induction of inﬂammatory cytokines by double- stranded and ...uncorrected proof induction of...

doi:10.1016/j.jmb.2005.03.013 J. Mol. Biol. (xxxx) xx, 1–12

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Induction of Inflammatory Cytokines by Double-stranded and Single-stranded siRNAs is Sequence-dependent and Requires Endosomal Localization

Mouldy Sioud*

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Department of ImmunologyMolecular Medicine GroupThe Norwegian RadiumHospital, Montebello, N-0310Oslo, Norway

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Abbreviations used: DC, dendriticdendritic cells; siRNA, small-interfeRNA interference; PKR, protein kinreceptor; IL, interleukin-; TNF-a, tuNF-kB, nuclear factor NF-kappa B;DOTAP, N-[1-(2,3-Dioleoyloxy)propammoniunmmethyl-sulfate.E-mail address of the author mos

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The potential induction of inflammatory cytokines and interferonresponses by small-interfering RNAs (siRNAs) represents a major obstaclefor their use as inhibitors of gene expression. Therapeutic applications ofsiRNAs will require a better understanding of the mechanisms that triggersuch unwanted effects, especially in freshly isolated human cells.Surprisingly, the induction of tumor necrosis factor (TNF-a) andinterleukin-6 (IL-6) in adherent peripheral blood mononuclear cells(PBMC) was not restricted to double-stranded siRNAs, because inductionwas also obtained with single-stranded siRNAs (sense or antisensestrands). The immunostimulatory effects were sequence-dependent, sinceonly certain sequences are prone to induce inflammatory responses whileother not. The induction of TNF-a, IL-6 and interferon a (IFN-a) waschloroquine sensitive and dependent more likely on endosomal Toll-likereceptor signaling. Indeed, no significant immunostimulatory effects weredetected when either double or single-stranded siRNAs were delivereddirectly to cytoplasm via electroporation. Both RNA types activated aNF-kB promoter-driven luciferase gene in transiently transfected humanadherent PBMC. Moreover, culture of immature dendritic cells with eitherdouble or single-stranded siRNAs stimulated interleukin-12 productionand induced the expression of CD83, an activation marker. Interestingly,several double-stranded siRNAs did not induced TNF-a, IL-6 and IFN-aproduction, however, their single-stranded sense or antisense did. Takentogether, the present data indicate for the first time that the inductioninflammatory cytokines and IFN-a responses by either double-stranded orsingle-stranded siRNAs in adherent PBMC is sequence-dependent andrequires endosomal intracellular signaling. The finding that endosomallocalization of self-RNAs (sense strands) can trigger Toll-like receptorsignaling in adherent human PBMC is intriguing because it indicates thatendosomal self-RNAs can display a molecular pattern capable foractivating innate immunity.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: RNA interference; siRNAs; innate immunity; Toll-like receptors;inflammatory cytokines

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R*Corresponding author

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OIntroductionRNA interference (RNAi) is an evolutionarily

lsevier Ltd. All rights reserve

cells; iDC, immaturering RNA; RNAi,ase R; TLR, Toll-likemor necrosis factor a;INF, interferon;yl]-N,N,N-trimethyl-

[email protected]

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conserved post-transcriptional gene-silencing pro-cess, resulting in specific mRNA degradation inseveral organisms.1,2 In this process, long double-stranded (ds) RNAs were processed by the RNaseIII-like enzyme Dicer to 21–25 nucleotides small-interfering RNAs (siRNAs), which are then incor-porated into a protein complex, the RNA-inducedsilencing complex (RISC). Thereafter, the RISC isremodulated into its active form, which contains theproteins necessary for cleaving the target mRNA atthe site, where the guide antisense siRNA strandbinds.2 RNAi and the related phenomenon of

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quelling and post-transcriptional gene silencing(PTGS) have been shown to exist in fungus(Neurospora), plants (Arabidopsis), invertebrates(Drosophila and C. elegans) and embryonic mouseand human cells.2,3 Notably, the cellular role ofRNAi is to maintain the integrity of the genome, todefend cells against viral infection, and to regulatethe expression of cellular genes.

In general RNAi was difficult to detect in somaticmammalian cells, since dsRNA structures greaterthan 30 bp stimulate the IFN pathway, whichrepresents a host response to viral infection whereseveral genes are activated.4 Much of the interferonresponses are caused by the activation of thedsRNA-dependent protein kinase R (PKR). Theantiviral effect of PKR is in part mediated throughthe phosphorylation of the alpha subunit of theeukaryotic translation initiation factor eIF2a, whichresults in the sequestration of the recycling factoreIF-2b in an inactive complex together with eIF2a-GDP.5 The net effect is global inhibition of proteinsynthesis. However, it has recently been shown thatsynthetic 21 nt siRNAs, the effector in RNAi,transfected into human somatic cells can effectivelybypass activation of the interferon pathway.6 Sincethen siRNAs have become a powerful tool forgenetic analysis and might serve as a potenttherapeutic tool for gene silencing.7,8 In additionto mRNA cleavage, siRNAs can induce chromatinmodification in different organisms. Interestingly, arecent report specified a function for siRNAs in

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Table 1. Sequences of the siRNAs used in this study

SiRNAs Sequence (5 0–3 0) (sense strand)

