PCDH20调节CD4+Th细胞亚群在克罗恩病发病中的作用研究

Our previous study have shown that the imbalances of CD4+ T helper (Th) cells which include Th1, Th9, Th17 and Treg cells, are obvious in patients with Crohn’s disease (CD). It suggested that rebuilding the balance of CD4+ Th cell subgroups might be a new therapeutic target for CD treatment. Recently, we found that PCDH20 was a novel and pivotal molecule regulating CD4+ Th cell subgroups. The expression of PCDH20 was down-regulated in the inflamed intestine and peripheral blood mononuclear cells collected from CD patients. Functional studies that either over-express or knockdown PCDH20 in naïve CD4+ Th cells by using lentivirus showed that the proportions of Th1, Th9, Th17 and Treg cells, the relevant transcription factors and cytokines changed significantly. Further mechanistic study showed that PI3K/AKT/mTOR pathway might be involved in the functions of PCDH20, while this pathway has been proven to be important in the regulation of the CD4+ Th cell subgroups. Based on these findings, we proposed the hypothesis that PCDH20 might regulate CD4+ Th cell subgroups via PI3K/AKT/mTOR pathway，thus playing a pivotal role in CD pathogenesis. To test this hypothesis, PCDH20-/- knockout mice will be built to identify its role in the development of CD-related intestinal inflammation. At the same time, multiple techniques including PCR, Western blot, immunohistochemistry, co-immunoprecipitation and flow cytometry will be employed to clarify that the role of PCDH20 in the regulation of CD4+ Th cell subgroups via PI3K/AKT/mTOR pathway in vivo and in vitro. Therefore, our project will provide further understanding for the role of PCDH20 in CD pathogenesis and inspire a novel potential therapeutic target for the treatment of CD patients.

我们前期研究结果显示：克罗恩病（CD）患者存在CD4+Th细胞亚群（Th1、Th9、Th17及Treg）紊乱，调节该群细胞免疫平衡有望成为CD治疗新的切入点。最近，我们发现PCDH20是调节该群细胞平衡新的关键分子：它在CD患者表达下调；过表达及干扰后，Th1、Th9、Th17及Treg细胞比例和相应转录因子及细胞因子表达均发生变化。进一步机制研究显示，PCDH20与PI3K/AKT/mTOR通路表达相关。由此，我们首次提出PCDH20可通过PI3K/AKT/mTOR通路调节CD4+Th细胞，进而在CD发病中发挥重要作用。本项目拟采用基因敲除小鼠阐明PCDH20在CD肠道炎症发生中的作用，并采用PCR、蛋白印迹、免疫组化、免疫共沉淀及流式细胞等技术阐明PCDH20通过PI3K/AKT/mTOR通路调节CD4+Th细胞亚群的机制，为明确PCDH20在CD发病中的作用、寻求新治疗手段打下基础。

免疫失衡及黏膜屏障破坏是克罗恩病（CD）发病的关键环节。钙黏蛋白家族是一类构成细胞连接的跨膜蛋白，在免疫调节及黏膜屏障功能调控中可能发挥重要作用。GEO数据库芯片结果发现，钙黏蛋白家族成员PCDH20在CD中表达明显下降，但在CD发病中的具体机制尚未明确。使用PCDH20全身性敲除鼠构建DSS及TNBS结肠炎模型，发现敲除PCDH20可加重结肠炎症，但在肠黏膜发挥促炎作用的Th1及Th17细胞比例则减少；而且我们扩大样本发现，在CD患者的外周血单个核细胞中PCDH20 mRNA与蛋白表达水平不一致，提示PCDH20在外周血免疫及黏膜免疫中的作用存在矛盾。鉴于结肠炎模型中PCDH20敲除对肠道黏膜造成显著损伤，因此我们重点关注PCDH20对肠上皮细胞黏膜屏障功能的调节作用。进一步研究发现，PCDH20在CD患者及小鼠肠炎模型的肠上皮中均呈低表达。肠特异性敲除PCDH20加重小鼠DSS结肠炎的炎症及肠道屏障破坏程度。细胞实验亦证明，下调PCDH20使肠上皮细胞屏障受损。通过数据库预测并经体外验证，转录激活因子6（ATF6）是PCDH20的下游相互作用蛋白。进一步体内外验证PCDH20通过正向调控ATF6维持肠道屏障功能及减轻肠道炎症。蛋白组学与磷酸化组学联合分析提示，PCDH20可能参与调节p-AKT/Casp-3凋亡通路。体内外实验验证PCDH20靶向ATF6调节p-AKT/Casp-3凋亡通路，进而影响肠黏膜屏障功能。本项目发现PCDH20在维持CD肠道屏障中起重要作用，其主要通过靶向作用下游蛋白ATF6调控p-AKT/Casp-3凋亡通路维持肠黏膜屏障功能。选择性激活PCDH20下游蛋白ATF6有望成为促进CD黏膜愈合精准治疗的新手段。