Nucleic Acid Recognition by the Innate Immune System

IY29CH08-Barton ARI 4 February 2011 16:27

Nucleic Acid Recognition bythe Innate Immune SystemRoman Barbalat,∗ Sarah E. Ewald,∗

Maria L. Mouchess,∗ and Gregory M. BartonDivision of Immunology & Pathogenesis, Department of Molecular and Cell Biology,University of California, Berkeley, California 94720-3200; email: [email protected]

Annu. Rev. Immunol. 2011. 29:185–214

First published online as a Review in Advance onJanuary 5, 2011

The Annual Review of Immunology is online atimmunol.annualreviews.org

This article’s doi:10.1146/annurev-immunol-031210-101340

Copyright c© 2011 by Annual Reviews.All rights reserved

0732-0582/11/0423-0185$20.00

∗Order of these authors is alphabetical.

Keywords

pattern-recognition receptors, Toll-like receptors, RIG-I-likereceptors, autoimmunity, virus

Abstract

Receptors of the innate immune system recognize conserved micro-bial features and provide key signals that initiate immune responses.Multiple transmembrane and cytosolic receptors have evolved to rec-ognize RNA and DNA, including members of the Toll-like receptorand RIG-I-like receptor families and several DNA sensors. This strat-egy enables recognition of a broad range of pathogens; however, in somecases, this benefit is weighed against the cost of potential self recogni-tion. Recognition of self nucleic acids by the innate immune systemcontributes to the pathology associated with several autoimmune orautoinflammatory diseases. In this review, we highlight our current un-derstanding of nucleic acid sensing by innate immune receptors anddiscuss the regulatory mechanisms that normally prevent inappropriateresponses to self.

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PRR: pattern-recognition receptor

TLR: Toll-likereceptor

LRR: leucine-richrepeat

INTRODUCTION

Nucleic Acids as a Signatureof Microbial Infection

Over the past 10 years, our understanding ofhow infectious microbes are initially recog-nized by the innate immune system has grownconsiderably (1, 2). The general theme that hasemerged from this effort is that the innate im-mune system utilizes receptors with fixed speci-ficities, often referred to as pattern-recognitionreceptors (PRRs), to recognize conserved fea-tures of microbes (3). In principle, this strategyallows a finite set of receptors to recognizean enormous diversity of potential pathogens.For the most part, the microbial featuresrecognized by innate receptors are commonto broad classes of microbes and representmolecules that are unequivocally foreign to thehost. One clear exception to this generalizationis the recognition of nucleic acids by innatereceptors. Because nucleic acids are commonto pathogen and host, these receptors have thepotential to respond to self ligands. Nonethe-less, this strategy is used by multiple families ofinnate immune receptors, and the recognitionof nucleic acids has been linked to severaldifferent signaling pathways with distinctoutcomes for the host response to infection.

The risk of self recognition by innate im-mune receptors makes the use of nucleic acidsto detect potential pathogens a curious strat-egy with potentially adverse consequences forthe host. Recognition of self nucleic acids byinnate immune receptors has been linked toseveral autoimmune and autoinflammatory dis-orders. The potentially negative consequencesare presumably outweighed by the benefit ofreliable microbial recognition. Innate immunedetection of viruses, in particular, appears torely almost exclusively on recognition of vi-ral nucleic acids. Indeed, nucleic acids may beone of the few viral features suitable for innateimmune recognition; that is, nucleic acids areshared by all viruses and not easily mutated toavoid recognition. Nucleic acid ligands of otherpathogen classes can also be recognized byinnate receptors.

In this review, we discuss the functions ofthe known innate receptors involved in nucleicacid recognition and the role played by eachin the host response to infection. In addition,we discuss the regulation of these recognitionsystems, with particular focus on how the hostavoids recognition of self nucleic acids.

Innate Receptors Involvedin Nucleic Acid Recognition

PRRs involved in nucleic acid recognitioncan be broadly divided into two groups basedon cellular localization. Several members ofthe Toll-like receptor (TLR) family monitorthe contents of endolysosomal compartments,whereas a collection of cytosolic recep-tors detects nucleic acids in the cytoplasm.Collectively, the receptors in each cellularcompartment can recognize multiple forms ofnucleic acid: single-stranded (ss) and double-stranded (ds) DNA and RNA. In some of thesecases, the precise molecular features targetedby the innate immune system are still beingdefined (see sections below). Nevertheless,receptors with specificity for different formsof genetic material exist within the transmem-brane and cytosolic receptor families. Thisstrategy enables the recognition of essentiallyany pathogen, provided pathogen-derived nu-cleic acids are accessible. Conceptually, thesetwo groups are often viewed as parallel systemsdiffering only in their compartments, althoughthere is now considerable evidence that expres-sion, signaling, and regulation of the two groupsare quite distinct. We first introduce the mainreceptors within these groups and discuss theirgeneral function within the innate immunesystem. A more detailed discussion of specificreceptors, their regulation, and the conceptualand functional differences between receptorfamilies follows in other sections of this review.

The TLRs are a family of transmembranereceptors (10 in human, 12 in mouse) consist-ing of an extracellular domain with multipleleucine-rich repeats (LRRs) linked by a trans-membrane domain to a conserved cytosolic sig-naling domain called the Toll/IL-1 receptor

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DC: dendritic cell

Plasmacytoid DC(pDC): a subset ofdendritic cellscharacterized by theability to produce largeamounts of type I IFNin response to TLR7and TLR9 ligands

Type I interferons(IFNs): a family ofgenes encodingcytokines with potentantiviral properties.The type I IFNsconsist of multiple Ifnagenes and a single Ifnbgene

RLR: RIG-I-likereceptor

homology (TIR) domain (1). Four TLR familymembers have been implicated in nucleic acidrecognition: TLR3, TLR7, TLR8, and TLR9.These receptors are localized to endosomes andlysosomes, where they monitor the lumen ofthese compartments for nucleic acids derivedfrom or associated with internalized cargo. Asdiscussed in greater detail below, this localiza-tion appears to be a key aspect of regulatingself/nonself discrimination by these receptors.

The TLRs involved in nucleic acid recog-nition are expressed in various immunologi-cally relevant cell types, including dendriticcells (DCs), macrophages, and B cells (4). Inthese cells, TLRs link nucleic acid recognitionto production of cytokines, induction of an-timicrobial effectors, and upregulation of MHCand costimulatory molecules. In general, thesedownstream effects contribute to the innate im-mune response as well as facilitate inductionof adaptive immunity. DCs are particularly im-portant for both of these aspects of TLR func-tion, so it is notable that the TLRs involvedin nucleic acid recognition are differentially ex-pressed among subsets of DCs. For example,plasmacytoid DCs (pDCs), a subset of DCs in-volved in antiviral immunity, express TLR7 andTLR9 but not TLR3 or TLR8 (5, 6). TLR7 andTLR9 expression in pDCs is highly related totheir role in antiviral defense, as pDCs producelarge amounts of type I interferons (IFNs) whenactivated by TLR7 or TLR9 ligands. Type IIFNs are a family of cytokines with potent an-tiviral activity (7, 8). In other cell types, TLR7and TLR9 activation generally does not lead totype I IFN production, but instead favors cy-tokines such as IL-12, IL-6, and TNF-α.

The cytosolic PRRs are more varied in theircomposition as well as their signaling. Variousforms of viral RNA are recognized by a familyof helicase domain–containing proteins calledRIG-I-like receptors (RLRs) (7). An analogousfamily of receptors responds to cytosolic DNA,although the molecular identification of theseproteins has been more difficult and in somecases controversial (9). Although the signalingpathways from these two families appear tohave some differences, both converge on the

transcription factor IRF3 (IFN regulatoryfactor 3) and potently induce type I IFNproduction in all cell types (9). In additionto these related signaling pathways, cytosolicDNA activates a multiprotein complex calledthe inflammasome. Inflammasome activationis conceptually distinct from the other innatepathways described above; activation results inprocessing of caspase-1, a protease required forcleavage (and eventual secretion) of pro-IL-1and other inflammatory cytokines (10).

