Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microrna mir-21

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1 Negtive regultion of TLR vi trgeting of the proinflmmtory tumor suppressor y the mirorna mir-1 Frederik J Sheedy 1, Ev Plsson-MDermott 1, Elizeth J Hennessy 1, Cr Mrtin,, John J O Lery,, Qingguo Run, Derek S Johnson, Youhi Chen & Luke A J O Neill 1 1 Nture Ameri, In. All rights reserved. The tumor suppressor is proinflmmtory protein tht promotes tivtion of the trnsription ftor NF- B nd suppresses interleukin 1 (IL-1). Here we found tht mie defiient in were proteted from lipopolyshride (LPS)-indued deth. The indution of NF- B nd IL- y LPS required, wheres LPS enhned IL-1 indution in ells lking. Tretment of humn peripherl lood mononuler ells with LPS resulted in lower expression, whih ws due to indution of the mirorna mir-1 vi the dptor MyD nd NF- B. Trnsfetion of ells with mir-1 preursor loked NF- B tivity nd promoted IL-1 prodution in response to LPS, wheres trnsfetion with ntisense oligonuleotides to mir-1 or trgeted protetion of the mir-1 site in Pdd mrna hd the opposite effet. Thus, mir-1 regultes expression fter LPS stimultion. Mny negtive regultory ontrol mehnisms exist to limit the toxi effets of lipopolyshride (LPS) 1. These inlude solule deoy reeptors, suh s solule Toll-like reeptor (TLR), nd splie vrints of signl-trnsdution proteins, inluding MyD-s, IRAK- M nd TAG 5, whih interfere with signl-trnsdution pthwys. The inhiitor of trnsription ftor NF-κB α-suunit (IκBα) is promptly resynthesized y NF-κB to lok exessive trnsription ftor tivity fter tretment with LPS. The prodution of nti-inflmmtory ytokines is lso indued y LPS signling, inluding interleukin 1 (IL-1), whih hs prrine effets on neighoring ells to negtively regulte the tion of NF-κB, nd proinflmmtory ytokines suh s IL- nd IL-1 (ref. 7). Nonoding RNA produts known s miro- RNAs (mirnas) hve lso een desried, suh s mir-1, whih is indued y LPS nd negtively trgets signling proteins suh s IRAK1 nd TRAF t the post-trnsriptionl level. ws first desried s protein indued y poptoti stimuli 9 tht ts s tumor suppressor 1. It is indued y ytokine tretment, onsistent with predited NF-κB site in its promoter 11. hs een shown to positively influene tumor nerosis ftor indued tivtion of NF-κB 1. It hs lso een shown to e trnsltionl inhiitor through intertion with memers of the eif fmily of eukryoti trnsltion-initition ftors 1,1. Trget mrnas inlude those enoding IL-1 nd IL- (ref. 15), whose prodution is therefore suppressed y. -defiient mie re resistnt to models of inflmmtory disese, suh s experimentl utoimmune enephlomyelitis nd streptozotoin-indued type II dietes. It is likely tht the proinflmmtory effet of is due to its role in NF-κB funtion nd its ility to suppress IL-1 trnsltion. is trgeted for protesoml degrdtion y β-trcp uiquitin ligses tivted y growth ftors during tumor promotion 1,17. The mirna mir-1 trgets Pdd mrna posttrnsriptionlly, loking prodution of protein 1. This mirna is upregulted in mny ners, inluding lymphom, leukemi nd solid tumors nd therefore hs een lled n onomir, whih my explin the loss of during neoplsti trnsformtion 1. Here we exmine the role of in the inflmmtory response to LPS. Similr to experimentl utoimmune enephlomyelitis nd type II dietes 15, we found -defiient mie were proteted from the lethlity of LPS, nd we exmined its role in LPS signling. We found tht LPS modulted the expression of through the indution of mir-1 nd otined evidene tht this modultion regulted NF-κB tivity while promoting IL-1 prodution. Our study identifies mir-1 s negtive regultor of TLR signling through the trgeting of. RESULTS is required for the lethlity of LPS To exmine the role of in TLR signling nd inflmmtion, we injeted -defiient nd wild-type ontrol mie with LPS nd monitored their survivl. -defiient mie were less suseptile to LPS, with lower mortlity thn wild-type mie (Fig. 1). Anlysis of irulting ytokine onentrtions h fter LPS injetion showed tht 1 Shool of Biohemistry & Immunology, Trinity College, Dulin, Irelnd. Deprtment of Pthology, Coome Women s Hospitl, Dulin, Irelnd. Deprtment of Histopthology, Shool of Mediine, Trinity College, Dulin, Irelnd. Deprtment of Pthology nd Lortory Mediine, Shool of Mediine, University of Pennsylvni, Phildelphi, USA. Correspondene should e ddressed to L.A.J.O. (loneill@td.ie). Reeived 11 Septemer; epted 1 Otoer; pulished online 9 Novemer 9; orreted online Deemer 9; doi:1.1/ni.1 nture immunology volume 11 numer ferury 1 11

2 1 Nture Ameri, In. All rights reserved. Survivl (%) 1 LPS (d) Pdd / IL- onentrtions were lower in -defiient mie treted with LPS (Fig. 1), onsistent with lower suseptiility. Anlysis of ytokine prodution t erlier times showed striking differenes etween - defiient mie nd wild-type mie in terms of prodution of the ntiinflmmtory ytokine IL-1 (Fig. 1). At 1 h fter LPS injetion, IL-1 serum onentrtions in -defiient mie were greter thn those in wild-type mie, n effet lso evident t h fter injetion. These dt indite tht hs proinflmmtory role in LPS signling. expression is regulted y TLR We investigted whether is trget of TLR signling. LPS resulted in higher expression of protein in RAW.7 mouse mrophges, evident t 1 h (Fig. ); this deresed nd ws olished t h. The effet t h ws onentrtion dependent nd ws evident t rnge of LPS onentrtions from.1 ng/ml to 1 ng/ml (Fig. ). A profound derese in Pdd mrna in response to LPS ws lso evident from h (Fig. ). We lso oserved the effet on protein in mouse primry one mrrow derived mrophges (BMDMs; Fig. d). LPS used slight inrese h fter stimultion, wheres t h nd h fter LPS, sustntil derese