Recognition of damage-associated molecular patterns related to nucleic acids during inflammation and vaccination.

1 MB·English

by  Jounai, Nao

REVIEWARTICLE
published:08January2013
CELLULAR AND INFECTION MICROBIOLOGY doi:10.3389/fcimb.2012.00168
Recognition of damage-associated molecular patterns
related to nucleic acids during inflammation and
vaccination
NaoJounai1,2,KoujiKobiyama1,2,FumihikoTakeshita1,2 andKenJ.Ishii1,2*
1LaboratoryofAdjuvantInnovation,NationalInstituteofBiomedicalInnovation,Osaka,Japan
2LaboratoryofVaccineScience,WPIImmunologyFrontierResearchCenter,OsakaUniversity,Osaka,Japan
Editedby: All mammalian cells are equipped with large numbers of sensors for protection
NelsonGekara,UmeaUniversity, from various sorts of invaders, who, in turn, are equipped with molecules containing
Sweden pathogen-associatedmolecularpatterns(PAMPs).Oncethesesensorsrecognizenon-self
Reviewedby: antigens containing PAMPs, various physiological responses including inflammation are
DarioS.Zamboni,Universidadede
induced to eliminate the pathogens. However, the host sometimes suffers from chronic
SãoPaulo,Brazil
WillemVanEden,Utrecht infection or continuous injuries, resulting in production of self-molecules containing
University,Netherlands damage-associated molecular patterns (DAMPs). DAMPs are also responsible for the
YanShi,UniversityofCalgary, eliminationofpathogens,butpromiscuousrecognitionofDAMPsthroughsensorsagainst
Canada
PAMPs has been reported. Accumulation of DAMPs leads to massive inflammation and
*Correspondence:
continuous production of DAMPs; that is, a vicious circle leading to the development of
KenJ.Ishii,LaboratoryofAdjuvant
Innovation,NationalInstitute autoimmunedisease.Fromavaccinologicalpointofview,theaccuraterecognitionofboth
ofBiomedicalInnovation, PAMPsand DAMPsis important for vaccine immunogenicity, because vaccine adjuvants
7-6-8Saito-Asagi,Ibaraki, are composed of several PAMPs and/or DAMPs, which are also associated with severe
Osaka567-0085,Japan.
adverseeventsaftervaccination.Here,wereviewastherolesofPAMPsandDAMPsupon
e-mail:[email protected]
infection with pathogens or inflammation, and the sensors responsible for recognizing
them, as well as their relationship with the development of autoimmune disease or the
immunogenicityofvaccines.
Keywords: PAMPs (pathogen-associatedmolecular patterns), DAMPs (damage-associatedmolecularpatterns),
nucleicacids,metabolites,innateimmunity,DNAsensors,uricacid,vaccineadjuvant
INTRODUCTION whereas continuous inflammatory responses owing to impaired
Host cells are equipped with numerous types of receptors to regulationofinflammatorysignalingresultsinchronicinflamma-
discriminateselffromnon-self.Whencellsareattackedbyinfec- torydiseaseorautoimmunedisease.Therefore,“bipolarsensors”
tiouspathogens,hostcellularreceptorssuchasToll-likereceptors for both PAMPs andDAMPs appear to be the mostly responsi-
(TLRs), nucleotide oligomerization domain (NOD)-like recep- blefordysregulatedinflammation.Here,wedescribethevarious
tors(NLRs),retinoicacid-induciblegene-I(RIG-I)-likereceptors types of DAMPs and their receptors, with a special focus on
(RLRs), C-type lectin receptors, andother non-classifiedrecep- nucleicacidsasDAMPs.
torsrecognizepathogen-associatedmolecularpatterns(PAMPs),
small molecular motifs conserved amongst microbes. Through LIPID-RELATEDDAMPs
the recognition of PAMP molecules, innate immune responses LIPOPOLYSACCHARIDE(LPS)
are induced, and inflammatory cytokines areproduced that aid A representative lipid for the induction of inflammatory
in the elimination of the pathogens. However, in some circum- responses is LPS, a PAMP present in gram-negative bacte-
stances host inflammatory responses can cause host cell death ria. Upon recognition by TLR4, LPS promotes the production
leading to tissue injury, and the release of host cellular com- of various inflammatory cytokines following bacterial infection
ponents to the extracellular environment. These cellular com- (Table1). However, Shi et al. reported that, TLR4 also rec-
ponents could be considered “messengers” for danger; they are ognizes endogenous fatty acids and can activate inflammatory
alsoknownas“damage-associatedmolecularpatterns”(DAMPs). responses in adipocytes and macrophages (Shi et al., 2006).
DAMPs include lipids, sugars, metabolites, and nucleic acids In addition, TLR4-deficient mice developed reduced inflamma-
such as RNA and DNA species. DAMPs are important for the tory cytokine production in response to a high fat diet (Shi
elimination of pathogens, but are also implicated in the devel- et al., 2006). Previous studies have revealed that saturated fatty
opment of autoimmune disease and chronic inflammatory dis- acids are released from hypertrophied adipocytes in the pres-
ease, and are used as adjuvants for vaccines. Interestingly, high enceofmacrophages,andthatreleasedfattyacidsaresensedby
numbersofPAMPreceptorsalsorecognizeendogenousDAMPs macrophages in a TLR4-dependent manner, following excessive
and can augment inflammatory responses against pathogens, production of inflammatory cytokines such as tumor necrosis
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Jounaietal. Immunesensorsfornucleicacids
Table1|AssociationofPAMPorDAMPsensorswithautoimmunediseases.
Receptor PAMP DAMP Autoimmunedisease
TLR1/TLR2 Lipopeptide SerumamyloidAprotein Atherosclerosis,rheumatoidarthritis,Crohn’sdisease
TLR4 LPS Fattyacid Obesity
Hyaluronicacid Rheumatoidarthritis,sarcoidosis,systemicsclerosis,
pancreaticcancer
NLRP3 Uricacid Uricacid Hyperuricemia,gout
ATP Unknown
RIG-I,MDA5,TLR7/8 VirusRNA ImmunocomplexofsnRNPs SLE
TLR9 BacterialDNA Self-DNA-containingimmune SLE
complexes,histone
RAGE −/? HMGB1 SLE
DAI,IFI16,AIM2,H2B, BacterialDNA,VirusDNA Self-DNA? SLE?
