TREM-2在日本血吸虫病巨噬细胞M2型极化中的作用及机制研究

The polarization of alternatively activated macrophages (M2 macrophages) is critical for granuloma formation and tissue repair of schistosomiasis japonicum. However, it remains unclear whether triggering receptor-2 expressed on myeloid cells (TREM-2) is implicated in regulating M2 polarization of macrophages during the infection of Schistosoma japonicum. In our previous study, we found that macrophages were typically skewed from M1 towards the M2 polarization, accompanied with the enhanced expression of TREM-2 in macrophages during Schistosoma japonicum infection. Moreover, the soluble egg antigens induced the upregulation of the expression of TREM-2 in vitro. It is speculated that the soluble egg antigens may upregulate TREM-2, and thereby induce M2 polarization via one or some signal pathways. In order to explore its role and the underlying mechanism, Trem-2-/- mouse model of schistosomiasis and macrophages lines are used. We also study the role and the underlying mechanism of TREM-2 on soluble egg antigens-induced M2 polarization of macrophages by Flow cytometry, reverse transcriptase (RT)-qPCR, Western blotting, Immunofluorescence, CRISPR/Cas9, RNA interference, ChIP, EMSA and so on. Desired outcomes not only go step further to deepen the understanding of the cause of M2 polarization of macrophages, but also provide the potential target that could prevent liver fibrosis in schistosomiasis japonicum.

巨噬细胞M2型极化在日本血吸虫病肉芽肿形成和组织修复过程中发挥着重要作用，但髓系细胞触发受体-2(TREM-2)是否调控其巨噬细胞M2型极化尚未清楚。本项目前期实验结果显示在日本血吸虫感染中随着巨噬细胞M1型优势向M2型优势偏移，巨噬细胞上TREM-2表达相应增加；SEA可诱导TREM-2表达上调。据此推测：SEA可能通过某种或某些信号通路上调TREM-2，诱导巨噬细胞向M2型极化。为了探明其机制，本项目将运用Trem-2-/-小鼠血吸虫病模型和巨噬细胞株，通过流式细胞术、RT-qPCR、Western blotting、免疫荧光、CRISPR-Cas9技术、RNA干扰、ChIP、EMSA等技术深入探讨TREM-2在SEA诱导巨噬细胞M2型极化中的作用及分子机制。预期结果将深化对日本血吸虫感染引起的巨噬细胞M2型极化成因的认识，为日本血吸虫病肝纤维化防治提供新的潜在靶点。

在血吸虫感染过程中，巨噬细胞可通过不同的表型调控血吸虫感染引起的炎症反应、肉芽肿形成及纤维化的发生发展。M2型巨噬细胞在血吸虫感染中重要作用之一是抑制急性期过度的Th1型免疫应答，对小鼠度过急性期至关重要。而髓系细胞触发受体-2（TREM-2）是否参与调控巨噬细胞M2型极化尚不清楚。.本项目运用Trem-2-/-小鼠血吸虫感染模型和巨噬细胞株，通过体内外实验，深入研究了TREM-2在血吸虫可溶性虫卵抗原（SEA）诱导巨噬细胞M2型极化中的作用及分子机制。结果显示，在日本血吸虫感染中随着巨噬细胞M1型优势向M2型优势偏移，巨噬细胞上TREM-2表达相应增加，TREM-2与巨噬细胞M2极化具有相关性；SEA可诱导TREM-2表达上调，TREM-2具有促进巨噬细胞M2极化，促进肝纤维化的作用；SEA通过IL-10/TREM-2信号通路促进巨噬细胞M2型极化；血吸虫可溶性成虫抗原（SWA）通过诱导TREM-1表达增加，促进巨噬细胞M1型极化。脂质运载蛋白2（Lipocalin 2）参与了SWA诱导巨噬细胞M1极化。日本血吸虫重组P40蛋白（rSjP40）通过促进YB1核易位和诱导BMP-7/Smad1/5/8通路抑制肝星状细胞的活化；rSjP40通过Ets-1抑制肝星状细胞中胶原表达；重组Y-box结合蛋白-1（rSJYB1）可下调α1胶原启动子活性抑制肝星状细胞中I型胶原蛋白的表达；SEA通过抑制TNF-α下调IL-34的表达；重组E16蛋白（rSjE16）可通过抑制肝星状细胞中α-平滑肌动蛋白和I型胶原蛋白的表达水平，抑制肝星状细胞活化。.本项目所观察到的现象及探究的机制尚未见报道，主要创新点：①首先观察到日本血吸虫病小鼠巨噬细胞TREM-2表达增加，与巨噬细胞M2型极化相关。②该研究揭示了TREM-2调控巨噬细胞M2型极化的分子机制，不仅可以从理论上深化对血吸虫病引起的巨噬细胞M2型极化成因的认识，还可在实践上为血吸虫病肝纤维化的治疗提供新的潜在靶点。