in ws evident. Similrly, tretment of primry BMDMs with other TLR lignds suh s Pm CSK ( TLR lignd) nd poly(i:c) ( TLR lignd) indued protein t erlier times. However, t h fter tretment, there ws muh less (Fig. d). Notly, in humn peripherl lood mononuler ells (PBMCs), LPS tretment inresed the expression of protein t h nd h, with derese ourring t h (Fig. e). regultes NF- B nd IL-1 To explin the proinflmmtory role of in LPS signling, we exmined its ility to ffet IL-1 prodution. Trnsfetion of Mouse ll- (ng/ml) Pdd / Mouse ll-1 (ng/ml) 1 Pdd / 1 h h Figure 1 -defiient mie re proteted from the lethlity of LPS. () Survivl of wild-type () ontrol mie nd -defiient (Pdd / ) mie 1 weeks of ge injeted intrvenously with LPS, monitored over period of 1 d; results re plotted s perentge of totl numers (n = 1 mie per group). (,) Enzyme-linked immunosorent ssy (ELISA) of mouse IL- () nd mouse IL-1 () in lood smples from wild-type nd -defiient mie (n = 1 () or 5 () mie per group), h () nd 1 h nd h () fter LPS injetion. Dt re representtive of three () or two (,) independent experiments. RAW.7 ells with smll interfering RNA (sirna) speifi for t onentrtion of 5 nm nd 1 nm deresed endogenous expression, using knokdown of 5%, s mesured y densitometry snning (Fig., top). LPS indued derese in protein oth in ells trnsfeted with ontrol sirna nd in ells treted with -speifi sirna. We oserved more LPSindued prodution of IL-1 in ells trnsfeted with inresing mounts of sirna speifi for (Fig., ottom). A similr inrese in IL-1 prodution ws lso evident in -defiient BMDMs fter h of tretment with LPS t onentrtions of 1 ng/ml nd 1 ng/ml (Fig. ). is known to inhiit p-dependent trnsltion of mrnas with omplex 5 untrnslted regions (UTRs), of whih Il1 is n exmple. To determine if this is the se, we nlyzed Il1 mrna undne in -defiient BMDMs. Unexpetedly, we found lower Il1 mrna expression in - defiient BMDMs tht produed more IL-1 protein thn did wildtype ells (Fig. ). At h fter LPS tretment, -defiient BMDMs expressed one-fourth less Il1 mrna thn did wild-type ells. Other genes tht re regulted y eife, whih re onsidered to e sensitive to tivity, lso show differenes in mrna undne when trnsltion is modulted,. To rule out the possiility of trnsriptionl effets of defiieny on IL-1 expression, we trnsfeted RAW.7 ells with n Il1 promoter luiferse onstrut longside sirna speifi for. LPS indued the tivity of this promoter t onentrtion of 1 ng/ml (Fig. d). However, when expression ws knoked down, we deteted no sustntil differene in Il1 promoter tivity. We lso exmined the role of in NF-κB tivtion y LPS with TLR-expressing humn emryoni kidney ells (HEK9- TLR ells) trnsfeted with n NF-κB luiferse reporter plsmid. Trnsfetion with sirna speifi for t onentrtion of nm deresed endogenous in HEK9-TLR ells (% knokdown, s mesured y densitometry snning; Fig. e, top). The knokdown of endogenous resulted in less tivtion of NF-κB t ll onentrtions of LPS tested, with 7% inhiition ourring t n sirna onentrtion of nm, equivlent to the degree of knokdown (Fig. e, ottom). We lso tested the indution of IL-, whih is NF-κB dependent, in -defiient BMDMs (Fig. f). This response ws impired in -defiient ells fter h of tretment with LPS t onentrtion of 1 ng/ml nd 1 ng/ml. We deteted signifintly less Il mrna in - defiient BMDMs thn in wild-type BMDMs fter h of LPS tretment (Fig. g), whih indited this impirment ours s result of less NF-κB-indued trnsription of Il. To explin this funtion of, we exmined erly signling events in wild-type nd -defiient BMDMs. Anlysis of IκBα protein y immunolot showed tht there ws more IκBα degrdtion t.5 h in the -defiient ells (Fig. h, top). Consistent with positive effet of on NF-κB tivtion, the Figure protein expression is regulted y LPS. () Immunolot nlysis of in RAW.7 ells treted for h (ove lnes) with LPS (1 ng/ml). () Immunolot nlysis of in RAW.7 ells treted for h with 1 ng/ml (ove lnes) of LPS. () Quntittive RT-PCR nlysis of Pdd mrna in RAW.7 ells treted for h (horizontl xis) with LPS, presented reltive to Pdd mrna in untreted ells. (d) Immunolot nlysis of in mouse BMDMs treted for h (ove lnes) with LPS (1 ng/ml), Pm CSK (1 ng/ml) or poly(i:c) (1.5 µg/ml). (e) Immunolot nlysis of in humn PBMCs treted for h (ove lnes) with LPS. expression serves s loding ontrol. Dt re representtive of three independent experiments (; men ± s.d.) or t lest three independent experiments (,,d,e). LPS (h) 1 LPS (ng/ml) Pdd mrna 1..5 LPS (h) 1 d LPS (h) LPS e LPS (h) Pm CSK Poly (I:C) 1 volume 11 numer ferury 1 nture immunology

3 1 Nture Ameri, In. All rights reserved. Ctrl 5 1 sirna (nm) LPS Bnd intensity (%) sirna (nm) Ctrl 5 1 IL-1 prodution IL-1 prodution Pdd / LPS (ng/ml) 1 1 NF-κB-dependent resynthesis of IκBα t lter times ws mrkedly impired in -defiient mrophges. These results indite tht the effet of on NF-κB tivtion is not due to modultion of the IKK omplex nd is proly indiret nd seondry to its primry funtion s repressor of trnsltion. is known to ffet the tivity of the trnsription ftor AP-1 in tumor progression. Here we nlyzed tivtion of the kinse Jnk y ssessing its phosphoryltion with n ntiody speifi for Jnk phosphoryltion. Jnk tivtion ws impired in response to LPS in -defiient BMDMs ompred with its tivtion in wild-type ells (Fig. h, middle). These dt onfirm tht hs proinflmmtory role in LPS signling events. Indution of mir-1 y LPS To explin the lower protein undne fter LPS tretment, we monitored expression of the -trgeting mirna mir- 1 fter LPS tretment. We used the indution of known LPSresponsive mirna, mir-1, s positive ontrol. LPS tretment indued mir-1 expression in RAW.7 mrophges, whih ws pprent from