RNApolIII
factor(TNF)-α(Suganamietal.,2007).Becausetheproduction SUGAR-RELATEDDAMPs
of pro-inflammatory or inflammatory cytokines is dysregulated Hyaluronic acid (HA) is a non-sulfated linear polysaccharide,
in obese adipose tissues, obesity can be thought of as a chronic andamajorcomponentoftheextracellularmatrix.Weigeletal.
inflammatory disease caused by fatty acids acting as DAMP revealedthatHAisinducedanddegradedduringinflammatory
molecules(BergandScherer,2005). responsesandthatitfunctionsinimmunecellactivationornew
bloodvessel formation (Weigel et al.,1986). Interestingly, small
SERUMAMYLOIDAPROTEIN(SAA) molecular weight HA (sHA), produced by the degradation of
Some lipoproteins can also act as DAMP molecules. In 1982, HA during inflammation, can induce the maturation of den-
Hoffman and Benditt revealed that the treatment of mice with driticcells(DCs)forpathogenelimination(Termeeretal.,2002).
LPS of Salmonella typhosa increased SAA levels (Hoffman and Bonemarrow-derivedDCsfrommiceexpressingnon-functional
Benditt, 1982). According to several studies, SAA functions in TLR4 could not be activated by sHA, while DCs from TLR2-
cholesterol transport as well as inthe production of proinflam- deficient mice retained the ability for sHA-mediated activation.
matory cytokines, suggesting that SAA is a DAMP molecule This suggests that sHA can act as a DAMP molecule signaling
that responds to bacterial endotoxins (Banka et al., 1995; He throughTLR4toinduceDCmaturationuponpathogeninfection
et al., 2003). In support of this, increased levels of SAA may (Termeeretal.,2002).Consistentwiththis,excessive sHAlevels
be closely related to various diseases such as atherosclerosis, appearedtobecloselyassociatedwithinflammatoryautoimmune
rheumatoidarthritis,andCrohn’sdisease(Chambersetal.,1983, diseasessuchasrheumatoidarthritis,sarcoidosis,systemicsclero-
1987; Malle and De Beer, 1996). SAA binds to two recep- sis,andpancreaticcancer(Hallgrenetal.,1985;Witteretal.,1987;
tors, TLR4 and TLR2, which also recognize bacterial PAMP Sugaharaetal.,2006;Yoshizakietal.,2008)(Table1).
molecules such as triacyl lipopeptides (in cooperation with
TLR1), diacyl lipopeptides or lipoteichoic acids (together with METABOLITE-RELATEDDAMPs
TLR6) (Schwandner et al., 1999; Takeuchi et al., 2001, 2002; URICACID
Cheng et al., 2008; Hiratsuka et al., 2008) (Table1). Recently, Uricacidisametabolite ofpurinenucleotidesandfreebasesin
Loser et al. showed direct evidence for the local production humans and other primates, and it functions as an antioxidant
of the SAA molecules myeloid-related protein-8 (Mrp8) and toprotecterythrocytemembranesfromlipidoxidation(Kellogg
+
Mrp14, which induced autoreactive CD8 T cells and sys- andFridovich,1977).However,itwaspreviouslyshownthatsol-
temic autoimmunity through TLR4 signaling in mice (Loser ubleuricacid-inducedinflammatorycytokinessuchasmonocyte
et al., 2010). Taken together, these findings suggest that TLR4 chemoattractant protein-1 in rat vascular smooth muscle cells
may be a key receptor in the discrimination of lipid PAMPs (Kanellis et al., 2003). Shi et al. also reported that uric acid is
from lipid DAMPs molecules, because promiscuous recogni- producedinultraviolet-irradiatedBALB/c3T3cells,andactivates
tion of lipids via TLR4 unfortunately causes inflammatory dis- DCs(Shietal.,2003).Inaddition,highlevelsofuricacidinthe
ease. Although a consensus recognition structure for TLR4 has bloodareassociatedwiththedevelopmentofhyperuricemiaand
not yet been identified, antagonists of TLR4 signaling by lipid- gout(Johnsonetal.,2005),suggestingthatitactsasaDAMPdur-
DAMPs might be candidate drugs for the treatment of chronic ing cell injury and can induce inflammatory responses that are
inflammatorydisease. relatedtoautoinflammatorydiseasessuchasgout(Table1).
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Jounaietal. Immunesensorsfornucleicacids
Receptors that recognize uric acid have been reported and purinemetabolites.Nucleicacidsexistinallorganismsincluding
Liu-Bryan et al. revealed that TLR2, TLR4, and their adaptor pathogens, and function as a store of genetic information for
moleculeMyD88areimportantforuricacid-mediatedinflamma- protein translation and synthesis. Bacterial genomic DNA can
tion(Liu-Bryanetal.,2005).Incontrast,theuricacid-mediated be recognized as a PAMP, as it contains unmethylated CpG
activation of DCs was shown to be TLR4-independent, sug- motifs whose frequency is higher in genomic DNA derived
gesting the possible existence of other receptors that recognize from pathogens compared with that of vertebrates. The earli-
uric acid in addition to TLR2 and TLR4 (Shi et al., 2003). est research related to bacterial genomic DNA as PAMPs was
To solve this question, Martinon et al. demonstrated that uric reported more than hundred years ago. Bruns et al. inves-
acid could be sensed by another receptor, NOD-like recep- tigated heat-killed gram-negative or gram-positive bacteria as
tor family, pyrin domain-containing 3 (NLRP3), and induced an immunotherapeutic agent termed Coley’s toxin, for cancer
to produce interleukin (IL)-1β through caspase-1 activation (Swain, 1895). Although LPS is a major factor in mediating
(Martinon et al., 2006). NLRP3 is a member of the NLR fam- anti-tumoreffects,otherfactorsmaybeconnectedwithitsphysi-
ily, and a component of the inflammasome, a platform that ological function,as gram-positive bacteria donotexpress LPS.
