TLR7 Is Involved in Sequence-Specific Sensing of Single-Stranded in Human

This information is current as Michael P. Gantier, Stephen Tong, Mark A. Behlke, Dakang of October 5, 2021. Xu, Simon Phipps, Paul S. Foster and Bryan R. G. Williams J Immunol 2008; 180:2117-2124; ; doi: 10.4049/jimmunol.180.4.2117 http://www.jimmunol.org/content/180/4/2117 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TLR7 Is Involved in Sequence-Specific Sensing of Single-Stranded RNAs in Human Macrophages1

Michael P. Gantier,* Stephen Tong,*† Mark A. Behlke,‡ Dakang Xu,* Simon Phipps,§ Paul S. Foster,§ and Bryan R. G. Williams2*

Human TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferred from studies of murine TLR7, this has yet to be established for hTLR7. We found that different short ssRNA sequences selectively induced either TNF-␣ or IFN-␣ in human PBMCs. The sequence-specific TNF-␣ response to ssRNAs observed in PBMCs could be replicated in activated human macro- phage-like (THP-1) cells pretreated with IFN-␥. Surprisingly, suppression of hTLR7 expression by RNA interference in this model reduced sensing of all immunostimulatory ssRNAs tested. Modulation of the relative expression ratio of hTLR7 to hTLR8 in THP-1 cells correlated with differential sensing of immunostimulatory sequences. Furthermore, the sequence-specific IFN-␣ Downloaded from induction profile in human PBMCs was accurately modeled by a sequence-specific activation of murine TLR7 in mouse macro- phages. Thus, we demonstrate for the first time that hTLR7 is involved in sequence-specific sensing of ssRNAs. We establish a novel cell model for the prediction of TNF-␣ induction by short RNAs in human macrophages. Our results suggest that differential sequence-specific sensing of RNA oligonucleotides between human and mouse macrophages is due to the modulation of TLR7 sensing by human TLR8. The Journal of Immunology, 2008, 180: 2117–2124.

oll-like receptors are a family of recognizing dif- Studies characterizing hTLR7 and 8 have typically used syn- http://www.jimmunol.org/ ferent pathogen-associated molecular patterns and form part thetic immune response modifiers as ligands, such as imidazo- T of the first line of defense against pathogens. Once engaged quinolines (7, 8), and show that hTLR7 agonists mainly stimulate by the TLRs, pathogen-associated molecular patterns trigger a signal- pDCs to produce IFN-␣, while hTLR8 agonists activate monocytes ing cascade resulting in specific expression profiles that stimu- to produce proinflammatory , notably TNF-␣ (9, 10). late the immune system to eliminate the invading pathogen (1). Consistent with this, PBMCs depleted of pDCs do not produce Among the 10 TLRs reported in humans, TLR3, 7, 8, and 9 constitute IFN-␣ upon RNA stimulation (11). a subgroup that can detect bacterial or viral nucleic acids. To date, the role of hTLR7 in RNA sensing remains poorly defined. 3 Human TLR7 (hTLR7) and TLR8 (hTLR8) are located in the Studies using an overexpression system in HEK293 cells failed to by guest on October 5, 2021 endosomes, are closely related in structure, and have been directly implicate hTLR7 in sequence-dependent sensing of ssRNA oligonucle- implicated in the detection of RNA by genetic complementation in otides (11, 12). In contrast, similar experiments demonstrated that HEK293 cells (2, 3). Among PBMCs, it is believed that hTLR7 is hTLR8 was involved in uridine-rich ssRNA sensing (11, 12). Be- expressed predominantly in plasmacytoid dendritic cells (pDCs) and cause the murine homolog of TLR7 (mTLR7) is required for se- B cells, while hTLR8 is mainly expressed in monocytes and myeloid quence-specific ssRNA sensing, Heil et al. (11) concluded that spe- dendritic cells (4, 5). However, microarray data mining shows that cies-specific differences exist in the sensing of RNA oligonucleotides hTLR7 is also expressed in monocytes, albeit at much lower levels where hTLR8 but not hTLR7 is the sequence-specific of relative to hTLR8 (6). Similarly, hTLR8 expression occurs in pDCs. ssRNAs in humans. Recent studies investigating the immunostimu- lation of small interfering RNA (siRNA) in immune cells have sug- gested a role for hTLR7 in sequence-dependent sensing of siRNA and its ssRNA components on the basis of IFN-␣ production by immune *Centre for Cancer Research, Monash Institute of Medical Research; and †Depart- cells. However, no mechanistic evidence was provided, showing that ment of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Austra- hTLR7 was involved in the responses observed (13–18). lia; ‡Integrated DNA Technologies, Coralville, IA 52241; and §Centre for Asthma and Respiratory Diseases, School of Biomedical Sciences, Faculty of Health, University In the present study, we use a human -like cell line of Newcastle, Newcastle, New South Wales, Australia and provide direct evidence that hTLR7 is involved in the se- Received for publication July 24, 2007. Accepted for publication December 6, 2007. quence-dependent sensing of ssRNAs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Cell isolation and culture 1 This work was supported by National Institutes of Health Grants RO1 A134039 and PO1 CA62220 (to B.R.G.W.). Fresh blood from healthy male donors was collected in heparin-treated 2 Address correspondence and reprint requests to Prof. Bryan R. G. Williams, Monash tubes, and submitted to Ficoll-Paque plus (17-1440-02; GE Healthcare) Institute of Medical Research, Monash Medical Centre, 246 Clayton Road, Clayton, gradient purification following the manufacturer’s guidelines. Isolated cells 5 Victoria 3168, Australia. E-mail address: [email protected] were plated in a 96-well plate at 2 ϫ 10 cells/well in RPMI 1640 plus 3 L-glutamine medium (11875; Invitrogen Life Technologies) complemented Abbreviations used in this paper: hTLR7/8, human TLR7/8; pDC, plasmacytoid ϫ ; mTLR7/8, mouse TLR7/8; siRNA, small interfering RNA; BMM, bone with 1 antibiotic/antimycotic (15204064; Invitrogen Life Technologies) marrow macrophage; DOTAP, N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammo- and 10% FBS (referred to as complete RPMI 1640), and incubated for4hat nium methylsulfate. 37°C in a 5% CO2 atmosphere before stimulation with TLR agonists. Human THP-1 and U937 cells were maintained in complete RPMI 1640 and subcul- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 tured in suspension every 2–3 days until passage ( p Ͻ 25). For experiments, www.jimmunol.org 2118 SEQUENCE-SPECIFIC SENSING OF ssRNAs BY hTLR7

