Academic Unit of Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Sir Henry Wellcome Laboratories, South Block, University Hospital Southampton, Southampton, United Kingdom.
PLoS One. 2013 Aug 23;8(8):e71278. doi: 10.1371/journal.pone.0071278. eCollection 2013.
Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.
致敏和暴露于变应性真菌链格孢属与哮喘和哮喘恶化的风险增加有关。首先遇到吸入过敏原的细胞是气道黏膜表面的上皮细胞。上皮屏障功能先前被报道在哮喘中存在缺陷。本研究调查了链格孢属蛋白酶对上皮屏障功能和炎症反应的贡献，并比较了来自严重哮喘供体和非哮喘对照的体外分化支气管上皮细胞培养物的反应。将极化的 16HBE 细胞或来自非哮喘或严重哮喘供体的气液界面 (ALI) 支气管上皮培养物用链格孢属提取物进行顶端挑战，并测量炎症细胞因子释放和跨上皮电阻 (TER) 的变化。对链格孢属提取物中的蛋白酶活性进行了表征，并使用蛋白酶抑制剂和热处理来检查选择性抑制蛋白酶活性对上皮反应的影响。在 16HBE 细胞中，链格孢属提取物刺激白细胞介素-8 和肿瘤坏死因子-α的释放，并伴有 TER 的降低；这些作用可通过提取物的热处理来预防。对蛋白酶抑制剂作用的研究表明，丝氨酸蛋白酶是介导这些作用的主要蛋白酶类。与健康对照组相比，来自哮喘供体的 ALI 培养物对链格孢属的 IL-8 反应降低，而 TSLP 释放均未增加。只有来自哮喘供体的培养物易受链格孢属的屏障减弱作用的影响。因此，严重哮喘个体的支气管上皮可能更容易受到链格孢属的有害影响。
