Bhushan Bharat, Homma Tetsuya, Norton James E, Sha Quan, Siebert Jason, Gupta Dave S, Schroeder James W, Schleimer Robert P
1 Division of Otolaryngology-Head and Neck Surgery, Ann & Robert H. Lurie Children's Hospital of Chicago and the Northwestern University Feinberg School of Medicine, Chicago, Illinois.
2 Division of Allergy and Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
Am J Respir Cell Mol Biol. 2015 Jul;53(1):87-95. doi: 10.1165/rcmb.2014-0333OC.
Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6 hours. Our results show that AF extracts suppressed expression of inflammatory cytokines in association with inhibition of the IFN-β signaling pathway and suppression of the formation of pSTAT1.
烟曲霉（AF）在免疫缺陷个体中常具有致病性，可导致危及生命的感染，如侵袭性曲霉病。肺部上皮细胞对AF感染的反应及其相关信号通路尚未得到充分研究。将BEAS-2B细胞或原代人支气管上皮细胞暴露于AF提取物中，并用IFN-β或Toll样受体3激动剂双链RNA（dsRNA）进行刺激。评估细胞因子释放（肿瘤坏死因子家族的B细胞活化因子[BAFF]、IFN-γ诱导蛋白10[IP-10]等）。使用超滤4离心力过滤器将AF提取物分离为低分子量（LMW）和高分子量（HMW）组分，以表征其活性。采用TaqMan方法进行实时PCR，并使用ELISA技术进行蛋白质定量。进行蛋白质印迹以评估信号转导和转录激活因子1（STAT1）的磷酸化。IFN-β和dsRNA在BEAS-2B细胞中诱导BAFF（分别为350倍和452倍[n = 3]）和IP-10（分别为1081倍和3044倍[n = 3]）的信使核糖核酸（mRNA）表达。当细胞先用AF提取物预处理1小时，然后用IFN-β或dsRNA刺激6小时时，相对于单独用IFN-β和dsRNA产生的水平，BAFF和IP-10 mRNA的诱导受到强烈抑制。与对照相比，在用1:320重量/体积的AF提取物处理的dsRNA刺激孔中，可溶性BAFF和IP-10蛋白水平受到最大程度的抑制（P < 0.005）。在进行分子大小分级分离后，AF提取物的LMW组分对IP-10 mRNA表达没有可测量的抑制作用。然而，AF提取物的HMW组分显著抑制了用dsRNA或IFN-β刺激的BEAS-2B细胞中IP-10的表达。当BEAS-2B细胞先用AF提取物预处理，然后用IFN-β刺激时，观察到pSTAT1水平降低，在4小时和6小时时抑制作用最大。我们的结果表明，AF提取物与IFN-β信号通路的抑制和pSTAT1形成的抑制相关，从而抑制炎症细胞因子的表达。