1 GGCCUUCCUACCUUCAGACTT2 GAUCAUCUUCUCAAAAUUCTT3 GUUCACCUGAGCCUAAUAGTT4 CUGAUGACCAGCAACUUGATT5 GACAACCAACUAGUGGUGCTT9 GAGGCUGAGACAUAGGCACTT7 GAACUGAUGACAGGGAGGCTT8 GAAGAAGUCGUGCUGCCUUTT9 GGUGACAAGAACAUCUCCATT10 GACCUCAUGUACCACAUUCTT11 GCCAUUGCACUGUGAAUACTT12 GUGAUCAUUCAGAGCCAGCTT13 GGCAUCUGGCUUAAGGUGATT14 GACCCUCGAGUCAACAGAGTT15 GCAUGCCUUGGAAUUCCUUTT16 GGCCGAUUGAUCUCAGCGCTT17 GGCCAAAUUUACAUUCUCCTT18 CCAACUAUGACCAGGAUAATT19 GUAGAUCAAUACCCUACACTT20 GGAGCGCACCAUCUUCUUCTT21 GACUUGAGCGAGCGCUUUUTT22 GAGAUGAUACCACCUGAAATT23 GCAGAUCUGCGUCGGCCAGTT24 GGUUCCAUCGAAUCCUGCATT25 GAGGCAAUCACCAAUAGCATT26 GAAGAUUUGCGCAGUGGACTT27 GUCCGGGCAGGUCUACUUUTT28 GGCAUGGAUCUCAAAGACATT29 CCAACGGCAUGGAUCUCAATT30 UGCCCUUCUACAACCAGGATT31 GCUGGAGUACAACUACAACTT32 GCUGGAGAUCCUGAAGAACTT

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DNAmethylation in mammalian cells.9 Contrary towhat was thought; recent experiments indicate thatexogenously delivered siRNAs can activate theinterferon pathway.10–15 However, there are con-flicting reports regarding the extent of off-targeteffects and interferon induction by siRNA inmammalian cells. For example, Rossi and col-leagues noted that none of the three testedchemically made siRNAs induced interferonresponses in human cells.16 These differencescould be due to several factors, including differ-ences in the cell types, reagent preparations andsiRNA sequences. Notably, in all studies only a fewsiRNAs were tested using the same experimentalconditions. To further address the question of whatdetermines siRNA stimulatory function, a compre-hensive analysis of 32 siRNAs targeting differentgenes was performed. The results indicate that thestimulating capacities of either double-stranded,single-stranded sense or antisense siRNAs aresequence dependent and require endosomal com-partments for intracellular signaling.

FResults and Discussion

OOThe non-specific effects of siRNAs aresequence-dependent

siRNA were initially thought to be small enoughto avoid double-stranded RNA responses.6

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RTarget gene

Mouse TNF-aMouse TNF-aHuman HIF-1Human HIF-1Mouse TNF-aMouse TNF-aScrambled siRNAScrambled siRNAHuman neuropilin-1Human PKC-aMouse Csf-1Mouse Csf-1 receptorMouse Csf-1 receptorMouse Csf-1Scrambled siRNAHuman TNF-aRat NG-2Human MMP-9Rat NG-2GFPHuman C20orf55Human CSPPHuman Frizzled-2Human Wnt-1Mouse BasiginHuman CSPPMouse TNF-aMouse TNF-aMouse TNF-aHuman AKT-1GFPHuman cDNA FLJ34902

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However, recent studies indicated that they couldinduce interferons and cytokine responses.10–15

Although the reported data provide a note ofcaution, the activation of the non-specific pathwaysby siRNAs is not well understood. We havepreviously examined the response of BALB/cmice to systemic delivery of siRNAs and foundinduction of TNF-a and IL-6 by larger RNAs andLPS, but not with a chemically synthesizedsiRNA.15 whilst the same siRNA preparationinduced inflammatory cytokine responses in

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adherent PBMC in vitro.15 To assess whether thesiRNA non-specific effects are sequence-dependentand to uncover themolecular mechanisms bywhichsiRNA activate innate immuniy genes, the induc-tion of TNF-a and IL-6 secretion by 32 differentsiRNAs was examined in adherent PBMC, anenriched monocyte population. Freshly isolatedcells were treated with the various siRNAsequences (see Table 1) for 18 hours. SpecificELISA on culture supernatants revealed thataround 50% of the tested siRNAs induced the

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Figure 1. Effects of siRNAs uponTNF-a and IL-6 production inadherent PBMC. Cells were trans-fected with the indicated siRNAsmolecules (100 nM) for 18 hoursand then TNF-a (a) and IL-6(b) were measured in the super-natants by ELISA. Results areshown as means of five indepen-dent experiments GSD. Thesequences of the used siRNAs areshown in Table 1. (c) Immunosti-mulatory siRNA induce TNF-aproduction in a dose dependentmanner. Adherent PBMC weretransfected with various concen-trations (5–200 nM) of eithersiRNA 27 for 18 hours. Subse-quently, TNF-a was measured inthe supernatants by ELISA. Allresults represent the mean of fouror more independent experiments.D, cells were incubated with onlyDOTAP and transfection buffer.

