Double-Stranded RNA and Live Rotavirus Epithelial Gene

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

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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 intracellular 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.

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 shown that TLR3 is also a receptor for dsRNA (27), supported by Confluent T84 cells on permeable support with transepithelial resistance of demonstration that expression of TLR3 confers cells with the abil- Ͼ1000 ⍀ cm2 were washed twice with SFM and stimulated with 100 ity to respond to polyinosinic acid:cytidylic acid (poly(I:C)) and ␮g/ml poly(I:C) either basolaterally, or apically or basolaterally pretreated that TLR3 null mice exhibit substantially reduced responses to (1 h before) with BFA on both sides. Supernatants were collected at different times and centrifuged at 15,000 ϫ g at 4°C for 10 min and subjected poly(I:C). Although there appears to be substantial overlap in some to agarose gel (0.9%) electrophoresis. of the genes activated by TLR3 and PKR, these pathways are nonetheless independent in that both PKR-null and TLR3-null Rotavirus infection Downloaded from mice still gave clear, albeit reduced, responses to poly(I:C) (27, Rhesus rotavirus (RRV) was cultivated in MA104 cells, then frozen and 28). As the relative importance of these receptors in response to thawed three times, clarified by low-speed centrifugation, and stored in different viral pathogens is only beginning to be elucidated (29), aliquots at Ϫ80°C until used (33). Trypsin-activated RRV was titrated by we investigated whether either of these pathways were important the fluorescent Ab method (34). Monolayers of T84 cells grown on 24-well for intestinal epithelial detection of dsRNA and rotavirus. As we plates were infected with RRV activated immediately before use with 10 ␮g/ml trypsin at 37°C for 30 min at different multiplicities of infection anticipated, similar to other epithelial cells, intestinal epithelia (MOI) ranging from 2.5 to 20. Trypsin was added to convert noninfectious

cells displayed robust responses to synthetic dsRNA. However, in virus into infectious virus via cleavage of virus spike protein VP4 into VP5 http://www.jimmunol.org/ contrast to the case for other epithelia, intestinal epithelial re- and VP8 (35). Before infection, cells were washed three times with SFM, sponses to dsRNA and rotavirus were mediated by PKR. and the desired amount of RRV, diluted in 1 ml of SFM, was added to the cells. Mock-infected cells (control) were treated with an equivalent amount

of trypsin-treated SFM. Cells were incubated for1hat37°C in 5% CO2, Materials and Methods followed by removal of the virus containing medium. Cells were washed Poly(I:C), poly(dI:dC), and poly(C) and anti-rabbit and mouse IgG-HRP three times with SFM, and the infection was continued for the indicated were purchased from Amersham Biosciences. Bafilomycin A1 (BFA), times in a 37°C incubator with 5% CO2. Viral infection was monitored and chloroquine, 2-aminopurine (2-AP), adenine, mouse anti-human ␤-actin, documented by viewing under a light microscope. At designated times, Ϫ and TRIzol reagent were purchased from Sigma-Aldrich. Affinity purified supernatants were removed and stored at 80°C.

and functional grade mAbs (clone 3.7) to human TLR3 were obtained from by guest on September 28, 2021 eBioscience. Matrix metalloproteinase (MMP)-7 Abs were procured from Microarray analysis Chemicon International. Rabbit anti-human STAT-1, STAT-1-phos-Y701 Model intestinal epithelial cells (T84) were prepared on 5-cm2 permeable eIF2-␣, eIF2-␣-phos-S51, PKR, and PKR-phos-T446 were obtained from filters and were used 8 days after plating and achieving a stable transepi- Cell Signaling Technology. IFN-␣, IFN-␤, and IFN-␥ were obtained from thelial resistance of Ͼ1000 ⍀ cm2. Cells were washed with SFM and National Institute of Allergy and Infectious Diseases Reference Reagent stimulated with 100 ␮g/ml poly(I:C) on both sides or 100 ng/ml flagellin Laboratory, Braton Biotech. Rabbit anti-human inducible NO synthase applied basolaterally (1 ml apically and 2 ml basolaterally). After 2 and (iNOS), TNF-␣, and IL-1␤ were purchased from R&D Systems. Human 48 h of stimulation, supernatants were collected for IL-8 assay, and RNA TLR3 primers (upstream, 5Ј-GATCTGTCTCATAATGGCTTG-3Ј, and was isolated and subjected to microarray analysis as described previously downstream, 5Ј-GACAGATTCCGAATGCTTGTG-3Ј) were obtained (36). Briefly, RNA was isolated from treated and untreated T84 cells via from Invitrogen Life Technologies (17). Flagellin was purified from Sal- TRIzol, subjected to DNase I digestion, and purified by using commer- monella typhimurium-conditioned medium by anion/cation exchange chro- cially available kit from Qiagen. Total cellular RNA (30 ␮g) was used to matography and purity verified as described previously (30). In brief, such synthesize cDNA labeled with fluorlink Cy5 dCTP. Universal human ref- flagellin does not activate any TLR other than TLR5 (13) and has Ͻ0.5 pg erence RNA (20 ␮g) was used to synthesize reference cDNA labeled with of LPS/␮g of flagellin (31). Mouse mAbs to human neutrophil-gelatinase- fluorlink Cy3 dCTP. Sample and reference cDNA were cohybridized on to associated lipocalin (NGAL) was clone 211.1 (32). the RG Human 11K gene chip purchased from the Vanderbilt MicroArray Cell culture Shared Resources (Vanderbilt University). Washed and dried chips were scanned using the GenePix 4100A scanner. Images were captured and an- Model human intestinal epithelia were prepared by culturing the colonic alyzed using the GenePix Pro 5.0 software. Ratio-based normalization was epithelial cell line T84 (passages 63–72) grown in DMEM:F-12 medium performed so that ratio of medians in the Cy5 and Cy3 channels equals one. supplemented with 10% FBS and HT-29 (passages 130–144), and Caco-2 Ratio of Cy5:Cy3 of untreated and treated samples was calculated to assess and Caco-2 brush border enhanced (passages 30–35) were grown in relative changes in gene expression. DMEM supplemented with 10% FBS, 1% nonessential amino acids, L- glutamine (2 mM), and D-glucose (4.5 g/L). Cells were grown in the pres- Analysis of TLR3 gene expression ␮ ence of 100 U/ml penicillin and 100 g/ml streptomycin in 5% CO2 at 37°C. All the tissue culture reagents were purchased from Invitrogen Life Confluent monolayers of T84 cells in six-well plates were washed with ␮ Technologies or Mediatech. SFM and treated with 100 g/ml poly(I:C). Total RNA was prepared using the TRIzol reagent. RT-PCR analysis of TLR3 gene expression was con- Poly(I:C) reconstitution ducted using 0.5 ␮g of total RNA/reaction (30 cycles) using Applied Bio- systems kit (Applied Biosystems). TLR3 primers yielded a product of 304 Lyophilized poly(I:C) was a potassium salt having a sedimentation coef- bp (17). Ten microliters of the PCR product were separated on a 1.5%

