Toxoplasma Polymorphic Virulence-associated Effectors Of GRA15_Ⅱ/ROP16_Ⅰ/Ⅲ:Causative Adverse Pregnancy Outcomes In Mice

Background:Toxoplasma gondii（T.gondii,Tg）is an obligate intracellular parasitic and opportunistic protozoan which infects most warm-blooded mammals worldwide,threatning human health and cattle husbandry.Clinical manifestations of toxoplasmosis vary depending on the virulence of Tg and genetic background of hosts.Curently,congenital vertically transmitted toxoplasmosis is one of the major causes of adverse pregnancy.Tg can infect the fetus via transplacental transmission.It was reported recently that Tg infection in mice may lead to adverse pregnancy outcomes,with a possible mechanism of reduction of regulatory T cell（Treg）and up-regulation of Th17.The virulence of Tg strains is closely correlated to polymorphic protein effectors originated from different genotypes of the parasite.Strains of Tg isolated from North America and Europe were mainly categorized into three genotypes,namely Types I,Ⅱ and ⅡI.Type Chinese 1（Toxo DB#9）is the dominant genotype in China according to the investigation of both animals and humans in recent years.Polymorphic proteins released directly into the host cells include rhoptry proteins（ROPs）,such as ROP16 and ROP18;dense-granule proteins,such as GRA15,GRA5,and GRA3.During the process of infection,Toxoplasma may subvert and escape from host immunity system.Therefore,macrophages（Mφ）and mononuclear phagocytes become the main effector cells inhibiting and eliminating Taxoplasma parasites.It has been reported that the parasite-excreted protein of ROP16_Ⅰ/Ⅲ activates the Mφ to develop the alternatively activated phenotype（AAM,M2）by phosphorylating STAT3 and STAT6,resulting in mass production of interleukin-4（IL-4）and interleukin-13（IL-13）;while GRA15_Ⅱ activates Mφ to polarize to the classically activated macrophage phenotype（CAM,M1）by phosphorylating of NF-κB p65,giving rise to massive release of proinflammatory factors,such as IL-23,IL-1β,and IL-6,as well as activation of T helper 17（Th17）and natural killer（NK）cells.The present study based on the fact that ROP16_Ⅰ/Ⅲ and GRA15_Ⅱ drive the macrophages to oppositely biased responses that might be responsible for the immune imbalance of maternal and child interface,finally leading to adverse pregnancy outcomes.Two pathways of activation of macrophages by different effector molecules are elaborated to find possible correlation between adverse pregnancy and the effector polymorphisms.Objective:To generate stably transgenic macrophage cell lines that express Tg ROP16_Ⅰ/Ⅲ or GRA15_Ⅱ;to test the effects of transfection on the phenotype and integrity of macrophage cells and the pregnancy outcome in mice,to discuss the relationship between ROP16_Ⅰ/Ⅲ,GRA15_Ⅱ and the adverse pregnancy,and to explore the changes of immune mechanisms in the process of adverse pregnancy.Methods:We amplified ROP16_Ⅰ/Ⅲ and GRA15_Ⅱ coding gene,then constructed lentiviral vector.The macrophages of mice were infected by lentiviral vector and control empty virus vector,respectively to obtain re GRA15_Ⅱ-RAW264.7,re ROP16_Ⅰ/Ⅲ-RAW264.7 and control empty virus stable expression cell strain.The cell surface markers of PD-L1 and PD-L2 were detected by Fluorescence Activated Cell Sorter（FACS）analysis.The content of nitric oxide（NO）,urea and cytokines（TGF-β,IL-10,IL-6,IL-23,IL-12,IL-1β,and IL-17A）in the supernatant of the cell culture were detected by enzyme-linked immunosorbent assay（ELISA）.Arg-1 and i NOS were examined by realtime-PCR（realtime fluorescence quantitative PCR,q PCR）.The content of STAT3,STAT6 and NF-κB were detected by Western blotting（WB）method to estimate the direction of polarization of macrophages.Three stable cell strains were analyzed through DNA microarrays for gene expression profiles.Magnetic absorption cell sorting（MACS）method was used to isolate the CD4+CD62L+ T lymphocytes from the spleen of the mice.The splenocytes after sorting and stable cell strains were subjected to co-culture.Flow cytometry,ELISA and q PCR were used to monitor the indexes of Th17 activation.The mice model of pregnancy was constructed.The 1x106 re GRA15_Ⅱ--RAW264.7 cell and empty virus vector-RAW264.7 were injected by cauda vein or 200 PRU tachyzoites were intraperitoneally injected on day 7.5 in first trimester pregnancy,repectively.The mice were sacrificed with euthanasia on day 14.5 in midtrimester.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/Th2/Th17 in spleen and placenta were monitored by FACS analyses.Results:Empty virus vector-RAW264.7,re GRA15_Ⅱ--RAW264.7 and re ROP16_Ⅰ/ⅢRAW264.7 were constructed successfully.The results of FACS show that surface factor（CD80 and CD86）were increased obviously.PD-L1,the surface marker of re GRA15_Ⅱ--RAW264.7,was notably upregulated.Nitric oxide and inflammatory factors（IL-6,IL-23,IL-1β,IFN-γ,IL-17）in the supernatant of cell culture were also elevated signicantly.NF-κB p65 was increased with NF-κB nuclear translocation.The expression of i NOS（inducible nitric oxide synthase）was upregulated.PD-L2,the surface marker of re ROP16_Ⅰ/Ⅲ--RAW264.7,and IL-13,IFN-γ and urea in the supernatant of culture solution were increased.The expression of Arg-1（Arginase-1）was upregulated obviously.IL-17 A from surface marker in lymphocytes and supernatant was increased when CD4+CD62L+ T lymphocytes and re GRA15_Ⅱ--RAW264.7 were co-cultured.C57BL/6 pregnant mice were injected with re GRA15_Ⅱ-RAW264.7,in comparison with the control group,the increased absorptivity of fetus was observed.Stillborn fetus,hemorrhage,and tissue organization were seen.The number of vital fetus was decreased.IL-17 expression on the surface of splenocytes in experimental mice was higher than that in normal and control pregnant mice.The cell number with expression of CD4+CD25+Fox P3（Tregs）was decreased.Conclusions:Three stable cell strains,re GRA15_Ⅱ--RAW264.7,re ROP16_Ⅰ/Ⅲ-RAW264.7 and empty virus vector-RAW264.7 were constructed successfully.The results showed that GRA15_Ⅱ is the key factor which mediated the polarization of macrophages to M1,whereas ROP16_Ⅰ/Ⅲ is the key factor that is responsible for mediating the M2 polarized resposne.Compared to the phenotype of macrophages infected with Tg,there was difference among them.T cells could differentiate to Th17 after activation induced by polarized macrophages driven by GRA15_Ⅱ.Injection of the above stable cell strains would lead to the adverse pregnancy of mice.Our results strongly implicate that teratogenic effect of Tg would be related to the polymorphic genotype-associated virulence factor GRA15_Ⅱ of T.gondii.