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Figure 2. Characterization ofsiRNA effects. (a) siRNA inducedTNF-a production required lipo-somal delivery of siRNAs. Adher-ent PBMC were incubated for 18hours with either siRNA liposomecomplexes or naked siRNA 27(100 nM) in X-VIVO 15 medium,a nuclease free medium. Subse-quently, TNF-a was measured inthe supernatants by ELISA.Results are shown as means offour independent experimentsGSD. (b) Inhibition of PKR.Adherent PBMC were pretreated,or not, with 2-aminopurine(10 mM) for one hour prior incu-bation with either siRNA 27 or 32(100 nM) for 18 hours. Subse-quently, TNF-a was measured inthe supernatants by ELISA.Results are shown as means offour independent experiments

GSD. (c) siRNA induced TNF-a production in blood monocytes. Purified CD14-positive cells were incubated witheither siRNA 27 or 32 (100 nM) for 18 hours, and then TNF-awas measured in the supernatants using ELISA. All resultsrepresent the mean of four or more independent experiments

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production of TNF-a(Figure 1(a)). Adherent PBMCproduced also IL-6 upon stimulation with siRNAscomplexed to DOTAP (Figure 1(b)). The profile ofIL-6 production is comparable to that of TNF-a.Notably, siRNAs varied dramatically in their abilityto induce TNF-a and IL-6 production, suggestingthat their activities are sequence-dependent. Onlysix of the siRNAs examined exhibited a strongimmunostimulatory effect, with siRNA-27 being themost effective under our experimental conditions.

To determine the immunostimulatory capacity ofsiRNAs, cells were transfected with siRNA 27 at arange of concentrations (5–200 nM). After 18 hoursin culture, secreted TNF-a was assessed by ELISA(Figure 1(c)). As shown, siRNA 27 stimulatedTNF-a production even at low concentrations.Taken together these data indicate that siRNAscan activate the expression of inflammatory cyto-kines, but their affinity and specificity for a potentialcellular target are more likely to be influenced bythe siRNA base composition. Sequence alignmentof the siRNA sequences revealed no significanthomology between the immunostimulatory siR-NAs, suggesting the involvement of RNA tertiarystructures and/or specific dinucleotides.

Intracellular siRNA delivery is required forcytokine induction

Having found that the non-specific effects ofsiRNAs are sequence-dependent, I have nextinvestigated whether intracellular siRNA deliverywas required. Therefore siRNAs were addeddirectly to the culture medium without complexingthem to DOTAP. siRNA 27 activated adherentPMBC to secrete TNF-a when complexed toDOTAP. In contrast, free siRNA did not lead to


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ROOsignificant cytokine production (Figure 2(a)).A nuclease resistant siRNA 27 gave similar results

as its unmodified version (data not shown). Theseresults argue that siRNAs target mainly intra-cellular compounds. In this respect, a role for PKRin the activation of interferons by siRNAs wasrecently suggested.12

To explore the involvement of the dsRNArecognition protein PKR in this process, the specificinhibitor of PKR, 2-aminopurine, was used. Cellswere pretreated with 2-aminopurine (10 mM), anormally used concentration, and subsequentlytreated with siRNA liposome complexes. Pretreat-ment with 2-aminopurine reduced, but did notabolish cytokine production by siRNAs (Figure2(b)). These results would indicate the involvementof other cellular targets in siRNA intracellularsignaling.

siRNAs activate TNF-a production in purifiedhuman monocytes

Monocytes are essential effector cells in chronicinflammatory disorders and infectious diseases. Toperform their function, monocytes need to beactivated, either via inflammatory cytokines pro-duced by the adaptive immune system or via directstimulation with microbial products. Although ahigh proportion of the adherent PBMC are mono-cytes, the capacity of siRNAs to activate TNF-aproduction in purified blood monocytes wasinvestigated. In these experiments, CD14-positivecell populations were prepared from PBMC bypositive selection with immunomagnetic beadscoated with anti-CD14 monoclonal antibodies(Dynal, Oslo, Norway), and the cells were subse-quently incubated with siRNAs (Figure 2(c)). There

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Figure 3. Expression of TLR3 by human monocytes and iDC. (a) Flow cytometry analysis. Freshly isolated monocytesand iDC, monocytes cultured for six days in the presence of GM-CSF and IL-4, were stained either with FITC-labeledanti-TLR3 monoclonal antibody (eBioscience) or control mouse IgG1. After washing, cells were analyzed by flowcytometry. The mean fluorescence of stained cells is shown in the upper-right corner of the individual dot plots. (b) RT-PCR for TLR3 and actin mRNAwas performedwith monocytes and iDC as described in Experimental Procedures. TLR3was not detected in human monocytes.