inducesIL-1βandIL-18production.NLRP3sensesvarioustypes A hundred years on from the discovery of Coley’s toxin, sev-
of pathogen infections or irritants such as Candida albicans, eral studies have shown that bacterial DNA can activate nat-
Legionella pneumophila, Listeria monocytogenes, Malaria hemo- ural killer (NK) cells or B cells, suggesting that the bacterial
zoin, alum, silica, and asbestos as well as uric acid (Kanneganti genomic DNA in Coley’s toxin could contribute to its anti-
et al., 2006; Martinon et al., 2006; Dostert et al., 2008, tumor activity by stimulating NK cells (Shimada et al., 1986;
2009; Eisenbarth et al., 2008; Gross et al., 2009). Collectively, Messina et al., 1991). Krieg et al. further revealed that bac-
these results revealed that NLRP3 is a promiscuous receptor terial genomic DNA contains unmethylated CpG motifs that
that senses PAMPs and DAMPs and can induce inflammatory can stimulate B cells and NK cells, and induce inflammatory
responses. cytokine production. Interestingly, methylated bacterial DNA
failed to stimulate immune cells, indicating that unmethylated
ADENOSINETRIPHOSPHATE(ATP) CpG motifs may act as PAMP molecules (Krieg et al., 1995;
ATP is an essential purine base required for almost all physi- Klinman et al., 1996). However, whether genomic DNA con-
cal responses such as glucose metabolism, muscle contraction, taining methylated CpG motifs is incapable of innate immune
biosynthesis, and molecular transfer. However, extracellular activationremainscontroversial.In1962,Glasgowetal.reported
ATP from injured cells or non-apoptotic cells also serves thatultraviolet-inactivatedvacciniavirus,aDNAvirus,resulted
as a danger signal through the activation of NLRP3 and in IFN production in mouse cells (Glasgow and Habel, 1962).
caspase-1 (Communi et al., 2000). Previous detailed research In addition, Suzuki et al. showed that viral DNA, vertebrate
has shown the importance of other ion channel molecules, DNA and bacterial DNA induced the upregulation of major
namely, P2X7 and pannexin-1, in inducing extracellular ATP- histocompatibility complex (MHC) class I expression and the
mediated caspase-1 activation following IL-1β maturation type I IFN-related activation of transcription factors such as
(Ferrari et al., 2006; Kanneganti et al., 2007). The formation STAT3 in rat thyroid cells, suggesting that genomic DNA also
of the NLRP3 inflammasome requires an adaptor molecule, activates innateimmunesignalingin aCpG-motif-independent
apoptosis-associated speck-like protein containing a carboxy- manner (Suzuki et al., 1999). Interestingly, the structure of
terminalcaspaserecruitmentdomain(ASC).ASC-deficientmice DNAstronglyaffects DNA-mediatedinnateimmuneactivation.
cannot activate caspase-1 and thus do not produce mature Double-stranded, right-handed B-form DNA, but not the left-
IL-1β following exposure to large amounts of ATP, suggest- handed Z-form DNA, strongly induced type I IFN production.
ing that ATP-mediated IL-1β production is dependent on the Genomic DNA has a high content of B-form DNA, indicating
NLRP3 inflammasome (Mariathasan et al., 2004). However, that it may also function as a PAMP or DAMP (Ishii et al.,
althoughextracellularATPhasbeensuggestedtoactasaDAMP 2006). Mitochondrial DNA has been also reported to function
molecule, there is no correlation between high amounts of as a DAMP molecule. Zhang et al. reported that cellular injury
extracellular ATP acting as DAMPs in vitro and physiological causedthereleaseofmitochondrialDNA,andinducedsystemic
conditions in vivo. Eckle et al. suggested that most extracel- inflammatory responses via p38 MAPK activation in a TLR9-
lular ATP might be immediately hydrolyzed by ectonucleoti- dependent manner. In addition, trauma patients had higher
dases (Eckle et al., 2007). Taken together, investigation into amountsofmitochondrialDNAthandidhealthyvolunteers,sug-
the roles of extracellular ATP in inducing pathological and gesting that mitochondrial DNA could be considered a marker
immuneresponses invivo may provideimportant cluesregard- of inflammatory disease (Zhang et al., 2010). When the clear-
ingthemechanismunderlyinginflammationinductionbyDAMP ance of mitochondrial DNA by autophagy was inhibited, IL-1β
molecule recognition or in the development of inflammatory productionwasaugmentedviatheNLRP3inflammasometoacti-
diseases. vatecaspase-1,indicatingthattheamountofmitochondrialDNA
DAMPactivity isregulated byautophagyto suppresserroneous
NUCLEICACID-RELATEDDAMPs activationofinnateimmunity(Nakahiraetal.,2011).Indeed,it
UNMETHYLATEDCpGMOTIFANDGENOMICDNA was revealedthatautophagynegatively regulates RNA-mediated
As described above, uric acid and ATP are products of purine typeIIFNproduction,possiblytomaintaincellularhomeostasis
metabolism. Nucleic acids such as adenine or guanine are also (Jounaietal.,2007).
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Jounaietal. Immunesensorsfornucleicacids
CORRELATIONBETWEENAUTOIMMUNEDISEASEANDDNADAMPs TLRs
BothDNAandRNAcanfunctionasPAMPsandDAMPs,andare AlargebodyofresearchexistsdemonstratingtheTLR-mediated
closelyconnectedwithinflammatoryresponsesandthedevelop- sensing of nucleic acids. TLR3 preferentially senses double-
ment of inflammatory disease. Direct evidence for DNA acting strandedRNA(dsRNA)species,whichcanoriginatefromsome
as a DAMP was shown using DNase-deficient mice. DNase I is viruses,andTLR3isassociatedwithinductionofinnateimmu-
presentinextracellularcompartmentssuchastheseraandurine, nityinresponsetoinfectionwithWestNilevirus,respiratorysyn-
andfunctionstodegradesingle-strandedDNA(ssDNA),double- cytialvirus, andencephalomyocarditis virus (Wang et al.,2004;
strandedDNA(dsDNA),orchromatin,whicharereleasedfrom Groskreutz et al., 2006; Hardarson et al., 2007) (Figure2). In
damaged or necrotic cells. Napirei et al. constructed DNase I- addition,artificialdsRNA,poly(I:C),hasbeenwell-characterized
deficientmice,andreportedthattheypresentedwiththeclassical asaligandfor TLR3.Althoughpathogen-relateddsRNAsact as
symptomsofsystemiclupuserythematosus(SLE)andglomeru- PAMPs,Karikoetal.reportedthathostmessengerRNAcouldbe
lonephritis(Napireietal.,2000).Inaddition,DNaseIIdeficient sensedbyTLR3toinduceinflammatoryresponses(Karikoetal.,
mice showed a similar phenotype to DNase I knockout mice. 2004).RNAreleasedfromnecroticcellscanalsoelicittypeIIFN
DNaseII inthelysosomes of macrophages degrades DNAfrom production,suggestingthathostRNAmightfunctionasaDAMP
apoptotic cells or nuclear genome DNA from liver erythrob- uponcellularinjury(Karikoetal.,2004).
