Abstract
BackgroundThe 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).MethodsThe 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.ResultsHere, 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.ConclusionsOur 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.
Highlights
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
MEK inhibitor (MEKi) reduces viral load in RV2 challenged airway epithelial cells (AECs) but not those challenged with RSVA2 The impact of inhibition of mitogen-activated protein kinase kinase (MEK) on viral load was assessed through the measure of total intracellular viral RNA (Fig. 1a)
Kit. c Cytotoxicity analysis of lactate dehydrogenase (LDH) release in the supernatants of AECs pretreated with DMSO, MEKi, or anti-IFN-β antibody for 1h followed by infection with or without RV2 or RSVA2 or UV-inactivated viruses for 24h (MOI 0.1). d Total RNA was extracted from AECs infected with RV2 for 2-6h (MOI 0.1)
Summary
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. The airway epithelium is directly exposed to respiratory viruses and provides a first-line of defence as a major component of the innate immune response system. This early immune response, which consists of inflammatory cytokines and interferons (IFN), is triggered upon infection by respiratory viruses [1], including rhinovirus (RV) or respiratory syncytial virus (RSV) both known to cause exacerbations in chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) [2]. The phosphoinositide-3kinase/protein kinase B (PI3K/AKT) pathway is a critical determinant of type I IFN response and the recruitment of PI3K following TLR3 and RIG-I activation significantly contributes to IRF3 activation and subsequent transcriptional activation of IFNs [6,7,8,9]. It has been proposed that an interaction between AKT and TBK1 is essential for optimal activation of IRF3 [10]
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