h (Fig., left). LPS tretment led to strong indution of oth mir-1 nd mir-1 t h with similr kinetis in this ell type. The effet of LPS on mir-1 indution fter h in RAW.7 ws dose dependent, with 1 ng/ml of LPS eing the optiml dose, nd gin we oserved similr pttern for mir-1 (Fig. ). Anlysis of the indution of mirna in primry mouse BMDMs gve result similr to tht otined with the RAW.7 ell line (Fig. ). We found tht mir-1 ws indued y LPS to n extent similr to tht of mir-1. We lso exmined the humn monoyti ell line THP-1 fter 1 h of tretment with LPS (Fig. d). We found tht mir-1 ws indued y LPS, onsistent with erlier reports ; however, we oserved no upregultion of mir-1 in this ell line. We lso oserved upregultion of Il1 mrna Pdd / d Il1 promoter luiferse.5 Ctrl sirna sirna Ctrl sirna e sirna (nm) Ctrl 5 1 Bnd intensity (%) Figure Regultion of TLR signling y. () Immunolot nlysis of (ove) nd ELISA of mouse IL-1 prodution (elow) in RAW.7 ells trnsfeted with 5 or 1 nm -speifi sirna nd then stimulted for h with LPS (1 ng/ml). Below lnes, densitometry of nd intensity reltive to tht of ells trnsfeted with ontrol sirna (Ctrl). () ELISA of IL-1 prodution in wild-type nd -defiient BMDMs stimulted for h with 1 or 1 ng/ml of LPS. () Quntittive RT-PCR nlysis of Il1 mrna in wild-type nd. nm Ctrl nm sirna nm sirna LPS (ng/ml) LPS (ng/ml) LPS (ng/ml) 1 1 NF-κB luiferse 1 9 h LPS (h) IκBα p-jnk Totl Jnk mir-1 y LPS in humn PBMCs (Fig. e). We found fourfold more mir-1, with similr effet for mir-1. Indution of mir-1 y LPS requires MyD nd NF- B We exmined the indution of mir-1 in immortlized BMDMs defiient in the TLR dptor proteins MyD nd TRIF (Fig. f). LPS used tenfold indution of mir-1 in wild-type ells fter 1 h. This effet ws olished in the sene of MyD nd ws only slightly impired in TRIF-defiient BMDMs (Fig. f, left). We noted similr dependeny on MyD for mir-1; however, in ontrst to mir-1 indution, mir-1 indution y LPS lso required TRIF (Fig. f, right). We next exmined the role of NF-κB in mir-1 indution y LPS, s the promoter region hs puttive NF-κB site loted t position (5 -GTGGGAGGTGCCT- ), s predited y the Genomtix MtInspetor softwre pkge. We found indution of mir-1 y LPS in wild-type mouse emryoni firolsts, ut this ws ompletely olished in mouse emryoni firolsts defiient in the NF-κB suunit p5 nd ws tully lower thn sl expression (Fig. g, left). We oserved similr dependeny on p5 for mir-1 (Fig. g, right), ut to lesser extent thn for mir-1. LPS dereses protein vi mir-1 indution Hving exmined the indution of mir-1 y LPS, we then exmined the ility of mir-1 to regulte undne fter TLR signling. We ssessed this effet y trnsfeting RAW.7 ells with ntisense oligonuleotides speifi to mir-1 (nti-mir-1) or ontrol ntisense RNA (Fig. 5). Tretment with LPS for h resulted in less in ells treted with ontrol ntisense RNA. Notly, pretretment with ntimir-1 loked the LPS-indued derese in, prtiulrly t nti-mir-1 onentrtions of 1.5 nm nd 5 nm, resulting in 1.-fold nd 1.57-fold more protein, respetively, thn in ontrol LPStreted ells, s mesured y densitometry snning. Notly, to verify f IL- prodution Pdd / g Il mrna Pdd / defiient BMDMs stimulted for h with 1 ng/ml of LPS. (d) Luiferse tivity (elow) in RAW.7 ells trnsfeted with n Il1 promoter luiferse reporter nd 1 nm -speifi sirna or 1 nm ontrol nontrgeting sirna nd then stimulted for h with 1 ng/ml of LPS (horizontl xis). Aove, immunolot nlysis of in unstimulted RAW.7 ells trnsfeted with ontrol or -speifi sirna. (e) Luiferse tivity in HEK9-TLR trnsfeted with n NF-κB-luiferse reporter nd 5 or nm -speifi sirna (key) nd then stimulted for h with 1 ng/ml of LPS (horizontl xis). Top, immunolot nlysis of protein in HEK9-TLR trnsfeted with ontrol sirna or 5 or nm -speifi sirna (ove lnes); elow lnes, densitometry of nd intensity reltive to tht of ells trnsfeted with ontrol sirna. (f) ELISA of IL- prodution in wild-type nd -defiient BMDMs stimulted for h with 1 or 1 ng/ml of LPS. (g) Quntittive RT-PCR nlysis of Il mrna in wild-type nd -defiient BMDMs stimulted for h with 1 ng/ml of LPS. (h) Immunolot nlysis of IκBα protein (top) nd Jnk phosphoryltion (p-jnk; middle) in wild-type nd -defiient BMDMs stimulted for 1 h (ove lnes) with LPS (1 ng/ml). serves s loding ontrol. In sirna experiments, ll ells were trnsfeted with n equl mount of totl RNA normlized with negtive ontrol sirna. Results for LPS-treted ells re presented reltive to those for wild-type ells or ells trnsfeted with negtive ontrol RNA only. P <.5, P <.1 nd P <.1 (two-tiled unpired t-test). Dt re representtive of three independent experiments (men nd s.d.). Pdd / nture immunology volume 11 numer ferury 1 1

4 Time (h) mirna indution (fold) mir-1 mir-1 1 mir-1 mir LPS (ng/ml) mirna indution (fold) mirna indution (fold) BMDM mir-1 mir-1 d mirna indution (fold) 1 1 THP-1 mir-1 mir1 e mirna indution (fold) 5 1 PBMC mir-1 mir-1 f mir-1 indution (fold) 1 mir-1 indution (fold) g mir-1 indution (fold) 1 Figure Indution of mir-1 y LPS tretment in mrophges. () Time-ourse nlysis of the indution of mir-1 nd mir-1 in RAW.7 ells stimulted for h (horizontl xis) with LPS (1 ng/ml). () Dose-response of the indution of mir-1 nd mir-1 in RAW.7 fter tretment for h with 1 1 ng/ml (horizontl xis) of LPS. ( e) Indution of mir-1 nd mir-1 in mouse BMDMs (), THP-1 ells (d) nd humn PBMCs (e) treted for h with LPS (1 ng/ml). (f) Indution of mir-1 (left) nd mir-1 (right) in immortlized Myd /, Trif / or wild-type (C57BL/) BMDMs stimulted for 1 h with LPS (1 ng/ml). (g) Indution of mir-1 (left) nd mir-1 (right) in wild-type nd p5-defiient (Rel / ) mouse emryoni firolsts stimulted for h with LPS (1 ng/ml). Results for