Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol

Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin.几丁质（chitin）由真菌、昆虫及其他多种生物大量合成，且与哮喘的发病机制密切相关。气道上皮细胞（airway epithelial cells）可直接接触环境颗粒物，作为抵御吸入性过敏原与病原体的第一道防线。然而气道上皮细胞在几丁质诱导的哮喘发病过程中的潜在贡献仍有待阐明。我们提出假说：几丁质可直接刺激气道上皮细胞释放促进2型免疫应答的细胞因子，并诱导先天免疫应答中发挥重要作用的分子表达。研究发现，在转化型人支气管上皮细胞BEAS-2B以及肺癌细胞A549、H292中，几丁质暴露可快速诱导三种关键的2型免疫促进型细胞因子——白细胞介素25（IL-25）、白细胞介素33（IL-33）及胸腺基质淋巴细胞生成素（TSLP）的表达。几丁质还可诱导关键模式识别受体Toll样受体2（TLR2）与Toll样受体4（TLR4）的表达。此外，几丁质可诱导miR-155（microRNA-155）、miR-146a（microRNA-146a）及miR-21（microRNA-21）的表达，上述三种微小RNA均已被证实可上调促炎细胞因子的表达；同时，miR-155的已知靶标——细胞因子信号抑制因子1（SOCS1）及SH2结构域包含肌醇5'-磷酸酶1（SHIP1）的表达可被几丁质处理所抑制。单萜酚类化合物香芹酚（carvacrol, Car）及其异构体百里香酚（thymol, Thy）存在于草本植物精油中，且已被证实可在哮喘模型中抑制过敏性炎症。我们发现，香芹酚/百里香酚可抑制几丁质对2型免疫促进型细胞因子释放的调控作用，以及对Toll样受体、SOCS1、SHIP1及微小RNA的表达诱导作用。香芹酚/百里香酚还可有效降低TLR4的蛋白水平，抑制几丁质联合香芹酚/百里香酚处理细胞中TLR2蛋白水平的升高，并上调TLR介导的炎症应答负调控因子SHIP1及SOCS1的蛋白水平。综上，几丁质对气道上皮细胞的直接作用可能参与哮喘等过敏性气道疾病的发病进程，而香芹酚/百里香酚可直接抑制气道上皮细胞针对几丁质的促炎应答。