| Objective Toxoplasma gondii (T. gondii) is an obligatory intracellular parasite thatinfects all warm-blooded animals including human. Infection with T.gondii duringpregnancy may result in preterm delivery,abortion, stillbirth and fetal malformations.Transplacental transmission is a major feature of Toxoplasma gondii infection and canaffect the postnatal growth and intellectual level[1]. Toxoplasma isolates in Europe andNorth America group primarily into one of three clonal lineages (types I, II, and III).Mouse macrophages infected with type I and III Toxoplasma strains are polarizedtoward an M2activation state, while type II infected macrophages resemble aspects ofM1activation. Parasite-specific factors that work independently of pattern recognitionreceptors to achieve M1or M2activation[2]. It was well-known that themicroenvironment of pregnancy was Th2-dominated.There were two Chinese isolatedToxoplasma gondii,TgCtwh3and TgCtwh6,which have the same genotypes butdifferences in virulence. Here,we explore M-1/M-2macrophages and the Th1/Th2paradigm after infection with these two kinds of parasite in pregnant mice and therelated mechanism of adverse pregnancy.Methods In vivo experiment: For mating purposes, four females werehoused overnight with two males.Ninty pregnant females were harvest andwere divided into TgCtwh3infection, TgCtwh6infection and uninfectedgroups as control randomly. On GD8, in the TgCtwh3infection group andTgCtwh6infection group, mice were injected intraperitoneally with200tachyzoites in0.1ml0.9%sterile saline solution. In the control group, themice were exposed to only0.9%sterile saline solution. Mice were killed bycervical dislocation on gestational day10,12,14,16,18. Peritoneal exudate cells (PECs) were harvested,placentas and pleen were carefully dissectedout.Some placenta were fixed in4%(w/v) buffered formaldehyde, paraffinembedded, and stained with H&E and TUNEL analysis.Prasites burdern inplacental tissues was detected by real-time RT-PCR. Arginase activity andNO concentration were measured in the supernatants of macrophages. iNOsand Arg-1protein expression level were analyzed by Westernblotting.Markers associated with either M1or M2on surface ofmacrophages were analyed by FACs.On GD14, fresh single-cell suspensionsof leukocytes from spleen were prepared, analyses of CD4+T cellsubpopulations by flow cytometry.In vitro experiment: This analysis wasperformed with a transwell system using cell culture plates with a0.4-μm-pore filter. Take placental tissues to primary trophoblast culture andperitoneal macrophages were also cultured on GD10to GD12days. We setfour time points,that were2h,6h,12h and24h.On each point, The placentaltrophoblast were divided into five groups on average (1×106) and thenadded in the lower wells remained uninfected.Group A:took macrophagesonly on the upper well(1×106); Group B: took macrophages and TgCtwh3parasites (1×106) on the upper well;Group C: took macrophages and TgCtwh6parasites (1×106) on the upperwell; Group D: took TgCtwh3parasites (1×106) only on the upper well;Group E: took TgCtwh6parasites (1×106) only on the upper well.Thenplacental trophoblast were co-cultured with the upper well on a24-wellplate at370Cfor the indicated time intervals. Apoptosis of placentaltrophoblast were measured through Annexin V-EGFP and TUNEL kit. M1cytokines levels were measured in the supernatants of macrophages.Analysis of iNOs,Arg-1protein expression level and Markers ofmacrophages as vivo experiment. Results Compared to TgCtwh6infection group, the trophoblast apoptosislevels of TgCtwh3infection group was higher.While these apoptosis levelsof TgCtwh6infection group was higher than control group.HE results showsthat there are abundant lymphocyte infiltration in placental tissues inTgCtwh6group during the early period of infection and this phenomenonwas not observed in other two groups. Levels of arginase in TgCtwh3infection group were significant higher than the other two groups. OnGD14,NO production by macrophages in TgCtwh6group began toappear.;however,no NO production was observed in TgCtwh3group andcontrol group.Expression of CD206,PDL-2and MHCⅡ in TgCtwh3groupwere significantly higher than the other two groups;In contrast, expressionof PDL-1,CD86and CD80in TgCtwh6group were higher than other twogroups.FCMs of spleen shown that the percentage of the Th1cells in twoinfection groups were dropped significantly and the percentage of the Th2cells was obviously rises only in TgCtwh3group.The change rules of levels of arginase in vitro experiment was assimilar as that in vivo experiment.Different from vivo experiment, thetrophoblast apoptosis levels of TgCtwh6infection group was higher thanTgCtwh3infection group. M1cytokines levels in TgCtwh6infection groupwere also higher than TgCtwh3infection group.Conclusion Infection with these two kinds of Toxoplasma gondii duringpregnancy both led to adverse pregnancy outcome.Macrophages inpregnance mouse infected with TgCtwh3tachyzoites are polarized towardan M2activation state,which leading the immune microenvironment tofurther Th2-bias that is favorable to reproduction of parasites. Host survivalmay already compromised due to uncontrolled parasite burden. WhileTgCtwh6tachyzoites infected macrophages are polarized toward an M1 activation state and secrete M1cytokines.Inflammatory cells are infiltrationin placental tissues during the early period of infection in order to controlreproduction of parasites.But with pregnancy lasts,Th2-dominated immunemicroenvironment is more advantageous to reproduction ofparasites.Infiltration of inflammatory cells and More and more parasitesalso increase apoptosis of trophoblast cells.The results suggest,infection with parasites, which have same genotypes butdifferent in virulence,can induce different polarization of macrophages(M1and M2).Or there still exist other different genetic type in Chinese1genotype(11locus points type).Wether these different parasites causeddifferent outcome of immune is relevant to the polymorphism of GRAs andROPs or not,is still to be further studied. |
PDF Full Text Request