ab4000 G IFN-α A A 3000 B-406-S C C α C G 2000 TNF- G C G G 1000 A A 800 A A 700 G U U 600 U U A 500 FIGURE 1. Sequence-dependent dif- 400 U ferential induction of IFN-␣ and TNF-␣ 300 U A SA C C (pg/ml) by ssRNA in human PBMCs. a and c, 200 C G Human PBMCs were treated with 90 nM 100 G C G G of indicated ssRNA complexed with 0 A A DOTAP and incubated for 16 h at 37°C. A A Cytokine production (IFN-␣ and TNF-␣) G U Mock U was measured by ELISA as described in si9.2-S 3M-002 Medium U A ␮ B-406-S U B-406-AS Lamin-AS Materials and Methods. 3M-002 (1 g/ ODN2216 STAT-2AS ml) and ODN2216 (3 ␮M) were used as GFP21-AS positive controls for induction of TNF-␣ Downloaded from U and IFN-␣, respectively. b, Schematic of c 4000 U A IFN-α C C base substitutions of ssRNA B-406-S. SB 3500 α C G The secondary structure was conserved TNF- G C between the ssRNAs, as predicted with 3000 G G A A mFOLD (24). The data (a and c) are in A A biological triplicate and are representa- 1500 G C tive of a minimum of three different A http://www.jimmunol.org/ 1250 U U A blood donors. 1000

U 750 U A SC C C Cytokine (pg/ml)Cytokine 500 C G G C 250 G G 0 A A A A SA SB SC

G C by guest on October 5, 2021

Mock A A G A 3M-002 Medium B-406-S B-406-AS ODN2216

80,000 THP-1 cells were differentiated for 16 h in conditioned medium with Isolation of bone marrow macrophages PMA at 20 ng/ml (in DMSO 524400; Calbiochem) per well of a 96-well plate TLR7-deficient mice (crossed to the BALB/c background for three generations (at 37°C in a 5% CO2 atmosphere). Priming of adherent PMA-treated THP-1 was conducted by rinsing the cells with 150 ␮l of complete RPMI 1640 sup- (N3), a gift from Dr. S. Akira, Research Institute for Microbial Diseases, ␥ Ͻ Osaka University, Osaka, Japan) were crossed to the BALB/c background for plemented with 100 U/ml human IFN- (IF002, ED50 0.05 ng/ml; Chemi- con International) or 50 ng/ml recombinant human IL-6 (a gift from B. J. a further four generations (N7). Wild-type BALB/c mice generated from the Jenkins, Monash Institute of Medical Research, Clayton, Victoria, Australia), original stock of TLR7-deficient mice were treated identically and used as controls. Mice were housed at the University of Newcastle and experiments for6hat37°C, 5% CO2. The cells were subsequently rinsed with 100 or 150 ␮l of complete RPMI 1640 before stimulation with TLR agonists. The RAW- approved under ACEC939. Bone marrow extraction and differentiation was ELAM cells, stably transfected with an ELAM promoter driving the expres- conducted following standard procedures (20). Briefly, femurs were flushed sion of luciferase (19), were cultured in DMEM complemented with 10% FCS with complete RPMI 1640, and cells were plated in complete RPMI 1640 and 1ϫ antibiotic/antimycotic. RAW-ELAM cells were plated to 90% con- supplemented with 104 U/ml recombinant human CSF-1 (a gift from D. A. fluency in a 96-well plate in 150 ␮l of culture medium for 7 h, and further Hume, Centre for Molecular Biology and University of Biotechnology, stimulated with TLR agonists. The supernatants were collected for TNF-␣ Queensland, Brisbane, Australia) on 10-cm bacteriological plastic plates for 7 analysis 14 h posttransfection. days at 37°C in a 5% CO2 atmosphere.

Table I. ssRNA sequences used in the study

Name of Sequence Sequence Uridines

B-406-S (reported as ␤gal-478-S (13)) 5Ј-GAAGGCCAGACGCGAAUUAUU-3Ј 4 B-406-AS (reported as ␤gal-478-AS (13)) 5Ј-UAAUUCGCGUCUGGCCUUCUU-3Ј 9 Lamin-AS 5Ј-UGUUCUUCUGGAAGUCCAGdTdT-3Ј 7 si9.2-S (reported as siRNA9.2-S (18)) 5Ј-AGCUUAACCUGUCCUUCAAdTdT-3Ј 6 STAT-2AS 5Ј-GUUCCAUUGGCUCUGGUGCUU-3Ј 9 GFP21-AS 5Ј-GAUGAACUUCAGGGUCAGCUU-3Ј 6 SA 5Ј-GAAGGCCUUACGCGAAUUAUU-3Ј 6 SB 5Ј-GAAGGCCUUACGCGAACAAUU-3Ј 4 SC 5Ј-GAAGGCCUUACGCGAACAAGA-3Ј 2 The Journal of Immunology 2119

a TLR7 a 120 TLR8 TLR7 14 100 TLR8 12 80

10 60

8 40 6 20 relative to siControl relative to treated cells 4 % of gene expression 0