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COwas a significant induction of TNF-a productionwith the immunostimulatory siRNA 27, whereas nosignificant activity was seen with the control siRNA32.
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UNTLR3 is not required for responsiveness toeither double-stranded or single-strandedsiRNAs in adherent PBMCNotably, the host sensors that initially detect viral

and bacterial antigens and trigger cytokine pro-duction has been investigated by several groups,


some of which have indicated the involvement ofToll-like receptors (TLRs). These receptors, whichare mammalian homologous of the Drosophila Toll,recognize specific structural motifs expressed bymicrobes.17–19 So far, ten TLRs have been describedin humans, and ligands have been defined for nineof them. TLR1, K2 and K6 are triggered bypeptidoglycan and other microbial products, TLR3by dsRNA, TLR4 by LPS, TLR5 by flagellin, TLR7and K8 by imidazoquinolines, and TRL9 byunmethylated CpG DNA motifs. Although TLR3is a receptor for dsRNA and cellular mRNA,20,21 it

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does not contain dsRNA binding motifs. While thiswork is underway, a recent study has indicated theinvolvement of TLR3 in siRNA signaling andsuggested the involvement of dsRNA-bindingprotein that has yet to be identified.14 In contrastto macrophages and dendritic cells, however,human monocytes do not express TLR3 as assessedby flow cytometry and RT-PCR (Figure 3(a) and (b)).These results agree with those of a recent studyreporting on the expression of TLR3 by humanmonocytes.22 In addition, treatment of either adher-ent PBMC or purified monocytes with anti-TLR3monoclonal antibody, known to blocks TLR3signaling,23 did not inhibit the production of TNF-a by immunostimulatory siRNAs (data not shown).

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Sense and antisense siRNA strands are highlystimulatory when compared to their double-stranded siRNA counterparts

The data described above indicate that a sub-group of double-stranded siRNAs can induce theproduction of inflammatory cytokines. During thisstudy, however, I have been surprised by thefinding that both single-stranded sense and anti-sense siRNAs can trigger the production of pro-inflammatory cytokines (e.g. TNF-a) andinterferons (IFN-a) in adherent PBMC (Figure 4 asa representative example). This is in contrast towhat have been reported using various cancer celllines.12,13 As for the double-stranded siRNAs, theinduction of TNF-a by single-stranded siRNAs wassequence dependent. In the case of siRNA 27, the

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After 18 hours transfection time, TNF-awas measured in thestranded and single-strand siRNAs. The test molecules (100 nelectroporation. To test the involvement of endosomes in siRNtwo hours with chloroquine prior to transfection with DOTAPsupernatants by ELISA. All results represent the mean of thr


sense strand exhibited the strongest effects, whereasin the case of siRNA 32 the antisense strand exertedthe strongest effect on TNF-a production (Figure4(a)). Notably, the single-stranded siRNAs werehighly immunostimulatory compared to theirdouble-stranded siRNA counterparts (e.g. siRNA32). As shown DOTAP alone did not induce eitherTNF-a or IFN-a production, indicating that theeffects are related to the RNA sequences. Directaddition of either naked double-stranded siRNAsor single-stranded siRNAs did not affect cytokineproduction (data not shown). Moreover, severaldouble-stranded siRNAs did not induce response,whereas their corresponding single-stranded senseor antisense strands did (Figure 4(a), as anillustration). It is worth noting that the samesingle-stranded siRNA preparations were used toprepare the annealed siRNAs. Taken together, theseobservations would indicate that the reportedsiRNA effects in adherent PBMC are related to thesiRNA sequences and ague against any possiblecontamination with endotoxins.

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Cytokine induction by double-stranded siRNAs,sense or antisense siRNA strands requiresendosomal acidification

Cationic liposome-delivered siRNAs areexpected to enter the cell via endocytosis. Becauseseveral Toll-like receptors, in particular TLR7,TLR8, TLR9, are localized in the endosomes, Ihypothesized that endocytically introduced RNAcould trigger the activation of these receptors,

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Figure 4. Effects of double-stranded and single-stranded siR-NAs upon TNF-a production inadherent PBMC. (a) Cells weretransfected with either double-stranded siRNAs (d), sensesiRNA strands (s) or antisensesiRNA strands (a) for 18 hours.Subsequently, TNF-a wasmeasured in the supernatants byELISA. All test RNA moleculeswere used at 100 nM and com-plexed with DOTAP at 10 mg/ml.(b) Inhibition of cytokine pro-duction by chloroquine in dose-dependent manner. Cells werepre-incubated with chloroquinefor two hours prior transfectionwith siRNAs (100 nM) for 18hours. Subsequently, TNF-a wasmeasured in the supernatants byELISA. (c) The test RNAmolecules(100 nM) were delivered to thecells either via DOTAP or electro-poration as indicated on the figure.

supernatants by ELISA. (d) Induction of IFN-a by double-M) were delivered to adherent PBMC via either DOTAP orA induction of interferons, cells were also pretreated for

. After transfection (20 hours), IFN-awas measured in theee or more independent experiments.

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leading MAPK kinases and NF-kB activation. Totest the involvement of endosomes in siRNAsignaling, cells were treated with an inhibitor oflysosomal acidification, chloroquine for two hoursprior to transfection with either double-strandedor single-stranded siRNAs. TNF-a secretion byadherent PBMCwere inhibited in a dose-dependentmanner by chloroquine (Figure 4(b)), indicating thatthe acidification of the endosomes is importantfor cellular response to either double-stranded orsingle-stranded siRNAs. A similar inhibition wasobtained with Bafilomycin, an additional inhibitorof endosomal acidification (data not shown).

