CCR5 Is Involved in Interruption of Pregnancy in Mice Infected with Toxoplasma gondii during Early Pregnancy

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ABSTRACT

 

INTRODUCTION

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii. T. gondii can infect humans and other warm-blooded animals and can cause embryonic death and resorption, fetal death, abortion, and stillbirth during pregnancy (1,–3). The outcome of T. gondii infection during pregnancy is thought to depend on the stage of pregnancy when the infection is contracted; however, the mechanism of abortion and fetal death remains unknown.

Changes in hormones and immune dynamics are closely associated with the maintenance of pregnancy, and hormones such as progesterone and estrogen, which are necessary during pregnancy, can modulate immune cell functions (4, 5). During T. gondii infection, the T helper 1 (Th1) immune response that results in the production of interferon gamma (IFN-γ) or interleukin-12 (IL-12) plays an important role in host defenses, but the level of production of IFN-γ in pregnant mice has been reported to be lower than that in nonpregnant mice during infection with T. gondii (6). IL-12 production is reduced by a high progesterone concentration, indicating that the downregulation of IL-12 and the Th1 immune response seems to be related to T. gondii susceptibility during pregnancy (7). Additionally, some studies suggested that T. gondii infection, particularly with type II strains that show moderate virulence in a murine host, induced apoptosis of human trophoblasts, which was associated with an increase in IFN-γ levels (8, 9). T. gondii infection during early pregnancy induced IFN-γ production or inflammation, and this was associated with apoptosis in mouse decidual cells, thereby resulting in fetal resorption (10, 11).

C-C chemokine receptor type 5 (CCR5) is a Th1-associated chemokine receptor, and the main CCR5 ligands are chemokine (C-C motif) ligand 3 (CCL3), CCL4, and CCL5 (also call RANTES). In addition to host ligands, secreted T. gondii cyclophilin 18 (TgCyp18) can bind to CCR5 (12, 13). Recombinant TgCyp18 has been reported to enhance RANTES expression in macrophages and to control their migration (14, 15). CCR5 is essential for controlling infection by T. gondii; CCR5-deficient (CCR5^−/−) mice showed high susceptibility to infection, severe tissue damage, low expression levels of IFN-γ and IL-12, and high parasite loads compared with wild-type mice (16).

Additionally, RANTES has been implicated as a physiological tolerogenic factor for successful implantation (17). RANTES production is induced by progesterone and can induce the apoptosis of maternal CD3⁺ lymphocytes (17, 18). The frequency of T cell apoptosis was significantly lower in recurrent spontaneous abortion patients than in fertile women (17). Furthermore, the epigenetically inadequate expression of chemokine genes, including the gene encoding RANTES, limits T cell access to the decidua (19).

1. gondiiinfection in pregnant animals can cause embryonic death, resorption, fetal death, abortion, and congenital transmission. Infection with T. gondiiduring early pregnancy has more severe consequences (e.g., decreased offspring survival rates and increased parasite transmission rates) than when it is contracted later in pregnancy (3, 20). Although the mechanism of abortion caused by T. gondii infection is poorly understood, the disturbance of many different factors, such as alterations in the immune response and hormone balance, may be associated with embryonic resorption and abortion. CCR5 and RANTES have been reported to be associated with the immune responses required for host defense against T. gondii and successful pregnancy. In this study, we investigated the role played by CCR5 in abortion caused by T. gondii infection using CCR5^−/− mice.

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RESULTS

Pregnancy rates decrease over time in wild-type mice, but not in CCR5^−/− mice, following infection with T. gondii.

In our preliminary experiments, inoculation of T. gondii PLK tachyzoites (5 × 10³) induced clinical signs, including weight decrease and rough coat, in nonpregnant wild-type mice and CCR5^−/− mice and induced the loss of embryos in wild-type mice inoculated at embryonic day 3 (E3) but not in CCR5^−/− mice inoculated at E3. Moreover, inoculation of both groups of mice with T. gondii tachyzoites (5 × 10³) at E7 did not induce a loss of embryos, when mice were euthanized at E18. Thus, to reveal when embryo loss occurred in wild-type mice, both groups of mice inoculated at E3 were euthanized at E6, E8, and E10 (Fig. 1). The pregnancy rate was 100% in both the wild-type (6/6 mice) and CCR5^−/− (6/6) groups at E6 (Fig. 1B). However, the rate decreased as pregnancy progressed in wild-type mice infected with T. gondii and decreased significantly at E10 (1/5 mice; 25%) compared with that at E6 (6/6; 100%) (Fig. 1B). In contrast, the pregnancy rate did not decrease at E8 (5/6 mice; 83.3%) and E10 (6/7; 85.7%) in CCR5^−/− mice (Fig. 1B). However, the average numbers of implantation sites were 8.7 ± 1.0 and 7.5 ± 1.3 at E6 and E8, respectively, in wild-type mice, and no normal implantation sites were observed at E10 (Fig. 1A and ​andC).C). Only one of five wild-type mice exhibited four implantation sites, and they were all degenerated. In contrast, there were no significant differences in the numbers of implantation sites in CCR5^−/− mice at each gestational stage (the average numbers of implantation sites were 8.8 ± 1.0, 9.4 ± 1.7, and 7.8 ± 1.3 at E6, E8, and E10, respectively) (Fig. 1C). These results showed that the loss of an embryo was not due to an inhibition of implantation in wild-type mice infected with T. gondii at E3. Furthermore, it was suggested that infection by T. gondii at E3 could affect embryos in wild-type mice after E8, and almost all embryos were lost at E10, whereas pregnancy was largely maintained in infected CCR5^−/− mice.