Fold induction to PMA- to induction Fold 2 Neg

0 siTLR7 siTLR8 siControl 0 25 50 75 100 IFN-γ + + + - IFN- γ (U/ml) b 1800 1400 ** siTLR8 b * 4000 1000 siTLR7 2500 NS 1000 600 *** siGAPDH 600 500

(pg/ml) 400 400 α 125 300 Downloaded from 100 TNF- 200 75 100 0 50 NS NS

relative to B-406-AS 3M-002 Loxoribine LPS Medium α 25 0 * c ***

% TNF- 125 Mock siTLR8 si9.2-S 3M-002 http://www.jimmunol.org/ Medium B-406-S B-406-AS Lamin-AS siTLR7 Loxoribine 100 STAT-2AS GFP21-AS siGAPDH FIGURE 2. Cytokine priming of THP-1 cells restores functional re- 75

sponsiveness to ssRNAs. a, PMA-differentiated THP-1 cells were treated (pg/ml) α for 8 h with the indicated amount of IFN-␥ and RNA extracted for real- 50 * time RT-PCR analysis. TLR7 and TLR8 mRNA levels were reported rel- TNF- ** ative to GAPDH expression, and further divided by the mean level of the 25 PMA-differentiated condition. Each data point is from biological tripli- 0

cates, and the data are representative of two independent experiments. b, SA

PMA-differentiated THP-1 pretreated with IFN-␥ were stimulated with 330 Mock by guest on October 5, 2021 Medium

nM of indicated ssRNA complexed with DOTAP and incubated for 16 h. STAT-2AS GFP21-AS ␮g/ml) and loxoribine (500 ␮M) were included as controls for 3M-002 (2 FIGURE 3. ssRNAs induce TNF-␣ release through TLR7- and TLR8- TNF-␣ production as measured by ELISA. The data are averaged from a dependent pathways in THP-1 cells. PMA-differentiated THP-1 cells were minimum of two independent experiments, where each treatment was con- transfected with the indicated siRNAs (17 nM), before further treatment ducted in triplicate and reported to the mean of B-406-AS condition. All with IFN-␥ and ssRNAs as detailed in Materials and Methods. a, Real-time ␣ ssRNA significantly induced TNF- production when compared with the RT-PCR analysis of TLR7 and TLR8 expression following8hofIFN-␥ p Ͻ mock control ( 0.001) unless otherwise noted (NS). priming, reported relative to GAPDH levels and expressed as a percent of expression of siControl treatment. The Neg condition indicates the levels of TLR7 and TLR8 independent of IFN-␥ priming. The data are from bio- Cell stimulation logical triplicates and are representative of two independent experiments. b and c, TNF-␣ levels were measured after treatment with TLR7 (loxoribine at ssRNAs were synthesized by Integrated DNA Technologies (IDT) with 900 ␮M), TLR8 (3M-002 at 1 ␮g/ml), and TLR4 (LPS at 500 ng/ml) agonists HPLC purification, and resuspended into duplex buffer (100 mM potassium (b) or ssRNAs (at 660 nM) in the presence of siRNAs (c). The data are av- acetate, 30 mM HEPES (pH 7.5), DNase-RNase-free H2O) to a concen- tration of 80 ␮M. ssRNAs from different syntheses were compared and no eraged from two (c) or three independent experiments in triplicate (b). Statis- immunostimulatory difference was observed. 3M-002 (TLR8 agonist; tlrl- tical analysis is as presented using siGAPDH treatment as a reference. c75), loxoribine (TLR7 agonist), and ODN2216 (TLR9 agonist; tlrl-hodna) were purchased from Invivogen and LPS was obtained from Sigma- Aldrich (L8274). ODN2216, loxoribine, 3M-002, and LPS were added directly to medium at the specified concentration. ssRNAs were transfected lowing the manufacturers’ guidelines, and the sequences were not supplied. into cells with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammonium siTLR7 sequences previously published (21) are as follows: TLR7-sense (S) 5Ј-GCCUUGAGGCCAACAACAUdTdT-3Ј, TLR7-antisense (AS) 5Ј- methylsulfate (DOTAP) (1811177; Roche). DOTAP was first diluted in Ј RPMI 1640 (75 ␮l) for 5 min before mixing with an equal volume of RPMI AUGUUGUUGGCCUCAAGGCdTdT-3 and were synthesized by IDT, 1640 containing the ssRNA. The resulting mix was incubated Ͼ10 min and resuspended into duplex buffer, annealed at 92°C for 2 min and left for 30 ␮ min at room temperature before aliquoting. All siRNAs were resuspended 50 l were added per well of a 96-well plate, resulting in a final volume of ␮ 150 or 200 ␮l. Transfections were conducted in triplicate in all experi- to a final concentration of 40 M. A total of 80,000 THP-1 cells were ␮ ␮ differentiated for 10 h with PMA at 20 ng/ml per well of a 96-well plate. ments. The ratios of DOTAP to ssRNA were as follows: 5.3 g/ l ssRNA ␮ (see Figs. 1 and 5b), 2.8 ␮g/␮l ssRNA (see Fig. 2), and 3.74 ␮g/␮l ssRNA Subsequently, the cells were rinsed with 100 l of RPMI 1640 plus L- (see Figs. 3, 4, 5c, and 6). When dose responses were conducted, the ratio glutamine medium (11875; Invitrogen Life Technologies) complemented of ssRNA to DOTAP was maintained constant. with 10% FBS without antibiotics. Lipofectamine RNAiMAX (13778-075; Invitrogen Life Technologies) was first diluted in Opti-MEM (51985-034; RNA interference 75 ␮l; Invitrogen Life Technologies) for 5 min before mixing with an equal volume of Opti-MEM containing the siRNA. After 20 min of incubation, siTLR8 was purchased from Dharmacon as an ON-TARGETplus SMART- 50 ␮l of the resulting RNAiMAX/siRNA was added directly onto the cells, pool (L-004715-00), and both siGAPDH (4631) and siControl 1 (4635) giving a final volume of 150 ␮l. The ratio of Lipofectamine RNAiMAX to ␮ were purchased from Ambion. Duplexed siRNAs were resuspended fol- siRNA was 5-1 l. Following a 14-h incubation at 37°C (5% CO2 2120 SEQUENCE-SPECIFIC SENSING OF ssRNAs BY hTLR7

a b c 7 2500 IFN-γ pretreatment1500 IL-6 pretreatment 2000 6 TLR7 1000 TLR8 1500 5 1000 500

