气道上皮细胞IL-33作为核因子在哮喘中的作用及其去泛素化修饰调控

Innate immunity plays a crucial role in the pathogenesis of asthma and the airway epithelial cell-derived interleukin-33 (IL-33) is one of the key factors. IL-33 has dual function in asthma: promote airway Th2 inflammation through binding to its receptor (ST2 and IL-1RAcP) as a cytokine, however the pathophysiological roles of IL-33 as a nuclear factor remain unclear. Our preliminary experiment has shown that IL-33 can regulate the transcription of IL-13 through binding to its promoter directly and this can be regulated by the deubiquitinase USP21. The aim of this study is to explore the function of IL-33 as a nuclear factor and its regulation by USP21 in asthma. (1) identify the target genes of IL-33 through ChIP-sequence in Flag-IL-33-16HBE stable cell lines; (2) identify the transcriptional complex of IL-33 through TAP purification combined with protein mass spectrometry in TAP-IL-33-16HBE stable cell lines; (3) in vitro, investigate the changes of subcellular localization and nuclear function of IL-33 in Flag-IL-33-16HBE cells after exposure to house dust mites or infected with shUSP21; (4) in vivo, investigate the function of IL-33 as a nuclear factor and its regulation by USP21 through sensitized and challenged the airway epithelial cell conditional USP21 knockout mice with OVA. This project will provide new understanding for innate immunity in asthmatic airway inflammation which could provide a unique therapeutic way for asthma.

近年来发现IL-33介导的固有免疫与哮喘发病紧密相关。气道上皮细胞来源IL-33具有双重功能：1）作为细胞因子激活胞外受体促进炎症反应；2）作为核因子调控基因转录，具体功能尚不清楚。我们研究发现核内IL-33结合IL-13启动子区并调节其转录，该功能受去泛素化酶USP21调控。本课题拟深入探讨哮喘发生发展中核因子IL-33及其去泛素化酶USP21介导的免疫调控机制：1)ChIP-sequence确定受IL-33直接调控的下游靶基因；2）蛋白亲和纯化鉴定转录复合体组成；3)体外过敏原刺激和shUSP21转染，检测气道上皮细胞IL-33亚细胞定位和核因子功能调节；4)利用我们已有USP21 fl/fl小鼠，构建气道上皮条件性基因敲除小鼠OVA哮喘模型，深入研究核因子IL-33及其去泛素化酶USP21在哮喘发生发展中的作用。该课题将为哮喘固有免疫发病机制提供理论创新，为药物早期干预提供潜在靶点。

气道上皮在哮喘的起病、发展和加重中都发挥关键作用，是固有免疫应答和适应性免疫应答之间的桥梁和调节者。气道上皮细胞在接受外界刺激时会分泌IL-33、IL-25、TSLP等，其中IL-33已被证明与哮喘的发病最为密切；一方面可以作为细胞因子被分泌至细胞外，通过配体受体结合的方式发挥作用（IL-33/ST2经典途径），另一方面可以作为核因子在细胞核内发挥调节基因转录的作用（核因子途径），但具体作用及其机制不甚清楚。.我们根据研究计划主要进行了以下四个方面的研究：①气道上皮细胞内IL-33作为核因子的下游靶基因研究；②气道上皮细胞内IL-33作为核因子的转录复合体成分鉴定；③去泛素化修饰对气道上皮细胞内IL-33作为核因子的调控；④动物模型的验证。.结果发现IL-33确实存在泛素化修饰，去泛素化酶USP21能与IL-33相互作用，并且介导IL-33的去泛素化，进而稳定IL-33的蛋白水平；首次发现IL-33能结合IL-13基因的启动子区，USP17可以在K48和K63位点去泛素化IL-33，调控IL-33与IL-13基因的启动子区结合的活性；首次发现IL-33存在PTRF依赖的可控性释放调节机制，即PTRF的磷酸化状态影响IL-33的细胞定位，LPS或HDM刺激使PTRF发生去磷酸化，去磷酸化的PTRF发生易位使得细胞核内的IL-33释放减少，从而减轻哮喘急性加重的表现。此外我们也研究了GATA3和FOXP3的泛素化调节及PIM2在哮喘发病中的机制，发现USP21、GATA3和FOXP3形成正反馈回路，导致哮喘患者Treg细胞功能失调，进而导致哮喘的发病。.上述发现拓展了我们对IL-33作为核因子的功能和泛素/去泛素化修饰调控基因转录的认识，考虑到IL-33、IL-13在哮喘免疫发病机制中的重要地位，对于今后哮喘防治有重要意义。该课题将为哮喘固有免疫发病机制提供理论创新，为药物早期干预提供潜在的靶点。