RNA:DNA hybrids are a novel molecular pattern sensed by TLR9
2014; Springer Nature; Volume: 33; Issue: 6 Linguagem: Inglês
10.1002/embj.201386117
ISSN1460-2075
AutoresRachel E. Rigby, Lauren M. Webb, Karen J. Mackenzie, Yangyang Li, Andrea Leitch, Martin A.M. Reijns, Rachel J. Lundie, Ailsa Revuelta, Donald J. Davidson, Sandra S. Diebold, Yorgo Modis, Alastair Macdonald, Andrew P. Jackson,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle21 February 2014Open Access RNA:DNA hybrids are a novel molecular pattern sensed by TLR9 Rachel E Rigby Rachel E Rigby MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lauren M Webb Lauren M Webb Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Search for more papers by this author Karen J Mackenzie Karen J Mackenzie MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yue Li Yue Li Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Andrea Leitch Andrea Leitch MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Martin A M Reijns Martin A M Reijns MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Rachel J Lundie Rachel J Lundie Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ailsa Revuelta Ailsa Revuelta MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Donald J Davidson Donald J Davidson MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Sandra Diebold Sandra Diebold Division of Immunology, Infection and Inflammatory Disease, King's College London, London, UK Search for more papers by this author Yorgo Modis Yorgo Modis Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Andrew S MacDonald Corresponding Author Andrew S MacDonald Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Search for more papers by this author Andrew P Jackson Corresponding Author Andrew P Jackson MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Rachel E Rigby Rachel E Rigby MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Lauren M Webb Lauren M Webb Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Search for more papers by this author Karen J Mackenzie Karen J Mackenzie MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Yue Li Yue Li Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Andrea Leitch Andrea Leitch MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Martin A M Reijns Martin A M Reijns MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Rachel J Lundie Rachel J Lundie Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ailsa Revuelta Ailsa Revuelta MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Donald J Davidson Donald J Davidson MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK Search for more papers by this author Sandra Diebold Sandra Diebold Division of Immunology, Infection and Inflammatory Disease, King's College London, London, UK Search for more papers by this author Yorgo Modis Yorgo Modis Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Andrew S MacDonald Corresponding Author Andrew S MacDonald Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK Search for more papers by this author Andrew P Jackson Corresponding Author Andrew P Jackson MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Rachel E Rigby1,6, Lauren M Webb2,7,‡, Karen J Mackenzie1,‡, Yue Li3, Andrea Leitch1, Martin A M Reijns1, Rachel J Lundie2, Ailsa Revuelta1, Donald J Davidson4, Sandra Diebold5, Yorgo Modis3, Andrew S MacDonald 2,7 and Andrew P Jackson 1 1MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK 2Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK 3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA 4MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK 5Division of Immunology, Infection and Inflammatory Disease, King's College London, London, UK 6Present address: MRC Human Immunology Unit, Radcliffe Department of Medicine, MRC WIMM, University of Oxford, Oxford, UK 7Present address: Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK ‡These authors contributed equally. *Corresponding author. Tel: +44 161 275 1504; E-mail: [email protected] author. Tel: +44 131 332 2471; Fax: +44 131 467 