One challenge in dissecting the function ofthe innate receptors involved in nucleic acidrecognition is the fact that any given pathogenis likely to be recognized by multiple recep-tors. This redundancy can complicate analysisof cells or mice lacking individual receptors orsignaling components. Despite the overlappingrecognition of certain nucleic acid ligands byTLRs and cytosolic PRRs, though, it is impor-tant to recognize that certain aspects of these re-ceptor families are conceptually quite distinct.For instance, TLR-mediated recognition doesnot require infection of a cell, which enablesTLR-expressing cells of the innate immune sys-tem to respond to pathogens in the absence ofthe manipulation that often occurs in infectedcells. In contrast, cytosolic PRRs detect nucleicacids that reach the cytosol, which typically in-dicates that the cell has been infected. CytosolicPRRs are expressed much more broadly thanTLRs and appear to function as general, cell-autonomous sensors of infection. Collectively,these two groups of PRRs facilitate recognitionof pathogen-derived nucleic acids in the multi-ple contexts associated with infection.

NUCLEIC ACID RECOGNITIONBY TOLL-LIKE RECEPTORS

Ligand Specificity andMicrobial Recognition

The nucleic acid–sensing TLRs represent a dis-tinct subfamily of receptors in terms of se-quence similarity, shared specificity for nucleicacids, and unique localization to endosomalcompartments of the cell. Unlike other TLR

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family members, the nucleic acid–sensing sub-family has poor intrinsic ability to discriminatebetween host- and pathogen-derived ligands.As is discussed in the following sections, alter-native mechanisms have evolved to determinethe origin of ligands in the absence of a molec-ular determinant.

Like all TLR family members, ligandbinding occurs in the luminal ectodomain,composed of 21–27 LRRs that form ahorseshoe-shaped solenoid and N-terminal andC-terminal caps, which are required for proteinfolding because they protect the hydrophobic

solenoid core (11). Whereas TLR7, TLR8,and TLR9 recognize nucleic acid ligands witha certain degree of sequence selectivity, TLR3recognition of dsRNA is largely sequence inde-pendent. TLR3 was the first TLR ectodomainto be crystallized and remains the only nucleicacid–sensing TLR for which the atomicstructure has been determined (12–15). Thesestudies indicate that ligand binding occurs onthe lateral face of the solenoid, sandwichingthe ligand between the TLR homodimers(Figure 1a). Sequence comparisons sug-gest that TLR7, TLR8, and TLR9 contain

CleavedTLR9

c

TLR3dimer

dsRNA

TLR3 ligand–binding sites

a b

N terminus

C terminus

N terminusC termini

Figure 1Structural motifs that mediate interaction between nucleic acid–sensing TLRs and their cognate ligands. (a) TLR3 dimer ( green, onlyectodomain shown) in complex with dsRNA (blue). (b) Two regions of the TLR3 ectodomain make contact with dsRNA. Site-directedmutagenesis has revealed an essential role for the residues His39, His60, and His108 in the N terminus and Asn541 and His539 in the Cterminus (residues indicated in magenta). Structures adapted from Liu et al. (14) [coordinates from the RCSB Protein Data Bank,(PDB) ID: 3CIY]. (c) Modeled structure of the cleaved form of the TLR9 ectodomain. Structure was generated by transposing theTLR9 coordinates on the structure of TLR3 from Choe et al. (12) (PDB ID: 1ZIW). Predicted residues involved in DNA binding areshown in yellow (193).

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ODN:oligodeoxynucleotides

Systemic lupuserythematosus(SLE): a clinicallyheterogeneousautoimmune diseaseassociated with hightiters of autoantibodiesspecific fornucleoproteins, DNA,and RNA. Theseautoantibodies incomplex with selfnucleic acid stimulateTLRs and induceimmune-mediatedtissue inflammationand damage

hydrophobic or basic amino acids correspond-ing to those identified for TLR3, and it ispredicted that ligand binding occurs via asimilar mechanism. In the following sections,we review the ligand specificity of each of thenucleic acid–sensing TLRs based on studieswith synthetic ligands and microbes.

TLR9. The observation that bacterial DNAcould elicit inflammatory cytokines and leuko-cyte proliferation was made before the discov-ery of the PRR families (16, 17). Subsequently,TLR9 was identified as the receptor thatrecognizes nonmethylated cytosine-guanosine(CpG) motifs in DNA (18). Although the stim-ulatory capacity of CpG motifs was originallydiscovered in the context of bacterial DNA,the immune system’s detection of these mo-tifs also enables it to recognize many viruses(19). Furthermore, TLR9’s preference for non-methylated CpG motifs reduces the likelihoodthat TLR9 will recognize self-DNA, given thatnonmethylated motifs are underrepresented inmammalian genomes compared with bacterialgenomes. Despite the suppression of stimula-tory motifs, however, mammalian genomes canstill stimulate TLR9 in certain contexts, whichsuggests that additional mechanisms must con-tribute to self/nonself discrimination.

Because TLR9 and TLR7 are potential tar-gets for vaccination and control of autoimmunediseases, the rules governing their ligand speci-ficity have been the focus of much research.Distinct classes of stimulatory oligodeoxynu-cleotides (ODN) have been defined based ontheir differential capacity to stimulate DCs orB cells (20, 21). Class A (CpG-A) ODN arecomposed of a natural phosphodiester back-bone with nuclease resistant phosphorothioate5′ and 3′ ends, as well as a poly-G tail. The poly-G tail leads to aggregation of ODN into largecomplexes, a process that is thought to enhanceuptake by macrophages and DCs but to inter-fere with B cell activation. CpG-A ODN arenotable in their ability to induce large amountsof type I IFN from pDCs. Class B (CpG-B)ODN consist entirely of a stabilized phospho-rothioate backbone without a poly-G tail and

induce potent B cell activation. CpG-A andCpG-B ODN can elicit inflammatory cytokinesfrom macrophages and DCs, although CpG-BODN are typically more potent in this regard(21). Swapping the CG motif for GC resultsin an ODN that binds TLR9 but inhibits acti-vation of the receptor. Independent inhibitorysequences have also been identified for TLR7and TLR9, and the ability of these inhibitoryODN to prevent TLR7 and TLR9 activationis being tested in the context of autoimmunediseases such as systemic lupus erythematosus(SLE) (22).

Although the precise mechanisms respon-sible for the different properties of each ODNclass remain unknown, uptake and intracellulartrafficking are thought to play a major role.The stimulatory capacity of a given ODNis also influenced by the sequence flankingthe CG motif. Notably, many of the rulesregarding specificity were defined using ODNwith a phosphorothioate backbone. Whereasthe CpG motif is absolutely required for recog-nition in the context of the phosphorothioatebackbone, non-CG motifs within a phospho-diester backbone can elicit TLR9 responses insome circumstances. These data have led to thesuggestion that the phosphodiester backboneitself may be the feature recognized directly byTLR9, although certain bases and/or modifica-tions may favor higher affinity interactions (23).While defining these rules regarding TLR9activation has been extremely fruitful in thedevelopment of therapeutics targeting thesereceptors, how well they apply to the detectionof pathogens is unknown. If TLR9 recognitionof pathogen DNA also favors certain motifs,then pathogens are likely to be under strongselective pressure to lose such motifs fromtheir genome. Nevertheless, this area ofinvestigation has received little attention.

