BRAP蛋白对支气管上皮细胞触发的免疫失平衡的“刹车”作用及机制

Disregulation of immune responses contributes to pathogenesis of asthma, a chronic airway disorder associated with airway hyperresponsiveness. Lining the surface of bronchial airways, bronchial epithelial cells (BECs) provide the first line of defense against pathogenic factors to which they are directly exposed. The activation threshold of BECs and their resulting ability to trigger immune responses are fine-tuned in order to avoid unnecessary allergic sensitization and excessive inflammatory and immune responses which are involved in development of airway hyperresponsiveness and asthma. Bombesin Receptor-Activated Protein (BRAP) was first identified as a protein that may interact with bombesin receptor subtype-3 (BRS-3) in our previous study. It is highly expressed in human BECs and we further explored that overexpression of BRAP inhibited NF-κB transcriptional activation in BECs and reduced BECs’ ability of antigen uptake and presentation. Based on those data, together with lines of evidence of its inhibitory effect on the crosstalk between BECs with immune cells, we hypothesize that BRAP is a tuner of the activation threshold of BECs to trigger immune responses. Inhibition of its expression in BECs may lower the activation threshold of BECs and thus trigger the disregulated immune responses in asthma. To test our hypothesis, we will perform the following experiments: 1. Construct gene knock out mice which are lack of homologous BRAP expression and induce asthma on those mice. Then examine the resulting airway immune state. 2. Establish the co-culture system of human BECs and dendritic cells and explore the effects of different levels of BRAP expression in BECs on the function of dendritic cells. 3. Screening proteins interacting with BRAP and investigate the signaling events of BRAP involved in regulation of BECs’ immune functions. Our proposed study will provide novel insights into mechanism of immune disregulation in asthma.

哮喘等气道高反应性疾病的发生发展与免疫失平衡状态密切相关，申请者认为支气管上皮细胞（BEC）作为呼吸系统抵御外界刺激损伤的第一道屏障，其功能紊乱能触发气道局部免疫调节失衡。BRAP是申请者所在团队前期发现的在人BEC大量表达但功能不明的新蛋白，前期实验发现BRAP抑制体外培养的BEC对外源性刺激产生的免疫应答反应，提示该蛋白很可能是一种对BEC触发的免疫失平衡起“刹车”作用的因子，可使气道对外源刺激产生的局部免疫应答具有容错功能，避免产生气道高反应和免疫失衡。本项目拟通过下列实验研究检验这一假说并探讨机制，为设计针对哮喘的干预策略提供新的思路，研究包括：①建立BRAP同源基因敲除小鼠并诱导哮喘，观察BRAP同源蛋白缺失对哮喘免疫状态的影响；②建立BEC与抗原递呈细胞共培养体系，观察BRAP对抗原递呈细胞功能的影响；③探讨BRAP在细胞内的超分子信号组装对BEC触发的气道免疫应激的调控。

本项目致力于研究Bombesin Receptor-Activated Protein （BRAP）在触发哮喘相关肺部免疫紊乱的支气管上皮细胞中的作用，构建了缺失小鼠BRAP同源蛋白表达的基因修饰小鼠BC004004-/-，并利用该小鼠建立哮喘疾病模型，在动物整体水平和细胞水平研究该蛋白对哮喘等呼吸系统疾病中存在的免疫状态的调控作用。项目基本按计划执行，部分未能按预定方案完成的探索性强的实验已按预备方案成功完成；研究已获得预期结果，发现在哮喘模型中由于支气管上皮细胞（bronchial epithelial cells，BEC）缺少BRAP的表达，明显加重哮喘病变，缺失BRAP的气道上皮层结构松散，细胞脱落明显，BEC释放更多的TSLP和IL-33，诱导Th2型辅助T细胞释放IL-4、IL-5、IL-13等细胞因子招募炎症细胞，促进IgE的合成，并增加肺组织TGF-β1的表达，从而促进气道重塑；在体外培养的正常人支气管上皮细胞中过表达或沉默BRAP，可降低或增强细胞内NF-κB的转录活性。这些证据证实BRAP对气道上皮触发的免疫失平衡具有重要的抑制作用，从而使气道对外源刺激的局部免疫具有容错功能。研究中还发现BRAP蛋白在成纤维细胞、皮肤鳞状上皮细胞等细胞等组织中也有表达，但功能不明，推测BRAP可能对机体不同部位、不同类型的免疫均可能有一定的调控作用。为进一步解析BRAP的功能和分子机制，我们建立了多种疾病模型，研究BRAP蛋白对不同疾病病理过程的影响，发现该蛋白可能影响肺纤维化、溃疡性结肠炎和皮肤银屑病样改变等多种病理过程，提示该蛋白在多个系统中均具有重要的免疫调节功能。