8456; Email: [email protected] The EMBO Journal (2014)33:542-558https://doi.org/10.1002/embj.201386117 See also: SB Jensen & SR Paludan (March 2014) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The sensing of nucleic acids by receptors of the innate immune system is a key component of antimicrobial immunity. RNA:DNA hybrids, as essential intracellular replication intermediates generated during infection, could therefore represent a class of previously uncharacterised pathogen-associated molecular patterns sensed by pattern recognition receptors. Here we establish that RNA:DNA hybrids containing viral-derived sequences efficiently induce pro-inflammatory cytokine and antiviral type I interferon production in dendritic cells. We demonstrate that MyD88-dependent signalling is essential for this cytokine response and identify TLR9 as a specific sensor of RNA:DNA hybrids. Hybrids therefore represent a novel molecular pattern sensed by the innate immune system and so could play an important role in host response to viruses and the pathogenesis of autoimmune disease. Synopsis Nucleic acids are potent ligands for the pattern recognition receptors of the innate immune system. In this work, RNA:DNA hybrids are established to be a novel class of immunostimulatory nucleic acid, binding and activating intracellular TLR9 in dendritic cells. TLR9 may therefore have a wider role in host response to microbial infection, including the sensing of RNA:DNA hybrid replication intermediates. RNA:DNA hybrids are a novel class of pattern recognition receptor ligand. RNA:DNA hybrids are detectable in cytoplasmic and endosomal fractions during retroviral infection. TLR9 is an intracellular sensor of RNA:DNA hybrids, binding with high affinity. As TLR9 senses both RNA:DNA hybrids and DNA, PRRs are not always restricted to detecting one type of nucleic acid. Introduction The innate immune system is a key component of host response to infection. It senses molecular patterns associated with pathogens and “danger” using a repertoire of germline-encoded pattern recognition receptors (PRRs) that permit detection of conserved microbial molecular motifs (Matzinger, 1994; Janeway & Medzhitov, 2002). Nucleic acids, as indispensable components of all pathogens, represent an important class of PRR ligand (Barbalat et al, 2011). Several families of nucleic acid-sensing PRRs have been characterised, including the membrane-associated Toll-like receptor (TLR) and cytosolic RNA-sensing RIG-I-like receptor (RLR) families (Desmet & Ishii, 2012). Five TLRs sense nucleic acids: TLR3 (double-stranded RNA, dsRNA), TLR7/8 (single-stranded RNA, ssRNA), TLR9 (bacterial DNA) and TLR13 (23S rRNA) (Hemmi et al, 2000; Alexopoulou et al, 2001; Diebold et al, 2004; Heil et al, 2004; Oldenburg et al, 2012). These TLRs localise to intracellular membranes, relocating from the ER to endolysosomes via UNC93B1-mediated trafficking (Latz et al, 2004; Kim et al, 2008), where they undergo proteolytic cleavage to generate a functional receptor that interacts with its nucleic acid ligand (Ewald et al, 2008; Park et al, 2008). Nucleic acid binding to TLR homodimers recruits the adapter proteins TRIF or MyD88 to trigger NF-κB and/or IRF signalling pathways, inducing pro-inflammatory cytokines and type I interferon (IFN) respectively (Blasius & Beutler, 2010). Viral genomes (Lund et al, 2003; Rehwinkel et al, 2010) and their replication intermediates (Lee et al, 2007) are detected as non-self nucleic acids in cytosolic and endosomal compartments by PRRs, which induce type I IFN production to establish a potent antiviral response (Samuel, 2001). In some circumstances, the same receptors bind self-nucleic acids, such as those released from damaged cells (Pisetsky & Fairhurst, 2007). This can occur when self-nucleic acids in complex with human cationic host defence peptides such as LL-37 are internalised by antigen presenting cells (APCs) which potently activate PRR signalling cascades and type I IFN production (Lande et al, 2007; Ganguly et al, 