Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo

BioMed Cen al Page 1 o 12 (page numbe no o ci a ion pu poses) Jou nal o In lamma ion Open Access Resea ch E icien deli e y o small in e e ing RNA o inhibi ion o IL-12p40 exp ession in i o Ma ion A Flynn†, Da id G Casey†, S ephen M Tod yk and Be na d P Mahon* Add ess: Ins i u e o Immunology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland Email: Ma ion A Flynn - ma ion.a. [email protected]; Da id G Casey - [email p o ec ed]; S ephen M Tod yk - s ephen. od yk@clinical- medicine.ox o d.ac.uk; Be na d P Mahon* - bpmaho[email p o ec ed] * Co esponding au ho †Equal con ibu o s Abs ac Backg ound: RNA in e e ence is an e olu iona y conse ed immune esponse mechanism ha can be used as a ool o p o ide no el insigh s in o gene unc ion and s uc u e. The abili y o e icien ly deli e small in e e ing RNA o modula e gene exp ession in i o may p o ide new he apeu ic app oaches o cu en ly in ac able diseases. Me hods: In i o, siRNA a ge ing IL-12p40 was deli e ed o he mu ine mac ophage cell line (J774A.1) encapsula ed in a liposome wi h an IL-12 inducing agen (LPS/IFN-γ) o e a numbe o ime poin s. Con ols included a a ie y o non- a ge speci ic siRNA eagen s. Supe na an s we e analyzed o cy okine p oduc ion while he cells we e emo ed o mRNA p o iling. In i o, siRNA- a ge ing IL-12p40 was deli e ed o he mu ine pe i oneal ca i y in a he apeu ic ashion, a e endo oxin (LPS) challenge. Cells om he pe i oneal ca i y we e emo ed by la age and analyzed by low cy ome y. Le els o IL-12 p esen in la age and in se um we e also examined by ELISA. Resul s: In his epo , we show ha IL-12p40 siRNA can speci ically silence mac ophage exp ession o IL-12p40 mRNA and IL-12p70 p o ein in i o. We ex end his inding o demons a e ha deli e y o liposome encapsula ed siRNA a ge ing IL-12p40 o he mu ine pe i oneal ca i y can modula e an in lamma o y s imulus in i o. Fu he mo e, speci ic siRNA can be used he apeu ically a e endo oxin challenge o educe bo h he local and sys emic in lamma o y esponse. Thus, he deli e y o siRNA can be used o elici speci ic non-pe manen inhibi ion o endogenous p o ein exp ession. Conclusion: In i o silencing o IL-12p40 using siRNA a selec ed doses leads o speci ic knockdown o IL-12p70 p o ein p oduc ion wi hou inducing ype I in e e ons. Fu he mo e, siRNA a ge ing mu ine IL-12p40 can be used he apeu ically o coun e an in lamma o y esponse in i o. Backg ound RNA in e e ence (RNAi) is an e olu iona y conse ed sequence-speci ic RNA silencing mechanism ound as an an i- i al esponse in in e eb a es, plan s and mamma- lian cells [1]. Al hough he mechanism o silencing is no comple ely unde s ood, he basic p emise o RNAi es s Published: 01 Oc obe 2004 Jou nal o In lamma ion 2004, 1:4 doi:10.1186/1476-9255-1-4 Recei ed: 28 June 2004 Accep ed: 01 Oc obe 2004 This a icle is a ailable om: h p://www.jou nal-in lamma ion.com/con en /1/1/4 © 2004 Flynn e al; licensee BioMed Cen al L d. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 2 o 12 (page numbe no o ci a ion pu poses) on he abili y o double s anded RNA (dsRNA) o speci - ically deg ade homologous messenge RNA (mRNA). The RNAi pa hway is igge ed in mammalian cells by he p esence o dsRNA o in he p esence o sho 19–22n dsRNA agmen s e med small in e e ing RNA molecules (siRNA). siRNA molecules ac i a e an RNA-induced silencing complex (RISC) ha unwinds he siRNA duplex [2]. The speci ici y o locus deg ada ion is guided by he an isense s and o he unwound siRNA, ollowed by sense s and siRNA binding o he complemen a y