Role of RIG-I, MDA-5, and PKR on the Expression of Inflammatory Chemokines Induced by Synthetic dsRNA in Airway Epithelial Cells

Abstract

Background: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and reported that dsRNA stimulates expression of inflammatory chemokines through a receptor of dsRNA Toll-like receptor (TLR) 3 in airway epithelial cells. In this study, we focused our study on the role of other receptors for dsRNA, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and double-stranded RNA-dependent protein kinase (PKR). Methods: Airway epithelial cell BEAS-2B was cultured in vitro. Expression of target RNA and protein were analyzed by PCR and ELISA. To analyze the role of receptors for dsRNA, knockdown of theses genes was performed with short interfering RNA (siRNA). Results: We first investigated the effects of chloroquine, an inhibitor of lysosomal acidification, on the expression of chemokines. Preincubation with 100 µM chloroquine significantly inhibited the expression of mRNA for RANTES, IP-10, and IL-8, stimulated by poly I:C, indicating that poly I:C may react with a receptor expressed inside the cells. RIG-I, MDA-5, and PKR are supposed to be expressed inside the airway epithelial cells. However, the expression of chemokines stimulated with poly I:C was not significantly inhibited for these putative receptors in the cells which were transfected with siRNA. Conclusions: Synthetic dsRNA poly I:C stimulates the expression of inflammatory chemokines in airway epithelial cells, but the putative receptors for dsRNA such as RIG-I, MDA-5, or PKR may not play pivotal roles in this process. TLR3 may play a major role as reported previously.

Rhinovirus-Induced Barrier Dysfunction in Polarized Airway Epithelial Cells Is Mediated by NADPH Oxidase 1

“…A synthetic dsRNA, poly(I:C), has been used extensively as a surrogate for viral dsRNA to study host responses. It provokes inflammatory and interferon (IFN) responses in airway epithelial cells, similar to viral dsRNA (4,30,49). Recently, dsRNA was also shown to decrease R T in immortalized nasal epithelial cells (35), but the underlying mechanism was not elucidated.…”

“…We demonstrate that RV-induced Rac1-dependent NOX1 activity is required to disrupt barrier function. We also show that polyinosinic-poly(C) [poly(I:C)], a synthetic analog of double-stranded RNA (dsRNA) that has been used to examine viral dsRNA-stimulated responses in airway epithelial cells (26,30,49), impairs barrier function by a mechanism similar to that of RV, indicating that dsRNA generated during replication contributes to the disruption of barrier function.…”

p53 Regulates Toll-Like Receptor 3 Expression and Function in Human Epithelial Cell Lines

“…4E), which demonstrated the essential role of TLR3 in the cell's response to poly(I-C). Several studies have confirmed the importance of TLR3, RIG-I, or MDA-5 in poly(I-C) recognition (19,21,26,27), and the question of whether RIG-I or TLR3 predominantly recognizes poly(I-C) in cells remains unresolved. Based on our results that p53 affects the expression of TLR3 but not that of MDA-5 or RIG-I, it is tempting to speculate that the activation of these pattern recognition receptors by poly(I-C) may, in part, depend on the p53 status of cells or tissues.…”

IL-10-Dependent S100A8 Gene Induction in Monocytes/Macrophages by Double-Stranded RNA

“…Expression was observed in our mice early in infection and was maximal at day 8 when strong immunoreactivity in epithelial cells lining the airways and in mononuclear cells was obvious. Pulmonary epithelial cells express TLR3 (46,68) and PKR (46,68,69). S100A8 is up-regulated in bronchial epithelial cells by LPS (3) and S100A8/S100A9 is expressed in tracheal epithelial cells from patients with cystic fibrosis (70).…”