RNA-sequencing of bronchial epithelial cells from an adult cohort including asthmatics, COPD and healthy controls, cultured with Rhinovirus 1A

Rhinovirus infections exacerbate chronic respiratory inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the primary site of rhinovirus replication and responsible for initiating the host immune response to infection. Numerous studies have reported that the anti-viral innate immune response in asthma is deficient leading to the conclusion that epithelial innate immunity is a key determinant of disease severity during a rhinovirus induced exacerbation. However, deficient rhinovirus-induced epithelial interferon production in asthma has not always been observed. We hypothesised that disparate in vitro airway epithelial infection models lacking genome-wide, time-course analyses have obscured the role of epithelial innate anti-viral immunity in asthma and COPD. To address this, we developed a low multiplicity of infection (MOI) rhinovirus model of differentiated primary epithelial cells obtained from healthy, asthma and COPD donors. Using genome-wide gene expression following infection, we demonstrated that gene expression patterns are similar across patient groups, but that the kinetics of induction are delayed in cells obtained from asthma and COPD donors. Rhinovirus-induced innate immune responses were defined by interferons (type-I, II and III), interferon response factors (IRF1, IRF3 and IRF7), TLR signalling and NF-kB and STAT1 activation. Induced gene expression was evident at 24 hours and peaked at 48 hours post-infection in cells from healthy subjects. In contrast, in cells from donors with asthma or COPD induction was maximal at or beyond 72-96 hours post infection. Thus, we propose that propensity for viral exacerbations of asthma and COPD relate to delayed (rather than deficient) expression of epithelial cell innate anti-viral immune genes which in turns leads to a delayed and ultimately more inflammatory host immune response. Overall design: Three participant groups: asthmatics, COPD and healthy controls. Cultured with/without virus over a timecourse with four timepoints (24, 48,72 and 96). Paired virus-infected and mock infected samples at each timepoint for each subject.

鼻病毒感染可加重哮喘、慢性阻塞性肺疾病（COPD）这类慢性呼吸道炎症性疾病。气道上皮细胞是鼻病毒复制的主要场所，亦是宿主抗感染免疫应答的启动位点。多项研究已证实，哮喘患者的抗病毒固有免疫应答存在缺陷，据此研究者提出上皮固有免疫是鼻病毒诱发病情急性加重时疾病严重程度的关键决定因素。但目前并非总能在哮喘患者中观测到鼻病毒诱导的上皮细胞干扰素生成缺陷现象。本研究团队推测，此前缺乏全基因组覆盖与时序分析的体外气道上皮感染模型，掩盖了上皮固有抗病毒免疫在哮喘与COPD发病机制中的作用。为解决这一研究局限，我们建立了基于低感染复数（MOI）的鼻病毒感染模型，所用细胞为从健康人群、哮喘患者及COPD患者体内分离的分化型原代上皮细胞。通过对感染后样本开展全基因组基因表达分析，我们发现不同患者组的基因表达模式整体相似，但哮喘与COPD患者来源的细胞中，基因诱导的动力学过程存在延迟。鼻病毒诱导的固有免疫应答以干扰素（I型、II型与III型）、干扰素调节因子（IRF1、IRF3及IRF7）、Toll样受体（TLR）信号通路，以及NF-κB与STAT1的激活为核心特征。健康受试者来源的细胞中，诱导基因的表达在感染后24小时即可被检测到，并于感染后48小时达到峰值。与之相反，哮喘或COPD患者来源的细胞中，诱导基因的表达峰值出现在感染后72至96小时及以后。据此我们提出，哮喘与COPD患者易发生病毒诱发的病情急性加重，其根源在于上皮细胞固有抗病毒免疫基因的表达延迟（而非表达缺陷），这继而会导致宿主免疫应答启动延迟，并最终引发更为剧烈的炎症反应。实验整体设计：共设置三个受试者组别，分别为哮喘患者组、COPD患者组与健康对照组。对细胞开展病毒感染与假感染处理，设置四个时间节点（24、48、72及96小时）的时序实验。每位受试者在每个时间点均设有病毒感染样本与假感染配对样本。