mRNA si e o clea age by RISC. The clea age o he sense s and siRNA and a ge mRNA esul s in he sel -ampli ying p o- duc ion o new siRNA in e media ies ha con inue mRNA a ge deg ada ion in an ATP dependen manne [3,4]. This phenomenon means ha low doses o siRNA can be mo e e ec i e han an isense he apy. Fu he - mo e, his app oach is p e e able o gene and an isense based he apies, in ha siRNA is non-he i able and does no equi e adeno i al ec o s, which limi he e ec i e- ness and accep abili y o use in child en. RNAi can be exploi ed as a ool o p o ide no el insigh s in o gene unc ion and s uc u e. The capaci y o e i- cien ly deli e siRNA o modula e gene exp ession in i o may p o ide new he apeu ic app oaches o cu en ly in ac able diseases. Like o he new gene ic echnologies, siRNA gene supp ession aces se e al me hodological lim- i a ions in i o. Fo emos among hese a e he e icien deli e y o siRNA o a ge cells [5,6], non-speci ic e ec s o pu a i e con ol duplexes [7-9] and he po en ial he a- peu ic p oblems o i al exp ession ec o s [10]. One app oach o o e coming hese obs acles is o deli e non- he i able siRNA duplexes in a model sys em and moni o he in luence upon expe imen ally induced in lamma- ion. This app oach would p o ide a me hod ha allows he apid sc eening o wha ha e been e med "d uggable" a ge s [11]. In e leukin-12 (IL-12p70) is a cy okine wi h a well-cha - ac e ized p o-in lamma o y unc ion [12] ha has been sugges ed as a a ge o he apeu ic in e en ion [13-15]. Bioac i e IL-12p70 is a he e odime o med by a hea y chain (p40) and a ligh chain subuni (p35), encoded by wo sepa a e genes whose exp ession is independen ly egula ed a he ansc ip ional le el [16]. The p35 sub- uni is cons i u i ely exp essed a low le els in mos cell ypes bu is up egula ed du ing cell ac i a ion. In con- as , he IL-12p40 gene is unde igh ansc ip ional con- ol only exp essed in mac ophages o o he APC ollowing ac i a ion by mic obial p oduc s [17]. P oduc- ion o IL-12p70 is enhanced by IFN-γ ia he IFN consen- sus sequence binding p o ein [18] bu educed by IL-10 [19]. IL-12p70 has pleio opic e ec s on a ge cells bu he majo ole is as a p o-in lamma o y cy okine in cell medi- a ed immuni y agains mic obial insul . In pa icula IL- 12p70 ac s upon T and NK cells o inc eases cy okine p o- duc ion, p oli e a ion, and cy o oxici y, unc ions ha become e iden se e al hou s a e exposu e o in ec ions agen s [19]. The IFN-γ subsequen ly p oduced, po en ia es an igen p esen a ion unc ions impo an in clea ing in ec ious agen s. These unc ions include inc eased co- s imula o y molecule exp ession, phagocy osis, and p o- duc ion o eac i e oxygen and ni ogen in e media es [19,20]. Howe e , IL-12p70 is no always p o ec i e o bene icial, indeed a a ie y o pa hological condi ions, including sepsis, a e associa ed wi h IL-12 d i en pa hol- ogy [21,22]. In addi ion o he well-cha ac e ized ole o IL-12p70, i is now known ha he IL-12p40 subuni is also biologically ac i e. This subuni may ac o an ago- nize he he e odime unc ion [23], o may ha e a b oade di ec ole, less dependen on IL-12p70 [24,25]. In o de o explo e he he apeu ic easibili y o RNA in e - e ence, we used siRNA o speci ically abla e IL-12p40 exp ession in i o and in i o. This app oach ex ends he powe o RNA in e e ence o gene exp ession s udies in li e animals wi hou he use o gene ic enginee ing, plas- mid DNA epo e sys ems [2,26] e o i al [27,28] o len- i i al siRNA exp ession ec o s [29] and opens he way o explo ing he use o siRNA in humans o ea disease. Ou esul s p o ide a desc ip ion o