Acute viral bronchiolitis (PBMC)

Background: A subset of infants are hyper-susceptible to severe/acute viral bronchiolitis (AVB), for reasons unknown. Purpose: To characterise the cellular/molecular mechanisms underlying infant AVB in circulating cells/local airways tissues. Methods: PBMC and nasal mucosal scrapings (NMS) were obtained from Infants (<18mths) and children (1.5-5yrs) during AVB and post-convalescence. Immune response patterns were profiled by multiplex analysis of plasma cytokines, flow cytometry, and transcriptomics (RNA-Seq). Molecular profiling of group-level data utilised a combination of upstream regulator and coexpression network analysis, followed by individual subject-level data analysis employing personalised N-of-1-pathways methodology. Results: Group-level analyses demonstrated that infant PBMC responses were dominated by monocyte-associated hyper-upregulated type I interferon signalling/pro-inflammatory pathways (drivers: TNF, IL6, TREM1, IL1B), versus a combination of inflammation (PTGER2, IL6) plus growth/repair/remodelling pathways (ERBB2, TGFB1, AREG, HGF) coupled with Th2 and NK-cell signalling in children. Age-related differences were not attributable to differential steroid usage or variations in underlying viral pathogens. Nasal mucosal responses were comparable qualitatively in infants/children, dominated by interferon types I-III, but the magnitude of upregulation was higher in infants (range 6-48-fold) than children (5-17-fold). N-of-1-pathways analysis confirmed differential upregulation of innate immunity in infants and NK cell networks in children, and additionally demonstrated covert AVB response sub-phenotypes that were independent of chronological age. Conclusions: Dysregulated expression of interferon-dependent pathways following respiratory viral infections is a defining immunophenotypic feature of AVB-susceptible infants and a subset of children. Susceptible subjects appear to represent a discrete subgroup who cluster based on (slow) kinetics of postnatal maturation of innate immune competence. Overall design: The study design consisted of PBMC from infants (<18months, n=15 pairs) and pre-school children (2-5yrs, n=16 pairs) sampled during severe acute viral bronchiolitis (acute visit = AV) and following recovery during convalescence (convalescent visit = CV). RNA-Seq profiles were generated by sequencing llumina HiSeq2500, 50bp single-end reads, v4 chemistry. Samples were sequenced across two lanes and collapsed prior analysis.

研究背景：部分婴儿对重症/急性病毒性细支气管炎（acute viral bronchiolitis, AVB）存在超高易感性，但其具体致病机制尚未阐明。
研究目的：旨在解析循环血细胞与局部气道组织中，婴儿罹患急性病毒性细支气管炎的细胞及分子机制。
研究方法：本研究于急性病毒性细支气管炎发病期与康复期，分别从婴儿（年龄<18月龄）与儿童（年龄1.5~5岁）体内采集外周血单个核细胞（peripheral blood mononuclear cell, PBMC）与鼻黏膜刮取物（nasal mucosal scrapings, NMS）。通过血浆细胞因子多重检测、流式细胞术及转录组测序（RNA sequencing, RNA-Seq）对免疫应答特征进行解析。针对群体水平数据的分子特征分析，采用上游调控因子分析与共表达网络分析相结合的策略；随后针对个体水平数据，采用个性化单个体通路分析（N-of-1-pathways）方法开展数据分析。
研究结果：群体水平分析结果显示，婴儿外周血单个核细胞的应答以单核细胞相关的高上调I型干扰素信号通路与促炎通路为主（核心调控因子：肿瘤坏死因子（tumor necrosis factor, TNF）、白细胞介素6（interleukin 6, IL6）、髓系细胞触发受体1（triggering receptor expressed on myeloid cells 1, TREM1）、白细胞介素1β（interleukin 1β, IL1B））；而学龄前儿童的应答则同时涵盖炎症通路（前列腺素E受体2（prostaglandin E receptor 2, PTGER2）、IL6）与生长/修复/重塑通路（红细胞肉瘤病毒癌基因同源物2（erythroblastic leukemia viral oncogene homolog 2, ERBB2）、转化生长因子β1（transforming growth factor β1, TGFB1）、双调蛋白（amphiregulin, AREG）、肝细胞生长因子（hepatocyte growth factor, HGF）），并伴随Th2与NK细胞信号通路活化。年龄相关的差异并非由糖皮质激素使用差异或潜在病毒病原体种类差异所引起。婴儿与儿童的鼻黏膜应答在定性层面具有一致性，均以I型、III型干扰素通路为主，但婴儿的通路上调幅度（6~48倍）高于儿童（5~17倍）。单个体通路分析（N-of-1-pathways）证实，婴儿体内先天免疫通路存在差异性上调，儿童体内则以NK细胞网络通路上调为主；此外还发现了与实际年龄无关的隐匿性急性病毒性细支气管炎应答亚表型。
研究结论：呼吸道病毒感染后干扰素依赖通路的表达失调，是急性病毒性细支气管炎易感婴儿的标志性免疫表型特征，同时也存在于部分易感儿童群体中。易感个体可归为一个独立亚群，其聚类特征与先天免疫功能的产后成熟缓慢动力学特征相关。
研究设计：本研究采集了两组样本：婴儿组（年龄<18月龄，共15对样本，即发病就诊期与康复随访期各1份）与学龄前儿童组（年龄2~5岁，共16对样本），分别于重症急性病毒性细支气管炎发病就诊期（acute visit, AV）与康复随访期（convalescent visit, CV）采集其外周血单个核细胞。转录组测序采用Illumina HiSeq2500平台，以50bp单端读长、v4测序化学试剂完成建库与测序。样本在两个测序泳道上完成测序，后续分析前对数据进行合并处理。