lasts.Interestingly,DNaseII-deficientmicepresentedwithlethal TLR7 and TLR8 recognize single-stranded RNA (ssRNA),
anemia owing to high levels of type I IFN production, caused andinduceanti-viralinnateimmuneresponsesagainstinfluenza
by the accumulation of non-degraded genomic DNA in liver virusorvesicularstomatitisvirus(Lundetal.,2004)(Figure2).
macrophages(Yoshidaetal.,2005). Insupportofthis,DNaseII Regardlessoftheircommonligands,thecellularandtissuedistri-
andIFNRa/bdoubleknockoutmiceshowedanon-lethalpheno- butionofTLR7expressionisincontrasttothatofTLR8.Human
type,butdevelopedrheumatoidarthritis-likesymptoms(Kawane TLR7ishighlyexpressedinplasmacytoidDCsthatpreferentially
et al., 2006), which could be attenuated by anti-TNF-α anti- inducetypeIIFNproduction,andisexpressedatlowerlevelsin
bodytreatment.Thissuggestedthattheaccumulationofgenomic myeloidcells.Conversely,thelevelofTLR8expressionishigherin
DNA in macrophages induced inflammatory cytokines, includ- monocytes andinmonocyte-derived DCsthanin plasmacytoid
ing type I IFNs and TNF-α, and the synergistic action of these DCs(Hornungetal.,2002).Furthermore,mouseTLR8didnot
inflammatorycytokinesresultedinlethalsystemicinflammation respond to ssRNA,but human TLR8 did, suggesting that TLR8
(Kawane et al., 2006). Furthermore, studies on DNase III, also might be inactivated in mice, although several papers have also
known as TREX1, also revealed that DNA could function as a linkedmouseTLR8withneuronalapoptosisandautoimmunity
DAMP.TREX1isthemajor3(cid:2) →5(cid:2) DNAexonucleaseforDNA (Heiletal.,2004;Gordenetal.,2006;Maetal.,2006).
editinginDNAreplicationorDNArepair.Moritaetal.showed In addition to the recognition of PAMPs, Vollmer et al.
that trex1-deficient mice had a reduced survival rate owing to revealed that promiscuous recognition through TLR7 or TLR8
high susceptibility to inflammatory myocarditis, although null causes the development of SLE with high levels of type I
mice showednospontaneous mutationsortumor development IFNs and TNF-α production (Vollmer et al., 2005). Because
(Moritaetal.,2004).Toexplainwhytrex1-deficientmicedevelop the sera from SLE patients contains high levels of autoantibod-
inflammatory myocarditis, Crow et al. demonstrated that the ies against self-antigens, such as small nuclear ribonucleopro-
mutationinthetrex1genethatabolishedTREX1enzymeactiv- tein particles (snRNPs) includingssRNA,TLR7, or TLR8 could
ity was responsible for the development of Aicardi-Goutieres recognize the immunocomplex of snRNPs with autoantibodies
syndrome (AGS), a severe neurological brain disease with high thorough Fc receptor-mediated internalization (Vollmer et al.,
levels of IFN-α in cerebrospinal fluid or serum, suggesting that 2005). Interestingly, TLR7 appears to be a specific sensor for
TREX1 is a suppressor of DNA DAMP-mediated inflammatory theinductionoftypeIIFNproductionfromplasmacytoidDCs,
responses (Crow et al., 2006). Furthermore, it was previously whereasTLR8isspecificforTNF-αproductionfrommonocytes
shown that the abolishment of interferon regulatory factor 3 inSLEpatients,suggestingthatplasmacytoidDCsandmonocytes
(IRF3) or IFN-α receptor 1 ameliorated the AGS symptoms in collaboratetodevelopinflammatoryresponsesinSLEviadistinct
trex1-deficientmice(Stetsonetal.,2008).Collectively,thesefind- sensors.
ings suggestthatthedysregulation ofself-DNAresults insevere TLR9 senses ssDNA containing unmethylated CpG motifs.
inflammatoryresponsessuchashighlevelsoftypeIIFNsleading Previous studies have revealed that TLR9 recognizes genomic
toautoinflammatorydisease. DNA from pathogens such as murine cytomegalovirus and
Herpes simplexvirustype1ortype2asPAMPs(Hemmi etal.,
2000; Lund et al., 2003; Krug et al., 2004a,b) (Figure2). With
NUCLEICACIDSENSORS regard to the development of autoinflammatory disease, TLR9
Host cells are equipped with numerous types of receptors to hasbeenalsobeenreportedtorecognizeself-antigenscomplexed
recognize nucleic acids as PAMPs or DAMPs. These recep- with autoantibodies. Leadbetter et al. revealed that autoreactive
tors function to protect the host from pathogen infection, but Bcellswereactivatedbyachromatin-autoantibodycomplexina
may also cause autoimmune disorders by inducing the con- TLR9-andMyD88-dependentmanner(Leadbetter etal.,2002).
stitutive activation of inflammatory responses (Figure1). In Inaddition,self-DNA-containingimmunecomplexes,whichare
this section, we introduce the well-characterized nucleic acid a well-characterized marker for SLE, were recognized by TLR9
sensors. throughFcγRIIA-mediatedinternalizationinplasmacytoidDCs
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Jounaietal. Immunesensorsfornucleicacids
FIGURE1|Autoimmunedisordersmaybeinducedbypromiscuoussensingofnucleicacids.
(Means et al., 2005). Thus, immune complexes containingself- mice developed attenuated lupus symptoms (Christensen et al.,
DNAmaysignalasDAMPsthroughTLR9,althoughextracellular 2006).Inaddition,arecentstudyrevealedthatTLR9suppressed
receptors such as FcγRIIA may be required for the delivery of the progression of autoinflammatory disease by antagonizing
autoimmunecomplexestotheTLR9-localizingcompartment. TLR7, suggesting that TLR9 counteracts TLR7 upon the recog-
As described previously, the subcellular localization of TLRs nition of self-immunocomplexes containing ssRNA or ssDNA
is important for the recognition of DNA, because TLR3, 7, 8 (Nickerson et al., 2010). To support the interaction between
and9localizeto theendosomal compartment. Previous studies TLR7andTLR9uponthedevelopmentofautoimmunedisease,
identifiedthreeadaptormolecules,Unc93B1,PRAT4A,andgp96, Fukuietal.generatedUnc93B1D34A/D34Aknock-inmicetoshow
whichareimportantforthetraffickingofTLRstositesforsens- that TLR9 competes with TLR7 for binding to Unc93B1 in the
ingtheirligands.Unc93B1functionstocontrolthetraffickingof healthystate,whileTLR7isconstitutivelyactivateduponautoin-
TLRs 3, 7, and 9 from the endoplasmic reticulum (ER) to the flammatory responses becauseTLR9hasalower affinityfor the
endosome. PRAT4A is localized in the ER and acts as a regula- Unc93B1-likeUnc93B1D34A/D34Amutant(Fukuietal.,2011).
torofthesubcellulardistributionofmostTLRsexceptforTLR3.