siRNA media ed sup- p ession o an endogenous immune gene in i o and desc ibe a no el he apeu ic and esea ch app oach o gene speci ic inhibi ion o an impo an cellula and immunological esponse. Ma e ials and Me hods Mice & Cell Lines Female BALB/c mice (Ha lan Limi ed, Bices e , UK) and IL-12p40 gene-dis up ed mice (IL12p40-/-) (Jackson Lab- o a o ies, Ba Ha bo , Maine) we e main ained unde he guidelines o he I ish Depa men o Heal h and he local bioe hics commi ee. All mice we e 12–14 weeks old a he ini ia ion o expe imen s and sac i iced on comple- ion. The mu ine mac ophage cell line (J774A.1) was used o in es iga e silencing o IL-12p40 cy okine gene exp ession. P epa a ion o siRNA siRNA oligonucleo ides wi h he ollowing sense and an i- sense sequences we e designed om he GenBank eposi- o y: accession numbe ; NM_008352, Mus musculus in e leukin 12b (IL12b), mRNA. IL-12p40 siRNA 5'-C CUC ACC UGU GAC ACG CCU dTdT-3' (sense) and 3'- dTdT G GAG UGG ACA CUG UGC GGA-5' (an isense); Mu an siRNA 5'-C CUC ACC UUC GAC ACG CCU dTdT- 3' (sense) and 3'-dTdTG GAG UGG AAG CUG UGC GGA- Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 3 o 12 (page numbe no o ci a ion pu poses) 5' (an isense); GFPsiRNA 5'-GGC UAC GUC CAG GAG CGC ACC dTdT-3' (sense) and 3'-dTdT CCG AUG CAG GUC CUC GCG UGG-5' (an isense). The an isense o he IL-12p40 siRNA duplex (As.RNA) was also used as a con- ol o in i o expe imen s. Each complemen a y RNA s and was dep o ec ed acco ding o manu ac u e 's ins uc ions. Fo he p oduc ion o he IL-12p40 siRNA duplex, sense and an isense siRNA s ands we e mixed in equimola a ios and ea ed by hea ing o 95°C o 1 min ollowed by annealing a 37°C o 1 h and allowed o cool slowly o e nigh o oom empe a u e. All siRNA oligonu- cleo ides we e syn hesized comme cially (Dha macon, La aye e, CO) using 2'ACE p o ec ion chemis y. In i o siRNA in e e ence Semi-con luen J774A.1 cells we e cul u ed a 1 × 105 cells/ml in an ibio ic ee, 8% ( / ) endo oxin-low e al- cal se um RPMI (Gibco-In i ogen, Paisley, UK) con ain- ing L-glu amine (Sigma, Poole, UK) 12–16 h be o e ans- ec ion. Fo siRNA ans ec ions 3 µl o a 20 µM siRNA duplex ( a ge o con ol) solu ion was mixed wi h 47 µl o Op i-mem (Gibco-In i ogen). In a second ube 3 µl o oligo ec amine (Gibco-In i ogen) was mixed wi h 12 µl o Op i-mem and incuba ed a oom empe a u e o 15 min. Solu ions we e combined o 40 min and b ough o a inal olume o 100 µl. The exp ession o IL-12p40 mRNA and IL-12p70 p o ein was induced by he addi ion o 1 µg/ml E. coli LPS Se o ype 0111:B4 (Sigma) and 10 ng/ml IFN-γ (Pha mingen, San Diego, CA.), o he las 12 h o each cul u e pos siRNA ans ec ion. RNA isola ion and semi-quan i a i e RT-PCR (sqRT-PCR) To al cellula RNA was isola ed om J774A.1 cells om in i o expe imen s wi h TRIZOL Reagen (Gibco-In i o- gen) ollowing he manu ac u e 's p o ocol and quan i- ied by spec opho ome y. RNA was e e se ansc ibed, and 100 ng o he complemen a y DNA p oduc ampli- ied by PCR as p e iously desc ibed [51] using 60 ng o gene speci ic ups eam and downs eam p ime s. Mu ine β-ac in p oduc was used o no malize RNA samples. PCR condi ions included a p e-incuba ion a 95°C o 5 min ollowed by 35 ampli ica ion cycles (95°C, 1 min; 1 min a annealing empe a u e; 2 min a 72°C, and a inal 10 min a 72°C). Ups eam and downs eam p ime s o IL- 12p40 we e speci ically designed o lank he IL-12p40 siRNA a ge egion; sense, 5'-AAACAGTGAACCTCACCT- GTGACAC-3' ; an isense, 5'-TTCATCAGCAAGTTCTT- GGGCG-3'. PCR p oduc s we e isualized by UV illumina ed aga ose gel elec opho esis. In i o siRNA in