Data_Sheet_1_The transcriptome landscape of 3D-cultured placental trophoblasts reveals activation of TLR2 and TLR3/7 in response to low Trypanosoma cruzi parasite exposure.PDF

Vertical transmission of Trypanosoma cruzi (T. cruzi) become a globalized health problem accounting for 22% of new cases of Chagas disease (CD). Congenital infection is now considered the main route of CD spread in non-endemic countries where no routine disease testing of pregnant women is implemented. The main mechanisms that lead to fetal infection by T. cruzi remain poorly understood. Mother-to-child transmission may occur when bloodstream trypomastigotes interact with the syncytiotrophoblasts (SYNs) that cover the placenta chorionic villi. These highly specialized cells function as a physical barrier and modulate immune responses against pathogen infections. To model the human placenta environment, we have previously used a three-dimensional (3D) cell culture system of SYNs that exhibits differentiation characteristics comparable to placental trophoblasts. Further, we have shown that 3D-grown SYNs are highly resistant to T. cruzi infection. In this work, we used RNA sequencing and whole transcriptome analysis to explore the immunological signatures that drive SYNs’ infection control. We found that the largest category of differentially expressed genes (DEGs) are associated with inflammation and innate immunity functions. Quantitative RT-PCR evaluation of selected DEGs, together with detection of cytokines and chemokines in SYNs culture supernatants, confirmed the transcriptome data. Several genes implicated in the Toll-like receptors signaling pathways were upregulated in 3D-grown SYNs. In fact, TLR2 blockade and TLR3/7 knockdown stimulated T. cruzi growth, suggesting that these molecules play a significant role in the host cell response to infection. Ingenuity Pathway Analysis of DEGs predicted the activation of canonical pathways such as S100 protein family, pathogen induced cytokine storm, wound healing, HIF1α signaling and phagosome formation after T. cruzi exposure. Our findings indicate that SYNs resist infection by eliciting a constitutive pro-inflammatory response and modulating multiple defense mechanisms that interfere with the parasite’s intracellular life cycle, contributing to parasite killing and infection control.

克氏锥虫（Trypanosoma cruzi, T. cruzi）的垂直传播已成为全球化公共卫生问题，约占恰加斯病（Chagas disease, CD）新增病例的22%。在未对孕妇开展常规疾病筛查的非流行国家，先天性感染现已被视为恰加斯病传播的主要途径。目前，克氏锥虫导致胎儿感染的核心机制仍不甚明晰。当血液中的锥鞭毛体与覆盖胎盘绒毛膜绒毛的合体滋养层细胞（syncytiotrophoblasts, SYNs）相互作用时，可能会发生母婴传播。这类高度特化的细胞既是物理屏障，亦可调控针对病原体感染的免疫应答。为构建人类胎盘环境的体外模型，本团队此前曾采用合体滋养层细胞的三维（3D）细胞培养体系，该体系的分化特征与胎盘滋养层细胞高度相似。后续研究证实，三维培养的合体滋养层细胞对克氏锥虫感染具有极强的抵抗能力。本研究中，我们借助RNA测序与全转录组分析，探究了调控合体滋养层细胞感染控制的免疫特征。研究发现，差异表达基因（differentially expressed genes, DEGs）中占比最高的类别与炎症及固有免疫功能密切相关。对筛选出的差异表达基因进行定量逆转录聚合酶链反应（quantitative RT-PCR, qRT-PCR）验证，并检测合体滋养层细胞培养上清中的细胞因子与趋化因子，结果证实了转录组分析的数据可靠性。在三维培养的合体滋养层细胞中，多条与Toll样受体（Toll-like receptors, TLRs）信号通路相关的基因表达上调。实际上，阻断Toll样受体2（TLR2）并敲低Toll样受体3/7（TLR3/7）后，克氏锥虫的增殖能力增强，提示这些分子在宿主细胞的抗感染应答中发挥重要作用。对差异表达基因进行Ingenuity通路分析（Ingenuity Pathway Analysis）后，预测得到克氏锥虫暴露后激活的经典通路包括S100蛋白家族通路、病原体诱导的细胞因子风暴、伤口愈合、缺氧诱导因子1α（HIF1α）信号通路以及吞噬体形成。本研究结果表明，合体滋养层细胞通过触发组成型促炎应答，并调控多种干扰寄生虫细胞内生命周期的防御机制，从而抵抗克氏锥虫感染，这有助于清除寄生虫并控制感染进程。