Role of endosomal and cytosolic pattern recognition receptors in dsRNA-induced cytokine expression in human airway smooth muscle cells

Background: Rhinovirus infections are major triggers of asthma exacerbations. Double-stranded RNA (dsRNA), a viral replication intermediate, is sensed by pattern recognition receptors (PRRs): endosomal TLR3 and cytosolic RIG-I-like receptors (RLRs). dsRNA induces cytokine expression in airway smooth muscle cells (ASMCs) via TLR3, but little is known about involvement of RLRs. Objective: Investigate if both TLR3 and RLRs are involved in dsRNA-induced cytokine expression in ASMCs. Methods: Cultured primary human ASMCs (n=8) were stimulated with dsRNA (10μg/ml) or dsRNA in complex with LyoVec (dsRNA/LV; 0,5μg/ml) that selectively distributes to the cytosol and activates RLRs. mRNA expression (3 and 24h) and protein production (24h) were analysed by RT-qPCR and ELISA respectively. Results: At 3h, dsRNA induced significantly higher mRNA expression of IL-8, TNFα and IFNβ (p<0.001) than dsRNA/LV (p<0.05). By 24h, TNFα and IFNβ expression returned towards baseline in dsRNA-treated cells while IL-8 further increased, as did all cytokines in dsRNA/LV-treated cells, particularly IFNβ (p<0.01). Chloroquine, an endosomal TLR3 inhibitor, reduced only dsRNA-induced effects. dsRNA produced more IL-8, but equal IFNβ protein compared to dsRNA/LV. dsRNA and dsRNA/LV also upregulated RLR (RIG-I and MDA5) mRNA expression at 24h (p<0.01). Conclusions: TLR3 largely mediated the prompt cytokine response to dsRNA in ASMCs. RLRs also played a role, especially in 24h antiviral IFNβ production. Upregulation of RIG-I and MDA5 further suggests involvement of cytosolic PRRs in addition to endosomal TLR3 and supports the immunomodulatory function of ASMCs in virus-infected airways.