2009). An inflammatory response in the absence of infection also occurs in the childhood onset single gene disorder Aicardi-Goutières syndrome (AGS)(Crow & Livingston, 2008). This genetic mimic of viral infection is caused by mutations in genes encoding four nucleic acid-metabolising enzymes, including Ribonuclease (RNase) H2 (Crow et al, 2006b). As RNase H enzymes hydrolyse the RNA strand of RNA:DNA heteroduplexes (Stein & Hausen, 1969), RNA:DNA hybrids are thought to accumulate in RNase H2-deficient AGS patient cells and induce type I IFN through PRR activation (Alarcon-Riquelme, 2006). Physiological sensing of RNA:DNA hybrids may also be immunologically advantageous, as many major pathogenic viruses (including HIV, CMV, EBV and Hepatitis B) generate RNA:DNA hybrid structures during their replication within an infected cell (Summers & Mason, 1982; Prichard et al, 1998; Rennekamp & Lieberman, 2011). Given the essential role of RNA:DNA hybrids in retroviral replication and the postulated accumulation of RNA:DNA hybrids in AGS, we hypothesised that RNA:DNA hybrids may represent an additional category of immunostimulatory nucleic acid species. Here we demonstrate that intracellular targeting of these molecules elicits an innate immune response and define viral-related RNA:DNA hybrid sequences that are sensed by both plasmacytoid and conventional dendritic cells (DCs). Finally, we identify MyD88 and TLR9 as the signalling adaptor and sensor required for this response, thereby establishing RNA:DNA hybrids as novel high-affinity ligands for TLR9. Results Synthesis and purification of RNA:DNA hybrids with viral sequence motifs To address whether RNA:DNA hybrids represent a novel class of molecular pattern sensed by PRRs, we generated a synthetic 60-bp hybrid containing a repetitive guanosine-uridine-(GU) RNA strand motif (Fig 1A), on the basis that GU-rich viral RNA sequences are established nucleic acid ligands (Diebold et al, 2004; Heil et al, 2004). Chemically-synthesised oligonucleotides were annealed to form a 60-bp duplex (“R:D60”) and hybridrisation confirmed by native polyacrylamide gel electrophoresis (PAGE) analysis (Fig 1B). However, low levels of contaminating nucleic acid species were also evident, including one with identical electrophoretic mobility to the constituent DNA oligonucleotide (ssDNA60) and also a high-molecular-weight species, likely to represent a multimeric form of the hybrid (Fig 1B, arrowheads). Fast performance liquid chromatography (FPLC) was used to remove the potentially immunostimulatory by-products by size-exclusion fractionation (Fig 1C). This resulted in a pure nucleic acid species of 60 bp (Fig 1D), which was confirmed to be an RNA:DNA hybrid by immunoblotting with the RNA:DNA hybrid-specific S9.6 monoclonal antibody (Boguslawski et al, 1986) and enzymatically by exhibiting sensitivity to RNase H (Fig 1E). Additional nucleic acid species were undetectable by PAGE analysis (Fig 1F), establishing that the purity of R:D60 was ≥ 97.5%, given that 1% ssDNA and 2.5% (w/w) ssRNA could be visualised by this method. Using this strategy, sufficient R:D60 was then purified for subsequent experiments to investigate whether RNA:DNA hybrids stimulate an innate immune response. Figure 1. Generation of purified RNA:DNA hybrids Schematic representation of the synthesis and purification of the 60-bp RNA:DNA hybrid “R:D60”. Equimolar amounts of single-stranded RNA and DNA oligonucleotides were heat-denatured and gradually cooled to form RNA:DNA hybrids. Contaminating nucleic acids were removed by size-exclusion FPLC. Annealing of ssDNA60 and ssRNA60 oligonucleotides generates a 60-bp RNA:DNA hybrid with low levels of contaminating nucleic acid by-products. Native PAGE analysis of 100 ng of each single-stranded oligonucleotide prior to hybridisation and 200 ng post-annealing. Contaminating nucleic acids are indicated by arrowheads. FPLC gel filtration separates R:D60 from contaminating nucleic acids. Top, OD280 readings of eluted fractions. Bottom, analysis of 100 μl