 

 

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FIG 1

Uteri (A), pregnancy rates (B), and numbers of implantation sites per litter (C) in T. gondii-infected mice. (A) Uteri of mice infected with T. gondii (5 × 10³ tachyzoites) at E3 and euthanized at E10. No normal implantation sites were observed in wild-type (WT) mice compared with CCR5^−/− mice. (B) Pregnancy rates measured for 6, 6, and 5 wild-type pregnant mice and 6, 6, and 7 CCR5^−/− pregnant animals at E6, E8, and E10, respectively. (C) Numbers of implantation sites per litter measured for 6, 4, and 1 wild-type dams and 6, 5, and 6 CCR5^−/− dams at E6, E8, and E10, respectively. Statistical comparisons were performed among different stages of pregnancy within each group of mice. * indicates significant differences as determined by the χ² test (P < 0.05).

Wild-type mice have a lower percentage of developed embryos and a higher percentage of embryo loss than do CCR5^−/− mice.

To evaluate the morphological changes and distribution of parasites, implantation sites were analyzed histopathologically and immunohistochemically (Fig. 2). Histopathological changes were most prominent in T. gondii-infected mice at E10. Although only one wild-type mouse had an implantation site at E10 (Fig. 1C), all of the fetoplacental tissues showed necrosis (Fig. 2A). In contrast, many normal-appearance embryos similar to those of the uninfected mice were observed in CCR5^−/− mice (Fig. 2A). In the uterine wall and adipose tissue attached to the uterus, there was mild to moderate inflammatory cell infiltration from mononuclear inflammatory cells and neutrophils, and there was evidence of parasitic infection in both groups of mice by immunohistochemistry (Fig. 2B). However, no inflammatory changes and no parasite antigens were detected in the fetoplacental tissues from either group (Fig. 2B). These results suggested that the loss of embryos was not induced directly by parasite proliferation or inflammation.

 

 

FIG 2

Histopathology (A) and immunohistochemistry for T. gondii (B) in fetoplacental tissues, including uteri, at E10. (A) Fetoplacental tissues consisted of embryos with a cavity structure and decidua in uninfected wild-type mice. Fetoplacental tissues of uninfected and infected CCR5^−/− mice were similar to those of uninfected wild-type mice. All embryos of wild-type mice infected with T. gondii showed necrosis (arrowheads). Inflammatory cell infiltration, including mononuclear inflammatory cells (open arrowheads) and neutrophils (arrow), was observed in the uterus and adipose tissue attached to the uterus. (B) T. gondii antigen was detected in the uterus (2) but not in the embryo or decidual tissues from wild-type and CCR5^−/− mice (1). The numbers of evaluated dams (numbers of embryos) were 3 (24) and 1 (4) for uninfected and infected WT mice and 2 (14) and 6 (47) for uninfected and infected CCR5^−/− mice at E10, respectively.

Although inflammatory changes in the uterine wall and adipose tissue were slight to mild in both groups at E6 and E8, there were variations in the growth stages and sizes of embryonic tissues in T. gondii-infected mice from both groups at E8 (Fig. 3). The embryonic tissues were classified into the three following categories: embryos with some structures, such as a head fold and amnion, as was observed for many of the embryos from the uninfected control mice at E8 (group A); small embryos with an irregularly folded appearance (group B); and no embryonic tissue in the decidual tissue (group C). Both groups of control mice showed a relatively high percentage of developed embryos (groups A and B), and there was no significant difference in embryo development between wild-type and CCR5^−/− mice. The infected wild-type mice had a lower percentage of developed embryos (group A) and a statistically significantly higher percentage of embryo loss (group C) than did the infected CCR5^−/− mice. These results suggested that infected wild-type mice had poorly developed embryos compared with those of infected CCR5^−/− mice.

 

 

FIG 3

Embryos of T. gondii-infected mice at E8. Some structures, including the head fold (arrow), amnion, or round cavity, which seemed to be the amniotic cavity, were observed in well-developed embryos of the uninfected control mice at E8. (A) Embryo that developed similarly to the well-developed embryos of the uninfected control mice. Embryos with some structures, such as the head fold or amnion, were considered group A. (B) Embryos were small and folded irregularly (arrowheads). (C) No embryo was observed in the decidual tissue. The table shows the numbers and percentages of embryos classified into each category. A signal in the TUNEL assay was observed around the embryos of both types of mice with or without inoculation. The numbers of embryos with predominant signals compared with those of the noninfected control were counted. * indicates significant differences between infected wild-type and CCR5^−/− groups as determined by the χ² test (P < 0.05). All images are of wild-type mice. The numbers of dams (numbers of embryos) were 2 (8) and 4 (15) for uninfected and infected wild-type mice and 3 (12) and 5 (26) for uninfected and infected CCR5^−/− mice at E8, respectively. HE, hematoxylin and eosin.

To evaluate whether apoptosis at the implantation site could be associated with the poor development of embryos, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed for the implantation sites at E8. In the TUNEL assay, a positive signal was observed around the embryos in infected and uninfected mice from both groups. Compared with control mice, the prominent signal was observed mainly in small embryos (group B) of infected mice. These results suggested the possibility that apoptosis of fetoplacental tissues may be associated with the poor development or loss of embryos in mice infected with T. gondii.

Wild-type mice show higher parasite burdens at implantation sites than do CCR5^−/− mice.