LPS-treted ells re presented reltive to those for untreted ells. P <.5, P <.1 nd P <.1 (two-tiled unpired t-test). Dt re from t lest three independent experiments (men nd s.d.). Myd / Trif / 1 1 Myd / Trif / Rel / mir-1 indution (fold) 5 1 Rel / 1 Nture Ameri, In. All rights reserved. this effet of mir-1 ws rought out through speifi nd diret trgeting of Pdd mrna, we designed morpholino oligonuleotide speifi to the mir-1 site of (morpho-1). This morpho- 1 oligonuleotide should trget speifilly the Pdd UTR t the mir-1 site nd lok the tivity of ny mirna t tht position. Tretment with LPS gin resulted in less protein expression in ontrol ells trnsfeted with the morpholino thn in unstimulted ells (Fig. 5). However, trnsfetion of morpho-1 followed y LPS stimultion proteted Pdd protein, prtiulrly t morpholino oligonuleotide onentrtion of 5 µm, whih resulted in twofold more protein thn in ontrol LPS-treted ells, s mesured y densitometry snning. LPS therefore regultes the trnsltion of Pdd mrna through the indution of mir-1. is regulted y the protesome protein hs een shown to e trgeted for degrdtion in tumor promotion through the tion of uiquitin ligses nd the S protesome 1,17. We therefore determined if LPS ould lso trget the mount of protein vi the protesome. Pretretment of RAW.7 ells with vehile ontrol nd susequent stimultion with LPS resulted in slight indution of LPS 1 h fter stimultion nd derese t h fter stimultion (Fig. 5). Pretretment with the protesome inhiitor MG1 loked the derese in t h. However, s prolonged MG1 tretment is toxi to ells, the effet of MG1 on the derese t h noted efore ould not e nlyzed. However, it ws ovious the protesome did ffet the mount of protein t erlier time points fter LPS stimultion. To explin this phenomenon, we exmined the ility of protein to eome uiquitinted in response to LPS y trnsfetion of HEK9-TLR ells with hemgglutinin (HA)-tgged uiquitin nd susequent LPS stimultion. We immunopreipitted nd susequently nlyzed HA-tgged uiquitin y immunolot (Fig. 5d). We deteted uiquitintion of endogenous t h fter LPS tretment in the presene of MG1. Thus, protesoml degrdtion tivted y LPS n degrde uiquitinted protein in the ell. Regultion of IL-1 prodution nd NF-κB tivtion y mir-1 To determine if mir-1, through its regultion of, ffets the prodution of IL-1, we trnsfeted RAW.7 ells with the mir-1 preursor pro-mir-1 (Fig. ). Trnsfetion of pro-mir-1, whih will generte mture mir-1, resulted in less, prtiulrly t onentrtion of 1 nm (Fig. ). LPS gin resulted in less protein (Fig. ). At the sme time, trnsfetion with promir-1 lso resulted in more prodution of IL-1 indued y LPS, with signifintly more IL-1 prodution evident t pro-mir-1 onentrtion of 1 nm (Fig., left). Trnsfetion of ells with nti-mir-1, shown ove to inrese during LPS signling (Fig. 5), lunted the prodution of IL-1 y LPS (Fig., middle). To onfirm tht the effet of mir-1 ws rought out speifilly through the trgeting of, we trnsfeted RAW.7 ells with inresing mounts of morpho-1, shown ove to protet from the LPS-indued derese (Fig. 5). Agin we exmined the effet of morpho-1 on LPS-indued prodution of IL-1 nd, s with nti-mir-1, found tht it signifintly inhiited IL-1 prodution (Fig., right). To onfirm tht the speifiity of the effet of mir-1 on IL-1 prodution ws due to trgeting of, we trnsfeted BMDMs from wild-type nd -defiient mie with pro-mir-1 or ontrol RNA nd monitored the effet on IL-1 prodution t h Anti-1 (nm) Ctrl Unstim LPS Ctrl Bnd intensity : Morpho-1 (µm) Ctrl Ctrl Unstim Bnd intensity : LPS Vehile MG1 LPS (h) 1 1 Bnd intensity : d LPS (h) IP: IB: α-ha EV HA-U Lystes IB: Figure 5 Regultion of expression in LPS signling. () Immunolot nlysis of in RAW.7 ells trnsfeted with vrious onentrtions of nti-mir-1 (ove lnes) nd then left untreted (Unstim) or treted for h with LPS (1 ng/ml). () Immunolot nlysis of in RAW.7 ells trnsfeted with vrious onentrtions of morpho-1 (ove lnes) nd then left untreted or treted for h with LPS (1 ng/ml). () Immunolot nlysis of in RAW.7 ells pretreted with 1 µm MG1 or vehile ontrol nd then treted for h (ove lnes) with LPS (1 ng/ml). Below lnes ( ), densitometry of nd intensity reltive to tht of ells trnsfeted with ontrol RNA (Ctrl;,) or no LPS (), set s 1. (d) Immunopreipittion (IP; with nti-) of endogenous together with overexpressed HA-tgged uiquitin (HA-U) from trnsfeted HEK9-TLR ells stimulted with LPS (1 ng/ml) in the presene of MG1 (1 µg/ml), followed y immunolot nlysis (IB) with nti-ha (α-ha). EV, empty vetor (ontrol). Bottom, immunolot nlysis of in lystes without immunopreipittion (loding ontrol). For mirna-morpholino trnsfetions, ll ells were trnsfeted with n equl mount of totl RNA normlized with negtive ontrol nti-mirna or morpholino trgeted to redundnt site in the mouse Pdd UTR. Dt re representtive of three independent experiments. 1 volume 11 numer ferury 1 nture immunology

