UP mice and placental and peripheral parasitemias. P values of 0.05 were considered to be significant.
RESULTS
P. chabaudi AS infection increased the incidence of resorptions and abortions in pregnant C57BL/6 mice. Our initial longitudinal experiment revealed that B6 mice infected on GD 0 with 1,000 P. chabaudi AS-infected erythrocytes were able to recover from infection (Fig. 1) with no significant differences in survival between IP and INP mice. Mortalities were observed only after GD/ED 11. Seven of 12 IP mice and 3 of 3 INP mice survived until GD/ED18 (P > 0.05 versus survival of IP mice). GD 18 was chosen as an appropriate time point for assessment of pregnancy since the duration of gestation in mice is 19 to 21 days. Although most IP mice recovered from infection, none of them went on to deliver live pups. Of the seven that progressed to GD 18, only three had any evidence of pregnancy, in the form of uterine resorption scars. This suggested that fetal loss had occurred at least several days earlier. (The pregnancy status of the other four animals could not be confirmed; thus, they were not included in any analysis.) The weight loss (Fig. 1E) and vaginal bleeding during ascending and peak parasitemia exhibited by IP mice suggested that they might have actively expelled their embryos during this time. Since fetal loss is one of the most severe and understudied consequences of malarial infection in pregnant women living in areas of low endemicity, the effects of malarial infection at different stages of fetal development were examined in greater depth.
To identify the stage of gestation at which mice start losing their pregnancies, an initial serial sacrifice study was performed by sacrificing mice on GD/ED 6, 9, 12, and 15. This study revealed that IP mice failed to maintain viable pregnancies up to GD 12 (Table 1), and the few IP mice that did proceed to GD 15 had only necrotic/resorbing embryos in their uteri (data not shown). To further characterize the events leading to fetal loss, a second, more-detailed serial sacrifice study was performed with mice being sacrificed on GD 6, 8, 9, 10, and 11, with heightened surveillance beginning on GD 8 to identify the mice in early stages of abortion. Abortions occurred at GD 10.3 ± 1.0 (mean ± standard deviation). The fetal outcome results pooled from the two serial sacrifice studies are presented in Table 1. At sacrifice, mice with vaginal bleeding were observed to have an open cervix with embryos in the cervix or vagina, demonstrating that they were actively expelling their embryos. IP mice had a significantly higher number of fetal resorptions and abortions compared to uninfected pregnant mice on GD 10 through 12. IP mice had 37.4% and 38.8% resorptions on GD 10 and 11 and 100% resorptions on GD 12 of pregnancy compared to 0, 5.1, and 3.3% in the UP group, respectively (P = 0.0001). Compared to UP mice, the number of viable fetuses was significantly reduced in IP mice on GD 12 (P = 0.0001). Thus, P. chabaudi AS infection abrogated pregnancy in B6 mice, with only nonviable fetuses and resorption scars being present in IP mice from GD 12 onwards.
Development of parasitemia and anemia is accelerated in P. chabaudi AS-infected pregnant mice. To study to what extent pregnancy impacts the course of P. chabaudi AS infection, development of parasitemia, anemia, and changes in body weight were studied in the four groups of mice. The results from the initial longitudinal study are presented in Fig. 1A, C, and E. Despite the fact that IP mice lost their pregnancies around GD 10, they were retained in this group for the purposes of analysis. Parasitemia was patent in IP mice by GD 6, was more than double that of INP mice on GD/ED 8 (P > 0.05), and peaked at 22.35% on GD 10. In contrast, peak parasitemia (19.62%) was observed on ED 12 in INP mice. However, parasitemia resolved after GD/ED 12 in all mice, decreasing to less than 2% on GD/ED 16. Since IP mice did not maintain pregnancy after GD 12, it is not possible to conclude from this study whether or not peak parasitemia and resolution of P. chabaudi AS infection differ in the context of pregnancy in B6 mice.