ﬁcient (S20.W) of 15.4. Based on the S20.W, the approximate m.w. and agarose gel electrophoresis and viewed under UV light. For quantitative average nucleotide length will be 3.5 ϫ 106 Da and 4,655 bp, respectively, real-time PCR, the following primers were used: PKR forward, 5Ј-TG although analysis of this poly(I:C) by agarose gel electrophoresis indicates GCGGTCTTCAGAATCAACATC-3Ј, and reverse, 5Ј-CAGCCATTTCT that it is substantially larger than our highest DNA standard (10,000 bp). It TCTTCCCGTATCC-3Ј; and GAPDH forward, 5Ј-ACCCAGAAGACT was dissolved in sterile PBS at 2 mg/ml and heated at 50°C water bath until GTGGATCG-3Ј, and reverse 5Ј-GGATGCAGGGATGATGTTCT-3Ј. solubilized and slowly cooled to room temperature for proper annealing. Reactions were performed in triplicate and values for PKR normalized to Similarly, poly(C) and poly(dI:dC) were reconstituted as described for GAPDH RNA using SYBR Green Super Mix purchased from Bio-Rad (36). 6324 EFFECT OF POLY(I:C) ON HUMAN INTESTINAL EPITHELIAL CELLS

Immunoblotting T84 cells grown in six-well plates were stimulated as described in figure legends, rinsed in ice-cold HBSS, lysed in radioimmunoprecipitation assay II buffer (20 mM Tris-HCl, 2.5 mM EDTA, 1% Triton X-100, 10% glyc- erol, 1% deoxycholate, 0.1% SDS, 50 mM NaF, 10 mM Na2P2O7, and 2 7 mM NaVO4 plus protease inhibitor mixture) at a concentration of 10 cells/ml cleared by centrifugation (10 min at 15,000 ϫ g at 4°C), and equal amounts of protein assayed for iNOS, total STAT-1, PKR, eIF2-␣, and phospho-STAT-1, PKR, and eIF2-␣ by 12% SDS-PAGE immunoblotting. Membranes were stripped and probed for ␤ -actin (control). T84 cell supernatants were used for SDS-PAGE immunoblotting NGAL and MMP-7 using 4–20% gels. Briefly, cells were stimulated as in figure legends, and supernatants were collected, centrifuged at (10 min at 15,000 ϫ g at 4°C), and used for SDS-PAGE immunoblotting. NGAL SDS-PAGE immunoblotting was conducted in both reducing (with 2-ME) and nonreducing (without 2-ME) conditions to distinguish homo- and heterodimers of NGAL. The immunoblots were visualized with the ECL system (Amer- sham Biosciences). Cytokine assays Human IL-8 was measured as described previously (37). Human IFN-␤ enzyme immunoassay kit (sensitivity 250–10,000 pg/ml) from R&D Sys- Downloaded from tems and used for the quantification of IFN-␤ in the cell-free supernatants, according to manufacturer’s instructions. Nitrite measurement Nitrite level was quantified in the supernatants using Griess Reagent Sys- tem (sensitivity 1.5–100 ␮M) (Promega), according to the manufacturer’s

