呼吸道干细胞炎症记忆在哮喘气道重塑过程中的作用与调控机制研究

Asthma is a chronic inflammatory airway disorder characterized by airway hyperresponsiveness and reversible airflow obstruction. Patients with severe asthma develop irreversible airflow obstruction and experience an accelerated rate of lung function decline, which are attributed to airway remodeling. The “inappropriate” structural changes in the airway epithelium and sub-epithelial compartments are referred to as airway remodeling, with key features including increased numbers of goblet cells (goblet cell hyperplasia), thickening of the reticular basement membrane, increased bronchial vascularity and hypertrophy of smooth muscle and submucosal glands. Goblet cell hyperplasia has a direct link to chronic mucus hypersecretion and drives consequent airflow obstruction and persistent hyperresponsiveness. Despite optimal treatment with corticosteroid and bronchodilator therapy, significant ongoing symptoms persist in severe asthmatics. Therefore, reversal of airway remodeling is bearing paramount therapeutic importance, and therapeutic agents or strategy working by way of mechanisms responsible for such remodeling are desirable for new asthma treatments. As well known, epithelial stem cells play a crucial role in establishment and maintenance of tissue homeostasis. Such cell population resides closely to the basement membrane of epithelium and stays quiescent at steady state, but responds vigorously to tissue injury by means of activating proliferation and differentiation. There are pieces of cues from studies of several chronic inflammatory conditions, such as chronic obstructive pulmonary disease and nasal polyps, suggesting that epithelial stem cells may contribute to the persistence of pathophysiological changes associated with the diseases by serving as repositories of inflammatory memories，which is in line with our previous findings on asthmatic bronchial stem cells. In the present study, we aim to further understand the incidence of and mechanisms underlying such event via systemically evaluating the cellular functions of patient-derived bronchial-bronchiolar stem cells from grouped moderate-severe asthmatics and their behaviors in response to cytokines, followed by a combinatorial application of chromatin-open-area sequencing and RNA sequencing. Potential therapeutic targets will be subsequentially examined for their efficacy on in vitro inflammation model and Crispr-based platforms. Improved knowledge on inflammatory memory gained from work above will lead to the development of a novel therapeutic strategy against airway remodeling, making beneficial for asthmatics at irreversible stage.

气道重塑是重症哮喘的典型特征，重塑的发生与上皮组织损伤和病理性修复有着密切关联。恢复气道复纤上皮正常细胞比例并抑制间质细胞增生是彻底扭转重塑的终极目标，离不开组织干细胞的参与。支气管基底细胞是传导气道专能干细胞，在建立和维系组织稳态、修复损伤过程中起着至关重要的作用。项目前期研究首次发现该类干细胞在脱离病理环境后仍呈现持久性功能改变（即炎症记忆），不利于组织正常修复。基于此，本课题拟通过对中重度哮喘患者支气管基底细胞分组进行单细胞克隆、体外3D培养及在体微气管构建系统性地对细胞炎症应答、分化潜能进行评估，明确干细胞炎症记忆的发生几率与规律；进而，对典型样本的表达谱、遗传谱进行RNA-Seq和ATAC-Seq分析，揭示干细胞发生遗传性功能改变的机制；然后，利用体外炎症模型、干细胞基因编辑对潜在靶点进行功能验证并作初步应用探索。相关成果将为治疗重症哮喘提供新思路、新手段。

气道重塑临床表现为小气道阻塞性通气功能障碍，是重度哮喘的一大特征，一旦发生难以逆转，是哮喘治疗的瓶颈。目前哮喘治疗多以控制炎症和舒张气道平滑肌为一线策略，然而重度哮喘患者通常对糖皮质激素抗炎治疗和气管舒张剂不敏感，半数或以上的重度哮喘患者对靶向免疫抑制疗法例如IgE单抗、IL5单抗、IL13单抗以及 IL5Rα受体拮抗剂响应度不佳，疗效波动性大。因此，解析哮喘气道重塑的发生根源并开发针对性新型治疗靶点具有临床迫切性。恢复气道复纤上皮正常细胞比例并抑制间质细胞增生是彻底扭转重塑的终极目标，离不开组织干细胞的参与。支气管基底细胞是传导气道专能干细胞，在建立和维系组织稳态、修复损伤过程中起着至关重要的作用。本项目聚焦在支气管基底细胞群体，通过对不同病程哮喘患者来源基底细胞进行单细胞测序、单克隆层面的体外类器官模型构建和在体微气管构建多角度地对细胞炎症应答、分化潜能进行评估，确证了哮喘环境下干细胞炎症记忆的发生并初步解析其发生范围和规律。结合scRNA 和单克隆bulk RNA测序结果，联想到气道基底细胞在另一气道慢性炎症性疾病骨化性气管支气管病中的表现，我们推测哮喘相关炎症记忆同样植根于表观基因组改变，从而体现为基底细胞长期而稳定地表型异化。此改变使气道重塑不再依赖环境刺激，是目前治疗手段难以触及的根本问题。根据表达谱分析所得，利用体外炎症模型和干细胞基因编辑技术对潜在靶点进行功能验证，初步筛选到能够逆转哮喘基底细胞分化倾向的关键基因。通过对一类内源性蛋白酶家族抑制剂的调控，有望实现粘膜上皮结构修复，并且改善局部免疫环境。综上发现，本项目研究为重度哮喘的控制和治疗带来了新思路，新靶点的进一步开发将具有明确应用价值。