in Functionally Disparate Signaling Events Distinct ...

RNA and Imidazoquinolines Are Sensed by Distinct TLR7/8 Ectodomain Sites Resulting in Functionally Disparate Signaling Events This information is current as of December 22, 2015.

Elif Colak, Alasdair Leslie, Kieran Zausmer, Elham Khatamzas, Andriy V. Kubarenko, Tica Pichulik, Sascha N. Klimosch, Alice Mayer, Owen Siggs, Andreas Hector, Roman Fischer, Benedikt Klesser, Anna Rautanen, Martin Frank, Adrian V. S. Hill, Bénédicte Manoury, Bruce Beutler, Dominik Hartl, Alison Simmons and Alexander N. R. Weber

Supplementary Material References Subscriptions Permissions Email Alerts

http://www.jimmunol.org/content/suppl/2014/05/08/jimmunol.130305 8.DCSupplemental.html This article cites 47 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/192/12/5963.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

J Immunol 2014; 192:5963-5973; Prepublished online 9 May 2014; doi: 10.4049/jimmunol.1303058 http://www.jimmunol.org/content/192/12/5963

The Journal of Immunology

RNA and Imidazoquinolines Are Sensed by Distinct TLR7/8 Ectodomain Sites Resulting in Functionally Disparate Signaling Events Elif Colak,*,1 Alasdair Leslie,†,1 Kieran Zausmer,† Elham Khatamzas,† Andriy V. Kubarenko,*,2 Tica Pichulik,‡ Sascha N. Klimosch,‡ Alice Mayer,† Owen Siggs,x Andreas Hector,{ Roman Fischer,† Benedikt Klesser,† Anna Rautanen,‖ Martin Frank,#,3 Adrian V. S. Hill,‖ Be´ne´dicte Manoury,**,†† Bruce Beutler,‡‡ Dominik Hartl,{ Alison Simmons,† and Alexander N. R. Weber*,‡

he detection of invading microorganisms by vertebrates involves TLRs, a family of pattern recognition receptors (1). TLRs recognize diverse structural classes of microbeassociated molecules. For example, TLR7 and TLR8 sense bacterial and viral RNA, and TLR9 detects bacterial and viral nucleic acids containing CpG motifs, respectively. TLR7 and 8 detect ligands present in endosomes by their extracellular domain (ECD), while their cytoplasmic Toll/IL-1R (TIR) domains relay an intracellular signal via the adaptor MyD88, IRAKs, and TNFRassociated factor (TRAF)6 (1–3). Depending on the cell type, this ultimately leads to the activation of MAPKs, NF-kB and/or IFN regulatory factor (IRF) transcription factors (1). For instance, in human plasmacytoid dendritic cells (pDC) TLR7 signaling induces

T

cell maturation, differentiation and type I IFN production via IRF5 and 7. In B lymphocytes, TLR7 activation triggers IL-10 and IL-6 production, Ag-specific stimulation and enhanced Ig G class switch DNA recombination required for humoral immune responses (1). Human monocytes and polymorphonuclear leukocytes exclusively express TLR8 and respond to its activation with TNF production and/or degranulation (4). The molecular basis for these disparate TLR7 or TLR8-mediated signaling events remains elusive. The majority of results regarding TLR7 or TLR8 have been generated not using natural RNA but rather synthetic G/U˚ rich RNA oligoribonucleotides (ORN; Mr ∼6,000,000, 60–80 A length), or low m.w. molecules of the imidazoquinoline family (5). Both types of ligands are able to trigger TLR7 and/or TLR8 and,

*Junior Research Group Toll-Like Receptors and Cancer, German Cancer Research Center, 69120 Heidelberg, Germany; †Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; ‡Department of Immunology, Interfaculty Institute for Cell Biology, University of T€ubingen, 72076 T€ubingen, Germany; xDepartment of Genetics, The Scripps Research Institute, La Jolla, CA 92037; {Department of Pediatrics I, University of T€ ubingen, 72076 T€ubingen, Germany; ‖Wellcome Trust Centre for Human Genetics, University of Oxford, Headington, Oxford OX3 7BN, United Kingdom; #Core Facility Molecular Structure Analysis, German Cancer Research Center, 69120 Heidelberg, Germany; **Institut National de la Sante´ et de la Recherche´ Me´dicale, Unite´ 1151, 75015 Paris, France; ††Universite´ Paris Descartes, Sorbonne Paris Cite´, Faculte´ de Me´decine Paris Descartes, 75015 Paris, France; and ‡‡Center for Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390

supported by the University of T€ubingen and the German Research Foundation (Grants We-4195-1 [to A.N.R.W.] and HA 5274/3-1 [to D.H.]), respectively. B.B. was funded by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grant HHSN272200700038C. Address correspondence and reprint requests to Alexander N.R. Weber, Interfaculty Institute for Cell Biology, Department of Immunology, University of T€ubingen, Auf der Morgenstelle 15, 72076 T€ubingen, Germany, or Alison Simmons, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, U.K., or Alasdair Leslie at the current address: Kwazulu Natal Research Institute for Tuberculosis and HIV, Durban 4001, South Africa. E-mail addresses: [email protected] (A.N.R.W.), alison.simmons@ndm. ox.ac.uk (A.S.), or [email protected] (A.L.)

1

Equal contribution.

The online version of this article contains supplemental material.

2

Current address: Department of Clinical Biochemistry, Institute for Clinical Chemistry and Pharmacology, University Hospital, Biomedical Centre, Bonn, Germany. 3

Current address: Biognos AB, Go¨teborg, Sweden.

Received for publication November 15, 2013. Accepted for publication April 4, 2014. This work was supported by German Research Foundation Grant We-4195-1 and the German Cancer Research Center (to E.C., A.V.K., and A.N.R.W.). A.L. is supported by the Howard Hughes Medical Institute. T.P., A.H., D.H., and S.N.K. were www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303058

Abbreviations used in this article: 3D, three dimensional; DC, dendritic cell; ECD, extracellular domain; HA, hemagglutinin; IRAK, IL-1R–associated kinase; IRF, IFN regulatory factor; LRR, leucine-rich repeat; MoDC, monocyte-derived DC; ORN, oligoribonucleotide; pDC, plasmacytoid DC; qPCR, quantitative PCR; TIR, Toll/ IL-1R; TRAF, TNFR-associated factor; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

TLRs 7 and 8 are pattern recognition receptors controlling antiviral host defense or autoimmune diseases. Apart from foreign and host RNA, synthetic RNA oligoribonucleotides (ORN) or small molecules of the imidazoquinoline family activate TLR7 and 8 and are being developed as therapeutic agonists. The structure-function relationships for RNA ORN and imidazoquinoline sensing and consequent downstream signaling by human TLR7 and TLR8 are unknown. Proteome- and genome-wide analyses in primary human monocyte-derived dendritic cells here showed that TLR8 sensing of RNA ORN versus imidazoquinoline translates to ligand-specific differential phosphorylation and transcriptional events. In addition, TLR7 and 8 ectodomains were found to discriminate between RNA ORN and imidazoquinolines by overlapping and nonoverlapping recognition sites to which murine lossof-function mutations and human naturally occurring hyporesponsive polymorphisms map. Our data suggest TLR7 and TLR8 can signal in two different “modes” depending on the class of ligand. Considering RNA ORN and imidazoquinolines have been regarded as functionally interchangeable, our study highlights important functional incongruities whose understanding will be important for developing TLR7 or 8 therapeutics with desirable effector and safety profiles for in vivo application. The Journal of Immunology, 2014, 192: 5963–5973.