The clearest evidence for TLR9 detectionof viral DNA comes from in vitro experiments.It has been more difficult to demonstrate theimportance of this receptor using in vivo in-fection models, possibly owing to redundancywith cytosolic detection pathways. Regardless,TLR9 has been shown to play an important


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Conventional DC(cDC): referscollectively to thenon-pDC subsets ofDCs. This designationis typically used todistinguish betweenDCs that produce typeI IFN in response toTLR7 and TLR9ligands (pDCs) andthose DCs that do not(cDCs)

Cytosolic sensors:proteins localized inthe cytosol of hostcells that detect DNA,RNA, or otherconserved microbialfeatures

role in detecting murine cytomegalovirus(MCMV), especially within pDCs (24–26).Herpes simplex virus (HSV)-1 and HSV-2 (27,28), human papilloma virus, and adenovirus(29) induce TLR9-dependent DC activationin vitro. Although the in vivo response to theseviruses is MyD88-dependent, TLR9 knockoutsdo not have a major defect in viral clearance,indicating redundancy with other TLRs, IL-1receptor family members, or possibly cytosolicsensing pathways (30, 31).

TLR7 and TLR8. Sequence comparisonsacross vertebrate TLRs indicate that TLR7and TLR8 are the most closely related ofthe nucleic acid–sensing TLRs and are moresimilar to TLR9 than to TLR3 (32). TLR7and TLR8 share a general specificity forguanosine/uridine-rich ssRNA and the small,synthetic antiviral compounds called imidazo-quinolines (33–36).

In humans, remiquimod can stimulateTLR7 and TLR8, yet the related imidazo-quinoline, imiquimod, only activates TLR7,demonstrating that there are differences inligand specificity between the receptors (37).TLR7 activation in response to small RNAsrequires uridine motifs, whereas guanosineresidues can be exchanged for cytosine withoutaffecting cytokine production (38). A study us-ing human peripheral blood monocytes showedthat a uridine-rich tetramer is the minimumsequence required to elicit TLR7 and TLR8responses. However, type I IFN productionby TLR7 (pDCs) is preferentially induced byGU-rich sequences (e.g., UUGU, GUUC),whereas AU-rich sequences (e.g., AUUU,UAUC) induce TNF responses from TLR8-expressing monocytes (39). Thus, in humansTLR7 and TLR8 recognize distinct sequencemotifs in ssRNA.

In mice, it is not clear whether the tlr8gene encodes a functional receptor. WhileTLR8 mRNA and protein are detected inmouse tissues, genetic deficiency in TLR7 issufficient to eliminate responses to syntheticligands and viral infection (33–36). Recently,a report identified a 5–amino acid region,

absent in murine TLR8 but present in allother species analyzed, that may account forthe observed inactivity (40). However, twoindependent studies have shown a MyD88-dependent, TLR7- and TLR9-independentresponse to stimulation with a combination ofimidazoquinoline and poly(dT) ODN (39, 41).Although this response has been attributedto TLR8, studies using TLR8-deficient miceare the definitive test of the function of thisreceptor in mice. Regardless, the specificities ofTLR7 and TLR8, and therefore the functionsof these receptors, appear to have divergedbetween humans and mice.

Although differences in ligand specificitymay translate into distinct functional roles forTLR7 and TLR8, the nonoverlapping expres-sion of these receptors in human cells maybe a greater determinant of differential func-tion. Whereas TLR7 and TLR9 are exclu-sively expressed in pDCs, TLR8 is expressedmore broadly in macrophages and conventionalDCs (cDCs) (5, 6). In contrast, TLR7 andTLR9 are expressed in macrophages and multi-ple DC subsets in mice. This differential expres-sion likely explains the distinct cytokine pro-files induced by TLR7 and TLR8 ligands inhuman cells (39). This overlapping expressionmay have obviated the need for TLR8 in mouseimmunity, such that an inactivating mutationin the receptor did not impart a significantdisadvantage.

TLR7 has been implicated in the recogni-tion of many RNA viruses as well as severalbacteria. pDCs from TLR7-deficient mice areunable to respond to vesicular stomatitis virus,influenza, or HIV-1 (33, 35, 36). For certainviruses, TLR7-dependent activation of pDCsrequires infection with live virus as well as func-tional autophagic machinery, suggesting thatviral particles are delivered from the cytosolto TLR7-containing compartments (42, 43).These data are consistent with the observationthat cytosolic sensors of RNA and DNA arenot functional within pDCs. TLR7-mediatedrecognition of bacterial RNA is much lessdocumented. Nevertheless, TLR7 plays a rolein the detection of group B streptococcus.

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Interestingly, TLR7 induces type I IFNproduction in infected cDCs but not inmacrophages or pDCs. In contrast, Listeriamonocytogenes and group A streptococcusare primarily detected by cytosolic sensingpathways owing to their ability to escapefrom phagosomes. Avirulent strains of thesepathogens that remain in the phagosome doactivate TLRs (44). Why cDCs but not othercell types can recognize bacterial RNA inphagosomes remains unclear. Most studiesexamining bacterial recognition have utilizedmacrophages, so it remains to be seen whetherRNA (or DNA) from other phagosome-resident pathogens can be recognized withincDCs.

TLR3. TLR3 was the first TLR implicatedin the sensing of viral nucleic acids (45). Itbinds dsRNA without a high degree of sequencespecificity, although a minimum length of 40base pairs is required for TLR3 responsiveness,with affinity increasing in proportion to dsRNAlength (46). TLR3 also recognizes the syn-thetic RNA analogs polyinosine-polycytidylicacid [poly(I:C)] and polyuridine [poly(U)] (45).Structural studies of the TLR3 ectodomainbound to dsRNA indicate that there are twoputative ligand-binding sites: one on the lowerlateral face within LRR19–LRR21 and anothercontaining a series of histidine residues (His39,His60, and His108) within LRR1–LRR3 that canplay redundant roles, as mutating all three isrequired to block signaling (14, 15, 46). Inter-estingly, mutational analysis of the TLR9 Nterminus indicates that an analogous positivelycharged region of this receptor may also be re-quired for TLR9 activation (47).

Somewhat surprisingly, TLR3 is not ex-pressed by pDCs (5, 6). Expression onmacrophages and several DC subsets has ledto the hypothesis that TLR3 may not only me-diate direct recognition of dsRNA within viralparticles, but may also enable detection of vi-ral nucleic acids within infected, apoptotic cellsafter phagocytosis (48). This strategy may al-low detection of a much broader collection ofviruses, as dsRNA is a common intermediate

in the replication of RNA viruses and often re-sults from transcription of overlapping readingframes within DNA viruses. Therefore, TLR3may act as a more general sensor of viral infec-tion in addition to a sensor for dsRNA viruses.

Consistent with this hypothesis, TLR3 hasbeen implicated in the host response to ss-RNA, dsRNA, and DNA viruses. TLR3-deficient mice mount impaired responses to en-cephalomyocarditis virus (49), West Nile virus(50), Semliki Forest virus (48), and MCMV(24). The precise role for TLR3 in the im-mune response to West Nile virus, a ssRNAflavivirus, is somewhat controversial, althoughTLR3 recognition of virus clearly occurs dur-ing infection (50, 51). Both studies examiningthis issue found increased viral titers in the pe-ripheral tissues of TLR3-deficient mice rela-tive to wild-type controls. However, in onestudy TLR3-deficient mice had much reducedmortality due to reduced inflammation andblood-brain barrier leakage (51), whereas an-other study did not observe such protection(50). These differing results may be due to dif-ferences in the viral stocks used for infectionor other details of each specific study. Stud-ies of human patients also support a broad rolefor TLR3 in antiviral host defense. Individualswith loss-of-function mutations in TLR3 suf-fer from HSV-1 encephalitis (52). This diseasehas also been linked to patients with mutationsin unc93b1, a gene required for proper localiza-tion of TLR3, TLR7, TLR8, and TLR9 (seediscussion below) (53).