To elucidate whether the number of parasites was associated with the poor development or loss of embryos, the parasite loads at implantation sites were determined by quantitative PCR (Fig. 4). The number of parasites found in the implantation sites increased as pregnancy progressed in both wild-type and CCR5^−/− mice. Moreover, the parasite load in wild-type mice was significantly higher than that in CCR5^−/− mice at E6 (3 days postinfection [dpi]). Although a higher parasite load was detected in wild-type mice at E10 (7 dpi), this result was obtained from only one pregnant mouse (Table 1). In nonpregnant mice, there were no significant differences in parasite burdens in the uteri between wild-type and CCR5^−/− mice at 3 dpi and 7 dpi (see Fig. S1 in the supplemental material). These results suggested that there are more parasites in the implantation sites of pregnant wild-type mice than in those of CCR5^−/− mice. Although parasite numbers increased at the implantation sites of both types of mice as pregnancy progressed, we were unable to differentiate whether this was due to the migration or proliferation of the parasite. Thus, the number of parasites at implantation sites at E6 (3 dpi) is a key factor for the poor development or loss of embryos.

 

FIG 4

Parasite load at implantation sites of T. gondii-infected mice at E6, E8, and E10. Each point represents data for one mouse, and the bars represent the average values from all data points. The numbers of investigated embryos were 28, 14, and 4 for wild-type (WT) mice and 27, 21, and 47 for CCR5^−/− (CCR5KO) mice, evaluated at E6, E8, and E10, respectively. * indicates significant differences between T. gondii-infected wild-type and CCR5^−/− mice as determined by the t test (P < 0.05).

TABLE 1

Mice and experimental design

Day of pregnancy (dpi)	Mouse type	Infection status	No. of mice	No. of pregnant mice	Pregnancy rate (%)	No. of implantation sites (mouse)a	No. of tissue samples used for histopathology (mouse)a,b	No. of tissue samples used for detection of no. of parasites (mouse)a,c	No. of tissue samples used for real-time RT-PCR analysis of mRNA (mouse)a,d	No. of serum samples used for detection of RANTES and IFN-γe
E6 (3)	Wild type	Uninfected	4	4	100	3 (1), 2 (2), 6 (3), 9 (4)	1 (1), 1 (2), 3 (3), 4 (4)	2 (1), 1 (2), 0 (3), 0 (4)f	2 (1), 1 (2), 0 (3), 0 (4)f	2f
		Infected	6	6	100	9 (1), 9 (2), 7 (3), 10 (4), 9 (5), 8 (6)	4 (1), 4 (2), 3 (3), 5 (4), 4 (5), 4 (6)	5 (1), 5 (2), 4 (3), 5 (4), 5 (5), 4 (6)	5 (1), 5 (2), 4 (3), 5 (4), 5 (5), 4 (6)	6
	CCR5−/−	Uninfected	4	4	100	10 (1), 6 (2), 9 (3), 10 (4)	5 (1), 3 (2), 4 (3), 5 (4)	5 (1), 3 (2), 0 (3), 0 (4)f	5 (1), 3 (2), 0 (3), 0 (4)f	2f
		Infected	6	6	100	8 (1), 10 (2), 10 (3), 8 (4), 9 (5), 8 (6)	4 (1), 5 (2), 5 (3), 4 (4), 4 (5), 4 (6)	4 (1), 5 (2), 5 (3), 4 (4), 5 (5), 4 (6)	4 (1), 5 (2), 5 (3), 4 (4), 5 (5), 4 (6)	6
E8 (5)	Wild type	Uninfected	2	2	100	8 (1), 8 (2)	4 (1), 4 (2)	4 (1), 4 (2)	4 (1), 4 (2)	2
		Infected	6	4	66.7	0 (1), 9 (2), 8 (3), 0 (4), 7 (5), 6 (6)	0 (1), 4 (2), 4 (3), 0 (4), 4 (5), 3 (6)	0 (1), 4 (2), 4 (3), 0 (4), 3 (5), 3 (6)	0 (1), 4 (2), 4 (3), 0 (4), 3 (5), 3 (6)	6
	CCR5−/−	Uninfected	3	3	100	8 (1), 9 (2), 8 (3)	4 (1), 4 (2), 4 (3)	4 (1), 5 (2), 4 (3)	4 (1), 5 (2), 4 (3)	3
		Infected	6	5	83.3	11 (1), 0 (2), 9 (3), 11 (4), 9 (5), 7 (6)	6 (1), 0 (2), 5 (3), 6 (4), 5 (5), 4 (6)	5 (1), 0 (2), 4 (3), 5 (4), 4 (5), 3 (6)	5 (1), 0 (2), 4 (3), 5 (4), 4 (5), 3 (6)	6
E10 (7)	Wild type	Uninfected	3	3	100	9 (1), 6 (2), 9 (3)	9 (1), 6 (2), 9 (3)	9 (1), 6 (2), 9 (3)	ND	3
		Infected	5	1	20	4 (1), 0 (2), 0 (3), 0 (4), 0 (5)	4 (1), 0 (2), 0 (3), 0 (4), 0 (5)	4 (1), 0 (2), 0 (3), 0 (4), 0 (5)	ND	5
	CCR5−/−	Uninfected	3	2	66.7	7 (1), 7 (2), 0 (3)	7 (1), 7 (2), 0 (3)	7 (1), 7 (2), 0 (3)	ND	2g
		Infected	7	6	85.7	10 (1), 8 (2), 7 (3), 0 (4), 6 (5), 8 (6), 8 (7)	10 (1), 8 (2), 7 (3), 0 (4), 6 (5), 8 (6), 8 (7)	10 (1), 8 (2), 7 (3), 0 (4), 6 (5), 8 (6), 8 (7)	ND	7

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^aNumbers in parentheses denote the mouse identification number per experimental group. ND, no data.

