MBD2调控Th2/Th17细胞分化与功能及其在不同年龄段小鼠哮喘气道炎症表型变化中的作用研究

Asthma is a heterogeneous disease with different phenotypes. In our daily clinical work, we found that a part of persons who have genetic background do not suffer from asthma. Moreover, some asthmatic patients show the age-related variation and different phenotypes of airway inflammation. The mechanism is not clear yet. Our previous experiments had confirmed that there were different phenotypes of airway inflammation in asthmatic mice of 4 weeks and 48 weeks. In clinical investigation, we discovered that remarkable differences existed in DNA methylation levels in the peripheral blood between asthmatics in childhood and asthmatics of the elderly. And there were also different in the expression of MBD2 in the above two age groups, which is the executor of DNA methylation’s regulatory effects. In addition, MBD2 had been suspected of being a regulator of Th2 type response. Our previous data demonstrated that MBD2 was involved in inducing the differentiation and function of Th17 cells in vitro. The above suggested that MBD2 could act as an inducer of phenotypic change involved in asthma airway inflammation of different ages through regulating Th2-type and/or Th17-type responses. The objective of this study is to investigate that whether MBD2 would regulate the asthma phenotypes by affecting the Th2-eosinophilic type and/or Th17-neutrophilic type airway inflammation based on experiments both in vitro and in vivo by using MBD2-/- mice for the first time. Moreover, this study may provide preliminary experimental basis for our further investigation of epigenetic mechanism in asthma phenotype and for finding target for individualized diagnosis and therapy of asthma in the future.

哮喘是异质性疾病，可呈现不同表型。在临床中我们也发现有些具有遗传学背景的人并未患病；而即使患病的也可表现出年龄差异性，呈现不同炎症表型，其机制至今不清。我们证实4周龄与48周龄小鼠哮喘存在气道炎症表型差异；而临床观察发现儿童与老年哮喘患者外周血DNA甲基化水平存在差异，且其效应执行者MBD2蛋白表达也不同。此外，研究发现MBD2可能影响Th2型应答，而我们的前期数据提示MBD2可调控Th17细胞的分化与功能。综上推断，MBD2可能通过调控Th2和/或Th17型应答而影响不同年龄小鼠哮喘气道炎症表型。本项目将利用MBD2-/-等小鼠为研究对象，结合体内外实验首次观察MBD2在不同年龄小鼠哮喘气道炎症表型变化中的作用，初步探讨MBD2是否通过影响Th2-嗜酸性粒细胞型和/或Th17-中性粒细胞型气道炎症从而调控哮喘表型；也为深入研究哮喘表型的表观遗传学机制及发掘哮喘个体化诊治靶点提供前期基础。

哮喘是一种异质性疾病，存在不同的气道炎症表型，且呈现年龄相关性：儿童青少年哮喘多表现为激素敏感型（嗜酸性粒细胞为主型），而中老年哮喘常表现为激素依赖甚至不敏感（混合细胞型或中性粒细胞为主型），其机制不清楚。我们的动物实验已发现：同等条件下，年轻小鼠呈现气道嗜酸性粒细胞浸润为主伴有明显气道高反应（Th2型应答）；老年小鼠则呈现气道中性粒细胞与嗜酸性粒细胞混合浸润（Th17-Th2），且以中性粒细胞为优势，并出现气道高反应性相对下降（Th17型应答），一定程度解释了临床现象和激素敏感与否问题。那可能的调节机制是什么？基于前期哮喘表观遗传学机制尤其是MBD2在免疫调节中的作用研究，我们拟探讨MBD2在调节Th2与Th17型应答及其在哮喘气道炎症表型中的作用。结果提示：1. 抑制DNA甲基化可促进Th2分化并提高IL-4水平；2.沉默MBD2可促进Th2分化和提高IL-4水平；加入过敏原OVA可诱导Th2分化和IL-4分泌。3.相反，降低DNA甲基化水平后，Th17分化及IL-17水平则降低；4.同样，沉默MBD2亦抑制Th17分化和IL-17分泌；加入过敏原OVA亦可增强刺激Th17分化和IL-17分泌，但程度不及Th2/IL-4明显。细胞实验结果印证了前期动物实验发现，其中，OVA对于Th2及Th17的协同正刺激和混合细胞浸润的气道炎症类型，提示是否存在另一种应答类型，实验发现Th2-Th17双阳细胞在OVA经典哮喘模型气道炎症中的作用得到了验证并可解释之，这也是研究过程中的有趣发现，更好解释了哮喘气道炎症表型的潜在演变特征。后续我们建立了不同周龄的中性粒细胞为主等哮喘动物模型，进一步观察MBD2的表达和Th17/IL-17作用变化，结果提示：与OVA经典哮喘（Th2/IL-4）比较，1.MBD2在气道局部的表达明显增高，并与周龄呈正相关；2.气道局部Th17细胞分布和IL-17表达及中性粒细胞浸润同样明显增高，且严重程度与周龄呈正相关。综上提示MBD2是中性粒细胞型浸润为主或Th17型应答为主型哮喘的潜在生物标记物，而团队后续临床研究结果也初步印证了这点，即与轻中度哮喘患者和健康人比较，重度患者外周血MBD2水平明显升高，且与Th17表达升高明呈显正相关，而与Th2表达水平呈负相关。综上所述，本项目研究结果初步阐明了哮喘气道炎症表型随年龄变化可能机制及其潜在临床意义。