5 IL-1 prodution Ctrl Ctrl Ctrl Pro-miR-1 (nm) Anti-miR-1 (nm) Morpho 1 (µm) Pro-miR-1 (nm) Ctrl 5 1 LPS Bnd intensity (%) 1 99 IL-1 prodution Pdd / Ctrl PromiR-1 d mir-1 luiferse Pro-miR-1 Anti-miR-1 (nm) (nm) enf-κb luiferse tivity Ctrl Ctrl 5 1 LPS Pro-miR-1 (nm) Ctrl Ctrl Anti-miR-1 (nm) Ctrl Ctrl Morpho-1 (µm) Figure Regultion of funtion y mir-1 in TLR signling () ELISA of mouse IL-1 in RAW.7 ells trnsfeted with vrious doses (horizontl xes) of pro-mir-1 (left), nti-mir-1 (middle) or morpho-1 (right) nd then stimulted for h with LPS (1 ng/ml). () Immunolot nlysis of in RAW.7 ells trnsfeted with 5 or 1 nm pro-mir-1 (ove lnes) nd then stimulted for h with LPS (1 ng/ml). () ELISA of IL-1 prodution in wild-type C57BL/ nd -defiient BMDMs trnsfeted with 1 nm pro-mir-1 nd then stimulted for h with LPS (1 ng/ml). (d) Luiferse tivity in HEK9-TLR ells trnsfeted with mir-1 luiferse reporter nd, 5 or 5 nm pro-mir-1 (left) or nti-mir-1 (right). (e) Luiferse tivity in HEK9-TLR trnsfeted with n NF-κB luiferse reporter nd 5 or 1 nm pro-mir-1 oligonuleotide (left), nti-mir-1 oligonuleotide (middle) or morpho-1 (right) nd then stimulted for h with LPS (1 ng/ml). In ll RNA-morpholino trnsfetions, ells were trnsfeted with n equl mount of totl RNA normlized with negtive ontrol pro-mirna, negtive ontrol nti-mirna or morpholino trgeted to redundnt site in the mouse Pdd UTR. In,d,e, results with LPS re presented reltive to those otined with negtive ontrol RNA. P <.5, P <.1 nd P <.1 (two-tiled unpired t-test). Dt re representtive of three independent experiments (,d,e; men nd s.d.), three experiments () or two independent experiments (; men nd s.d. of triplite smples). 1 Nture Ameri, In. All rights reserved. fter LPS (Fig. ). There ws slightly more IL-1 prodution in BMDMs trnsfeted with pro-mir-1 thn in ontrol ells. This effet ws not present, however, in BMDMs from -defiient mie, whih indited tht the effet of pro-mir-1 ws speifilly due to trgeting of Pdd mrna. Agin we exmined the effet of mir-1 on NF-κB tivity with HEK9-TLR ells trnsfeted with nti-mir-1, s well s those trnsfeted with pro-mir-1 or morpho-1, nd mesured NF-κB-linked luiferse tivity in response to LPS. We ssessed the tivity of nti-mir-1 nd pro-mir-1 in these ells with mir-1- dependent luiferse onstrut (whih ontins syntheti mir-1 inding site in the UTR of the luiferse gene). Trnsfetion of ells with pro-mir-1 resulted in less luiferse tivity (Fig. d, left), wheres nti-mir-1 resulted in more mir-1-dependent luiferse tivity (Fig. d, right). Trnsfetion of pro-mir-1 lso negtively regulted the tivtion of NF-κB, with 5% inhiition ourring t pro-mir-1 onentrtion of 1 nm (Fig. e). Notly, we oserved the opposite trend when we trnsfeted nti-mir-1 into ells. We oserved douling in LPS-indued NF-κB tivity with trnsfetion of 5 nm nti-mir-1 reltive to the tivity in ells trnsfeted with ontrol ntisense RNA (Fig. e, middle). Similrly, trnsfetion of ells with 1 µm morpho-1 resulted in more LPSindued NF-κB tivity thn tht in ells trnsfeted with ontrol morpholino. Together these results demonstrte tht mir-1 ffets IL-1 prodution nd lso NF-κB tivtion through its effet on expression. DISCUSSION Here we hve found tht the ontrol of expression is key step in the negtive regultion of the inflmmtory response to LPS, ting s moleulr swith etween the proinflmmtory (NF-κB) nd nti-inflmmtory (IL-1) response. This swith is derese in protein undne, whih is rought out through the indution of mir-1. This proess positively influenes IL-1 prodution while negtively regulting NF-κB tivity, presumly to ontrol the LPS response tht n e lethl. Our study hs demonstrted upregultion of mir-1 y LPS in mny ell types, inluding mrophges, mouse emryoni firolsts nd PBMCs. We found tht the indution of mir-1 y LPS ws dependent on MyD nd lso dependent on NF-κB, onsistent with the presene of n NF-κB-inding site in the mir-1 promoter 5,. There is upregultion of mir-1 in vriety of disese sttes. Overexpression of mir-1 hs een reported in mny types of ner 7,. Also, mir-1 hs een reported to e upregulted in mny inflmed sttes, inluding the inflmed lung in LPS-treted mie 9, llergi irwy inflmmtion, osteorthritis 1, psorisis nd topi ezem, disese-tive ulertive olitis tissue, Helioter pylori ssoited gstri ner, rdi musle injury 5 nd rdi hypertrophy. Therefore, mir-1 my e n inditor of inflmmtion nd, given its role in tumorigenesis, might e n importnt link etween ner nd inflmmtion. Evidene is now emerging inditing tht TLR tivtion ffets the expression of mny mirnas. The first LPS-responsive mirna reported ws mir-1, nd here we hve shown tht its indution ws similr to tht of mir-1 in mrophges. Studies hve shown onsiderle upregultion of mir-155 in mrophges 7 s well s dendriti ells, to n extent higher thn reported here for mir-1 nd mir-1. Experiments with nimls genetilly defiient in mir-155 hve onfirmed its importne in the genertion of dptive immunity 9,. Additionl TLR-responsive mirnas inlude mir- 1 (ref. ), mir-9 (ref. 1), mir-17 (ref. ) nd mir- (ref. ); these re upregulted in vrious ell types fter stimultion with TLR lignds. Some mirnas hve een reported to e downregulted fter LPS tretment, inluding let-7i, whih is thought to trget TLR itself, nd mir As mny of those mirnas regulted y TLR signling re lso dysregulted in ner 7, it is possile tht mirnas form key link etween inflmmtion nd ner nd tht the indution of speifi mirnas, inluding mir-1, y TLRs my e key step in tumor progression. hs een demonstrted to funtion s n inhiitor of p-dependent trnsltion of omplex mrnas through its intertion with the eukryoti initition ftors eif nd eifg 1,1. Gene produts inhiited y this mehnism inlude growth ftors nd ytokines, inluding IL-1. Furthermore, hs een linked to NF-κB tivtion y n unknown mehnism 1. We hve demonstrted here tht ws required for NF-κB tivtion nd ttenuted IL-1 prodution in LPS signling nd ws involved in the lethlity of LPS. Here we hve shown tht the suppressive effet of on IL-1 ourred t the trnsltionl level, s -defiient mrophges hd more IL-1 protein yet less Il1 mrna, onsistent with pulished reports tht IL-1 trnsltion is highly regulted nd tht more eife-direted trnsltion n led to more mrna turnover,. In ddition, we hve shown tht ffeted NF-κB tivtion y n undefined mehnism tht promotes Il trnsription, whih my involve positive effet on Jnk. It is likely tht other trgets for exist in TLR signling tht re regulted t the trnsltionl level. nture immunology volume 11 numer ferury 1 15

