Toxoplasma Type Chinese 1 WH3 Strain With ?rop16_?/? Facilitates Abnormal Pregnant Outcomes In Murine Model

Background:Toxoplasma gondii is an extensive intracellular parasitic protozoon that is capable of affecting almost all vertebrate animals including humans and leading to reproductive failure in its hosts.Primary infection with T.gondii during pregnancy,particularly in the first trimester,can cause miscarriages,stillbirths or fetal abnormalities in humans.T.gondii parasite can invade the fetus via placental transmission.It has been reported that neither parasites nor their DNAs,are detectable in some diseased samples with abnormal pregnancy outcomes,suggesting that an immunity-related pathogenesis induced by T.gondii might be involved.We previously reported that type Chinese 1strains dominantly prevalent in China share both GRA15_IIand ROP16_I/IIIeffectors.Polymorphic difference of theses virulence-associated effectors of Chinese 1 strains from those of the archetypical genotypes of T.gondii in Europe and North America strongly suggests that strains of Chinese 1?ToxoDB#9,WH3 strain?prevalent in Chinese mainland may have the pathogenesis differing from the strains with the archetypical types in the other continents.The parasite with ROP16_I/IIIdeficiency carried in type Chinese 1 strains may induce imbalance at maternal-fetal interface of pregnant women and account for the failure of pregnancy caused by Toxoplasma infection in some cases.Toxoplasma virulence effectors possess the genotype-associated polymorphism.ROP16_I/IIIand GRA15_IIcarried by types I and III or type II strains drive macrophages to M2 or M1,respectively,inducing a remarkably different immune consequences.It is hypothesized that strains with genotype Chinese 1?WH3?prevalent in China may have the pathogenesis differing from the strains with archetypical types due to the fact that WH3 is characterized by sharing ROP16_I/IIIand GRA15_II.It has been reported that T.gondii infection in mice lead to adverse pregnancy outcomes,with a possible mechanism of reduction of regulatory T cell?Tregs?and up-regulation of Th17.The physiological balance of Th1/Th2 is believed to play a crucial role in pregnancy of mammals including humans.In normal pregnancy,Th1-type response is down-regulated and Th2 cytokines are up-regulated for ensuring immune tolerance at the maternal–fetal interface.Previous investigations indicate that the parasite rhoptry protein of ROP16_I/IIIdrives macrophages to M2 polarization at early phase of infection while GRA15_IIinduces M1 polarization,subsequently activating NK,Th1 and Th17 cells.In this study,we observed the effect of ROP16_I/IIIdeletion on adverse pregnancy outcomes by constructing a T.gondii WH3?rop16 deficient strain based on CRSPR/Cas9 technology.Objective:To explored the potential of rop16^-/-_I/IIIparasite with gra15_IIbackground in subverting the maternal immune tolerance which may facilitate the process of adverse pregnancy.Methods:T.gondii WH3?rop16 deficient strain was constructed based on CRISPR/Cas9technology.Monoclone was detected and confirmed by PCR and Western blotting.The invasion of WH3 WT or WH3?rop16 strain in inoculated HFF cells were visualized by Giemsa staining.C57BL/6 mice were infected with 1×10³tachyzoites of WH3 WT or WH3?rop16,respectively.The animals were monitored daily for manifestations following infection and the survival rate was recorded.Control macrophages and WH3WT or WH3?rop16 tachyzoites infected macrophages were added to the upper well separately.The placental trophoblasts were co-cultured in the lower wells.After co-culturing,we detected the iNOS,NO and TNF-?by qRT-PCR and ELISA.The placental trophoblasts were collected for detection of apoptosis by FCM.The mouse model of pregnancy was constructed.C57BL/6 pregnant mice were intraperitoneally injected with 400 tachyzoites of WH3 WT or WH3?rop16 on day 8 in first trimester pregnancy,respectively.The mice were sacrificed with euthanasia on day14 in mid-trimester.The placenta and fetal animals were taken for the morphological and histopathological examinations.The number of vital fetus and absorptivity were calculated.The expressions of Th1 cytokine?IFN-??,Th2?IL-4?,Th17?IL-17?,and Tregs in spleen and placenta were monitored by FACS analyses.The mRNA relative expressions of the cytokines in splenocytes and placenta were examined by quantitative real time reverse-transcriptase polymerase chain reaction?qRT-PCR?.Simultaneously,the concentrations of the cytokines in the spleen and placenta were tested by enzyme-linked immunosorbent assay?ELISA?.Results:The rop16_I/IIIdefect strain of T.gondii has been successfully constructed by the CRISPR-Cas9 technology.Giemsa staining results indicated that the number of parasites per parasitophorous vacuole of WH3 WT infected cells was more than that of the WH3?rop16 infected cells.Mouse passages showed no difference of virulence between WH3 WT and WH3?rop16 strain.Compared with the WH3 WT group,macrophages infected with WH3?rop16 strain produced a high level of NO,iNOS and TNF-?.To elucidate the placental trophoblasts apoptosis,we detected cell apoptosis of placental tissues by FITC/PI staining assay.Compared with the WH3 WT group,the number of total,early,and late apoptotic cells of trophoblasts which co-culture with WH3?rop16strain infected macrophages were obviously increased.C57BL/6 pregnant mice injected with the tachyzoites of WH3 WT or WH3?rop16 presented increased absorptivity of fetus in comparison with the control animals.Stillborn fetus,hemorrhage,and tissue organization were seen.The number of vital fetuses was decreased.Additionally,C57BL/6 pregnant mice injected with WH3?rop16 parasites showed more serious abortion than those with the WH3 WT innoculated groups.In C57BL/6 pregnant mice injected with WH3?rop16 tachyzoites,both IL-17 and IFN-?expressions in splenocytes and placenta tissues was notably higher than in WH3 WT group infected.The cell number with expression of CD4⁺CD25⁺FoxP3?Tregs?was decreased.In contrast,expression of IL-4,which belongs to Th2 cytokine,was lower than that in WH3 WT group.Meanwhile,in comparison with WH3 WT group,the mRNA relative expressions of splenocytes and placenta of mice injected with WH3?rop16 showed a down-expression of IL-10 and TGF-?1,and an up-expression of IFN-?and IL-12examined by q RT-PCR and ELISA.Conclusions:Toxo-rop16^-/-_I/IIIstrain with gra15_IIgenetic background of Chinese 1 genotype,similar to type II strain ofPRU or ME49,may cause imbalance of immune tolerance on the maternal-fetus interface and in system immunity,leading to adverse pregnancy outcomes during pregnancy,which is attributed to the Th1 biased response induced by Toxo-rop16^-/-_I/IIIstrain with GRA15_IIbackground.The results will provide a deep insight into the pathogenesis of abnormal pregnancy caused by Toxoplasma genotype associated molecules rather than the live parasite predominantly circulating in China.