Hematocrit values measured on alternate days were used as an indicator of anemia. Both IP and INP mice experienced profound reductions in hematocrit during the course of infection (Fig. 1C). Development of malarial anemia was accelerated in the IP group compared to the INP group, with the percent hematocrit values being significantly lower in IP mice compared to the UP and UNP groups on GD/ED 10. However, the hematocrits of both infected groups reached their nadir on GD/ED 12 (13.67% for INP versus 14.0% for IP mice; P > 0.05) and were significantly lower than those in the UP and UNP groups (P < 0.05). These significantly lower hematocrits persisted throughout the remainder of the experiment. Interestingly, the hematocrits of the UP mice were also significantly lower than those of UNP mice on GD/ED 6 (P < 0.05). This was likely due to hemodilution associated with normal pregnancy (18).
Body weights of the mice were recorded daily and are represented in Fig. 1E. UP mice exhibited a steady increase in body weight starting on GD 10, from which time their body weights were significantly higher than all other groups (P < 0.05, GD/ED 10 to 18). IP mice also exhibited an increase in body weight during the initial stages of pregnancy, presumably due to the initial fetal development. However, both infection groups lost weight as parasitemia rose and hematocrits fell. Also, some reduction in body weight in IP mice was likely due to fetal resorption. IP and INP groups regained weight as they started to resolve the infection. As expected, UNP mice maintained their body weights throughout the experimental period.
In the initial longitudinal study, the clinical parameters such as development of parasitemia and anemia were monitored only on alternate days. To further characterize the course of infection, the development of parasitemia and anemia were monitored daily in the serial sacrifice study starting from GD/ED 8, and the experiments were terminated on GD/ED 12, the time point at which no viable fetuses were evident in IP mice (Table 1). Similar to the longitudinal study, the development of parasitemia was accelerated in IP mice and was significantly different from INP mice on GD 8 (P = 0.0004) (Fig. 1B). However, peak parasitemia was observed on GD/ED 11 in both IP (27.17%) and INP (25.04%) mice (P > 0.05). The discrepancy in the results for peak parasitemia between this and the longitudinal study is likely due to our failure to prepare blood smears on GD/ED 11. Anemia also developed earlier in IP mice and was significantly different from all the groups on GD 8, 9, and 10 (P < 0.05). However, hematocrit values for all infected mice were statistically significantly lower than that of UP and UNP mice on GD/ED 10 postinfection and reached their nadir on day 12 postinfection (INP, 11.88%; IP, 11.33%; P < 0.05 versus uninfected mice).
As in the longitudinal study, IP mice exhibited an initial increase in body weight which was significantly different from INP mice on GD/ED 6 through 9 (P < 0.05). By GD/ED 8, IP mice gained, on average (± standard deviation), 1.2 ± 1 g (6.8% ± 6.3% of average GD 0 body weight). However, as the infection progressed, both infected groups lost weight compared to UP mice on GD 9 through 12 (P < 0.05). The lowest body weights for IP and INP mice were recorded on GD 12 (8.8% ± 15.3% and 13.4% ± 5.4% loss in IP and INP mice, respectively, compared to average GD 0 weight). Compared to peak body weight on GD 8, IP mice lost 13.1% ± 9.8% by GD 12. This loss of body weight in IP mice could be due to a combined effect of malaria-induced cachexia and fetal loss. In contrast to IP mice, UP mice exhibited a steady increase in body weight starting at GD 6, increasing 9.1% ± 5.7% and 31.1% ± 13.1% at GD 8 and 12, respectively, relative to starting weight.
