Trem2/PPARγ/STAT6 axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via decidual macrophages

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.

图1. 刚地弓形虫（Toxoplasma gondii, T. gondii）抑制髓系细胞触发受体2（Trem2）及其下游效应分子的表达。(A) 磷酸盐缓冲液（PBS）处理及刚地弓形虫感染的野生型（WT）小鼠在妊娠第17.5天（G 17.5）的子宫及胎仔代表性图像。(B) 采用流式细胞术（Flow cytometry）鉴定妊娠第17.5天PBS处理组与刚地弓形虫感染组小鼠胎盘中CD11b+ 蜕膜巨噬细胞（CD11b+ DMs）的占比，展示代表性直方图。(C) 采用流式细胞术检测妊娠第17.5天PBS处理组与刚地弓形虫感染组小鼠胎盘中CD11b+ 蜕膜巨噬细胞表面Trem2的平均荧光强度（MFI），展示代表性直方图。(E-G) PBS处理组与刚地弓形虫感染组小鼠胎盘中Trem2（E）、磷酸化STAT6（P-STAT6）及过氧化物酶体增殖物激活受体γ（PPARγ）（G）的免疫组化（Immunohistochemistry, IHC）染色代表性图像。图2. 刚地弓形虫抗原抑制Trem2、P-STAT6及PPARγ的表达。图2. (A) 采用蛋白质印迹（Western blot）检测Trem2的表达水平。(B) 采用蛋白质印迹检测PPARγ、磷酸化（P）及总STAT6的表达水平。(C-D) 采用细胞免疫荧光染色检测P-STAT6与PPARγ的表达水平。图3. 小鼠感染刚地弓形虫期间，Trem2缺失对其下游通路的影响。(A) 野生型（WT）及Trem2基因敲除（Trem2-/-）小鼠在感染或未感染刚地弓形虫时，妊娠第17.5天的胎仔代表性图像。胎儿发育情况通过胎体尺寸（CRL×OF）及胎儿体重进行评估（n=5~7只生物学重复小鼠）。(B) 采用流式细胞术鉴定野生型及Trem2基因敲除感染组小鼠胎盘中CD11b+ 蜕膜巨噬细胞的占比，展示代表性直方图。(C) 采用蛋白质印迹分析Trem2基因敲除及野生型感染组小鼠胎盘中P-STAT6、STAT6及PPARγ蛋白的表达水平。(D-E) 采用蛋白质印迹检测P-STAT6、STAT6及PPARγ的表达水平。图4. 骨髓源性巨噬细胞（Bone Marrow-Derived Macrophages, BMDMs）中，刚地弓形虫抗原（TgAg）刺激期间Trem2缺失对其下游通路的影响。(A) 从野生型及Trem2基因敲除小鼠中提取的骨髓源性巨噬细胞按照示意图中描述的实验流程进行处理，该示意图由BioRender.com平台制作。(B-C) 采用蛋白质印迹检测骨髓源性巨噬细胞中P-STAT6、STAT6、PPARγ及Trem2的表达水平。(D) 采用共聚焦显微镜（Confocal microscopy）检测野生型小鼠骨髓源性巨噬细胞中P-STAT6、PPARγ及Trem2的表达水平。图5. 刚地弓形虫感染通过影响PPARγ/STAT6信号通路下调Trem2的表达。(A) 采用蛋白质印迹评估PPARγ、P-STAT6、STAT6及Trem2的表达水平。(B) 采用蛋白质印迹检测PPARγ、P-STAT6及STAT6的表达水平。图S1. P-STAT6或STAT6与PPARγ存在相互作用。(A-B) STAT6与PPARγ的共免疫沉淀（Co-immunoprecipitation, Co-IP）实验。分别将flag-STAT6、myc-PPARγ或flag-STAT6+myc-PPARγ转染至人胚肾293T细胞（HEK293T）。以myc或flag抗体孵育全细胞裂解液，通过免疫印迹分析flag-STAT6（A）或myc-PPARγ（B）的存在情况。(C) P-STAT6与PPARγ的共免疫沉淀实验。(D) 经或不经20 ng/ml白细胞介素4（Interleukin 4, IL-4）处理1 h的RAW264.7细胞中，P-STAT6、PPARγ及Trem2表达情况的共聚焦显微镜成像。