instructions. http://www.jimmunol.org/ Results Poly(I:C) induces changes in epithelial gene expression Although intestinal viruses such as rotavirus have long been ap- preciated as activators of intestinal epithelial cell proinflammatory FIGURE 1. Poly(I:C) induces IL-8 in intestinal epithelial cells. A, Con- gene expression, little is known regarding either the viral or host fluent monolayers of T84 cells were stimulated with increasing doses of molecules that mediate this response. Because rotavirus is a poly(I:C) (100 ␮g/ml) or poly C (100 ␮g/ml) or poly(dI:dC) (100 ␮g/ml) dsRNA virus and dsRNA activates cytokine expression in retinal or flagellin (Flag) (100 ng/ml) in SFM for 48 h. B, T84 cells were stimu- pigment and lung epithelia (17–19, 38), dsRNA seemed a strong lated with either poly(I:C) (100 ␮g/ml) or Flag (100 ng/ml). Supernatants by guest on September 28, 2021 candidate to be a viral activator of gut epithelial cells. To inves- were taken at various time points and assayed for IL-8 by ELISA. Values Ϯ tigate this notion, we stimulated T84 intestinal epithelial cells with are mean SD obtained from duplicate samples of three representative a range of doses of synthetic dsRNA (poly(I:C) 2–100 ␮g/ml) and experiments. measured changes in epithelial gene expression. First, we measured secretion of IL-8 because this gene is known to be induced by rotavirus (6–8) and is commonly used as a general indicator of a panel of genes known to play roles in host defense in general epithelial proinflammatory gene expression. As shown in Fig. 1A, (e.g., lipocalin-2/NGAL, hereafter referred to as NGAL and poly(I:C) induced IL-8 secretion in a concentration-dependent MMP-7) and in particular a number genes associated with antivi- manner with 100 ␮g/ml (a common dose used in other studies) ral/IFN responses. Although a couple of these general host-defense eliciting similar levels of IL-8 secretion as a maximal (100 ng/ml) genes (e.g., NGAL) were also moderately induced by flagellin, the concentration of flagellin. Poly(C) and poly(dI:dC) did not induce antiviral associated genes were not induced substantially by flagel- IL-8 secretion, indicating that the cells were responding to dsRNA lin at either time point. Conversely, consistent with its fast action, rather than DNA or simply a bolus of nucleotides. However, as flagellin potently up-regulated a panel of genes at 2 h, whose ex- shown in Fig. 1B, poly(I:C) was a relatively slow-acting inducer of pression had returned toward baseline by 48 h (Table II). Most of IL-8 expression compared with flagellin. Specifically, poly(I:C)- the genes exhibiting the highest levels of activation in response to induced IL-8 secretion was not detectable until 12 h (vs3hfor flagellin were not induced by poly(I:C) at either time point. Thus, flagellin) and with half-maximal induction requiring over 24 h (vs while flagellin and poly(I:C) are both potent activators of epithelial 8 h for flagellin). gene expression in general, the specific patterns of genes activated We next used cDNA microarray analysis to globally assess the by these agonists are very different, suggesting intestinal epithelial changes in epithelial gene expression induced by synthetic dsRNA cells can appropriately tailor their responses to specific classes of again comparing responses to those induced by flagellin, the best microbes. characterized microbial activator of gut epithelial cells. Specifi- We next sought to determine whether changes measured in cally, model epithelia were treated with buffer only (control) or the mRNA would in fact be manifested at the level of protein expres- above-defined maximal doses of poly(I:C) or flagellin. RNA was sion. In consistent with the microarray data, poly(I:C) induced rel- isolated 2 and 48 h later to assess short-term and long-term effects atively slow changes in expression of both NGAL and MMP-7 on gene expression. RNA was subjected to cDNA microarray anal- (Fig. 2A, i–iii). Such NGAL induction contained only 25-kDa ysis using RG Human 11K gene chips. Table I lists the 30 genes monomer rather than the homo- (46 kDa) or heterodimers (135 most up-regulated in response to poly(I:C) in comparison to con- kDa) observed in human neutrophils by nonreducing SDS-PAGE trol. Consistent with its slow induction of IL-8, poly(I:C) did not immunoblotting as described in Ref. 32. In light of poly(I:C)-ac- induce substantial changes at 2 h but at 48 h potently up-regulated tivating genes associated with both NF-␬B and IFN regulatory The Journal of Immunology 6325

Table I. Poly(I:C) (100 ␮g/ml) up-regulated genes in T84 cellsa

Accession No. Gene Name 2 h/Control 48 h/Control

1. N33920 Ubiquitin D 1.00 (ND)* 12.73 (ND) 2. AA400973 Lipocalin 2 (oncogene 24p3) 1.08 (1.22) 11.61 (3.12) 3. NM࿝002423 Matrix metalloproteinase 7 (matrilysin, uterine) 4.11 (1.24) 11.46 (1.27) 4. R95691 Unknown 0.89 6.50 5. AA457042 IFN-inducible protein p78 3.04 (0.66) 6.43 (0.98) 6. AA410188 IFN-induced protein 1.58 (1.03) 6.40 (1.01) 7. N55205 ␤-globin pseudogene 1.00 (1.48)* 5.86 (0.12) 8. AA401441 B-factor, properdin 1.17 (1.12) 5.70 (1.68) 9. AA458965 NK cell transcript 4 1.18 (1.10) 5.40 (0.72) 10. AA286908 Myxovirus (inﬂuenza virus) resistance 2 (mouse) 1.00 (0.93)* 5.34 (1.06) 11. AA464246 Major histocompatibility complex, class I, C 0.90 (0.81) 5.13 (1.04) 12. H48533 Apoptosis inhibitor 2 3.68 (7.10) 4.85 (2.05) 13. AA448478 IFN, ␣-inducible protein (clone IFI-6–16) 1.00 (0.79) 4.79 (1.18) 14. AA292074 Ubiquitin-conjugating enzyme E2L 6 1.02 (0.70) 4.54 (1.09) 15. AA489640 IFN-induced protein 56 1.00 (1.19)* 4.49 (1.00) 16. AA644657 Major histocompatibility complex, class I, A 0.90 (0.80) 4.44 (1.04) 17. AA443090 Interferon regulatory factor 7 0.61 (0.75) 4.03 (1.04) 18. AA677534 Laminin, ␥2 0.77 (2.19) 4.01 (1.00) 19. AA459401 Kallikrein 10 0.58 (1.49) 3.97 (0.72) Downloaded from 20. AA487921 KIAA0152 gene product 1.36 (0.68) 3.89 (1.57) 21. AA476272 TNF, ␣-induced protein 3 1.00 (1.65)* 3.66 (1.10) 22. H69561 Mannosidase, ␣ class 2A, member 1 1.70 (16.35) 3.54 (2.24) 23. T64469 P8 protein (candidate of metastasis 1) 1.00 (0.83)* 3.50 (1.01) 24. AA479882 Keratin 10 2.37 (1.58) 3.40 (0.85) 25. AA058323 IFN-induced transmembrane protein 1 1.45 (1.66) 3.34 (0.33)

