Toll-like receptors (TLRs) are the basic signaling receptors of the innate immune system. They are activated by molecules associated with pathogens or injured host cells and tissue. TLR3 has been shown to respond to double stranded (ds) RNA, a replication intermediary for many viruses. Here we present evidence that heterologous RNA released from or associated with necrotic cells or generated by in vitro transcription also stimulates TLR3 and induces immune activation. To assess RNA-mediated TLR3 activation, human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-κB-dependent luciferase reporter were generated. Exposing these cells to in vitro transcribed RNA resulted in a TLR3-dependent induction of luciferase activity and interleukin-8 secretion. Treatment with in vitro transcribed mRNA activated nuclear factor-κB via TLR3 through a process that was dose-dependent and involved tyrosine phosphorylation. Furthermore, in vitro transcribed natural or 2′-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-α, and interleukin-1 receptor-associated kinase-M mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-α secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses. Toll-like receptors (TLRs) are the basic signaling receptors of the innate immune system. They are activated by molecules associated with pathogens or injured host cells and tissue. TLR3 has been shown to respond to double stranded (ds) RNA, a replication intermediary for many viruses. Here we present evidence that heterologous RNA released from or associated with necrotic cells or generated by in vitro transcription also stimulates TLR3 and induces immune activation. To assess RNA-mediated TLR3 activation, human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-κB-dependent luciferase reporter were generated. Exposing these cells to in vitro transcribed RNA resulted in a TLR3-dependent induction of luciferase activity and interleukin-8 secretion. Treatment with in vitro transcribed mRNA activated nuclear factor-κB via TLR3 through a process that was dose-dependent and involved tyrosine phosphorylation. Furthermore, in vitro transcribed natural or 2′-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-α, and interleukin-1 receptor-associated kinase-M mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-α secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses. Mammalian Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; DC, dendritic cell; ds, double stranded; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; IRAK, interleukin-1 receptor-associated kinase; IRF-1, IFN regulatory factor-1; LPS, lipopolysaccharide; luc, luciferase; mAb, monoclonal antibody; NF-κB, nuclear factor-κB; PKR, dsRNA-activated protein kinase; ss, single strand; TRIF, TIR domain-containing adapter inducing IFN-β. 1The abbreviations used are: TLR, Toll-like receptor; DC, dendritic cell; ds, double stranded; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; IRAK, interleukin-1 receptor-associated kinase; IRF-1, IFN regulatory factor-1; LPS, lipopolysaccharide; luc, luciferase; mAb, monoclonal antibody; NF-κB, nuclear factor-κB; PKR, dsRNA-activated protein kinase; ss, single strand; TRIF, TIR domain-containing adapter inducing IFN-β. play a key role in host defense during pathogen infection by regulating and linking innate and adaptive immune responses. TLRs belong to a family of receptors that recognize pathogen-associated molecular patterns (for review, see Refs. 1Armant M.A. Fenton M.J. Genome Biol. 2002; http://genomebiology.com/2002/3/8/reviews/3011PubMed Google Scholar, 2Barton G.M. Medzhitov R. Curr. Top. Microbiol. Immunol. 2002; 270: 81-92Crossref PubMed Scopus (249) Google Scholar, 3Gordon S. Cell. 2002; 111: 927-930Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 4Werling D. Jungi T.W. Vet. Immunol. Immunopathol. 