The Mechanism Of Toxoplasma Gondii Excreted-secreted Antigens On The Suppression Of Foxp3 Expression In Regulatory T Cells

Objective To investigate the effect of Toxoplasma gondii ESA on the number and function of Treg as well as the expression of Foxp3 and its upstream signaling molecules in Treg,and to study the role of Smad2/3/4 and JAK3/Stats signaling pathway on the expression of Foxp3.Methods 1.Female 6~8 week old and male 8~10 week old C57BL/6 mice were paired in the evening,and the next morning,confirmation of a vaginal plug was used to define day 0 of pregnancy(G0).Pregnant mice at gestational day 5(G5)were injected with T.gondii ESA of Chinese 1 strain,PBS,and RU486intraperitoneally.Then mice were euthanized at gestational day 18(G18)and the percentage of abortion was calculated.The structure of the placenta was observed by HE staining;The percentage of CD4~+CD25~+Foxp3~+T cells in mouse spleen cells was assessed by flow cytometry;The expression of Foxp3 protein in mouse placenta was detected by Western blot;The primary Treg was isolated from mice,and evaluated the function of CD4~+CD25~+Foxp3~+T cells in inhibiting lymphocyte proliferation(experimental group:PBS,ESA and RU486).2.EL4 cells were treated with the ESA.The expression of Foxp3 was detected with the methods of Western blot,qRT-PCR and immunofluorescence;The expression of Smad2,P-Smad2,Smad3,P-Smad3,Smad4,IL-2R?,JAK3,Stat3,P-Stat3,Stat5 and P-Stat5 were detected by Western blot;The expression of P-Smad3,P-Stat3 and P-Stat5 in EL4 cells was detected by immunofluorescence assay(experimental group:OVA,Control and ESA).3.Smad2 siRNA,expression plasmid of pcDNA3.1-Smad2,pcDNA3.1-Smad3,pcDNA3.1-Smad4,pcDNA3.1-Stat3,and pcDNA3.1-Stat5 were transfected into EL4 cells.The effects of ESA on the expression of Foxp3 and its upstream signal molecules were detected by Western blot(experimental group:OVA,Control,ESA,empty plasmid/nonspecific siRNA,empty plasmid/nonspecific siRNA+ESA,overexpression plasmid/specific siRNA,overexpression plasmid/specific siRNA+ESA).4.The T?RII agonist(TGF-?1),CD3,and CD28 were exposed to EL4 cells to increase the expression of T?RII.The expression of T?RII,Smad2,P-Smad2,Smad3,P-Smad3,Smad4 and Foxp3 in EL4 cells were detected by Western blot.Primary Treg were treated with the ESA in vitro.The mRNA expression levels of TGF?RII,Smad4 and Foxp3 were detected by realtime PCR;The expression of TGF-?1 in the culture supernatant of EL4 cells was detected by ELISA(experimental group:OVA,Control,ESA,TGF-?1,TGF-?1+ESA).The TGF-?neutralizing antibody were exposed to EL4 cells to block the expression of TGF-?1.The expression of TGF?RII was detected by Western blot(experimental group:OVA,Control,ESA,ESA+TGF-?1 neutralizing antibody,TGF-?1,TGF-?1+TGF-?1neutralizing antibody).5.The IL-2R?agonist(IL-2)were exposed to EL4 cells to increase the expression of IL-2R?.The expression of IL-2R?,JAK3,Stat3,P-Stat3,Stat5,P-Stat5 and Foxp3 in EL4 cells were detected by Western blot(experimental group:OVA,Control,ESA,IL-2,IL-2+ESA).The expression of IL-2 in the culture supernatant of EL4 cells was detected by ELISA(experimental group:OVA,Control,ESA,IL-2,IL-2+ESA).The IL-2R?neutralizing antibody were exposed to EL4 cells to block the expression of IL-2.The expression of IL-2R?was detected by Western blot(experimental group:OVA,Control,ESA,ESA+IL-2R?neutralizing antibody,IL-2,IL-2+IL-2R?neutralizing antibody).Results 1.ESA of Chinese 1 strain of Toxoplasma gondii led to abortion and placental structural changes in early pregnancy,due to the decrease in the number and the suppressive activity of regulatory T cells.2.ESA could negatively moderate Foxp3 via the suppression of P-Smad2,P-Smad3,and Smad4.Over expression of Smad2,Smad3 and Smad4 partially abrogated the inhibitory effect of ESA on Foxp3 expression,whereas Smad2 siRNA cooperated with ESA to further suppress the level of Foxp3.3.ESA elicited an inhibitory effect on the expression of Foxp3 by directly inhibiting the expression of TGF?RII rather than TGF-?1.TGF?RII agonist could abrogate the ESA-induced reduction in Foxp3.Neutralization of TGF-?1 cooperated with ESA further reduced TGF?RII expression.4.ESA elicited an inhibitory effect on the expression of IL-2R?.Then,ESA could negatively moderate Foxp3 via the suppression of JAK3,P-Stat3,and P-Stat5.Overexpression of Stat3and Stat5 partially abrogated the inhibitory effect of excreted-secreted antigens on Foxp3 expression.TGF?RII agonist could abrogate the ESA-induced reduction in Foxp3,whereas neutralization of IL-2R?cooperated with ESA to suppress the level of IL-2R?.Conclusions 1.ESA of Chinese 1 strain of Toxoplasma gondii led to the decrease in the number and the suppressive activity of regulatory T cells.2.ESA of Chinese 1 strain of Toxoplasma gondii inhibited the expression of Foxp3 via Smad2/3/4 signaling pathway.3.ESA of Chinese 1 strain of Toxoplasma gondii inhibited the expression of Foxp3 via JAK3/Stats signaling pathway.