Accumulation of P. chabaudi AS-infected erythrocytes in the placentae of pregnant mice. Sequestration of P. chabaudi AS-infected erythrocytes has been reported in the heart, liver, lungs, and spleen of infected mice (13, 23), and placental sequestration of P. falciparum is associated with poor fetal outcome in human pregnancy (49). Thus, it was of interest to determine whether the observed pregnancy loss in P. chabaudi AS-infected pregnant mice is associated with an accumulation of iRBCs in placentae. To investigate this possibility, placental parasitemias scored on Giemsa-stained, 2-μm-thick placental sections were compared with the corresponding peripheral parasitemias. Indeed, there was massive accumulation of P. chabaudi AS-infected erythrocytes in the maternal sinusoids of the placentae (Fig. 2) with placental parasitemias in GD 10 and 11 mice being statistically significantly higher than in the peripheral blood (41.9% ± 12.7% versus 22.4% ± 8.0%; P = 0.003). To characterize the dynamics of placental accumulation of iRBCs, parasitemias were determined in mice on GD 9 (a day before abortions were observed), GD 10 (both aborting and nonaborting), and GD 11 (all aborting) of pregnancy (Fig. 3). Whereas GD 9 parasitemias were low in both placental and peripheral blood, parasitemias in placental sections from mice undergoing abortions on GD 10 and 11 were >40% higher than in the peripheral blood (47.1% and 42.4% versus 27.1% and 24.4%, respectively). A trend toward higher placental parasitemia in GD 10 nonaborting mice was not statistically significant (P > 0.05). These data demonstrate that there is an accumulation of P. chabaudi AS-infected RBCs in the placentae of infected mice, with statistically significantly higher levels than in the peripheral blood being found only in mice undergoing abortion. Contrary to what has been reported in the placentae of malaria-infected pregnant women (22, 45, 74), preliminary histopathological analysis revealed little accumulation of monocyte/macrophages in the placentae of IP mice (J. Poovassery et al., unpublished data).
Splenic function in P. chabaudi AS-infected pregnant mice. Pregnancy-associated immunomodulation is thought to play an important role in the increased susceptibility of pregnant women to malarial infection. To assess the immunocompetence of IP mice, changes in spleen size and cellularity as well as the proliferative response of splenocytes to mitogens at different stages of infection were evaluated. To compensate for significant differences in the body weights of the pregnant and nonpregnant mice, splenomegaly was evaluated using a spleen index (see Materials and Methods). For UNP mice, spleen parameters were recorded only on ED 6, 9, and 12. A comparison of the spleen indexes among the four groups is presented in Fig. 4A. Although both pregnant groups showed some level of persistent splenomegaly beginning on GD/ED 6 (both versus UNP, P < 0.05 for days 6, 9, and 12), P. chabaudi AS infection, regardless of pregnancy, resulted in large, progressive increases in the splenic index beginning on GD/ED 9 (compared to uninfected mice, days 9 and 12 [P < 0.05]).
Spleen cell number was also high in infected mice (Fig. 4B), increasing dramatically from GD/ED 8 compared to uninfected mice. Whereas splenocyte count in INP mice persisted at an elevated but constant level from days 8 to 11, spleen cellularity peaked on GD 9 in IP mice ([9.46 ± 1.3] x 107); this value was significantly different from UP ([2.93 ± 0.4] x 107), UNP ([1.53 ± 0.3] x 107), and INP ([4.93 ± 0.8] x 107) mice (P < 0.05). This enhancement of spleen cell number in IP mice was at least in part pregnancy related, since UP mice also had a higher splenocyte count compared to UNP mice, beginning at GD/ED 6 (GD/ED 6 and 12, P < 0.05). Overall, with the exception of a transient, accelerated expansion of spleen cellularity at GD/ED 9 in IP mice, increases in both spleen weight and cellularity were comparable in IP and INP mice.
Splenocyte function was assessed by the proliferative response to mitogenic stimulation (Fig. 5). At day 6 of infection, responses were comparable among the four experimental groups. However, at time points corresponding to ascending and peak parasitemia, responses in infected mice decreased relative to uninfected mice. Proliferation in response to LPS stimulation was significantly higher in UNP mice compared to INP mice on ED 9 and 12 (P < 0.05) and to IP mice on GD/ED 9 (P < 0.05). Proliferation of splenocytes from the IP and INP groups in response to ConA and PWM was also statistically significantly lower than that of the UNP group on GD/ED 12 (P < 0.05). A tendency for IP mice to have a higher LPS response compared to INP mice on GD 12 was not statistically significant (P > 0.05).