of selected FPLC fractions (peaks 1–4) by native PAGE. Contaminating nucleic acids are indicated by arrowheads. FPLC-purified R:D60 is free from contaminating nucleic acids. Native PAGE analysis of concentrated R:D60 from “peak 3” fractions alongside single-stranded constituent oligonucleotides. Purified R:D60 is an RNA:DNA hybrid as demonstrated by immunoblotting with an RNA:DNA hybrid-specific antibody. Immunoblotting with the S9.6 monoclonal antibody detects intact R:D60 but not R:D60 that has been enzymatically digested by the RNA:DNA hybrid-specific enzyme RNase H1. Native PAGE analysis is sensitive enough to detect ≤ 1% ssDNA and ≤ 2.5% (w/w) ssRNA within the purified R:D60 hybrid. Download figure Download PowerPoint Dendritic cells are phenotypically activated and secrete cytokines in response to intracellular RNA:DNA hybrids Nucleic acid-sensing PRRs of the innate immune system are widely expressed across a range of cell types, including non-immune cells such as fibroblasts. Therefore R:D60 was initially transfected into primary mouse embryonic fibroblasts (MEFs). However, this failed to induce the expression of genes encoding type I IFNs, in contrast to the robust response induced by the TLR3 ligand poly(I:C) and the TLR9 ligand CpG ODN (ODN1585, Type A CpG ODN) (Supplementary Fig S1A). This led us to consider whether the detection of RNA:DNA hybrids might be restricted to specialised APCs. Transfection of R:D60 into bone-marrow derived macrophages (BMDMs) failed to induce the secretion of a range of cytokines by these cells, including IFN-α, IL-6 and TNF-α (Supplementary Fig S1B). In contrast, transfection of R:D60 into Fms-like tyrosine kinase 3-ligand (Flt-3)-differentiated bone marrow-derived dendritic cells (FLDCs) resulted in substantial production of IL-6 (P = 0.0010), IFN-α (P = 0.0156) and TNF-α (P = 0.0458) (Fig 2A). To confirm that viral RNA:DNA hybrids were immunostimulatory, we generated a second RNA:DNA hybrid containing 45 bp of the HIV-1 group-associated antigen (gag) gene (“R:D45”) (Supplementary Fig S2). Transfection of this hybrid into FLDCs also induced a substantial inflammatory cytokine response, comparable to that stimulated by R:D60 (Fig 2B). Figure 2. Intracellular RNA:DNA hybrids induce cytokine production and activation of dendritic cells A. Transfection of R:D60 into FLDCs stimulates IL-6, IFN-α and TNF-α secretion. Bone marrow-derived FLDC cultures were transfected with R:D60 complexed with Lipofectamine LTX. Supernatant cytokine levels were quantified 18 h later by ELISA. Data shown are the mean of at least five independent experiments ± s.e.m. ***P = 0.001023 (IL-6), *P = 0.015561 (IFN-α), *P = 0.45789 B. Stimulation of cytokine production in FLDCs by a 45-bp RNA:DNA hybrid containing sequence from the HIV-1 gag gene. FLDCs were transfected with R:D45 or R:D60 using Lipofectamine LTX. Data shown are from four independent experiments ± s.e.m.; *P = 0.0122 (IL-6), *P = 0.0201 (IFN-α). C, D. R:D60 induces phenotypic activation of FLDCs. Unsorted FLDC cultures were stimulated with R:D60 as described for (A). Expression levels of the co-stimulatory molecules CD40, CD80 and CD86 on CD11c+ B220+ pDCs (C) and CD11c+ B220− cDCs (D) were quantified by flow cytometry. Below, median fluorescence intensity (MFI) values for CD40 and CD86 from six independent experiments ± s.e.m.; ***P = 0.000003 (pDCs CD86), ***P = 0.0001 (pDCs CD40), ***P = 0.00009 (cDCs CD40), ***P = 0.000000003 (cDCs CD86). CpG ODN added to the culture medium was included as a control. (CpG A could be added to cultures without complexing to Lipofectamine, as it generates large macromolecular aggregates due to unusual self-aggregating properties, sufficient to stimulate spontaneous cellular uptake (Wu et al 2004)). E. R:D60 stimulates IL-6 secretion by cDCs and IFN-α production by pDCs. Day 8-FLDC cultures were sorted into CD11c+ B220+ PDCA1+ (pDC) and CD11c+ B220− PDCA1− (cDC) populations and stimulated as described for (C) and (D). Cytokine concentrations were quantified 18 h later by ELISA. Data shown are from three (IL-6) or four (IFN-α) independent experiments ± s.e.m. F. R:D45 delivered into the cell by LL-37 stimulates cytokine production and phenotypic activation of FLDCs. 