Deciphering the preeclampsia-specific immune microenvironment and the role of pro-inflammatory macrophages at the maternal-fetal interface

Preeclampsia (PE), a major cause of maternal and perinatal mortality with highly heterogeneous causes and symptoms, is usually complicated by gestational diabetes mellitus (GDM). However, a comprehensive understanding of the immune microenvironment in the placenta of PE and the differences between PE and GDM is still lacking. In this study, Cytometry by time of flight (CyTOF) indicated that the frequencies of memory-like Th17 cells (CD45RA-CCR7+IL-17A+CD4+), memory-like CD8+ T cells (CD38+CXCR3-CCR7+Helios-CD127-CD8+) and pro-inflam Macs (CD206-CD163-CD38midCD107alowCD86midHLA-DRmidCD14+) were increased, while the frequencies of anti-inflam Macs (CD206+CD163-CD86midCD33+HLA-DR+CD14+) and granulocyte myeloid-derived suppressor cells (gMDSCs, CD11b+CD15hiHLA-DRlow) were decreased in the placenta of PE compared with that of NP, but not in that of GDM or GDM&PE. The pro-inflam Macs were positively correlated with memory-like Th17 cells and memory-like CD8+ T cells but negatively correlated with gMDSCs. Single-cell RNA sequencing revealed that transferring the F4/80+CD206- pro-inflam Macs with a Folr2+Ccl7+Ccl8+C1qa+C1qb+C1qc+ phenotype from the uterus of PE mice to normal pregnant mice induced the production of memory-like IL-17a+Rora+Il1r1+TNF+Cxcr6+S100a4+CD44+ Th17 cells via IGF1-IGF1R, which contributed to the development and recurrence of PE. Pro-inflam Macs also induced the production of memory-like CD8+ T cells but inhibited the production of Ly6g+S100a8+S100a9+Retnlg+Wfdc21+ gMDSCs at the maternal-fetal interface, leading to PE-like symptoms in mice. In conclusion, this study revealed the PE-specific immune cell network, which was regulated by pro-inflam Macs, providing new ideas about the pathogenesis of PE.

Methods
CyTOF analysis of placental-derived CD45+ immune cells from normal pregnancy (NP), preeclampsia (PE), gestational diabetes mellitus (GDM), and GDM complicated with PE (GDM_PE).

RNA transcriptome analysis of CD45+F4/80+CD206- pro-inflammatory macrophages (Macs) and CD45+F4/80+CD206+ anti-inflammatory Macs isolated from the uterus and placentas of mice with reduced uterine perfusion pressure (RUPP).

single-cell RNA transcriptome analysis of CD45+ immune cells in the uterus and placenta from mice injected with pro-inflammatory Macs or anti-inflammatory Macs isolated from the uterus and placentas of RUPP mice.

子痫前期（Preeclampsia, PE）是孕产妇及围产儿死亡的主要诱因之一，其病因与症状具有高度异质性，且常合并妊娠糖尿病（gestational diabetes mellitus, GDM）。但目前学界对子痫前期胎盘的免疫微环境，以及子痫前期与妊娠糖尿病的免疫差异仍缺乏全面认知。
本研究通过飞行时间流式细胞术（Cytometry by time of flight, CyTOF）分析发现：与正常妊娠（normal pregnancy, NP）胎盘组织相比，子痫前期胎盘中记忆样Th17细胞（CD45RA-CCR7+IL-17A+CD4+）、记忆样CD8+T细胞（CD38+CXCR3-CCR7+Helios-CD127-CD8+）及促炎巨噬细胞（CD206-CD163-CD38midCD107alowCD86midHLA-DRmidCD14+）的占比显著升高；而抗炎巨噬细胞（CD206+CD163-CD86midCD33+HLA-DR+CD14+）与粒细胞髓系来源抑制细胞（granulocyte myeloid-derived suppressor cells, gMDSCs, CD11b+CD15hiHLA-DRlow）的占比显著降低。上述变化仅见于子痫前期胎盘，在妊娠糖尿病或GDM_PE胎盘组织中未观察到。
促炎巨噬细胞与记忆样Th17细胞、记忆样CD8+T细胞呈正相关，而与粒细胞髓系来源抑制细胞呈负相关。单细胞RNA测序结果显示，从子痫前期小鼠子宫中分离获得的F4/80+CD206-表型促炎巨噬细胞，其特征为高表达Folr2、Ccl7、Ccl8、C1qa、C1qb与C1qc；将这类细胞移植至正常妊娠小鼠体内后，可通过IGF1-IGF1R通路诱导产生记忆样IL-17a+Rora+Il1r1+TNF+Cxcr6+S100a4+CD44+ Th17细胞，进而参与子痫前期的发生与复发。此外，促炎巨噬细胞还可诱导记忆样CD8+T细胞生成，并抑制母胎界面处Ly6g+S100a8+S100a9+Retnlg+Wfdc21+ 粒细胞髓系来源抑制细胞的产生，最终诱导小鼠出现子痫前期样症状。
综上，本研究揭示了由促炎巨噬细胞调控的子痫前期特异性免疫细胞网络，为阐明子痫前期的发病机制提供了全新的研究思路。

## 研究方法
1. 飞行时间流式细胞术分析：采集正常妊娠（NP）、子痫前期（PE）、妊娠糖尿病（GDM）及合并子痫前期的妊娠糖尿病（GDM_PE）患者的胎盘来源CD45+免疫细胞进行检测。
2. RNA转录组分析：对子宫灌注压降低（reduced uterine perfusion pressure, RUPP）模型小鼠的子宫及胎盘组织中分离得到的CD45+F4/80+CD206-促炎巨噬细胞与CD45+F4/80+CD206+抗炎巨噬细胞进行转录组测序分析。
3. 单细胞RNA转录组分析：将从子宫灌注压降低模型小鼠子宫及胎盘组织中分离的促炎巨噬细胞或抗炎巨噬细胞注射至正常小鼠体内，随后对受体小鼠子宫及胎盘组织中的CD45+免疫细胞进行单细胞RNA转录组测序分析。