2003; 91: 1-12Crossref PubMed Scopus (333) Google Scholar). DCs are the primary antigen-presenting cells and the only antigen-presenting cells capable of sensitizing naive T cells. TLRs expressed by immature DCs, upon binding their respective ligands, deliver activation and maturation signals that cause the DCs to migrate to lymphoid tissue and switch from antigen acquisition to antigen presentation. Ligands for most of the TLRs have been identified and consist of bacterial and viral constituents such as unmethylated CpG DNA, dsRNA, lipopolysaccharide (LPS), and flagellin (2Barton G.M. Medzhitov R. Curr. Top. Microbiol. Immunol. 2002; 270: 81-92Crossref PubMed Scopus (249) Google Scholar, 3Gordon S. Cell. 2002; 111: 927-930Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 4Werling D. Jungi T.W. Vet. Immunol. Immunopathol. 2003; 91: 1-12Crossref PubMed Scopus (333) Google Scholar, 5Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4849) Google Scholar). Host-derived ligands for the TLRs have also been identified, and these include heat shock proteins, extracellular matrix breakdown products, chromatin-IgG complexes, pulmonary surfactant, and necrotic cells (for review, see Refs. 3Gordon S. Cell. 2002; 111: 927-930Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 6Vabulas R.M. Wagner H. Schild H. Curr. Top. Microbiol. Immunol. 2002; 270: 169-184Crossref PubMed Scopus (238) Google Scholar, and 7Beg A.A. Trends Immunol. 2002; 23: 509-512Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar).Upon binding of ligand, TLRs have been shown to activate a variety of signaling pathways, including phosphoinositide 3-kinase, Jun N-terminal kinase, p38, NF-κB, extracellular signal-related kinase, and interferon (IFN) regulatory factor-3 (IRF-3), each leading to the induction of numerous target genes involved in inflammation, cellular differentiation, and direct antimicrobial activity (8Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1011) Google Scholar, 9Shinobu N. Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Fukuhara Y. Amano F. Fujita T. FEBS Lett. 2002; 517: 251-256Crossref PubMed Scopus (43) Google Scholar). A TLR3/4-specific antiviral gene program mediated by the adaptor protein TRIF and involving the activation of IRF-3 has recently been identified (8Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1011) Google Scholar), with TLR3 being the more potent mediator (10Doyle S.E. O'Connell R. Vaidya S.A. Chow E.K. Yee K. Cheng G. J. Immunol. 2003; 170: 3565-3571Crossref PubMed Scopus (154) Google Scholar). IRF-3 transactivates a set of primary genes including IFN-β. Secreted IFN-β then participates in an autocrine/paracrine loop leading to the production of a set of secondary genes that are involved in antiviral and antimicrobial responses (10Doyle S.E. O'Connell R. Vaidya S.A. Chow E.K. Yee K. Cheng G. J. Immunol. 2003; 170: 3565-3571Crossref PubMed Scopus (154) Google Scholar). Thus, TLRs are capable of recognizing both pathogen-associated molecular patterns, as well as certain endogenous stimuli, which may function as danger signals (11Matzinger P. Annu. Rev. Immunol. 1994; 12: 991-1045Crossref PubMed Scopus (3935) Google Scholar, 12Matzinger P. Ann. N. Y. Acad. Sci. 2002; 961: 341-342Crossref PubMed Scopus (220) Google Scholar). In this report, we identify free and cell-associated RNA as a new host-derived ligand of TLR3 and demonstrate its importance in modulating the phenotype of activated DCs.EXPERIMENTAL PROCEDURESPlasmids and Reagents—NF-κB-dependent ELAM-1-luciferase reporter plasmid (pELAM-luc) (Jesse Chow, Eisai Research Institute, Andover, MA) and pTEVluc (Daniel Gallie, University of California at Riverside) were used. Generation of pTEVgag (p55 core protein of HIV-1) was described previously (13Weissman D. Ni H. Scales D. Dude A. Capodici J. McGibney K. Abdool A. Isaacs S.N. Cannon G. Karikó K. J. Immunol. 