Induction of Type I IFN by NucleicAcid–Sensing TLRs

The nucleic acid–sensing TLRs recognize lig-ands and initiate signal transduction within en-dolysosomal compartments. Only two of thefour TIR-containing signaling adaptors areused by these receptors: TLR7, TLR8, andTLR9 signal through MyD88, whereas TLR3signals through TRIF (TIR domain–containingadapter inducing IFN-β). These adaptors re-cruit multiprotein signaling complexes and ul-timately lead to the production of cytokines as


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well as the induction of many genes importantfor the initiation of the immune response. Thesignaling networks responsible for these effectsare not the focus of this article and have beendiscussed in detail in several recent reviews (1).Instead, we briefly discuss how TLR3, TLR7,TLR8, and TLR9 induce type I IFN produc-tion, as this link is critical for their role in an-tiviral immunity.

There are two major pathways of TLR-dependent type I IFN production in responseto nucleic acids (7). The first involves TLR3-dependent activation of IRF3 and occurs incDCs and macrophages. TLR3 activation in theendolysosome induces association with TRIF,which serves as a platform for a multiproteincomplex that includes TRAF3 (TNF receptor–associated factor 3). TRAF3 induces TANK(TRAF family member–associated NF-κBactivator)-binding kinase 1 (TBK1) and IKKi(inhibitor of NF-κB kinase, also referred to asIKKε) dimerization (54, 55). The TBK1/IKKicomplex is responsible for phosphorylation ofIRF3 (56, 57), which then translocates to thenucleus and initiates transcription of the Ifnbgene. Notably, this TRIF-dependent pathwayis also activated by TLR4.

The second pathway of type I IFN in-duction involves TLR7- or TLR9-dependentactivation of IRF7. The mechanism of IRF7activation is quite distinct from the aforemen-tioned TRIF-dependent pathway, given thatTLR7 and TLR9 signal via MyD88. Althoughthe mechanistic details of signaling componentslinking MyD88 to IRF7 are not completely de-fined, considerable progress has been made inrecent years. Originally, investigators thoughtthis pathway only exists within pDCs, as thesecells are unique in their ability to produce largeamounts of type I IFN, especially IFN-α, inresponse to TLR7 and TLR9 activation. pDCsdo express high levels of IRF7 and appear tohave distinct wiring that links MyD88 to IRF7(58, 59). However, recent work suggests that,although certain specialized features of pDCsmay favor type I IFN production, the ability tolink TLR7 and/or TLR9 activation to type IIFN production is shared by other DC subsets

as well as by macrophages (60). The outcomeof TLR7/9 activation appears to rely on thesubcellular compartment from which signalingis initiated. Macrophages or cDCs in whichstimulatory ODN are artificially retainedin early endosomes produce type I IFN,whereas lysosome-initiated signaling inducesinflammatory cytokine production but nottype I IFN (60). Typically, the latter outcomeis favored in cDCs and macrophages, whereasthe former is favored in pDCs. How thesecompartments are established and maintainedis poorly understood.

Localization and Trafficking ofNucleic Acid–Sensing TLRs

Before the identification of TLRs, investiga-tors’ observation that inhibition of endosomalmaturation blocked responses to immunostim-ulatory nucleic acids suggested that the rel-evant receptors were localized intracellularly(61). Subsequent analysis verified that nucleicacid–sensing TLRs were not detectable at thecell surface but instead resided within internalcompartments. Using immunofluorescence mi-croscopy, several groups could demonstrate in-tracellular receptors but were unable to detectTLR9 on endosomes. Instead, most if not all ofthe receptor appeared to be localized to the en-doplasmic reticulum (ER), an observation thatwas difficult to reconcile with recognition of nu-cleic acid released from microbes within phago-somes (62, 63). Even more unusual was the find-ing that upon stimulation TLR9 appeared tobypass the Golgi and translocate directly fromthe ER to endosomes. As is discussed below,this confusion in the field seems to have beenresolved by the identification of a proteolyticfragment of the receptor that is only present inendolysosomal compartments (64). Analysis ofTLR9 glycosylation indicates that the cleavedform of TLR9 as well as a small amount of thefull-length receptor traffic through the Golgiapparatus en route to the endolysosome (64,65). We have observed similar trafficking andlocalization for TLR3 and TLR7 (S.E. Ewaldand G.M. Barton, unpublished observations).

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An important step toward understanding themechanisms responsible for the distinct traf-ficking and localization of nucleic acid–sensingTLRs has been the identification of Unc93b1as an ER-resident protein necessary for TLR3,TLR7, and TLR9 function. In a forward ge-netic screen, Beutler’s group generated micewith a point mutation in the ninth membrane-spanning region of this twelve-pass transmem-brane protein (66). Unc93b1 appears to controlexit of TLR3, TLR7, and TLR9 from the ER.The Unc93b1 point mutant cannot exit the ER,and overexpression of wild-type Unc93b1 leadsto increased trafficking of TLR7 and TLR9to endosomes (64, 67, 68). Although the pre-cise mechanism through which Unc93b1 con-trols TLR trafficking is not clear, it may actas a chaperone in the ER or mediate tran-sit through the secretory pathway, as Unc93b1colocalizes with TLR7, TLR9, and nucleic acidligands (67). Recently, an asparagine residue(Asp34) was identified in the N-terminal cy-toplasmic tail of Unc93b1 that is required fora bias toward TLR9 activation at the expenseof TLR7 activation (75). The mechanismsbehind these differential responses are notclear.

Studies using chimeric receptors, gener-ated by swapping domains of the nucleic acid–sensing TLRs for those of surface-localizedproteins (e.g., TLR4, CD4, CD25), have re-vealed an important role for the transmembranedomain in controlling TLR7 and TLR9 lo-calization (69–72). By contrast, TLR3 appearsto depend on the linker region between thetransmembrane and TIR domains for properlocalization (72, 73). Additionally, there is ev-idence that TLR9 may first traffic to the cellsurface before being internalized into endolyso-somal compartments. Mutation of a tyrosine-based internalization motif in the cytosolic tailof TLR9 results in receptor stabilization at thecell surface (65, 74). Although it is not en-tirely clear how the structural motifs in theseTLRs relate to regulation by Unc93b1, recentwork indicates that the TLR3 and TLR9 trans-membrane domains are required for interactionwith Unc93b1 (67). The reciprocal motifs in

Unc93b1 that mediate this interaction have notbeen defined.

Receptor Proteolysis LimitsNucleic Acid Recognition by TLRsto Endolysosomes

Recent work has made apparent that the activa-tion of TLR9 and possibly of the other nucleicacid–sensing TLRs requires receptor proteol-ysis in the endolysosome. Three groups haveindependently shown that, when TLR9 reachesendolysosomal compartments, the N terminusof TLR9 is removed by acid-dependentproteases. The resulting cleaved receptorconsists of roughly half the ectodomain, themembrane-spanning region, and the cytosolicdomain (64, 76, 77). The cleaved form ofTLR9 is functional: It can bind DNA directly,it is the exclusive form of the receptor thatassociates with the signaling adaptor MyD88,and it co-elutes with MyD88 from stimulatedcell lysates (78, 79). Furthermore, expressionof a precleaved form of TLR9 complementsTLR9-deficient cells (79, 80). This finding isparticularly compelling because it suggests thatthe nucleic acid–sensing TLRs have evolved amechanism to absolutely limit their activationto intracellular compartments. This mechanismmay be particularly important in light of theobservation that TLR9 may pass through thecell surface before reaching the endolysosome(65). Coupling TLR9 activation to processingthat occurs only in the endolysosome by res-ident proteases would prevent receptors fromresponding to nucleic acids they encounter atthe cell surface (see discussion sections below).