26. R44417 Apoptosis inhibitor 1 0.87 (3.05) 3.34 (1.22) http://www.jimmunol.org/ 27. AA487893 Transmembrane 4 superfamily member 1 1.19 (3.41) 3.22 (0.48) 28. NM࿝013671 Mn superoxide dismutase 1.07 (2.56) 3.22 (1.08) 29. AA406020 IFN, ␣-inducible protein 0.85 (0.91) 3.09 (0.93) 30. AA464250 Keratin 19 1.07 (0.82) 3.08 (0.64)

a Values are ratios of expression from poly(I:C)-treated to untreated (control) state. The numbers in parentheses corresponds to induction by ﬂagellin; ND, not detected. Results are representative of two independent experiments. Genes shown in bold are associated with IFN/antiviral responses. * Gene expression was basally undetectable; thus, data are given as fold induction above values of minimum reliable detection.

Table II. Flagellin (100 ng/ml) up-regulated genes in T84 cellsa by guest on September 28, 2021

Accession No. Gene Name 2 h/Control 48 h/Control

1. AA043551 UDP-GlcNAc:␤Gal ␤-1,3-N-acetylglucosaminyltransferase 5 18.52 (1.16) 1.31 (1.12) 2. H69561 Mannosidase-␣ class 2A, member 1 16.34 (1.70) 2.23 (3.53) 3. W56300 I␬B␣ 10.40 (2.16) 2.02 (2.53) 4. W46900 Gro-␣ 7.84 (1.33) 1.63 (1.00) 5. AA002126 Apoptosis inhibitor 2 7.1 (3.68) 2.05 (4.85) 6. H69683 Chromosome 13 open reading frame 18 5.45 (0.81)* 1.00 (1.00) 7. AA148737 Syndecan 4 5.39 (1.39)* 1.00 (2.34) 8. AA449440 IFN-␥ receptor 2 4.97 (0.92) 0.92 (0.53) 9. R44417 Apoptosis inhibitor 1 4.62 (0.87) 1.16 (3.33) 10. AA490466 Gap junction protein ␤2 26kDa 4.62 (0.67) 1.21 (1.51) 11. T61649 SOD 2, mitochondrial 4.57 (ND) 1.69 (ND) 12. AA443688 GTP cyclohydrolase 1 (dopa-responsive dystonia) 4.21 (0.65)* 1.00 (0.98) 13. N32768 Pregnancy speciﬁc ␤-1-glycoprotein 3 4.13 (ND) 0.81 (ND) 14. AA436152 Semaphorin 5A 4.05 (ND) 0.89 (ND) 15. T94279 Fibrinogen, ␥ polypeptide 3.78 (0.44) 0.75 (0.49) 16. R70685 Jagged - 1 (Alagille syndrome) 3.75 (0.34) 0.79 (0.88) 17. R64600 Oxysterol binding protein-like 3 3.73 (1.00) 0.99 (0.85) 18. AA495790 Ras homologue gene family, member B 3.72 (0.88) 0.29 (1.51) 19. R67336 Homo sapiens LOC158525 (LOC158525), mRNA 3.71 (ND)* 1.00 (ND) 20. AA017383 EBNA-2 coactivator (100-KD) 3.67 (1.86) 1.06 (1.85) 21. AA458884 S100 calcium binding protein A2 3.66 (ND)* 1.00 (ND) 22. AA457705 Immediate early response 3 3.64 (1.64) 0.80 (2.97) 23. AA284669 Plasminogen activator, urokinase 3.51 (1.26) 0.70 (1.26) 24. AA487893 Transmembrane 4-super family member 1 3.41 (ND) 0.48 (ND) 25. N92502 Unknown 3.41 (1.98) 0.84 (0.56) 26. T72596 Homo sapiens cDNA clone IMAGE:5288160, partial cds 3.41 (ND) 1.15 (ND) 27. T95748 Pregnancy speciﬁc ␤-1-glycoprotein 1 3.34 (0.86) 1.01 (1.34) 28. T89996 Fos-related antigen 1 3.21 (1.41) 1.15 (2.02) 29. N33214 Matrix metalloproteinase 14 (membrane-inserted) 3.19 (1.28)* 1.00 (1.00) 30. W42723 Chemokine (C-X-C motif) ligand 1 3.19 (ND)* 1.00 (ND)

a Values are ratios of expression from ﬂagellin-treated to untreated (control) state. The numbers in parentheses correspond to induction by poly(I:C), ND, not detected. Results are representative of two independent experiments. * Gene expression was basally undetectable; thus, data are given as fold induction above values of minimum reliable detection. 6326 EFFECT OF POLY(I:C) ON HUMAN INTESTINAL EPITHELIAL CELLS Downloaded from http://www.jimmunol.org/ FIGURE 2. Poly(I:C) up-regulates NGAL, MMP-7, and iNOS expression. Conﬂuent monolayers of T84 cells were stimulated with NGAL protein expression (Ai) at various doses of 10–100 ␮g/ml poly(I:C) (Aii). Supernatants from wells treated with 100 ␮g/ml poly(I:C) collected at dif- FIGURE 3. Poly(I:C) up-regulates STAT-1 and induces tyrosine phos- ferent times and subjected to SDS-PAGE immunoblotting. NGAL (Aii), phorylation. Epithelial cells were stimulated with 100 ␮g/ml poly(I:C) or MMP-7 (Aiii), and T84 (Aiv) cell lysates were prepared from six-well 200 IU/ml IFN-␣, IFN-␤, or IFN-␥ as indicated times. Cells were lysed and plates treated with 100 ␮g/ml poly(I:C) and subjected to SDS-PAGE im- assayed for total and phosphorylated STAT-1 by SDS-PAGE immunoblot- munoblotting for iNOS. B, Nitrite levels were measured in supernatants ting. Total STAT-1 (A), STAT-1 tyrosine phosphorylation (B), and ␮ from cells stimulated with 100 g/ml poly(I:C) at different time points (left ␮

STAT-1 tyrosine phosphorylation (C) in the presence of 10 g/ml cyclo- by guest on September 28, 2021 panel) and at various doses of poly(I:C) (right panel). The ﬁgure shown is heximide (CHX). a representative of two experiments.