1 μg/ml R:D45 complexed with 25 μg/ml LL-37 or scrambled LL-37 (scLL-37) peptide was added to the medium of FLDC cultures. Supernatant cytokine levels and expression of co-stimulatory molecules were determined. Data shown are representative of three independent experiments ± s.e.m. (n = 3 replicates); **P = 0.0025 (CD86), **P = 0.0090 (IL-6). Download figure Download PowerPoint FLDCs are a heterogeneous population that can be subdivided into conventional DC (cDC) and plasmacytoid DC (pDC) populations by surface expression of B220/CD45R (Brasel et al, 2000; Brawand et al, 2002), which differentially sense viral nucleic acids (Kato et al, 2005). We therefore investigated which DC subtype was responding to RNA:DNA hybrids. Transfection of R:D60 into FLDCs induced phenotypic activation of both FACS-purified pDC (CD11c+ B220+) and cDC (CD11c+ B220−) populations, as determined by upregulated surface expression of the costimulatory molecules CD40, CD80 and CD86 (Fig 2C, D). Next, we sought to determine which subset of FLDCs was responsible for the cytokine response to intracellular R:D60, as phenotypic activation can occur in the absence of cytokine production. FLDCs were sorted into pDC (CD11c+ B220+ PDCA-1+) and cDC (CD11c+ B220− PDCA-1−) populations by flow cytometry and transfected with R:D60. This induced a pro-inflammatory cytokine response (IL-6) exclusively in cDCs (Fig 2E, left panel) whereas type I IFN (IFN-α) was produced solely by pDCs (Fig 2E, right panel). The complexing of R:D60 with a liposomal transfection reagent (Lipofectamine) was essential for cytokine production, in keeping with intracellular detection or RNA:DNA hybrids by PRRs (Fig 2A). LL-37, a naturally occurring inflammatory product of neutrophils, epithelial cells and macrophages (Beaumont, 2013), is known to bind and internalise nucleic acids into mammalian cells (Sandgren et al, 2004; Lande et al, 2007; Ganguly et al, 2009; Lai et al, 2011). Internalisation of R:D45 using LL-37 but not a scrambled peptide control (scLL37) was also able to induce cytokine production and activation of FLDCs (Fig 2F). We therefore concluded that intracellular RNA:DNA hybrids induce cytokine secretion and phenotypic activation of dendritic cells in vitro. Intracellular targeting of RNA:DNA hybrids stimulates cytokine secretion in vivo in mice and ex vivo in human PBMCs As FLDC cultures represent an in vitro model of steady-state splenic DC populations (Brawand et al, 2002; Naik et al, 2005) we next investigated whether RNA:DNA hybrids could be detected by splenic DCs in vivo. R:D45 was injected intraperitoneally into C57BL/6 mice either alone or complexed to the cationic liposome Invivofectamine. Analysis of splenic DC populations 12 h post injection by flow cytometry showed a significant upregulation of CD40, CD80 and CD86 expression by cDCs and pDCs when the hybrid was administered in a liposomal complex (Fig 3A). A comparable level of activation was seen between cDC subsets (Supplementary Fig S3). Furthermore, R:D45 complexed to Invivofectamine induced a robust cytokine response, with significantly elevated levels of both IL-6 and IFN-α in the serum of these mice (Fig 3B). Consistent with in vitro FLDC experiments, liposomal delivery was essential for RNA:DNA hybrid stimulation for cytokine secretion and DC activation (Fig 3A, B). Figure 3. R:D45 activates DCs and induce a systemic cytokine response in vivo in mice and ex vivo in human cells Delivery of R:D45 complexed to Invivofectamine in vivo phenotypically activates DCs. C57BL/6 mice were injected intraperitoneally with 80 μg R:D45 or 80 μg R:D45 complexed to Invivofectamine and the activation of splenic DC populations was analysed 12 h later by flow cytometry. Left, representative histograms comparing cell surface expression of the indicated marker on DCs from mice treated with