Effects Of The Culture Supernatant Of Toxoplasma Gondii On The CD4～+CD25～+ Regulatory Cells In Vivo

Toxoplasma gondii is a highly prevalent intracellular protozoanpathogen. Its infection often causes widespread tissue damage inimmuno-compromised host, which can be fatal if not appropriatelytreated; while in immuno-competent host infection often goes unnoticed.During pregnancy, a state of immunological tolerance, acutetoxoplasmosis may lead to abortion, premature birth, abnormal embryo orfetal death. It is reported that, the culture supernatant of Toxoplasmagondii injected into the peritoneal can induce abortion or result in severedevelopmental disorders. More and more studies in human and mice haveproved that during pregnancy, the number of CD4~+CD25~+ regulatory Tcells diminished in abortion individuals while in normal individuals it iselevated. Mice can't initiate or maintain pregnancy without CD4~+CD25~+regulatory T cells. But when abortion-prone mice were transferredpregnancy-induced CD4~+CD25~+ regulatory T cells, rejection can beprevented. This indicated that CD4~+CD25~+ regulatory T cells played acritical role in mediating maternal tolerance to the fetus and maintainingpregnancy. Considering the importance of CD4~+CD25~+ regulatory T cells inpregnancy, and there is no evidence saying that the functional andquantitative changs of CD4~+CD25~+ regulatory T cells will cause abortionin human or animal models in infection with Toxoplasma gondii or afterinjected with its culture supernatant, we observed the fetus development,the quantitative and functional changes of CD4~+CD25~+ regulatory T cellsafter treated with the culture supematant of Toxoplasma gondii in vivoand in vitro.At fitst we observed the fetus development and the percentage ofCD4~+CD25~+ Foxp3~+T cells and CD4~+CD25~+T cells in the monocyte ofspleen after the culture supernatant of Toxoplasma gondii injected to thepregnant mice. Then, to examine the functional change of CD4~+CD25~+regulatory T cells treated by the culture supernatant of Toxoplasma gondii,CD4~+CD25~+ regulatory T cells were separated from the mice spleen usingmagnetic beads. The other immunocytes that were isolated by removingCD4~+CD25~+ regulatory T cells were co-cultured with or withoutCD4~+CD25~+ regulatory T cells. The inhibitory function of CD4~+CD25~+regulatory T cells was assessed by the [~3H] thymidine incorporationmethod with the stimulation of ConA. The percentages of splenicCD4~+CD25~+CTLA-4~+ T cells and CD4~+CD25~+GITR~+ T cells inCD4~+CD25~+T cells at a serial of treated time were measured by flowcytometry. The gene expression of CTLA-4 in CD4~+ T cell was detectedby general RT-PCR. Finally, the apoptosis of CD4~+CD25~+ regulatory Tcells treated by different ways were detect by flow cytometry.The main results we got are as follows:1. The culture supernatant of Toxoplasma gondii can induceabortion in pregnant mice. The pregnant mices were injected withthe culture supernatant of Toxoplasma gondii at gestational day 8, and were killed at gestational day 14. We dissected the uterus andfound that all the embryos of the experimental mice were necrosis orabsorbed, but all the embryos of the control mice developed well.2. The culture supernatant of Toxoplasma gondii can downreglatethe percentage of CD4~+CD25~+Foxp3~+ T cells and CD4~+CD25~+ Tcells in spleen monocytes of pregnant mice. The splenocyte atgestational day 14 were isolated from the the normal pregnant miceand the pregnant mice injected with the culture supernatant ofToxoplasma gondii at gestational day 8. FCM detected CD4~+CD25~+Foxp3~+T cells and CD4~+CD25~+ T cells. The results of FCMshowed that, the percentage of the CD4~+CD25~+Foxp3~+ T cells andCD4~+CD25~+ T cells in the splenic monocyte in experimental group is(0.73±0.03)% and (1.34±0.11)%, and the control is (3.39±0.46)% and (3.79±0.59)%.3. The culture supernatant of Toxoplasma gondii decreased thesuppression function of CD4~+CD25~+ regulatory T cells.CD4~+CD25~+ regulatory T cells from normal mice treated by differentways have suppression function on the immunocytes to ConAstimulation. However, the culture supernatant of Toxoplasma gondiiattenuated the suppression function. At a serial treated time, 79%,30% and 15% inhibition were observed when the immune cells wereco-cultured with CD4~+CD25~+ regulatory T cells treated by theculture supernatant of Toxoplasma gondii, but 77%,61% and 57%inhibition occurred in control. 4. The culture supernatant of Toxoplasma gondii candownregulate the expression of CTLA-4 and GITR on CD4~+CD25~+ T cells. Compared with the RPMI-1640, the percentage ofCD4~+CD25~+CTLA-4~+ T and CD4~+CD25~+GITR~+ T in CD4~+CD25~+ T cells treated by the culture supernatant of Toxoplasma gondiidecreased. CTLA-4, which has a higher affinity for B7 than CD28,may activate CD4~+CD25~+ regulatory T cells to deliver a negativesingal to other immunocytes for activation and proliferation. Thus,the expression of gene CTLA-4 may help CD4~+CD25~+ regulatory Tcells to suppress the immune response. The mechanisms of GITRcontribute to the suppression of CD4~+CD25~+ regulatory T cells isunclear, but a lot of evidences suggested that GITR played animportant role in the suppression of CD4~+CD25~+ regulatory T cells.5. The culture supernatant of Toxoplasma gondii can downregulatethe mRNA expression of CTLA-4 on the CD4~+ T cells. Thedata clearly demonstrated that the gene CTLA-4 in the CD4~+ T cellstreated by the culture supernatant of Toxoplasma gondii isdownregulated compared with the control. Besides, a typical markersfor CD4~+CD25~+ regulatory T cells, CTLA-4 is a marker of activationmolecular, it's expression in CD4~+T cells may strongly emphasizethat the culture supernatant of the Toxoplasrna gondii candownregulate the gene CTLA-4 expression.6. The culture supernatant of Toxoplasma gondii can promote theapoptosis of CD4~+CD25~+ regulatory T cells. The Annexin-vpositive rate of CD4~+CD25~+ T cells as incubated with the culturesupematant of Toxoplasrna gondii for 10 hours increased (13.6±2.15)%, which is higher than that of the control cells as incubatedwith RPMI-1640.In summary, we first provide evidence about the relationshipbetween Toxoplasma gondii-caused abortion and CD4~+CD25~+ regulatoryT cells. The in vivo and in vitro experiments all indicate that the culture supernatant of Toxoplasma gondii can mediate the mice abortion bydecreasing the numbers of CD4~+CD25~+ regulatory T cells and inhibitingtheir suppressive ability. These observations illustrate how the culturesupernatant of Toxoplasma gondii can affect immune response and openimportant avenues for future research on the mechanism of abortioncaused by Toxoplasma gondii infection.