Foxp3介导孕期尼古丁暴露致子代调节性T细胞发育编程改变及哮喘易感的表观遗传机制

Our and others' researches indicated that prenatal nicotine exposure contributes to the increased susceptibility of asthma in offspring. But the underlying mechanism remains to be investigated. Epidemiological data revealed a close connection between programming alteration in the development of thymic regulatory T cells (Tregs) and allergic inflammatory diseases (e.g. asthma) risk. Tregs are characterized by the expression of the Foxp3, which is also critical in modulating Tregs development and functions. Our previous studies demonstrated that prenatal nicotine exposure significantly decreased the expression of Foxp3 in fetal thymus and offspring’s spleen. The stable expression of Foxp3 in Tregs depends on the level of DNA demethylation at the Treg-specific demethylated region (TSDR) of Foxp3 gene. Based on the data from ours and literatures, we hypothesized that nicotine could combine with α7 nicotinic acetylcholine receptor (α7 nAChR) on the cell surface of fetal thymic Tregs, which in turn suppress signal transducer and activator of transcription 5 (STAT5), weaken the DNA demethylation of the TSDR and inhibit the expression of Foxp3, and ultimately block the mature of fetal thymic Tregs. These effects may lead to decreased peripheral Tregs numbers and down-regulation of Tregs-mediated suppression after birth, resulting in the increased susceptibility to asthma. In the present study, we will use experimental animals to confirm the critical role of Foxp3 in offspring's programming alteration in thymic Tregs development induced by prenatal nicotine exposure. Further, we will elucidate the epigenetic mechanisms of nicotine-induced down-regulation of fetal thymic Foxp3 at cellular and molecular levels. This study is important in providing novel mechanistic insights into the developmental origins of asthma caused by prenatal nicotine exposure.

本室及他室研究提示，孕期尼古丁暴露可致子代哮喘易感，但机制不清。流行病学调查显示，哮喘等过敏性炎性疾病的发生与胸腺调节性T细胞（Tregs）的发育编程改变密切相关。本室前期发现，孕期尼古丁暴露可抑制胎胸腺及成年子代小鼠脾脏中Tregs 发育和功能的关键调控分子—Foxp3的表达。Tregs中Foxp3的稳定表达依赖于其TSDR区的DNA去甲基化程度。综合文献，我们推测，尼古丁与胎胸腺Tregs细胞上的α7尼古丁受体结合，通过抑制STAT5使Foxp3的去甲基化程度减弱，抑制Foxp3表达，进而阻碍Tregs分化成熟，子代出生后外周Tregs数量和免疫抑制功能降低，易发哮喘。本项目拟利用动物实验证实胎胸腺Foxp3在孕期尼古丁暴露所致子代Tregs发育编程改变中的重要作用，进一步在细胞和分子水平阐明尼古丁抑制胎胸腺Foxp3表达的表观遗传机制，以期解析孕期尼古丁暴露致子代哮喘的发育起源。

哮喘患者出生前后所受的影响为其致病的重要因素之一，其发病风险增高与孕期不良环境暴露所致胎儿免疫系统发育编程性改变有关。流行病学调查显示，孕期接触烟草烟雾与子代哮喘发病率增加密切相关，本项目研究了烟雾的主要活性成分尼古丁在其中所起的作用及机制。我们观察到孕期尼古丁暴露（PNE）子代气道阻力显著增加，肺组织病理改变明显，不同时间点胸腺、脾脏及肺脏中Tregs细胞比例、免疫抑制功能显著降低，进而导致Th17细胞数量升高，Tregs/Th17比例降低，这种改变可从新生儿期一直持续至成年，是引起哮喘炎症反应的重要原因。机制研究显示，新生儿时期子代胸腺Tregs中Foxp3表达已显著降低，关键发育通路IL-2/p-STAT5信号分子表达降低。流式分选4天龄和6周龄子代胸腺和脾脏CD4+CD25+Tregs细胞，发现Foxp3基因CNS2区域甲基化程度显著高于同期对照。进一步建立胸腺Tregs细胞体外培养体系，分别给予IL-2及pSTAT5特异性抑制剂匹莫齐特处理，观察到尼古丁可抑制IL-2激活的Foxp3表达，且Tregs细胞比例及Tregs表面分子CD25、CD122、CD132、核内蛋白pSTAT5表达均明显降低。单独用尼古丁或匹莫齐特处理后，Tregs细胞Foxp3基因CNS2区的DNA去甲基化明显下降，这些变化与二者同时处理的作用相当，证实了IL-2/pSTAT5通路介导尼古丁抑制Foxp3基因CNS2区DNA去甲基化。进一步加入α7nAChR受体阻断剂α-BTX后，尼古丁降低IL-2R及Foxp3表达的作用被阻断，说明尼古丁通过激活Tregs表面的α7nAChR发挥作用。本项目首次从Tregs宫内发育编程角度阐明了尼古丁暴露致哮喘的发育起源机制，将孕期不良影响和出生后免疫失衡联系起来，有助于促进人们对哮喘诱因及预防的认识。另外，生命早期免疫系统的发育不同于成年阶段，目前人们对Tregs在胎儿宫内发育时期的研究还很有限，本项目以尼古丁作为孕期暴露因素，借助毒理学思路与手段促进了对Tregs在胎胸腺的产生、分化发育及其影响因素的认识。