4 600 60 500 50 (pg/ml) 3 (pg/ml) α α 400 40

treated cells treated ** 2 300 30 * TNF- TNF- 200 20

Fold induction to PMA- to induction Fold 1 100 10 0 0 0 0 25 50 75 100 200 400 600 800 1000 200 400 600 800 1000 IL-6 (ng/ml) ssRNA (nM) ssRNA (nM)

de500 4000 STAT-2AS 3500 400 si9.2-S 3000 SA 2500 300 B-406-S

(pg/ml) 2000 (pg/ml)

α 3M-002 α 200 1500 Downloaded from Loxoribine IFN- TNF- 1000 100 Mock 500 Medium ND ND ND ND 0 0 200 400 600 800 200 400 600 800 ssRNA (nM) ssRNA (nM) FIGURE 4. Differential sensing of ssRNAs correlates with differential expression ratio of hTLR7 to hTLR8. a, PMA-differentiated THP-1 cells were treated for 8 h with the indicated amount of IL-6, and RNA was extracted for real-time RT-PCR analysis. TLR7 and TLR8 mRNA levels are shown reported http://www.jimmunol.org/ relative to GAPDH expression, and further divided by the mean level of the PMA-differentiated condition. Each data point is from biological triplicates, and the data are representative of two independent experiments. b and c, PMA-differentiated THP-1 were primed with 100 U/ml IFN-␥ (b) or 50 ng/ml IL-6 (c) for 8 h before further stimulation with the indicated ssRNA/DOTAP complexes. Sixteen hours after treatment, the levels of TNF-␣ were analyzed by ELISA. The data are averaged from two independent experiments in triplicate and are representative of four independent experiments. Statistical analysis comparing SA and si9.2-S is presented. d and e, Human PBMCs were subjected to a similar dose-response treatment of ssRNAs as in b and c, and the supernatants were analyzed for IFN-␣ and TNF-␣ after 16 h of treatment. Each treatment was conducted in triplicate, and the data presented are from two different blood donors. No IFN-␣ was detectable (ND) with SA and B-406-S (e). 3M-002 (1 ␮g/ml) (b–e) and loxoribine (900 ␮M) (b and c) were used as positive controls for TLR7/8 activation. Mock control corresponds to the highest amount of DOTAP used with 1 ␮M ssRNA. The dose responses were

conducted with a constant ratio of DOTAP/ssRNA. by guest on October 5, 2021 atmosphere), the cells were further rinsed with 150 ␮l of complete RPMI in PBS) at a final 108 cells/ml buffer. The cells were incubated with 100 ␮l 1640 supplemented with 100 U/ml IFN-␥. No cell death was observed of StemSep Human CD14 Positive Selection Cocktail (14758; StemCell during this procedure and the cells were further treated as described above. Technologies) per milliliter of cell suspension for 10 min at 4°C. A total of 60 ␮l of magnetic colloid were subsequently added per milliliter of cell Real-time RT-PCR suspension and incubated for another 10 min at 4°C. The cell suspension was rinsed with 16 ml of magnetic buffer per milliliter of cell suspension cDNA was synthesized from column-purified RNA (NucleoSpin RNAII ϫ columns; 740955; Macherey-Nagel) using the SuperScript III First-Strand and pelleted at 300 g for 8 min. The pellet was resuspended in 4 ml of magnetic buffer per milliliter of cell suspension, before loading onto an LS kit (18080-051; Invitrogen Life Technologies), with oligo-dT(20) priming and following the manufacturer’s instructions. Real-time PCR was con- column (130-042-401; Miltenyi Biotec) according to the manufacturer’s ducted with the iQ SYBR Green Supermix (170-8882; Bio-Rad) on a Bio- recommendations. The purity of eluted cells from positive magnetic puri- fication was assessed by flow cytometer analysis using BD Pharmingen Rad iCycler. hTLR7 (NM_016562) and hTLR8 (NM_138636) were ampli- Ͼ fied using the following primer pairs (5Ј–3Ј): TLR7 forward (FWD): CCT CD14 PE-conjugated Ab (BD Biosciences), and was 97%. TTCCCAGAGCATACAGC; TLR7 reverse (REV): GGACAGAACTCCC ACAGAGC; TLR8-FWD: CAGAGCATCAACCAAAGCAA; TLR8- Statistical analyses REV: GCTGCCGTAGCCTCAAATAC. hGAPDH (NM_002046) was used as a reference gene and amplified with the following primer pair: Statistical analyses were conducted using GraphPad Instat version 3.05. GAPDH-FWD: CATCTTCCAGGAGCGAGATCCC; GAPDH-REV: TTC Unpaired t tests with Welch correction were used to compare the signifi- ACACCCATGACGAACAT. Each amplicon was sequence-verified and cance of the results. Error bars on each figure represent the SEM. Symbols .p Ͻ 0.0001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء :used to generate a standard curve for the quantification of gene expression. used Detection of cytokines Human IFN-␣ in culture supernatants was quantified by sandwich ELISA Results using mouse monoclonal (0.5 ␮g/ml, 21112-1; PBL Biomedical) and rabbit Sequence-dependent differential induction of IFN-␣ and polyclonal Abs (0.5 ␮g/ml, 31130-1; PBL Biomedical). A goat anti-rabbit TNF-␣ by ssRNA HRP-conjugated Ab (0.8 ␮g/ml, 31460; Pierce) was used for detection. Hu- man and mouse TNF-␣ were measured using the OptEIA ELISA sets (555212 Different ssRNA sequences were screened for immunostimulatory and 558874, respectively; BD Biosciences). In both IFN and TNF-␣ ELISAs, activity in human PBMCs (6 of 14 sequences tested are shown in tetramethyl benzidine substrate (T0440; Sigma-Aldrich) was used for quanti- Fig. 1a with the detail of the sequences in Table I), and it was fication of the cytokines on the Fluostar OPTIMA (BMG Labtech) plate observed that some sequences strongly induced both IFN-␣ and reader. TNF-␣ (such as B-406-AS, Lamin-AS, and STAT-2AS), while ϩ Magnetic purification of CD14 monocytes others preferentially induced IFN-␣ (see si9.2-S and GFP21-AS). After extraction of PBMCs by Ficoll-Paque, cells were pelleted at 350 ϫ Because it has been previously suggested that IFN-␣ induction g for 7 min and resuspended in magnetic buffer (2 mM EDTA, 0.5% BSA may be more specific to hTLR7 and that TNF-␣ may reflect more The Journal of Immunology 2121