Cationic liposomes such as DOTAP both protectthe siRNA and mediate internalization via endo-cytosis. After entry, the siRNA must escape theendolysosomes and enter the RNAi pathway.The requirement for acidification of endosomes inadherent PBMC was further examined via electro-poration of siRNAs into adherent PBMC. In contrastto DOTAP, the electroporation method open uppores in the cell, so siRNA molecules can enter thecell directly into the cytoplasm. As shown in Figure4(c) and (d), no TNF-a and IFN-a induction wasobtained when either double-stranded or single-stranded siRNAs were delivered via electropora-tion. Therefore, endosome compartments arerequired for both double-stranded and single-stranded siRNA intracellular signaling andsubsequent cytokines and interferon productionby adherent PBMC. Notably, pretreatment of PBMCwith chloroquine prior to transfection with siRNAabrogated IFN-a production (Figure 4(d)), indicat-ing for the first time the involvement of endosomesin siRNA induction of interferons.

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Endosomal siRNA signaling supports theinvolvement of the Toll-like receptor 8

Toll-like receptors (TLRs) represent a class ofpattern recognition receptors (PPRs) that detectmicrobes or their components.19 TLRs are pre-dominantly expressed on the cell surface; however,a subset (TLR7, TLR8, TLR9, and in some cases,TLR3) is retained in intracellular compartments.Notably, TLR9, TLR7 and TLR8 require endosomalacidification for signaling.19 Because the inductionof inflammatory cytokines by siRNAs is chloro-quine-sensitive, the data would support the invol-vement of these receptors in siRNA intracellularsignaling. The recent identification of single-stranded RNAs as a ligand for human TLR824,25

would support our initial observation15 and thepresent elaborated results suggesting the involve-ment of endosomal TLRs in single-stranded siRNAsignaling. This novel observation invites the ques-tion of what serves as the endosomal receptors fordouble-stranded siRNAs. In acidic microenviron-ment such as the endosomes, double-strandedsiRNA might dissociate and generate single-stranded siRNAs for intracellular signaling. Insupport of this idea is the observation that certaindouble-stranded siRNAs are very less effective ininducing inflammatory cytokine production thantheir corresponding single-stranded sense or anti-sense strand counterparts (Figure 4). Depending ofthe relative internal stability, certain siRNAduplexes may exist in equilibrium with their singlestrand forms, which can activate TRL8 and/or otherendosomal TLRs (Figure 5). Alternatively, double-stranded siRNAs may signal through endosomalTLR3, known to bind double-stranded RNAs.21

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Figure 5. A schematic diagramillustrating the intracellular siRNAsignaling. A complex of eitherdouble-strand or single-strand siR-NAs or cationic lipids bind to theplasma membrane electrostaticallyand are internalized via endocyto-sis. Within the endosomes RNAmolecules can bind to endosomalToll-like receptors, in particularTLR8 and 7, which can activatethe transcription factor NF-kB,leading to inflammatory cytokineproduction. TLR7, 8 and 9 useMyD88 as primary adaptor toactivate interferon production viaundefined signaling intermediates.TLR3 uses the adaptor proteinTRIF to activate interferon regulat-ory factor-3 (IRF-3). The activationof Toll receptors may activate PKRdirectly.19,11

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Figure 6. Immunostimulatory siRNAs activate theNF-kB transcription factor. A NF-kB-dependent luciferasereporter gene was co-transfected in human adherentPBMC with a control plasmid containing the Renillaluciferase gene under the control of the CMV promoterfor 24 hours. Subsequently; cells were treated with eithersiRNA 27 or 32. The sense (S) and the antisense strand (A)of siRNA 27 and 32, respectively, were also tested.Eighteen hours later, luciferase activity was determinedusing Dual-Luciferase Reporter Assay System (Promega).The data show the relative firefly luciferase activitynormalized to that of Renilla luciferase. Results areshown as means of three independent experiments GSD.

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Given that single-stranded siRNA are highlyimmunostimulatory compared to their correspond-ing double-stranded siRNAs, during siRNAannealing it is important to make sure that allsiRNA molecules exist as siRNA duplexes.Although all double-stranded siRNA preparationsused in this study were checked by PAGE for thepresence of unannealed single-stranded siRNAs,some of the double-stranded siRNA effects couldoriginate from tiny traces of free single-strandedsiRNAs. Whatever the nature of double-strandedsiRNA receptors; however, the present data indicatefor the first time that induction of inflammatorycytokines and interferon-a by either double-stranded or single-stranded siRNAs requires endo-somal acidification in fleshly isolated PBMC. There-fore, the development of carriers that deliver siRNAdirectly into the cytoplasm should overcome theproblem of inflammatory cytokine induction bychemically synthesized siRNA, a major obstacle forsystemic administration of siRNA in patients.Similarly to siRNA liposome complexes, nakedsiRNAs delivered via other techniques in vitro andin vivo are more likely to be taken up by cells viaendocytosis.