5964

Materials and Methods Reagents and cells Chemicals and cell culture reagents were from Sigma-Aldrich, unless otherwise stated. HEK293T cells from A. Dalpke (University of Heidelberg, Heidelberg, Germany) were cultured as described previously (10). MoDC with .97% purity (CD11c+DC-SIGN+) were prepared (16) from fresh buffy coats (U.K. national blood service, informed consent obtained) by density gradient centrifugation, CD14 magnetic bead purification (Miltenyi Biotec) and culture with IL-4 and GM-CSF (PeproTech) for 5 d. MoDC were then stimulated with RNA40 (precomplexed using LyoVec; InvivoGen) and R848 (InvivoGen or Axxora). For experiments in HEK293T cells, RNA40 ORN (IBA) were complexed with DOTAP (N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl sulfate; Roche). The following Abs were used: anti-hemagglutinin (HA) (Sigma-Aldrich and Cell Signaling Technology), anti-V5 (Invitrogen), anti-AU1 (Bethyl), anti–b-tubulin (SigmaAldrich), anti-rabbit-HRP (Vector Laboratories), and anti-mouse-HRP (Promega). Anti–phospho-Abs were from Cell Signaling Technology, p-ERK (3179), p-NF-kB (3039), p-MEK (9154), STAT3 (4904), and STAT6 (9362).

Plasmids and site-directed mutagenesis Untagged hTLR7 and hTLR8 expression plasmids were from A. Dalpke (University of Heidelberg) and used to generate hTLR7/8-HA, hTLR7-YFP, and hTLR7/8-Renilla, further information on request. Rab7a-CFP and ERCFP were gifts from H. Huseby (Institute of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway) and H. Stanmark (Department of Biochemistry, Norwegian Radium Hospital, Oslo, Norway), respectively. Point mutations introduced according to the QuikChange II XL Kit (Stratagene) were verified by DNA sequencing and backcloned into the original backbones to avoid unwanted mutations. A list of all mutants and PCR and mutagenesis primer sequences are available upon request.

Reporter gene experiments HEK293T cells in 24-well plates (7.5 3 104 cells/well) were transfected using CaPO4 (13) with a firefly luciferase reporter (Stratagene), Renilla luciferase control reporter (pRL-Tk), and pC1-EGFP (BD Clontech) and 24 h later stimulated with 12 mg/ml DOTAP-complexed RNA40 or indicated concentrations of R848 (usually 0.75 mg/ml for TLR7 and 2.5 mg/ml for TLR8) for 18 h. Luciferase activities were determined using the Dual Luciferase Reporter Assay System (Promega) on a Fluostar Optima Instrument (BMG Labtech). Mean values of triplicates (6SD) are shown throughout.

Microarray analysis For microarray analysis, cells were prepared as above and stimulated 4 h, lysed and mRNA extracted using a microRNeasy (Qiagen) kit. RNA quality was verified by Bioanalyser 2100 (Agilent). Samples from five separate donors were submitted for microarray analysis (Agilent; G3 gene-expression chip). TIFF image files were processed using Agilent’s Feature Extraction software (version 10.7.3.1) using protocol GE2_107_Sep09. Data were imported into GeneSpring and log2 ratios calculated as Cy3(sample)/Cy5 (reference). ANOVA was used to compare the three groups (RNA40, R848, and control) for differentially expressed genes and, at the same time, a fold change analysis conducted. The Benjamini–Hochberg multiple testing correction was applied and a corrected p , 0.01 taken to be significant. All analysis was carried out in GeneSpring GX version 11.5.

Quantitative PCR RNAwas extracted as above, DNA removed by incubation with DNAse I (Ambion) and reverse transcribed using High-Capacity RNA-to-cDNA Kit (ABI). Quantitative PCR (qPCR) was carried out on a 7500 Fast real-time PCR (ABI), using 50 ng template, TaqMan gene expression MasterMix (ABI), and the following TaqMan probes: IFN-b1 (Hs00277188_s1), IL-6 (Hs00985639_m1), DDX58 (Hs00204833_m1), IL8 (Hs00174103_m1), CXCL1 (Hs00236937_m1), IL10 (Hs00961622_m1), and HHEX (Hs00242160_m1. All gene expression value were normalized against GAPDH (Hs99999905_m1) and 2 2DCT values plotted, each dot representing one donor and means indicated by horizontal bars. Alternatively, fold ratios were calculated over media for each ligand. In each experiment a minimum of seven donors were analyzed. LyoVec twice the amount used to complex RNA40 (condition “RNA40”) was used as an additional control in Figs. 1B, 2A, and 2B and was nonsignificantly different to “media” for all targets. In Fig. 2C, differences in fold ratios (RNA-R848) over media were plotted for RNA40 versus R848. Donors with a difference , 0 are shown as red indicating higher induction by R848, donors with a difference . 0 as blue indicating higher induction by RNA40.

Phosphoproteomic screen For protein studies, unless otherwise stated, 5 3 106 MoDC from each biological replicate were stimulated with 5 mg of either RNA40, R848, or Lyovec control in 1 ml media for 10 min at 37˚C. The stimulation was terminated by addition of ice-cold HBS lysis buffer (20 mM HEPES, 150 mM NaCl, and 1% phosphatase inhibitor mixture I [Sigma-Aldrich] [pH 7.4]). Cells were washed twice in ice-cold HBS lysis buffer and then lysed using the CHAPS lysis buffer from the Qiagen Phosphoprotein purification kit with additional Phosphatase inhibitors as above. Cells were lysed for 40 min at 4˚C, cell debris removed by 30 min centrifugation at 16,000 3 g and the equal quantities (as quantified by Bradford assay) of protein run over a phosphoprotein purification column (Qiagen). Eluates showed comparable protein amounts as assessed by Bradford assay. For immunoblot analysis using phospho-specific Abs, equal amounts were loaded for SDS-PAGE. For mass spectroscopy analysis, phospho-enriched samples from three biological replicates were cleaned by methanol:chloroform precipitation, and trypsinized. Tryptic digests were desalted and subjected to LC-MS/MS analysis using a Thermo LTQ Orbitrap Velos. Samples were

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

because of their ability to induce type I IFN, are considered antiviral and antitumor therapeutic molecules. For example, imiquimod (also known as R837, tradename Aldara; Mr 240,000, ˚ ), a human and murine TLR7 agonist, has been licensed for 9A the treatment of genital warts and malignant tumors of the skin (6). Another imidazoquinoline, R848 (also known as resiquimod; ˚ ), is a dual TLR7 and TLR8 agonist. ImidazoquiMr 314,000, 9 A nolines are being actively being pursued for therapeutic use (5). Both classes of TLR7 and/or TLR8 ligands, namely RNA ORN and imidazoquinolines, have been considered functionally congruent and interchangeable because so far no significant differences in the outcome of TLR7 or TLR8 activation by RNA ORN versus R848 have been reported. Thus, R848 has frequently been used as mimic for viral RNA detection via TLR7 or TLR8 in various functional studies (e.g., Ref. 7). Recognition of both RNA ORN and imidazoquinolines involves the TLR7 or 8 ECD, which consist of ∼25 tandem leucine-rich repeat (LRR) motifs forming a horseshoe-shaped solenoid (3). Some TLRs, including TLR7 and TLR8, contain “irregular” LRRs with amino acid insertions thought to protrude from the LRR backbone (3). In TLR8 and 9, these insertions were recently shown to play a role in receptor function (8–10). Nucleic acid sensing by TLR3 and TLR9 involves N- and C-terminal charged binding patches (9, 11, 12). On the sequence level, TLR7 and TLR8 are most closely related to TLR9 and TLR3; hence an RNA recognition mechanism similar to TLR9 and TLR3 appears likely. The small size of imidazoquinolines has led to the proposal that their recognition by TLR7 and TLR8 may be altogether different to that of RNA ORN and involve insertion of a single imidazoquinoline molecule into a small binding interface (13). However, until recently, no systematic studies addressing the recognition principles for RNA ORN and imidazoquinolines by TLR7/8 have been conducted. Given the therapeutic importance of RNA ORN- and imidazoquinolines as TLR7 or 8 agonists, we sought to determine functional outcomes of TLR8 activation by RNA ORN versus R848 in terms of gene transcription and phospho-proteomics in primary human monocyte-derived DCs (MoDCs). These potent APCs play an important role as sentinels of infection (14) and as antiviral or cancer vaccines (15). We observed striking differences in transcription and phosphorylation patterns in MoDC stimulated with RNA ORN versus R848. These were further analyzed in terms of ligand recognition of both classes of ligands combining threedimensional structural modeling, model-guided mutagenesis and the study of naturally occurring murine and human TLR7 mutations/ variants, respectively.

