BioMed Cen al
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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.
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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-
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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.
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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.
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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.

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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.
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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.
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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.
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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.