a 25 CD14+ b 400 +IFNγ 20 U937 300 NS Medium 15 200

10 100 * (pg/ml) 4 α 60 40 TNF- non-treated cells non-treated

Fold induction to 2 20 0 0

TLR7 TLR8 SA Mock si9.2-S c STAT-2AS 40 STAT-2AS 30 si9.2-S 3M-002 γ (pg/ml) 20 +IFN α Loxoribine Mock Downloaded from TNF- 10 Medium STAT-2AS +Medium 0 250 500 750 1000 ssRNA (nM) FIGURE 5. TLR7/8 induction by IFN-␥ in monocytic cells correlates with sequence-specific sensing of ssRNAs. a, CD14ϩ monocytes and U937 cells http://www.jimmunol.org/ were treated with 100 U/ml IFN-␥ for 6 and 8 h, respectively, and RNA was extracted for real-time RT-PCR analysis. TLR7 and TLR8 mRNA levels are shown, reported relative to GAPDH expression, and further divided by the mean level of nontreated cells. The data for CD14ϩ cells are from one blood donor in biological triplicate and are representative of two blood donors. The data for U937 cells are averaged from three independent experiments in triplicate. b, CD14ϩ monocytes were purified as presented in Materials and Methods, plated in complete RPMI 1640 at 50,000–100,000 cells/well of a 96-well plate and primed (ϩIFN-␥) or not (Medium) for 6 h with 100 U/ml IFN-␥. After rinsing with fresh complete RPMI 1640 medium, the cells were incubated with the indicated ssRNA (90 nM) complexed with DOTAP. Sixteen hours after treatment, the levels of TNF-␣ and IFN-␣ (data not shown) were analyzed by ELISA. The data are averaged from two independent experiments in triplicate, from two different blood donors. Statistical analyses comparing conditions si9.2-S and SA are presented (NS, nonsignificant). c, U937 cells were plated in growing medium complemented with 100 U/ml IFN-␥ (ϩIFN-␥) or not (ϩMedium) at 80,000 cells/well of a 96-well plate. After an 8-h incubation, the indicated ssRNAs complexed with DOTAP were added and incubated for 16 h before analysis of TNF-␣ levels via ELISA. The data are representative of two independent experiments in triplicate. by guest on October 5, 2021 of an hTLR8 response (9), we speculated that preferential hTLR7 A human macrophage cell model reproduces functional sensing or hTLR8 signaling by RNA ligands may be sequence specific. of ssRNAs To further characterize the differential activation of IFN-␣ and Because human macrophage-like THP-1 (25) cells naturally ex- TNF-␣ observed with different ssRNAs, the impact of base sub- press hTLR7 and 8 and have been successfully used for studying stitutions on a sequence that was not immunostimulatory (B- synthetic hTLR7/8 agonists (26–28), they provide a natural model 406-S, Fig. 1a) was assessed. It has been previously proposed that to study endogenous TLR signaling rather than the overexpression hTLR7/8 sensing of ssRNAs is dependent on the presence of uri- of the receptors in HEK293 cells. We found that ssRNA stimula- dine ribonucleotides, as observed in mice with mTLR7 (11, 16, 22, tion of THP-1 cells with or without PMA differentiation did not 23). Accordingly, we determined whether the degree of immuno- promote detectable induction of TNF-␣ on ELISA, although PMA stimulation was simply a reflection of the number of uridines treatment strongly induced sensitivity of the cells to the TLR4 present, or whether the location of the uridines in the ssRNA rel- ative to its secondary structure had any influence on immuno- ligand LPS (data not shown). Given the functionality of the TLR4/ stimulation. To do this, three ssRNAs were designed where the MyD88-signaling pathway in PMA-differentiated THP-1 cells, we number of uridines was varied and their location relative to a cen- reasoned that the absence of ssRNA sensing was related to low tral stem loop changed, without disrupting the predicted secondary levels of hTLR7/8. In accord with this, PMA-differentiated THP-1 structure (Fig. 1b) (24). As expected, a significant induction of TNF-␣ cells stimulated with synthetic hTLR7 agonists failed to induce ␣ was observed with SA ( p Ͻ 0.0001, compared with B-406-S), which TNF- production (data not shown; Ref. 27), suggesting TLR7 ␥ contains two more uridine residues than B-406-S, in the stem loop was not expressed at a functional level in these cells. IFN- prim- (Fig. 1, b and c). In contrast, however, SB induced more TNF-␣ than ing of PMA-differentiated THP-1 cells has been proposed to se- B-406-S ( p ϭ 0.0217), even though both have the same number of lectively induce both hTLR7 and hTLR8, with a predominance for uridines. This is in accord with immunostimulation being affected by hTLR8 (26). Accordingly, IFN-␥ priming (at 50–100 U/ml) of the location of the uridines within the ssRNA. PMA-differentiated THP-1 cells robustly induced hTLR8 (ϳ11- Unexpectedly, neither SA nor SB induced IFN-␣ production fold) and hTLR7 (ϳ3-fold) mRNA (Fig. 2a). More importantly, (Fig. 1c). This contrasts with Lamin-AS, which, although it has a IFN-␥ priming of the cells restored the sequence-specific TNF-␣ similar amount of uridine residues to SA (Table I), stimulates the response observed in PBMCs (Fig. 2b and data not shown). B-406- release of both TNF-␣ and IFN-␣ (Fig. 1, a and c). Taken together, AS, Lamin-AS, and STAT-2AS significantly induced TNF-␣, but these results suggested that sequence-specific sensing of ssRNAs not B-406-S and si9.2-S, in agreement with the findings in PBMCs in PBMCs is mediated differentially by hTLR8 and hTLR7. (compare Figs. 1a and 2b, TNF-␣). In fact, screening of all 14 2122 SEQUENCE-SPECIFIC SENSING OF ssRNAs BY hTLR7 sequences in the IFN-␥-primed THP-1 showed good agreement with the response profiles seen in PBMCs (data not shown), with a 1600 STAT-2AS a few exceptions. For example, GFP21-AS induced TNF-␣ in 1400 si9.2-S THP-1 but not in PBMCs, where it potently induced IFN-␣ (Fig. 1200 SA 1a). This suggested that TNF-␣ production in the THP-1 model 1000 B-406-S could also be indicative of sequences that preferentially induce (pg/ml) 800

