弓形虫调控巨噬细胞m6A去甲基化酶FTO诱导TNF-α效应机制

Toxoplasma gondii is an opportunistic intracellular protozoan parasite with a worldwide distribution. The host immune status directly affects the development and final outcome of toxoplasmosis. It is well-known that T. gondii infection up-regulates the TNF-α expression of macrophage. TNF- α is of paramount importance in the resistance against T. gondii infection. Interestingly, T. gondii infection in mouse and human differs in their mechanism to trigger TNF-α production. In the mouse, T. gondii induces TNF-α expression at the transcriptional level through up-regulating the mRNA of TNF-α. However, when human macrophages are exposed to T. gondii tachyzoites, TNF-α expression is induced as a result of promoted mRNA translation, and until now the mechanism has not been elucidated. In eukaryotic cells, mRNA N6-methyladenosine (m6A) modification is critical for managing the mRNA translation. It is newly reported that m6A modification plays a vital role in anti-infection immunity. Our previous work demonstrated that FTO, as an m6A demethylase, was highly inhibited in T. gondii-infected human macrophage. Moreover, there are several m6A modification sites with high confidence in TNF-α mRNA. Based on these results, we proposed the hypothesis that T. gondii enhanced TNF-α expression through inhibiting FTO and up-regulating m6A levels in TNF-α mRNA transcript. Hence, in this proposal we will focus on elucidating the role of m6A demethylase FTO and m6A modification in T. gondii-induced TNF-α expression and its mechanism. The results will shed light on the role of m6A modification in pathogen-host interactions and reveal its mechanism.

弓形虫是重要机会性致病胞内寄生原虫，宿主免疫状态决定了感染的发展和转归。巨噬细胞分泌的TNF-α在抗弓形虫感染中起重要作用。已知人和小鼠感染弓形虫后诱导TNF-α效应机制存在差异：不同于小鼠中TNF-α表达调控发生在转录水平，人体内弓形虫对TNF-α合成的调控发生在蛋白翻译水平，但机制未明。新近报道mRNA上m6A修饰是调控蛋白翻译的重要方式，可介导免疫细胞感知并快速应答入侵病原体。我们前期研究发现，人TNF-α mRNA存在m6A高频修饰位点，弓形虫感染人巨噬细胞，可明显下调m6A去甲基化酶FTO的表达。由此我们假设，弓形虫感染人巨噬细胞可通过抑制FTO影响TNF-α mRNA的m6A修饰，上调TNF-α表达。本项目拟揭示弓形虫对FTO和TNF-α m6A修饰的调控作用，阐明m6A修饰调控TNF-α表达的分子机制。该研究为进一步丰富m6A修饰在病原体感染免疫中的作用及机制提供了帮助。

在弓形虫诱导的巨噬细胞免疫应答中，多效性细胞因子TNF-α在控制感染中发挥重要作用。前期研究发现人和小鼠感染弓形虫后诱导TNF-α表达的机制存在较大差异，但在人体其详细机制至今尚不明确。因此，本研究首先证实了弓形虫速殖子感染人THP-1源性巨噬细胞后，可诱导炎症相关基因的表达，影响炎症相关信号通路，促进炎症因子TNF-α、IL-1β和IL-6分泌。进一步研究发现，弓形虫感染会影响m6A修饰蛋白的表达，主要表现为甲基转移酶WTAP和去甲基化酶FTO表达上调。此外，弓形虫感染对人巨噬细胞mRNA的m6A修饰谱造成了显著影响，3'UTR区的m6A甲基化修饰增加，而5'UTR区的m6A修饰下降。弓形虫感染后，m6A修饰显著变化的基因主要富集在炎症介质对TRP通道的调节通路、癌症信号通路等。最后，本研究阐明了弓形虫感染人巨噬细胞调控TNF-α表达的分子机制：弓形虫感染人巨噬细胞，可上调去甲基化酶FTO的表达，减少TNF-α mRNA上5'UTR区和3'UTR区m6A修饰位点的修饰程度。敲低FTO后，弓形虫感染诱导的TNF-α mRNA m6A修饰下降的现象基本消失，以TNF-α为代表的巨噬细胞抗虫免疫应答效应减弱，弓形虫速殖子增殖增强。m6A识别蛋白YTHDF2可通过与mRNA的结合介导其发生降解，弓形虫感染人巨噬细胞后，由于TNF-α mRNA m6A修饰下降，YTHDF2结合的TNF-α mRNA相比于未感染组显著下调，提示弓形虫感染可通过抑制TNF-α mRNA的降解上调TNF-α的表达。本研究拓展了对TNF-α表达调控机制的认识，是深入认识弓形虫感染免疫特征的重要内容。此外，本研究揭示了m6A修饰调控抗虫免疫应答的机制，研究结果进一步加深和拓展了对m6A修饰调控感染免疫应答的认知。