The Different Roles Of High And Low Virulence Toxoplasma Gondii Excreted-secreted Antigens On IFN-γ–inducing Apoptosis Of CD4~+T Cells

Toxoplasma gondii is an obligate intracellular protozoan which is highly prevalentin warm-blooded vertebrates, including humans. In an immunocompetent hostinfection often goes asymptomatic and unnoticed. However, maternal infection ofT.gondii is associated with increased adverse pregnancy outcomes such as abortion,fetal death, stillbirth and congenital defect.The majority of Toxoplasma strains can be grouped into three main clonallineages (types I, II and III) that differ genetically by1%or less. Most of what isknown regarding the immune response against T.gondii is based on data obtainedfrom laboratory mice infected with the parasites from these three haplogroups. Inlaboratory mice, type I strains are categorically lethal, with an LD100=1, whereasthe LD50of type II and III strains are103~105, respectively.The excreted-secreted antigens (ESA) are expressed at both the tachyzoite andencysted bradyzoite stages of T. gondii. They represent the majority of the T. gondiicirculating antigens in sera from hosts with acute T. gondii infection. ESA havebeen shown as important components in the process of invasion and replication of T.gondii within host cells. Recent evidence in pregnant mice indicates that T. gondiiESA may result in abortion and reduction of CD4+CD25+regulatory T cells, whichis similar to that of the T. gondii infection. Up to now, few studies involve in theimpact of Pru strain ESA in immune cells and the different effects between RHstrain and Pru strain ESA. Based on the references above, our study focus on thefunctional roles of IFN-γ caused by T. gondii ESA treatment and the changes ofCD4+T cells in mice treated with T. gondii ESA.In this study, C57BL/6murine model were established and treated with T. gondiiRH strain(type I) and Pru strain (type II) ESA. The apoptosis of splenic CD4+Tcells in all splenic cells were measured by flow cytometry. To detect the levels ofIFN-γ, ELISAs were conducted. After that, the influences of different straints T.gondii ESA on CD4+T cells in different cell culture system were assessed by flowcytometry in vitro. Flow cytometric analysis was performed to investigate theapoptosis of splenic CD4+T cells, expression levels of bcl-2and Fas. To furtherconfirm the expression of bcl-2and bax presenting CD4+T cells, Western-blot was performmed.The main results we got are as follows：1. Treatment with different ESA induces apoptosis of the splenic CD4~+T cellsAt3days post-treatment, the apoptotic rates of splenic CD4~+T cells in allsplenic cells tended to increased in RH strain and Pru strain ESA treated micecompared with that in control mice(P<0.01), and there was statistically significantdifference between the two groups(P<0.05). In vitro,the apoptotic rates of thesplenic CD4~+T cells increased obviously when treated with RH strain and Prustrain ESA for3days compared with that of control group(P<0.0001) and therewas statistically significant difference of apoptotic rates in group of treatment withRH strain ESA between Pru strain ESA treated group(P<0.05).2. Treatment with different ESA induces production of IFN-γThe expression levels of IFN-γ in serum samples caused by different T. gondiistrains ESA treatment were tested by ELISA. The levels tended to increased in RHstrain and Pru strain ESA treated mice compared with that in control mice(P<0.01),and there was statistically significant difference between the two strains ESAgroups (P<0.05). In vitro, the level of IFN-γ by RH strain ESA treatment washigher than that of Pru strain ESA treatment(P<0.05).3. The different roles of high and low virulence Toxoplasma gondii excreted-secreted antigens on IFN-γ-inducing apoptosis of CD4~+T cellsWe investigated the implication of IFN-γ in the apoptosis of CD4~+T cells in T.gondii ESA treated groups. Addition of anti-IFN-γ antibody to culture resulted insignificant decrease in apoptosis of CD4~+T cells. Because Fas interactions havebeen shown by many investigators to be involved in T cell death, we examined therole of this molecules in the death of CD4~+T cell populations by two differentstrains ESA treatment. To do this, we assessed the magnitude of apoptosis in CD4~+Tcells in the presence or absence of anti IFN-γ mAb. The treatment of CD4~+T cellswith anti IFN-γ mAb resulted in decline in expression levels of Fas (P<0.0001).The members of Bcl-2regulate apoptosis via their capacity to modulatemitochondrial membrane pore (MMP). To further confirm the apoptosis occurred inCD4~+T cells, we analysed the expressions of Bcl-2/bax. After an initial increasepost treatment, the expression of bcl-2in splenic CD4~+T cells from Pru strainESA-treated mice increased further(P<0.05). Then, we cultured the two strains ESAtreatment CD4~+T lymphocytes in the presence of anti-IFN-γ mAb (10ng/ml) for aperiod of72h and determine the percentage of CD4~+T cells that were Bcl-2positive.Antiapoptotic Bcl-2expression was significantly less in anti-IFN-γ mAbuntreatment group compared to that of anti-IFN-γ mAb treated group(P<0.05). Theapoptotic process in T cells from T. gondii ESA treated group is associated withaltered induction of Bcl-2or Bax. The relative intensity of Bcl-2and Bax protein expression was determined by Western blot. A higher expression of Bcl-2wasobserved in T cells from Pru strain ESA treated cells than that in RH strain ESAtreated cells. Also, increased expression of Bcl-2/Bax was observed in CD4~+T cellsfrom mice treated with Pru strain ESA compared to cells treated with RH strainESA. We proposed that IFN-γ promotes apoptosis of CD4~+T cells during RH strainESA and Pru strain ESA treatments probably by the following mechanisms:1) byactivating cell-extrinsic apoptosis signals that kill CD4~+T cells and2) bysensitizing CD4~+T cells to apoptosis by inducing intracellular apoptosis molecules.