Multiple proteases have been implicated inTLR processing. In 2008, a study from theMiyake group traced a defect in TLR9 andTLR7 signaling in Baf/3 cells to a deficiencyin cathepsin (cts) B and ctsL (81). A subsequentstudy reported that TLR9 activation is defectivein ctsK-deficient DCs and that blocking ctsKactivity ameliorates autoimmune inflammationin a TLR9-dependent manner (82). How-ever, several groups have reported that TLR9proteolysis is unaffected in ctsB-, ctsL-, or


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ctsK-deficient macrophages and DCs. Onlytreatment with pan-cathepsin inhibitors ap-pears to have a significant effect on processing(64, 76). Although initial reports have suggestedthat cathepsins are responsible for TLR9 pro-cessing in macrophages, a subsequent publica-tion identified asparagine endopeptidase (AEP)as a key protease required for TLR9 process-ing in DCs (77). Given that the protease reper-toire varies greatly between macrophage andDC subsets, the proteases that cleave TLR9 arelikely to be somewhat specific to a given celltype (83). However, AEP has been implicatedin the processing of procathepsins to their activeforms, suggesting that these two findings maynot be mutually exclusive (84). Allowing diverseproteases to cleave TLR9 may ensure that thereceptor will be activated in multiple cell typesand throughout the endosomal system. Clearly,future studies are necessary to work out the in-tricacies of receptor processing in the cell typesthat endogenously express TLR9 and the othernucleic acid–sensing TLRs.

How processing, ligand binding, and dimer-ization are coordinated to induce signal trans-duction by the nucleic acid–sensing TLRs is stillunclear. Although the cleaved form of TLR9has been shown to bind DNA, it is well es-tablished that the full-length forms of TLR9and TLR3 can directly bind their cognate lig-ands (14, 63). At this point, the precise rolesplayed by the full-length receptor versus thecleaved receptor during ligand recognition inthe cell is not entirely clear. An elegant studyfrom the Golenbock group used chemical cross-linking to show that TLR9 mainly exists asa homodimer that assembles in the ER (85).Using TLR9 molecules bearing the N termi-nus or the C terminus of split-GFP, the samegroup showed that a membrane-proximal con-formational change accompanies ligand bind-ing and brings the TIR domains together (85).These studies were completed before the obser-vation that TLR9 is cleaved. Given that TLR9cleavage is required for MyD88 recruitment,the cleavage process likely participates in thisconformational change, although this possibil-ity has not been tested directly.

CYTOSOLIC SENSORSOF NUCLEIC ACIDS

Nucleic acid–sensing TLRs sample the lume-nal contents of endolysosomal compartmentsand respond to ligands released from degradedmicrobes. Accordingly, TLRs enable the detec-tion of viruses before productive infection of acell. A different family of receptors is requiredto detect nucleic acids derived from microbesthat enter the cytosol. In the past few years,several proteins involved in cytosolic nucleicacid sensing have been described. These path-ways are expressed in many more cell types thanthose that express TLRs, which presumably en-ables any cell to sense a cytosolic pathogen in acell-autonomous fashion. In the following sec-tion, we review our knowledge of the nucleicacid cytosolic sensors and their known ligandspecificity. (See Figure 2 for an overview ofthe known pathways involved in this process.)

Recognition of RNAby Cytosolic Receptors

After the identification of the nucleic acid–sensing TLRs and their cognate ligands, in-vestigators soon realized that another class ofreceptors must exist. Although the responseto Poly(I:C) was reduced in TLR3-deficientcells, it was not completely abrogated (45).Subsequent studies from Reis e Sousa’s groupdemonstrated that delivery of Poly(I:C) into thecytosol of cells induced type I IFN in a TLR3-independent manner (86). This observation ledto the identification of an entire family of re-ceptors that are required for the sensing of viralRNA products in the cytosol of infected cells.

RIG-I-like receptors. In a cDNA screento identify genes that enhance activation ofan IFN-β promoter in response to cytosolicPoly(I:C), the Fujita lab cloned a gene, retinoicacid inducible gene-I (RIG-I), which becamethe founding member of the RLR family ofreceptors (87). Interestingly, this initial RIG-Iclone encoded a protein truncated after thetwo N-terminal caspase recruitment domains

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RIG-I

Proteolytic cleavage

RIG-I only

Mitochondrion Peroxisome

ER

5’ triphosphate RNA

Short Poly(I:C) Long Poly(I:C) DNA DNA

CARD RDHelicase

MDA5

CARD RDHelicase

AIM2

Pyrin HIND200

MAVSCARDTM CARD TM

DAI

STING

DNAsensor(s)

STING

Procaspase-1

Type IIFN

Antiviralstate

Type I IFN

IL-1β processing/secretion

Caspase-1CARD

Active caspase-1

Figure 2Recognition of cytoplasmic RNA and DNA. A schematic of the known pathways involved in innate immune recognition of cytosolicRNA and DNA. RIG-I and MDA5 (left) recognize different synthetic ligands. RIG-I recognizes short dsRNAs and RNAs with 5′triphosphate ends, whereas MDA5 recognizes long dsRNA. Both receptors signal through the essential adapter MAVS, which localizesto at least two distinct organelles: mitochondria and peroxisomes. When associated with mitochondria, MAVS leads to type I IFNproduction; when peroxisome associated, MAVS leads to induction of a distinct set of antiviral genes. Signaling via RIG-I also requiresthe membrane-bound protein STING. DAI and unidentified cytoplasmic receptor(s) recognize cytoplasmic DNA (middle). Afterrecognition, these DNA sensors require STING to induce type I IFNs. In addition, AIM2 can recognize cytoplasmic DNA (right).AIM2 links recognition of DNA to activation of the inflammasome and caspase-1-dependent processing of IL-1β into its active form.(Abbreviations: AIM2, absent in melanoma 2; CARD, caspase recruitment domain; DAI, DNA-dependent activator of IRFs;MAVS, mitochondrial antiviral signaling; MDA5, melanoma differentiation–associated gene 5; RIG-I, retinoic acid-inducible gene I;STING, stimulator of IFN gene.)

(CARDs) and was able to activate IRF3 in-dependently of ligand. Full-length RIG-I alsocontains a central DEAD box helicase/ATPasedomain whose ATPase function is necessary forIRF3 activation, and a C-terminal regulatorydomain that prevents constitutive activation ofthe protein.

The two other members of the RLR familyare MDA5 and LGP2. MDA5 has a domainstructure similar to RIG-I: N-terminal CARDdomains, a central DEAD box helicase/ATPasedomain, and a C-terminal regulatory domain(88). Similar to RIG-I, MDA5 activates theIRF3 pathway in response to Poly(I:C). Incontrast, LGP2 lacks the N-terminal CARDdomains necessary for IRF3 activation and

consists only of the central DEAD box he-licase/ATPase domain and the C-terminaldomain (88). Lack of a CARD domain sug-gests that LGP2 cannot induce downstreamsignaling, and an initial report supported theconclusion that LGP2 functions as a negativeregulator of the RIG-I/MDA5 pathways (88).However, more recent work using LGP2-deficient mice suggests that LGP2 acts as acoreceptor for some RIG-I and MDA5 ligands(89). These conflicting results are likely dueto the use of different ligands and different celltypes and to the difficulty with interpretingprotein overexpression data.

A recent report has implicated NOD2 inthe cytosolic sensing of ssRNA (90), which is


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surprising because NOD2 is also a sensorof muramyldipeptide (MDP) derived frompeptidoglycan (91, 92). NOD2 induces aclassic proinflammatory signature in responseto MDP, but it also induces type I IFN inresponse to ssRNA. NOD2 has an N-terminalCARD domain, a central nucleotide-bindingdomain, and a C-terminal LRR domain. Thedouble life of NOD2 raises several questionsrelevant to other innate immune sensors. Howcan one receptor recognize molecules withsuch distinct molecular structures (such asMDP and ssRNA)? Moreover, as discussedin the previous section regarding TLR7 andTLR9, how does recognition of distinct ligandsby the same receptor lead to such differentsignaling pathways?

