Early Respiratory, Inflammation & Autoimmunity, R&D BioPharmaceuticals, AstraZeneca, Gothenburg, Sweden.
Precision Medicine, R&D Oncology, AstraZeneca, Gothenburg, Sweden.
Cell Commun Signal. 2019 Jul 18;17(1):78. doi: 10.1186/s12964-019-0378-7.
The airway epithelium is a major target tissue in respiratory infections, and its antiviral response is mainly orchestrated by the interferon regulatory factor-3 (IRF3), which subsequently induces type I (β) and III (λ) interferon (IFN) signalling. Dual specificity mitogen-activated protein kinase kinase (MEK) pathway contributes to epithelial defence, but its role in the regulation of IFN response in human primary airway epithelial cells (AECs) is not fully understood. Here, we studied the impact of a small-molecule inhibitor (MEKi) on the IFN response following challenge with two major respiratory viruses rhinovirus (RV2) and respiratory syncytial virus (RSVA2) and a TLR3 agonist, poly(I:C).
The impact of MEKi on viral load and IFN response was evaluated in primary AECs with or without a neutralising antibody against IFN-β. Quantification of viral load was determined by live virus assay and absolute quantification using qRT-PCR. Secretion of cytokines was determined by AlphaLISA/ELISA and expression of interferon-stimulated genes (ISGs) was examined by qRT-PCR and immunoblotting. A poly(I:C) model was also used to further understand the molecular mechanism by which MEK controls IFN response. AlphaLISA, siRNA-interference, immunoblotting, and confocal microscopy was used to investigate the effect of MEKi on IRF3 activation and signalling. The impact of MEKi on ERK and AKT signalling was evaluated by immunoblotting and AlphaLISA.
Here, we report that pharmacological inhibition of MEK pathway augments IRF3-driven type I and III IFN response in primary human AECs. MEKi induced activation of PI3K-AKT pathway, which was associated with phosphorylation/inactivation of the translational repressor 4E-BP1 and activation of the protein synthesis regulator p70 S6 kinase, two critical translational effectors. Elevated IFN-β response due to MEKi was also attributed to decreased STAT3 activation, which consequently dampened expression of the transcriptional repressor of IFNB1 gene, PRDI-BF1. Augmented IFN response translated into inhibition of rhinovirus 2 replication in primary AECs but not respiratory syncytial virus A2.
Our findings unveil MEK as a key molecular mechanism by which rhinovirus dampens the epithelial cell's antiviral response. Our study provides a better understanding of the role of signalling pathways in shaping the antiviral response and suggests the use of MEK inhibitors in anti-viral therapy against RV.
呼吸道上皮细胞是呼吸道感染的主要靶组织，其抗病毒反应主要由干扰素调节因子 3（IRF3）协调，随后诱导 I 型（β）和 III 型（λ）干扰素（IFN）信号。双特异性丝裂原激活蛋白激酶激酶（MEK）通路有助于上皮防御，但它在调节人原代气道上皮细胞（AEC）中的 IFN 反应中的作用尚不完全清楚。在这里，我们研究了一种小分子抑制剂（MEKi）对两种主要呼吸道病毒鼻病毒（RV2）和呼吸道合胞病毒（RSVA2）以及 TLR3 激动剂聚肌苷酸（poly[I:C]）刺激后 IFN 反应的影响。
在存在或不存在针对 IFN-β的中和抗体的情况下，用 MEKi 处理原代 AEC 以评估病毒载量和 IFN 反应。通过实时病毒测定法和绝对定量 qRT-PCR 测定病毒载量。通过 AlphaLISA/ELISA 测定细胞因子的分泌，并通过 qRT-PCR 和免疫印迹法检测干扰素刺激基因（ISGs）的表达。还使用 poly（I:C）模型进一步了解 MEK 控制 IFN 反应的分子机制。使用 AlphaLISA、siRNA 干扰、免疫印迹法和共聚焦显微镜法研究 MEKi 对 IRF3 激活和信号转导的影响。通过免疫印迹法和 AlphaLISA 评估 MEKi 对 ERK 和 AKT 信号转导的影响。
在这里，我们报告称，MEK 通路的药理学抑制增强了原代人 AEC 中 IRF3 驱动的 I 型和 III 型 IFN 反应。MEKi 诱导 PI3K-AKT 通路的激活，这与翻译抑制剂 4E-BP1 的磷酸化/失活以及蛋白质合成调节剂 p70 S6 激酶的激活有关，这两个关键的翻译效应子。由于 MEKi 导致 IFN-β反应增加，还归因于 STAT3 激活减少，从而降低了 IFNB1 基因转录抑制因子 PRDI-BF1 的表达。增强的 IFN 反应转化为抑制原代 AEC 中的鼻病毒 2 复制，但对呼吸道合胞病毒 A2 没有作用。
我们的发现揭示了 MEK 是鼻病毒抑制上皮细胞抗病毒反应的关键分子机制。我们的研究提供了对信号通路在塑造抗病毒反应中的作用的更好理解，并表明使用 MEK 抑制剂治疗 RV 抗病毒治疗。