e e ence Con ol mice (BALB/c & IL12p40-/-) ecei ed 200 µl Op i- mem in a-pe i oneal (i.p.) con aining oligo ec amine alone. In addi ion LPS posi i e con ol mice ecei ed 1 µg E. coli LPS. Fo each expe imen al adminis a ion, 10 µl siRNA duplexes (IL-12p40 o con ols a equimola con- cen a ion) we e p emixed wi h 40 µl o Op i-mem. Sepa- a ely, 6 µl o oligo ec amine was mixed wi h 24 µl o Op i-mem and incuba ed a oom empe a u e o 15 min. These solu ions we e mixed a oom empe a u e o 40 min. Fo co-injec ion expe imen s, hese we e com- bined wi h LPS (1 µg/mouse) and o mula ed as abo e. Fo he apeu ic silencing, mice ecei ed 1 µg LPS, in he absence o siRNA duplexes, 1 h p io o adminis a ion o siRNA (IL-12p40 o con ols) as abo e. A a ious ime poin s, blood se um, pe i oneal cells o la age luid we e sampled o u he analysis. Pe i oneal La age & Se um p epa a ion Pe i oneal cells we e ha es ed by washing he pe i oneal ca i y wi h 1 ml o s e ile PBS. This was cen i uged o 5 min a 400 g, la age supe na an was emo ed o analysis and cells analysed by low cy ome y. Se um was p epa ed by ca diac punc u e. Se a and la age supe na an s we e assayed wi hou delay o s o age. Flow Cy ome y Pheno ypic analysis o siRNA- ans ec ed cells was pe - o med using a FACScalibu ™ wi h associa ed Cellques ™ so wa e (Bec on Dickinson, San Jose, CA). Fo wa d and side sca e we e measu ed om pe i oneal la age p epa- a ions a 12, 24 and 48 h in esponse o simul aneous deli e y o IL-12p40 siRNA and LPS, and a 24 h o hose mice ecei ing he apeu ic IL-12p40 siRNA pos LPS adminis a ion. Cell su ace ma ke analysis o CD11b, CD14, CD40, CD80, CD86, F4/80 and MHC class II by J774A.1 cells was pe o med as p e iously desc ibed [52], con ol samples included cells incuba ed wi h iso ype ma ched, di ec ly conjuga ed, con ol an ibodies as app op ia e. Analysis o cy okine p oduc ion Cy okine p oduc ion om in i o expe imen s was assayed using comme cially a ailable immunoassays o mouse IL-12p70, IFN-γ, IFN-β, IL-10, and IL-4 (Pha min- gen). Mouse IL-12p40 in blood se um and pe i oneal la - age luid was assayed using mu ine IL-12p40 ELISA (R&D sys ems, Abingdon, UK) acco ding o he manu ac u e 's ins uc ions. S a is ical analysis One-way ANOVA was used o de e mine signi icance o cy okine p oduc ion be ween g oups; pos es analyses we e no pe o med. The s uden -Tes was used o de e - mine he signi icance o di e en luo escen in ensi ies ob ained by low cy ome y. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 4 o 12 (page numbe no o ci a ion pu poses) siRNA in e e ence o IL-12 ansc ip ion and ansla ion in i oFigu e 1 siRNA in e e ence o IL-12 ansc ip ion and ansla ion in i o. J744A.1 cells we e ans ec ed wi h IL-12p40 siRNA o di e - en pe iods (24–72 h). Fo he las 12 h o cul u e, cells we e s imula ed wi h LPS/IFN-γ o wi h PBS (-LPS, he ea e e med uns imula ed). Exp ession o IL-12p40 (A) was de e mined by sqRT-PCR. Samples we e no malized o β-ac in exp ession (lowe panel A). Con ol siRNA ans ec ions included naked siRNA o IL-12p40, mu an siRNA o IL-12p40 and GFP (B). IL- 12p70 p o ein exp ession was de e mined by ELISA (C). Da a a e ep esen a i e o a leas ou independen expe imen s; IL- 12p70 p o ein concen a ion is exp essed as he mean (+/-SEM) om iplica e cul u es (n = 3) on each occasion. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 5 o 12 (page numbe no o ci a ion pu poses) Resul s IL-12 p40 siRNA knocks down IL-12 exp ession in i o To in es iga e silencing o cy okine gene exp ession in i o, he mu ine mac ophage-like cell line J774A.1 was ansien ly ans ec ed wi h siRNA a ge ing IL-12p40 o he ime poin s shown in Fig. 1 (24, 48, 72 h). These cells we e s imula ed o he inal 12 h o each expe imen , wi h LPS and IFN-γ (LPS/IFN-γ), a p o ocol ha induces IL-12p70 [30]. T ans ec ion wi h IL-12p40 siRNA esul ed in a signi ican supp ession o p40 mRNA and a conse- quen loss o de ec able IL-12p70 in cell cul u e supe na- an (Fig. 1A,1B,1C). A educ ion in IL-12p40 mRNA was obse ed a 24 h, bu silencing was mo e p onounced a 48 h. T ans ec ion o 72 h wi h IL-12p40 siRNA was in e- io o ei he 24 o 48 h, as IL-12p40 mRNA exp ession and IL-12p70 p o ein syn hesis began o eco e by his ime (Fig. 1A &1C). Thus siRNA silencing was ansien in his sys em. Con ol siRNA ans ec ions included siRNA o IL-12p40 wi hou ans ec ion agen (naked siRNA), siRNA o IL-12p40 whe e he 10 h and 11 h bases we e e e sed (mu an siRNA), and siRNA a ge ing GFP, a p o- ein ha does no na u ally occu in J774A.1 cells. These con ol siRNAs did no induce IL-12p40 mRNA exp es- sion (Fig. 1B). Ou esul s show sequence-speci ic siRNA media ed inhibi ion o IL-12p40 mRNA syn hesis in i o a 48 h pos siRNA incuba ion (Fig. 1A). ELISA con i med he siRNA media ed silencing o IL-12p70 p o ein exp es- sion (Fig. 1C), e lec ing he signi ican inhibi ion o IL- 12p40 mRNA syn hesis (p < 0.001, compa ed o LPS/IFN- γ g oup). Supe na an s om uns imula ed cells, o cells incuba ed wi h con ol siRNAs, showed no IL-12p70 p o- ein p oduc ion. Supp ession o IL-12p70 was ansien , wi h le els eco e ing a he emaining ime poin s. mRNA exp ession p o iling o he in lamma o y cy okines IFN-β, IL-12p35, IL-23p19, IL-6, IL-10 and IFN- γ in IL-12p40 o con ol silenced cells, showed no non- speci ic siRNA silencing a he doses employed (Table 1). Con ol siRNA deli e ed by he same p o ocol did no induce mRNA o IFN-β, IL-12p35, IL-23p19, IL-6, IL-10 and IFN-γ. Likewise, cells ans ec ed wi h IL-12p40 siRNA showed no modula ion o he p o ein le els o IL-4, IL-5, IL-6, IL-10, and TNF-α ( esul s no shown). One cy okine did no ollow his pa e n. Al hough IL-12p40 siRNA ans ec ion o s imula ed mac ophages did no esul in a de ec able educ ion o IFN-γ mRNA (Table 1 and Fig. 2A), a educ ion o de ec able IFN-γ p o ein was obse ed (Fig. 2B). This disc epancy be ween IFN-γ mRNA and p o- ein de ec ion may e lec he ole o IL-12p40 in pos - ansc ip ional egula ion o IFN-γ sec e ion [31] and is consis en wi h he iming o IFN-γ p o ein syn hesis and sec e ion p e iously obse ed in LPS challenged IL-12p40- /- mice in i o [32]. Silencing IL-12p40 educes LPS/IFN- γ d i en mac ophage ac i a ion in i o To de e mine whe he silencing IL-12p40 had b oade e ec s on mac ophages, he exp ession o he ac i a ion/co- s imula o y ma ke s CD40, CD80, CD86, and MHC class II was examined ollowing simul aneous exposu e o J774 cells o LPS/IFN-γ and ei he con ol o IL-12p40-speci ic siRNA. Exp ession o CD14, a componen o he LPS ecog- ni ion machine y was also examined. LPS/IFN-γ s imula- ion alone (24 h) esul ed in inc eased CD40, CD86 and MHC class II exp ession (Table 2), bu had no e ec on CD80 o CD14 as expec ed. IL-12p40-speci ic siRNA did no ac i a e mac ophages in he absence o LPS/IFN-γ (Table 2). In he p esence o LPS/IFN-γ, siRNA a ge ing IL- 12p40 p e en ed inc eased exp ession o CD40, and CD86, sugges ing ha silencing IL-12 in e e ed wi h mac ophage ac i a ion. The exp ession o CD80, CD14 and MHC class II we e no a ec ed (Table 2). In con as , Mu .siRNA did no p e en CD86 up egula ion when cells we e s imula ed wi h LPS/IFN-γ bu a he esul ed in inc eased exp ession, sugges