Sequence determinants of innate immune activation by short interfering RNAs
© Goodchild et al; licensee BioMed Central Ltd. 2009
Received: 22 December 2008
Accepted: 24 July 2009
Published: 24 July 2009
Short interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated.
A total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs. There was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex. Using siRNAs specifically designed to analyse the effect of base substitutions and hybridisation of the two strands, we found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity.
The data presented favour a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.
The mammalian innate immune system utilises a number of protein receptors for the identification of microbial molecules. Amongst these are several receptors specific for foreign ribonucleic acids (RNAs), including Toll-like receptor 3 (TLR3), TLR7, TLR8, RIG-I, MDA5 and PKR (reviewed in ). These receptors are specifically activated by different microbial RNA features, including double-strandedness (TLR3, RIG-I, MDA5, PKR) [2–4], extracellular or endosomal localization (TLR3, TLR7, TLR8) [5, 6] and/or the presence of 5'-triphosphates (RIG-I, PKR) [7–9]. The engagement of these receptors by their cognate ligands induces the expression of anti-viral genes, including cytokines and type I interferons (IFNs).
Short interfering RNAs (siRNAs) are routinely used in laboratory settings, and hold promise for a range of therapeutic applications [10, 11]. Although initially thought to bypass the innate immune system by virtue of their size [12, 13], siRNAs have more recently been shown to induce immune stimulation in a variety of in vivo, and in vitro settings (reviewed in [14–16]). Synthetic siRNA duplexes have now been show to activate PKR in glioblastoma cells , RIG-I in glioblastoma cells and primary human monocytes [7, 18], TLR3 in HEK293 cells and murine models [19, 20], TLR7 in murine leukocytes and human plasmacytoid dendritic cells , and TLR8 in human monocytes . In addition, a number of groups have demonstrated innate immune stimulation by siRNAs in human peripheral blood mononuclear cells (PBMCs) without defining the cell or receptor specificity [23–26].
Whilst the activation of RIG-I, PKR and TLR3 described above does occur with specific types of siRNA and in specific cellular contexts, it appears that the major innate immune response to standard 21 mer siRNAs (i.e. without 5'-triphosphates and with 3'-overhangs) is activation of TLR7 and/or TLR8 in leukocytes [21–26]. In isolated human leukocytes immune stimulation has been shown to be mediated by TLR7/8 activation since it is dependent upon endosomal maturation (excluding activation of cytosolic receptors such as RIG-I), and mediated by single-stranded RNA in a sequence-dependent fashion (excluding TLR3) [21, 22, 24, 26]. Sequence-dependent activation of TLR7 by such siRNA has been demonstrated in primary human pDCs, murine leukocytes and in in vivo murine models . Sequence-dependent activation of human monocytes through an endosomal pathway has also been reported , presumably through activation of TLR8 which is the primary sensor of single-stranded RNA in these cells . Stimulation of human PBMCs with siRNA can lead to production of either IFNα or TNFα or both simultaneously, leading to the hypothesis that differences in the sequence specificity of TLR7 and TLR8 may cause a given immunostimulatory sequence to activate pDCs through TLR7 and/or monocytes through TLR8 .
TLR7 and TLR8 are both activated by single-stranded RNA. The precise sequence requirements for their activation have not yet been elucidated, however, it has been demonstrated that G and U rich sequences tend to stimulate TLR7 causing IFNα production from pDCs, and A and U rich sequences tend to stimulate TLR8 causing production of both IFNα and TNFα from monocytes [28–30]. It has not yet been determined whether these base preferences also affect the activation of TLR7/8 by siRNA (it should be noted that for an siRNA duplex of a given length, the total G+U content is identical irrespective of the sequence and is equal to the length of the siRNA). However, it has been shown that TLR7/8 immunostimulatory duplex siRNAs are less active than their component single strands, suggesting that activation of TLR7/8 by siRNAs is induced by the latter . In support of this hypothesis, specific G and U rich TLR7 stimulatory motifs (GUCCUUCAA and UGUGU) have been identified in the single strand components of two siRNA duplexes [21, 24].
Thus, it appears that the primary mechanism of innate immune stimulation by siRNAs is the activation of TLR7/8 by a single strand of the siRNA duplex (which may become denatured in the endosome, allowing release of the immunostimulatory strand). In the present study, we have investigated the sequence requirements for siRNA stimulation of TLR7/8 by screening 207 siRNA sequences for stimulation of human PBMCs. We found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity. In addition, we have identified a number of highly immunogenic RNA sequences.