Protein kinase R (PKR) has also been impli-cated in dsRNA recognition (93). The subse-quent identification of RIG-I and MDA5, how-ever, has raised questions regarding the rolePKR may play in dsRNA sensing. Little workhas been done to understand how PKR fitswithin the framework of these newly discov-ered RLRs, but a recent paper suggests thatPKR is necessary for stabilizing IFN-α/β tran-scripts downstream of MDA5 signaling but notof RIG-I signaling (94). This observation mayexplain why PKR-deficient cells and animals re-spond poorly to virus, but how PKR is activatedis still not understood. PKR contains a dsRNA-binding domain, but whether it functions as aPRR independently of RIG-I and MDA5 is stillunclear.

Little is known about how RIG-I, MDA5, orNOD2 recognize RNA. Although none of theproteins contain classic RNA-binding domains,a substantial amount of work has defined whichdomains of RIG-I are important for bindingRNA. Two reports have demonstrated that theC-terminal regulatory domain of RIG-I, whichis necessary to prevent constitutive activation(see discussion above), also binds RNA (95, 96).The mechanism of RLR activation remains in-complete, but a model has been proposed inwhich the regulatory domain maintains RIG-Iin an inactive form until RNA is bound (97).Upon binding RNA, a conformational change

occurs that frees the N-terminal CARD do-mains and allows recruitment of downstreamadapters.

Ligand specificity of RLR proteins. The dis-covery of RIG-I and MDA5 raised the centralquestion of how these receptors discriminatebetween self- and nonself-RNA. Unlike en-dolysosomes or phagosomes, the cytosol con-tains many self-RNAs, which RIG-I and MDA5must somehow ignore while retaining the abil-ity to respond to pathogen-derived molecules.Most of the studies in the field have used syn-thetic ligands to define the ligand specificity ofRIG-I and MDA5. Although the use of such lig-ands does not lead to better understanding ofhost-pathogen interactions, it can address howRIG-I and MDA5 can discriminate betweenself- and nonself-RNAs.

When first characterized, RIG-I andMDA5 were both implicated in recogni-tion of Poly(I:C). With the generation ofgene-targeted mice, though, the roles ofthese two receptors have been dissected withmore precision. Initial reports suggested thatMDA5, but not RIG-I, recognizes cytosolicPoly(I:C) (98, 99), but a more recent report hasimplicated both family members in Poly(I:C)sensing (100). These conflicting results may beexplained by the observation that long (>2 kb)polymers of Poly(I:C) are preferentially recog-nized by MDA5, whereas smaller polymers (asshort as 70 bp) are recognized by RIG-I (100).Thus, the preferential recognition of differentsizes of RNA may be an important functionaldifference between RIG-I and MDA5, althoughthe relevance of these distinct specificities forpathogen recognition remains unclear.

Another synthetic ligand recognized byRIG-I is short, uncapped 5′-triphosphatessRNA (101, 102). Whereas host mRNAsare capped with a 7-methyl-guanosine group,many viral RNAs remain uncapped, provid-ing a potential mechanism for self/nonselfdiscrimination by RIG-I. Recent work has sug-gested that uncapped 5′-triphosphate ssRNAscannot activate RIG-I without small regionsof base pairing (103, 104). To explain these

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discrepancies, the more recent reports suggestthat T7 transcribed RNAs (which were usedin the earlier studies) contain a small amountof dsRNA. Additional complications to under-standing the requirements for RIG-I activationare reports that short dsRNAs without 5′-triphosphate can also activate RIG-I (96, 100).

In parallel with efforts to understand themolecular mechanisms underlying RNA de-tection by RIG-I and MDA5, attention hasbeen paid to the role these receptors play inimmunity to specific viral pathogens. Virusescan generally be divided into three categories:Those only recognized by RIG-I (flavivirus,orthomyxovirus), those only recognized byMDA5 (picornavirus), and those recognized byboth RIG-I and MDA5 (paramyxovirus, re-ovirus, flavivirus) (98, 99, 105, 106). For manyof the viruses listed above, in vivo work has con-firmed the importance of these receptors in hostdefense (98, 99, 107–109). While functional in-teractions between different families of virusesand different receptors have been described, lit-tle work has been done to understand why someviruses activate certain receptors and not others.The simplest explanation is that certain viruseseither lack the appropriate ligand for RIG-Ior MDA5 or have evolved evasion strategiesfor specific RLR family members. Ultimately,more comparative analysis is needed betweendifferent viruses and different receptors to un-derstand the nature of these specificities.

RLR accessory proteins. The initial experi-ments that elucidated the function of the RLRsused downstream events such as type I IFN pro-duction or IRF3 activation as a proxy for RLRactivation. Although there is substantial over-lap between the multiprotein complexes thatTLRs and RLRs used to activate IRF3 (e.g.,TBK1, IKKi, and TRAF3) (54, 55, 57), as re-viewed below, many of the proximal adaptersused by RLRs to activate IRF3 are unique tothe RLR family of receptors.

MAVS. MAVS (mitochondrial antiviral sig-naling, also known as IPS-1, CARDIF, andVISA) is an essential adapter that connects

RIG-I/MDA5/NOD2 activation with IRF3phosphorylation. Identified by four differentgroups simultaneously using a combinationof candidate gene approaches and cDNAscreening (110–113), MAVS contains anN-terminal CARD domain that mediates itsassociation with RLRs and is essential foractivation of the IRF3 axis. In addition to aCARD domain, MAVS contains a C-terminaltransmembrane domain that targets MAVS tothe mitochondrial outer membrane (112). Thismitochondrial localization is essential for cy-tosolic MAVS to activate IRF3 (112). However,the underlying mechanism for this localizationrequirement remains unclear. To further com-plicate matters, Dixit et al. (114) have recentlydemonstrated that MAVS is also localizedto peroxisomes. Moreover, the subcellularlocalization of MAVS appears to have func-tional consequences: MAVS signaling from theperoxisomes induces antiviral genes withouttype I IFN, whereas MAVS signaling fromthe mitochondria leads to production of typeI IFN (114). The mitochondrial response isdelayed compared with signaling from the per-oxisome, suggesting that MAVS signaling fromdistinct compartments may occur with distinctkinetics. The mechanisms responsible for thistranscriptional specificity remain unclear.

Despite these recent breakthroughs, manyquestions remain about the cell biology ofRLR signaling. Why does MAVS requiremembrane association for signaling? RIG-Iand MDA5 are cytosolic, so why must thedownstream signaling events take place onspecific cellular organelles? One possibilityis that MAVS associates with other signalingcomponents that only localize to specificorganelles. However, it is difficult to reconcilethis model with the observation that MAVS cansignal from the cytosol after being artificiallydimerized (115). It will be important to exam-ine whether additional intracellular receptorsrequire association with specific organelles toorganize signaling complexes. Currently, onlythe nucleic acid–sensing intracellular receptorsseem to require this level of organization (seebelow for DNA).


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TRIM25. TRIM25 (tripartite motif-containing 25) is an E3 ligase that directly ubiq-uitinates RIG-I (116). TRIM25-dependentubiquitination of RIG-I is necessary forrecruitment of MAVS and subsequent signaltransduction. Although these data couldsuggest that ubiquitination of RIG-I is suf-ficient to activate the IRF3 pathway, RIG-Iis ubiquitinated independently of activation,indicating that additional steps are required toinitiate signaling. Thus, TRIM25-dependentubiquitination might serve to prime RIG-Ibefore it binds viral RNA.

