A novel 2B4 receptor leads to worse pregnancy outcomes by facilitating TNF-α and IFN-γ production in dNK cells during Toxoplasma gondii infection

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-alpha], interferon gamma [IFN-gamma]), 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-alpha and IFN-gamma expression and elevated dNK cell cytotoxicity were found in 2B4(-/-) pregnant mice during T. gondii infection. In contrast, reduced TNF-alpha and IFN-gamma 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-alpha and IFN-gamma 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-alpha and IFN-gamma in NK-92 cells. In addition, Fyn can bind to 2B4 and p-ERK after activation of 2B4, thereby inhibiting TNF-alpha and IFN-gamma 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.