Results and Discussion
In order to determine the determinants of immunogenicity of siRNAs, we initially sought to develop a sensitive and relatively high-throughput assay for the detection of innate immune stimulation by short duplex RNAs. The logical choice of target cells for such an assay is primary human PBMCs, which have been shown to produce pro-inflammatory cytokines and IFN in response to siRNAs, through activation of TLR7 and/or TLR8 [21, 22, 24–26]. These primary cells have the additional advantage that, unlike TLR7 transfected immortalised cell lines, they appear to fully recapitulate in vivo TLR signalling . We and others [32, 33], had shown that the human hepatoma cell line Huh7, harbouring an HCV replicon that expresses a luciferase reporter, is a sensitive and easily assayed system for detection of IFN. Therefore, we chose to employ an assay system in which innate immunostimulation of PBMCs by siRNAs is detected using inhibition of HCV replication as a surrogate marker for IFN production. Such assay systems have been shown to be dependent on immune stimulation of the leukocytes (since treatment of Huh7 cells with a broad range of TLR agonists in the absence of leukocytes does not inhibit replication of the HCV replicon) and to be dependent upon secretion of IFNα from the activated leukocytes (as shown by neutralizing mAb experiments) [32, 33].
In order to demonstrate that the PBMC/Huh7-Luc assay system was capable of measuring differences in immunostimulatory activity between different siRNAs, we assessed the dose responses of 4 of the above mentioned siRNA: 2 strong immunostimulants (siCyan1 and siCyan2) and 2 moderate immunostimulants (siGFP19+2 and siNP_1496). Each of these duplexes induced a dose-dependent response from PBMC, with clear differences between the activity of each detectable in this assay (Figure 1B–E). This response was dependent upon endosomal maturation (and was therefore not mediated by cytosolic RNA receptors), since pre-treatment of the PBMCs with chloroquine inhibited IFNα production in a dose-dependent manner.
Screening siRNA duplexes for TLR7/8 activation
Although apparently affected by characteristics of the duplex (such as the free energy of hybridisation as discussed above), immune activation of PBMCs by siRNAs generally appears to be mediated by a single U rich strand of the siRNA [21, 31]. We compared the U content of both strands in each of the 207 duplexes, and selected the strand with the higher U content as being the probable immunostimulatory strand. Consistent with this model, there was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex (Figure 2C). G content has also been postulated to promote activation of TLR7/8 , however, we saw no correlation between combined U+G content and activity (Figure 2D).
In the present study we have used a broad-based screening strategy to identify sequence determinants of TLR7/8 activation by standard format duplex siRNA molecules. We found that sequence motifs contained in a single, U-rich strand of the siRNA duplex were a major determinant of activity, as demonstrated by the profound increase in immunostimulation caused by the introduction of even a single G-nucleotide into a poly-U sequence. We also found that the thermodynamic properties of the duplex affected TLR7/8 activation, and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity. Thus, our findings suggest a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. In the course of these studies, we have also identified several RNA species that stimulate TLR7/8 more strongly than any previously identified agent that we are aware of. These findings have relevance to the design of siRNAs (particularly for in vivo or clinical applications) and to the understanding of the physiological mechanisms of TLR7/8 activation. The highly immunostimulatory RNA species that we have identified may also be useful in the development of pharmacological agents with anti-viral or vaccine adjuvant applications.
Human hepatoma cells (Huh7) containing a stable pFK-I389/NS3-3'/5.1 based, Luciferase expressing HCV replicon (Huh7-Luc)  were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad CA) containing 10% fetal calf serum (FCS, Sigma-Aldrich, St. Louis, MO) and 750 μg/mL G418 (Invitrogen) at 37°C in a 5% CO2 incubator.
Buffy coats were obtained from the Australian Red Cross Blood Service. Buffy coat (50 mL) was diluted 1:3 with DM-L wash buffer (PBS + 2% FCS). This mixture was split into 6 separate 25 mL aliquots and each 25 mL of diluted cells layered over 15 mL of RosetteSep DM-L (Stemcell Technologies, Vancouver, Canada) solution. Following centrifugation for 20 min at 1200 g, erythrocytes were discarded and the mononuclear band washed with 40 mL DM-L wash buffer. Cells were pooled in 10 mL DM-L wash buffer prior to addition of 25 mL Ammonium Chloride solution (Stem Cell Technologies). Following 5 min incubation on ice the cells were centrifuged at 300 g for 5 min and resuspended in 50 mL DM-L wash buffer.