2000; 165: 4710-4717Crossref PubMed Scopus (137) Google Scholar). pUNO-TLR3 and pSFV1 were purchased from InvivoGen (San Diego, CA) and Invitrogen, respectively. Expression plasmid pEF-BOS-TRIFΔNΔC for dominant negative TRIF was a gift of S. Akira (Osaka University, Osaka, Japan). Plasmid containing Renilla luciferase-encoding sequences (pSVren) were generated from p2luc (John Atkins, University of Utah) (14Grentzmann G. Ingram J.A. Kelly P.J. Gesteland R.F. Atkins J.F. RNA (N. Y.). 1998; 4: 479-486Crossref PubMed Scopus (43) Google Scholar) following the removal of the firefly luciferase coding sequence with BamHI and NotI digestions, end-filling, and religation. Cells were treated with the following reagents: 1 ng/ml TNF-α (R&D Systems, Minneapolis, MN); 0.1–1 μg/ml LPS (Escherichia coli 055:B5) (Sigma); CD40L trimer (a kind gift from Elaine Thomas, Immunex, Seattle, WA); poly(A), poly(C), poly(G), and poly(U) single strand (ss) RNA and poly(I)·poly(C) dsRNA (Sigma); 2 μg/ml lipoteichoic acid, 5 μm CpG oligodeoxynucleotide, and 1 μg/ml R-848 (InvivoGen).Cell Culture—Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA) were propagated in Dulbecco's modified Eagle's medium supplemented with glutamine (Invitrogen), and 10% fetal calf serum (Hyclone, Ogden, UT) (complete medium). A cell line, TLR3-293, derived from HEK293 cells, which was first stably transformed with pELAM-luc and then with pCMV6-XL5 containing human TLR3 cDNA (Origene Technologies, Rockville, MD) was generated. TLR3-293 cells were grown in complete medium with 125 μg/ml Zeocin and 400 μg/ml G418 (Invitrogen). Leukophoresis samples were obtained from HIV-uninfected volunteers through an IRB-approved protocol. Peripheral blood mononuclear cells were purified by Ficoll-Hypaque density gradient purification. DCs were produced as described previously (13Weissman D. Ni H. Scales D. Dude A. Capodici J. McGibney K. Abdool A. Isaacs S.N. Cannon G. Karikó K. J. Immunol. 2000; 165: 4710-4717Crossref PubMed Scopus (137) Google Scholar). The resulting immature DCs were used between 6 and 9 days after the initial culture of monocytes.Treatment of TLR3-293 Cells—TLR3-293 cells were seeded into 96-well plates (5 × 104 cells/well) 1 day prior to stimulation and cultured without antibiotics. The following day, the cells were exposed to RNA without or with prior complexing to Lipofectin (Invitrogen) as described previously (15Karikó K. Kuo A. Barnathan E.S. Langer D.J. Biochim. Biophys. Acta. 1998; 1369: 320-334Crossref PubMed Scopus (38) Google Scholar). When the treatment was performed in the absence of Lipofectin, cells were exposed to 50 μg/ml mRNA (F-mRNA1 and F-mRNA2) containing 2′-deoxy-2′-fluoro-substituted nucleotides or mRNA1, or poly(I)·poly(C) that was added to complete medium and incubated for 8 or 16 h. When Lipofectin-complexed RNA was used, cells were exposed to 5 μg/ml poly(C), poly(A), poly(U), poly(G), poly(I)·poly(C), or in vitro transcribed mRNA with or without a cap structure or poly(A) tail, for 1 h. The RNA was then removed, and the cells were incubated further in complete medium for 7 or 15 h. Where noted, cells were pretreated with 3 nm staurosporine, 0.5 μm genistein, 5 mm 2-aminopurine (Sigma), or 40 μm TIRAP peptide (Novagen, Madison, WI) for 30 min prior to stimulation with RNA. Supernatants were collected for IL-8 measurement, and cells were lysed with luciferase lysis buffer (Promega, Madison, WI) and analyzed using luciferase assay system (Promega) in a Dynatech MLX luminometer (Chantilly, VA).Transient Transfection of HEK293 Cells—HEK293 cells in 96-well plates at 60–80% confluence were transiently transfected with pUNO-TLR3 and with either pEF-BOS-TRIFΔNΔC (8Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1011) Google Scholar) or pEF-BOS control plasmid using FuGENE 6 (Roche Diagnostics) per the manufacturer's instructions. 