^bSee Fig. 3.

^cSee Fig. 4.

^dSee Fig. 5.

^eSee Fig. 6.

^fBecause two of four uninfected mice in each group were added after experimental infection, only samples collected in the first experiment were analyzed.

^gBecause no implantation sites were detected in one of the three uninfected mice, this animal was removed from consideration.

CCR5 expression levels at implantation sites are increased significantly in wild-type mice infected with T. gondii.

To evaluate the possible involvement of CCR5 and its ligands in embryo loss due to T. gondii infection, the mRNA expression levels of CCR5, CCL3, CCL4, and RANTES (CCL5) in the implantation sites were quantitated by real-time reverse transcriptase PCR (RT-PCR) (Fig. 5). CCR5 expression levels in the implantation sites increased significantly in wild-type mice infected with T. gondii at E6 (3 dpi) and E8 (5 dpi) compared with those in uninfected wild-type mice. In contrast, compared with uninfected wild-type mice, the expression level of CCL3 decreased in T. gondii-infected CCR5^−/− mice at E6, whereas CCL4 expression levels decreased in both types of infected animals. Moreover, the expression levels of IFN-γ and tumor necrosis factor alpha (TNF-α), which are cytokines associated with apoptosis, were evaluated. Although the IFN-γ expression level increased in both groups of mice infected with T. gondii at E8, there were no significant differences among the experimental groups. Compared with uninfected wild-type mice, the expression of TNF-α was downregulated in T. gondii-infected CCR5^−/− mice at E6, whereas decreased expression levels of TNF-α were seen in both types of infected animals at E8. Additionally, we evaluated the mRNA expression levels of IFN-γ, TNF-α, CCL3, CCL4, RANTES (CCL5), and CCR5 in the uteri of nonpregnant mice at 3 dpi (see Fig. S2 in the supplemental material). However, there was no significant difference in the mRNA expression levels of these target genes in the uteri of nonpregnant mice at 3 dpi. Since we observed increased expression levels of CCR5 in infected wild-type mice, decreased expression levels of TNF-α and CCL3 in infected CCR5^−/− mice, and decreased expression levels of CCL4 in both types of infected mice at E6, compared with uninfected pregnant wild-type mice, we suggest that these reactions may be specific for infected pregnant mice. Together, these results suggest that the lack of the CCR5 gene and the downregulation of TNF-α and CCL3 expression at E6 (3 dpi) are important factors for the maintenance of pregnancy following T. gondii infection.

 

 

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FIG 5

Relative mRNA expression levels of IFN-γ, TNF-α, CCL3, CCL4, RANTES (CCL5), and CCR5 at implantation sites of T. gondii-infected wild-type (WT) and CCR5^−/− (KO) mice at E6 and E8. Data are mean values ± SD. * and # indicate significant differences as determined by one-way ANOVA plus Tukey-Kramer post hoc analysis for four experimental groups and by the t test for two experimental groups (P < 0.05). * and # indicate comparisons between animals of different and the same lineages, respectively. The numbers of investigated implantation sites were 3, 8, 28, and 27 at E6 and 8, 13, 14, and 21 at E8 for wild-type controls, CCR5^−/− controls, wild-type T. gondii-infected mice, and CCR5^−/− T. gondii-infected mice, respectively. cont, control (uninfected); Tg, T. gondii infected.

CCR5^−/− mice have higher RANTES levels than do wild-type mice.

To evaluate the serum component that could affect pregnancy, the serum levels of RANTES and IFN-γ were quantified (Fig. 6). Serum RANTES levels increased in T. gondii-infected CCR5^−/− mice at E6 (3 dpi), E8 (5 dpi), and E10 (7 dpi) (Fig. 6A). Particularly at E8 and E10, the levels were significantly higher than those of the wild-type group (Fig. 6A). High levels of serum RANTES were observed in nonpregnant CCR5^−/− mice infected with T. gondii as well as in infected pregnant CCR5^−/− mice (see Fig. S3 in the supplemental material). Furthermore, the serum IFN-γ level rose in both groups of infected mice to similar levels and then increased significantly at E10 compared with the levels in uninfected pregnant mice (Fig. 6B). Similar patterns of IFN-γ levels were also observed for nonpregnant mice at 3 and 7 dpi (Fig. S3). In the case of progesterone and estradiol, there was no difference between wild-type and CCR5^−/− mice or between infected and uninfected mice of the same strain (data not shown). These results suggested that IFN-γ and hormones involved in the maintenance of pregnancy were not associated with embryo loss in wild-type mice inoculated with T. gondii at E3.

 

FIG 6

Serum levels of RANTES (A) and IFN-γ (B) at E6, E8, and E10. Data are mean values ± SD. * and # indicate significant differences as determined by one-way ANOVA plus Tukey-Kramer post hoc analysis (P < 0.05). * and # indicate comparisons between animals of different and the same lineages, respectively. The numbers of dams used were 2, 2, 6, and 6 at E6; 2, 3, 6, and 6 at E8; and 3, 2, 5, and 7 at E10 in wild-type uninfected mice, uninfected CCR5^−/− mice, wild-type T. gondii-infected mice, and CCR5^−/− T. gondii-infected mice, respectively. WT, wild type; CCR5KO, CCR5^−/−; No inf., uninfected; Tg, T. gondii infected.