Invivofectamine alone (grey) and R:D45 complexed to Invivofectamine (black). Isotype control shaded grey. Right, MFI values for CD40 (P = 0.0000013), CD80 (P = 0.000106) and CD86 (P = 0.00000024). Data shown are from one experiment ± s.e.m. (n = 5 mice per group), representative of a total of three independent experiments. Large-scale R:D45 hybrid synthesis was performed for each experiment, with purity estimated by PAGE at 96%, 91%, and 98% hybrid, respectively. R:D45 complexed to Invivofectamine induces cytokine production in vivo. C57BL/6 mice were injected with R:D45 as described for (A). Serum levels of IL-6 (***P = 0.000526) and IFN-α (***P = 0.0000026) were determined 12 h post-injection. Data pooled from three independent experiments ± s.e.m. (10–15 mice total per condition). R:D45 induces cytokine production when transfected ex vivo in human PBMCs. Freshly isolated PBMCs were transfected with R:D45 complexed to Lipofectamine LTX. Supernatant cytokine levels were quantified 18 h later by ELISA. Data pooled from two independent experiments ± s.e.m., **P = 0.00166 (IL-6), ***P = 0.00011 (IFN-α) (7 donors in total). Download figure Download PowerPoint To investigate whether RNA:DNA hybrids were also able to induce a cytokine response in human cells, we used ex vivo peripheral blood mononuclear cells (PBMCs) that comprise a mixed population of cells including lymphocytes, monocytes, cDCs and pDCs. Transfection with R:D45 induced significant production of both IL-6 and IFN-α by PBMCs (Fig 3C), establishing that the innate immune sensing of RNA:DNA hybrids is not species-specific. In summary we concluded that the detection of RNA:DNA hybrids within an intracellular compartment occurs in mice (in vivo) and in humans (ex vivo). Consequently, we next sought to identify the cellular pathway involved in the sensing of RNA:DNA hybrids. MyD88 is essential for FLDC activation by RNA:DNA hybrids Many nucleic acid-sensing PRRs require the binding of an adaptor molecule to mediate downstream signalling and subsequent cytokine production. To identify the PRR-adaptor pathways sensing RNA:DNA hybrids, FLDCs derived from mice lacking the adaptor proteins IPS-1, TRIF or MyD88 were transfected with R:D60. The cytokine response of Ips-1−/− FLDCs was indistinguishable from that of C57BL/6 control FLDCs, however R:D60-induced production of both IL-6 and IFN-α was undetectable in cells from mice lacking both the TRIF and MyD88 adaptor molecules (Fig 4A, B). FLDCs deficient in MyD88 alone failed to produce cytokines in response to R:D60 (Fig 4C), while cytokine production was intact in Trif−/− FLDCs (Supplementary Fig S4A). Similarly, phenotypic activation of both pDCs and cDCs was abrogated in cells lacking MyD88 (Fig 4D) but intact in Trif−/− FLDCs (Supplementary Fig S4B), thereby confirming that MyD88 is essential for downstream signalling following PRR detection of RNA:DNA sensing in both DC subtypes. Figure 4. Detection of RNA:DNA hybrids requires MyD88 and TLR9 A, B. The cytokine response to R:D60 is absent in Myd88−/−Trif−/− but not Ips-1−/− mice. FLDCs derived from MyD88−/−;Trif−/−, Ips-1−/− and wild-type (C57BL/6) control mice were transfected with R:D60. Supernatant levels of IL-6 (A) and IFN-α (B) are represented as percentage of cytokine produced by C57BL/6 wild-type controls included in each experiment. Data shown are the mean of three (Ips-1−/−) or two (Myd88−/−;Trif−/−) independent experiments ± s.e.m. (one-sample t-test). ns P = 0.9442 (IPS-1 IFN-α), **P = 0.0102 (MyD88/TRIF IFN-α), ns P = 0.5790 (IPS-1 IL6), *P = 0.0396 (MyD88/TRIF IL6). C, D. MyD88 is essential for cytokine secretion and phenotypic activation of FLDCs by R:D60. FLDCs derived from MyD88−/− and C57BL/6 control mice were transfected with R:D60. Supernatant cytokine levels (C) and surface expression of co-stimulatory molecules were determined 18 h post-transfection, with CD86 shown as representative (D). Data shown are the mean of 2 independent experiments ± s.e.m. (C) or ± s.d. (D) (unpaired t-test). In (C), **P = 