24 h later, cells were stimulated with Lipofectin-complexed mRNA1 as described above for TLR3-293 cells, and supernatants were collected 24 h later for IFN-β measurement.In Vitro RNA Transcription—Messenger RNAs encoding gag, firefly luciferase, Renilla luciferase, and the nonstructural polyprotein of Semliki Forest virus were obtained from corresponding plasmids (NdeI, NdeI, SspI, and SpeI-linearized pTEVluc, pTEVgag, pSVren, and pSFV1, respectively) using in vitro transcription assays (MessageMachine and MegaScript kits; Ambion, Austin, TX) as described previously (15Karikó K. Kuo A. Barnathan E.S. Langer D.J. Biochim. Biophys. Acta. 1998; 1369: 320-334Crossref PubMed Scopus (38) Google Scholar). The transcripts (TEVluc, TEVgag, Renilla) were designated mRNA1, mRNA2, and mRNA3, respectively. In certain instances, the Dura-Script kit (Epicentre, Madison, WI) was used to generate nuclease-resistant, 2′-F-deoxycytidine and 2′-F-deoxyuridine nucleotide-containing mRNAs (TEVluc and SFV1, designated as F-mRNA1 and F-mRNA2) as described by the manufacturer. All mRNA contained a cap structure and poly(A) tail unless otherwise noted. Assays for LPS in RNA preparations using the Limulus Amebocyte Lysate gel clot assay were negative with a sensitivity of 3 pg/ml (Department of Genetics, Cell Center Service Facility, University of Pennsylvania). Transcribed mRNA was treated with DNase I and then precipitated with LiCl to remove any trace amount of DNA. The transcripts were also analyzed by agarose gel electrophoresis to confirm that the mRNAs were intact, migrate with the expected size, and were free of DNA contaminants.Secondary Structure Analysis of the RNA—RNA secondary structures for mRNA1, mRNA2, and mRNA3 were predicted using mFOLD 3.1 (www.bioinfo.rpi.edu/applications/mfold), which utilizes a free energy minimization algorithm established by Zuker and Stiegler (16Zuker M. Stiegler P. Nucleic Acids Res. 1981; 9: 133-148Crossref PubMed Scopus (2589) Google Scholar, 17Zuker M. Stiegler P. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10097) Google Scholar). The longest uninterrupted self-complementary sequences in F-mRNA2 were determined by DotPlot analysis using MegAlign 5.5 (DNASTAR Inc., Madison, WI). 20 μg of in vitro transcript was digested with S1 nuclease (Promega) according to the manufacturer's recommendation. S1 nuclease-resistant RNA products were recovered by ethanol (2.5 v/v) precipitation performed in the presence of glycogen carrier and 0.75 m LiCl at -20 °C for 20 h. Precipitate was washed with 75% ethanol and reconstituted in water. Using agarose gel electrophoresis only very low molecular weight cleavage products were detected as a diffuse band capable of retaining ethidium bromide.Analysis of DC Maturation—DCs were treated with the indicated ligands for 24 h. Supernatants were collected for cytokine quantitation, and cells were harvested for flow cytometry. Isotype control or anti-TLR3 antagonistic mAbs (40 μg/ml) (3.7) (eBioscience, Inc., San Diego, CA) were added 30 min prior to stimulation, when indicated. Necrotic cells were prepared by freeze-thawing HEK293 cells four times. Then, necrotic cells were incubated with or without 1–10 units/106 cells of Benzonase (Novagen) at 4 °C for 6–24 h and added to DCs at a ratio of 2:1. DCs were stained with CD83-phycoerythrin mAb (Research Diagnostics Inc., Flanders, NJ) and HLA-DR-fluorescein isothiocyanate mAb and analyzed on a FACScalibur flow cytometer using CellQuest software (BD Biosciences).Enzyme-linked Immunosorbent Assays for Cytokines—Supernatants of activated DCs, and HEK293 and TLR3-293 cells were collected at the indicated times for cytokine measurement. The levels of IL-12 (p70) (Pharmingen), IFN-α, IFN-β, and IL-8 (BIOSOURCE International, Camarillo, CA) were measured in supernatants by sandwich enzyme-linked immunosorbent assay. Cultures were performed in duplicate to quadruplicate and measured in duplicate.Northern Blot Analysis—DCs plated in 48-well plates (3.3 × 105 cells/well) were treated for 8 or 16 h, as indicated, with the exception of samples containing Lipofectin, which either alone or within an RNA complex was left on the cells for 1 h followed by replacement with fresh medium. Where