靶向Siglec-9诱导中性粒细胞程序性细胞死亡和治疗重症哮喘作用机制的研究

Severe asthma comprises 5% of the patients with asthma, but it is responsible for most asthma attacks and emergency visits. Medical care for severe asthma costs more than half of the economic burden associated with the disease. Clinical evidence shows that severe asthma is associated with recruitment and activation of neutrophils in the airways, which are resistant to corticosteroid treatment. In order to improve the clinic management of patients with severe asthma, it is imperative to understand the underlying mechanisms in neutrophil recruitment, activation and programmed cell death (PCD) in severe asthma and to develop novel therapies more effective in treating neutrophilia. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are members of the immunoglobulin gene family. Among them, Siglec-9 is mainly expressed by human neutrophils and it plays a key role in regulating neutrophil recruitment and PCD. We speculate that Siglec-9 is a promising target, through which new biologicals can be used to target neutrophils in severe asthma. In a combined effort, we have generated humanized anti-Siglec-9 monoclonal antibodies (mAbs) that are able to induce apoptosis in human cells in vitro and we also created two humanized Siglec-9-expressing mouse lines: Siglec-9 Knock-In (KI) mice and Siglec-9 BAC transgenic (Tg) mice. In this application, we propose to use our humanized Siglec-9 mAbs to determine whether these antibodies have anti-inflammatory effects against neutrophils in Siglec-9-expressing mouse lines subjected to allergen-induced severe asthma models. The severe asthma mouse model was generated by exposing the mice to a mixture of several human disease relevant allergens, including house dust mite, Alternaria alternata, Aspergillus, and LPS, through intratracheal instillation (i.t.). We will explore whether any beneficial effects of humanized Siglec-9 mAbs are through induction of neutrophil apoptosis and autophagy by using a variety of techniques, including Western Blot and Flow Cytometry. Using this unique set of tools in the combined in vivo and in vitro studies, we will be able to investigate the mechanisms of neutrophil recruitment, activation and PCD in severe asthma and to test new approaches of using humanized monoclonal antibodies as biologicals as potentially more effective therapies for severe asthma. The findings from these studies using humanized mAbs and mouse lines will pave the way for the future clinical trials for patients with severe asthma.

重症哮喘虽然仅占哮喘患者的5%，但其急诊就医率和住院率分别为轻、中度哮喘的15倍和20倍，是导致哮喘治疗费用骤增的瓶颈。现公认，气道中性粒细胞大量募集浸润和活性增强是重症哮喘典型的病理生理特征之一。探索重症哮喘发病过程中中性粒细胞募集浸润和程序性死亡的调控机制，是精准治疗重症哮喘的必要前提，具有重要的临床意义。唾液酸结合的免疫球蛋白样凝集素9（Siglec-9）特异性表达于中性粒细胞表面，在调控中性粒细胞募集浸润和程序性死亡的过程中起关键作用，可能是一种治疗重症哮喘的新靶点。本项目制备了人源Siglec-9单抗，应用免疫印迹、流式细胞术等方法，探讨人源Siglec-9单抗诱导中性粒细胞凋亡和自噬的发生机制；制备了Siglec-9基因敲入、Siglec-9 BAC 转基因小鼠和Siglec-E基因敲除小鼠的重症哮喘动物模型，体内外实验分析人源Siglec-9单抗对重症哮喘的治疗作用及其机制。

中性粒细胞是重症哮喘的主要“元凶”。唾液酸结合的免疫球蛋白样凝集素9（Siglec-9）特异性表达于中性粒细胞，在调控中性粒细胞募集浸润和程序性死亡的过程中起关键作用。本项目制备了可特异性结合和诱导中性粒细胞凋亡的人源Siglec-9单抗，以及人源化Siglec-9 BAC Tg小鼠。研究发现：（1）Siglec-9替代Siglec-E可保护小鼠肺功能；（2）瓜氨酸组蛋白H3在Siglec-E KO小鼠体内表达；（3）人源化Siglec-9 Tg小鼠脾细胞表达Siglec-9；（4）建立新型Siglec-E KO小鼠；（5）TLR-4协同Siglec-E调控肺部炎症和肺气肿；（6）制备OVA和TNF-α诱导的慢性中性粒细胞重症哮喘小鼠模型；（7）Siglec-9与Siglec-E介导气道炎症细胞凋亡；（8）Siglec-9/E调控上皮细胞间质样转化（EMT）并抑制气道重塑；（9）DMSO诱导HL-60细胞分化为类中性粒细胞；（10）AECOPD患者外周血中性粒细胞高表达Siglec-9；（11）合成可吸入式Siglec-9靶向诊疗一体化纳米探针。第一发明人获国家发明专利1项（专利号ZL201510870684.5）。获2018年国家自然科学基金重点国际（地区）合作研究项目1项（批准号81820108001）和国家重点研发计划子课题1项（批准号2018YFC1313602）。第一作者和（或）通讯作者发表SCI论文3篇、中华杂志论文10篇，另有5篇SCI论文已接受、3篇高质量SCI论文待发表。均标注“国家自然科学基金资助项目（编号81670029）”。参编专著2本。主译专著2本。