Gp96isamemberoftheheatshockprotein(HSP)90family,and RIG-I-LIKERECEPTORS(RLRs)
residesintheERwhereitcontrolsthematurationofTLRs2,4,5, Although TLRs can sense both non-self and self nucleic acids,
7,and9(SaitohandMiyake,2009).BecauseTLR7andTLR9are fibroblasts, and endothelial cells that do not express TLRs also
regulatedbythesamemolecularmachinery,thecrosstalkbetween producetypeIIFNsinresponsetoinfectionwithpathogens,indi-
TLR7 and TLR9 may affect the sensing of auto-nucleic acids cating the existence of other receptors that sense nucleic acids.
and the development of autoinflammatory disease. Christensen Yoneyamaetal.determinedthatacytoplasmicDExD/HboxRNA
et al. showed that a deficiency of TLR9 results in malignant helicase, RIG-I, senses infection by RNA viruses as well as arti-
symptomsinamousemodeloflupus,despitethelevelsofanti- ficial dsRNA, and induces innate antiviral immune responses
body production specific for DNA and chromatin being down- mediated by type I IFNs (Yoneyama et al., 2004) (Figure2). In
regulated (Christensen et al.,2005). In contrast, TLR7-deficient addition to RIG-I, melanoma differentiation factor-5 (MDA5)
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Jounaietal. Immunesensorsfornucleicacids
FIGURE2|Intracellularsensorsfornucleicacids.
and laboratory of genetics and physiology-2 (LGP2) were also (Satoetal.,2009;Nakashimaetal.,2010). Accompanying these
identified; these receptors were classified as RLRs because their observations, loss of function single nucleotide polymorphisms
protein structures were similar to that of RIG-I (Yoneyama have been found in RIG-I and IPS-1 that are closely related
et al., 2005). To induce an anti-pathogen immune response, a to the development of autoimmune disease (Pothlichet et al.,
CARDdomaininRIG-IandMDA5transmitsdown-streamsig- 2011),suggestingthatinhibitionofRLRsignalingmaybeimpor-
nals through homophilic interactions with the CARD adaptor tant in the progression of autoimmune disease. However, as
molecule, IFN-β promoter stimulator-1 (IPS-1, also known as describedearlier,excessiveproductionofinflammatorycytokines
MAVS,Cardif,orVISA)(Kawaietal.,2005;Meylanetal.,2005; includingtypeIIFNsappearstoresultinautoinflammatorydis-
Seth etal.,2005; Xuet al.,2005). Thefunction of LGP2 iscon- ease. In contrast, the dysfunction of RLRs induces poor type I
troversial. Some in vitro studies showed that LGP2 negatively IFN production, but leads to autoimmune disease (Nakashima
regulatesRIG-I-orMDA5-mediatedinnateimmuneresponsesby et al., 2010; Pothlichet et al., 2011). One possibility to explain
competingforbindingwiththeirRNAligands(Yoneyamaetal., this phenomenon is that non-functional RLRs result in an
2005; Bamming and Horvath, 2009). However, in vivo studies increased susceptibility against various types of virus infec-
usinglgp2-deficientmicerevealedthatLGP2isacofactorofRLR- tions, and the subsequent virus-mediated cell death may cause
mediated innate immune signaling (Venkataraman et al., 2007; the release of DAMPs and signaling through DAMP receptors.
Satohetal.,2010). Support this possibility, the loss of MDA5 function increased
RLRssensepathogen-derivedRNAspeciesasPAMPstoinduce the susceptibility of beta cells to viral infection with picor-
type I IFN production, while MDA5 has been detected as an navirus or encephalomyocarditis virus-D, and resulted in type
autoantigen in clinically amyopathic dermatomyositis patients 1 diabetes, whose types of diabetes are often caused by virus
(Sato et al., 2009; Nakashima et al., 2010). Although it is infection or autoimmunity (Colli et al.,2010; McCartney et al.,
not clear how extracellular MDA5 is produced, the accumula- 2011). Further analyses are required to elucidate the cross-talk
tion of immunocomplexes containing MDA5 is a marker for between RLR signaling and the development of autoimmune
the frequency of rapidly progressive interstitial lung disease disease.
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Jounaietal. Immunesensorsfornucleicacids
ABSENTINMELANOMA2(AIM2)-LIKERECEPTORS(ALRs) (Rozzoetal.,2001). Interestingly, p202 levels are varied among
AlthoughvariousNLRfamilymembersthatcaninducetheacti- mousespecies,whileAIM2isexpressedatthesamelevel,indicat-
vation of caspase-1 and maturation of IL-1β, IL-18, and IL-33 ingthatp202expressionistightlycorrelatedtoSLEdevelopment.
in response to a wide range of PAMP and DAMP molecules Furthermore, Ravichandran et al. revealed that ablation of the
havebeenidentified,nosensorofintracellulardsDNAforIL-1β aim2geneleadstohigherexpressionofp202andtypeIIFNsin
maturation has been identified. However, four research groups mice, andaim2-deficientmice areproneto SLE (Panchanathan
concurrently reported a role for the novel intracellular DNA et al., 2010). Taken together, these findings suggest that mouse
sensor,AIM2,intheactivationofcaspase-1followingIL-1βpro- p202mightbehomologous tohumanIFI16. Insupportofthis,
duction (Burckstummer et al., 2009; Fernandes-Alnemri et al., expression levels of IFI16 and anti-IFI16 autoantibodies were
2009; Hornungetal.,2009; Roberts etal.,2009). AIM2belongs dramaticallyincreasedinSLEpatients,indicatingthatIFI16has
to a family of hematopoietic interferon-inducible nuclear pro- similarfeaturestop202(Mondinietal.,2006).
