Single-cell heterogeneity in interferon induction potential is heritable and governed by variation in cell state II

Type I and III interferons (IFNs) are among the first lines of defense against viral infections, yet they are generally only produced by a tiny fraction of infected cells. Here, we show that variability in tonic cell signaling significantly influences cells' ability to produce IFN upon stimulation with the synthetic double-stranded RNA, polyinosinic:polycytidylic acid (pIC). Using single-cell approaches, we found that members of the activator protein (AP)-1 transcription factor were implicated in IFNL1 expression predisposition. This guided us to investigate the role of the mitogen-activated protein kinase (MAPK) pathway, specifically the c-Jun N-terminal kinase (JNK), in IFNL1 production. We found that inhibition of JNK signaling severely altered the nature of the innate antiviral response upon pIC stimulation, independently of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Together, our study emphasizes the influence of intrinsic stochasticity in cell state on heterogeneity in IFN expression. Overall design: Bulk RNA-seq of A549 cells treated with ruxolitinib, JNK-IN-8, both inhibitors, or DMSO. Cells were pretreated with the inhibitors for 4 hours before either being treated with pIC or only lipofectamine. The cells were then incubated for 16 hours then the RNA was extracted for sequencing.

I型和III型干扰素(IFNs)是对抗病毒感染的第一道防线之一，但通常仅由极少数受感染细胞产生。本研究发现，基础细胞信号传导的异质性可显著影响细胞在受到合成双链RNA聚肌胞苷酸（polyinosinic:polycytidylic acid，pIC）刺激后产生干扰素的能力。通过单细胞实验方法，我们发现激活蛋白(AP)-1转录因子家族与IFNL1基因的表达易感性相关，这引导我们进一步探究丝裂原活化蛋白激酶(MAPK)通路，尤其是c-Jun氨基末端激酶(JNK)在IFNL1产生过程中的作用。研究结果显示，抑制JNK信号通路会显著改变pIC刺激下的先天抗病毒应答性质，且该调控过程不依赖贾纳斯激酶/信号转导与转录激活子(JAK/STAT)通路。综上，本研究强调了细胞固有状态的随机性对干扰素表达异质性的重要影响。整体实验设计：对A549细胞开展批量RNA测序(Bulk RNA-seq)，处理分组包括：鲁索替尼(ruxolitinib)处理组、JNK-IN-8处理组、两种抑制剂联合处理组，以及二甲基亚砜(DMSO)对照组。细胞先经抑制剂预处理4小时，随后分别接受pIC刺激或仅用脂质体转染试剂(lipofectamine)处理，继续孵育16小时后提取RNA，用于测序分析。