Plasmacytoid dendritic cell purification
Plasmacytoid dendritic cells (pDCs) were purified by negative selection using a plasmacytoid dendritic cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Briefly, fresh PBMCs were resuspended in MACS buffer (PBS pH 7.2, 0.5% BSA, 2 mM EDTA) and 100 μL PDC biotin antibody cocktail added per 108 cells. Following incubation on ice for 10 min the cells were washed twice with 10 mL MACS buffer per 108 cells. Cells were incubated with 100 μL anti-biotin microbeads for 15 min on ice and washed in MACS buffer prior to being loaded on an LS column (Miltenyi Biotec). The unlabelled fraction containing the enriched pDC fraction was collected and cells stained with CD303-FITC (Miltenyi Biotec) and CD45-PerCP (Becton Dickinson, Mountain View, CA). Analysis of pDC purity was performed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson).
Stimulation of TLR signalling in immune cells
The following small molecule TLR ligands were dissolved in DMSO and stored at -20°C until use: Resiquimod (Alexis Biochemicals, Lausen, Switzerland); CL075 and CL087 (both from Invivogen, San Diego, CA). Chloroquine (Sigma-Aldrich, St Louis, MO) and all siRNAs (see Additional File 1) were resuspended in nuclease-free water and stored at -20°C until required. Unless otherwise indicated IFNα-2b (R&D Systems, Minneapolis, MN) was used at a concentration of 100 U/mL. Where indicated PBMCs were treated with up to 10 μM chloroquine and incubated at 37°C in a 5% CO2 incubator for 1 h prior to addition of siRNA.
Duplex siRNAs for testing were sourced from 3 different research groups within Johnson & Johnson Research Pty Ltd, and had been designed to a number of targets from different species. RNA oligos and duplex siRNAs were purchased from Sigma-Aldrich).
PBMCs or pDCs (84.5% CD303+, data not shown) were stimulated with either siRNA or known TLR agonists for 24 h prior to supernatant transfer to Huh7-Luc cells, following previously determined experimental protocols [32, 33]. Unless otherwise indicated 50,000 PBMCs plated in 96-well plates in 80 μL antibiotic-free 10%FCS-RPMI were stimulated with TLR agonist (final volume of 100 μL/well; maximum 1% DMSO per well) or transfected with siRNA complexed with DOTAP (Roche) (final volume of 100 μL/well; maximum 100 nM siRNA per well). For complexations 10 pmol siRNA was complexed with 0.7 μg DOTAP in 10%FCS-RPMI. The mock transfection control used the highest concentration of DOTAP (0.7 μL DOTAP per well). After addition of stimuli the plates were incubated at 37°C in a 5% CO2 incubator for 24 h.
For measurement of IFN production, Huh7-Luc cells were seeded at 7500 cells/well in 80 μL antibiotic-free media (10%FCS-DMEM) in white 96-well plates (Greiner Bio-one, Frickenhausen, Germany). Media was removed 24 h post-seeding and 100 μL PBMCs pre-treated for 24 h with TLR agonist or siRNA was added to each well. After addition of PBMC supernatant the plates were incubated at 37°C in a 5% CO2 incubator for 24 h. PBMCs were subsequently removed and proliferation of Huh7 or Huh7-Luc cells determined using the Cell Titre Blue (CTB) assay (Promega, Madison, WI). Following measurement of fluorescence (Fluostar Optima plate reader, BMG Labtech, Offenburg, Germany) the CTB reagent was removed and cells lysed with 30 μL 1× Passive Lysis Buffer (Promega) at room temperature for 10 min. Luminescence was measured every 0.5 s for 5 s using automatic substrate injection (Luciferase assay system, Promega) and average luminescence calculated for each well. TNF-α production was measured by Quantikine ELISA (R&D systems) according to the manufacturer's instructions.
Bioinformatics and statistical analysis
Statistical analysis was performed using Prism (GraphPad Software, San Diego, CA) and Pipeline Pilot (Accelrys Software, San Diego, CA) software. Logos were generated using Phylo-mLogo 2.3 software . Unless otherwise indicated treatments were performed in triplicate and error bars represent standard deviation.
The authors are grateful to the Australian Red Cross Blood Service (ARCBS) for the provision of buffy coats.
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