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DISCUSSION

Infection with T. gondii during early pregnancy can cause fetal resorption in mice (10, 11). In this study, implantation sites were observed in all wild-type and CCR5^−/− mice at E6; however, the number of mice without embryos increased as pregnancy progressed, and no normal embryos were observed at E10 in wild-type mice (Fig. 1). Bonfá et al. showed previously that CCR5^−/− mice were susceptible to oral T. gondii infection (ME-49 strain) compared with wild-type C57BL/6 mice (16), but Khan et al. then reported that CCR5^−/− mice in the C57BL/6 background survived longer than did wild-type mice during T. gondii (76K strain) infection (21). In the present study, pregnant mice were used, and the infection route and strain of parasite were different from those of the previous reports (oral infection of cysts): it is not easy to compare our data with those results. However, the present results suggest that CCR5 is associated with the loss of embryos following T. gondii infection during early pregnancy. Additionally, RANTES was induced, and the levels correlated with IFN-γ levels in pregnant mice infected with the Gram-negative bacterium Brucella abortus; neutralization of RANTES decreased the number of aborted fetuses in B. abortus-infected mice (22). Therefore, while CCR5 is essential for the control of T. gondii infection, it is possible that interactions between CCR5 and RANTES during infection are associated with negative outcomes during pregnancy.

Some studies on nonpregnant mice have shown that CCR5^−/− mice had high T. gondii loads in various tissues, including the liver and spleen (16, 21), and that they experienced severe tissue damage compared with wild-type mice (16). In contrast to those findings, the parasite loads in the implantation sites were significantly higher in wild-type mice than in CCR5^−/− mice in our study of pregnant mice (Fig. 4). Additionally, there were no significant differences in parasite loads in nonpregnant uteri between wild-type and CCR5^−/− mice (see Fig. S1 in the supplemental material). As a potential cause of the high parasite load, an increase in parasite proliferation at the implantation site or the migration of infected cells should be considered. If enhanced parasite proliferation occurs locally, prominent tissue damage is likely to be observed in wild-type mice. However, no histopathological differences were found between wild-type and CCR5^−/− mice in the present study (Fig. 2). CCR5 expression levels in the implantation sites of the wild-type mice increased at E6 and E8 (Fig. 5). Although it is not known whether the high parasite loads in the wild-type mice were uterus specific, CCR5 may be associated with the migration of T. gondii-infected cells to the uterus in pregnant mice. Previous studies have shown that the regulation of dendritic cells and macrophage migration depends on CCR5 and that inoculation of dendritic cells or peripheral leukocytes infected with T. gondii enabled such cells to disseminate more rapidly than what occurred following inoculation with free parasites (14, 23, 24). However, we did not observe a prominent infiltration of phagocytes into the uterus. In the presence of some pathogen-associated molecular pattern (PAMP) stimuli, such as lipopolysaccharide and peptidoglycan, trophoblast cells restrained early monocyte migration and reduced CCR5 and RANTES expression levels in monocytes (25). Although these PAMPs were derived from bacteria and viruses, PAMPs from parasites may also be associated with the restrained migration of monocytes in trophoblasts. Thus, factors other than parasite proliferation and migration of infected cells may be involved in embryo loss during early pregnancy following infection with T. gondii.

Our data showed that there were no significant differences in the numbers of implantation sites at E6 (Fig. 1C), but the number of parasites in wild-type mice was higher than that in CCR5^−/− animals at E6 (Fig. 4). The number of implantation sites in wild-type mice is likely to be lower than that in CCR5^−/− animals if parasite infection affected embryo survival. Therefore, these findings suggest that embryo loss is related to functional changes in the uterus or fetoplacental tissues, such as disorder of decidual cells and trophoblasts via the disturbance of immune cells (e.g., natural killer cells, regulatory T cells [Treg cells], and macrophages) that are possibly involved in the immune tolerance of embryos. Furthermore, it is worth considering the role of T. gondii secretory molecules other than those that cause tissue damage related to parasite proliferation in fetoplacental tissues. Senegas et al. detected T. gondii cysts in decidual tissue at 10 days postcoitum in Swiss Webster mice inoculated orally during early pregnancy; however, no necrosis in the implantation sites was observed (10). Swiss Webster mice are relatively resistant to T. gondii infection and show a lower Th1 response, including IFN-γ production, and less tissue damage than do C57BL/6 mice during T. gondii infection (26, 27). Additionally, embryonic resorption caused by the inoculation of T. gondii antigen during early pregnancy suggests that embryonic resorption could be related to a mechanism other than the direct proliferation of T. gondii in the uterus (28). Treg cells involved in maternal-fetal tolerance, and CCR5 expression in Treg cells, play an important role in the accumulation of Treg cells in the pregnant uterus (29). Furthermore, mRNA levels of CCL4, but not RANTES, were elevated in pregnant uteri, and CCL4 attracted CCR5⁺ Treg cells in vitro. In mice inoculated with T. gondii or T. gondii-excreted-secreted antigens, Treg cell apoptosis was induced at the fetal-maternal interface or the spleen, and fetal loss could be prevented partly by the adoptive transfer of Treg cells from normal pregnant mice (28, 30, 31). These findings suggest that a decrease in Treg cell function could be involved in fetal loss following T. gondii infection. CCR5 may be associated with both proinflammatory and anti-inflammatory reactions, and a decrease in the frequency of CCR5⁺ Treg cells with a suppressive function may obstruct maternal-fetal tolerance or enhance proinflammatory reactions in T. gondii-infected mice. However, in the present study, no significant difference was observed in mRNA expression levels of CCL4 in fetoplacental tissues between wild-type and CCR5^−/− mice infected with T. gondii (Fig. 5). Additionally, no significantly difference was observed in mRNA expression levels of Foxp3, a marker of Treg cells, at implantation sites between wild-type and CCR5^−/− mice at E6 (data not shown). Thus, the role of Treg cells in embryo loss remains unclear.

