ATF3-mediated inhibition of Trem2 by Toxoplasma gondii contributes to adverse pregnancy outcomes

Abstract

Background: Approximately one in three people worldwide have been exposed to Toxoplasma gondii (T. gondii). Primary infection with T. gondii during pregnancy can cause severe complications. Our previous study demonstrated that deficiency of triggering receptor expressed on myeloid cells 2 (Trem2) exacerbates pregnancy-related complications in T. gondii-infected mice. However, understanding the mechanisms by which T. gondii modulates Trem2 expression in macrophages remains an unmet challenge.

Methods: A mouse pregnancy model of T. gondii infection and an in vitro cellular stimulation model using soluble T. gondii antigens (sTgAg) were used to assess Trem2 expression. Recombinant plasmids containing the full-length Trem2 promoter were constructed to evaluate the effect of sTgAg on promoter activity, followed by the construction of truncated promoter plasmids to identify key regulatory regions. Transcription factors potentially binding to the Trem2 promoter were predicted using PROMO and JASPAR, with ATF3 identified as responsive to sTgAg stimulation via western blot analysis. The binding of ATF3 to the Trem2 promoter was validated by chromatin immunoprecipitation (ChIP) assays. Finally, ATF3 knockdown experiments were performed to determine its role in mediating the inhibitory effect of sTgAg on Trem2 expression.

Results: T. gondii significantly suppressed Trem2 expression in both mouse placentas and cellular models, with truncated promoter assays identifying key regulatory regions of the Trem2 promoter inhibited by sTgAg. ATF3 was identified as a transcription factor responsive to sTgAg stimulation, which bound to the Trem2 promoter. Importantly, knockdown of ATF3 restored Trem2 expression, demonstrating its critical role in mediating the inhibitory effect of sTgAg.

Conclusions: We identified that sTgAg may target and inhibit Trem2 expression through the transcription factor ATF3, and inhibition of ATF3 activity may help maintain Trem2 expression in macrophages, providing a potential therapeutic approach to avert negative effects on pregnancy related to T. gondii infection.

Keywords: ATF3; Toxoplasma gondii; Trem2 promoter; Adverse pregnancy outcomes; Macrophages.

Fig. 1

Soluble T. gondii antigens inhibit Trem2 expression in vitro. A Trem2 expression in the placentas of T. gondii-infected and normal pregnancy (PBS) mice were assessed using western blotting. Each data point corresponds to the placenta isolated from one pregnant mouse (n = 6 mice). B The expression of Trem2 was assessed utilizing western blotting after Raw264.7 cells were exposed to sTgAg (5 µg/ml) for 48 h. The analysis results were obtained using Image J. C The mRNA level of Trem2 in Raw264.7 cells was assessed using real-time quantitative PCR following the stimulation of Raw264.7 cells with 5 µg/ml of sTgAg for 24 h, using GAPDH as an internal control. D Immunostaining of Trem2 was performed in Raw264.7 cells exposed to sTgAg (5 µg/ml) for 48 h. Scale bars, 20 µm. Two-tailed unpaired Student’s t-tests used for all data; n = 3 independent experiments; mean ± SD; sTgAg, soluble T. gondii antigens; CON, the untreated control group. Quantifications normalized to GAPDH for western blot

Fig. 2

The activity of the Trem2 promoter is inhibited by soluble T. gondii antigens. A The luciferase reporter gene vectors incorporating the entire length of Trem2 promoter (p-Trem2), along with the pRL-TK, were co-transfected into Raw264.7 cells for 24 h. After Raw264.7 cells were exposed to 5 µg/ml of sTgAg for 24 h, the luciferase activity was assessed. B Schematic plasmid construction containing truncated fragments of the Trem2 promoter, designated as p-Trem2-A (−1542 to +200), p-Trem2-B (−851 to +200), and p-Trem2-C (−448 to +200). C Luciferase activity was assessed at 24 h post-transfection after co-transfecting the recombinant plasmids with pRL-TK into Raw264.7 cells. D The recombinant plasmids and pRL-TK were co-transfected into Raw264.7 cells for 24 h, which were then exposed to 5 µg/ml of sTgAg, and luciferase activity was assessed at 24 h post-stimulation. (mean ± SD; n = 3 independent experiments; two-way ANOVA with Sidak’s multiple comparisons test (A, D); one-way ANOVA with Tukey’s multiple comparisons test (C). sTgAg, soluble T. gondii antigens; CON, the untreated control group

Fig. 3

ATF3 binds to the −1542 to −851 region of the Trem2 promoter. A Raw264.7 cells were exposed to 5 µg/ml of sTgAg for 48 h and screened by western blotting to identify transcription factors that can bind to the Trem2 promoter. Analysis was performed using Image J. B Immunostaining and quantification of ATF3 (red) was conducted following stimulation of Raw264.7 cells with 5 µg/ml of sTgAg for 48 h. Scale bar: 20 μm. C ChIP analysis was assayed to confirm that the putative ATF3 transcription factor can bind to the Trem2 promoter. RPL30 Primer served as a positive control group. Data are shown as the mean ± SD. A two-tailed unpaired Student’s t-test was used (A, B); n = 3 independent experiments; sTgAg, soluble T. gondii antigens; CON, the untreated control group. Quantifications normalized to GAPDH for western blot analysis

Fig. 4

Soluble T. gondii antigens repress Trem2 promoter activity in an ATF3-dependent manner. A ATF3 was knocked down by transfecting Raw264.7 cells with small interfering ATF3 (20 nM) and the negative control siNC (20 nM). After 6 h of transfection, cells were exposed to sTgAg at 5 µg/ml for 48 h. Trem2 expression was assessed using western blotting, with analysis performed with Image J. B The luciferase reporter gene vector containing the Trem2 promoter (p-Trem2) was co-transfected into Raw264.7 cells along with pRL-TK after the transfection with siATF3 (20 nM) or siNC (20 nM) in Raw264.7 cells. Following 6 h of transfection, luciferase activity in Raw264.7 cells was measured after 24 h of incubation with 5 µg/ml of sTgAg. (C) Schematic representation of ATF3 binding site-specific mutations at −1364 to −1354 and −1134 to −1124 in the Trem2 promoter sequence are shown. The p-Trem2-A (−1542 to +200) and p-Trem2-A-MUT luciferase reporter gene vectors were transfected into Raw264.7 cells for 24 h, which were subsequently exposed to 5 µg/ml of sTgAg, and luciferase activity was assessed after 24 h post-stimulation. A two-way ANOVA with Sidak’s multiple comparisons test was used (A, B, and C); n = 3 independent experiments; mean ± SD; TSS, transcription start site; sTgAg, soluble T. gondii antigens; CON, the untreated control group. Quantifications normalized to GAPDH for western blot analysis