去泛素化酶USP21对支气管哮喘Treg细胞FOXP3/GATA3平衡及免疫抑制功能的调控

It has been demonstrated that the deficiency of regulatory T cells (Treg) leads to unleashed Th2 responses in asthma, but the underlying mechanism is still not fully understood. GATA3,Th2 specific transcription factor, was recently found to express in Treg cells and interact with FOXP3, so FOXP3/GATA3 balance may be involved in regulating the function of Treg cells. Our preliminary studies in Jurkat T and HEK 293T cell line showed that the E3 deubiquitinase USP21 can stabilize GATA3 by preventing its ubiquitination and degradation. Meanwhile,USP21 mRNA expression of PBMCs in asthmatics was significantly higher than that in healthy controls and was positively correlated with GATA3 mRNA expression in PBMCs. The goal of this study is to explore whether USP21 plays a crucial role in the pathogenesis of asthma by regulating the function of Treg cells: (1) The first specific aim is to confirm the relationship of Treg USP21 expression (mRNA and protein) with its FOXP3/GATA3 balance and immunosuppression function by comparing asthmatics with healthy controls; (2) The next is to confirm the interaction between GATA3 and USP21 in Treg cells by endogenous immunoprecipitation; (3) The role of USP21 in FOXP3/GATA3 balance and immunosuppression function of Treg cells is further confirmed by infecting primary human Treg cells with pseudovirus containing USP21 shRNA (shUSP21), sensitizing and challenging conditional USP21 knockout mice with OVA. This study will deepen our understanding regarding the regulation of Treg cells on Th2 responses and provide potential target for the management of asthma.

支气管哮喘时Treg细胞免疫抑制功能缺陷机制仍不甚清楚。最新研究显示GATA3可表达于Treg细胞，并与FOXP3相互作用，推测FOXP3/GATA3平衡与Treg细胞功能有关。我们的预实验发现，去泛素化酶USP21可阻止GATA3泛素化和降解。为此本研究进一步探究USP21是否通过稳定GATA3，从而影响Treg细胞免疫抑制功能。拟通过：①比较哮喘患者与健康对照Treg细胞USP21、FOXP3、GATA3 mRNA和蛋白表达与免疫抑制功能的差异，初步确定USP21表达与FOXP3/GATA3平衡、Treg细胞免疫抑制功能的相关性。②用免疫沉淀确证Treg细胞中USP21与GATA3存在相互作用。③用shUSP21基因沉默原代Treg细胞，以及OVA诱发USP21条件性基因敲除小鼠，进一步明确USP21对Treg细胞免疫抑制功能的影响及其机制，籍此为哮喘防治提供新策略和有价值的线索。

项目开展期间根据项目标书完成研究。首先比较了哮喘患者与健康对照Treg细胞USP21、FOXP3、GATA3 mRNA和蛋白表达与免疫抑制功能的差异，结果提示哮喘患者外周血PBMC USP21 mRNA水平相对较高，伴随GATA3及FOXP3 mRNA的水平升高；哮喘患者外周血中CD25+CD4+T细胞占CD4+T细胞的比例降低，哮喘患者外周血中CD25+CD4+T细胞ki67表达下降，表面分子CLTA4及GITR表达下降，表达IL-4的CD25+CD4+T细胞比例增加，提示哮喘患者Treg抑制功能障碍；同时哮喘患者外周血CD25+CD4+T细胞中Foxp3+及GATA3+比例均增加，但Foxp3/GATA3比值下降，提示哮喘患者可能存在Treg稳定性改变并向Th2细胞转换。.分子层面研究探索了USP21表达与FOXP3/GATA3平衡、Treg细胞免疫抑制功能的相关性，结果提示USP21与GATA3之间存在相互作用，并通过His-pull down实验证明USP21降低GATA3的泛素化修饰水平，从而稳定GATA3蛋白；且USP21增加GATA3诱导的IL-4及IL-5转录活性；另一方面，FOXP3结合USP21启动子区域，并促进USP21的转录；即Foxp3－USP21－GATA3三者之间存在信号回路，在Treg功能及稳定性中起到重要作用。.条件性敲除小鼠相关研究中，完成了FOXP3-CRE USP21fl/fl小鼠的构建，并在该条件性敲除小鼠中建立哮喘模型。结果提示哮喘小鼠肺组织USP21表达增加，FOXP3-CRE USP21fl/fl小鼠中气道炎症水平与野生型小鼠差异不明显。.我们的研究结果证实了在细胞分子水平Foxp3－USP21－GATA3三者之间的信号回路，及其在Treg功能中的作用。