The finding that several siRNA sense strands(mRNA sequences) can be highly immunostimula-tory indicates that endosomal Toll-like receptorscan recognize self-RNA. Thus, how can the immunesystem distinguish between self and non-self RNAs(e.g. viral RNAs,) and how to mount and amplify animmune response against invading RNA in aspecific manner? The present data argue that theimmune system uses the endosomes or lysosomalcompartments as molecular recognition signaturefor RNA in general (whether self or non-self).In contrast to viral RNAs, self-RNAs exist in thecytoplasm and therefore cannot enter the endoso-mal compartments; hence in normal situation theimmune system cannot be activated by self-RNAs.Similar to single-stranded siRNAs, liposomaldelivery of short single-stranded RNA (21 nt)derived from HIV-1 TAT mRNA or human TNF-amRNA also induced TNF-a and IFN-a productionin adherent PBMC (data not shown). Thus, endo-somal location of RNA seems to function as a“danger signal” for TLR recognition, and sub-sequent activation of innate immunity genes. Theobservation that certain sequences are more stimu-latory than others would indicate that endosomalTLRs, in particular TLR8, have preferences for aparticular nucleotides and/or structures that needfurther investigation.

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UNCsiRNA treatment can activate the NF-kB

promoter

The core TLR signaling pathway uses myeloiddifferentiation factor 88 (MyD88) as the primaryadaptor protein and results in NF-kB activation,cytokine production and expression of costimula-tory molecules, such as the class II major histocom-patibility complex. To address the involvement of


ED PNF-kB, promoter activation studies using fireflyluciferase as a reporter gene was performed.

Adherent PBMC were transiently transfected withNF-kB-luciferase gene along with plasmid DNAwith the Renilla luciferase gene for 24 hours.Subsequently, the cells were either mock transfectedor transfected with a stimulatory siRNA, and 18hours later, cells were harvested, and equalamounts of cell extracts were subjected to luciferaseassay (Figure 6). The data showed that double-stranded siRNA 27 could induce around 8-foldincrease in NF-kB activity, whereas 22-fold increasewere obtained with the single-stranded siRNA(S27), again identifying single-stranded siRNAs asan important inducer of NF-kB activation.

Single-stranded and double-stranded siRNAsstimulate monocyte-derived immature dendriticcells to produce cytokines and up-regulatecostimulatory molecules

If we view the induction of inflammatorycytokines as a beneficial mediator in cancer andinfectious diseases, then stimulatory siRNAs couldemerge as a viable agent to knockdown specificgenes and activate innate and acquired immunityagainst tumor cells. Notably, stimulation of imma-ture dendritic cells (iDC) by microbial productsinduces the production of inflammatory cytokines

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Figure 7. Immature DC activatedwith siRNA upregulated theexpression of CD83 and secretedIL-12. Immature dendritic cellswere culture for 48 hours in thepresence of either siRNA 27 or 32(100 nM) and then stained withFITC-labeled monoclonal anti-bodies specific for CD83 (a). Cellswere also stained with the corre-sponding isotype control. Themean fluorescence of stained cellsis shown in the upper-right cornerof the individual dot plots. Resultsare shown as means of threeindependent experiments GSD.

(b) After 48 hours stimulation with siRNAs, IL-12 was measured in the supernatants using ELISA. Results are shownas means of three independent experiments GSD.

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such as TNF-a and IL-12, which can inducedifferentiation of T cells into a T helper cell type1.26 In addition, these stimuli are known to up-regulate certain costimulatory molecules such asCD40, CD80, CD83 and CD86. This processunderlies DC maturation, and it strongly poten-tiates the ability of DC to activate naive T cells.26,27

Therefore, the effects of siRNAs upon the matu-ration of DC were investigated. To generate iDCs,freshly isolated monocytes were cultured for sixdays in the presence of GM-CSF (50 ng/ml) andIL-4 (100 ng/ml). Subsequently, they were bothtransfected with siRNA 27 or siRNA 32 and thenanalyzed by flow cytometry (Figure 7(a)). Addingstimulatory siRNA 27 to the culture mediumupregulated the expression of CD83, whereas nosignificant effects were obtained with the controlsiRNA 32. Treatment with siRNA 27 also upregu-lated the expression of CD86 (data not shown).Furthermore, siRNA 27-treatment resulted in IL-12production (Figure 7(b)). Similar results wereobtained with single-stranded siRNA 27. Theseresults suggest that stimulatory siRNAs canenhance immunity by inducing cytokine pro-duction and DC maturation. Thus, the use ofstimulatory double-stranded or single-strandedsiRNAs as a new generation of adjuvants mayfacilitate the design of effective vaccines.

The ability to enhance or augment the innateimmune response clearly represents a potentialpowerful way to prevent or treat infections as wellas a way to develop cancer vaccines. Recently, it hasbeen discovered that unmethylated CpG dinucleo-tides in particular base contexts are recognized bythe immune system as danger signals.28 In animalmodels, deoxyoligonucleotides with stimulatoryCpG motifs have been shown to be of therapeuticvalue as adjuvants for conventional and therapeuticvaccines against infectious diseases and tumors.29

As shown in the present study, the stimulatoryRNAs, in particular single-stranded siRNAs, acti-vated human monocytes and dendritic cells toproduce TNF-a and IL-12, two key cytokines inimmune regulation. In addition, the induction of


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other cytokines such as IL-6 and interferons is anessential response for the clearance of viral infec-tions.30 Natural killer cells are activated by cyto-kines and chemokines, including type I IFN andIL-12, which are secreted by dendritic cells.31