DISCUSSION
In nonimmune pregnant women, malarial infection during the first or second trimester has been reported to be associated with high rates of abortion (42). No detailed study has been done, however, to investigate antimalarial immune responses during early pregnancy in the absence of preexisting immunity or the effect of these responses on fetal outcome. Some progress toward this end has been made using nonimmune, pregnant rhesus monkeys infected with Plasmodium coatneyi (16). In this model, infected monkeys experienced increased rates of abortion and intrauterine growth retardation associated with placental pathology (15, 16). Also, the leukocyte profiles were altered in infected pregnant monkeys, with lower CD4+ and CD8+ T-cell and B-cell counts that were suggestive of pregnancy-associated immunomodulation (17). As an alternative approach, we have developed a mouse model for the study of adverse fetal outcomes associated with malaria. The short gestation time, availability of inbred, congenic, and gene knockout mice, and the well-characterized immune system of the laboratory mouse allow us to address questions and undertake experiments that, due to ethical, financial, and biological constraints, cannot easily be performed in humans or nonhuman primates.
In this model system, both IP and INP B6 mice survived P. chabaudi AS infection. Although the development of parasitemia and anemia was accelerated in IP mice, there was no significant increase in peak parasitemia or reduction in hematocrit as a function of pregnancy. Regardless of whether or not mice were pregnant at the beginning of the experiment, all were able to ultimately control parasitemia, reducing it to less than 2% by ED 16. The 2- to 3-day delay observed in development of patent and peak parasitemia in this study compared to most published literature on P. chabaudi AS infection in B6 mice is likely due to the difference in the size of the inoculum (103 versus 106) and the difference in the route of infection (intravenous versus intraperitoneal) (10).
The clinical outcome observed in IP mice was different from previous observations made in other rodent model systems for malaria during pregnancy, which showed a more severe course for pregnant animals (19, 29, 48, 72). Differences in rodent strain and parasite species, inoculum size, and the gestation day on which infection was initiated may be responsible for the observed differences between those studies and the current results. P. chabaudi AS infection has been shown to result in nonlethal infections in BALB/c, C57BL/6, and C57BL/10 mice and lethal infections in A/J and DBA/2 mice (14, 38, 61). B6 mice develop moderate levels of acute primary parasitemia and resolve the infection by 4 to 5 weeks postinfection by mounting a Th1 cytokine-biased immune response early during the course of infection (14, 63, 82). In contrast, susceptible A/J mice mount an early Th2 cytokine-biased immune response and succumb to infection by 10 to 12 days postinfection (53, 63).
It has been well demonstrated that successful pregnancy requires a bias against Th1-type and toward Th2-type cytokines (24, 52, 75). Since the survival rate of IP mice was comparable to INP mice, it is likely that the former also developed a proinflammatory/Th1 cytokine-biased immune response early during the infection to control the parasitemia, but at the expense of their pregnancies. It is noteworthy that malaria-induced abortion in Plasmodium vinckei-infected mice was shown to be dependent on proinflammatory tumor necrosis factor alpha (11). The results of the present study are consistent with that finding in that viable pregnancy in IP mice was completely abolished. Most failed to carry their fetuses beyond mid-gestation, with higher rates of fetal resorption and abortion compared to UP mice, and none delivered live, term pups. Spontaneous abortions during the first trimester have been reported in P. coatneyi-infected, nonimmune, pregnant rhesus monkeys (16) and in P. falciparum-infected, nonimmune pregnant women (42, 47, 58, 79). However, the immunologic mechanisms that lead to fetal loss during such nonimmune malarial infections remain to be fully elucidated.