ing ha his sequence may con ibu e o mac o- phage ac i a ion no seen wi h IL-12p40 speci ic siRNA. The exp ession o he mac ophage pheno ypic ma ke s CD11b and F4/80 we e unchanged in all expe imen s, no signi ican di e ence was seen in le els o apop osis be ween g oups (da a no shown). Table 1: IL-12p40 siRNA speci ically silences mRNA o IL-12p40 and no o he cy okines. Ta ge Silencing by ea men a LPS/IFN-γ:- + + SiRNA: IL-12p40 Mu .siRNA IL-12p40 β-Ac in - - - IL-12p40 - - + IL-12p35 - - - IL-23p19 - - - IFN-γ--- IFN-β--- TNF-α--- IL-4 - - - IL-5 - - - IL-6 - - - IL-10 - - - aJ774 cells we e incuba ed wi h o wi hou LPS/IFN-γ and speci ic o con ol siRNA as desc ibed in he ma e ials and me hods sec ion. mRNA o di e en a ge s we e de ec ed by sqRT-PCR. In his able silencing (+) is de ined as he loss o a isible band om s imula ed cul u es; he - symbol indica es ei he no loss o a isible band om s imula ed (siRNA + LPS/IFN-γ) cul u es, o no isible al e a ion (induc ion o loss) in uns imula ed (siRNA – LPS/IFN-γ) cul u es. All esul s ep esen a leas wo expe imen s pe o med in iplica e. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 6 o 12 (page numbe no o ci a ion pu poses) Silencing IL-12p40 in luences IFN-γ p o ein exp ession.Figu e 2 Silencing IL-12p40 in luences IFN-γ p o ein exp ession. The in luence o silencing IL-12p40 on he exp ession o IFN-γ was de e mined using he condi ions desc ibed in Fig. 1, by sqRT-PCR o IFN-γ mRNA (A) o by ELISA o IFN-γ p o ein p esen in cul u e supe na an (B). Da a a e ep esen a i e o a leas ou independen expe imen s; IFN-γ p o ein concen a ion is exp essed as he mean (+/-SEM) om iplica e cul u es (n = 3) on each occasion. Le els o IFN-γ a e signi ican ly educed in he p esence o IL-12p40 siRNA o 24, 48, and 72 h (p < 0.0001) when compa ed o s imula ed non-silenced cul u es. Table 2: LPS/IFN-γ d i en mac ophage exp ession o CD40 and CD86 is modula ed by IL-12p40 siRNA. T ea men Mean Fluo escen In ensi y (+/-SEM) LPS/IFN-γsiRNA CD40 CD86 CD80 CD14 MHCII - - 22 (5) 9 (4) 34 (8) 44 (11) 19 (8) - IL-12p40 16 (9) 14 (1) 29 (2) 49 (4) 18 (8) + - 290 (27) 41(5) 102 (21) 86 (11) 46 (5) + IL-12p40 94 (17)* 7 (1)* 66 (13) 47 (9) 46 (5) + Mu .siRNA 241 (18) 72 (6) 102 (4) 86 (16) 43 (5) Da a a e he mean ± SEM o nea es whole numbe o MFI o 3 independen expe imen s, each pe o med in iplica e (n = 3), * s a is ical signi icance (α = 0.05) compa ed o cells s imula ed wi h LPS/IFN-γ in he absence o IL-12p40 siRNA. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 7 o 12 (page numbe no o ci a ion pu poses) siRNA a ge ing IL-12p40 speci ically educes LPS d i en in lamma ion in i o We in es iga ed he possibili y o silencing IL-12 by RNA in e e ence in i o, using a well-es ablished mu ine model o LPS d i en pe i oneal in lamma ion [33,34]. The deli e y o LPS i.p. esul ed in inc eased ac i a ed phagocy ic cells de ec able a 12, 24 and 48 h by la age, compa ed o con ols (Fig. 3A g oups I & II). This e ec was g ea ly educed in IL-12p40-/- mice. Simul aneous deli e y o a con ol i ele an siRNA (GFPsiRNA) o a mu an IL-12p40 siRNA (Mu .siRNA) duplex con aining wo misma ches o he IL-12p40 speci ic sequence had no in luence on LPS d i en in lamma ion. Likewise, a con ol siRNA ha was he an isense o he unc ional duplex (As.siRNA) did no esul in a signi ican educ ion in he le el o ac i a ed phagocy ic cells. Howe e , deli e y o IL- 12p40 siRNA d ama ically educed he le els o in lam- ma ion (Fig. 3A) a 12, 24 and 48 h. Deli e y o encapsu- la ed siRNA did no esul in inc eased in cell dea h o pe i