Increased CD11b⁺ Gr‐1⁺ cell population in the placenta after infection with Toxoplasma gondii

ABSTRACT

Toxoplasma gondii is an obligate intracellular protozoan pathogen that can cross the placenta, resulting in congenital toxoplasmosis with severe fetal brain abnormalities. The molecular mechanisms of immune responses against T. gondii infection in the placenta have largely remained unclear. An analytical method for characterizing phenotypes of immune cells in the placenta by flow cytometry was established and it was found that numbers of CD11b⁺ Gr‐1⁺ cells in the placenta increased significantly after T. gondii infection. These results suggest that innate immune responses play an important role in immunity against T. gondii infection via the feto‐maternal interface.

Abbreviations

FSC

forward scatter

PE

phycoerythrin

SSC

side scatter

Toxoplasma gondii is an obligate intracellular protozoan pathogen. This infection can be acquired through oral ingestion of infected undercooked meat or contaminated food or water. Following oral infection, the parasite crosses the intestinal epithelium and disseminates widely. IFN‐γ derived from Th1 and NK cells plays an important role in immune responses against T. gondii 1. In addition, Gr‐1⁺ inflammatory monocytes, but not neutrophils, are also required for mucosal resistance to T. gondii 2, 3. It has been reported that neutrophils also play an important role in host protection against T. gondii by acting as a source of IFN‐γ 4. Moreover, neutrophils also release neutrophil extracellular traps that limit T. gondii infection 5. However, there is evidence that suggests that neutrophils are invaded by T. gondii and then play a role as their motile reservoirs, leading to parasitic spread in the body 6.

T. gondii can cross the placenta, resulting in congenital toxoplasmosis with fetal vision and hearing loss, mental and psychomotor retardation, seizures and hematological abnormalities 7-10. Despite the fact that T. gondii continues to be a severe threat to the fetus during pregnancy, immunological control against T. gondii infection and molecular mechanisms of immune responses in the placenta have largely remained unclear.

To examine the immune responses against T. gondii infection at the feto‐maternal interface, we used a pregnant murine model with T. gondii infection to establish a method for investigating immune cells in the placenta. Mice were infected or not with 30 cysts of T. gondii (PLK strain; avirulent cyst‐forming [type 2] strain) by oral inoculation on the seventh day of gestation (G7). Previous studies have shown that abortion occurs around G16 when mice are inoculated with cysts on G7 11, suggesting that T. gondii infection affects homeostasis at the feto‐maternal interface until G16. We therefore obtained placental tissue from which the embryos had been totally removed on G16 to investigate the effects of T. gondii infection on immune cell populations in the placenta. The placental tissue was minced into ∼1 mm³ fragments and then digested for 45 min at 37 °C under agitation in PBS with 1 mg/mL collagenase type D (Roche Applied Science, Mannheim, Germany) (Fig. 1a). The resultant cell suspensions were filtered through sterile stainless‐steel 100 nylon mesh and resuspended in PBS with 2% FBS. Placental cells were then simultaneously stained with PE‐conjugated anti‐CD45.2 mAb (BD Biosciences, San Jose, CA, USA), a hematopoietic marker, and FITC or allophycocyanin‐conjugated mAbs against lineage markers, including CD3, CD4, CD8, B220, NK1.1, Gr‐1 and/or CD11b (BD Biosciences).

Figure 1

Flow cytometry analysis of immune cells in placentas from infected and non‐infected mice on day 9 (G16) after T. gondii inoculation (a) The day of plug detection was considered day 0 of gestation (G0). T. gondii infected mice were infected orally with 30 cysts of T. gondii on G7. Placental tissue was obtained on G16 (9 days after infection). (b) Flow cytometry analyses of CD11b⁺ Gr‐1⁺, CD11b⁺ Gr‐1⁻ and CD11b⁻ Gr‐1⁻ cells among CD45.2 cells in placentas from infected and non‐infected mice on day 9 (G16) after T. gondii inoculation. (c) The percentages of CD11b⁺ Gr‐1⁺, CD11b⁺ Gr‐1⁻ and CD11b⁻ Gr‐1⁻ cells in CD45.2 cells in placentas from infected (n = 6) and non‐infected (n = 6) mice on day 9 (G16) after T. gondii inoculation. Error bars indicate SD. Data are representative of two independent experiments. ^**P < 0.01; ^***P < 0.001. (unpaired Student's t‐test).