To begin to address this issue, we assessed the development of splenomegaly as well as the splenocyte proliferative response in the presence of mitogens in infected and uninfected mice. In murine models it has been shown that during acute malarial infections the spleen plays important roles in parasite clearance (77), development of pathogen-specific T- and B-cell responses (36, 81), and provision of strong hematopoietic support (71, 78). Spleen cellularity and architecture also change dramatically during malarial infection (27, 62). Depending on the mouse strain and parasite species, these changes have been shown to be associated with either resistance or susceptibility to infection. Thus, development of massive splenomegaly has been found to correlate with resistance to P. chabaudi AS infection in resistant B6 mice but not in susceptible A/J mice (62). In agreement with this, both IP and INP B6 mice developed massive splenomegaly and, in a longitudinal study, survived the infection, albeit, for IP mice, in the absence of viable pregnancy. Also, contrary to expectation, IP mice had the highest splenocyte counts at one time point corresponding to ascending parasitemia. However, after the initial peak on day 9, both IP and INP mice exhibited a decrease in splenocyte count after peak parasitemia, as reported in the case of P. chabaudi AS-infected BALB/c mice (27).
In vitro proliferation of splenocytes isolated from both IP and INP mice during the acute phase of the infection was significantly reduced compared to uninfected mice. Malaria patients frequently show reduced immune responses not only to the malaria parasite but also to unrelated antigens (31, 70, 80), suggesting that an active immunosuppressive mechanism may be operating during the course of malarial infection. One possibility is nitric oxide-mediated suppression of splenocyte proliferation, as was reported for responses to ConA in P. chabaudi AS-infected B6 mice (34). This has also been demonstrated in other protozoan infections (28, 76). Furthermore, it is noteworthy that NO has been linked to pregnancy loss in mice (26). Recently, it was suggested that CD4+ CD25+ regulatory T cells may be involved in the immunosuppression observed with Plasmodium yoelii strain 17XL infection in BALB/c mice (30). Apart from the mechanisms proposed in nonpregnant mice, pregnancy-specific immunosuppression may be also operating in IP mice prior to pregnancy loss (69).
Despite low splenocyte proliferative responses, both IP and INP mice survived P. chabaudi AS infection, suggesting that the in vitro proliferative response of splenocytes is not a good indicator of the ability of infected mice to mount an effective immune response to malarial infection. As demonstrated in the case of nonpregnant B6 mice, an early proinflammatory/Th1 cytokine-biased immune response may be relatively more important for protection in IP and INP mice (64). This, however, may not translate to immune responses that can clear parasites from the placenta. Ultimately, it will be necessary to investigate in detail the immunological events occurring in the placental environment and in the spleen of P. chabaudi AS-infected pregnant mice to fully elucidate the protective and pathogenic immune mechanisms at play in pregnant mice. Furthermore, development of an experimental system that allows IP mice to progress to term pregnancy will be necessary for investigation of pregnancy-associated alterations in disease course and immune patterns throughout gestation. In this context, it will also be of value to assess the course of infection and outcome of pregnancy in previously malaria-exposed mice; all of these issues are currently being addressed in our laboratory.
Although the peak peripheral parasitemia was not significantly different between IP and INP mice, placental parasitemia was >40% higher than peripheral parasitemia in IP mice at the time of abortion, which effectively translates to a higher total parasite load in these animals. Sequestration of infected erythrocytes in several organs has been reported in P. chabaudi AS-infected nonpregnant mice (13, 23). Furthermore, the in vitro binding of the P. chabaudi AS-infected RBCs to endothelial cells has been shown to be mediated through CD36 (a well-characterized receptor utilized by P. falciparum-infected erythrocytes to bind to endothelial cells) (32), but other receptors are likely to be involved as well (46). Sequestration of P. falciparum-infected erythrocytes in the placental intervillous blood space is a key feature of malarial pathogenesis in pregnant women (49, 74) and is thought to be mediated largely through interaction with chondroitin sulfate A (21). The accumulation of P. chabaudi AS-infected erythrocytes in the placentae of infected mice, which has not been described before, may be a manifestation of specific placental sequestration. It is noteworthy that, like human trophoblasts, murine trophoblasts express a low-sulfated chondroitin sulfate (C. Gowda, personal communication). Clearly, further detailed studies of the interactions between P. chabaudi AS iRBCs and fetal trophoblast cells are required to define the biological mechanisms of placental parasite accumulation in mice and the pathogenic implications thereof. Our laboratory is currently investigating the impact of iRBC binding to trophoblasts on immunopathological events at the human (39) and murine maternofetal interfaces.