TLR7/8 LIGAND SENSING AND SIGNALING

The Journal of Immunology randomized and analyzed in triplicates using Gas Phase Fractionation. Peptides were detected and quantified with Progenesis LC-MS software (version 3.1.4003.30577) using default settings (no deconvolution/deisotoping, 200 most intense MSMS peaks). A merged peak list generated by Progenesis LC-MS was searched against the IPI human database (version 3.80, 86719 entries) using Mascot (http://www.matrixscience.com/) version 2.3.01. Significantly regulated proteins were identified by LIMMA analysis conducted in the R software package.

Confocal cell imaging HEK293T cells seeded to 8-well chamber slides (Lab-Tek; Nunc) and transfected with FuGENE HD (Promega) for 16 h cells and then media exchanged to imaging media (Life Technologies). Live cell imaging was conducted by using an inverted laser scanning microscope (LSM 780, Zeiss, Germany), equipped with an Axio Observer Z1. Images were sequentially digitally acquired and further analyzed by using the Axiovision software. Further settings are available upon request.

RNA40 pull-down assay and Immunoblot

RNA40 pulldown assay combined with Renilla measurement HEK293 cells were prepared as described for the Ligand-precipitation by using the passive lysis buffer (Promega) for lysis and all washing steps but instead of TLR7-HA TLR7 fused to Renilla luciferase was transfected. The washed RNA40–biotin-coated streptavidin beads remained in 50 ml lysis buffer and were measured for bound luciferase activity (Promega) in a white microplate, in parallel with 50 ml reference whole cell lysates (raw luciferase activity) using a FLUOstar OPTIMA luminescence plate reader (BMG Labtech). Binding was calculated as the bound:raw ratio for each transfection.

Sequence and structural files, homology modeling, comparison of model, and TLR8 crystal structure At the time of initiating this study, structural information on the hTLR7 (NP_057646) and hTLR8 (NP_619542) ECD was not available. Therefore, three-dimensional (3D) models were generated by homology modeling (15) based on the related TLR3 ECD structures 2a0z and 1ziw as recently done successfully for TLR9 (12, 13). GROMACS molecular dynamics, quality analysis tools (ANOLEA, VERIFY_3D, and ERRAT), N-glycan analysis (GlyProt server), visualization/analysis tools (SwissPBD Viewer and PyMol) and software for the computation of surface charges were employed as described in Ref. 15 and citations therein. All mutations were selected on the basis of these models. For TLR8, a crystal structure of the ECD was published recently (17). Although the curvature of the TLR8 ECD model and this crystal structure differ (see below), the spatial organization of sensing sites is highly congruent. For example, site 2 residues D543, F568, and H566 are surface exposed and in close proximity to one another and the R848 ligand. The differences in curvature can be explained by the use of molecular dynamics to sterically and energetically optimize the TLR8 ECD model, leading to a more open, “relaxed” conformation, compared with the TLR8 ECD crystal structure which did not undergo molecular dynamics simulation. Similar differences were observed for TLR3 (12). We consider both crystal structure and molecular dynamicsoptimized models to reflect the conformational space assumed by TLR ECD. Thus spatial distances for site 1–site 2 distances may be approx˚ in both model and crystal, imations varying in distance around 60-70 A which matches the estimated dimension of the agonist molecules investigated in this study.

Polymorphism information, analysis and genotyping A list of reported SNPs in the human TLR7 gene (Gene ID: 51284) was obtained from the National Center for Biotechnology Information dbSNP at http://www.ncbi.nlm.nih.gov (as of 2009). rs numbers were as follows and the number of submissions or individual dbSNP submitter IDs are given in (): TLR7 Q11L rs179008 (multiple submissions), V222D rs55907843 (5 submitters), A448V rs5743781 (multiple submissions), N576D rs34501186 (ss43702473), F580S rs35160120 (ss43684376), Q599H rs36076482 (ss43784597), M603I rs55835602 (ss86353189, ss76868495), S610C rs36110053 (ss43920081), S620T rs34729893 (ss43674426), R627I rs34014664 (ss43741106), L634* rs34557368 (ss43945191), and R636T rs35337229 (ss43637315). Genotyping in different study collectives was carried out as described in Ref. 10.

Mice experiments N-Ethyl-N-nitrosourea mutagenesis, macrophage isolation, TLR stimulation, and TNF measurement by an L-929 cell cytotoxicity bioassay based on propidium iodide absorption (18). Tlr7rsq1 (TLR7T68I; Ref 19) and Tlr7rsq2 (TLR7N182Y; this study) mice were generated on a pure C57BL/6J background and maintained as a homozygous/hemizygous stock. C57BL/6J mice were obtained from The Scripps Research Institute breeding colony. All animal procedures were in accordance with institutional animal care guidelines.

Data analysis and statistical testing Experimental data were analyzed using Microsoft Excel 2007 or GraphPad Prism 5. For luciferase assays, p values were determined using the Student t test by comparing to the respective wild-type condition. For microarray and proteomics data, significance testing ANOVA was used, and for qPCR validation, the Wilcoxon matched pair signed rank-sum test: *p , 0.05 and ** p , 0.01.

Results R848 and RNA ORN differ in the transcriptional events triggered via the same receptor, TLR8 Both synthetic RNA ORN and imidazoquinolines have thus far been considered functionally interchangeable as in pDC R848 and RNA ORN both induce IFN-a via TLR7, or TNF via TLR8 in monocytes (1). Given the fundamental differences in size and shape between the two classes of ligands, we sought to compare cellular responses elicited through the same receptor (in this study TLR8) in primary human immune cells. Primary human primary MoDC express TLR8, but neither contain TLR7 mRNA (Fig. 1A) nor respond to imiquimod, a strict TLR7 agonist (Fig. 1B). Comparison of the prototypical imidazoquinoline R848 and the 20-mer synthetic RNA ORN RNA40 (20) in an initial microarray analysis of stimulated MoDC revealed that R848 significantly regulated 1140 genes with a bias toward a proinflammatory and proapoptotic response, compared with 240 for RNA40 biased toward antiviral response genes (see Fig. 1C, 1D, Supplemental Table I). Only 185 genes were regulated by both (Fig. 1C). Validation of selected genes by qPCR analysis (Fig. 2) at equimolar ligand concentrations further corroborated the differences between RNA40 and R848. In terms of relative mRNA levels (Fig. 2A, left graphs), CXCL1, IL6, and IL8 were strongly upregulated by R848 but significantly less by RNA40. IL10 and HHEX were strongly regulated by R848 but hardly by RNA40. Conversely, RNA40 induced IFNb1 and DDX58 more strongly than R848 (Fig. 2B, left graphs). This was strikingly illustrated by a donor-by-donor analysis of mRNA fold changes (Fig. 2A, 2B, right graphs). Blockage of IFNb1, IL6, IL8, and IL15RA gene induction by bafilomycin, an inhibitor of the endosomal ATPase, ruled out involvement of cytosolic RNA sensors or cell surface TLR but confirmed the role of an endosomal TLR (i.e., TLR8) (data not shown). Summarized clearly by the plotted fold differences of gene induction (Fig. 2C), the gene profiles induced by RNA40 versus R848 differ despite acting via the same receptor (TLR8) and at equimolar ligand concentrations.

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

HEK293T cells were transfected as before with 10 mg indicated plasmids or empty vector (pcDNA3.1+) and 1 mg pC1-EGFP. Forty-eight hours later, cells were scraped in lysis buffer containing protease and phosphatase inhibitor mixtures (Roche). Protein concentrations were adjusted and cleared by centrifugation. For the RNA40 pull-down assay 800 ml cell lysates were incubated with 15 mg 39-Biotin-RNA40 (IBA) at 4˚C and 2.5 ng/ml recombinant V5 control protein for 2 h. Subsequently 30 ml streptavidin–agarose beads (Pierce) were added for 2.5 h. Beads were washed three times in lysis buffer, boiled and equal volumes analyzed on 3–8% Tris-Acetate gradient gels (Invitrogen). Blocked membranes were probed using anti-HA, anti-AU1, or anti–b-tubulin Abs (Sigma-Aldrich) and antimouse or anti-rabbit HRP conjugates (Promega and Vector Laboratories, respectively). For anti–phospho-immunoblot, analysis was conducted using polyvinylidene difluoride membrane and Abs as decribed above. For confirmation of phospho-screen data western blots were run on phosphoenriched samples. Time course experiments were carried out on whole cell lysates, generated using the same lysis buffer. Visualization was carried out using ECL reagents (Pierce). Blots in Fig. 3F and 4E were quantified using ImageJ relative to wild-type (WT) TLR7 or TLR8.

