组蛋白乙酰化修饰在妊娠子宫巨噬细胞极化中的功能及不明原因反复流产相关机制研究

The maternal-fetal interface is a privileged site for coordinating the process of immune tolerance to protect the semi-allogeneic embryo from attack by the maternal immune system. Uterine macrophages and balance of polarized M1 and M2 subpopulations are key players in establishing and maintaining pregnancy, and the dysregulation of this balance is correlated with recurrent spontaneous abortion (RSA) in human. We have separated uterine M1 and M2 macrophage subpopulations by fluorescence-activated cell sorting during early pregnancy in mice. By using a low-dose lipopolysaccharide (LPS)-induced mouse abortion model, we have found that M1 preponderance was significantly exaggerated at 12 h of LPS treatment, when abortion did not occur. Importantly, adoptive transfer of M2 macrophages, but not of M1, partially rescued LPS-induced abortion in mice. By RNA sequencing analysis and qPCR validation of candidate differentially expressed genes (DEGs) in M1 versus M2 subpopulations, we have found several interesting histone modifying enzymes that are involved in histone methylation and acetylation, among which Hdac9 was the most significant DEG. Based on these results, we aim to investigate the regulatory landscape by histone acetylation in macrophage polarization in the pregnant uterus, mechanism of RSA pathogenesis, and in vivo therapeutic function in mouse abortion models, by using conventional and conditional knockout mouse models, in vitro macrophage polarization, mouse abortion models, human RSA samples and high-throughput omics approaches. We are keen to recognize the molecular basis of M1-M2 balance/imbalance and immune tolerance/rejection during embryo implantation and abortion, to explain macrophage-based immunologic mechanisms relevant to RSA, and to discuss the possibilities of development of useful histone acetylation targets in macrophages for RSA intervention.

成功的胚胎着床和妊娠依赖于母胎免疫耐受，子宫巨噬细胞M1和M2亚群的极化与平衡调节母体免疫容受，该平衡失调与反复自发性流产（RSA）密切相关。我们通过流式分选获得小鼠妊娠早期子宫M1和M2亚群；利用低剂量脂多糖（LPS）诱导的小鼠流产模型，发现LPS处理12小时、流产发生前即出现M1优势显著增强；过继转移实验表明仅M2能有效挽救小鼠流产表型；子宫M1和M2的转录组测序分析和验证发现了显著差异表达的组蛋白去乙酰化酶Hdac9。基于以上前期基础，我们将利用全基因和条件性基因敲除小鼠、体外巨噬细胞极化、小鼠流产模型、人临床材料和组学等技术，研究组蛋白乙酰化修饰在小鼠妊娠子宫M1和M2极化中的功能、在RSA中的作用及作为治疗靶点的可能性，以深入认识正常胚胎着床和流产过程中M1-M2优势平衡与失衡、免疫耐受与免疫排斥的分子基础，为揭示RSA的发生机制和探索靶向巨噬细胞的治疗策略提供理论基础和新思路。

母胎免疫耐受是成功的胚胎着床和妊娠的基础，子宫巨噬细胞M1和M2亚群的极化与平衡调节母体免疫容受，该平衡失调导致流产。我们通过流式分选获得小鼠妊娠早期子宫M1（CD45+F4/80+CD206-）和M2（CD45+F4/80+CD206+）亚群。利用低剂量脂多糖（LPS）诱导的小鼠流产模型，发现LPS处理6小时后M1优势即开始出现，并延续至处理后24小时。过继转移实验表明仅M2能有效挽救小鼠流产表型。子宫M1和M2的转录组测序分析发现了1837个差异表达基因（DEG），其中M2相较于M1有629个基因上调，1208个基因下调。经RT-qPCR实验验证，发现了子宫M1/M2显著差异表达的组蛋白去乙酰化酶Hdac9，其在M2中的表达比M1中显著增高。值得注意的是，Hdac9在小鼠脾脏M1/M2以及体外小鼠腹腔、骨髓和RAW 264.7诱导的M1/M2巨噬细胞中也有相似的差异表达模式。在Hdac9敲除的RAW 264.7和THP-1源巨噬细胞中，M1标记分子的表达不变或下降，但M2标记分子的表达明显增强，对巨噬细胞诱导极化过程中的细胞增殖能力没有影响。而且，Hdac9敲除导致RAW 264.7 M2的荧光微球吞噬能力显著上升，THP-1诱导M1的吞噬能力显著下降。Hdac9基因敲除（KO）与野生型（WT）Raw264.7巨噬细胞的比较转录组分析发现了Cxcl2、Cxcl10、Cd86、Cd80等DEG，RIG-I样受体信号通路、Toll样受体信号通路等被显著富集的KEGG通路；H3K9Ac的ChIP-Seq分析表明WT有2045个特异peaks，KO有2228个特异peaks，Hdac9敲除导致H3K9Ac在intron和TSS上游2 kb的差异peak相关基因数目减少。以上结果表明，Hdac9敲除导致小鼠和人巨噬细胞倾向于向M2表型分化，为揭示母胎免疫耐受中M1/M2极化与平衡的表观遗传调控机制提供了新思路。