701. Specifically, while such poly(I:C)-induced activation of factor pathways, e.g., IL-8 and MHC class I, respectively, we also STAT-1 required at least 5 h, IFNs ␣, ␤, and ␥ activated STAT-1 measured whether poly(I:C) might regulate expression of iNOS within 15 min of stimulation (Fig. 3B). Similar kinetics was ob- because this gene is known to play a central role in host defense served for total STAT-3 and its tyrosine phosphorylation (data not and a variety of immunopathologic events (39). Poly(I:C) induced shown). This time course of poly(I:C)-induced STAT-1 activation expression iNOS at a level sufficient to be detected by both im- suggested poly(I:C) might induce the synthesis of IFN or other munoblot and by a functional assay (Fig. 2, Aiv and B). Although proteins that might mediate STAT activation as occurs in LPS- the concentration dependence of iNOS induction was similar to treated macrophages or in epithelial cells in response to flagellin that of IL-8 and NGAL, activation of iNOS expression was some- (40, 41). However, sensitive ELISA kits failed to detect IFN-␤ what faster appearing to be maximal by 12 h and almost returned expression, and moreover, global blockade of protein synthesis to baseline by 24 h (Fig. 2Aiv). Lastly, we measured whether indicated that poly(I:C)-induced STAT activation did not require poly(I:C) had similar effects on model epithelia made from other new protein synthesis. Specifically, we observed that treating T84 intestinal epithelial cells lines. Although some cell lines tested cells with cycloheximide under conditions that block protein syn- (Caco-2 and its more differentiated subclone Caco-2 brush border thesis and subsequently STAT activation in response to flagellin enhanced) did not respond to poly(I:C) (data not shown), such as (Ref. 41 and data not shown) had no effect on poly(I:C)-induced T84 cells, HT29 cells made copious amounts of IL-8 and NGAL STAT-1 tyrosine phosphorylation (Fig. 3C), indicating that with similar kinetics and concentration dependence in response to poly(I:C) activation of STAT signaling does not require new pro- poly(I:C), indicating the response to this dsRNA analogue was not tein synthesis. a cell line-specific phenomenon (data not shown). In light of the relatively slow time course of poly(I:C)-induced Epithelial detection of poly(I:C) occurs intracellularly gene expression and the observation that flagellin was activating Epithelial responses to bacterial products and cytokines are often some genes thought to be regulated by IFN/STAT pathways, we highly polarized, consistent with the role of these cells as an in- next investigated the involvement of these molecules in epithelial terface between lumenal microbes and immune cells (13, 30). De- responses to poly(I:C). Poly(I:C) induced a modest (2-fold) in- fining the polarity of such responses can provide insights into both crease in expression of total STAT-1 (Fig. 3A) with a time course the mechanism of response and its physiological role. While unlike of response paralleling the above-described changes in induced microbial surface component products, such as flagellin, dsRNA gene expression. Preceding increases in total STAT-1 were a con- would not be expected to be readily available for detection, “un- comitantly slow activation of STAT-1 phosphorylation of tyrosine packaged” dsRNA can be envisioned to be released upon host cell The Journal of Immunology 6327 lysis. Epithelial responses were indeed polarized to poly(I:C) in though BFA alone induced a modest level of IL-8 secretion, it that epithelia exhibited greater secretion of both IL-8 and NGAL in nonetheless completely blocked subsequent IL-8 expression in- response to basolateral than apical application of poly(I:C) (Fig. 4, duced by poly(I:C). In contrast, BFA had no effect on flagellin- A and B). Such greater responses to basolateral poly(I:C) could be induced IL-8 expression (IL-8 secretion by flagellin and BFA was observed whether IL-8 or NGAL were measured in the apical or additive) (Fig. 4C). Similarly, BFA ablated poly(I:C)-induced ex- basolateral reservoir. Epithelia exhibiting greater responses to ba- pression of NGAL (Fig. 4D). These results suggests that, in con- solateral poly(I:C) could result from either preferential basolateral trast to flagellin, which signals through a surface receptor, poly(I: expression of a poly(I:C) receptor, analogous to the basolaterally C)-induced activation of epithelial gene expression may require polarized expression of TLR5 that mediates the polarity of re- internalization of this viral mimetic. sponse to flagellin (13) or, alternatively, might result from a ba- Although approaches to directly measure uptake of poly(I:C) solaterally preferential uptake of poly(I:C) and subsequent deliv- have not yet been successfully developed, we examined levels of ery to an intracellular receptor. Thus, the role for internalization of poly(I:C) in epithelial supernatants to allow an indirect assessment ϩ poly(I:C) was investigated via BFA, which inhibits vacuolar H - of poly(I:C) uptake by epithelia. The commercially available ATPases and subsequently blocks endocytosis (35, 42, 43). Al- poly(I:C) we used is a large polymer whose m.w. is not well defined. We observed that it does not consistently penetrate agarose gels (even following ethanol precipitation to desalt) and thus appears on the edges of the wells where it was loaded (Fig. 5). Fol- lowing 12 h of incubation with the basolateral surface of epithelia,