5965

5966

TLR7/8 LIGAND SENSING AND SIGNALING

In primary human MoDC R848 and RNA trigger different phosphorylation events We hypothesized that different transcriptional outcomes might be due to differences in upstream events, for example, 1) phosphorylation of signaling mediators, 2) ligand trafficking, and/or 3) recognition via the receptor ECD. To address the first possibility,

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

FIGURE 1. In MoDC R848 and RNA ORN trigger different transcriptional profiles exclusively via TLR8. (A) qPCR of basal expression levels of different PRR in MoDC (left) and PBMC (right). TLR7 is highly expressed in PMBC but not in MoDC. (B) qPCR analysis of cytokine induction upon stimulation with Imiquimod, a strict TLR7 agonist, in comparison with RNA40 shows unresponsiveness to TLR7 stimulation in purified MoDC but not PBMC. In (A) and (B), relative expression is shown as 22DCT values where each dot represents a different biological replicate. Means 6 SEM are also shown. Overview (C) and specific list of genes (D) significantly (p , 0.01) regulated in a microarray analysis of MoDCs stimulated with RNA40 or R848 (see Materials and Methods and Supplemental Table I). Heat maps show antiviral, inflammatory, and apoptotic gene sets identified by pathway analysis (DAVID). RNA40 and R848 elicit an antiviral signature, but RNA40 lacks the strong proinflammatory and proapoptotic signature induced by R848.

we conducted a global phospho-proteomics analysis in MoDC to identify proteins whose phosphorylation status changed most significantly within 10 min of R848 or RNA40 treatment. Using three biological replicates, each run in triplicate allowed the use of statistics (see Materials and Methods). Under stringent correction for multiple comparisons, disparate phosphorylation events were

The Journal of Immunology

5967 RNA40 caused de-phosphorylation (Fig. 3A). These proteomics results were confirmed by immunoblot analysis of the phosphoenriched lysates fraction of stimulated MoDCs (Fig. 3B). In this study, STAT3 showed R848-induced phosphorylation but no dephosphorylation in response to RNA40. A time course analysis of ERK1/2 and MEK1/2 phosphorylation confirmed that RNA40 induced dephosphorylation of both ERK1/2 and MEK1/2 compared with control between 10 min and 1 h following stimulation, whereas both were strongly phosphorylated by R848 up to 30 min poststimulation (Fig. 3C). These findings were further validated by phospho-flow cytometry (A. Leslie, unpublished data). As a control, NF-kB was phosphorylated by both stimuli, albeit with a slightly delayed time course for RNA40 (Fig. 3C). Thus, our data suggest differences in protein signaling between RNA40 and R848 that may contribute to the observed transcriptional differences. Differences between RNA40 and R848 extend to recognition by the TLR7 and TLR8 ECD

identified between the two ligands. Whereas R848 caused differential phosphorylation of 32 proteins, only 6 were significantly modified for RNA40 (Supplemental Table II). Even among the three proteins regulated by both stimuli, the classical TLRassociated signaling proteins ERK1/2, MEK1/2, and STAT3, R848 stimulation augmented their phosphorylation, whereas

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

FIGURE 2. R848 and RNA ORN disparately induce specific immunerelated genes. qPCR analyses in individual donors (n = 7) show that whereas CXCL1, IL6, IL8, IL10, and HHEX (A) are more strongly regulated by R848 (red), IFNb1 and DDX58 (B) are more strongly regulated by RNA40 (blue) at equimolar concentrations. Relative expression is shown as 2^2DCT values (left graphs) or fold changes over media (right graphs). As HHEX is downregulated (,1), fold changes are inverted (1/mRNA fold). Each dot represents one donor. (C) Differences in fold ratios plotted against each other. Donors showing a positive difference (more strongly RNA40-regulated) are shown in blue, those with negative difference (R848regulated) in red throughout. Differences were tested by Wilcoxon matchedpairs signed rank test.

Disparate phosphorylation and transcription events may be due to differences in uptake kinetics, subcellular localization or actual differences in ligand sensing at the receptor level. We focused in this study on the latter because this has not been addressed in detail (see also Discussion). This was explored for RNA40 and R848 responses via TLR8 but to investigate whether any differences were exclusive for TLR8 or might extend to the closely related TLR7, the latter receptor was also included in the analysis. On the basis of 3D TLR7 and 8 ECD homology models (Ref. 12, see also Materials and Methods), charged or proline residues in TLR7 and TLR8 that had positions similar to site 1 and site 2 in TLR3 or TLR9 (9, 10, 12) on the glycan-free sides of both ECDs (greatest ˚ ) were preferentially selected for mutagenesis dimension ∼135 A (see Fig. 4A, 4B). These TLR7 or TLR8 mutants were then assayed for their ability to activate NF-kB in HEK293T cells, a well-characterized model system for such structure–function analyses. TLR7 mutation at position R186E (site 1a in Fig. 4A), H304E (site 1b), R553, D555, or Y579 (site 2) resulted in attenuated RNA40 NF-kB activation (Fig. 4C). TLR7 L116, a residue integral to LRR stability, served as a negative control. For TLR8, mutation at position R53, K185 (site 1 in Fig. 4B), D543, Y567, and F568 (site 2) significantly reduced NF-kB activation (Fig. 4D). Immunoblot of HA-tagged constructs, confirmed similar expression levels for all mutants and WT TLR7 or TLR8 (Fig. 4E, 4F). Live cell microscopy of YFP-tagged TLR7 constructs confirmed localization to intracellular membranes like the endoplasmic reticulum identical to WT TLR7 (data not shown). Thus, in both TLR7 and 8 residues mapping to N-terminal (red/ purple, site 1a/b) as well as the central part of their ECDs (blue, site 2) are involved in sensing of RNA40, and their distance (∼60– ˚ ) fits the anticipated dimensions of an RNA40 molecule 70 A ˚ , see Fig. 4A, 4B). To contrast RNA ORN with R848 (∼60–80 A recognition, we investigated whether any of the point mutations rendering TLR7 or TLR8 defective in RNA40 sensing would also impact R848 recognition. Fig. 5A and 5B show that single mutations in both the N-terminal (site 1) as well as the central (site 2) RNA ORN sensing site of TLR7 and TLR8 resulted in drastically reduced R848-triggered NF-kB activation, which was unexpected for a small ligand. Similar results were obtained for the imidazoquinolines CL264 and imiquimod (data not shown). Thus, despite their small size, imidazoquinolines appear to require distal ECD sites for full receptor activation. Given that H304E, R553E, and D555A in TLR7 and K185E, D543A/K, Y567A/F, and F568S in TLR8 failed to fully activate NF-kB in response to both RNA40 and R848, it also can be concluded that imidazoquinoline and

5968

TLR7/8 LIGAND SENSING AND SIGNALING

Functionally important human and murine TLR7 alleles map to the identified sensing sites

FIGURE 3. R848 and RNA induce differential downstream signaling factor phosphorylation. (A) Box plots of MoDC quantitative mass spectrometry data for MEK1/2, ERK1/2, and STAT3, which were enriched (phosphorylated) by R848 treatment but depleted (dephosphorylated) by RNA40 relative to Lyovec control (see also Supplemental Table II) in phospho-enriched lysate fractions. Both ligands were used at 1 mg/ml for optimal pathway stimulation. “No change” (fold = 1.0) marked by dashed line. (B) Densitometric analysis (above) of immunoblots (below) for phospho-MEK1/2, ERK1/2, and STAT3 done on phospho-enriched lysate fractions. Equal total protein amounts were used for phosphoenrichment and subsequently loaded (as assessed by Bradford assay). Two donors were analyzed for each protein. (C) Immunoblot analysis of a time course stimulation of MoDCs using phospho-MEK, -ERK, and -NFkB (p65, control) Abs and actin as a loading control. Blots are run on whole-cell lysates. One representative out of two independent experiments shown.