Although there were no differences in parasite infection and inflammatory changes in the fetoplacental tissues between wild-type and CCR5^−/− mice, wild-type animals displayed poorly developed fetoplacental tissues compared with those of CCR5^−/− animals at E8 (Fig. 3). In the TUNEL assay, the positive signal detected around the embryos probably included trophoblasts, and this signal was observed predominantly in small embryos, indicating the involvement of apoptosis in embryo loss (Fig. 3). Apoptosis at the maternal-fetal interface occurs during normal implantation and gestation and is associated with appropriate tissue remodeling of the decidua and invasion of the developing embryo. Effective clearance of apoptotic cells and cellular debris by macrophages is thought to be important for preventing the release of intracellular contents, which may cause tissue damage and inflammatory reactions and promote the production of proliferative factors for trophoblasts (32, 33). Therefore, the imbalance between apoptosis and the clearance of apoptotic cells by macrophages may affect the establishment and maintenance of pregnancy. As described previously, TgCyp18 enhances RANTES expression in macrophages and controls macrophage migration (14, 15). Dense granule protein 15 (GRA15), a secretary protein of T. gondii, from type II parasites can activate the NF-κB pathway, and this pathway is involved in inflammation, immune responses, and antiapoptosis in various cell types, including macrophages and embryonic fibroblasts in vitro (34). Therefore, T. gondii infection can affect macrophage function via secretary proteins and may induce functional aberrations in various types of cells at the implantation site. Furthermore, human extravillous trophoblasts displayed high apoptosis indexes after treatment with the supernatant from macrophages with or without T. gondii infection (35). However, an increase in IL-6 production in monocytes treated with the supernatant from trophoblast cells suppressed T. gondii proliferation in monocytes in vitro (36). Further investigations are required to elucidate the mechanism of embryo loss following parasite infection, including the role of macrophages and T. gondii-derived molecules.

Senegas et al. reported that IFN-γ-dependent apoptosis of placental cells was involved in embryo resorption in T. gondii-infected mice during early pregnancy and that serum and uterine IFN-γ levels were increased compared with those in uninfected controls (10). In the present study, the serum IFN-γ level was increased in both wild-type and CCR5^−/− mice; however, there was no significant difference between these groups as the pregnancies progressed (Fig. 6). Additionally, no significant differences were found in serum progesterone and estradiol levels between wild-type and CCR5^−/− mice (data not shown). These results suggest that IFN-γ and pregnancy-associated hormones have little association with apoptosis or embryo resorption associated with CCR5.

The expression levels of TNF-α and CCL3 at implantation sites were significantly lower in infected and uninfected CCR5^−/− mice than in uninfected wild-type mice at E6, and both groups of mice showed a significant downregulation of TNF-α at E8 in the present study. Coutinho et al. showed increased serum TNF-α levels and necrotic implantation sites in C56BL/6 mice infected with the T. gondii ME-49 strain; these results suggested that an impaired outcome of pregnancy due to T. gondii infection may be associated with high TNF-α levels (11). In contrast, Bonfá et al. reported lower tissue expression levels of Th1 cytokines, including TNF-α, and extensive tissue damage in livers of CCR5^−/− mice infected with the T. gondii ME-49 strain compared with wild-type mice (16); tissue expression of TNF-α does not correlate well with tissue damage in T. gondii infection. Although the serum TNF-α level was not evaluated, there was no prominent difference in inflammatory changes in uteri between wild-type and CCR5^−/− mice in the present study. The uptake of apoptotic cells suppresses the secretion of proinflammatory cytokines, including TNF-α, from macrophages and promotes the release of anti-inflammatory cytokines during normal pregnancy (32). TNF-α can inhibit interactions between trophoblast-like cells and maternal endothelial cellular networks in vitro (37). Although it is unclear whether CCL3 has adverse effects except for an inflammatory response at the implantation site, the downregulation of TNF-α and CCL3 at implantation sites may have or reflect a positive effect on the maintenance of pregnancy in CCR5^−/− mice infected with T. gondii.

Interestingly, CCR5^−/− mice showed higher levels of serum RANTES than did wild-type mice (Fig. 6). Although the mRNA expression level of RANTES in the implantation sites increased in both infected wild-type and infected CCR5^−/− mice at E8, no significant difference was found between these mouse groups (Fig. 5). However, the CCR5 expression levels in the implantation sites were significantly higher in infected wild-type mice than in uninfected controls (Fig. 5). Although not in serum, CCR5 expression in apoptotic neutrophils and T cells was reported to clear CCL3 and RANTES in peritoneal exudates (38). In a mouse model of proteoglycan-induced arthritis, the serum RANTES level increased significantly in CCR5^−/− mice compared with that in wild-type mice (39). This indicates that CCR5 expression in cells can control the level of circulating RANTES (39). In our study, RANTES may have been consumed by the increased level of CCR5 in wild-type mice infected with T. gondii, because the RANTES expression level was increased in infected wild-type mice, but the serum RANTES level did not increase after infection. Regarding the relationships between CCR5, RANTES, and apoptosis, RANTES has been shown to induce the apoptosis of CCR5-expressing T cell lines (40). In trophoblast cells, anti-RANTES antibodies decreased the apoptosis of a trophoblast cell line (Swain 71 cells, with no expression of CCR5) in coculture with peripheral blood mononuclear cells from recurrent spontaneous abortions (17), suggesting that CCR5-independent RANTES signaling is needed for apoptosis induction. Thus, RANTES may play a role in apoptosis induction in embryos following T. gondii infection. However, the CCR5-RANTES interaction can inhibit virus-induced apoptosis of macrophages (41). Therefore, further investigations will be needed to determine whether these findings are consistent across several cell types.