teins with a 200-amino acid repeat (HIN-200), known as the A recent article described a correlation between psoriasis
p200orPYHINfamily.Currently,fourHIN-200familymolecules symptoms and AIM2 activation. Psoriasis is a chronic autoin-
havebeenidentifiedinhumans,andsixinmice.HIN-200fam- flammatorydiseasecausedbyincreasedIL-1βproductionleading
ilymolecules sharesimilarstructural features,includingapyrin to Th17 cell maturation (Ghoreschi et al., 2010). Dombrowski
domain at the NH terminus, and a HIN-200 domain at the et al. observed increased levels of cytosolic DNA fragments in
2
COOHterminus.SimilartotheroleofNLRP3inIL-1βproduc- skin lesions from psoriatic patients, which could be sensed by
tion, AIM2 causes oligomerization of the inflammasome upon AIM2(Dombrowskietal.,2011).Interestingly,thoseDNAfrag-
DNAbinding.TheAIM2inflammasomerecruitsASC,anessen- ments, which might be released from skin lesions in psoriatic
tialadaptormolecule,andinducesNLRP3inflammasomeforma- patients,wereinternalizedthroughbindingtotheantimicrobial
tionthroughhomophilicinteractionsbetweenthepyrindomain peptide LL-37(Dombrowski etal.,2011). Previous studies have
inAIM2andthatinASC(Figure2).TheimportanceoftheAIM2 shownthatthecomplexofself-DNAwithLL-37canactivateplas-
inflammasome upon PAMP recognition has been confirmed by macytoid DCs to produce type I IFNs, and complex-mediated
infectionexperimentsusingaim2-deficientmacrophagesinfected type I IFN production is closely related with skin lesion devel-
withFrancisella tularensis,L.monocytogenes,vacciniavirus,her- opmentinpsoriasis(Nestleetal.,2005;Landeetal.,2007).AIM2
pes simplex virus-1 and mouse cytomegalovirus (Fernandes- isaninterferon-induciblegene,suggestingthatLL-37complexes
Alnemrietal.,2010;Rathinametal.,2010). withself-DNAactivateplasmacytoidDCstoproducetypeIIFNs,
A second ALR, interferon-inducible protein 16 (IFI16) in and that the subsequent upregulation of AIM2 leads to IL-1β
humans (a homologue of p204 in mice), has been also investi- production, and finally, psoriatic skin lesions occur because of
gated as an intracellular dsDNA sensor. However, while AIM2 the increased levels of type I IFN production as well as IL-1β
induces IL-1β production in response to intracellular dsDNA production.
binding, IFI16 is a sensor for type I IFN production upon
recognition of intracellular dsDNA (Unterholzner et al., 2010). HIGHMOBILITYGROUPBOX1(HMGB1)
Although IFI16 also contains a pyrin domain, the pyrin in HMGB1 has been reported to be a major DAMP molecule.
IFI16 is quite distinct from that in AIM2 as it has a lower Goodwin et al. first identified HMGB1 from calf thymus chro-
affinity for ASC. Consistent with these different features of matin as a non-histone DNA-binding protein (Goodwin et al.,
pyrin,IFI16-mediated typeIIFNproductionuponintracellular 1973).However,Wangetal.showedthatamousemacrophagecell
dsDNA stimulation was not affected by ASC deficiency, sug- linereleasedHMGB1inresponsetoLPSstimulation.Inaddition,
gesting that the two HIN-200 family molecules regulate both LPS-treated mice developed increased serum levels of HMGB1,
IL-1βandtypeIIFNproductionupontherecognitionofintra- similartohumanpatientswithsepsis,suggestingthatHMGB1isa
cellular dsDNA (Unterholzner et al., 2010). Although AIM2- DAMPmoleculeinregardtosepsissymptoms(Wangetal.,1999).
mediated signaling appears to be distinct from IFI16-mediated Accumulating evidence suggests that cellular injury results in
type I IFN production, recent research has revealed that IFI16 thereleaseofHMGB1leadingtoinflammation(Abrahametal.,
negatively regulates the AIM2-mediated activation of caspase-1 2000; Scaffidi et al., 2002). Consistent with these observations,
(Veerankietal.,2011).Asincreasedinflammatorycytokinepro- numerous studies have showed a correlation between HMGB1
duction is closely related to the development of autoinflam- and autoimmune/inflammatory diseases such as atherosclero-
matory disease, the regulation between AIM2-mediated innate sis, diabetes, SLE, rheumatoid arthritis and Sjögren syndrome
immune signaling and IFI16 might be deregulated in patients (Taniguchi et al., 2003; Porto et al., 2006; Urbonaviciute et al.,
withautoimmunedisease. 2008;Devarajetal.,2009).
Roberts et al. identified p202 and AIM2 as cytosolic DNA Asdescribedpreviously,higherserumlevelsofimmunocom-
bindingproteinsinmice.p202isanotherALRmoleculewithout plexes of self-DNA with autoantibodies is a hallmark of SLE.
apyrindomain,indicatinganinabilitytobindASCforinflam- Previous research has shown that HMGB1 is also contained in
masome formation (Roberts et al., 2009). p202 appears to be a immunocomplexesandcanelicitinflammatorycytokineproduc-
negativeregulatorforAIM2-mediatedsignaling,asthereduction tion, suggesting thatHMGB1may bea carrier of DNADAMPs
ofp202resultsinhigherAIM2-mediatedactivationofcaspase-1 (Tian et al., 2007; Urbonaviciute et al., 2008). Furthermore,
in response to intracellular DNA. However, elevated levels of HMGB1 appears to promiscuously bind numerous molecules
p202 have been reported to induce SLE-like symptoms in mice such as LPS, IFN-γ, IL-1β, and CXCL12 to induce synergistic
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Jounaietal. Immunesensorsfornucleicacids
physiological responses (Sha et al., 2008; Youn et al., 2008; dsDNA.PreviousreportsshowedthathistonesactasDAMPs,and
Campanaet al.,2009). Moreover, HMGB1 can sensepathogen- that excessive intracellular dsDNA induces type I IFNs through
derivednucleicacids,whichinducetypeIIFNproduction(Yanai H2B(Kobiyamaetal.,2010).Inconfirmationofthis,H1orH2
et al., 2009). Collectively, HMGB1 might be a promiscuous arereleasedfromthenucleusafterDNAdamage,andaretranslo-
carrier that enhances innate immune responses against PAMPs cated to mitochondria following the induction of apoptosis. In
andDAMPs. addition, H1, H2A, and H2B may act as antimicrobial proteins
The receptors for HMGB1 have been investigated, but are incertainanimals,suggestingthatH2BisanintracellulardsDNA
still controversial. A well-studied receptor for HMGB1 is the sensor thatrecognizes dsDNAPAMPs andDAMPs (Kawashima
receptor for advanced glycation end products (RAGE). Similar et al., 2011). Histones may be related to autoimmune diseases
to HMGB1, RAGE is a promiscuous receptor that can bind to as anti-histone antibodies were detected in patients with such
various ligands including DNA, RNA, SAA protein, HSPs and diseases.Furtheranalysesarerequiredtoclarifytherelationship
prionprotein,suggestingthatRAGEmaysenseavarietyofDAMP betweenhistonesandautoimmunedisease.
