Toll-like receptor 4 (TLR4) deficiency impedes Toxoplasma gondii excreted-secreted antigens (ESA)-induced abortion

Toxoplasma gondii is an opportunistic intracellular parasite that is a major pathogenic factor in miscarriage, especially when it occurs early in pregnancy. We have previously demonstrated that the regulation of forkhead box transcription factor (Foxp3) is associated with abortion in early pregnancy caused by excretory-secretory antigen (ESA) of strain China 1. We aimed to reveal the underlying mechanism of miscarriage caused by ESA. A TLR4-/- pregnant mouse model was successfully constructed. Pregnant mice at gestational day 5 (G5) were injected with ESA. All animals were sacrificed on G13, pregnancy outcomes were observed, and abortion rates were calculated. Placental status observed by Hematoxylin-eosin staining; gene expression was measured by IHC; flow cytometry analysis was used to determine the number and function of regulatory T cells. In EL4 cells, real-time PCR and Western blot were used to evaluate gene expression and cytokines assay. In vivo studies revealed that ESA injection caused 83% abortion in pregnant mice but only 35% abortion in TLR4-/- pregnant mice. In addition, ESA attenuated the number and function of regulatory T cells, further suppressed Foxp3, FOXO1 levels, and upregulated CD127 expression. TLR4-/- mice partially reversed this inhibitory effect on regulatory T cells. Furthermore, in vitro studies revealed that ESA inhibited TLR4/ NF-κB signaling pathway expression and that TLR4 agonists significantly restored the ESA-induced decrease in Foxp3. These findings suggest that ESA suppresses Foxp3 expression by blocking TLR4/NF-κB signaling, resulting in miscarriage. More importantly, the results indicated that miscarriage caused by ESA is TLR4 dependent.