We next sought to determine whether N- and C-terminal recognition sites were of general physiological importance in vivo. Therefore, reported loss-of-function mutations in murine Tlr7 and single-nucleotide polymorphisms in human TLR7 or TLR8 that mapped to these sites were functionally tested. R848-unresponsive mouse strains generated during N-ethyl-N-nitrosourea-mutagenesis (18) include the rsq2 strain (Mouse Genome Informatics accession number: 3811335). Indeed rsq2 peritoneal macrophages failed to respond to R848 (Fig. 7A, 7B) like the “functionally null” Tlr7 strain rsq1 (mutation T68I; Ref. 19). rsq2 mice contained a N182Y mutation in Tlr7, which maps to the proposed N-terminal sensing site 1 (Fig. 7C). The equivalent mutation in human TLR7, N182Y, was also hyporesponsive to R848 in HEK293T cells (Fig. 7D). Thus, mutation in the N-terminal recognition site of human and murine TLR7 appear to lead to R848 hyporesponsiveness in vivo. In addition, dbSNP, a database of reported human genetic variants, yielded several reported nonsynonymous TLR7 variants mapping to the central part of the ECD, that is, site 2 (Fig. 7C) but with an unknown frequency (see Materials and Methods). Genotyping in collectives of United Kingdom (n = 502), Kenyan (n = 369), and Indian origin (n = 393 (10)) failed to identify homo- or heterozygous carriers (see Discussion). Consequently, cells from allele carriers were unavailable for functional analysis but experiments in HEK293T cells (Fig. 7E, 7F) showed that M603I, S610C, N576D, and R627I resulted in .50% or complete loss of function despite normal expression levels. N576D and R627I also showed a dominant-negative effect over WT TLR7 (data not shown). Because TLR7 is X-chromosomal, these findings suggest that hemizygous male carriers of site 2 variant alleles may have reduced TLR7 function and even in heterozygous female carriers the function of the WT allele may be impaired. Collectively, these data imply that the N- and C-terminal sensing sites in at least TLR7 bear physiological relevance.

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

RNA ORN sensing share overall structure–function principles. However, when we compared the levels of NF-kB activation for both ligands within the same experiment relative to their respective WT (Fig. 5C, 5D), most notably, TLR7 R186E and Y579A, and TLR8 R53E responded normally to R848 but not RNA40, whereas the response of TLR8 H566E, and to a lesser extent TLR7 K328E, was greater for RNA40 than R848 (mutations highlighted in green in Fig.4A, 4B). Titration experiments showed this was not dependent on ligand concentration (data not shown). Thus, despite overlapping sensing areas, recognition of either ligand appears to involve several unique residues not required for the detection of the other ligand. Additional experiments demonstrated that the effect of mutagenesis was due to abolished recognition rather abolished binding (at least for RNA40): pull-down of HA- (Fig. 6A–D, Supplemental Fig. 1A, Supplemental Fig. 1B) or Renilla-tagged (Fig. 6E, Supplemental Fig. 1C) TLR7 and/or TLR8 receptors by biotinylated RNA40 confirmed normal binding (i.e., similar quantities of precipitated and lysate TLR7- and TLR8-HA/-Renilla levels) for constructs containing single (Fig. 6A, 6B) or multiple mutations (Fig. 6C–F), excluding the possibility that N- and C-terminal sites cooperate in binding as for TLR3 (11). Because mutation at a single site was sufficient to completely abolish RNA40 responsiveness but not binding (even if combined with other muta˚ ) sites tions), we conclude that the spatially distal (distance ∼60 A identified in this study are essential for ligand recognition but not ligand binding in TLR7 and TLR8, at least in terms of RNA recognition.

The Journal of Immunology

5969

Discussion In this study, we investigated similarities and differences between RNA40 versus R848 downstream signaling and receptor recognition using both primary cells and model systems. Several novel findings warrant further discussion: To our knowledge, the first unexpected discovery was that in primary human MoDC both types of TLR8 ligands, RNA ORN and R848, at equimolar concentrations elicited different transcriptional events via the same receptor (compare Figs. 1, 2). These may result from differential ligand uptake kinetics, subcellular localization or actual differences in ligand sensing at the receptor level. For example, for both TLR4 and TLR9 it was shown that the subcellular

location from which signaling is initiated strongly influenced the balance between NF-kB– versus IRF-dependent signaling for the same ligand (e.g., LPS and CpG, respectively) (21–24). Thus, it is conceivable that, if RNA ORN and IMQ concentrate in or transit through different endosomal compartments disparately, this could lead to differences in the relative strength of downstream signals and thus phosphorylation and transcriptional differences. Unfortunately, according to our knowledge no direct comparisons have been published for RNA ORN and IMQ. However, because IMQ and RNA ORN can functionally counterinfluence each other (25, 26), we consider it highly likely that the uptake and trafficking profiles of IMQ and RNA ORN overlap significantly in a time

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

FIGURE 4. Structure-guided analysis identifies residues in the human TLR7 and 8 ECD essential for receptor function. 3D models of the imidazoquinoline imiquimod, RNA, human TLR7 (A) and TLR8 (B) ECD (gray) with mutated residues shown as spheres as indicated. Residues of functional importance for the sensing of both RNA ORN and R848 are colored red (site 1/1a), magenta (site 1b) or blue (site 2). Residues in green show differential importance for RNA ORN or R848 sensing. N-glycans are in orange. All structures to scale. (C and D) Signaling activity of TLR7 and TLR8 mutants in Nor C-terminal patches is compromised. HEK293T cells were transfected with pcDNA, WT or mutant constructs of TLR7 (C) or TLR8 (D), stimulated with 12 mg/ml RNA40 or DOTAP only (unstimulated) and NF-kB-activation measured by dual luciferase assay (triplicate means + SD). (E and F) TLR7 or TLR8 mutants express to similar levels as WT. HEK293T cells transfected with WT or mutant TLR7-HA (E) or TLR8-HA (F) constructs or an empty vector were analyzed by anti-HA and anti-tubulin immunoblot (loading control). Bands were quantified and intensities relative to the WT signal for each blot calculated. One representative out of three independent experiments each shown.

5970

TLR7/8 LIGAND SENSING AND SIGNALING

frame relevant for receptor recognition and therefore that the differences observed in this study are likely to result from disparate recognition at the receptor ECD level. Disparate ligand recognition was reported for TLR3 and TLR4 that, depending on subtle ligand differences (e.g., length of dsRNA or phosphorylation status of Lipid IVa moiety, respectively), activate downstream signaling disparately (27, 28). Our ECD mutagenesis data provide good evidence that this applies to TLR7 and TLR8 and that both receptors, like TLR3 and TLR4, are able to operate in more than one signaling “mode” in a particular cell type, depending on the class of ligand (RNA ORN or imidazoquinolines). Different signaling modes may thus be a general feature of TLR signaling and can be mediated either directly by the receptor ECD (nucleic acid sensors TLR3, TLR7, and TLR8) or via coreceptors (MD-2 with TLR4, TLR1/6 with TLR2). Mechanistically, two classes of ligands inducing different ECD conformations may result in different TIR-TIR and/or Myddosome postreceptor complexes (29) and subsequent downstream mediator phosphorylation or dephosphorylation events (compare Supplemental Table II)—evidenced in this study for ERK1/2 or MEK1/2 (compare Fig. 3)— details of which will now be interesting to address in future studies. For the regulation of TNF in monocytes or IFN-a in pDC, different signaling modes may be of no consequence. However,

regarding other genes (e.g., CXCL1, HHEX, IL6, IL8, IL10, and IFNb1) and in certain cell types (e.g., MoDC [this study and Ref 30]) or neutrophils (4), RNA ORN- and R848-induced gene transcription is interchangeable. We suspect similar results for TLR7mediated R848 versus RNA ORN signaling may emerge in pDC or B cells (which exclusively express TLR7) if a wider panel of activated genes is assessed. Our study thus cautions the extrapolation from one class of TLR7/8 ligand to the other but rather suggests that both types of ligands need to be assessed separately. Therefore, studies in which R848 has been used as a surrogate for RNA to “simulate” the response of cells to “viral infection” may have to be re-evaluated. In contrast, the insights reported in this study suggest that use of either RNA ORN or imidazoquinolines could be suitable for generating MoDC-based cellular vaccines with particular effector profiles (14). Further studies in this direction appear mandatory to anticipate beneficial effects and potentially harmful off-target effects linked to the use of RNA ORN and imidazoquinolines (5). Second, our data provide evidence that both TLR7 and TLR8 use spatially distal N-terminal and central ECD sensing sites for ligand sensing. For TLR7 and 8 the N-terminal site 1 involves LRRs 2–5 and their insertions—with contributions from residues in LRR 10/ 11 at least for TLR7 (site 1b)—and the central site 2 LRRs 14–18