this high-m.w. poly(I:C) completely disappeared from the epithe- Downloaded from lial supernatant, indicating that the poly(I:C) had been taken up by the cells and/or degraded it to a lower m.w. species (Fig. 5A). Indeed, the disappearance of the high-m.w. poly(I:C) corresponded with appearance of a smear of lower m.w. nucleic acids, suggesting degradation had occurred. This same pattern of events was

exhibited by poly(I:C) added to the apical reservoir but occurred http://www.jimmunol.org/ with considerably less efficiency (i.e., slower), still being incom- plete by 48 h (Fig. 5B). As both the disappearance of the high-m.w. species and appearance of the low-m.w. species were both potently blocked by BFA (Fig. 5C), it is likely that such disappearance of the high-m.w. poly(I:C) and appearance of low-m.w. nucleic acids reflect uptake of the poly(I:C) by epithelial cells, possibly followed by release of intracellularly degraded poly(I:C). Accordingly, the much slower disappearance of apical than basolateral intact poly(I:C) likely represents a less efficient uptake of poly(I:C) from by guest on September 28, 2021 this surface, thus suggesting that the polarity of response to poly(I:C) (as assessed by secretion of IL-8 and NGAL) may be underlied by the polarity of uptake of this viral mimetic. In contrast, flagellin (100 ng/ml) applied basolaterally remained intact even after 48 h (assessed by immunoblotting; data not shown), consistent with it acting on surface receptors. While the more rapid degradation of basolateral poly(I:C) may yet possibly occur via a BFA-sensitive mechanism that does not directly involve poly(I:C) internalization, these results nonetheless demonstrate that a greater response to basolateral poly(I:C) does not necessarily indicate the presence of a specific receptor.

FIGURE 4. Enhanced basolateral response to poly(I:C). Polarized epithelial cells cultured on 5-cm2 filters were stimulated with 100 ␮g/ml poly(I:C) either basolaterally or apically. After 48 h, basolateral and apical FIGURE 5. Enhanced basolateral uptake of poly(I:C). Polarized epithe- supernatants were collected separately and assayed for IL-8 secretion (A) lial cells grown on 5-cm2 filters were stimulated with 100 ␮g/ml poly(I:C) and NGAL secretion (B). Epithelial cells grown on plastic were stimulated either basolaterally (A) or apically (B) or basolaterally (C) in the presence with 20 ␮g/ml poly(I:C) or 10 ng/ml flagellin with or without 100 nM BFA of BFA (100 nM) on both sides. Supernatants were taken at various times for 48 h. Supernatants were assayed for IL-8 (C) and NGAL (D). Data in and separated on 0.9% agarose gel containing ethidium bromide and this figure are mean Ϯ SD obtained from duplicate samples of three rep- viewed under UV. Data are from representative of two independent resentative experiments. experiments. 6328 EFFECT OF POLY(I:C) ON HUMAN INTESTINAL EPITHELIAL CELLS

Epithelial detection of poly(I:C) and rotavirus uses PKR PCR indicated that PKR mRNA levels were up-regulated 3.5-fold Ϯ We next investigated whether epithelial recognition of poly(I:C) ( 0.5). Furthermore, consistent with such changes in PKR mRNA involved either TLR3 or PKR because both of these molecules are levels, we observed easily detectable expression of PKR by im- capable of independently (of each other) mediating responses to munoblotting (Fig. 7). Use of a phospho-specific and total PKR dsRNA. Microarray experiments did not observe detectable levels Abs further revealed an increase in levels of both phospho-PKR of TLR3 mRNA (with or without poly(I:C) treatment) in model and total PKR within 18 h of poly(I:C) treatment and persisting for ␣ epithelia, suggesting that TLR3 may not be abundantly expressed at least 48 h (Fig. 7). As eIF2- is known to serve as a downstream ␣ by these cells. However, there is likely at least some expression of effector of PKR, we also measured phospho- and total eIF2- . ␣ TLR3 by these cells as using the more sensitive assay of RT-PCR While basal levels of phospho-eIF2- were easily detectable, we we did observe detectable levels of TLR3 transcript (inset of Fig. nonetheless observed a consistent, albeit modest, increase in levels 6A). Such detectable expression of TLR3 is consistent with the of this phosphoprotein, consistent with the activation of PKR, ␣ possibility that TLR3 might mediate poly(I:C) detection in these while levels of total eIF2- did not appear to change in response cells. Thus, a potential role for TLR3 in mediating intestinal epi- to poly(I:C) (Fig. 7). We next examined the requirement for PKR thelial responses to poly(I:C) was investigated via a commercially in mediating responses to poly(I:C) using two well-studied inhib- available mAb to TLR3 that has been demonstrated to inhibit (by itors of this kinase, namely 2-AP and adenine (48, 49). Both 2-AP 60%) surface-expressed, TLR3-mediated signaling (44) but not in- and adenine potently inhibited poly(I:C)-induced IL-8 secretion in tracellularly expressed TLR3 signaling (45). This Ab did not in- a concentration-dependent manner (Fig. 8, A and B). Neither com- hibit poly(I:C)-induced IL-8 secretion from model epithelia (Fig. pound had a significant effect on IL-8 secretion induced by flagel- 6A). Because such an Ab might not be able to inhibit intracellular lin, indicating these PKR inhibitors were not acting nonspecifi- Downloaded from TLR3, we measured the effect of chloroquine on poly(I:C)-induced cally. A similar result was obtained using NGAL as a readout responses because this compound is known to inhibit endosomal further suggesting the importance of PKR for generating epithelial acidification and block signaling of TLR expressed intracellularly responses to dsRNA (Fig. 8C). Taken together, these results indi- (45–47). Chloroquine had no effect on poly(I:C)-induced epithelial cate that PKR is activated by poly(I:C) and is required for activa- IL-8 production, suggesting intracellular TLR are not required for tion of epithelial gene expression in response to this mimetic of generating this response (Fig. 6B). viral dsRNA. http://www.jimmunol.org/ We next investigated the possibility that the other known de- In light of the apparent role for PKR in mediating epithelial tector of dsRNA, PKR, might mediate intestinal epithelial re- responses to poly(I:C), a synthetic mimetic of viral dsRNA, we sponses to poly(I:C). Our microarray experiments indicated that next investigated whether PKR was important for epithelial re- PKR is expressed and up-regulated 2-fold in response to (48 h) sponses to intact live rotavirus. First, we defined how our polarized poly(I:C). In accordance with this result, quantitative real-time model epithelia would respond to RRV, a common laboratory adapted strain of rotavirus. Epithelia were mock infected or infected with live trypsin-activated rotavirus at MOI of 2.5–20, and supernatants were collected at 48 h. Although all MOI tested elicited substantial induction of both IL-8 (Fig. 9A) and NGAL (Fig. by guest on September 28, 2021 9B, i and ii), an MOI of 10 was chosen for further study as a potent but not saturating MOI. Furthermore, at this MOI, the response required the RRV to be preactivated by trypsin, indicating the response requires epithelial interaction with infectious virus rather than the presence of only virions with uncleaved VP4 protein that are poorly infectious (data not shown). In addition, BFA had no effect on RRV-elicited IL-8 secretion (data not shown), consistent with knowledge that RRV does not enter the cells by endocytosis (35, 50, 51). Having defined conditions for measuring rotaviral- induced activation of epithelial cells, we next measured the effect of PKR inhibition on RRV-induced IL-8 and NGAL expression.