6 1 Nture Ameri, In. All rights reserved. We hve lso demonstrted here tht modultion of mir-1 hd effets opposite to those of in LPS signling. It ttenuted NF-κB tivtion nd promoted IL-1 prodution in response to LPS. Notly, through the use of trget protetion of the mir-1 site nd through the trnsfetion of -defiient BMDMs with mir-1, we hve demonstrted tht mir-1 hs n importnt role in negtively regulting these proesses speifilly through the trgeting of, therey limiting exessive inflmmtion. Highlighting the importne of the removl of for the pproprite development of inflmmtory responses ws the finding tht n lso e trgeted for protesoml degrdtion in LPS signling. We hve demonstrted here tht inhiition of the S protesome y MG1 stilized protein t erly time points fter LPS stimultion. In ddition, we hve identified s key trget protein for uiquitintion downstrem of TLR signling. The existene of multiple mehnisms to ontrol protein expression highlights the importne of this moleule in the immune response. Degrdtion of the pre-existing pool of protein ourred through uiquitintion nd the tion of the S protesome t erly times fter LPS tretment ( h). This ourred in ddition to the post-trnsriptionl silening of Pdd mrna through the tion of mir-1 to lok the trnsltion of new protein, whih, euse of the dely in mir-1 indution, hd its effet t lter times ( h). As for the indution of protein t erlier time points fter LPS tretment (1 h), this ws not ompnied y n inrese in Pdd mrna, whih egn to derese h fter LPS dministrtion. It is likely tht t erlier time points, euse of lower sl mounts of mir-1, trnsltion of n our without hlting. undne ws high initilly nd filitted NF-κB tivtion while suppressing IL-1. LPS deresed vi mir-1 to limit NF-κB tivity while promoting IL-1 prodution, therey providing negtive regultory loop for TLR signling. The link etween mir-1 nd nd etween NF-κB nd IL-1 is diret, s shown y use of morpholino oligonuleotide direted speifilly to the mir-1 site of mouse Pdd mrna. This stilized protein nd hd the opposite effet in terms of enhned NF-κB nd deresed IL-1 reltive to tht in ells treted with sirna speifi for or -defiient ells. It is therefore resonle to onlude tht mir-1 limits in LPS signling, leding to derese in NF-κB nd n inrese in IL-1, whih in turn regultes inflmmtory proesses indued y LPS. In onlusion, our study hs identified n xis involving mir-1 nd tht provides importnt new insight into the negtive regultion of TLR signling. In terms of the importne of TLR in vine djuvny 9 or for inflmmtion in proesses suh s sepsis, rheumtoid rthritis nd llergi sthm 5, this finding might present new opportunities for oosting or limiting TLR tivtion therpeutilly. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Aknowledgments We thnk D. Golenok (University of Msshusetts) for wild-type, MyD- defiient nd TRIF-defiient BMDMs immortlized y retrovirus; R. Hy (University of St. Andrews) for p5-defiient nd mthed wild-type ontrol mouse emryoni firolsts nd for HA-tgged uiquitin; R. Hofmeister (Universitet Regensurg) for 5 NF-κB reporter luiferse plsmid; A.M. Cheng (Amion) for the pmir-report mir-1 reporter luiferse plsmid; nd A. Bowie (Trinity College, Dulin) for the Il1 promoter luiferse plsmid. Supported y Siene Foundtion Irelnd nd the Irish Reserh Counil for Siene, Engineering nd Tehnology (RS/5/19). AUTHOR CONTRIBUTIONS F.J.S. did the funtionl experiments on nd mir-1 nd owrote the mnusript; E.P.-M. did experiments on degrdtion y the protesome nd experiments on signls in -defiient ells; E.J.H. helped with experiments on mir-1; C.M. nd J.J.O. provided dvie on mirna profiling experiments; Q.R., D.S.J. nd J.Y.C. did the experiments on the lethlity of LPS in defiient mie nd supplied BMDMs from the mie; nd L.A.J.O. direted the work nd owrote the mnusript. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Liew, F.Y., Xu, D., Brint, E.K. & O Neill, L.A. Negtive regultion of toll-like reeptormedited immune responses. Nt. Rev. Immunol. 5, 5 (5).. Iwmi, K.I. et l. Cutting edge: nturlly ourring solule form of mouse Toll-like reeptor inhiits lipopolyshride signling. J. Immunol. 15, ().. Jnssens, S., Burns, K., Vermmen, E., Tshopp, J. & Beyert, R. MyDS, splie vrint of MyD, differentilly modultes NF-κB- nd AP-1-dependent gene expression. FEBS Lett. 5, 1 17 ().. Koyshi, K. et l. IRAK-M is negtive regultor of Toll-like reeptor signling. Cell 11, 191 (). 5. Plsson-MDermott, E.M. et l. TAG, splie vrint of the dptor TRAM, negtively regultes the dptor MyD-independent TLR pthwy. Nt. Immunol. 1, (9).. Sun, S.C., Gnhi, P.A., Bllrd, D.W. & Greene, W.C. NF-κB ontrols expression of inhiitor IκBα: evidene for n induile utoregultory pthwy. Siene 59, (199). 7. Murry, P.J. STAT-medited nti-inflmmtory signlling. Biohem. So. Trns., 1 11 ().. Tgnov, K.D., Boldin, M.P., Chng, K.J. & Bltimore, D. NF-κB-dependent indution of mirorna mir-1, n inhiitor trgeted to signling proteins of innte immune responses. Pro. Ntl. Ad. Si. USA 1, 11 1 (). 9. Shihr, K. et l. Isoltion of novel mouse gene MA- tht is indued upon progrmmed ell deth. Gene 1, 97 1 (1995). 1. Yng, H.S., Knies, J.L., Strk, C. & Colurn, N.H. Pdd suppresses tumor phenotype in JB ells y inhiiting AP-1 trnstivtion. Onogene, 71 7 (). 11. Onishi, Y., Hshimoto, S. & Kizki, H. Cloning of the TIS gene suppressed y topoisomerse inhiitors. Gene 15, 5 59 (199). 1. Yng, H.S. et l. A novel trnsformtion suppressor, Pdd, inhiits AP-1 trnstivtion ut not NF-κB or ODC trnstivtion. Onogene, 9 7 (1). 1. Yng, H.S. et l. The trnsformtion suppressor Pdd is novel eukryoti trnsltion initition ftor A inding protein tht inhiits trnsltion. Mol. Cell. Biol., 7 (). 