Therefore, treatment of cancer and infectious dis-eases might benefit from the induction of cytokineand dendritic cell activation by immunostimulatorysiRNAs. TLR activators are already used asadjutants to boost immune responses in vaccines.However, the success of a given vaccine maydepend on appropriate activation of TLR. There-fore, single-stranded sense RNA-27 should beexplored as an adjuvant for vaccination andimmunotherapy. On the other hand, the develop-ment of TLR antagonist holds promise as a newclass of anti-inflammatory agents. Although furtherstudy is required, some of the non-stimulatoryRNAs may function as antagonists.
ESelection of siRNA against human TNF-a
One of the key cytokines crucial to manybiological processes is TNF-a. Low levels of TNF-a produced by monocytes and tissue macrophagesunder physiological conditions are expected to beinvolved in maintaining cellular and tissue homeo-static. The production of TNF-a increases inresponse to microbial infection and tissue injury.Because of its important role in the pathogenesis ofa variety of inflammatory and immune diseases,TNF-a has been identified as a key target forpharmacological modulation.32 Therefore, thedevelopment of agents that specifically inhibitTNF-a may provide clinician with a valuablealternative to traditional disease-modifying anti-inflammatory drugs. In this respect, we have shownthat siRNA-targeting mouse TNF-a can delay theonset of LPS-induced sepsis.33 Out of 12 recentlytested siRNA targeting mouse TNF-a, siRNA 29(see Table 1), exhibited the greatest protective effect.To select, siRNAs against human TNF-a, several

siRNAs were designed (Table 2) and tested for theirinhibitory effect on siRNA 27-induced TNF-a

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Table 2. Sequences of siRNAs targeting human TNF-a

SiRNAs Sequence (5 0–3 0) (Sense strand)

H1 GAGUGACAAGCCUGUAGCCTTH2 CUCAGCGCUGAGAUCAAUCTTH3 CUAGUGGUGCCAGCCGAUGTTH4 GCGUGGAGCUGAGAGAUAATT

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production in adherent PBMC (Figure 8(a)). SomesiRNAs were effective in inhibiting TNF-a pro-duction, whereas others had no effects, thussuggesting the specificity of the inhibitory effects.When tested alone, the siRNA inhibitors signifi-cantly reduced LPS-induced TNF-a production byadherent human PBMC (Figure 8(b)). The utility ofthis selection assay may be extended to otherinflammatory cytokines and type I interferon toselect effective siRNAs in human blood cells. It isworth noting that the immunostimulatory effects ofsiRNAs are more pronounced in human PBMC ascompared to other human cell lines. This maydepend on the expression level of Toll-like recep-tors, which is under investigation.

Collectively, the present study underscores forthe first time the importance of the endosomecompartments in siRNA intracellular signaling inadherent PBMC. The results can easily be explainedby the interaction of RNA molecules with endo-somal Toll-like receptors and subsequent inductionof inflammatory and interferon responses (seeFigure 5). The finding that endosomal localizationof self RNA can trigger Toll-like receptors signalingin adherent PBMC is intriguing because it indicates

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Figure 8. Selection of siRNAs against human TNF-a.(a) Inhibitory effects of TNF-a siRNAs on TNF-aproduction by the immunostimulatory siRNA 27. Adher-ent PBMC cells were co transfected with siRNA 27 andvarious siRNA targeting the human TNF-a (H1-5).Eighteen hours later supernatants were analyzed forTNF-a contents by ELISA. Results are shown as means ofthree independent experiments GSD. (b) Blockage ofLPS-induced TNF-a expression by the inhibitory siRNAs.The cells were transfected for 18 hours with either siRNA32, H2, H3 or H4 (50 nM). Subsequently, cells werestimulated with LPS (20 ng/ml) for eight hours and thensupernatants were analyzed for TNF-a contents byELISA. Results are shown as means of three independentexperiments GSD.


that endosomal self RNA can display a molecularpattern capable for triggering innate immunityactivation.

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Experimental Procedures

siRNAs

The siRNAs used in this study correspond to variousmolecules targeting genes related to other ongoingprojects. All siRNAs were chemically synthesized byEurogentec (Seraing, Belgium), dissolved in water andannealed in the transfection buffer (20 mM Hepes,150 mM NaCl, pH 7.4) at 20 mM. Analysis of LPS/endo-toxin levels in siRNA stocks was found to be less than0.01 EU/ml (PyrogentR, CAMBREX). The sequencesof the sense strands and the target genes are shown inTable 1.

Preparation of human cells

Human mononuclear cells were prepared from buffycoats by density gradient centrifugation (Lymphoprep,Nycomed Pharm, Oslo), washed, and then resuspendedin RPMI 1640 containing 10% heated inactivated fetal calfserum (FCS). Enriched monocyte populations wereisolated by plastic adherence. After three hours incu-bation at 37 8C, non-adherent cells were removed byrepeated gentle washing with warm medium. More than75% of the obtained cells by this technique are CD14Ccells. In addition, CD14C cell population was preparedfrom PBMC by positive selection with immunomagneticbeads coated with anti-CD14 monoclonal antibodies(Dynal, Oslo, Norway).