α 3M-002 ␣ high IFN- in PBMCs, thus revealing a possible overlap of hTLR7 600 LPS and hTLR8 signaling pathways in THP-1 cells. TNF- 400 Mock Medium hTLR7 is required for sequence-specific sensing by macrophages 200 0 To investigate the respective contributions of hTLR7 and hTLR8 100 200 300 400 500 600 700 in sequence-specific sensing in IFN-␥-primed THP-1 cells, RNA ssRNA (nM) interference was used to selectively down-regulate hTLR7 or b +/+ hTLR8 before priming. Fig. 3a shows that significant knockdown 1500 TLR7 80 nM TLR7 +/+ 500 nM of mRNA expression was achieved for both hTLR7 and 8. Atten- 1250 TLR7 -/- 80 nM uation of IFN-␥-driven hTLR8 up-regulation by siTLR8 function- 1000 TLR7 -/- 500 nM ␣ 750 ally resulted in a 4-fold decrease of TNF- production with the 300 (pg/ml) hTLR8 agonist 3M-002 (Fig. 3b). siRNA silencing of hTLR7, but α 250 200 not 8, abrogated the response to loxoribine, a synthetic hTLR7 *** TNF-

150 Downloaded from agonist. In contrast, silencing of both receptors had no effect on 100 NS 50 LPS signaling, a TLR4 ligand (Fig. 3b). Interestingly, hTLR7 0 down-regulation also affected 3M-002 sensing (2.5-fold decrease, SA LPS Mock si9.2-S compare siTLR7 vs siGAPDH conditions). This is in agreement 3M-002 Medium B-406-S with some cross-reactivity of 3M-002 to hTLR7, as reported by the STAT-2AS manufacturer at the concentration used (Invivogen). FIGURE 6. ssRNAs induce TNF-␣ in a TLR7-dependent manner in The effect of down-regulating hTLR7 and hTLR8 was next as- mouse macrophages. a, RAW-ELAM cells were treated with the indicated http://www.jimmunol.org/ sessed in the sensing of three ssRNA sequences (STAT-2AS, SA, doses of ssRNA/DOTAP complexes and incubated for 14 h before TNF-␣ and GFP21-AS). These were chosen on the basis of their different analysis of the medium via ELISA. Mock control corresponds to the high- cytokine induction profiles in PBMCs (Fig. 1), where SA produced est amount of DOTAP used with 750 nM ssRNA. The data are represen- ␣ tative of three independent experiments in triplicate. b, BMMs from only a TNF- response (thus indicating a possible preference for Ϫ/Ϫ ϩ/ϩ ␣ TLR7 and TLR7 littermates were treated for 16 h with the indicated hTLR8) while GFP21-AS preferentially induced IFN- (suggest- ssRNA/DOTAP doses (80 or 500 nM) and TNF-␣ was measured via ing its preference for hTLR7), whereas STAT-2AS induced both ELISA. The Mock control for both 80 and 500 nM ssRNA is shown. cytokines. Unexpectedly, hTLR7 down-regulation by siTLR7 sig- 3M-002 (1 ␮g/ml) and LPS (500 ng/ml) were used as controls, and were nificantly affected sensing of all three ssRNAs (Fig. 3c), demon- not complexed with DOTAP (a and b). Statistical analyses shown are com- by guest on October 5, 2021 strating direct involvement of this receptor in the sensing of all pared with Mock 500 nM (NS: nonsignificant). The data are averaged from types of immunostimulatory RNA oligonucleotides identified in three TLR7ϩ/ϩ and three TLR7Ϫ/Ϫ mice, in triplicate, conducted in two this study. The sensing of SA, a putative hTLR8 ligand, was sig- independent experiments. nificantly decreased by hTLR7 down-regulation and this effect was more than that seen with down-regulation of hTLR8. Nevertheless, hTLR8 down-regulation by siTLR8 also affected sensing of duction profiles in PBMCs, on IFN-␥- and IL-6-primed THP-1 ssRNA (Fig. 3c, STAT-2AS), although to a lesser extent than cells. Although STAT-2AS strongly induced TNF-␣ in both sys- with hTLR7 knockdown. tems in a dose-dependent manner, discrepancies were observed between si9.2-S, SA (that both have a similar amount of uridines, Variation of relative levels of endogenous hTLR7 to hTLR8 see Table I), and B-406-S (Fig. 4, b and c). In accordance with the affects sequence-dependent sensing preferential induction of hTLR7, the ability of si9.2-S to promote Recently, possible interaction between hTLR7 and hTLR8 was immunostimulation was significantly higher than that of SA and inferred with the finding that both receptors could be immunopre- B-406-S in IL-6-primed cells (Fig. 4c). This correlated with a cipitated following overexpression in HEK293 cells (27). A neg- dose-response induction of IFN-␣ with si9.2-S, whereas neither ative-regulatory role for hTLR8 on hTLR7 sensing of synthetic SA nor B-406-S potently induced IFN-␣ in PBMCs (Fig. 4e). Con- agonists was also suggested (27). To further investigate the pos- versely, SA was significantly more immunostimulatory than sible impact of hTLR8 on the sequence-specific detection of si9.2-S and B-406-S in IFN-␥-primed THP-1 cells and in PBMCs ssRNA through hTLR7, we investigated how the modulation of as measured by TNF-␣ production (Fig. 4, b and d). expression levels of hTLR8 relative to hTLR7 related to ssRNA Induction of hTLR7 and 8 further correlated to sequence-spe- ϩ sensing, in the THP-1 model. Having shown that IFN-␥ priming of cific ssRNA sensing in primary CD14 monocytes pretreated with PMA-differentiated THP-1 favored hTLR8 over hTLR7 expres- IFN-␥ (Fig. 5, a and b). Although no immunostimulation could be ϩ sion (thus mimicking the balance observed in monocytes) (Fig. seen without IFN-␥ pretreatment of CD14 cells for SA and 2a), we searched for a treatment resulting in favored hTLR7 ex- si9.2-S, pretreatment resulted in specific sensing of SA over pression compared with hTLR8 (thus modeling the balance seen in si9.2-S (Fig. 5b). pDCs). In agreement with a previous report (26), IL-6 priming of PMA-differentiated THP-1 cells resulted in a preferential up-reg- Interspecies conservation of ssRNA sensing in macrophages ulation of hTLR7 (ϳ6-fold) vs hTLR8 (ϳ2-fold) (Fig. 4a), thereby via TLR7 mimicking the ratio of hTLR7 to hTLR8 observed in pDCs. To mTLR8 is not involved in the detection of ssRNAs in mouse mac- determine the effect of this on immunostimulation, we compared rophages, but rather this has been proposed to rely solely on the activities of four ssRNAs sequences with distinct cytokine in- mTLR7 from studies using TLR7Ϫ/Ϫ and TLR8Ϫ/Ϫ mice (11, 18). The Journal of Immunology 2123

