泛素化调节依赖性EGF/EGFR轴介导的母胎界面CXCR4+NK细胞促滋养细胞侵袭的分子机制

Immune cells exert critical modulatory effect on the behavior of trophoblast, though the specific mechanism is unclear. Our previous study discovered that CXCR4+NK cells, the unique NK cell subset characterized with high production of IL-4 and the potential to induce Th2 differentiation at the maternal-fetal interface, contribute to the establishment and maintenance of immune tolerance in pregnancy. Recently, further investigation by our team showed that CXCR4+/- NK cells display different gene expression profiles and significant expression genes enrichment involve in the degradation of extracellular matrix, cell migration and invasion. Compared to CXCR4- NK cells, CXCR4+ NK cells had a significant higher expression of mmp9 and promoting effect on trophoblast invasion showed by our preliminary experiment. EGFR was also significantly higher expressed on CXCR4+ NK cells than that on CXCR4- NK cells. Moreover, rhEGF interacted with EGFR led to significant upregulation of mmp9 in NK cells. Coincidently, EGFR degradation associated E3 ubiquitin ligases were significantly lower expressed in CXCR4+ NK cells. Therefore, we speculated that significant lower expression of EGFR E3 ubiquitin ligases in CXCR4+NK cells inhibited the ubiquitination-dependent degradation of EGFR, ensuring the activation of signal pathway of EGF/EGFR and mmp9 production, which was the underlying mechanism of CXCR4+ NK cells facilitating trophoblast invasion. In this project we plan to analyze the functional molecules including mmp9, EGFR on CXCR4+/- NK cells in decidua derived from normal early pregnancy and spontaneous abortions. Explore the effect of CXCR4+/-NK cells on the behavior of throphoblast by the co-culture system. Further study is designed to investigate the specific mechanism of CXCR4+/- NK cells-exerted differential modulation on trophoblast invasion via E3 ubiquitin ligases /EGFR/EGF/MMP9 axis. We attempt to provide new insights for the diagnosis and treatment of pregnancy associated diseases including recurrent spontaneous abortion and preelampsia.

母胎界面免疫细胞调控滋养细胞生物学行为的确切机制仍不明晰。最近我们发现，CXCR4+/-NK差异表达基因在细胞侵袭迁移功能显著富集。CXCR4+NK显著高表达MMP9促滋养细胞侵袭。EGF通过与CXCR4+NK细胞表面显著高表达EGFR相互作用促MMP9表达。此外，EGFR泛素化E3连接酶在CXCR4+NK显著低表达，推测E3连接酶差异表达调节EGFR泛素化依赖性降解及EGF/EGFR信号介导的MMP9表达调控可能是CXCR4+/-NK差异调节滋养细胞侵袭的分子机制。本研究拟利用正常早孕和自然流产标本，分析相关功能分子在CXCR4+/-NK差异表达；建立CXCR4+/-NK与滋养细胞共培养体系，探究其对滋养细胞生物学行为的差异调节作用。进一步采用过表达、Si-RNA、IP等手段解析CXCR4+/-NK差异调节滋养细胞侵袭的分子机制，为反复自然流产、子痫前期等疾病防治提供新思路。

NK细胞因其功能多样性及可塑性在母-胎耐受、胎盘血管形成、子宫内膜蜕膜化等多个关键生物学事件中发挥至关重要的调节作用。本研究以早孕期蜕膜局部NK细胞为研究对象，围绕NK细胞参与或调控的妊娠建立和维持的关键生物学事件，应用组学分析、信息学、分子生物学及免疫学技术，利用临床样本与动物模型进行体内、外实验，发现了具有特异表型和功能的妊娠早期NK细胞亚群分型（CXCR4+/-NK细胞），深入解析了CXCR4+NK细胞的来源、表型、功能，揭示其与滋养细胞及其他免疫细胞之间的交互对话从而参与母胎免疫耐受、滋养细胞生物学行为调控的作用及分子机制。研究发现：1）早孕期滋养细胞通过SDF-1趋化外周CXCR4highCD56dim NK细胞至蜕膜局部，并进一步训导其CXCR4降表达，CD56高表达，形成蜕膜局部特异表型CXCR4midCD56highNK细胞。蜕膜局部CXCR4+CD56highNK细胞呈现低活化、低杀伤活性免疫耐受表型，并通过高表达IL-4诱导naïve T细胞向Th2型细胞分化。反复自然流产患者蜕膜局部CXCR4+CD56highNK细胞数量减少，CXCR4表达丰度显著降低，且CXCR4+CD56highNK细胞诱导Th2型细胞分化能力减弱，过继转输蜕膜CXCR4+NK细胞可以缓解NK缺失小鼠胚胎丢失，并促进蜕膜局部Th2型细胞分化；2）CXCR4+NK细胞较CXCR4-NK细胞显著高表达EGFR，滋养细胞通过分泌EGF与CXCR4+NK细胞EGFR结合并相互作用，活化ERK1/2信号通路，促进MMP9表达。CXCR4+NK细胞高表达MMP9促进滋养细胞侵袭。反复自然流产患者滋养细胞EGF表达显著下降，蜕膜CXCR4+NK细胞EGFR和MMP9的表达亦显著降低；3）RhoB/SEMA3A信号介导妊娠早期母胎界面免疫平衡与基质细胞蜕膜化；4）蜕膜CCR1+ Mφ的来源、表型及调控滋养细胞侵袭、迁移的作用机制；5）妊娠肥胖对母胎免疫应答及妊娠结局的影响及机制探究；6）米非司酮（RU486）诱导流产的免疫学机制探究。该研究进一步丰富母胎交互对话机理，为复发性自然流产等免疫调节紊乱性疾病的防治提供新型预测与防治靶点。