Culture of dendritic cells

Enriched population of CD14 positive monocytes wereisolated from human PBMC as described above andcultured for five days in RPMI 1640 supplemented with10% FCS and antibiotics, in the presence of 50 ng/ml GM-CSF and 100 ng/ml IL-4 to obtain monocyte- derivedimmature dendritic cells (iDC). After five days in culture,the cells were either mock transfected or transfected witheither siRNA 27 or 32 for 48 hours, and then cells werestained with FITC-conjugated monoclonal antibodiesspecific for CD83 (Immunotech). Cells were also stainedwith FITC-conjugated normal mouse IgG1, an isotypecontrol.

FACS staining and analysis

Briefly, 2!105 cells were incubated in the recom-mended FITC-labeled antibody dilutions for 30 minutesat 4 8C in staining buffer (PBS containing 0.5% FCS and0.1% azide). Cells were also stained with the correspond-ing Ab isotype controls. Subsequently, the cells werewashed twice with the staining buffer and then analyzedon a FACScan using Cell-Quest software (BD Bio-sciences). Cells were gated in forward/side scatter andthe quadrants were set on the relative isotype controls.

Transfection and ELISA

Cells were seeded at 5!104/well/200 ml in 96-wellplates. After an overnight incubation with siRNAs/lipo-some complexes, culture supernatants were collected and

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cytokine contents were assessed by ELISA. Cells weretransfected in RPMI 1640 medium (PAA LaboratoriesGmbH) supplemented with 10% heat-inactivated FCS,2 mM L-glutamine, 100 mg/ml penicillin and 100 mg/mlstreptomycin using DOTAP (10 mg/ml) as described.33

Each assay was carried out in triplicate. When nakedsiRNAs were used, cells were incubated in X-VIVO 15medium without adding FCS (CAMBREX). In the caseof monocytes, 2!104 cells were used per well. Theinactivation of PKR was carried out as follows: Cells wereincubated with 2-aminopurine (10 mM), a specific inhibi-tor of PKR, for one hour prior to addition of the siRNA/liposome complexes. After an overnight transfectionwith the tested molecules, the levels of TNF-a, IL-6 andIL-12p70 in the culture supernatants were measured byavailable ELISA kits according to the manufacture’sinstructions (R&D Pharmingen). Samples were run intriplicate. Human IFN-a was measured using ELISA kit(PBL Biomedical Laboratories).

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Transfection and reporter assays

An enriched monocyte population (2!106 cells) wereseeded onto a 24-well plate and transfected the followingday with firefly luciferase gene (1 mg) under the control ofthe NF-kB-promoter and pRL-TK DNA (1 mg, Promega)encoding the Renilla luciferase gene. After 24 hours, thecells were transfected with siRNAs and then cells wereharvested 18 hours later. Luciferase activities weremeasured using the Dual-Luciferase Reporter AssaySystem according to the manufacture’s instructions(Promega).

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RT-PCR analysis

Total RNA was extracted from freshly isolated mono-cytes and monocytes-derived iDC using total RNAisolation kit (Pharmacia Biotech). DNAse I-treated RNA(10 mg) was reverse-transcribed with NotI-d(T)18 primerusing the first-strand cDNA synthesis Kit (PharmaciaBiotech) according to the manufacturer’s instructions.Polymerase chain reaction (PCR) was performed on 3 mlof cDNA in 50 ml of final volume. The TLR3 primers are:

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C5 0-ATTGGGTCTGGGAACATTTCTCTTC-3 0 (forwardprimer)

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5 0-GTGAGATTTAAACATTCCTCTTCGC-3 0 (reverseprimer)

As a control, b actin mRNA was also amplified usingthe following primers:

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R5 0-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3 0 (forward primer)

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NCO50-CGTCATACTCCTGCTTGGTGATCCACATCTGC-30
After 35 cycles of amplification (one minute at 92 8C;one minute at 56 8C; one minute at 72 8C, samples wereanalyzed by electrophoresis on 1.5% agarose gel andvisualized by ethidium bromide staining.
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Acknowledgements

I thank Drs Per Ole Iversen and Anne Dybwadfor critical reading of the manuscript, and LiseForfang for excellent technical assistance with flowcytometry. This work was supported by theNorwegian Cancer Society.

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Induction of Inflammatory Cytokines by Double-stranded and Single-stranded siRNAs is Sequence-dependent and Requires Endosomal LocalizationIntroductionResults and DiscussionThe non-specific effects of siRNAs are sequence-dependentIntracellular siRNA delivery is required for cytokine inductionsiRNAs activate TNF-alpha production in purified human monocytesTLR3 is not required for responsiveness to either double-stranded or single-stranded siRNAs in adherent PBMCSense and antisense siRNA strands are highly stimulatory when compared to their double-stranded siRNA counterpartsCytokine induction by double-stranded siRNAs, sense or antisense siRNA strands requires endosomal acidificationEndosomal siRNA signaling supports the involvement of the Toll-like receptor 8siRNA treatment can activate the NF-kappaB promoterSingle-stranded and double-stranded siRNAs stimulate monocyte-derived immature dendritic cells to produce cytokines and up-regulate costimulatory moleculesSelection of siRNA against human TNF-alpha

Experimental ProceduressiRNAsPreparation of human cellsCulture of dendritic cellsFACS staining and analysisTransfection and ELISATransfection and reporter assaysRT-PCR analysis

Uncited ReferenceAcknowledgementsReferences

induction of inﬂammatory cytokines by double- stranded and ...uncorrected proof induction of...

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