1. Loh, P.G. et l. Struturl sis for trnsltionl inhiition y the tumour suppressor Pdd. EMBO J., 7 5 (9). 15. Hillird, A. et l. Trnsltionl regultion of utoimmune inflmmtion nd lymphom genesis y progrmmed ell deth. J. Immunol. 177, 95 1 (). 1. Dorrello, N.V. et l. SK1- nd βtrcp-medited degrdtion of promotes protein trnsltion nd ell growth. Siene 1, 7 71 (). 17. Shmid, T. et l. Trnsltion inhiitor Pdd is trgeted for degrdtion during tumor promotion. Cner Res., 15 1 (). 1. Asngni, I.A. et l. MiroRNA-1 (mir-1) post-trnsriptionlly downregultes tumor suppressor Pdd nd stimultes invsion, intrvstion nd metstsis in oloretl ner. Onogene 7, 1 1 (). 19. Frnkel, L.B. et l. Progrmmed ell deth () is n importnt funtionl trget of the mirorna mir-1 in rest ner ells. J. Biol. Chem., 1 1 ().. Lu, Z. et l. MiroRNA-1 promotes ell trnsformtion y trgeting the progrmmed ell deth gene. Onogene 7, 7 79 (). 1. Cmrik, J.L. et l. Differentilly expressed protein Pdd inhiits tumor promoter-indued neoplsti trnsformtion. Pro. Ntl. Ad. Si. USA 9, 17 1 (1999).. Rousseu, D., Kspr, R., Rosenwld, I., Gehrke, L. & Sonenerg, N. Trnsltion initition of ornithine deroxylse nd nuleoytoplsmi trnsport of ylin D1 mrna re inresed in ells overexpressing eukryoti initition ftor E. Pro. Ntl. Ad. Si. USA 9, (199).. Grff, J.R. et l. Trnsltion of ODC mrna nd polymine trnsport re suppressed in rs-trnsformed CREF ells y depleting trnsltion initition ftor E. Biohem. Biophys. Res. Commun., 15 (1997).. Tlott, F. et l. An utoregultory loop medited y mir-1 nd ontrols the AP-1 tivity in RAS trnsformtion. Onogene, 7 (9). 5. Fujit, S. et l. mir-1 Gene expression triggered y AP-1 is sustined through doule-negtive feedk mehnism. J. Mol. Biol. 7, 9 5 ().. Loffler, D. et l. Interleukin- dependent survivl of multiple myelom ells involves the Stt-medited indution of mirorna-1 through highly onserved enhner. Blood 11, 1 1 (7). 7. Clin, G.A. & Croe, C.M. MiroRNA signtures in humn ners. Nt. Rev. Cner, 57 ().. Cho, W.C. OnomiRs: the disovery nd progress of mirornas in ners. Mol. Cner, (7). 1 volume 11 numer ferury 1 nture immunology

7 1 Nture Ameri, In. All rights reserved. 9. Moshos, S.A. et l. Expression profiling in vivo demonstrtes rpid hnges in lung mirorna levels following lipopolyshride-indued inflmmtion ut not in the nti-inflmmtory tion of gluoortioids. BMC Genomis, (7).. Lu, T.X., Munitz, A. & Rothenerg, M.E. MiroRNA-1 is up-regulted in llergi irwy inflmmtion nd regultes IL-1p5 expression. J. Immunol. 1, 99 5 (9). 1. Iliopoulos, D., Mlizos, K.N., Oikonomou, P. & Tsezou, A. Integrtive mirorna nd proteomi pprohes identify novel osteorthritis genes nd their ollortive metoli nd inflmmtory networks. PLoS ONE, e7 ().. Sonkoly, E. et l. MiroRNAs: novel regultors involved in the pthogenesis of psorisis? PLoS ONE, e1 (7).. Wu, F. et l. MiroRNAs re differentilly expressed in ulertive olitis nd lter expression of mrophge inflmmtory peptide-α. Gstroenterology 15, 1 15 ().. Zhng, Z. et l. mir-1 plys pivotl role in gstri ner pthogenesis nd progression. L. Invest., 15 1 (). 5. Ji, R. et l. MiroRNA expression signture nd ntisense-medited depletion revel n essentil role of MiroRNA in vsulr neointiml lesion formtion. Cir. Res. 1, (7).. Cheng, Y. et l. MiroRNAs re errntly expressed in hypertrophi hert: do they ply role in rdi hypertrophy? Am. J. Pthol. 17, 11 1 (7). 7. O Connell, R.M., Tgnov, K.D., Boldin, M.P., Cheng, G. & Bltimore, D. MiroRNA- 155 is indued during the mrophge inflmmtory response. Pro. Ntl. Ad. Si. USA 1, 1 19 (7).. Ceppi, M. et l. MiroRNA-155 modultes the interleukin-1 signling pthwy in tivted humn monoyte-derived dendriti ells. Pro. Ntl. Ad. Si. USA 1, 75 7 (9). 9. Thi, T.H. et l. Regultion of the germinl enter response y mirorna-155. Siene 1, (7).. Rodriguez, A. et l. Requirement of i/mirorna-155 for norml immune funtion. Siene 1, 11 (7). 1. Bzzoni, F. et l. Indution nd regultory funtion of mir-9 in humn monoytes nd neutrophils exposed to proinflmmtory signls. Pro. Ntl. Ad. Si. USA 1, 5 57 (9).. Liu, G. et l. mir-17, mirorna tht is indued upon Toll-like reeptor stimultion, regultes murine mrophge inflmmtory responses. Pro. Ntl. Ad. Si. USA (9).. Alsleh, G. et l. Bruton s tyrosine kinse is involved in mir--relted regultion of IL-1 relese y lipopolyshride-tivted rheumtoid firolst-like synovioytes. J. Immunol. 1, (9).. Chen, X.M., Splinter, P.L., O Hr, S.P. & LRusso, N.F. A ellulr miro-rna, let-7i, regultes Toll-like reeptor expression nd ontriutes to holngioyte immune responses ginst Cryptosporidium prvum infetion. J. Biol. Chem., 99 9 (7). 5. Tili, E. et l. Modultion of mir-155 nd mir-15 levels following lipopolyshride/ TNF-α stimultion nd their possile roles in regulting the response to endotoxin shok. J. Immunol. 179, 5 59 (7).. Nor, H., Altin, J.G. & Young, I.G. TCR-dependent nd -independent signling mehnisms differentilly regulte lymphokine gene expression in the murine T helper lone D1.G.1. J. Immunol. 15, (199). 7. Nemeth, Z.H. et l. Adenosine ugments IL-1 prodution y mrophges through n AB reeptor-medited posttrnsriptionl mehnism. J. Immunol. 175, 7 (5).. Shrm, A. et l. Posttrnsriptionl regultion of interleukin-1 expression y hs-mir-1. Pro. Ntl. Ad. Si. USA 1, (9). 9. vn Duin, D., Medzhitov, R. & Shw, A.C. Triggering TLR signling in vintion. Trends Immunol. 7, 9 55 (). 5. Cook, D.N., Pisetsky, D.S. & Shwrtz, D.A. Toll-like reeptors in the pthogenesis of humn disese. Nt. Immunol. 5, (). nture immunology volume 11 numer ferury 1 17

