doi: 10.1371/journal.ppat.1012543.
eCollection 2024 Sep.
Trem2/Syk/PI3K axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via modulating decidual macrophages
Qing Wang
1
, Yining Cao
1
, Songyi Ye
2
, Maoyuan Ding
1
, Wenliang Ge
2
, Yuejin Liang
3
, Jinling Chen
1
Affiliations
- PMID: 39250507
- PMCID: PMC11412541
- DOI: 10.1371/journal.ppat.1012543
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Trem2/Syk/PI3K axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via modulating decidual macrophages
Qing Wang et al.
PLoS Pathog.
.
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Abstract
Decidual macrophages residing at the maternal-fetal interface have been recognized as pivotal factors for maintaining normal pregnancy; however, they are also key target cells of Toxoplasma gondii (T. gondii) in the pathology of T. gondii-induced adverse pregnancy. Trem2, as a functional receptor on macrophage surface, recognizes and binds various kinds of pathogens. The role and underlying mechanism of Trem2 in T. gondii infection remain elusive. In the present study, we found that T. gondii infection downregulated Trem2 expression and that Trem2-/- mice exhibited more severe adverse pregnancy outcomes than wildtype mice. We also demonstrated that T. gondii infection resulted in increased decidual macrophages, which were significantly reduced in the Trem2-/- pregnant mouse model as compared to wildtype control animals. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. Concurrently, Trem2 deficiency in bone marrow-derived macrophages (BMDMs) heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells, accompanied by higher pro-inflammatory factors (IL-1β, IL-6 and TNF-α) but a lower chemokine (CXCL1) in T. gondii antigens-treated BMDMs. Furthermore, compelling evidence from animal models and in vitro cell experiments suggests that T. gondii inhibits the Trem2-Syk-PI3K signaling pathway, leading to impaired function of decidual macrophages. Therefore, our findings highlight Trem2 signaling as an essential pathway by which decidual macrophages respond to T. gondii infection, suggesting Trem2 as a crucial sensor of decidual macrophages and potential therapeutic target in the pathology of T. gondii-induced adverse pregnancy.
Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
(A) Representative image of the placentas and fetuses of wildtype mice at G 17.5 infected with T. gondii. Red arrow indicated fetal demise. (B and C) Fetal development assessed by fetal size (×ばつOF) and fetal weight. Each data points represent individual fetuses (n = 5–7 mice). CRL: crown-rump length; OF: occipito-frontal diameter. (D) Representative HE staining showing pathological features of mouse placentas. The normal mouse placental structure is composed of decidua zone (De), junctional zone (JZ), and labyrinth zone (LZ). Pathological necrosis in wildtype mice infected with T. gondii mainly occurs in the De. (E) Representative flow cytogram of CD11b+ F4/80+ decidual macrophages, comparing decidual macrophage proportions in normal or infected placentas in mice. The placentas of each mouse were divided into three groups for technically repeated experiments. Data point represents the placenta of a single pregnant mouse (n = 7–8 mice). (F) Representative MFI of Trem2 on the surface of CD11b+ F4/80+ decidual macrophages in normal or infected placentas in mice. The placentas of each mouse were divided into three groups for technically repeated experiments. Data point represents the placenta of a single pregnant mouse (n = 7–8 mice). MFI: mean fluorescence intensity. (G) Decidual macrophages were labeled with F4/80, and the colocalization between Trem2 (red) and F4/80 (green) was imaged by immunohistofluorescence. (H) Immunohistochemistry of normal and infected mouse placentas, which was immunostained with anti-Trem2 and counterstained with hematoxylin. (I) Immunoblot of Trem2 expression and statistical analysis on normal and infected mouse placentas. Each data point represents the placenta of an individual pregnant mouse (n = 8 mice). Data were presented as mean ± SD. Statistical analysis was conducted using two-tailed unpaired Student’s t-test (B, C, E, F and I). *: P < 0.05.
