Protein Kinase R Mediates Intestinal Epithelial Gene Remodeling in Response to Double-Stranded RNA and Live Rotavirus This information is current as Matam Vijay-Kumar, Jon R. Gentsch, William J. Kaiser, of September 28, 2021. Niels Borregaard, Margaret K. Offermann, Andrew S. Neish and Andrew T. Gewirtz J Immunol 2005; 174:6322-6331; ; doi: 10.4049/jimmunol.174.10.6322 http://www.jimmunol.org/content/174/10/6322 Downloaded from References This article cites 59 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/174/10/6322.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Protein Kinase R Mediates Intestinal Epithelial Gene Remodeling in Response to Double-Stranded RNA and Live Rotavirus1 Matam Vijay-Kumar,* Jon R. Gentsch,† William J. Kaiser,‡ Niels Borregaard,§ Margaret K. Offermann,‡ Andrew S. Neish,* and Andrew T. Gewirtz2* 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 ␮g/ml) and displayed slower kinetics com- ϳ Downloaded from pared with IL-8 induced by bacterial flagellin (ET50 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 intra- cellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. http://www.jimmunol.org/ 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. The Journal of Immunology, 2005, 174: 6322–6331. otaviruses are the single most important etiologic agents tigenemia and viremia (10). In addition to a potential role in im- of severe dehydrating diarrheal disease in young chil- peding viral dissemination, epithelial gene expression in response R dren, accounting for as many as 100 million cases and to rotavirus may also be important for regulating the adaptive im- Ͼ440,000 deaths annually (1–4). Rotavirus has a rather limited mune response analogous to processes thought to occur in re- tissue tropism, primarily infecting only the epithelial cells lining sponse to bacterial colonization of epithelia. Such adaptive re- by guest on September 28, 2021 the villi of the small intestine consistent with gastroenteritis being sponses to rotavirus result in lasting protection against reinfection its major clinical manifestation (5). Rotaviral infection of such (11). Thus, in light of its potential importance, we sought to de- epithelial cells induces a substantial induction of epithelial gene termine the mechanism by which gut epithelia might detect rota- expression, including the activation of a panel of chemokines that virus and, subsequently, regulate remodeling of gene expression. promote the recruitment and activation of immune cells (6–8). While a number of studies have observed rotaviral-induced activa- Although such immune cell recruitment in response to rotavirus tion of epithelial chemokines secretion in well-defined model systems occurs on a much smaller scale than that seen in response to bac- and have carefully examined the roles of host transcription factors in terial pathogens (e.g., Salmonella, Shigella) (9), the fact that rota- regulating these responses (6–8, 12), little is known in regarding the virus infection is localized generally to the gastrointestinal tract primary host receptor(s) or other sensing mechanism that initiate these suggests that, nonetheless, rotaviral-induced immune cell recruit- responses. Based on the emerging paradigm that epithelial sensing of ment may be important for preventing viral spread throughout the bacteria is largely based on a series of intracellular and extracellular host. Rotavirus indeed has the potential to infect extraintestinal pattern recognition receptors (13, 14), we reasoned that epithelia may sites as rotaviral RNA has been found in cerebral spinal fluid and also have receptors capable of directly recognizing viral products. As serum of some rotavirus-infected children, possibly associated an- rotavirus is a nonenveloped, 11-segmented dsRNA virus (15, 16) and several epithelial cell types (e.g., retinal pigment, lung) have been observed to respond to dsRNA (17–19), this seemed an especially *Department of Pathology and Laboratory Medicine, Epithelial Pathobiology Unit, Emory University School of Medicine, Atlanta, GA 30322; †Viral Gastroenteritis likely molecule to be promoting activation of intestinal epithelial gene Team, Respiratory and Enteric Viruses Branch, National Center for Infectious Dis- expression in response to rotavirus. Thus, we sought to define the eases, Centers for Disease Control and Prevention, Atlanta, GA 30333; ‡Winship effects of dsRNA on gut epithelial gene expression using a well-de- Cancer Institute, Emory University, Atlanta, GA 30322; and §Department of Hema- tology, Rigshospitalet, Copenhagen, Denmark fined polarized model system. Received for publication December 17, 2004. Accepted for publication March 7, 2005. Two distinct mechanisms by which mammalian cells can rec- ognize dsRNA have been described. One mechanism is the acti- The costs of publication of this article were defrayed in part by the payment of page 3 charges. This article must therefore be hereby marked advertisement in accordance vation of dsRNA-dependent protein kinase R (PKR) . PKR is with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants DK061417 and 3 Abbreviations used in this paper: PKR, protein kinase R; poly(I:C), polyinosinic R24 DK064399. acid:cytidylic acid; eIF2-␣, eukaryotic initiation factor 2 ␣; BFA, bafilomycin A1; 2 Address correspondence and reprint requests to Dr. Andrew T. Gewirtz, Pathology- 2-AP, 2-aminopurine; MMP, matrix metalloproteinase; iNOS, inducible NO synthase; WBRB 105H, 615 Michael Street, Emory University, Atlanta, GA 30322. E-mail NGAL, neutrophil-gelatinase-associated lipocalin; SFM, serum-free medium; RRV, address: [email protected] Rhesus rotavirus; MOI, multiplicity of infection. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6323 activated upon binding dsRNA to undergo dimerization and auto- poly(I:C). Throughout this study, we used poly(I:C) from the same lot (no. phosphorylation. This 68-kDa, cytoplasmic serine/threonine ki- 3074729011). nase phosphorylates its physiological substrate eukaryotic initia- Cell stimulation and chemokine secretion tion factor 2-␣ (eIF2-␣) and inhibits translation and perhaps other substrates that results in activation of a panel of genes that ulti- On the day of stimulation, confluent cells were washed twice with serum- mately leads to cessation of virus replication in cells (20–22). It free medium (SFM) and stimulated as described in figure legends. For inhibition studies, TLR3 mAbs (affinity purified and functional grade), has also been shown that PKR regulates other pathways, including BFA, 2-AP, and adenine were added 1 h before addition of the stimuli. those activating p53, p38, IFN regulatory factor-1, and NF-␬B After stimulation, supernatants were collected and centrifuged at 15,000 ϫ (23–25). Induction of NF-␬B has a relevant role in mediating PKR g at 4°C for 10 min and stored at Ϫ80°C for IL-8 analysis. functions, and NF-␬B activation by PKR is involved in IFN-␤ induction in response to dsRNA (26). More recently, it has been Poly(I:C) uptake studies