STING. The transmembrane protein STING(stimulator of IFN gene, also known as MITAand ERIS) has also been implicated in the RLRsignaling pathway, although its precise role issomewhat confusing (117–120). STING is re-quired for maximal RIG-I signaling but not forMDA5 signaling (117). These data are difficultto reconcile with the observations that overex-pression of MAVS leads to STING-dependentIRF3 activation, yet MDA5 activation of IRF3is MAVS-dependent but STING-independent(117). Another confusing aspect of STING bi-ology is its localization; several groups havedemonstrated that STING is ER resident, al-though this finding is controversial (118). How-ever, STING also has been shown to interactwith MAVS (120), which is primarily foundon the mitochondrial membrane (see above).Why activation of the IRF3 axis requires sig-naling across so many different organelles isnot understood, but understanding these re-quirements may provide insight into antiviralimmunity.

EYA4. EYA4 (Eyes absent 4) is an enigmaticprotein involved in the RLR response (119).Similar to other members of the EYA familyof proteins, EYA4 can enhance the type IIFN response when overexpressed. EYA4 isa phosphatase whose activity is required foroptimal type I IFN production; therefore,identifying the targets of EYA4 will likelyprovide important information about the RLRsignaling pathway.

Recognition of DNAby Cytosolic Receptors

In 2006, the Medzhitov and Akira groupsdemonstrated the existence of an additionalpathway of DNA recognition (121, 122). Al-though no receptor for this pathway was iden-tified in these initial studies, the two reportsdemonstrated that a DNA sensor(s) in the cy-toplasm of cells could lead to the activation ofthe IRF3 pathway. Both groups demonstratedthat multiple sources of DNA could activate theDNA sensor, but they each focused on differentDNA molecules. While the Akira group used along polymer of a poly(dA-dT) · poly(dT-dA)DNA, the Medzhitov lab used a much smaller(45 mer) dsDNA oligonucleotide (called im-munostimulatory DNA, or ISD) that lackedCpG motifs. Both ligands activate similar path-ways in cells, but it is unclear if the same recep-tors recognize both ligands.

To date, little is known about the specificityof putative cytosolic DNA sensors for substrateDNA molecules. As is discussed in the follow-ing section, mice that lack DNaseII die froman autoimmunity syndrome before birth, likelybecause of the presence of self-DNA in thecytosol. This observation suggests that DNAsensor(s) cannot discriminate between self- andnonself-DNA that gains access to the cytosol(123). This lack of specificity is probably tol-erated because the cytosolic compartment nor-mally does not contain DNA.

Finally, it has been demonstrated that trans-fected poly(dA-dT) can be transcribed by RNApolymerase III (PolIII) to generate RNA lack-ing a 5′ cap. This transcribed RNA can ac-tivate a RIG-I-dependent cytosolic response(125, 126). Although the role for PolIII in hostdefense is not well understood, this pathway hascertainly made the analysis of the DNA sen-sor(s) more complicated.

DNA sensor(s) and accessory proteins.Many of the primary receptors and adapters forthe sensing of cytosolic RNA were discoveredrelatively quickly, but the receptors for thesensing of cytosolic DNA have remained

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controversial and elusive. Using a candidategene approach, the Taniguchi group identifiedDAI (DNA-dependent activator of IRF), alsoknown as DLM-1 and ZBP1) as the putativeDNA sensor. In L929 cells, knocking downDAI led to a decrease in type I IFN in responseto transfected dsDNA (127). Additionally, DAIwas shown to bind DNA and interact with thekey transcription factor IRF3. Although thesedata certainly suggested that DAI is the DNAsensor, subsequent analyses of mice lackingDAI found no defect in the response to trans-fected DNA in mouse embryonic fibroblasts orin DCs (128). The lack of a phenotype suggestseither that DAI is not a DNA sensor or thatthere is a functional redundancy in certain celltypes. More recent work by the Taniguchigroup has argued the latter point, showing thatmany cell types possess multiple DNA sensors(129).

The only gene product that is known to beessential for the activation of the IRF3 path-way in response to cytoplasmic DNA is STING(130). Although STING is necessary for maxi-mal RIG-I signaling, there is stronger evidencefor its role in sensing DNA (130). STINGis absolutely essential for type I IFN produc-tion in response to cytosolic DNA. Preciselyhow STING mediates signal transduction isunknown. It is presumably not a sensor, as ithas not been shown to bind DNA and doesnot contain an obvious DNA-binding domain.STING has multiple transmembrane domainsthat are necessary for its ability to activate theIRF3 pathway, suggesting that it may be anadapter or scaffold protein (117). Interestingly,several groups have noted that STING translo-cates from the ER to punctate perinuclear struc-tures upon stimulation, yet the significance ofthis translocation is not understood (117, 131).

Cyclic dinucleotides. Investigators have re-cently shown that bacterial-derived cyclic dinu-cleotides that enter the host cytosol activate cy-tosolic innate receptors. These small moleculesare used by bacterial cells as secondary messen-gers (132). Initial reports indicated that c-di-GMP is immunostimulatory when injected into

mice (133) and that this immunostimulatoryactivity depends on the delivery of c-di-GMPto the cytosol of cells but is independent of allknown cytosolic-sensing pathways (134). Therelevance of cyclic dinucleotides as ligands forinnate sensors was unclear until quite recently,when it was shown that secreted c-di-AMPfrom Listeria monocytogenes activates a cystolicimmune surveillance program in infected cells(135). Interestingly, many bacteria have beenreported to activate a type I IFN response uponaccessing the cytosol of eukaryotic cells (136).It will be interesting to see if other bacteriaare sensed via the presence of various bacterialcyclic dinucleotides in the cytosol of host cells.

Inflammasome activation by cytosolicDNA. All of the cytosolic RNA- and DNA-sensing pathways discussed thus far inducetype I IFN production, yet another nucleicacid–sensing receptor was recently reportedthat leads to a different cellular response—activation of the inflammasome. The inflam-masome cleaves pro-IL-1β to its active formand induces a form of rapid cell death, termedpyroptosis (10). For several years, activationof the inflammasome was known to be in-duced either by the presence of cytosolic flagellathat activate the IPAF/NAIP5 inflammasomeor by several forms of extracellular particlesthat activate the NALP3 inflammasome (10). In2008, the Tschopp group demonstrated the ex-istence of an additional inflammasome respon-sible for sensing cytosolic DNA (137). Usingvarious ligands, they have demonstrated thatthe DNA-sensing inflammasome responds todsDNA more than 250 bp long (137). Althoughthe inflammasome DNA sensor responds to along polymer of poly(dA-dT) · poly(dT-dA), itdoes not recognize the synthetic ISD ligand(used previously by the Medzhitov group), sug-gesting that the inflammasome DNA sensor hasan overlapping but unique specificity relative tothe DNA sensor(s). It remains unclear how thedecision is made to induce type I IFN or activatethe inflammasome.

Using a candidate gene approach, sev-eral groups have identified AIM2 (absent in


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ANA: antinuclearantibodies

melanoma 2) as the sensor that activates theinflammasome upon the introduction of cy-tosolic DNA (138–141). The two domains thatare important for AIM2 function (and thatwere used to identify AIM2) are the HIN2000DNA-binding domain and the pyrin domainthat interacts with the inflammasome adapterprotein ASC (apoptotic speck protein con-taining a CARD). Whereas the role of AIM2was first demonstrated by siRNA knockdown,AIM2-deficient mice have recently been gener-ated that are unable to activate the inflamma-some in response to DNA (142–144). Little isknown about the specificity of AIM2. It seemsthat AIM2 can recognize DNA in a sequence-independent manner and that both bacterialand viral DNA can activate AIM2 (144, 145).This apparent lack of specificity is not surpris-ing because host DNA is normally not found inthe cytoplasm of eukaryotic cells.