noted, cells were treated with 2.5 μg/ml cycloheximide (Sigma) 30 min prior to the stimulation and throughout the entire length of incubation. At the end of the 8- or 16-h incubation, cells were lysed in 100 μl of guanidinium thiocyanate (Master Blaster, Bio-Rad), and RNA was extracted according to the manufacturer. To enhance the RNA yield, 70 μg of glycogen (Roche Diagnostics) was added as carrier, and the precipitation was performed in siliconized tubes at -20 °C overnight. The RNA pellet was reconstituted in 4 μl of nuclease-free water (Promega) and stored at -20 °C. 2-μl RNA samples (∼1 μg) were denatured then separated in denaturing, 1.4% agarose, 0.22 m formaldehyde gel submerged into MESA buffer (Sigma) supplemented with 0.22 m formaldehyde. RNA was transferred to NYTRAN SuperCharge filters (Schleicher & Schuell) and UV cross-linked. The filters were prehybridized at 68 °C for 1 h in MiracleHyb (Stratagene). To probe the Northern blots, 50 ng of DNA was labeled using Redivue [α-32P]dCTP (Amersham Biosciences) with a random prime labeling kit (Roche Applied Science). The filters were hybridized at 68 °C for 20 h with MiracleHyb containing the labeled and denatured probe. The filters were washed and exposed to Kodak MS film using an MS intensifier screen at -70 °C for 2–72 h.Probes—Templates for human TNF-α and GAPDH probes were excised and gel purified from plasmids (pE4 and pHcGAP, respectively) obtained from ATCC. Coding sequences for human IRF-1 were excised from a plasmid (accession number BC009483) purchased from Open Biosystems (Huntsville, AL). Probes for human TLR3 and IL-1 receptor-associated kinase (IRAK)-M were purified cDNAs derived from plasmids generated by TOPO TA cloning (Invitrogen). The TLR3- and IRAK-M-specific PCR products were generated from RNA of phorbol 12-myristate 13-acetate-treated U937 cells. For TLR3, the 5′ primer (5′-AAGGAAAGGCTAGCAGTC-3′) and 3′ primer (5′-TCGGAGCATCAGTCGT-3′) of the coding sequence of human TLR3 were used (Accession: U88879). This probe detected two transcripts (∼3 and 5 kb) in RNA samples. The IRAK-M-specific 5′-primer (5′-TTTCAACCCAAACTAACTGAT-3′) and 3′-primer (5′-CAAGAATGCCCTGGAGCTTC-3′) of human IRAK-M CDS were used (accession number AF113136). This probe detected a 2.5-kb RNA species and two additional spurious transcripts (8 and 9 kb) in the RNA samples. The specificity of all probes was confirmed by sequencing (DNA Sequencing Facility, University of Pennsylvania).RESULTSIn Vitro Transcribed mRNA Activates the NF-κB Signaling Pathway through TLR3 in TLR3-293 Cells—We demonstrated previously that mRNA containing a poly(A) tail signaled and activated DCs. Two mechanisms were described. The first acted through a P2Y nucleotide receptor and was mediated by the poly(A) tail. The second induced TNF-α and was independent of the poly(A) tail (18Ni H. Capodici J. Cannon G. Communi D. Boeynaems J.M. Karikó K. Weissman D. J. Biol. Chem. 2002; 277: 12689-12696Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). TLR3 has been demonstrated to signal in response to poly(I)·poly(C) dsRNA but not ssRNA (poly(C) homopolymer) (5Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4849) Google Scholar). Although mRNA is a single strand, it differs from synthetic poly(C) homopolymer in that it contains considerable secondary structure, including double stranded regions. This led us to test whether mRNA signaled through TLR3 and might be the TNF-α-inducing activity observed in our earlier experiments (18Ni H. Capodici J. Cannon G. Communi D. Boeynaems J.M. Karikó K. Weissman D. J. Biol. Chem. 2002; 277: 12689-12696Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). mRNA is intrinsically unstable in tissue culture because of the presence of RNases, so different formulations of ssRNA and dsRNA were complexed to Lipofectin, which provides protection against RNase-mediated degradation. These Lipofectin-complexed RNAs were added to HEK293 cells stably