共刺激分子B7-H3调控中性粒细胞肺内聚集在支气管哮喘中的作用及其分子机制

Asthma is the most common chronic inflammation disease in children and related to polymorphonuclear neutrophils (PMNs). Severe and refractory asthma is associated to pulmonary neutrophils accumulation which caused by its migration and apoptosis. Our previously work has demonstrated that B7-H3 is associated with Th17 differentiation and delayed apoptosis of PMNs. Recently, we discovered that anti-B7-H3 antibody could reduce the level of PMNs and IL-17 in alveolar lavage fluid. Thus, we hypothesized that B7-H3 is the key factor of pulmonary PMNs acculumation in asthma patients and play an important role in asthma development through regulating Th17/IL-17 axis and delaying apoptosis of PMNs.Therefore,the specific objectives of this research are to determine whether B7-H3 levels in the clinical samples from patients with asthma correlates to PMNs apoptosis and activity, thus functioning as prognosis marker during the development of asthma; and to examine the role of B7-H3 in regulating Th17 differentiation and PMNs apoptosis, and the possible downstream signal molecules involved depending on anti-B7-H3 antibody, B7-H3 fusion protein and B7-H3 knock-out mice as well as to clarify the role of intervention of B7-H3 in airway inflammation of asthma. In all, we aim to provide potential new immunological method to cure severe or refractory asthma.

哮喘是儿童最常见的慢性炎症性疾病，中性粒细胞参与其中。重症或难治性哮喘发病与中性粒细胞肺内聚集密切相关，而中性粒细胞的聚集与其迁移和凋亡有关。本项目组前期发现B7-H3可以调控Th17细胞分化和拮抗中性粒细胞凋亡。同时，应用抗B7-H3单克隆抗体可以降低OVA/LPS诱导的哮喘小鼠肺泡灌洗液中IL-17和中性粒细胞的水平。因此，我们推测B7-H3是调控哮喘中性粒细胞肺内聚集的关键分子，其可通过调控Th17/IL-17路径和拮抗中性粒细胞凋亡而在哮喘中发挥作用。鉴此，本项目组拟进一步分析B7-H3在哮喘进程中的临床意义和与气道中性粒细胞凋亡、活性之间的相关性；以及利用B7-H3单克隆抗体、融合蛋白以及敲基因小鼠等手段，研究B7-H3调控Th17细胞分化和拮抗中性粒细胞凋亡的作用以及涉及的信号分子机制，并探讨B7-H3干预治疗的临床应用价值，从而为重症或难治性哮喘的免疫治疗提供新手段。

哮喘是儿童最常见的慢性炎症性疾病，中性粒细胞参与其中。重症或难治性哮喘发病与中性粒细胞肺内聚集密切相关。本项目以B7-H3 为切入点，紧紧抓住B7-H3调控中性粒细胞肺内聚集这一新线索，通过收集哮喘患儿外周血及肺泡灌洗液（BALF），检测外周血及BALF中B7-H3及细胞因子表达水平，并分析B7-H3与这些细胞因子和BALF中中性粒细胞比例（NEU%）的相关性。发现中性粒细胞哮喘患儿血浆及BALF中B7-H3及炎症因子明显升高，中性粒细胞哮喘患儿BALF中NEU%与B7-H3表达正相关，且哮喘患儿B7-H3水平与IL-17显著正相关。通过建立哮喘动物模型，给予B7-H3单克隆抗体干预，检测小鼠BALF中细胞总数及分类计数、外周血及BALF中细胞因子水平；小鼠肺组织病理切片行HE、PAS染色及B7-H3免疫组化。结果显示使用抗B7-H3单克隆抗体后BALF中细胞总数中性粒细胞数减少，小鼠外周血及BALF中炎性细胞因子明显降低，肺组织染色示炎性细胞浸润减少，肺组织病理改变减轻，气道黏液分泌减少。提示抗B7-H3单克隆抗体能减轻小鼠气道中性粒细胞炎症、减轻气道炎症细胞因子表达、减轻肺部炎症及气道黏液分泌，同时能减轻小鼠全身炎症细胞因子表达。此外，我们还积极寻找B7-H3上游调控分子，通过筛选哮喘患儿外周血单核细胞差异表达的miRNAs，寻找到能与下游B7-H3结合的miRNA miR-29c。发现miR-29c在哮喘患儿体内低表达，且能负向调控单核-巨噬细胞的B7-H3表达。miR-29c作用于巨噬细胞表面可以调控Th细胞分化，下调miR-29c能使Th细胞向Th2及Th17方向分化。本研究为全面认识B7-H3在哮喘尤其是重症及难治性哮喘中的作用提供新的视角，为我们哮喘的干预治疗提供了思路及依据。本项目共计发表论文共计35篇，其中SCI13篇；团队获得国家级课题4项；获得奖项7项，其中国家级奖项3项。多次参加国内外学术会议，并进行学术交流。培养江苏省青年医学人才2名，苏州市姑苏卫生人才1名，培养博士研究生3名，硕士研究生10名。