(A) Representative images of fetuses at G17.5 from wildtype and Trem2-/- pregnant mice with or without T. gondii infection. Fetal development was assessed by fetal size (×ばつOF) and fetal weight. Data point represents the placenta of a single pregnant mouse (n = 5–7 mice). (B) Representative HE staining shows the pathological characteristics of wildtype and Trem2-/- mouse placentas, with hemorrhage and necrosis lesions present in the De of the infected group. (C) Representative flow cytogram of CD11b+ F4/80+ decidual macrophages, comparing decidual macrophage proportions in wildtype and Trem2-/- mouse placentas with or without T. gondii infection. The placentas of each mouse were divided into three groups for technically repeated experiments. Data point represents the placenta of a single pregnant mouse (n = 6–8 mice). (D) T. gondii burden in the placentas of wildtype and Trem2-/- mice was measured by real-time PCR (n = 8 mice). (E) mRNA levels of IL-1β, IL-6, IL-10, IL-12, IFN-γ, TNF-α, TGF-β and G-CSF in the mouse placentas were assayed by real-time PCR (n = 8 mice). Data were presented as mean ± SD. Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons test (A, C, D and E). *: P < 0.05.
(A) Syk and PI3K protein levels in normal and infected mouse placentas were analyzed by immunoblot. Each data point represents the placenta of an individual pregnant mouse (n = 8 mice). (B) Immunohistochemistry of normal and infected mouse placentas, which were immunostained with anti-Syk antibody and counterstained with hematoxylin. (C) Syk and PI3K protein levels in wildtype and Trem2-/- pregnant mouse placentas with or without T. gondii infection were analyzed by immunoblot. Data point represents the placenta of a single pregnant mouse (n = 6 mice). Data were presented as mean ± SD. Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons (A and C). *: P < 0.05.
(A) RAW264.7 cells were stimulated with solvent control and TgAg for 48 h respectively. The expressions of Trem2, Syk and PI3K proteins were detected by immunoblot, and the statistical analysis was conducted by Image J. Data represent the results of three independent experiments. (B) Immunofluorescence images of the co-localization Trem2 (red) and PI3K (green) in RAW264.7 cells stimulated with either control or TgAg were shown. (C) RAW264.7 cells were treated with different stimuli (control, TgAg, HSP60 and TgAg+HSP60) for 48 h. The expressions of Trem2, Syk, and PI3K were shown, as analyzed by immunoblot and quantification. Data represent the results of four independent experiments. (D) The lentiviral control vector and Trem2 lentiviral overexpression vector were transfected into RAW264.7 cells, respectively. After 12 h of overexpression, TgAg was added for stimulation for 48 h. The expressions of Trem2, Syk, and PI3K were measured by Immunoblot and quantified by Image J. Data represent the results of six independent experiments. (E) RAW264.7 cells were transfected with the vector plasmid and Syk recombinant plasmid, respectively. After 12 h of overexpression, TgAg was added for stimulation for 48 h. The expressions of Trem2, Syk, and PI3K were measured by Immunoblot and quantified by Image J. Data represent the results of six independent experiments. (F) RAW264.7 cells were transfected with the vector plasmid and PI3K recombinant plasmid, respectively. After 12 h of overexpression, TgAg were added for stimulation for 48 h. The expressions of Trem2, Syk, and PI3K were measured by Immunoblot and quantified by Image J. Data represent the results of six independent experiments. (G and H) HEK-293T cells were transfected separately or co-transfected with myc-tagged Syk and flag-tagged p85 for 48 h. Cell lysates were immunoprecipitated with myc antibody (G) or flag antibody (H), and the immunoblots with myc and flag antibodies are shown. Data were presented as mean ± SD. Statistical analysis was conducted using two-tailed unpaired Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparisons test (C, D, E and F). *: P < 0.05. TgAg: T. gondii antigens.