molecules in an HMGB1-dependent or -independent manner
(Simsetal.,2010).Experimentswithrage-deficientmicerevealed Ku70
that HMGB1-mediated DNA sensing requires RAGE for inter- Ku70 functions in DNA repair, V(D)J recombination and in
nalization of DNA complexes to produce type I IFNs via TLR9 retaining the telomere. Zhang et al. showed that various DNA
(Tian et al., 2007). Interestingly, RAGE could associate with species-induced the production of type III interferon, IFN-λ1,
TLR9uponrecognitionoftheAtypeofCpG-HMGB1complex, andidentified Ku70 as a novel DNAsensor by pull-down assay
indicating a possible function for RAGE as a bridge molecule from the nucleus compartment (Zhang et al., 2011a). While
between theextracellular HMGB1-DNAcomplex andtheTLR9 other DNA sensors are important for the production of type I
compartment (Tian et al., 2007). In contrast to this observa- IFNs,Ku70appearstobeimportantfortypeIIIIFNproduction
tion,nucleosomescouldsenseHMGB1complexesindependently through IRF1 and IRF7. Furthermore, Ku70-mediated type III
of RAGE. Instead of RAGE, TLR2 appears to be important for IFNproductionisrestrictedwhenthelengthofintracellularDNA
the recognition of HMGB1-nucleosome complexes, suggesting stimuliisgreaterthan500basepairs.
that the sensing machinery of the HMGB1-nucleosome com-
plex mightbedistinct fromthatof theHMGB1-DNAcomplex, RNAPOLYMERASEIII
as the HMGB1-nucleosome complex could not elicit produc- As described above, RIG-I senses intracellularRNAspecies, but
tion of type I IFNs even though TNF-α or IL-10 were induced may also recognize intracellular dsDNA. siRNA treatment of a
(Urbonaviciuteetal.,2008).Furthermore,recentresearchidenti- human hepatoma cell line, Huh7, suppressed dsDNA-mediated
fiedanovelligandforRAGE,complementC3a,thatbindshuman type I IFN production. Subsequently, Chiu et al. showed that
stimulatory CpG DNA to induce type I IFNs in an HMGB1- RIG-IsensesthetranscribedRNAbyproductsofDNAtemplates
independent manner. This suggests that RAGE-mediated DNA that are generated by RNA polymerase III (as is the case for
sensing may involve numerous ligands (Ruan et al., 2010). poly(dA·dT)·poly(dT·dA)andEBVgenomicDNA)andinduces
Although there are many varieties of HMGB1- or RAGE- production of type I IFNs (Chiu et al., 2009). An inhibitor of
mediated DNArecognition, both molecules arestrongly associ- RNApolymeraseIII suppressedDNA-mediatedtypeIIFNpro-
atedwiththeinductionofinflammationandthedevelopmentof duction, suggesting that RNA polymerase III is a distinct DNA
chronicinflammatorydisease. sensor.However, RNApolymeraseIII-mediated dsDNAsensing
isrestricted tosequences ofDNAstimulicontainingless dA·dT
DNA-DEPENDENTACTIVATOROFIFN-REGULATORYFACTORS(DAI) thandG·dC.
DAI has been identified as a molecule that recognizes intra-
cellular DNA. Previous studies have revealed that DAI senses DHX9ANDDHX36
Z-type DNA; however, it may also bind to B-type DNA and AlthoughtheDExD/Hbox RNAhelicasefamilycontainsRIG-I
induce type I IFN production through associations with TBK1 andMDA5,whichfunctionasRNAsensors,recentreportshave
andIRF3(Takaokaetal.,2007).Interestingly,DAI-deficientmice revealedasimilarRNAhelicasefamilyofmolecules(DExDcfam-
respondednormallytocytosolicdsDNAstimulation,suggesting ily) that contain DHX9 and DHX36, which function as ssDNA
thatDAImayfunctionasoneofanumberofDNAsensorsina sensors in plasmacytoid DCs (Kim et al., 2010). Interestingly,
celltype-specificfashion(Ishiietal.,2008).Currently,thefunc- whileDHX36sensesCpG-A,DHX9sensesCpG-BinaMyD88-
tionofDAIiscontroversial,althoughthegeneticadjuvanticityof dependent manner. This may suggest that ssDNA PAMPs or
DAIhasbeenshowntoinducestrongcytotoxicTcellresponses DAMPs are recognized by either DHX9 or DHX36, but recent
(Lladser et al., 2011). Although the ability of DAI to recognize researchhasshownthatDHX9collaborateswithIPS-1torecog-
DNADAMPshasnotbeendeterminedyet,DAImightbealink nizedsRNAinmyeloidDCs,indicatingthepromiscuoussensing
betweenthedevelopmentofautoimmunediseaseandhostDNA ofDHX9(Zhangetal.,2011b).
immunecomplexes.
LEUCINE-RICHREPEATFLIGHTLESS-INTERACTINGPROTEIN1(Lrrfip1)
HISTONES Some sensor molecules such as TLRs or NLRs share common
HistoneH2B(H2B)isacomponentofchromatin,andKobiyama molecularpatterns,suchasleucinerichrepeats(LRRs),whichare
et al. identified that H2B also functions to sense intracellular importantforligandrecognitionorprotein–proteininteractions.