Because flow cytometry analyses showed that the hematopoietic cells among the placental cells were CD45.2⁺ cells (Fig. 1b), we analyzed the cells gated on the CD45.2⁺ cell population. On G16, 5.73 ± 3.4% of CD45.2⁺ placental cells were CD11b⁺ Gr‐1⁺ cells in non‐infected mice (Fig. 1b,c). We then investigated the placentas of mice on day 9 (G16) after T. gondii inoculation and found that the percentage of CD45.2⁺ cells of the placenta that were CD11b⁺ Gr‐1⁺ cells was significantly higher (23.3 ± 8.2%) in mice with T. gondii infection than in non‐infected mice (Fig. 1b,c). By contrast, a significantly smaller percentage of CD45.2⁺ hematopoietic cells were CD11b⁻ Gr‐1⁻ cells in the placentas from T. gondii‐infected mice (36.5 ± 8.0%) than in those from non‐infected mice (60.1 ± 13.1%) (Fig. 1b,c). However, the percentage of CD11b⁺ Gr‐1⁻ cells was comparable between infected and non‐infected mice (32.3 ± 10.8% vs. 37.0 ± 9.2%) (Fig. 1b,c). We then analyzed the lymphocyte population of the lymphocyte gate (FSC^low, SSC^low) in CD45.2⁺ cells (Fig. 2). The percentages of CD4⁺ T, CD8⁺ T, NK and B cells were all comparable between infected and non‐infected mice on day 9 (G16) after T. gondii inoculation (Fig. 2a,b).

Figure 2

Flow cytometry analyses of lymphocyte subsets in placentas from infected and non‐infected mice on day 9 (G16) after T. gondii inoculation (a) Flow cytometry analyses of lymphocyte subsets in placentas. Cells in placentas from pregnant T. gondii‐infected and non‐infected mice obtained on G16 were stained with specific antibodies against CD45.2, CD4, CD8, NK1.1 and B220 and analyzed by flow cytometry. (b) The percentage of CD4⁺ CD3⁺ T, CD8⁺ CD3⁺ T, NK1.1⁺ CD3⁻ NK and B220⁺ CD3⁻ B cells in the lymphocyte gate (FSC^low, SSC^low) in the placentas of T. gondii‐infected (n  = 6) and non‐infected (n = 6) mice on G16. Error bars indicate SD. Data are representative of two independent experiments. ^**P < 0.05; ^***P < 0.001. (unpaired Student's t‐test).

Because CD11b and Gr‐1 are expressed on both Ly6G⁺ neutrophils and Ly6C⁺ inflammatory monocytes, it remained undetermined whether the cells the percentage of which had increased were neutrophils, inflammatory monocytes or both. Nevertheless, CD11b⁺ Gr‐1⁺ cells are able to secret proinflammatory cytokines and chemokines, nitric oxide and/or reactive oxygen species 12, 13, which are involved in immunity against T. gondii. However, it is also possible that CD11b⁺ Gr‐1⁺ cells are involved in spreading T. gondii to the fetus by acting as motile reservoirs 6. At present, the role of the increased percentage of CD11b⁺ Gr‐1⁺ cells in the placenta after T. gondii infection remains unclear. Further investigation is required to clarify the pathophysiology of T. gondii infection in pregnant mice. Flow cytometry analysis of immune cells in the placenta may be a useful technique for analyzing immune responses in the placenta against various pathogens that cause congenital infection via placenta, including cytomegalovirus, rubella and parvovirus, as well as T. gondii.

ACKNOWLEDGMENTS

This research was supported in part by grants provided by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to A.S and K.S).

DISCLOSURE

No authors have any conflict of interests to disclose.