Activation of Innate Immune Defense Mechanisms by Signaling through RIG-I/IPS-1 in Intestinal Epithelial Cells

Abstract

Intestinal epithelial cells (IECs) are a first line of defense against microbial pathogens that enter the host through the intestinal tract. Moreover, viral pathogens that infect the host via the intestinal epithelium are an important cause of morbidity and mortality. However, the mechanisms by which viral pathogens activate antiviral defense mechanisms in IECs are largely unknown. The synthetic dsRNA analog polyinosinic-polycytidylic acid and infection with live virus were used to probe the molecules that are activated and the mechanisms of signaling in virus-infected human IECs. Polyinosinic-polycytidylic acid activated IFN regulatory factor 3 dimerization and phosphorylation, increased activity of the IFN-stimulated response element, induced a significant increase in IFN-β mRNA transcripts and IFN-β secretion, and up-regulated the expression of IFN-regulated genes in IECs. Those responses were dependent upon activation of the dsRNA binding protein retinoic acid inducible gene I (RIG-I) and the RIG-I interacting protein IFN promoter stimulator-1, but not on dsRNA-activated protein kinase or TLR3, which also were expressed by IECs. Virus replication and virus-induced cell death increased in IECs in which RIG-I was silenced, consistent with the importance of the RIG-I signaling pathway in IEC antiviral innate immune defense mechanisms.

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

“…Response to poly(I-C) and dsRNA could also be mediated by the cytoplasmic viral sensors, the RNA helicases retinoic inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) (13,28). It was recently reported by Hirata et al that the recognition of poly(I-C) in intestinal epithelial cells is mediated by RIG-I but not by TLR3 (17). The different cell lines used may account for the conflicting findings: whereas, we used the HCT116 cell line, which expresses wild-type p53, Hirata et al utilized cell lines with mutated p53 (17).…”

“…It was recently reported by Hirata et al that the recognition of poly(I-C) in intestinal epithelial cells is mediated by RIG-I but not by TLR3 (17). The different cell lines used may account for the conflicting findings: whereas, we used the HCT116 cell line, which expresses wild-type p53, Hirata et al utilized cell lines with mutated p53 (17). We observed that HCT116 p53 ϩ/ϩ and p53 Ϫ/Ϫ cells showed the same expression levels of RIG-I and MDA-5, which indicated that the regulation of their expression might not be dependent on p53 (data not shown).…”

RIG-I/MDA5/MAVS Are Required To Signal a Protective IFN Response in Rotavirus-Infected Intestinal Epithelium

“…Definitive evidence for the role of RIG-I and MDA5 in signaling activated by dsRNA viruses was obtained using mice or cells from mice deficient in either RIG-I or MDA5 (21,30). Using an IEC line (HT-29), we observed that RIG-I was important for the activation of type I IFN production after cells were transfected with the dsRNA analog polyinosinic-polycytidylic acid [poly(I:C)], and this also appeared to be the case when those cells were infected with vesicular stomatitis virus or a strain of rotavirus (31).…”

The Nucleotide-Binding Oligomerization Domain-Like Receptor NLRC5 Is Involved in IFN-Dependent Antiviral Immune Responses

“…Transfection of siRNA was performed 24 h prior to transfection of luciferase vectors. Intracellular delivery of poly I:C was performed as previously described (19). Dual luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega) and a MicroLumatPlus Luminometer (Berthold Technologies, Bad Wildbad, Germany).…”