The Roles Of Different Strains Of Toxoplasma Gondii Excreted-secreted Antigens (ESA) On The Subpopulations Of Murine Immune Cells

Toxoplasma gondii is an obligate intracellular protozoan parasite,which is alsoan important zoontic and veterinary pathogen. In an immunocompetent host infectionoften goes asymptomatic and unnoticed. However, maternal infection of T.gondiiduring pregnancy is associated with increased adverse pregnancy outcomes such asabortion, fetal death, stillbirth and congenital defects. The excreted-secreted antigens(ESA) are expressed at both the tachyzoite and encysted bradyzoite stages of T.gondii. They represent the majority of the T. gondii circulating antigens in sera fromhosts with acute T. gondii infection. ESA have been shown as important componentsin the process of invasion and replication of T. gondii within host cells.T. gondii is an important opportunistic pathogen,whose pathogenicity is relatedto the virulence of the parasite and the immune state of the host. T. gondii can bedivided into different strains, based on their invasion, proliferation, mortality andformation of cysts after infection. The high virulence strain (RH strain) parasites aresensitive to mice, the infection usually causes mice death in3-5days, while the lowvirulence strain (PRU strain) parasites prefer to form cysts in the tissues and sustainthe chronic infection.The outcome of T. gondii infection is determined by the interaction between thehosts and the parasites. The harm to host is correlated with the immune state of thehost and the cell-mediated immunity which is regarded as the predominant mechanism to resist T.gondii infection. The cell-mediated immunity includesinteraction of many immunocytes and cytokines which play an important role ininhibiting the proliferation of parasite, cleaning up the parasite and controllingparasitemia. At present, more and more studies in human and mice have proved thatCD4~+CD25~+regulatory T cells play an important role in regulating the immunity ofpathogen infection. These studies have found that CD4~+CD25~+regulatory T cells canrestrain excessive immune response by reducing its sensitiveness in the infectionlocated, which can lead to the pathogen surviving in the hosts.Our previous study has demonstrated that treatment with T. gondii ESA mayresult in abortion, increase of NK cells and reduction of CD4~+CD25~+regulatory Tcells, which is similar to that of the T. gondii infection. Up to now, few studiesinvolve in the impact of this pooled ESA in immune cells. So is there any differentinfluence of immune cells treated with different strains of T. gondii ESA? Is there anyconnection between different state of immune cells and different endings withinfection of different strains of T. gondii? Our study focus on the different effects ofsubpopulations of murine immune cells caused by different strains of T. gondii ESAtreatment and the changes of NK cells, CD4~+CD25~+T cells. At the same time, wediscuss the preliminary mechanism, and provide the theory basis for the futureprevention and immune intervention of T. gondii infection.In this study, C57BL/6normal murine model were used and treated withdifferent strains of T. gondii ESA. The numbers of splenic NK cells and CD4~+CD25~+regulatory T cells in all splenic cells were measured by flow cytometry. NK cellswere isolated by MACS, their cytotoxicity on YAC-1cells were detacted by LDHmethod. The expression level of Granzyme B of NK cells were detected by RT-PCR.At the same time, CD4~+CD25~+regulatory T cells were isolated by MACS, their suppression function was detected by3H-TdR. The IFN-γ levels in serum was testedby ELISA.The main results we got are as follows:1. First, we prepared the serum-free ESA from T.gondii RH strain and PRUstrain, respectively. Then, the protein concentrations were determined by BCAmethod. To test the protein dosages used in the experiments, different concentrationsof ESA (0.05mg,0.01mg,0.002mg and0.004mg) were injected into mice,respectively. The splenocyts were isolated and detected by flow cytomety. The resultsshowed that under the dosages of0.002mg and higher, the NK cell numbers wereincreased significantly compared with that of PBS control. Therefore, we chosed0.002mg dosage of ESA to be used in the following experiments.2. Different strain ESA increased the number of NK cells. The results showedthat, by flow cytometry analyses, the numbers of NK cells in both RH ESA and PRUESA treated groups were increased significantly compared with that of PBS control.In addition, the increasement in RH ESA treated group was higher than that in PRUESA treated group, and the difference with statistical significance.3. Different strain ESA elevated the sera IFN-γ. In this experiment, ELISAmethod was used to detect the sera levels of IFN-γ. The results showed that the levelsof IFN-γ in both RH ESA and PRU ESA treated groups were increased significantlycompared with that of PBS control. Also, the increasement in RH ESA treated groupwas higher than that in PRU ESA treated group, and the difference with statisticalsignificance.4. Different strain ESA decreased the percentages of splenic CD4~+CD25~+regulatory T cells. The results showed that the ratio of splenic CD4~+CD25~+regulatory T cells in splenocytes in both RH ESA and PRU ESA treated groups were increased significantly compared with that of PBS control. Importantly, thereduction in RH ESA treated group was higher than that in PRU ESA treated group,and the difference with statistical significance.5. Different strain ESA enhanced the cytotoxicity of NK cells. In thisexperiments, NK cells were obtained from RH ESA and PRU ESA treated mice,respectively, and isolated by MACS, their cytotoxicity on YAC-1cells was evaluatedby LDH method. The results showed that the cytotoxic effects in both RH ESA andPRU ESA treated groups were increased obviously compared with that of PBScontrol. As expected, the enhancement was higher in RH ESA treated group than thatin PRU ESA treated group, and the difference with statistical significance.6. Different strain ESA enhanced the suppresive functions of CD4~+CD25~+regulatory T cells. In this experiment, CD4~+CD25~+T cells were obtained from RHESA and PRU ESA treated mice, respectively, and isolated by MACS. Thesuppresive functions of CD4~+CD25~+regulatory T cells on proliferation ofCD4+CD25-T cells were analysed by3H-TdR method. The results showed that theCD4~+CD25~+T cells from both RH ESA and PRU ESA treated mice could suppressthe proliferation of CD4+CD25-T cells after anti-CD3stimulation significantlycompared with that from PBS control. Also, the enhancement in RH ESA treatedgroup was higher than that in PRU ESA treated group, and the difference withstatistical significance.7. Different strain ESA increased the cytotoxicity of NK cells on CD4~+CD25~+Tcells. In this experiment, nomal NK cells and CD4~+CD25~+T cells were isolated frommous splenocytes by MACS, and cultured with RH ESA and PRU ESA, respectively.The cytotoxic effects of NK cells on CD4~+CD25~+T cells were detected by LDHmethod. The results showed that the ESA-treated NK cells had more potential to killthe CD4~+CD25~+T cells and the efficacy in RH ESA treated group was higher than that in PRU ESA treated group.In conclusion, the results indicated that there were obvious differences betweenRH ESA and PRU ESA on effecting the number and function of NK cells andCD4~+CD25~+regulatory T cells of mice. The efficacy was usually higher in RH ESAtreated group than that in PRU ESA treated group. These phenomenon may explain,in part, the virulence differences between RH and PRU strains of T. gondii infections.