背景:感染是人类生殖健康的主要威胁,因为它们会导致妊娠失败,包括复发性流产,死产,和早产。弓形虫(T.gondii)感染可通过影响某些免疫分子和细胞因子而导致不良妊娠结局。然而,弓形虫导致妊娠失败的详细机制尚不清楚.
方法:建立弓形虫感染的野生型(WT)妊娠小鼠和2B4敲除(2B4-/-)妊娠小鼠进行体内研究。培养人蜕膜自然杀伤(dNK)细胞进行体外研究。观察异常妊娠结局,和2B4的表达,功能分子(CD69,CD107a,肿瘤坏死因子α[TNF-α],干扰素γ[IFN-γ]),和信号分子(SHP-2,Fyn,p-ERK,通过流式细胞术检测dNK细胞中的p-P38),蛋白质印迹,逆转录聚合酶链反应(RT-PCR),和/或免疫荧光。通过NK-92细胞中的共免疫沉淀(co-IP)验证了直接相互作用(2B4与SHP-2和Fyn相互作用;SHP-2与p-P38和2B4相互作用;Fyn与p-ERK和2B4相互作用)。
结果:这里,结果表明,弓形虫感染后2B4显著下调。随后,与感染的WT妊娠小鼠相比,感染的2B4-/-妊娠小鼠表现出更差的妊娠结局。此外,在弓形虫感染期间,在2B4-/-妊娠小鼠中发现TNF-α和IFN-γ表达增加以及dNK细胞毒性升高。相比之下,在弓形虫感染期间2B4激活后,发现TNF-α和IFN-γ表达降低,人dNK细胞活性降低。有趣的是,结果表明,2B4与接头SHP-2或Fyn结合,然后在弓形虫感染期间触发不同的信号通路来调节dNK细胞中TNF-α和IFN-γ的表达。Further,SHP-2在2B4激活后直接结合2B4和p-P38,在NK-92细胞中产生TNF-α和IFN-γ的抑制信号。此外,在弓形虫感染后,Fyn可以与2B4和p-ERK结合,从而抑制NK-92细胞中TNF-α和IFN-γ的表达。
结论:这些数据表明,2B4可能是一种新的危险信号分子,与弓形虫感染期间妊娠失败有关。解开2B4调节dNK细胞活性的机制将提供新的见解,以帮助我们理解弓形虫诱导的不良妊娠结局。
BACKGROUND: Infections are a major threat to human reproductive health because they can induce pregnancy failure, including recurrent abortion, stillbirth, and preterm birth. Toxoplasma gondii (T. gondii) infection can result in adverse pregnancy outcomes by affecting certain immune molecules and cytokines. However, the detailed mechanisms behind T. gondii-induced pregnancy failure are poorly understood.
METHODS: Toxoplasma gondii-infected wild-type (WT) pregnant mice and 2B4 knockout (2B4-/-) pregnant mice were established for in vivo study. Human decidual natural killer (dNK) cells were cultured for in vitro study. Abnormal pregnancy outcomes were observed, and the expression of 2B4, functional molecules (CD69, CD107a, tumor necrosis factor alpha [TNF-α], interferon gamma [IFN-γ]), and signaling molecules (SHP-2, Fyn, p-ERK, p-P38) in dNK cells were detected by flow cytometry, Western blot, reverse transcriptase polymerase chain reaction (RT-PCR), and/or immunofluorescence. The direct interactions (2B4 interacts with SHP-2 and Fyn; SHP-2 interacts with p-P38 and 2B4; Fyn interacts with p-ERK and 2B4) were verified by co-immunoprecipitation (co-IP) in NK-92 cells.
RESULTS: Here, results showed that 2B4 was significantly downregulated after T. gondii infection. Subsequently, infected 2B4-/- pregnant mice displayed worse pregnancy outcomes compared with infected WT pregnant mice. Also, increased TNF-α and IFN-γ expression and elevated dNK cell cytotoxicity were found in 2B4-/- pregnant mice during T. gondii infection. In contrast, reduced TNF-α and IFN-γ expression and decreased human dNK cell activity were found following 2B4 activation during T. gondii infection. Interestingly, results showed that 2B4 binds to adaptor SHP-2 or Fyn, which then triggers different signaling pathways to regulate TNF-α and IFN-γ expression in dNK cells during T. gondii infection. Further, SHP-2 binds 2B4 and p-P38 directly after 2B4 activation, which generates an inhibitory signal for TNF-α and IFN-γ in NK-92 cells. In addition, Fyn can bind to 2B4 and p-ERK after activation of 2B4, thereby inhibiting TNF-α and IFN-γ expression in NK-92 cells following T. gondii infection.
CONCLUSIONS: These data suggest that 2B4 may be a novel danger-signaling molecule that is implicated in pregnancy failure during T. gondii infection. Unraveling the mechanism by which 2B4 regulates dNK cell activity will provide novel insights to aid our understanding of T. gondii-induced adverse pregnancy outcomes.