Trem2/PPARγ/STAT6 axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via decidual macrophages
Description
Figure 1. T. gondii inhibited the expressions of Trem2 and its downstream effectors. (A) Representative images of uteri and fetuses at G 17.5 in PBS and T. gondii-infected WT mice. (B) Flow cytometry was employed to identify the percentage of CD11b+ DMs in PBS and T. gondii-infected mouse placentas at G 17.5 and representative histograms were shown . (C) Flow cytometry was employed to assay the MFI of Trem2 on DMs in PBS and T. gondii-infected mouse placentas at G 17.5 and representative histograms were shown . (E-G) Representative IHC staining images of Trem2 (E), P-STAT6 (F), and PPARγ (G) in PBS and T. gondii-infected mouse placentas. Figure 2. T. gondii antigens inhibited the expressions of Trem2, P-STAT6, and PPARγ. Figure 2. (A) Trem2 expression was determined with Western blot. (B) Expressions of PPARγ, phosphorylated (P), and total STAT6 were assayed by Western blot. (C-D) P-STAT6 and PPARγ expression were measured by cellular immunofluorescence staining. Figure 3. The effect of Trem2 deficiency on its downstream pathway during T. gondii infection in mice. (A) Representative images of fetuses at G 17.5 from WT and Trem2-/- mice with or without T. gondii infection. Fetal development was assessed by fetal size (CRL×OF) and fetal weight (n = 5-7 biologically independent mice). (B) Flow cytometry was utilized to identify the percentage of CD11b+ DMs in WT and Trem2-/--infected mouse placenta and representative histograms were shown. (C) P-STAT6, STAT6, and PPARγ proteins were analyzed by Western blot in mouse placentas from Trem2-/- and WT-infected mice. (D-E) Expressions of P-STAT6, STAT6, and PPARγ were assayed by Western blot. Figure 4. The effect of Trem2 deficiency on its downstream pathway during TgAg stimulation in BMDMs. (A) BMDMs extracted from WT and Trem2-/- mice were experimented with following the protocol described in the schematic. The graphic was created with BioRender.com. (B-C) Expressions of P-STAT6, STAT6, PPARγ, and Trem2 in BMDMs were assayed by Western blot. (D) Confocal microscopy was employed to assay the expression of P-STAT6, PPARγ, and Trem2 in BMDMs from WT mice. Figure 5. T. gondii infection down-regulated Trem2 by affecting the PPARγ/STAT6 signaling pathway. (A) Western blot was utilized to evaluate the expression of PPARγ, P-STAT6, STAT6, and Trem2. (B) Expressions of PPARγ, P-STAT6, and STAT6 were measured by Western blot. Figure S1. P-STAT6 or STAT6 interacts with PPARγ. (A-B) Co-immunoprecipitation of STAT6 with PPARγ. HEK293T cells were transfected with flag-STAT6, myc-PPARγ, or flag-STAT6+myc-PPARγ, respectively. Whole-cell lysates were incubated with myc or flag antibodies, and the presence of flag-STAT6 (A) or myc-PPARγ (B) was analyzed by immunoblotting. (C) Co-immunoprecipitation of P-STAT6 with PPARγ. (D) Confocal microscopy of the expression of P-STAT6, PPARγ, and Trem2 in Raw264.7 cells treated with or without IL-4 (20 ng/ml) for 1 h.