FIGURE 6. TLR3-independent IL-8 secretion. Conﬂuent monolayers of epithelial cells were stimulated with 20 ␮g/ml poly(I:C) in the absence or presence of 20 ␮g/ml TLR3 Ab (A)or50␮M chloroquine (B) as described FIGURE 7. Poly(I:C) induces PKR activation. Conﬂuent monolayers of in Materials and Methods. Supernatants assayed for IL-8 at indicated time T84 cells were stimulated with 100 ␮g/ml poly(I:C) for different time periods. (Inset: A, Constitutive expression of TLR3 transcript.) Data in A periods, and lysates were prepared as described in Materials and Methods and B are mean Ϯ SD obtained from duplicate samples of three represen- and subjected to SDS-PAGE immunoblotting. Total PKR (i), phospho- tative experiments. PKR (ii), total eIF2-␣ (iii), phospho-eIF2␣ (iv), and ␤-actin (v). The Journal of Immunology 6329 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. PKR inhibitors block IL-8 secretion. Conﬂuent epithelial cells were stimulated with 20 ␮g/ml poly(I:C) or 10 ng/ml ﬂagellin in the presence of 2-AP (A) or adenine (B) (1 h prior treated) for 48 h. Superna- tants were collected and assayed for IL-8. C, Poly(I:C) induced NGAL expression in the presence of inhibitors. Values are mean Ϯ SD obtained from duplicate samples of three representative experiments.

As described above, verification that these compounds had no effect on IL-8 expression induced by flagellin served as a negative control for these inhibitors. Paralleling their ablation of responses to poly(I:C), both 2-AP and adenine significantly attenuated both IL-8 expression (Fig. 9C) and NGAL expression (Fig. 9D) induced by RRV, although the absolute level of inhibition was somewhat less for live rotavirus than for poly(I:C), suggesting that rotavirus uses both activation of PKR and other pathways in activating ep- FIGURE 9. RRV induces IL-8 and NGAL. Epithelial cells were in- ithelial gene expression. fected with RRV or mock as described in Materials and Methods in the presence or absence of 2-AP or adenine. After 48 h, supernatants were collected and assayed for IL-8 (A) and NGAL (B, i and ii) secretion in Discussion MOI-dependent secretion and IL-8 (C) and NGAL (D) secretion in the Most viruses first encounter their hosts at mucosal surfaces and presence of PKR inhibitors, respectively. thus the first cells contacted and infected are often epithelial cells. Although such epithelial cells serve as an important barrier to microbes, the epithelium is also a vigorous participant in generating pecially the predominant role for bacterial flagellin in mediating immune responses, especially via their activation of proinflamma- activation of epithelial gene expression in response to motile bac- tory and immunomodulatory gene expression. Although a number teria, we hypothesized that intestinal epithelial cells might sense of mechanistic studies of how epithelia sense bacteria have been the products of the intestinal pathogen rotavirus by detection of the investigated, relatively little is known regarding the primary mech- one known microbial pattern displayed by rotavirus, namely its anisms by which epithelia might sense viruses. Based on the dsRNA. Herein, we observed that, indeed, intestinal epithelial cells emerging paradigms governing bacterial-epithelial interactions, es- are capable of sensing a synthetic analogue of viral RNA. Such 6330 EFFECT OF POLY(I:C) ON HUMAN INTESTINAL EPITHELIAL CELLS sensing of dsRNA occurs intracellularly, uses the dsRNA-depen- system, at least as a primary sensor of poly(I:C). However, it certainly dent kinase PKR, and was required for optimal epithelial sensing remains possible that TLR3 may be expressed in inflammatory con- of live activated rotavirus. Consistent with poly(I:C) serving as a ditions and/or in response to stimulation by poly(I:C) as has been major mediator of rotaviral activation of epithelial cells, we ob- demonstrated recently in endothelial cells (54, 55) and analogous to served that one intestinal epithelial cell line (Caco-2) that was the observation that gut epithelial cells display functional TLR4 only previously shown not to respond to rotavirus (6, 7) also does not under inflammatory conditions (56). The reason for lack of apparent respond to poly(I:C) despite responding robustly to the bacterial TLR3-mediated detection of poly(I:C) is not clear. Although lack of product flagellin. We have observed that Caco-2 cells express sim- available specific antiserum prevents us from determining the precise ilar levels of PKR as T84 cells (data not shown) and are currently level of TLR3 expression and microarray studies herein observed no investigating whether this pathway is operable in this cell line. detection of TLR3, previous microarray analysis using our “home- Although some genes were activated by both synthetic dsRNA made” chip suggests our epithelial cells have similar mRNA levels for (i.e., poly(I:C)) and the bacterial stimulus flagellin, the overall pat- TLR3, TLR4, and TLR5 (36) but yet are exquisitely sensitive to terns of gene expression activated by these agonists as assayed by flagellin, exhibit undetectable responses to LPS, and appear not to use microarray analysis were markedly different. Specifically, only one TLR3 in response to poly(I:C). Nonetheless, such lack of TLR3-me- gene (apoptosis inhibitor 1) was among the 30 most up-regulated diated responses could result from insufficient expression of TLR3 genes induced by both poly(I:C) and flagellin at either time point and/or any potential coreceptors as has been shown for TLR4 in gut assayed. This is in marked contrast to our expression profiling epithelial cells (57, 58) or perhaps TLR3 could signal in a nonclassical studies comparing flagellin to TNF-␣, in which these stimuli in- manner, analogous to the way TLR2 ligands alter status of epithelial duced very similar patterns of epithelial gene expression with in- tight junctions rather than activates proinflammatory gene expression Downloaded from duction of only a few genes, showing differential activation in (59). However, as little is known regarding levels of nonpathogenic response to these agonists (36). Although the precise role of each (i.e., commensal) viruses in the gut, at present, a precise role for TLR3 up-regulated gene in responding to pathogens is largely unknown, in regulating intestinal homeostasis is difficult to envision. the fact that poly(I:C)-induced genes included genes known to be In contrast to bacterial flagellin, which is an overriding determinant associated with IFN/antiviral responses validates the use of this for intestinal epithelial responses to S. typhimurium (13), blocking