Our finding of a direct role for hTLR7 in sequence-specific sensing PBMCs (Figs. 1a and 2b), as observed with si9.2-S which induces of ssRNAs in THP-1 macrophages, in conjunction with hTLR8, TNF-␣ in PBMCs when used at doses higher than 200 nM (Fig. 4d). led us to investigate whether sequences that were immunostimu- To directly study the involvement of hTLR7 and 8 in the re- latory in humans were also immunostimulatory in mouse macro- sponse observed in these macrophage-like cells, we used RNA phages. In agreement with a conservation of sequence-specific interference to knockdown hTLR7 or hTLR8 in THP-1 cells. This sensing of ssRNAs between human and mouse macrophages (11, altered IFN-␥-induced hTLR7 and hTLR8 up-regulation (Fig. 3a) 18), all the sequences that induced IFN-␣ in human PBMCs acti- and resulted in a significant decrease of TNF-␣ production by syn- vated NF-␬B and induced TNF-␣ production in mouse macro- thetic hTLR7/8 ligands, but not the TLR4 ligand LPS (Fig. 3b). phage RAW-ELAM cells, in a dose-dependent manner (Fig. 6 and Unexpectedly, down-regulation of both hTLR8 and hTLR7 func- data not shown). This sensing of ssRNAs was TLR7 dependent, as tionally altered ssRNA sensing (Fig. 3c, STAT-2AS). The down- demonstrated by the lack of immunostimulation in TLR7Ϫ/Ϫ bone regulation of hTLR7 also resulted in a stronger impairment of marrow macrophages (BMMs) (Fig. 6b). sensing of all three ssRNAs tested when compared with hTLR8 However, two ssRNA sequences inducing TNF-␣ but not IFN-␣ RNA interference (Fig. 3c), probably as a result of more efficient in human PBMCs (hence showing a possible preference for siTLR7 inhibition of hTLR7 induction by IFN-␥ (Fig. 3a). hTLR8) did not activate mTLR7 in mouse macrophages, even at Taken together, these results demonstrate a direct involvement high concentration (Fig. 6, see SA, and data not shown). These of hTLR7 in the induction of TNF-␣ by ssRNAs in the macro- results are in accord with the findings by Heil et al. (Ref. 11, see phage-like THP-1 cell line. Consistent with this, sequence-depen- ssRNA42), and suggest that the discrepancies in ssRNA sensing dent ssRNA detection by murine BMMs was also dependent on between human and mouse macrophages (as shown with SA) rely TLR7, as illustrated by the ablation of the TNF-␣ response in on the lack of mTLR8 function, while TLR7 function and se- TLR7Ϫ/Ϫ BMMs (Fig. 6b and Refs. 11 and 18). Furthermore, all Downloaded from quence-specific sensing is conserved between species. inducers of IFN-␣ in human PBMCs were also immunostimulatory in mouse macrophages. It should be noted however, that the sens- Discussion ing of a sequence favored by an overexpression of hTLR8 vs The potential of RNA oligonucleotides for use as therapeutic hTLR7 in THP-1 cells and CD14ϩ monocytes (Figs. 4, b and d, agents is currently under intense investigation. Several reports and 5b, SA) and which did not induce IFN-␣ in PBMCs, is not have implicated activation of hTLR7 and 8 by siRNA duplexes conserved in mouse BMMs (Fig. 6, a and b, SA). http://www.jimmunol.org/ complexed with cationic liposomes in human blood cells (13–16, Based on our findings, we propose a model where TLR7 is 18). The induction of innate was sequence dependent involved in sequence-specific sensing of RNA oligonucleotides in and was also observed with at least one of the two strands of the both mouse and human macrophages; nevertheless, sensing of immunostimulatory duplex, when used separately (15, 16, 18). ssRNAs by hTLR7 would be complemented by hTLR8 in human Although investigating the mechanisms underlying the immune but not in mouse macrophages. In cytokine-primed THP-1 cells, response invoked by short ssRNAs in human PBMCs, we observed the relative expression of hTLR7 to hTLR8 modulates the se- differential cytokine induction profiles (Fig. 1a). Although some quence-dependent sensing of ssRNAs with an identical amount of sequences potently promoted both IFN-␣ and TNF-␣ induction uridine residues (Fig. 4, b and c, and Table I, compare SA and by guest on October 5, 2021 (B-406-AS, Lamin-AS, STAT-2AS), others were restricted to in- si9.2-S). We also show that primary CD14ϩ monocytes produce duction of IFN-␣ (si9.2-S, GFP21-AS) at the concentration used. TNF-␣ in response to some