Human rhinoviruses (HRVs) are associated with acute exacerbations in patients with chronic obstructive pulmonary disease (COPD) and asthma, which are accompanied by mucus hypersecretion. Whereas, various studies have shown that HRVs increase epithelial mucin production and thus may directly contribute to mucus hypersecretion. The effects of drugs used in the treatment of COPD and asthma on HRV-induced mucin production in epithelial cell cultures have not been studied. In the present study, we assessed effects of HRVs on mucin production and secretion in well-differentiated primary human bronchial epithelial cells (PBEC) and studied the effect of the inhaled corticosteroid fluticasone propionate and the long-acting muscarinic antagonist tiotropium bromide on this process. Differentiated PBEC that were cultured at the air-liquid interface (ALI-PBEC) were infected with HRV-A16 and HRV-1B. Quantitative PCR, immunofluorescence staining, ELISA, periodic acid-Schiff (PAS) staining and immunostaining assays were used to assess the effects of HRV infection. Here we demonstrate that both HRV-A16 and HRV-1B increased mucin (MUC5AC and MUC5B) gene expression and protein release. When exploring this in more detail in HRV-A16-infected epithelial cells, mucin expression was found to be accompanied by increases in expression of SAM-pointed domain-containing Ets-like factor (SPDEF) and SPDEF-regulated genes known to be involved in the regulation of mucin production. We also found that pre-treatment with the purinergic P2R antagonist suramin inhibits HRV-enhanced MUC5AC expression and protein release, implicating involvement of purinergic signaling by extracellular ATP. We furthermore found that both fluticasone and tiotropium decreased HRV-induced mucin production without affecting viral replication, and obtained evidence to suggest that the inhibitory effect of fluticasone involved modulation of SPDEF-regulated genes and extracellular ATP release. These data show that both tiotropium and fluticasone inhibit HRV-induced epithelial mucin production independent of viral clearance, and thus provide insight into the mechanisms underlying beneficial effects of tiotropium and fluticasone in the treatment of COPD, asthma and accompanying exacerbations in these patients. Furthermore, our findings provide additional insight into the mechanisms by which HRV increases epithelial mucin production.

人鼻病毒（HRV）与慢性阻塞性肺疾病（COPD）和哮喘患者的急性加重相关，并伴有粘液分泌过多。然而，各种研究表明，HRV可增加上皮粘蛋白的产生，因此可能直接导致粘液分泌过多。尚未研究用于治疗COPD和哮喘的药物对HRV诱导的上皮细胞培养物中粘蛋白产生的影响。在本研究中，我们评估了HRVs对分化良好的原代人支气管上皮细胞（PBEC）中粘蛋白产生和分泌的影响，并研究了吸入皮质类固醇氟替卡松丙酸酯和长效毒蕈碱拮抗剂噻托溴铵在此过程中的作用。在气液界面（ALI-PBEC）上培养的分化PBEC感染了HRV-A16和HRV-1B。定量PCR，免疫荧光染色，ELISA，高碘酸-希夫（PAS）染色和免疫染色法用于评估HRV感染的影响。在这里，我们证明了HRV-A16和HRV-1B均可增加粘蛋白（MUC5AC和MUC5B）的基因表达和蛋白质释放。当在受HRV-A16感染的上皮细胞中进行更详细的研究时，发现粘蛋白表达伴随着SAM指向含域结构域的Ets样因子（SPDEF）和SPDEF调控的基因的表达增加，这些基因已知与之相关。粘蛋白生产的调节。我们还发现，用嘌呤能P2R拮抗剂苏拉明预处理可抑制HRV增强的MUC5AC表达和蛋白质释放，暗示嘌呤能信号传导参与细胞外ATP。我们还发现，氟替卡松和噻托溴铵都降低了HRV诱导的粘蛋白生成，而不影响病毒复制，并获得证据表明氟替卡松的抑制作用涉及SPDEF调控基因的调节和细胞外ATP的释放。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。我们还发现，氟替卡松和噻托溴铵都降低了HRV诱导的粘蛋白生成，而不影响病毒复制，并获得证据表明氟替卡松的抑制作用涉及SPDEF调控基因的调节和细胞外ATP的释放。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。我们还发现，氟替卡松和噻托溴铵都降低了HRV诱导的粘蛋白生成，而不会影响病毒复制，并获得证据表明氟替卡松的抑制作用涉及SPDEF调控基因的调节和细胞外ATP的释放。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。并获得证据表明氟替卡松的抑制作用涉及SPDEF调控基因的调节和细胞外ATP的释放。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。并获得证据表明氟替卡松的抑制作用涉及SPDEF调控基因的调节和细胞外ATP的释放。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。这些数据表明，噻托溴铵和氟替卡松均抑制HRV诱导的上皮粘蛋白生成，而与病毒清除率无关，从而提供了噻托溴铵和氟替卡松在治疗这些患者的COPD，哮喘和加重病情中潜在作用的潜在机制的见解。此外，我们的发现为HRV增加上皮粘蛋白产生的机制提供了更多的见解。