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Jounaietal. Immunesensorsfornucleicacids
AnLRR-containingmolecule,Lrrfip1,hasbeenreportedtosense antigen-presentingcells aswell asantigen-specificantibody and
intracellular DNA or RNA (Yang et al., 2010). Interestingly, cytotoxic T cell responses. This suggests that self-DNA DAMPs
whereas other DNA sensors often regulate type I IFN-related can activate innate immune responses that induce acquired
transcriptionfactorssuchasIRF3/7orcaspase-1toinducemat- immunoresponses. Recently, Marichal et al. demonstrated that
uration of IL-1β, Lrrfip1 stimulates β-cateninand CBP/p300 to the adjuvanticity of alum was dependent on self-DNA released
enhanceifnb1transcription,indicatinganovelpathwayinvolving from cells at the alum inoculation site (Marichal et al., 2011).
β-cateninfortypeIIFNproductionuponcytosolicDNAsensing. NLRP3 appears to be a key sensor in the induction of alum-
Because Wnt/β-catenin signaling is also linked to tumor devel- mediatedinnateimmunity,althoughitsfunctionisonlypartially
opment,furtheranalysesmayidentifythemachineryinvolvedin dependent upon alum adjuvanticity. Intraperitoneal inocula-
the regulation of type I IFN signaling by Lrrfip1 under tumor tion ofmice with aluminducedtherecruitment of neutrophils,
development. and the resulting alum deposits contained high amounts of
genomicDNA.BecausetreatmentwithDNaseIattenuatedalum
STING(STIMULATOROFINTERFERONGENESPROTEIN) adjuvanticity, the alum-mediated release of genomic DNA may
The major function of MHC class II is antigen presentation, account for its potent adjuvanticity. In addition, the alum-
whilemonoclonalantibodiesagainstMHCclassIIcancausecell mediated induction of antibody production is dependent on
activation or apoptotic cell death. Jin et al. identified a novel TBK1andIRF3asdemonstratedusingknockoutmice, suggest-
tetraspanin family molecule, MPYS, associated with MHC-II- ingthatalum-mediatedgenomicDNAinduceshighadjuvantic-
mediated cell death (Jin et al., 2008). Three research groups ity of alum via the TBK1/IRF3 pathway, while alum-mediated
performing cDNA library screening to identify molecules asso- uric acid production is less related to alum adjuvanticity via
ciated with activation of the typeI IFN promoter identifiedthe NLRP3 (Marichal et al., 2011). Furthermore, self-DNAs from
samemolecule,STING(alsoknownasMITA,andERIS).STING alum inoculation can activate inflammatory monocytes, and
isanoveladaptormoleculethatactivatesinnateimmunesignal- homodimers of IL-12p40 are more important than type I IFN
ingmediated byintracellularnucleicacidstimuli (Ishikawaand productionuponalumadjuvanticity.Takentogether,thesefind-
Barber,2008; Zhonget al.,2008; Sunetal.,2009). Surprisingly, ings suggest that self-DNA DAMPs are important for pathogen
theBarberresearchgroupfurtherrevealedthatSTINGisessen- elimination, the development of autoimmune disease and the
tialfortheinductionoftypeIIFNproductionfollowingsensing adjuvanticity of alum. Further analyses are required to elu-
ofcytosolicdsDNA,usingSTING-deficientmice.Basedontheir cidate which types of cells produce self-genomic DNA after
imaginganalysis,STINGappearstolocalizetotheERduringthe adjuvant inoculation, and which sensors recognize extracellular
steadystate,buttranslocatestotheGolgiapparatusuponintra- genomicDNAs.
cellular dsDNA stimulation to activate down-stream molecules In addition to alum adjuvant, there are many licensed adju-
such as TBK1.This suggests thatSTING is an essential adaptor vants such as MF59®, AS03®, and AS04®. Both MF59® and
molecule for cytosolic dsDNA-mediated type I IFN production AS03®areemulsionsofoil/watercontainingsqualene.Although
inmice. bothadjuvantselicitantibodyresponsesaswellascell-mediated
Cyclic-di-GMPandc-di-AMParesmallmoleculesthatfunc- immuneresponsesspecificforantigens,theirmodeofactionhas
tionassecondmessengersandareimportantforcellsurvival,dif- notbeenidentified.Informationonthereceptorsforandsignal-
ferentiation,colonization,andbiofilmformation.Recentresearch ing induced by these adjuvants is needed, because unfortunate
has revealed that the cytosolic delivery of c-di-GMP or c-di- sideeffectscanbeexpectedmoreeasily.
AMP-induced type I interferon (IFN) production from bone
marrowmacrophages,suggesting thatc-di-GMP andc-di-AMP CONCLUDINGREMARKS
arebacterialPAMPmolecules(McWhirteretal.,2009;Woodward Many sorts of nucleic acid species exist in the environ-
et al., 2010). As type I IFN production by c-di-GMP or c-di- ment. These species affect all organisms such as the evolu-
AMPrequirestheirinternalization,liveinvasivebacteriapossibly tion of organisms, the inflammatory response, and the advent
produce these second messenger molecules after internalization of drug-resistant microorganisms. To prevent pathogen infec-
intocells. tion, mammalian cells have equipped themselves with many
Recentreports haverevealedthatSTINGisadirectsensorof sorts of sensors to recognize exogenous nucleic acid species as
bacterialsecondmessengermolecules,suchasc-di-GMPorc-di- PAMPs, while those sensors are also stimulated by endogenous
AMP (Burdette et al., 2011; Jin et al., 2011). This indicates the nucleic acids species as DAMPs. Dysfunction of the machiner-
novelpossibilitythatcytosolicdsDNAstimulationmightproduce ies sensing both PAMPs and DAMPs is strongly associated
c-di-GMP/c-di-AMPorrelatedmoleculesthatcanbesensedby with chronic inflammatory disease or autoimmunity. In addi-
STINGandinducetypeIIFNproduction. tion, both PAMPs and DAMPs underlie the action of vaccines,
because most modern vaccines contain adjuvants, which are
ADJUVANTICITYTHROUGHDNADAMPs composed of both PAMP- and DAMP-associated molecules.
Although DNA DAMPs are closely associated with the devel- Therefore, the machinery responsible for sensing nucleic acids
opment of autoimmune disease, DNA DAMPs also contribute species should be further elucidated to help us understand
to the activation of acquired immune responses following vac- machineryofchronicinfection,autoimmunedevelopment,iden-
cination with alum adjuvant. Previous studies have shown that tifying the side effects of vaccines, and developing safe vaccine
genomic DNA from dying cells induces the maturation of adjuvants.
FrontiersinCellularandInfectionMicrobiology www.frontiersin.org January2013|Volume2|Article168|9
Jounaietal. Immunesensorsfornucleicacids
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