In conclusion, this study has shown that CCR5 is involved in the loss of embryos in mice infected with T. gondii during early pregnancy. Additionally, apoptosis around an embryo, rather than direct damage to the fetoplacental tissue from infection, can be associated with poor embryo development and embryo loss in the presence of CCR5. Although IFN-γ seemed not to be involved in the embryo loss associated with CCR5, wild-type mice had increased levels of CCR5 and RANTES expression at implantation sites and low serum RANTES levels, indicating the possibility of an interaction between CCR5 and RANTES. In addition, our results suggested that the lack of the CCR5 gene and the downregulation of TNF-α and CCL3 expression at E6 (3 dpi) may be key factors involved in the maintenance of pregnancy following T. gondii infection. However, the existence of a direct relationship between CCR5 and apoptosis requires further investigation. Future studies will address the molecular mechanisms of embryo loss, including the role of CCR5, RANTES, and T. gondii-derived molecules in the apoptosis of fetoplacental tissue.

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MATERIALS AND METHODS

Ethics statement.

This study was performed in strict accordance with the recommendations in the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan (43). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Obihiro University of Agriculture and Veterinary Medicine (permit numbers 28-57, 25-59, 24-15, and 23-61). All surgery for sampling was performed under isoflurane anesthesia, and all efforts were made to minimize animal suffering.

Mice and experimental design.

C57BL/6 mice were obtained from Clea Japan (Tokyo, Japan). CCR5^−/− mice (B6 129P2-Ccr5 ^tmlKuz/J) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained under specific-pathogen-free conditions in the animal facility of the National Research Center for Protozoan Diseases at the Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan. The mice were used according to the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan (43).

Virgin female mice were housed with males at 10 to 11 weeks of age, and when a visible vaginal plug was noted, we designated this day 0 of pregnancy (E0). Female mice of both groups were inoculated intraperitoneally with T. gondii strain PLK tachyzoites (5 × 10³ tachyzoites/mouse) at day E3 and then euthanized at E6 (3 dpi), E8 (5 dpi), and E10 (7 dpi) (Table 1). Blood and uteri were collected from the mice for quantitative analysis of serum IFN-γ and RANTES levels and histopathological analysis. The number of implantation sites was counted. The pregnancy rate was calculated as the ratio of the number of mice with implantation sites to the total number of mice with a vaginal plug in each group. About half (E10) and half the number (E6 and E8) of implantation sites (fetoplacental tissues) were fixed with a 4% paraformaldehyde solution for histopathological analysis, and of the others, half were frozen at −80°C prior to DNA and RNA extraction for quantitation of the parasite burden and RT-PCR analysis, respectively (Table 1).

Preparation of T. gondii tachyzoites.

5. gondii(strain PLK; type II) tachyzoites were propagated in monkey kidney adherent fibroblasts (Vero cells) cultured in Eagle's minimum essential medium (EMEM; Sigma, St. Louis, MO, USA) supplemented with 8% heat-inactivated fetal bovine serum. To purify the tachyzoites, parasites and host cell debris were washed in ice-cold phosphate-buffered saline (PBS), and the final pellet was resuspended in cold PBS and passed through a 27-gauge needle and a 5.0-μm-pore-size filter (Millipore, Bedford, MA, USA).

Histopathological and immunohistochemical analyses.

After fixation with a 4% paraformaldehyde solution, the tissues from the implantation site were routinely embedded in paraffin wax and prepared as 4-μm-thick sections. The sections were stained with hematoxylin and eosin.

Immunohistochemistry of T. gondii in tissues from the implantation site was performed by using an anti-T. gondii polyclonal antibody (catalog number ab15170; Abcam, Cambridge, UK). Briefly, after deparaffinization, the tissues were treated with 0.01 M citrate buffer (pH 6.0) and heated in a microwave (200 W for 5 min, twice) for antigen retrieval. After blocking of endogenous peroxidase with 3% hydrogen peroxide and of nonspecific protein with 10% normal goat serum (Histofine SAB-PO kit; Nichirei Corp., Tokyo, Japan) for 30 min at room temperature, the sections were incubated with the primary antibody (dilution of 1:50) overnight at 4°C. After washing, the sections were incubated with the secondary antibody (EnVision⁺ K4003; Dako, Burlingame, CA, USA) for 40 min at 37°C. The sections were treated with 3,3′-diaminobenzidine (DAB) (Impact DAB; Vector Laboratories Inc., Burlingame, CA, USA), and the chromogenic reaction was stopped with H₂O. The sections were then counterstained with Mayer's hematoxylin.

TUNEL analysis of the implantation sites.

Cell death in the implantation sites was identified by TUNEL using an In Situ Cell Death Detection kit (Roche Diagnostics GmbH, Mannheim, Germany) as recommended by the manufacturer.