transfected with human TLR3 and the ELAM-luc reporter plasmid, which responds to NF-κB activation by production of luciferase. Neither Lipofectin alone nor poly(C) nor poly(U) homopolymer complexed to Lipofectin activated TLR3. However, HIV gag-encoding mRNA complexed to Lipofectin induced luciferase activity, demonstrating signaling through TLR3. The presence of a poly(A) tail or cap structure on the mRNA was not required for TLR3 signaling (Fig. 1A). The addition of mRNA to HEK293 cells that contained only the ELAM-luc reporter plasmid demonstrated no signaling, but the addition of poly(I)·poly(C) dsRNA yielded low level luciferase production (data not shown). A subsequent Northern blot analysis confirmed the presence of a small amount of endogenous TLR3 mRNA in the parental HEK293 cells (data not shown).The instability of mRNA in tissue culture systems required the use of Lipofectin as a stabilization reagent to be able to measure mRNA signaling. Lipofectin did not seem to affect TLR3 signaling because similar induction was seen with both Lipofectin-complexed and free poly(I)·poly(C) dsRNA (Fig. 1, A and B). However, it is conceivable that Lipofectin complexing might lead to structural alteration of mRNA, causing it to signal through TLR3. To exclude this possibility, we generated RNase-resistant mRNA containing 2′-deoxy-2′-fluoro-substituted nucleotides (2′-F-deoxycytidine and 2′-F-deoxyuridine). Two mRNAs, encoding luciferase and the Semliki Forest virus nonstructural genes, were made with fluoro-substituted nucleosides. We found that neither of these mRNAs was translated in HEK293 cells. 2K. Karikó, unpublished observation. These RNAs, delivered without complexing to Lipofectin and in the presence of 10% fetal calf serum, signaled TLR3-293 cells with about half the efficiency of poly(I)·poly(C) (Fig. 1B).We were also concerned about the use of a synthetic promoter reporter system. Transcriptional regulation, even of the same promoter, can differ considerably between a stably integrated promoter versus the endogenous locus because of differences in chromatin structure and other variables in the promoter environment (19Smith C.L. Wolford R.G. O'Neill T.B. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar, 20Weinmann A.S. Mitchell D.M. Sanjabi S. Bradley M.N. Hoffmann A. Liou H.C. Smale S.T. Nat. Immunol. 2001; 2: 51-57Crossref PubMed Scopus (145) Google Scholar). Additionally, the luciferase transcription unit has been reported to confer ambiguous promoter-independent responsiveness in certain cell lines (21Plevy S.E. Gemberling J.H. Hsu S. Dorner A.J. Smale S.T. Mol. Cell. Biol. 1997; 17: 4572-4588Crossref PubMed Scopus (273) Google Scholar). Therefore, it was important to demonstrate that stimulation of TLR3 by mRNA could induce transcription of an endogenous locus. It was reported previously that HEK293 cells transfected with TLR4 produced IL-8 in response to specific ligand (22Yang H. Young D.W. Gusovsky F. Chow J.C. J. Biol. Chem. 2000; 275: 20861-20866Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Therefore, we analyzed supernatants from TLR3-expressing HEK293 cells for IL-8 production after stimulation with RNA. Interestingly, in the absence of Lipofectin, these cells exhibited a high base-line secretion of IL-8 (typically 100–400 pg/ml) with a significant increase in IL-8 after poly(I)·poly(C) or mRNA stimulation. In the presence of Lipofectin, endogenous IL-8 secretion was greatly reduced (less than 10 pg/ml), although ligand-induced secretion remained intact. We found that mRNAs encoding HIV gag or Renilla luciferase induced the secretion of IL-8 at levels comparable with that observed for poly(I)·poly(C) (Fig. 1C). Using IL-8 as a readout of TLR3 signaling allowed us to observe more easily concentration-dependent induction by mRNA (Fig. 1D). These data demonstrate that mRNAs can efficiently signal through TLR3 and are functionally different from synthetic ssRNA homopolymers, which