TLR9配体CpG-ODN调控TSLP-DC-OX40L途径对阻滞变应性鼻炎发展为支气管哮喘的机制研究

Recent evidence demonstrates that epithelial cells of asthma produce thymic stromal lymphopoietin (TSLP), TSLP activates immature dendritic cells (DCs), TSLP-activated DCs express OX40 ligand(OX40L), which can trigger T-helper type 2 (Th2) inflammatory responses. Therefore,TSLP and OX40L pathway palys an important rule in the initiation of DCs-mediated allergic inflammation. Our previous studies have proved that dexamethasone-treated DCs inhibited the Th2-type reactions, and the addition of budesonide(BUD) reduced the expression of DCs surface markers including CD40,CD80,CD86 and TSLPR, and the level of IL-5 in the culture supematants of DC-T coculture, which demonstrated that the therapeutic activity of BUD in asthmatic mice may be related to modulation of Th1 and Th2 cell functions and this effect is probably mediated through the TSLP-DC pathway, and neutralization of TSLP also inhibited airway remodeling in a murine model of allergic asthma. Asthma is epidemiologically linked to allergic rhinitis (AR), and management of AR improves asthma control. However, whether or not the TSLP-DC-OX40L pathway is crucially involved in the interaction between upper and lower airways remains to be identified. Because toll like receptor 9 ligand CpG oligodeoxynucleotides (CpG-ODN) induce Th1-type cytokines, which can suppress the Th2-type responses that cause many of the manifestations of allergic disease, we consider that they may be useful in preventing or reversing the inflammation of AR and asthma. Our preliminary tests show that intranasal administration of CpG-ODN and/or BUD on AR model can inhibit the inflammatory response of asthmatic model, we therefore speculate brockade TSLP-DC-OX40L pathway by CpG-ODN and/or BUD of AR may prevent the development of asthma. In order to prove the hypothesis, we plan to perform murine model based on adoptive transfer of bone marrow-derived DCs experiments in vivo for DCs pulsed with ovalbumin(OVA)-driven AR and asthma,which can leave the other inflammatory cells uninvoloved, to explore intranasal administration of CpG-ODN and/or BUD on brockade the progression from AR to bronchial asthma by analyzing not only the airway inflammation, bronchial hyperresponsiveness and airway remodeling of murine model, but also the underlying molecular and cellular pathways. In the present study, we aims to explain the natural processes pathogenesis of upper and lower airway inflammatory disease,to control asthma development from its source driver, and to explore the new treatment pathways for steroid-resistant asthma.

TSLP-DC-OX40L是哮喘Th2反应重要途径。我们前期研究证实激素预处理DC减轻哮喘Th2反应，抑制DC表型并下调TSLP受体，阻滞TSLP抑制气道重构。而变应性鼻炎（AR）与哮喘流行病学相关，干预AR有助哮喘控制，但TSIP-DC-OX40L途径在AR与哮喘发生发展中的作用尚不明确。预试验我们发现免疫佐剂TLR9 配体CpG-ODN以及激素（BUD）干预AR后减轻下呼吸道炎性反应，推测CpG-ODN、BUD可能调控上气道TSLP-DC-OX40L而阻滞AR发展为哮喘。为证实该假说，项目组拟复制AR/哮喘联合模型，采用DC过继转移排除其他炎症细胞参与，从整体-细胞-分子三层次探讨干预AR模型 TSLP-DC-OX40L途径对下呼吸道气道炎症、气道高反应性及气道重构影响。旨在阐明上下气道炎性疾病自然进程的发病机制，从源头调控哮喘发生发展的驱动因素，为激素抵抗哮喘治疗新途径提供理论依据。

本项目重点关注了TSIP-DC-OX40L途径在变应性鼻炎-哮喘综合征（CARAS）发生发展中的作用。变应性鼻炎（AR）和哮喘均为慢性气道炎症性疾病，流行病学密切相关，“变应性鼻炎-哮喘综合征”、“变应性联合气道疾病”等相关概念体现了两者的相关性及一致性。AR常先于哮喘发病，干预AR有助哮喘控制，但两者密切相关的分子学机制有待阐明。项目组制备了急性和慢性联合CARAS模型，探讨了CpG-ODN和/或激素干预AR后对下气道TSLP-DC-OX40L途径及发展为哮喘Th2反应、气道重塑等影响。项目组发现CpG-ODN鼻内应用较皮下注射更有效地通过抑制AR而阻滞CARAS鼠鼻部症状、上下气道炎性反应（HE染色示嗜酸性粒细胞浸润减少）、降低气道重塑（Masson染色示胶原增生缓解，免疫组化和免疫荧光示α-SMA及TGF-β1减少）和减低气道高反应性，且与激素具有协同性，其机制在于CpG-ODN抑制胸腺基质淋巴生成素(TSLP)-树突细胞(DC)-OX40L途径：CpG-ODN下调AR鼻粘膜和哮喘肺组织及支气管肺泡灌洗液中TSLP表达，抑制TSLP驱动的DC表达OX40L、CD86、CD80等共刺激分子、及其分泌IL-12 p70，从而减轻AR和哮喘的炎性反应、Th2优势反应和气道重塑的相关指标。临床研究中发现吸烟哮喘控制率低，项目组进一步动物实验研究发现CpG-ODN对烟草烟雾暴露后以嗜酸性和嗜中性粒细胞混合的气道炎症哮喘也可通过TSLP-DC途径抑制其Th2/Th17优势反应。项目组已发表相关SCI论著3篇和中文核心期刊2篇，已接收核心期刊1篇，待发表SCI论著1篇。本项目研究阐明了上下气道炎性疾病自然进程的发病机制，有助于从源头上调控哮喘发生发展的驱动因素，首创免疫佐剂CpG-ODN作为以TSLP-DC为主要靶点哮喘干预措施，为CpG-ODN鼻喷剂研发提供理论参考，为寻找早期、安全、有效的哮喘干预方式和难治性哮喘治疗提供了理论和实验依据。