well-defined model to mechanistically understand rotaviral inter- recognition of dsRNA only partially reduced activation of gene ex- http://www.jimmunol.org/ action with gut epithelia. Consistent with poly(I:C) activating an pression in response to challenge with live rotavirus. This suggests antiviral gene program, poly(I:C) potently activated the STAT- epithelial cells have alternate mechanisms of recognizing viral patho- signaling pathway, and unlike STAT activation described in re- gens. Both the delayed kinetics of responses to RRV and that RRV- sponse to TLR agonists (40, 41), poly(I:C)-induced STAT activa- induced response requires activated virus suggest such responses, like tion was independent of new protein synthesis. Conversely, the responses to poly(I:C), also require viral entry into epithelial cells. relatively delayed course of poly(I:C)-induced IL-8 secretion com- Although poly(I:C)-induced responses required endocytosis as indi- pared with both STAT activation and iNOS induction suggests that cated by their inhibition with BFA, RRV-induced responses were not while both flagellin and poly(I:C) induce IL-8 expression, that in- reduced by BFA likely due to RRV entry of epithelial cells using lipid duced by poly(I:C) might be a secondary response (i.e., IL-8 tran- rafts but yet not being mediated endocytosis (35, 50, 51). That PKR- by guest on September 28, 2021 scription dependent on new protein synthesis). independent activation of epithelial gene expression also occurs in- Although the role of the presumed antiviral genes in response to tracellularly suggests that such viral recognition is also not occurring poly(I:C) makes clear physiologic sense, the role of some poly(I: through a cell surface pattern recognition receptor and rather occurs C)-induced genes is less clear. For example, while several studies intracellularly. Such PKR-independent recognition of RRV could per- have shown intestinal epithelial cells make IL-8 in response to haps occur through another TLR or an as yet unidentified intracellular RRV and, herein, we show poly(I:C) recapitulates this response, pattern recognition receptor. Regardless, considering that the epithe- the role of induction of this gene is not clear in the pathophysiol- lial response to RRV required viral entry and its active replication in ogy of RRV-induced diarrhea in that a characteristic feature of cytoplasm, which generates abundant dsRNA, it would seem difficult RRV-induced diarrhea is the absence of intestinal inflammation. for RRV to avert detection by PKR. Thus, such epithelial PKR-me- Another potently up-regulated gene in response to both poly(I:C) diated recognition of dsRNA likely plays a broad role in recognizing and RRV for which an obvious role in response to RRV is not clear intestinal viruses. is NGAL. Specifically, the best defined role for NGAL in host defense is to bind bacterial siderophores, thus preventing their use Acknowledgments of iron (52, 53). Although it is certainly possible that IL-8 and/or We thank R. I. Glass for the helpful discussion. We greatly appreciate NGAL may serve an as yet unappreciated role in retarding RRV, S. Malkapuram for microarray analysis and Susan Voss and Sean Lyons for alternatively, epithelial activation of antibacterial genes and genes technical assistance. We acknowledge National Institute of Allergy and that would promote neutrophil infiltration may be a nonspecific Infectious Diseases Reference Reagent Laboratory, Braton Biotech (Gaith- means to reduce the possibility of opportunistic infection that ersburg, MD) for providing World Health Organization standards IFN-␣, might otherwise occur following immune-mediated destruction of IFN-␤, and IFN-␥. virally infected epithelial cells. In light of the recent attention focused on TLR and observations Disclosures that epithelia express some TLR and exhibit TLR-mediated re- The authors have no financial conflict of interest. sponses, we hypothesized that intestinal epithelial recognition of poly(I:C) would be mediated by TLR3. 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