Department of Pathogen Biology, School of Medicine, Nantong University, 19 Qixiu Road, Nantong, Jiangsu, 226001, People's Republic of China.
ZhenJiang Provincial Blood Center, Zhenjiang, Jiangsu, 212000, People's Republic of China.
BMC Infect Dis. 2024 May 13;24(1):490. doi: 10.1186/s12879-024-09375-0.
Toxoplasma gondii (T. gondii) is capable of infecting nearly all warm-blooded animals and approximately 30% of the global population. Though most infections are subclinical in immunocompetent individuals, congenital contraction can lead to severe consequences such as spontaneous abortion, stillbirth, and a range of cranio-cerebral and/or ocular abnormalities. Previous studies reported that T. gondii-infected pregnancy mice unveiled a deficit in both the amount and suppressive functions of regulatory T (Treg) cells, accompanied with reduced levels of forkhead box p3 (Foxp3). Recently, accumulative studies have demonstrated that microRNAs (miRNAs) are, to some extent, relevant to T. gondii infection. However, the link between alterations in miRNAs and downregulation of Foxp3 triggered by T. gondii has been only sporadically studied.
Quantitative reverse transcription polymerase chain reaction (RT-qPCR), protein blotting and immunofluorescence were employed to evaluate the impact of T. gondii infection and antigens on miRNA transcription and Foxp3 expression. Dual-luciferase reporter gene assays were performed to examine the fluorescence activity in EL4 cells, which were transfected with recombinant plasmids containing full-length/truncated/mutant microRNA-142a-3p (miR-142a) promoter sequence or wild type/mutant of Foxp3 3' untranslated region (3' UTR).
We found a pronounced increase in miR-142a transcription, concurrent with a decrease in Foxp3 expression in T. gondii-infected mouse placental tissue. Similarly, comparable findings have been experimentally confirmed through the treatment of EL4 cells with T. gondii antigens (TgAg) in vitro. Simultaneously, miR-142a mimics attenuated Foxp3 expression, whereas its inhibitors markedly augmented Foxp3 expression. miR-142a promoter activity was elevated upon the stimulation of T. gondii antigens, which mitigated co-transfection of mutant miR-142a promoter lacking P53 target sites. miR-142a mimics deceased the fluorescence activity of Foxp3 3' untranslated region (3' UTR), but it did not affect the fluorescence activity upon the co-transfection of mutant Foxp3 3' UTR lacking miR-142a target site.
In both in vivo and in vitro studies, a negative correlation was discovered between Foxp3 expression and miR-142a transcription. TgAg enhanced miR-142a promoter activity to facilitate miR-142a transcription through a P53-dependent mechanism. Furthermore, miR-142a directly targeted Foxp3 3' UTR, resulting in the downregulation of Foxp3 expression. Therefore, harnessing miR-142a may be a possible therapeutic approach for adverse pregnancy caused by immune imbalances, particularly those induced by T. gondii infection.
刚地弓形虫（T. gondii）能够感染几乎所有温血动物，大约全球 30%的人口受到感染。尽管大多数感染在免疫功能正常的个体中呈亚临床状态，但先天性感染可能导致严重后果，如自发性流产、死产以及一系列颅面和/或眼部异常。既往研究报道，感染 T. gondii 的妊娠小鼠的调节性 T（Treg）细胞数量和抑制功能均出现缺陷，同时 Foxp3 水平降低。最近，越来越多的研究表明，微小 RNA（miRNA）在一定程度上与 T. gondii 感染相关。然而，T. gondii 感染导致的 miRNA 改变与 Foxp3 下调之间的联系仅得到了零星研究。
采用定量逆转录聚合酶链反应（RT-qPCR）、蛋白质印迹和免疫荧光技术评估 T. gondii 感染和抗原对 miRNA 转录和 Foxp3 表达的影响。通过双荧光素酶报告基因检测，转染含有全长/截短/突变 microRNA-142a-3p（miR-142a）启动子序列或野生型/突变型 Foxp3 3'非翻译区（3'UTR）的重组质粒的 EL4 细胞，检测荧光活性。
我们发现 T. gondii 感染小鼠胎盘组织中 miR-142a 转录显著增加，同时 Foxp3 表达降低。同样，体外用 T. gondii 抗原（TgAg）处理 EL4 细胞也得到了类似的发现。同时，miR-142a 模拟物降低 Foxp3 表达，而其抑制剂则显著增加 Foxp3 表达。T. gondii 抗原刺激后 miR-142a 启动子活性升高，同时共转染缺失 P53 靶位的突变 miR-142a 启动子时则减轻了该作用。miR-142a 模拟物降低了 Foxp3 3'UTR 的荧光活性，但共转染缺失 miR-142a 靶位的突变 Foxp3 3'UTR 时则不影响其荧光活性。
在体内和体外研究中，均发现 Foxp3 表达与 miR-142a 转录之间呈负相关。TgAg 通过 P53 依赖性机制增强 miR-142a 启动子活性，促进 miR-142a 转录。此外，miR-142a 直接靶向 Foxp3 3'UTR，导致 Foxp3 表达下调。因此，利用 miR-142a 可能是治疗由免疫失衡引起的不良妊娠的一种可行方法，尤其是由 T. gondii 感染引起的免疫失衡。