oneal cells (apop osis o nec osis) compa ed o con- ols a he ime poin s selec ed (da a no shown). Con ol wild ype and IL-12p40-/- mice showed no in lam- ma o y esponse o siRNA ans ec ion eagen s alone (Fig. 3). LPS challenged wild ype mice, and mice co-challenged wi h con ol siRNAs displayed a ypical in lamma o y cell esponse in he pe i oneal ca i y wi h inc eased numbe s o ac i a ed phagocy ic cells, a 24 h (19.8%, 16.8 %, 22.01%) and 48 h (22.53%, 17.95% 16.64%) compa ed o con ol unchallenged mice (2.17% and 3.46%). Simila esul s we e obse ed in mice co-challenged wi h LPS and As.siRNA a 24 and 48 h (18.9% and 23.64% espec i ely). Howe e , mice co-adminis e ed LPS and speci ic IL-12p40 siRNA displayed educed numbe s o ac i a ed cells (6.3%), mi o ing he educed in lamma o y esponse seen in LPS challenged IL-12p40-/- mice (7.3%) a 24 h. How- e e , modula ion o he in lamma o y esponse in he LPS- IL-12p40 siRNA challenged mice was no pe manen . An inc ease in ac i a ed phagocy ic cells (11.10%) was seen a 48 h, al hough le els we e s ill lowe han he LPS chal- lenged BALB/c mice (22.53%), (Fig. 3A &3C). Thus, siRNA- media ed silencing o IL-12p40 mRNA in his model has a signi ican bu non-pe manen e ec on he abili y o medi- a e a esponse o LPS challenge in i o. siRNA a ge ing o he p oin lamma o y cy okine IL-12p40 can be used as a he apeu ic in e en ion agains in lamma ion d i en by mic obial p oduc s In o de o explo e he po en ial use o siRNA in a mo e he apeu ic con ex and based on he indings abo e, we deli e ed IL-12p40 siRNA by di ec injec ion in o he pe i oneal ca i y, 1 h pos LPS challenge. Adminis a ion o IL-12p40 siRNA pos LPS challenge (Fig. 3B I-VI) esul ed in a dec ease in he numbe o ac i a ed phago- cy ic cells, (4.22%) a 24 h, when compa ed o mice ecei ing LPS only (16.64%), con ol siRNAs (Mu .siRNA and GFPsiRNA) o As.siRNA, (25.03%, 17.07% and 12.63% espec i ely). These da a demons a e ha IL- 12p40 siRNA can be used he apeu ically o speci ically silence a cy okine-d i en in lamma o y esponse in i o, i deli e ed a a sui able momen . In pa allel expe imen s, he local and sys emic e ec s o siRNA media ed silencing we e assessed. IL-12p40 siRNA co-deli e ed wi h LPS o adminis e ed pos LPS insul , had bo h local and sys emic an i-in lamma o y e ec s (Fig. 4). Con ol BALB/c mice gi en siRNA ans ec ion eagen s alone, showed low le els o IL-12p40 p o ein exp ession in blood se um and pe i oneal la age samples (103 pg/ml and 75 pg/ml espec i ely). Howe e , mice challenged wi h LPS, o co-challenged wi h LPS and con ol siRNAs (GFP- siRNA, Mu .siRNA)(Fig. 4) showed signi ican inc eases in IL-12p40 p o ein de ec ed in bo h se um and la age com- pa ed o con ol (p < 0.05). Deli e y o As.siRNA did esul in educed se um IL-12p40 p o ein bu only when admin- is e ed he apeu ically (Fig. 4B). S ikingly, deli e y o IL- 12p40 siRNA deli e ed simul aneous o, o 1 h pos LPS adminis a ion, esul ed in a signi ican educ ion in he le els o IL-12p40 p o ein de ec ed in all se um and pe i o- neal la age samples compa ed o LPS alone (p < 0.05, in each case) (Fig. 4). Deli e y o nega i e con ol siRNAs showed no such educ ion. Ou indings demons a e ha well designed sequence speci ic siRNA can p o ide a signi - ican he apeu ic e ec and elici local and sys emic p o ec- ion agains in lamma ion. Discussion The abili y o e icien ly deli e small in e e ing RNA o modula e gene exp ession in i o may p o ide new he apeu ic app oaches o cu en ly in ac able diseases. We ini ially demons a e ha in i o IL-12p40 siRNA spe- ci ically silenced