cannot signal through TLR3.mRNA Treatment Induces IRF-1, IRAK-M, TNF-α, and TLR3 mRNAs in Human DCs—To confirm the role of extracellular mRNA as a TLR3 ligand in primary cells, we treated DCs with various RNA constructs and used Northern blot analysis to monitor changes in the expression of genes known to be involved in dsRNA-mediated signaling. First, we examined IRF-1, a transcription factor whose mRNA accumulates in response to stimulation by various cytokines, including type I IFN and dsRNA (for review, see Refs. 23Kroger A. Koster M. Schroeder K. Hauser H. Mueller P.P. J. Interferon Cytokine Res. 2002; 22: 5-14Crossref PubMed Scopus (226) Google Scholar, 24Sato M. Taniguchi T. Tanaka N. Cytokine Growth Factor Rev. 2001; 12: 133-142Crossref PubMed Scopus (131) Google Scholar, 25Taniguchi T. Ogasawara K. Takaoka A. Tanaka N. Annu. Rev. Immunol. 2001; 19: 623-655Crossref PubMed Scopus (1274) Google Scholar). Fibroblasts from IRF-1 knock-out mice exhibit defective production of type I IFN after poly(I)·poly(C) treatment, suggesting the involvement of IRF-1 in type I IFN induction by dsRNA (26Matsuyama T. Kimura T. Kitagawa M. Pfeffer K. Kawakami T. Watanabe N. Kündig T.M. Amakawa R. Kishihara K. Wakeham A. Potter J. Furlonger C.L. Narendran A. Suzuki H. Ohashi P.S. Paige C.J. Taniguchi T. Mak T.W. Cell. 1993; 75: 83-97Abstract Full Text PDF PubMed Scopus (553) Google Scholar). To test whether TLR3 stimulation with different RNA constructs would lead to the transcriptional up-regulation of IRF-1, Northern blot analysis was performed on DCs that were treated for 8 h with RNA. These RNAs included in vitro transcribed conventional and 2′-deoxy-2′-fluoro-substituted mRNA, poly(C) homopolymer, and poly(I)·poly(C). We observed that IRF-1 mRNA was induced in response to both mRNA and poly(I)·poly(C), but not to RNA homopolymer (Fig. 2A). IFN-α is known to induce IRF-1, but we found that addition of cycloheximide did not block IRF-1 induction (Fig. 2A), suggesting that IRF-1 induction by mRNA does not require new protein synthesis and is therefore a direct result of primary signal transduction.Fig. 2Up-regulation of IRF-1, IRAK-M, TNF-α, and TLR3 mRNAs in DCs stimulated with mRNA. Monocyte-derived DCs were stimulated with the indicated ligands for 8 or 16 h, after which total cellular RNA was analyzed for expression of IRF-1, IRAK-M, TNF-α, TLR3, and GAPDH genes by Northern blotting. A, cells were treated with 5 μg/ml Lipofectin-complexed RNA (mRNA1) or 2′-deoxy-2′-fluoro-substituted mRNA (F-mRNA1), poly(C), or poly(I)·poly(C); or Lipofectin alone (none). The experiment was performed in the presence or absence of 2.5 μg/ml cycloheximide (CHX) as described under “Experimental Procedures.” RNA was harvested after an 8-h incubation and analyzed for IRF-1 expression. B, DCs were exposed to several TLR ligands: 0.1 μg/ml LPS, 50 μg/ml poly(I)·poly(C), 1 μg/ml R-848, 5 μm CpG oligodeoxynucleotide (ODN), and 2 μg/ml lipoteichoic acid (LTA) or to 2 ng/ml TNF-α for 16 h. Expression of IRAK-M, TNF-α, IRF-1, and GAPDH mRNAs was analyzed by Northern blotting. C, cells were exposed to 50 μg/ml conventional RNA (mRNA1) or fluoro-substituted RNA (F-mRNA1), or poly(I)·poly(C) without Lipofectin complexing in the presence or absence of 2.5 μg/ml cycloheximide for 8 h. Cellular RNA was analyzed for expression of IRAK-M, TNF-α, and GAPDH genes. D, DCs were exposed to Lipofectin alone (none), RNA complexed to Lipofectin (5 μg/ml mRNA1 or poly(C)), or to uncomplexed RNA (50 μg/ml poly(I)·poly(C), poly(C), poly(U), or poly(A)) added directly to the medium as described under “Experimental Procedures.” In addition, cells were unstimulated or exposed to 2 ng/ml TNF-α or 1 μg/ml CD40L, 10% serum with 1 μg/ml LPS, or serum alone. Cellular RNA, harvested 16 h after initiation of the treatments, was tested for expression of TLR3 and GAPDH mRNAs. Experiments were performed in triplicate.View Large Image Figure ViewerDownload Hi-res image