(A) Schematic diagram of migration assay of HTR-8 cells co-cultured with M0-type macrophages in a transwell system. (B) Migration assays for detection of the migratory capacity of HTR-8 cells co-cultured with control or M0-type macrophages with or without TgAg treatment for 24 h. Statistical analysis was conducted on the number of HTR-8 cells. Data represent the results of three independent experiments (n = 5 fields of view / group). (C) Migration assays for detection of the migratory capacity of HTR-8 cells co-cultured with control or TgAg-pretreated M0-type macrophages with or without HSP60 stimulation for 24 h. Statistical analysis was conducted on the number of HTR-8 cells. Data represent the results of three independent experiments (n = 5 fields of view / group). (D) Schematic diagram of invasion assay of HTR-8 cells co-cultured with M0-type macrophages in a transwell system. (E) Matrigel invasion assays for detection of the invasive capacity of HTR-8 cells co-cultured with control or M0-type macrophages with or without TgAg treatment for 24 h. Statistical analysis was conducted on the number of HTR-8 cells. Data represent the results of three independent experiments (n = 5 fields of view / group). (F) Matrigel invasion assays for detection of the invasive capacity of HTR-8 cells co-cultured with control or TgAg-pretreated M0-type macrophages with or without HSP60 stimulation for 24 h. Statistical analysis was conducted on the number of HTR-8 cells. Data represent the results of three independent experiments (n = 5 fields of view / group). (G) Schematic diagram of proliferation assay of HTR-8 cells co-cultured with M0-type macrophages in a transwell system. (H) Flow cytometry for the evaluation of proliferation of CFSE-dyed HTR-8 cells, co-cultured with control or M0-type macrophages with or without TgAg treatment for 24 h. Statistical analysis was conducted on the MFI of HTR-8 cells. Data represent the results of three independent experiments. (I) Flow cytometry for the evaluation of proliferation of CFSE-dyed HTR-8 cells, co-cultured with control or TgAg-pretreated M0-type macrophages with or without HSP60 stimulation for 24 h. Statistical analysis was conducted on the MFI of HTR-8 cells. Data represent the results of three independent experiments. (J) Migration assays for detection of the migratory capacity of HTR-8 cells co-cultured with control or TgAg-pretreated M0-type macrophages transfected with siRNA-NC (si-NC) or siRNAs against Trem2 (si-Trem2) for 24 h. Statistical analysis was conducted on the number of HTR-8 cells. Data represent the results of three independent experiments (n = 5 fields of view / group). (K) Matrigel invasion assays for detection of the invasive capacity of HTR-8 cells co-cultured with control or TgAg-pretreated M0-type macrophages transfected with si-NC or si-Trem2 for 24 h. Statistical analysis was conducted on the number of HTR-8 cells. Data represent the results of three independent experiments (n = 5 fields of view / group). Data were presented as mean ± SD. Statistical analysis was conducted using one-way ANOVA Tukey’s multiple comparisons test (B, C, E, F, H, I, J, and K). *: P < 0.05. TgAg: T. gondii antigens. The schematic diagram is created with Biorender.com .
(A) Migration assays for detection of the migratory capacity of MPCTs co-cultured with control or TgAg-pretreated BMDMs from wildtype mice and Trem2-/- mice. Statistical analysis was conducted on the number of MPCTs. Data represent the results of three independent experiments (n = 5 fields of view / group). (B) Matrigel invasion assays for detection of the invasive capacity of MPCTs co-cultured with control or TgAg-pretreated BMDMs from wildtype mice and Trem2-/- mice. Statistical analysis was conducted on the number of MPCTs. Data represent the results of three independent experiments (n = 5 fields of view / group). (C) mRNA levels of IL-1β, IL-6, IL-10, IL-12, TNF-α, TGF-β, G-CSF, CXCL1 and CXCL5 in the BMDMs were assayed by real-time PCR. Data represent the results of three independent experiments. Data were presented as mean ± SD. Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons test (A, B and C). *: P < 0.05. TgAg: T. gondii antigens.
T. gondii infection results in adverse pregnancy outcomes in wildtype mice, paralleled by the down-regulation of Trem2 on the surface of decidual macrophages. Under normal pregnancy conditions, Trem2 signaling becomes activated and initiates the recruitment of downstream Syk and PI3K signaling, thereby activating the Trem2-Syk-PI3K pathway. However, T. gondii infection inhibits Trem2-Syk-PI3K pathway in decidual macrophages, leading to the impaired migration, invasion and proliferation of trophoblast cells. Furthermore, deficiency of Trem2 aggravates adverse pregnancy outcomes induced by T. gondii infection. The schematic diagram is created with Biorender.com .
References
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- Qiu J, Zhang R, Xie Y, Wang L, Ge K, Chen H, et al.. Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner. Frontiers in Immunology. 2018;9:1102. doi: 10.3389/fimmu.2018.01102 - DOI - PMC - PubMed
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