i s mRNA homologue leading o an- sien silencing o IL-12p40 p o ein and consequen knockdown o IL-12p70 exp ession. This app oach did no a ge o he p oin lamma o y cy okines (IL-6, IL-23, IL-10, TNF-α), o RNA-induced gene silencing, no did con ol siRNA induce hese cy okines o ype I in e e on a he concen a ions employed. Fu he mo e, we demon- s a e ha his app oach can be ex ended in i o by show- ing ha silencing o IL-12p40 esul s in he non- pe manen supp ession o IL-12 in a mu ine model o pe i oneal in lamma ion. Such silencing is e iden in he educed le els o IL-12 de ec able locally in pe i oneal la - age and sys emically in blood se um. Finally, we show ha siRNA can be used he apeu ically a e he ini ia ion o an in lamma o y esponse o knockdown IL-12 exp es- sion and o educe he obse ed in lamma o y in il a e seen in his model. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 8 o 12 (page numbe no o ci a ion pu poses) Silencing o IL-12 by siRNA in e e es wi h he in lamma o y esponse in i oFigu e 3 Silencing o IL-12 by siRNA in e e es wi h he in lamma o y esponse in i o. siRNA was deli e ed wi h LPS (A) o he apeu i- cally 1 h pos LPS s imula ion (B). Mice ecei ed ans ec ion eagen s only (no siRNA) (g oup I), LPS alone (g oup II) o we e co-injec ed wi h LPS and con ol siRNAs (g oup III-V, As.siRNA, GFPsiRNA, and Mu .siRNA espec i ely) o LPS and IL-12p40 speci ic siRNA (g oup VI). In lamma ion was cha ac e ized by low cy ome y o pe i oneal la age a 12, 24 and 48 h. The ypi- cal in lamma o y cell esponse in he pe i oneal ca i y is shown in he enclosed egion. Con ol IL-12p40-/-mice showed he cha ac e is ic ge m-line knockou esponse o LPS h oughou he expe imen (g oup VII). IL-12p40 siRNA was also deli e ed he apeu ically (B) 1 h pos LPS challenge, and in lamma ion measu ed a 24 h. Mice ecei ing con ol siRNAs (g oups III-V) dis- played a simila in lamma o y esponse o mice ecei ing LPS insul alone (g oup II). Mice ecei ing IL-12p40 siRNA (g oup VI) displayed a educed numbe o ac i a ed phagocy ic cells a he same ime poin (enclosed egion). Da a a e ep esen a i e o a leas h ee independen expe imen s (G oups I-VI) o wo expe imen s (G oup VII). In each expe imen , n = a leas 4 mice on each occasion. The absolu e numbe s o cells p esen in la age luid, ep esen ed by he enclosed egion in A, a e illus a ed (C). Da a in ba cha s ep esen he mean numbe o cells (+/-SEM)/ml la age luid om ou mice a he ime poin s indica ed. Jou nal o In lamma ion 2004, 1:4 h p://www.jou nal-in lamma ion.com/con en /1/1/4 Page 9 o 12 (page numbe no o ci a ion pu poses) siRNA silencing IL-12p40 educes local and sys emic le els o IL-12p40 p o einFigu e 4 siRNA silencing IL-12p40 educes local and sys emic le els o IL-12p40 p o ein. LPS deli e y o he pe i oneal ca i y wi h simul- aneous (whi e ba s), o he apeu ic adminis a ion (black ba s) o IL-12p40 speci ic siRNA educed he appea ance o IL- 12p40 p o ein in se um (A) and pe i oneal la age (B) as measu ed by IL-12p40 speci ic ELISA. Se um and la age we e sampled a 6 h pos LPS challenge. IL-12p40 obse ed in se um o la age om LPS challenged mice o mice co-challenged wi h LPS and ei he GFPsiRNA o Mu .siRNA was signi ican ly g ea e han con ol (p < 0.05, in bo h cases). IL-12p40 siRNA when deli - e ed simul aneously o he apeu ically signi ican ly educed IL-12p40 p o ein p oduc ion compa ed o LPS challenge alone (p < 0.05, in each case). Da a a e ep esen a i e o h ee independen expe imen s whe e n = 4 on each occasion; IL-12p40 p o ein concen a ion is exp essed as he mean (+/-SEM) om iplica e samples.