METHODS: Pregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9-/- pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting.
RESULTS: Toxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy.
CONCLUSIONS: Toxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.
方法:记录弓形虫感染的C57BL/6和Lgals9-/-妊娠小鼠模型的妊娠结局。Gal-9,c-Jun氨基末端激酶(JNK)的表达,磷酸化JNK(p-JNK),用蛋白质印迹法检测叉头盒蛋白O1(FOXO1),流式细胞术或免疫荧光。通过染色质免疫沉淀-聚合酶链反应(ChIP-PCR)确定FOXO1与Lgals9启动子的结合。细胞外信号调节激酶(ERK)的表达,磷酸化ERK(p-ERK),cAMP反应元件结合蛋白(CREB),磷酸化CREB(p-CREB),在T细胞中表达的T-box(T-bet),白细胞介素10(IL-10),用蛋白质印迹法测定dNK细胞中的干扰素γ(IFN-γ)。
结果:弓形虫感染增加了dMφ中p-JNK和FOXO1的表达,由于FOXO1与Lgals9启动子的结合升高,导致Gal-9减少。Gal-9的下调增强了ERK的磷酸化,抑制dNK细胞中p-CREB和IL-10的表达,促进T-bet和IFN-γ的表达。在小鼠模型中,Lgals9基因敲除加重了妊娠期间弓形虫感染引起的不良妊娠结局.
结论:弓形虫感染通过激活JNK/FOXO1信号通路抑制dMφ中Gal-9的表达,Gal-9的减少通过Gal-9/Tim-3相互作用导致dNK功能障碍。本研究为弓形虫不良妊娠结局的分子机制提供了新的见解。