DNA isolation and quantitative PCR analysis of parasite numbers.

DNA from the fetoplacental tissues was extracted by using Tri reagent (Sigma). Amplification of parasite DNA was performed by using primers specific for the T. gondii B1 gene (5′-AAC GGG CGA GTA GCA CCT GAG GAG A-3′ and 5′-TGG GTC TAC GTC GAT GGC ATG ACA AC-3′), which is present in all known strains of this parasite species (42). The PCR mixture (25 μl) contained 1× SYBR green PCR buffer, 2 mM MgCl₂, 200 μM each deoxynucleoside triphosphate (dNTP), 400 μM dUTP, 0.625 U of AmpliTaq Gold DNA polymerase, 0.25 U of AmpErase uracil-N-glycosylase (UNG) (AB Applied Biosystems, Carlsbad, CA, USA), 0.5 μM each primer, and 50 ng of genomic DNA. Amplification was performed according to a standard protocol recommended by the manufacturer (2 min at 50°C, 10 min at 95°C, 40 cycles at 95°C for 15 s, and 1 min at 60°C). Amplification, data acquisition, and data analysis were carried out with an ABI 7900HT Prism sequence detector (AB Applied Biosystems), and the cycle threshold (C_T) values were exported to Microsoft Excel for analysis. Parasite loads were estimated by comparison with internal controls, with the level of the internal control calculated per parasite. Briefly, parasite numbers were calculated by interpolation on a standard curve, with the C_T values plotted against a known concentration of parasites. After amplification, the PCR product melting curves were acquired via a stepwise temperature increase from 60°C to 95°C. Data analyses were conducted with Dissociation Curves version 1.0 f (AB Applied Biosystems).

Real-time RT-PCR analysis.

Total RNA was extracted from fetoplacental tissues with Tri reagent (Sigma) according to the manufacturer's instructions. First-strand cDNA synthesis was performed by using an oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). PCR was performed as described above, using the SYBR green PCR system and an ABI 7700 Prism sequence detector instrument. The relative mRNA amounts were calculated by using the ΔΔC_T method according to the manufacturer's instructions (guide to performing relative quantitation of gene expression using real-time quantitative PCR; AB Applied Biosystems) (44). The primer sequences (sense and antisense sequences) designed by using Primer Express software (Applied Biosystems, Foster City, CA, USA) were as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer 5′-TGT GTC CGT CGT GGA TCT GA-3′, GAPDH antisense primer 5′-CCT GCT TCA CCA CCT TGT TGA T-3′, mouse IFN-γ sense primer 5′-GCC ATC AGC AAC AAC ATA AGC GTC-3′, mouse IFN-γ antisense primer 5′-CCA CTC GGA TGA GCT CAT TGA ATG-3′, mouse TNF-α sense primer 5′-GGC AGG TCT ACT TTG GAG TCA TTG C-3′, mouse TNF-α antisense primer 5′-ACA TTC GAG GCT CCA GTG AA-3′, mouse CCL3 sense primer 5′-CCA GCC AGG TGT CAT TTT CCT-3′, mouse CCL3 antisense primer 5′-TCC AAG ACT CTC AGG CAT TCA GT-3′, mouse CCL4 sense primer 5′-CAA CAC CAT GAA GCT CTG CG-3′, mouse CCL4 antisense primer 5′-GCC ACG AGC AAG AGG AGA GA-3′, mouse RANTES (CCL5) sense primer 5′-CCA ATC TTG CAG TCG TGT TTG T-3′, mouse RANTES (CCL5) antisense primer 5′-CAT CTC CAA ATA GTT GAT GTA TTC TTG AAC-3′, mouse CCR5 sense primer 5′-GAC ATC CGT TCC CCC TAC AAG-3′, and mouse CCR5 antisense primer 5′-TCA CGC TCT TCA GCT TTT TGC AG-3′. Gene-specific expression values were normalized against the level of expression of the GAPDH housekeeping gene. The optimal reference gene was selected based on the Cotton EST database (http://150.216.56.64/index.php).

Measurement of serum RANTES and IFN-γ levels.

Serum samples were collected for measurement of RANTES and IFN-γ levels. Serum RANTES levels were measured with a Quantikine mouse RANTES immunoassay kit (R&D Systems, Minneapolis, MN, USA). The serum IFN-γ level was measured with an OptEIA mouse IFN-γ enzyme-linked immunosorbent assay (ELISA) kit (BD Bioscience, San Jose, CA, USA). Each assay was performed according to the manufacturer's instructions.

Statistical analyses.

GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analyses. Data are presented as means ± standard deviations (SD). Statistical analyses were performed by using the Student t test and one-way analysis of variance (ANOVA), followed by the Tukey-Kramer test for group comparisons. The significance of the differences in the pregnancy rates and embryo structures was analyzed by a χ² test. The levels of statistical significance are presented with asterisks and are defined in each figure legend, together with the name of the statistical test that was used. A P value of <0.05 was considered statistically significant.

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SUPPLEMENTARY MATERIAL

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ACKNOWLEDGMENTS

We thank Youko Matsushita, Megumi Noda, and Yoshie Imura for their excellent technical assistance with the experiments. English language editing for the manuscript was provided by Edanz Group, Japan.

This research was supported by the Japan Society for the Promotion of Science through the Funding Program for Next Generation World-Leading Researchers (Next Program), initiated by the Council for Science and Technology Policy (2011/LS003).

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FOOTNOTES

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00257-17.

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