8 1 Nture Ameri, In. All rights reserved. ONLINE METHODS Regents. Ultrpure TLRgrde LPS ws from Alexis. Pm CSK ws from Cliohem nd poly(i:c) ws from Amershm Biosienes. MG1 ws from Cliohem. SMARTpool sirnas speifi for humn nd mouse nd negtive ontrol SMARTpool sirna were from Dhrmon. Pro-miR-1, nti-mir-1, speifi to humn nd mouse mirna, nd the respetive negtive ontrol RNAs were from Amion. Morpholino oligonuleotides with the following sequenes were designed in ssoition with GeneTools: -mir-1 (morpho-1), 5 -AAGTAGCTTATCAGAACAC CCACAC-, nd -ontrol (morpho-trl), 5 -GATCAGGTCCTAAA CATGGCACTTA-. Cell ulture nd niml hndling. RAW.7 nd THP-1 ells were from the Europen Colletion of Cell Cultures. HEK9-TLR ells (HEK9-TLR- MD-CD1) were from Invivogen. Wild-type C57BL/ mie were housed nd mintined t Trinity College, Dulin. Wild-type, MyD-defiient nd TRIF-defiient BMDMs immortlized y retrovirus were provided y D. Golenok. -defiient mie were housed nd mintined t the University of Pennsylvni. The p5-defiient nd mthed wild-type ontrol mouse emryoni firolsts were provided y R. Hy. All ells were mintined in either RPMI medi or DMEM (Invivogen) supplemented with 1% (vol/vol) FCS nd peniillin nd streptomyin. For BMDM extrtion, nimls were killed humnely ording to the regultions of the Europen Union nd the Irish Deprtment of Helth. Bone mrrow ws extrted from femurs nd tiis nd red lood ells were lysed with Red Blood Cell Lysing Buffer (Sigm), nd the resulting ells were grown in medium onditioned with mrophge olony-stimulting ftor. For nlysis of humn PBMCs, mononuler ells were isolted from whole lood with Fioll grdient (Sigm) nd were grown in RPMI medi s desried ove. For nlysis of the lethlity of LPS, mie were injeted intrperitonelly with LPS t dose of mg per kg ody weight. The survivl of mie ws monitored for 1 d, fter whih ll surviving mie were killed humnely in ordne with the Animl Reserh Committee of the University of Pennsylvni. RNA extrtion nd PCR. Cells were grown to 5 1 to 1 1 ells nd RNA ws extrted with the RNesy Kit (Qigen), modified to otin smll RNA speies. For mirna nlysis, individul mirna TqMn ssys for the endogenous referene RNA RNUB, mir-1 nd mir-1 were done ording to the mnufturer s instrutions (Applied Biosystems). For nlysis of gene expression, DNA ws prepred with the High-Cpity DNA Arhive kit ording to mnufturers instrutions (Applied Biosystems), nd individul mrnas were monitored with the following inventoried TqMn ssys (Applied Biosystems): mouse Gpdh (glyerldehyde phosphte dehydrogense) ssy, mouse Pdd ssy (Mm1_m1), mouse Il ssy (Mm99999_m1) nd mouse Il1 ssy (Mm99999_m1). The AB79FAST pltform (Applied Biosystems) ws used for ll PCR, done in triplite. Chnges in expression were lulted y the hnge in threshold ( C T ) method with RNUB s the endogenous ontrol for mirna nlysis nd Gpdh s n endogenous ontrol for gene-expression nlysis nd were normlized to results otined with untreted ells. Trnsient trnsfetion. For trnsfetion of mirna nd morpholino oligonuleotides, 5 1 RAW.7 ells were trnsfeted with the pproprite RNA oligonuleotides with % Lipofetmine (Invitrogen). For sirna trnsfetion, 5 1 RAW.7 ells were trnsfeted with % Lipofetmine RNAiMx (Invitrogen). Cells were llowed to reover for h efore tretment with LPS for vrious times. For ll RNA trnsfetion, equl totl onentrtions of RNA were used for eh retion, with negtive ontrol RNA moleules used for normliztion. For luiferse ssys, 5 1 HEK9-TLR ells were trnsfeted with endotoxin-free 5 NF-κB reporter luiferse plsmid ( gift from R. Hofmeister) nd prl-tk, the renill luiferse reporter, with % GeneJuie (Novgen). Cells were llowed to reover for h efore eing trnsfeted with RNA oligonuleotides s desried ove. Reporter gene tivity ws mesured with the Dul-Luiferse kit (Promeg) 1 h fter LPS tretment. The pmir-report mir-1 reporter luiferse plsmid ws from A.M. Cheng. The Il1 promoter luiferse plsmid ws gift from A. Bowie. HA-tgged uiquitin ws gift from R. Hy. ELISA. Cytokine onentrtion in superntnts were mesured with ELISA DuoSet Development systems ording to the mnufturer s instrutions (R&D Systems). Coimmunopreipittion of uiquitinted. HEK9-TLR ells (.5 1 ) were grown to 7% onflueny nd were trnsfeted with µg plsmid enoding HA-tgged uiquitin with % GeneJuie (Novgen). Cells were llowed to reover for h efore stimultion for vrious times with 1 ng/ml of LPS, then were lysed s desried 51. For ssistne in the detetion of uiquitinted proteins, protesoml degrdtion ws prevented y tretment of the ells with MG1 (1 µg/ml) for h efore lysis. Anti- ( µg; -1-95; Roklnd) ws preinuted for 1 h with protein A/G PLUS- Agrose eds (s_; Snt Cruz Biotehnology) nd susequently wshed in lysis uffer. ws isolted y inution of eds with ell lystes for h t C. After eds were wshed, immune omplexes were eluted with 5 µl Lemmli smple uffer nd seprted y SDS-PAGE nd proteins were deteted y immunolot nlysis with nti-ha (HA-11R; Sigm). Immunolot. Cells (5 1 5 to ) were lysed with low-stringeny lysis uffer omplete with protese inhiitors. Smples loded with 5 denturing smple uffer were seprted y 1% SDS-PAGE. Proteins were trnsferred to polyvinylidene difluoride memrne y stndrd tehniques nd were susequently nlyzed y immunolot with the relevnt ntiodies. Protein undne ws lulted y densitometry snning of the lot with IMAGE-J softwre; undne ws normlized to undne nd is presented reltive to results otined with the ontrol smple (perentge or frtion), set s 1.. The monolonl ntiody used to detet ws from Sigm (AC-15). Sttistil tests. All sttistil nlyses used the Student s t-test, unpired for norml distriutions of t lest three independent experiments. 51. Bowie, A. et l. AR nd A5R from vini virus re ntgonists of host IL-1 nd toll-like reeptor signling. Pro. Ntl. Ad. Si. USA 97, (). nture immunology doi:1.1/ni.1