瘦素调控Th17细胞分化促进肥胖型哮喘糖皮质激素抵抗的机制研究

Obesity-associated asthma is a distinct phenotype of asthma. Patients of obesity-associated asthma tend to present with severe clinical manifestations and resistance to glucocorticoid therapy, but the underlying molecular mechanisms remain unknown yet. It is well recognized that Th17 cells play a critical role in glucocorticoid resistance of asthma patients through inducing neutrophilic inflammation in airway. Interestingly, our previous studies demonstrated that Exogenous recombinant human leptin stimulated the differentiation of Th17 cells in human peripheral blood, and IL-17 concentrations in the sputum and plasma of patients with obesity-associated asthma were significantly higher compared with non-obese asthmatic patients, which suggested that hyperleptinemia in patients of obesity-associated asthma could promote the differentiation of Th17 cells and may be the molecular basis for glucocorticoid resistance. In this project, we aim to perform a clinical observational study and analyze the correlation between plasma leptin level and the sensitivity to glucocorticoid therapy in asthmatic patients. Furthermore, by using pharmacological intervention and gene knockout technique, we will investigate how the activation or inhibition of leptin and its downstream pathways regulate Th17 cells differentiation, airway inflammation and glucocorticoid response in vitro and in vivo. In summary, we try to elucidate the regulatory role of leptin in the glucocorticoid resistance of obese-associated asthma and its underlying molecular mechanism, thus providing novel therapeutic targets for obesity-associated asthma.

肥胖型哮喘是一种特殊的哮喘表型，因其严重的临床表现与对糖皮质激素治疗的抵抗性而备受关注，但其发生机制尚未明确。已知Th17细胞通过诱导气道中性粒细胞性炎症在哮喘患者的糖皮质激素抵抗中发挥重要作用。而我们的前期研究发现，外源性的瘦素刺激可促进人外周血T淋巴细胞向Th17细胞分化，且肥胖型哮喘患者的痰与血浆标本的IL-17浓度明显高于非肥胖型哮喘患者。提示肥胖型哮喘患者体内的高水平瘦素促进Th17细胞分化，可能是肥胖型哮喘糖皮质激素抵抗表型的细胞免疫学基础。本项目拟通过临床观察性研究，分析哮喘患者体内瘦素水平与其对糖皮质激素治疗敏感性之间的相关性；同时，运用药理学干预与基因敲除技术，通过细胞实验和肥胖型哮喘动物模型的构建，探索激活或抑制瘦素及其下游通路对Th17细胞分化、气道炎症反应及糖皮质激素治疗反应的影响。据此阐明瘦素在肥胖型哮喘糖皮质激素抵抗表型中的作用及分子机制，以期为肥胖型哮喘的临床治疗提供新思路。

肥胖相关性哮喘是一种气道中性粒细胞过多浸润的非过敏性哮喘。肥胖可通过分泌瘦素等脂肪相关因子，在慢性炎症反应中起到至关重要的作用。然而，瘦素/瘦素受体(obR)信号通路如何参与肥胖相关哮喘中性粒细胞气道炎症的发病机制尚不明确。我们使用高脂饲料诱导小鼠肥胖，使用LPS/OVA致敏和OVA激发建立了中性粒细胞气道炎症小鼠模型。组织病理学分析（苏木精和伊红(H&E)、支气管肺泡灌洗液(BALF)炎症细胞计数和肺部炎症细胞因子用于分析气道炎症的严重程度。使用蛋白质印迹、流式细胞术、定量实时逆转录聚合酶链反应和酶联免疫吸附测定(ELISA)来评估潜在机制。使用骨髓来源的巨噬细胞(BMDMs)和骨髓来源的中性粒细胞进行体外实验。我们发现，肥胖小鼠血清瘦素水平明显高于痩小鼠。在中性粒细胞气道炎症小鼠中，与瘦小鼠相比，肥胖小鼠支气管周围炎症、BALF中性粒细胞浸润、肺部Th1/Th17相关炎症因子(IFN-γ、TNF-α、IL-1β、IL-6 和 IL-17A)，肺部M1巨噬细胞极化而非M2巨噬细胞极化，长型瘦素受体磷酸化水平明显增加。瘦素受体的抑制剂或瘦素受体的缺失可显着逆转肥胖相关中性粒细胞气道炎症小鼠模型BALF中的中性粒细胞浸润、支气管周围气道炎症、肺部Th1/Th17相关炎症因子和肺部M1巨噬细胞极化。巨噬细胞耗竭和过继转移研究表明，肥胖小鼠中性粒气道炎症与瘦小鼠中性粒气道炎症的严重程度的差异与促炎型M1巨噬细胞有直接相关性。体外实验证实瘦素可协同LPS/IFN-γ促进长型瘦素受体的磷酸化以及上调JNK/STAT3/AKT信号通路磷酸化来增加M1巨噬细胞的活化，此作用可被瘦素受体及相应通路抑制剂所逆转。此外，瘦素/瘦素受体信号通路通过促进M1巨噬细胞分泌CXCL2，从而促进了中性粒细胞的募集。临床研究显示，与非肥胖相关哮喘患者相比，肥胖相关哮喘患者血清中瘦素水平更高，诱导痰中M1巨噬细胞数量而非M2巨噬细胞数量升高；且哮喘患者诱导痰M1巨噬细胞数量与血清瘦素的含量成正相关。我们的研究结果表明，瘦素/瘦素受体信号通过促进M1巨噬细胞极化，在肥胖相关中性粒细胞气道炎症的发病机制中发挥重要的作用。

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