As reported in the case of P. chabaudi AS-infected nonpregnant B6 mice, both IP and INP mice experienced profound anemia (14). Although the lowest hematocrit levels and the day on which these levels were reached were not different between IP and INP mice, the development of anemia was faster in IP mice. Because UP mice also developed some anemia, it is likely that the faster rate in IP mice was not entirely malaria specific. Pregnancy-associated hemodilution has been reported in rats (18, 37) and pregnant women (7). In general, several factors may contribute to the complex process of anemia during malarial infection. Sequestration of infected RBCs (2), rupture of iRBCs during schizogony, development of autoantibodies (1, 25, 43), and ineffective erythropoiesis (12, 51, 83) may all contribute. Furthermore, it was recently suggested that the proinflammatory cytokine macrophage migration inhibitory factor (MIF) may play an important role in malarial anemia (40). It is of interest that MIF expression is massively upregulated in the placentae of malaria-infected pregnant women (9) and is specifically secreted by trophoblasts bound by P. falciparum-infected RBCs (8). Further study will be required to determine the relative roles of all of these factors, particularly that of MIF, in anemia and other protective and pathogenic immune mechanisms during pregnancy in B6 mice.
In addition to immune responses and placental accumulation of parasites, anemia may play a role in the observed pregnancy loss in this model. However, most of the abortions occurred between days 10 and 11, which is 1 to 2 days before peak anemia. Additionally, while anemia has been shown to be associated with low birth weight (6) and preterm labor (65), it is not associated with fetal loss in rodents or in humans. Furthermore, for cases of low birth weight among women with severe malarial anemia (<7 g/dl; 36% below normal level), Brabin and Piper (6) calculated that anemia alone can account for only about 10% of infant low birth weight cases, whereas malaria (with all associated immunopathogenic effects) can account for 40%. Thus, factors other than anemia likely play dominant roles in inducing pregnancy loss in P. chabaudi AS-infected B6 mice. We are actively pursuing this line of investigation.
In conclusion, this study shows that P. chabaudi AS infection leads to poor pregnancy outcomes in B6 mice. Although the splenocytes from both IP and INP mice exhibited reduced proliferation in response to mitogens compared to UNP mice, both IP and INP mice exhibited a comparable increase in spleen size and cell number during the course of infection and all survived the infection, albeit in the absence of viable postmidgestational pregnancy for IP. This suggests that IP mice develop peripheral antimalarial immune responses that are sufficient to control parasitemia during primary infection. Despite effective control of peak parasitemia, IP mice experienced massive accumulation of iRBCs in their placentae and failed to maintain their pregnancies beyond GD 12. This suggests that while peripheral parasitemia is controlled immunologically, this response is not sufficient to control localized placental parasitemia but may, paradoxically, contribute to fetal loss. Continued characterization of this model will contribute significantly to our understanding of the molecular and cellular immunological mechanisms involved in fetal loss during malarial infection.
ACKNOWLEDGMENTS
This work was supported by a Faculty Grant from the University of Georgia Research Foundation and State of Georgia funds (Veterinary Medical Experiment Station) as well as National Institutes of Health grant no. AI50240 to J.M.M. Jayakumar Poovassery is a recipient of a University of Georgia Department of Infectious Diseases Doctoral Assistantship.
We thank Liliana Jaso-Friedmann and Daniel G. Colley for critical review of the manuscript.
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