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

FIGURE 5. R848 follows overall RNA ORN recognition principles but certain TLR7 and TLR8 residues discriminate between the two structural different ligands. (A and B) HEK293T cells were transfected with pcDNA, WT or mutant constructs of TLR7 (A) or TLR8 (B), stimulated with RNA versus R848 and analyzed for NF-kB-activation by dual luciferase assay. One representative out of three independent experiments shown (triplicate means + SD). (C and D) HEK293T cells were transfected as before and stimulated side-by-side with R848 (black, 0.75 mg/ml for TLR7 and 2.5 mg/ml for TLR8) and RNA40 (hashed, 12 mg/ml) and the NF-kB-activation data measured by dual luciferase assay determined and normalized to the level of WT (as 100%). As evident R186E and Y579A in TLR7, and R53E and H566E in TLR8 show significantly different activation between R848 and RNA40 treatment. One representative out of three independent experiments each shown (relative triplicate means + SD).

The Journal of Immunology

(compare Fig. 4A, B). Regarding both site 1 and 2, our data are in good agreement with earlier bioinformatics and experimental approaches (8, 13, 31) and a recent TLR8 ECD crystal structure

(17), which shows two sites of R848 recognition (see also Materials and Methods). For the central TLR7 ECD sensing site, several reported hyposensitive TLR7 alleles described in this study indicate a functional importance (compare Fig. 7C, 7E), albeit indirectly, as the frequency and functional phenotype of these variants will require further verification (see also below). Our mutagenesis data and the R848 hyposensitivity of rsq2 mice (mutated N-terminally at position 182; compare Fig. 7A–C) implicate the TLR7/8 ECD N-termini in ligand sensing. It has been suggested that the reported requirement for endopeptidase cleavage for human TLR7 and TLR9 require to gain functionality (discussed in Ref. 3) argued for the N-termini of endosomal TLRs to be nonessential for ligand recognition. However, recent biochemical (17) and cell biological evidence (32–34) clearly demonstrates that despite ECD cleavage, the N- and C-terminal fragments remain associated after backbone cleavage in a position between the two sensing sites proposed in this study. In good agreement with our data, both parts were also found to be strictly necessary for function. In HEK293T cells (this study and Ref. 9) and human neutrophils (35), TLR7–9 cleavage is not observed and thus appears redundant in some cell types. Collectively, therefore, our data strongly support a direct functional and physiologically relevant role of the TLR7/8 N-termini in the process of ligand sensing. TLR7 and TLR8 thus follow a general principle of nucleic acid sensing by TLRs proposed by us earlier (9), namely that ligand recognition involves N- and C-terminal sensing sites. In terms of RNA recognition this appears plausible ˚ ; comas the distance between N-terminal and central site (∼60 A pare Fig. 4A, 4B) also fits the minimum TLR7 stimulatory RNA ˚ ) reported in Ref. 36. sequence of 19 nt (corresponding to 77 A Interestingly, whereas site 2 residues are fully conserved between TLR7 and TLR8, N-terminal recognition residues are less conserved (data not shown), which may explain differences in ligand preference among different imidazoquinolines. Of note, the sites identified in this study do not appear critical for ligand binding. Earlier studies showed a general promiscuity of TLR7 for both RNA and DNA (37) and in an ORN pull-down assay we found TLR7 binding was stronger for activating versus nonactivating ORN, but not altogether abrogated (19), indicative of a basal level of (nonspecific) binding for nucleic acids. Similarly to TLR9 (10, 38), RNA ORN binding by TLR7 and TLR8 thus appears to be relatively nonspecific with ligand recognition being a separate event dependent on the presence of activation determinants (e.g., G/U-richness). Such nonspecific binding used by TLRs 7–9 may be useful for sampling the endosomal nucleic acid content for low-abundance viral or bacterial RNA or DNA molecules. The finding that not only sensing of an elongated RNA ORN ligand but also small molecular imidazoquinolines by TLR7 and TLR8 was influenced by two spatially distant sites (distance ˚ ) was unexpected as R848 in between site 1a and site 2 of ∼60 A ˚ (compare Fig. 4A, 4B). its longest dimension only spans ∼9 A Previously, it was proposed that the imidazoquinoline mechanism of receptor activation was the insertion of one imidazoquinoline at a single defined ECD site (13, 17). Our functional data argue for R848 and other imidazoquinolines to require multiple or extended contact sites with the receptor. This is confirmed by the TLR8 crystal structure which shows two imidazoquinolines “wedged” by two TLR8 ECD (17). On the basis of the structural resemblance of imidazoquinolines to RNA nucleobases, imidazoquinoline sensing also could involve a multimeric, aggregated and/or sequentially arrayed “RNAlike” R848 structure that could span spatially distal areas of the ECD. Interestingly, concentration-dependent, .100-fold

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

FIGURE 6. RNA binding is not compromised in TLR7 and TLR8 lossof-function mutants. TLR7 and TLR8 single point mutants (A and B) or mutants harboring multiple mutations (C–E) are precipitated in similar ratios compared with WT (A–D). Receptor pull-down using 39-biotinylated RNA40 reveals intact binding for loss-of-function mutants of TLR7 and TLR8. HEK293T cells transfected as indicated with pcDNA, WT or mutant constructs of TLR7- (A and C) or TLR8-HA (B and D). Cells were lysed, incubated with 39-biotinylated RNA40 and complexes precipitated with Streptavidin-beads (SA). Isolated complexes and untreated lysates were analyzed by anti-HA immunoblot. In (C) and (D) a biotinylated V5tagged control protein was added to lysates prior to RNA pull-down to verify equal bead carryover. (E) RNA pull-down of Renilla-tagged TLR7 WT and mutant receptors. HEK293T cells transfected with TLR7-Renilla or Renilla-Renilla fusion constructs were lysed. The sample was split and one aliquot used for immediate raw Renilla activity measurement (triplicates). From the remainder, TLR7 or TLR8 were precipitated with biotinlabeled RNA40. Biotin complexes were captured with streptavidin beads and washed beads then measured for bound Renilla luciferase activity (triplicates). Plots represent the relative light units (RLU) of bound versus raw Renilla for each samples (see Supplemental Fig. 1C and Supplemental Fig. 1D), normalized to WT as 100%. One single experiment shown. (F) Overview of multiple point mutants used.