ssRNAs, and that sequence-specific The addition of a UU motif within a predicted stem loop region of sensing is enhanced with IFN-␥ priming (Fig. 5b, compare STAT- a nonstimulatory ssRNA (B-406-S giving SA) resulted in specific 2AS, SA and si9.2-S); of note, we did not detect IFN-␣ production TNF-␣ induction independent of IFN-␣, even at very high doses in in these cells (data not shown). Enhanced TNF-␣ levels in CD14ϩ PBMCs (Figs. 1c and 4, d and e). Although this supports previous cells correlated with an induction of both hTLR7 and 8 mRNA claims that RNA immunostimulation is directly dependent on the levels, with a predominance of hTLR8 (Fig. 5a). The observation amount of uridine residues in human PBMCs (11, 16), we found that all monocytic/macrophage cells used in this study induced that localization of the uridines within the ssRNA was also important both hTLR7 and 8 in response to IFN-␥ priming, which correlated (Fig. 1, b and c, compare B-406-S and SB). Assuming that IFN-␣ with sequence-specific sensing of ssRNAs, indicates that expres- production is a readout of pDC activation and that of TNF-␣ mono- sion of the two TLRs is functionally related in human macro- cyte activation, it is possible that some ssRNAs preferentially recruit phages. This adds credence to a role for hTLR7, together with hTLR7 (in pDCs) or hTLR8 (in monocytes) in a similar manner to hTLR8 in ssRNA sensing in human macrophages. Indeed, IFN-␥ that of synthetic immune response modifiers (9, 10, 16). inducibility of hTLR7 and 8 is most likely relevant to a complex To further characterize the involvement of hTLR7 and hTLR8 feedback of cytokines produced by stimulated PBMCs. NK cell in sequence-specific ssRNA sensing, we established a macro- activation by hTLR7/8 agonists has recently been shown to be phage-like cell model (THP-1 cells) (25) reproducing functional dependent on the production of IL-12 by activated macrophages detection of immunostimulatory RNA oligonucleotides (Fig. 2). and to result in IFN-␥ production (29, 30). It is therefore likely that TNF-␣ production by ssRNA-stimulated cells was only observed the hTLR7/8 responsiveness to IFN-␥ in monocytes/macrophages after IFN-␥ priming of the cells, which induced both hTLR7 and observed here is part of a loopback mechanism, further increasing hTLR8 expression—with a preference for hTLR8 (Fig. 2a). As sensing of hTLR7/8 agonists by macrophages. In support of this is shown in Fig. 5, a and c, this was replicated in another monocytic/ the observation that the levels of TNF-␣ from unprimed CD14ϩ macrophage-like cell line (U937 cells, see STAT-2AS condition) cells treated with ssRNAs were Ͼ10-fold lower than that observed (25), although the THP-1 model appeared to be more sensitive to with PBMCs, even though the number of monocytes was ϳ2- to ssRNAs (and yielded much higher TNF-␣ levels). The sequence- 4-fold higher (compare Figs. 5b and 1a, STAT-2AS). specific cytokine induction profile in THP-1 cell model paralleled The finding of a requirement for hTLR7 in ssRNA sensing by TNF-␣ production observed in PBMCs (Figs. 1a and 2b). One THP-1 cells was unexpected given the prevalence of hTLR8 in- sequence inducing IFN-␣ but not TNF-␣ in PBMCs (GFP21-AS) duction by IFN-␥ in this cell type. Furthermore, the previous dem- was found to induce TNF-␣ in this model. This is likely related to the onstration that transient overexpression of hTLR8 in HEK293 and higher concentration of ssRNAs used in THP-1 compared with U20S cells reconstitutes responsiveness to ssRNAs via activation 2124 SEQUENCE-SPECIFIC SENSING OF ssRNAs BY hTLR7 of NF-␬B (11, 31) indicates that hTLR8 does not require hTLR7 9. Gorden, K. B., K. S. Gorski, S. J. Gibson, R. M. Kedl, W. C. Kieper, X. Qiu, for signaling when expressed artificially. The discrepancies be- M. A. Tomai, S. S. Alkan, and J. P. Vasilakos. 2005. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J. Immunol. 174: tween these results and the data presented here are likely related to 1259–1268. overexpression of TLRs in these studies and the saturation doses of 10. Jurk, M., A. Kritzler, B. Schulte, S. Tluk, C. Schetter, A. M. Krieg, and ssRNAs used (ϳ3-fold higher than the highest dose used here). In J. Vollmer. 2006. 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