title: Protein kinase R mediates intestinal epithelial gene remodeling in response to double-stranded RNA and live rotavirus.
abstract: As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.
authors.affiliation: Department of Pathology and Laboratory Medicine, Epithelial Pathobiology Unit, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Matam
authors.initials: M
authors.lastname: Vijay-Kumar
authors.forename: Jon R
authors.initials: JR
authors.lastname: Gentsch
authors.forename: William J
authors.initials: WJ
authors.lastname: Kaiser
authors.forename: Niels
authors.initials: N
authors.lastname: Borregaard
authors.forename: Margaret K
authors.initials: MK
authors.lastname: Offermann
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Neish
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Gewirtz
mesh_terms: D000276:Adjuvants, Immunologic / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D005408:Flagellin / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism / Q000502:physiology; D016209:Interleukin-8 / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000201:enzymology / Q000276:immunology / Q000378:metabolism* / Q000821:virology*; D007424:Intracellular Fluid / Q000276:immunology / Q000378:metabolism / Q000821:virology; D008562:Membrane Glycoproteins / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D011070:Poly I-C / Q000138:chemical synthesis / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000502:physiology*; D012367:RNA, Viral / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000502:physiology; D012401:Rotavirus / Q000502:physiology*; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D014775:Virus Activation / Q000276:immunology; D019892:eIF-2 Kinase / Q000502:physiology*
chemical_list: D000276:Adjuvants, Immunologic; D016207:Cytokines; D018836:Inflammation Mediators; D016209:Interleukin-8; D008562:Membrane Glycoproteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011956:Receptors, Cell Surface; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D005408:Flagellin; D019892:eIF-2 Kinase; D011070:Poly I-C