Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils

Purified blood neutrophils were transmigrated in vitro as previously described (Dobosh et al, STAR Methods) using ALI/ARDS mBAL supernatant obtained by mechanical dissociation on ice using an 18G needle and syringe, followed by differential centrifugation at 800g and 3,000g to obtain the cell- and bacteria-free supernatant. A2 neutrophils were transmigrated, as described above, into ALI/ARDS mBAL supplemented with either 10𝜇g/mL of IgG control (Biolegend) or 10𝜇g/mL of Anifrolumab (anti-IFNAR1, Creative biolabs). Treatment with IgG control antibody or Anifrolumab was continued at the same concentration for the first hour of incubation for SARS-CoV-2. RNA from non infected neutrophils was isolated using the Nucleospin RNA kit (Takara). RNA isolated from in vitro samples was sequenced on the Illumina NextSeq500 at 75bp paired-end with a target of 20 million reads per sample. FASTQ files were checked for quality and raw sequencing data was aligned to the human reference genome (GRCh38) using STAR (Version 2.5.2) and quantmode was used to generate raw transcript counts.参照此前文献（Dobosh 等，《STAR Methods》）的方法开展体外跨迁移实验：将纯化的血液中性粒细胞（neutrophils）以急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）小鼠支气管肺泡灌洗液（mBAL）上清液作为迁移诱导介质，该上清液的制备流程为：使用18G针头与注射器在冰上进行机械解离，随后以800g和3000g先后实施差速离心，最终获得无细胞且无细菌的上清液。将A2型中性粒细胞按照上述方法进行跨迁移，所用的ALI/ARDS mBAL上清液分别添加了10μg/mL的IgG同型对照抗体（IgG control，Biolegend）或10μg/mL的阿尼鲁单抗（Anifrolumab，抗IFNAR1抗体，Creative Biolabs）。在针对新型冠状病毒（SARS-CoV-2）的孵育初始1小时内，继续以相同浓度添加IgG对照抗体或阿尼鲁单抗进行处理。使用Nucleospin RNA提取试剂盒（Takara）从未感染的中性粒细胞中提取总RNA。从体外实验样本中提取的RNA，采用Illumina NextSeq500测序平台进行75碱基对双端测序，设定每个样本的目标测序读段数为2000万。对FASTQ格式文件进行质量质控，使用STAR（版本2.5.2）将原始测序数据比对至人类参考基因组（GRCh38），并通过定量模式（quantmode）生成原始转录本计数结果。