Toxoplasma Type Ⅰ Strain With Δrop16_Ⅰ/Ⅲ Lead To Abnormal Pregnant Outcomes Associated With Macrophage Polarization In Murine Model

BackgroundToxoplasma gondii is an obligate intracellular parasite.The vast majority of warm-blooded animals,including human infections,can cause toxoplasmosis.After infection in normal individuals,there are generally no obvious symptoms and signs,leading to chronic infection.People with impaired immune function（such as the cancer patients with long-term chemotherapy,or AIDS patients with immune suppression,etc.）may present activated toxoplasmosis,even death.Toxoplasma infection in pregnant women,particularly in the early trimester,or middle pregnancy,may cause abnormal pregnant outcomes due to spreading of the parasite to the fetus through the placenta.At the same time,Toxoplasma infection in pregnant women has been found to be able to activate the decidual immune cells,causing rejection and intrauterine retarded growth of the fetus.Under this circumstances,intrauterine growth restriction may occur and result in,abortion,stillbirth and fetal congenital toxoplasmosis or other adverse pregnancy consequences.When human is infected with Toxoplasma gondii,macrophage is one of the preferential host cells of Toxoplasma and is also the main way to general dissemination during acute infection.In normal pregnancy,macrophages,accounting for 20%-30%of the total number of white blood cells,have shown to paly a key role in maintaining a normal microenvironment of pregnancy on the maternal-fetal interface by inducing immunotolerant status in the presence of alternatively activated macrophage（M2）.Additionally,T.gondii infection may induce decidual macrophages polarization from M2 to M1,leading to the subversion of M2 dominant inmmune tolerance,changes of the maternal-fetal interface microenvironment,and finally,the serious adverse pregnancy outcomes.I has been known that Toxoplasma roptry protein 16（ROP16）is one of the genotype-associated effectors in modulation of host immunity during early stage of parasite invasion.ROP16,as a kinase,is featured by driving macrophages to alternatively activated polarization via phosphorylation of Stat3/6,which is responsible for subsequent induction of Th2 dominant response.Based on this characterization of ROP16I,we proposed that Toxoplasma ROP16_I/III/III might be invloved in maintaining the physiological immunity on the maternal-fetal interface and beneficial to normal development of fetus.In doing this,we successfully constructed the RH strain rop16_Ⅰ/Ⅲ/III gene-deficient strain RHΔrop16 by using CRISPR-Cas9 technology.RHΔrop16 strain was used to infect pregnant mice.We found that the RHΔrop16 strain infection of early pregnant mice caused the imbalance of maternal-fetal immune tolerance.The macrophages in the unterine tissues skewed from M2 to M1,which highly expressed iNOS and IL-12,thereby changed the immune microenvironment at the maternal-fetal interface,which is responsible for the adverse pregnancy outcomes in mice.ObjectiveThe aim of this study was to investigate the role of Toxoplasma-derived effector ROP16_I/IIIin maintaining the physiological balance of general and maternal-fetal interface by keeping M2/Th2 response.Defeciency of ROP16I/III may result in adverse pregnancy outcomes.It suggests that ROP16_I/III/III may play a protective part in abnormal pregnancy caused by Toxoplasma gondii type I strain although Toxoplasma infection is believed to cause abortion in pregnancy through direct invasion and multiplication of the parasite.MethodsThe pregnant mice model was constructed using C57BL/6 in a ratio of 2:1 for females and males,and a normal control group was used as control.After 7.5 days of gestation,the mice were injected with sterile 200μl PBS.The infected groups were injected with400 tachyzoites of RH wild type（WT）strain and of RHΔrop16 strain,respectively,at7.5 days of gestation.Mice were sacrificed under anesthesia on day 6 after infection of pregnant mice.Eye blood was collected and serum was taken to detect the expression of TNF-αand IL-12 by ELISA.The uterus,placenta and fetal tissues were collected,and the placental hemorrhage and absorption were observed.The fetus and placenta weight were weighed and recorded.The changes of fetal and placental weight in the normal group mice and the mice with RH strain infection or RHΔrop16 infection were compared.Placental tissue was taken for histopathological examination and HE staining,and pathology in placental bleeding was observed.The changes of Th1（IFN-r）and Th2（IL-4）cytokines in splenocytes were detected by flow cytometry.The expression levels of molecular markers of M1（CD80,CD86）or M2（CD206）phenotype macrophages in placental decidual cells were detected by flow cytometry.Western blotting（WB）was used to detect the protein expression level of M1（iNOS）and M2（Arg-1）in decidual tissue of each group.The expression of M1（TNF-α,IL-12）or M2（TGF-β1）in spleen and placenta tissues of each group was detected by qPCR.The expression of cytokines TNF-αand IL-12 in M1 and spleen and placenta were detected by ELISA.ResultsCompared with the normal control group,visual examination revealed that different degrees of placental hemorrhage absorption occurred after infection with T.gondii,fetal and placental body weight decreased significantly,and the abortion rate increased greatly.In comparison with the RH WT infected group,the placental hemorrhage and the fetal placental absorption were more serious in the fetus of the RHΔrop16 infected group.By measurement of cytokine expression of IFN-?in Th1 cells by flow cytometry,we found that the expression of IFN-?significantly increased both in the RH-infected group and the RH-Δrop16-infected group,however,the increase of expression of IFN-?in the RHΔrop16-infected was more pronounced compared with the normal group.The expression of Th2 cytokine IL-4 was down-regulated in the RH-infected group and the RHΔrop16 infected group,but more obviously in the RHΔrop16-infected group.Flow cytometry detection of the M1 phenotype CD80,CD86 and M2phenotype CD206 of decidual macrophages showed that the expression of CD80 and CD86 increased after infection of RH WT strain as well as RHΔrop16 strain,while the expression of CD206 was decreased.Among them,the expression of CD80 and CD86in RHΔrop16 infection group was more remarkable,and the decrease of CD206 was also obvious.By WB test,compared with the normal control group,the expression of iNOS was the highest in RHΔrop16 infection group,followed by RH infection,while the expression of Arginase-1 was the lowest in RHΔrop16 infection group,second to RH infection group.The results of ELISA and qPCR showed that the expression of cytokines TNF-αand IL-12 secreted by M1 cells in spleen and placenta tissues was significantly increased in the infected group,and the cytokine of M1 in the RHΔrop16-infected group was obviously elevated.TGF-β1 production in the spleen and placenta tissues was detected by qPCR.Compared with the normal control group,the expression of TGF-β1 in the infected group was down-regulated,which was more apparent in the RHΔrop16-infected group.ConclusionThe above experimental results show that Toxoplasma gondii infection leads to adverse pregnancy outcomes,not only caused by the massive tissue damage of Toxoplasma parasite,but also due to the imbalance of maternal-fetal physiological immunotolerance evoked by the infection-induced immune bias.Among this,ROP16_I/III/III effector carried by type I strain of T.gondii may play a beneficial role in reversion of the abnormal pregnacy by inducing the decidual macrophage M2 bias and dampening expression of iNOS,IL-12 and TNF-αand increasing Arginase-1 and TGF-β1 expression.The present study revealed a new molecule of Toxoplasma which might be helpful for creating the microenvironment of maternal-fetal immunity.