0.0015 (IL-6); *P = 0.0271 (IFN-α). In (D): ***P = 0.00072 (pDCs CD86); **P = 0.00444 (cDCs CD86). E, F. The cytokine response to R:D60 is significantly impaired in TLR9-deficient but not TLR7-deficient FLDCs. Cultures derived from Tlr9−/−, Tlr7−/− and C57BL/6 mice were transfected with R:D60/poly U, or stimulated by the addition of CpG ODN to the culture medium. Supernatant cytokine levels were determined by ELISA. Levels of IL-6 and IFN-α3 are represented as percentage of cytokine produced by wild-type controls to the TLR9 ligand CpG ODN (E) or the TLR7 ligand poly U (F). Data are the mean of six and three independent experiments ± s.e.m., respectively. ***P = 0.0003 (Tlr9−/− IL-6), *P = 0.0514 (Tlr9−/− IFN-α), ns P = 0.3761 (Tlr7−/− IL-6), ns P = 0.1224 (Tlr7−/− IFN-α). G. R:D45-induced cytokine production is TLR9-dependent. FLDCs derived from Tlr9−/− and C57BL/6 mice were transfected with R:D45 or poly U. Cytokine levels were determined 18 h post-transfection. Phenotypic activation as determined by CD40/80/86 expression was also entirely TLR9-dependent. Data are representative of two independent experiments ± s.d. of duplicate samples. H. Cytokine production by both cDCs and pDCs in response to R:D60 is impaired in Tlr9-deficient FLDCs. Day 8-FLDC cultures were sorted into CD11c+ B220+ PDCA1+ (pDC) and CD11c+ B220− PDCA1− (cDC) populations and transfected with R:D60 or stimulated by the addition of CpG ODN. Data shown are representative of three independent experiments ± s.d. of replicate samples. *P = 0.0355 (cDCs, IL-6), *P = 0.0165 (pDCs, IFN-α). I. The cytokine response to R:D60 is sensitive to chloroquine treatment. FLDCs derived from C57BL/6 mice were treated with 10 μM chloroquine prior to stimulation with R:D60 and CpG ODN as described for (H). Data shown are representative of three independent experiments ± s.d. of replicate samples. **P = 0.0025 (IL-6), *P = 0.0151 (IFN-α). Download figure Download PowerPoint TLR9 senses RNA:DNA hybrids Given that response to RNA:DNA hybrids was independent of TRIF, TLR3 and the DDX1/DDX21/DHX36 complex (Yamamoto et al, 2002; Zhang et al, 2011) were ruled out as candidate sensors of these hybrids. Likewise, the cytosolic RNA sensors RIG-I and MDA5 were excluded as both are dependent on binding to IPS-1 for downstream signalling (Kawai et al, 2005). However, TLR7 and TLR9 both require MyD88 for downstream signalling (Schnare et al, 2000; Hemmi et al, 2002) and so represented strong candidates for the intracellular sensor of RNA:DNA hybrids. FLDCs derived from Tlr7−/− and Tlr9−/− mice were transfected with R:D60. Production of both IL-6 and IFN-α was found to be significantly reduced in Tlr9−/− FLDCs (Fig 4E). Similarly, cytokine production in response to R:D45 was undetectable (Fig 4G). Conversely, Tlr7−/− FLDCs displayed normal cytokine responses to RNA:DNA hybrids (Fig 4F). Analysis of cytokine production in FACS-sorted FLDCs confirmed that IL-6 production by TLR9-deficient cDCs was completely abolished and IFN-α production by pDCs significantly impaired (Fig 4H). Therefore, TLR9 appears to be the sole RNA:DNA hybrid-sensing receptor in cDCs and represents the major receptor for hybrids in pDCs. Residual IFN-α secretion by Tlr9−/− pDCs in response to R:D60 could suggest an additional hybrid-sensing receptor in this cell type, in which case DHX9 or DHX36 could be plausible candidates given that they have been reported to be MyD88-dependent sensors (Kim et al, 2010). Chloroquine treatment of wild-type C57BL/6 FLDCs also impaired cytokine secretion following subsequent R:D60 transfection (Fig 4I). Since cholorquine is an established inhibitor of endosomal TLR-nucleic acid binding (Hacker et al, 1998) this indicated that hybrids activate TLR9 in an endosomal compartment. Together, these data established that endosomal detection of RNA:DNA hybrids by TLR9 leads to activation of downstream signalling and cytokine production. Intact RNA:DNA hybrids stimulate cytokine secretion by FLDCs S
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