5971

5972

TLR7/8 LIGAND SENSING AND SIGNALING

accumulation of imidazoquinolines in acidified compartments was reported (8, 25) and is evidenced in molecular dynamics simulations (data not shown). Imidazoquinolines and nucleic acids were also shown to interact (25, 39), so that aggregation may be stabilized by natural RNA or DNA fragments originating from cellular debris through base-pairing or intercalation (26). Whichever precise stoichiometry and molecular mechanism applies to this novel observation regarding imidazoquinoline sensing by TLR7 and TLR8, it is of fundamental interest and warrants future experimental investigation, not least as aggregation/sequential arrangement would influence the pharmacological properties of imidazoquinolines. Our genotyping data argue that for wider therapeutic use of TLR7/8 agonists, hypofunctional alleles are unlikely to pose a problem as the TLR7 alleles studied here emerged with estimated frequencies of ,1% in the several ethnic groups investigated. A TLR7 and TLR8 evolutionary genetics study (40) recently proposed low nonsynonymous allele frequencies in TLR7 and TLR8 may reflect purifying selection, based on the assumption that viral infection has profoundly influenced human allelic inheritance. This was also applied to loss-of-function variants in other critical TLR pathway genes like TIRAP, MYD88, or TLR9 (10, 41–44). Although two children deficient in UNC93B1, an ER protein required for efficient TLR3, 7, 8, and 9 trafficking and thus functionality, appeared only susceptible to herpes simplex infection, other reports imply TLR7/8 in surveillance against HIV and endogenous retroviruses (e.g., Refs. 45 and 46) and in autoimmune diseases like systemic lupus erythematosus (47) so that TLR7/8 functional alleles may have an impact in these diseases. Larger genotyping studies in such

patient collectives may yield carriers of TLR7 loss-of-function alleles and open the way for further clinical and ex vivo studies in primary cells. This could shed more light on the physiological importance of the proposed sensing sites and also clarify the general role of TLR7/8 in human immunity. In conclusion, our data reveal fundamental principles of RNA and imidazoquinoline recognition by human TLR7 and TLR8 and hint to a model of activation shared by other nucleic acid–detecting TLRs and involving multiple signaling modes. This structure– function framework will be vital to dissect further to better understand the role of these receptors in antiviral immunity or clinical autoimmunity. Considering the use of TLR7 or TLR8 ligands in vivo, our study suggests each class (ORN versus imidazoquinolines) may lead to a distinct combination of cell type– specific favorable and unwanted cellular effects. Structure-guided approaches using chemically modified RNA ORN and/or imidazoquinolines may yield TLR7 and TLR8 agonists, which elicit clinically desirable receptor outcomes. Proteome and genome wide profiling of downstream signaling events as used in this study may be required for sufficient resolution of ligand-specific differences.

Acknowledgments We thank J. Willemsen, M. Helm, L. Weber, A. Naumann, A. Dalpke, B. Kaiser, T. Schmidt, the DKFZ light microscopy core facility, and Zeiss for helpful discussions, critical reading of the manuscript, and technical assistance, respectively.

Disclosures The authors have no financial conflicts of interest.

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

FIGURE 7. Functionally important murine and human TLR7 alleles map to N- and C-terminal ectodomain sensing sites. Peritoneal macrophages from C57BL/6J (filled circles), rsq1 (open circles) and rsq2 (Tlr7N182Y, gray diamonds) mutant mice were stimulated with different TLR ligands (A), or a dose titration of R848 (B). TNF secretion was measured by an L-929 cytotoxicity bioassay based on propidium iodide absorption. One single experiment each shown. (C) TLR7 ECD 3D model (gray) with mutations corresponding to naturally occurring TLR7 mutations/SNPs. Mutations leading to altered receptor function are shown in red or blue, the rsq1 mutation reported earlier (19) in orange. (D) The rsq2-equivalent mutation in human TLR7, N182Y, leads to abrogated receptor responsiveness in transfected HEK293T cells stimulated with R848 and analyzed for NF-kB-activation by dual luciferase assay. One representative out of two independent experiments shown (triplicate means + SD). (E) The naturally occurring TLR7 variants N576D, M603I, S610C and R627I (blue) lead to reduced NF-kB-activation, despite similar expression levels (F) compared with WT. HEK293T cells were transfected with pcDNA, WT or TLR7 SNP-equivalent mutants, stimulated with R848 and analyzed for NF-kB-activation by dual luciferase assay (E) or lysates probed using anti-HA and anti-Tubulin (loading control) Abs (F). One representative out of three independent experiments shown (triplicate means + SD).

The Journal of Immunology

References

26. Jurk, M., A. Kritzler, B. Schulte, S. Tluk, C. Schetter, A. M. Krieg, and J. Vollmer. 2006. Modulating responsiveness of human TLR7 and 8 to small molecule ligands with T-rich phosphorothiate oligodeoxynucleotides. Eur. J. Immunol. 36: 1815–1826. 27. Mata-Haro, V., C. Cekic, M. Martin, P. M. Chilton, C. R. Casella, and T. C. Mitchell. 2007. The vaccine adjuvant monophosphoryl lipid A as a TRIFbiased agonist of TLR4. Science 316: 1628–1632. 28. Pirher, N., K. Ivicak, J. Pohar, M. Bencina, and R. Jerala. 2008. A second binding site for double-stranded RNA in TLR3 and consequences for interferon activation. Nat. Struct. Mol. Biol. 15: 761–763. 29. Gay, N. J., M. Gangloff, and L. A. O’Neill. 2011. What the Myddosome structure tells us about the initiation of innate immunity. Trends Immunol. 32: 104–109. 30. Ma¨kela¨, S. M., P. Osterlund, and I. Julkunen. 2011. TLR ligands induce synergistic interferon-b and interferon-l1 gene expression in human monocytederived dendritic cells. Mol. Immunol. 48: 505–515. 31. Zhu, J., R. Brownlie, Q. Liu, L. A. Babiuk, A. Potter, and G. K. Mutwiri. 2008. Characterization of bovine Toll-like receptor 8: ligand specificity, signaling essential sites and dimerization. Mol. Immunol. 46: 978–990. 32. Toscano, F., Y. Estornes, F. Virard, A. Garcia-Cattaneo, A. Pierrot, B. Vanbervliet, M. Bonnin, M. J. Ciancanelli, S. Y. Zhang, K. Funami, et al. 2013. Cleaved/associated TLR3 represents the primary form of the signaling receptor. J. Immunol. 190: 764–773. 33. Onji, M., A. Kanno, S. Saitoh, R. Fukui, Y. Motoi, T. Shibata, F. Matsumoto, A. Lamichhane, S. Sato, H. Kiyono, et al. 2013. An essential role for the Nterminal fragment of Toll-like receptor 9 in DNA sensing. Nat. Commun. 4: 1949. 34. Kanno, A., C. Yamamoto, M. Onji, R. Fukui, S. Saitoh, Y. Motoi, T. Shibata, F. Matsumoto, T. Muta, and K. Miyake. 2013. Essential role for Toll-like receptor 7 (TLR7)-unique cysteines in an intramolecular disulfide bond, proteolytic cleavage and RNA sensing. Int. Immunol. 25: 413–422. 35. Lindau, D., J. Mussard, B. J. Wagner, M. Ribon, V. M. Ro¨nnefarth, M. Quettier, I. Jelcic, M. C. Boissier, H. G. Rammensee, and P. Decker. 2013. Primary blood neutrophils express a functional cell surface Toll-like receptor 9. Eur. J. Immunol. 43: 2101–2113. 36. Hornung, V., M. Guenthner-Biller, C. Bourquin, A. Ablasser, M. Schlee, S. Uematsu, A. Noronha, M. Manoharan, S. Akira, A. de Fougerolles, et al. 2005. Sequence-specific potent induction of IFN-a by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11: 263–270. 37. Haas, T., J. Metzger, F. Schmitz, A. Heit, T. M€uller, E. Latz, and H. Wagner. 2008. The DNA sugar backbone 29 deoxyribose determines Toll-like receptor 9 activation. Immunity 28: 315–323. 38. Ashman, R. F., J. A. Goeken, E. Latz, and P. Lenert. 2011. Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding. Int. Immunol. 23: 203–214. 39. Gorden, K. K., X. Qiu, J. J. Battiste, P. P. Wightman, J. P. Vasilakos, and S. S. Alkan. 2006. Oligodeoxynucleotides differentially modulate activation of TLR7 and TLR8 by imidazoquinolines. J. Immunol. 177: 8164–8170. 40. Barreiro, L. B., M. Ben-Ali, H. Quach, G. Laval, E. Patin, J. K. Pickrell, C. Bouchier, M. Tichit, O. Neyrolles, B. Gicquel, et al. 2009. Evolutionary dynamics of human Toll-like receptors and their different contributions to host defense. PLoS Genet. 5: e1000562. 41. George, J., A. V. Kubarenko, A. Rautanen, T. C. Mills, E. Colak, T. Kempf, A. V. Hill, A. Nieters, and A. N. Weber. 2010. MyD88 adaptor-like D96N is a naturally occurring loss-of-function variant of TIRAP. J. Immunol. 184: 3025– 3032. 42. Knezevic´, J., D. Pavlinic´, W. A. Rose, II, C. A. Leifer, K. Bendelja, J. Gabrilovac, M. Parcina, G. Lauc, A. V. Kubarenko, B. Petricevic, et al. 2012. Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells. J. Biol. Chem. 287: 24544–24553. 43. George, J., P. G. Motshwene, H. Wang, A. V. Kubarenko, A. Rautanen, T. C. Mills, A. V. Hill, N. J. Gay, and A. N. Weber. 2011. Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly. J. Biol. Chem. 286: 1341–1353. 44. von Bernuth, H., C. Picard, Z. Jin, R. Pankla, H. Xiao, C. L. Ku, M. Chrabieh, I. B. Mustapha, P. Ghandil, Y. Camcioglu, et al. 2008. Pyogenic bacterial infections in humans with MyD88 deficiency. Science 321: 691–696. 45. Beima-Sofie, K. M., A. W. Bigham, J. R. Lingappa, D. Wamalwa, R. D. Mackelprang, M. J. Bamshad, E. Maleche-Obimbo, B. A. Richardson, and G. C. John-Stewart. 2013. Toll-like receptor variants are associated with infant HIV-1 acquisition and peak plasma HIV-1 RNA level. AIDS 27: 2431–2439. 46. Yu, P., W. L€ubben, H. Slomka, J. Gebler, M. Konert, C. Cai, L. Neubrandt, O. Prazeres da Costa, S. Paul, S. Dehnert, et al. 2012. Nucleic acid-sensing Tolllike receptors are essential for the control of endogenous retrovirus viremia and ERV-induced tumors. Immunity 37: 867–879. 47. Nickerson, K. M., S. R. Christensen, J. Shupe, M. Kashgarian, D. Kim, K. Elkon, and M. J. Shlomchik. 2010. TLR9 regulates TLR7- and MyD88-dependent autoantibody production and disease in a murine model of lupus. J. Immunol. 184: 1840–1848.

Downloaded from http://www.jimmunol.org/ by guest on December 22, 2015

1. Kawai, T., and S. Akira. 2011. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34: 637–650. 2. Gay, N. J., M. Gangloff, and A. N. Weber. 2006. Toll-like receptors as molecular switches. Nat. Rev. Immunol. 6: 693–698. 3. Botos, I., D. M. Segal, and D. R. Davies. 2011. The structural biology of Tolllike receptors. Structure 19: 447–459. 4. Berger, M., C. Y. Hsieh, M. Bakele, V. Marcos, N. Rieber, M. Kormann, L. Mays, L. Hofer, O. Neth, L. Vitkov, et al. 2012. Neutrophils express distinct RNA receptors in a non-canonical way. J. Biol. Chem. 287: 19409–19417. 5. Panter, G., A. Kuznik, and R. Jerala. 2009. Therapeutic applications of nucleic acids as ligands for Toll-like receptors. Curr. Opin. Mol. Ther. 11: 133–145. 6. Hemmi, H., T. Kaisho, O. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T. Horiuchi, H. Tomizawa, K. Takeda, and S. Akira. 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3: 196–200. 7. Cros, J., N. Cagnard, K. Woollard, N. Patey, S. Y. Zhang, B. Senechal, A. Puel, S. K. Biswas, D. Moshous, C. Picard, et al. 2010. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity 33: 375–386. 8. Gibbard, R. J., P. J. Morley, and N. J. Gay. 2006. Conserved features in the extracellular domain of human Toll-like receptor 8 are essential for pHdependent signaling. J. Biol. Chem. 281: 27503–27511. 9. Peter, M. E., A. V. Kubarenko, A. N. Weber, and A. H. Dalpke. 2009. Identification of an N-terminal recognition site in TLR9 that contributes to CpG-DNAmediated receptor activation. J. Immunol. 182: 7690–7697. 10. Kubarenko, A. V., S. Ranjan, A. Rautanen, T. C. Mills, S. Wong, F. Vannberg, M. Neumaier, I. Bekeredjian-Ding, A. V. Hill, P. Ahmad-Nejad, and A. N. Weber. 2010. A naturally occurring variant in human TLR9, P99L, is associated with loss of CpG oligonucleotide responsiveness. J. Biol. Chem. 285: 36486–36494. 11. Liu, L., I. Botos, Y. Wang, J. N. Leonard, J. Shiloach, D. M. Segal, and D. R. Davies. 2008. Structural basis of Toll-like receptor 3 signaling with double-stranded RNA. Science 320: 379–381. 12. Kubarenko, A. V., S. Ranjan, E. Colak, J. George, M. Frank, and A. N. Weber. 2010. Comprehensive modeling and functional analysis of Toll-like receptor ligand-recognition domains. Protein Sci. 19: 558–569. 13. Govindaraj, R. G., B. Manavalan, S. Basith, and S. Choi. 2011. Comparative analysis of species-specific ligand recognition in Toll-like receptor 8 signaling: a hypothesis. PLoS ONE 6: e25118. 14. Serbina, N. V., T. Jia, T. M. Hohl, and E. G. Pamer. 2008. Monocyte-mediated defense against microbial pathogens. Annu. Rev. Immunol. 26: 421–452. 15. Murthy, V., A. Moiyadi, R. Sawant, and R. Sarin. 2009. Clinical considerations in developing dendritic cell vaccine based immunotherapy protocols in cancer. Curr. Mol. Med. 9: 725–731. 16. Hodges, A., K. Sharrocks, M. Edelmann, D. Baban, A. Moris, O. Schwartz, H. Drakesmith, K. Davies, B. Kessler, A. McMichael, and A. Simmons. 2007. Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication. Nat. Immunol. 8: 569–577. 17. Tanji, H., U. Ohto, T. Shibata, K. Miyake, and T. Shimizu. 2013. Structural reorganization of the Toll-like receptor 8 dimer induced by agonistic ligands. Science 339: 1426–1429. 18. Siggs, O. M., M. Berger, P. Krebs, C. N. Arnold, C. Eidenschenk, C. Huber, E. Pirie, N. G. Smart, K. Khovananth, Y. Xia, et al. 2010. A mutation of Ikbkg causes immune deficiency without impairing degradation of IkBa. Proc. Natl. Acad. Sci. USA 107: 3046–3051. 19. Iavarone, C., K. Ramsauer, A. V. Kubarenko, J. C. Debasitis, I. Leykin, A. N. Weber, O. M. Siggs, B. Beutler, P. Zhang, G. Otten, et al. 2011. A point mutation in the amino terminus of TLR7 abolishes signaling without affecting ligand binding. J. Immunol. 186: 4213–4222. 20. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, G. Lipford, H. Wagner, and S. Bauer. 2004. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303: 1526–1529. 21. Sasai, M., M. M. Linehan, and A. Iwasaki. 2010. Bifurcation of Toll-like receptor 9 signaling by adaptor protein 3. Science 329: 1530–1534. 22. Kagan, J. C., T. Su, T. Horng, A. Chow, S. Akira, and R. Medzhitov. 2008. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferonb. Nat. Immunol. 9: 361–368. 23. Blasius, A. L., C. N. Arnold, P. Georgel, S. Rutschmann, Y. Xia, P. Lin, C. Ross, X. Li, N. G. Smart, and B. Beutler. 2010. Slc15a4, AP-3, and Hermansky-Pudlak syndrome proteins are required for Toll-like receptor signaling in plasmacytoid dendritic cells. Proc. Natl. Acad. Sci. USA 107: 19973–19978. 24. Chockalingam, A., W. A. Rose, II, M. Hasan, C. H. Ju, and C. A. Leifer. 2012. Cutting edge: a TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production. J. Immunol. 188: 527–530. 25. Kuznik, A., M. Bencina, U. Svajger, M. Jeras, B. Rozman, and R. Jerala. 2011. Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. J. Immunol. 186: 4794–4804.

5973