MECHANISMS OF SELF/NONSELFDISCRIMINATION BY NUCLEICACID RECEPTORS

Innate immune recognition of nucleic acidsplays an important role in the effective detec-tion of pathogens, but this strategy introducesthe potential for self recognition leading toautoimmune or autoinflammatory disorders.Multiple regulatory mechanisms have evolvedto ensure that self/nonself discrimination oc-curs reliably. Much of our understanding of thisregulation has been gained from the study ofgene-targeted mice or from the study of mousemodels of autoimmune diseases, especiallySLE. In particular, the MRL strain of miceas well as an MRL strain lacking functionalFas (MRL/lpr) develop SLE-like disease andhave served as valuable models for evaluatingthe role of innate immune receptors in thisdisease (146). In our discussion of self/nonselfdiscrimination by nucleic acid–specific recep-tors, we emphasize regulation of TLR activity,although similar regulatory mechanisms mayapply to cytosolic sensors and are likely toemerge as our understanding of these pathwaysincreases.

One of the signatures of SLE that indicatesa break in tolerance to nucleic acids is the pres-ence of antibodies with specificity for nucleicacids or nucleic acid–associated proteins, of-ten referred to as antinuclear antibodies (ANA)based on their specificity for nuclear structures.These antibodies are commonly used as an in-dicator of SLE-like autoimmunity in mousemodels. TLR9 and TLR7 have been impli-cated in the development of ANA against DNAand RNA, respectively (147–151). Despite theirsimilarity in function and specificity, however,TLR7 deficiency is protective on an MRL/lprbackground, whereas TLR9-deficient mice suf-fer accelerated disease (148, 152). The reasonfor this unexpected difference in the roles ofTLR7 and TLR9 remains unclear, although re-cent work has demonstrated that the increasedpathology in TLR9-deficient mice still requiresTLR7 function (152). Future work will surelyfocus on understanding the mechanism(s) be-hind TLR9’s protective effects. Further evi-dence for the primary role of TLR7 in SLEpathology comes from the discovery that a du-plication of the tlr7 gene is responsible for theY chromosome autoimmune accelerator (Yaa)locus phenotype in various strains of mice (153–155), and overexpression of TLR7 throughtransgenesis is sufficient to induce SLE-likedisease (156).

As discussed in greater detail below, theself nucleic acids that activate TLRs (or otherinnate receptors) during autoimmune diseaseare typically released from dead or dying cells.TLR-mediated pathology is primarily drivenby activation of antigen-presenting cells (APCs)such as DCs, macrophages, and B cells; thesecells express TLRs and can acquire nucleic acidligands, either by receptor-mediated uptake orby endocytosis/phagocytosis. Recently, a typeI IFN signature has been described in SLEpatients as well as in mice with SLE-like dis-ease (157, 158). Activation of pDCs via TLR7and/or TLR9 contributes significantly to thiscytokine profile. The production of type I IFNenhances many aspects of the immune response,including B cell activation and the productionof autoantibodies (159). In addition, TLR

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Immune complexes(ICs): antibodymolecules bound toantigen. In this review,we use this term torefer to antibodiesbound to nucleic acidsor nucleic acid/proteincomplexes

activation in macrophages and other DCsubsets induces production of inflammatorycytokines such as TNF-α that can exacer-bate disease. Thus, linking viral nucleic acidrecognition by innate receptors to inductionof type I IFN and inflammatory cytokines canpotentially amplify the risk of autoimmunityassociated with self nucleic acid recognition.

For an innate immune response against selfnucleic acids to occur, multiple checkpointsmust be overcome. These checkpoints collec-tively function to establish a threshold for acti-vation that ensures self/nonself discrimination.One such checkpoint can be that the specificityof innate receptors favors recognition of for-eign nucleic acids. Second, mechanisms are inplace to reduce the levels of self ligands. Finally,the accessibility of receptors is regulated by sub-cellular compartmentalization. In the followingsections, we discuss examples of each of thesecheckpoints, with particular emphasis on theirrelevance to TLR activation.

The Nature of Self NucleicAcid Ligands

In principle, the simplest mechanism to avoidself recognition is to target sequence motifs orchemical modifications unique to microbes. ForTLR9, evidence suggests that such mechanismsmay reduce the likelihood of self-DNA recog-nition. Unmethylated CpG DNA is a muchmore potent activator of TLR9 than are methy-lated motifs, and in mammalian genomes CpGmotifs are largely methylated (20). Such sup-pression of stimulatory motifs in the genomedecreases the frequency of TLR9-activatingligands, yet it is quite clear that the remain-ing motifs are sufficient to activate TLR9 whenadditional regulatory mechanisms fail. As dis-cussed above, defining motif preferences forTLR7 and TLR8 has been more difficult.While certain sequences may be more likely toactivate TLR7, many self-RNAs appear capableof stimulating the receptor. Thus, self/nonselfdiscrimination by TLR7 and TLR9 cannotbe assured simply based on the sequence ofligands.

Despite the stimulatory potential inherentto host DNA and RNA, these ligands are typ-ically not accessible to TLRs. The topologyof TLRs requires ligands to be extracellularor within vesicles whose lumen is topologicallyequivalent to the extracellular space (i.e., en-dosomes and lysosomes). Thus, for any TLRto respond to self nucleic acid, DNA or RNAmust be released from a cell. Typically, releaseof self nucleic acid only occurs during cell death,although there are some interesting exceptions,such as release of DNA “nets” by neutrophils,which is not discussed here but has been else-where (160, 161). Conceptually, necrosis is themost likely form of cell death to result in releaseof stimulatory self-DNA and -RNA. In addi-tion, pyroptosis, a form of cell death that resultsfrom inflammasome activation, can also lead tonuclear content release. In contrast, apoptosisleads to laddering of the genome and packagingof cellular contents into apoptotic bodies, whichare typically noninflammatory. However, re-cent evidence suggests that DNA from apop-totic cells retains the ability to stimulate TLR9.The average length of the DNA found in theDNA:anti-DNA antibody immune complexes(ICs) in the blood from lupus patients is simi-lar to the size of DNA generated from cleavedchromatin during apoptosis (162). Moreover,repeated injection of apoptotic cells can lead toautoantibody production, suggesting that apop-totic cells can stimulate autoreactive B cells withspecificities for nuclear antigens (163). Perhapsthe strongest evidence that apoptotic nucleicacids remain potential self ligands comes fromthe observation that failure to clear apoptoticcells in vivo is associated with several autoim-mune disorders. As discussed in greater detailbelow, apoptotic cells that are not efficientlycleared can undergo secondary necrosis, whichalso leads to release of self nucleic acids.

Mechanisms of Self Ligand Clearance

Most, if not all, self nucleic acid ligands asso-ciated with innate immune activation duringautoimmunity originate from dying cells.Accordingly, various regulatory mechanisms


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function to remove these stimulatory ligandsbefore they engage innate receptors.

Clearance of apoptotic cells is a key home-ostatic process in all multicellular organ-isms. Macrophages recognize features on apop-totic cells and engulf them via phagocytosis.The most well-characterized and evolutionarilyconserved “eat me” signal is phosphatidylser-ine (PS), a component of the inner leafletof the plasma membrane that becomes ex-posed in apoptotic cells (Figure 3). The known

secreted molecules that bind to PS and facili-tate apoptotic cell uptake are MFG-E8, Gas6,and Protein S (164). MFG-E8 binds αvβ3 in-tegrins, which are found on phagocytic cells,whereas Gas6 and Protein S bind the recep-tor tyrosine kinase family members Tyro3, Axl,and Mer (TAM receptors). A nonredundantrole for MFG-E8 has been demonstrated usingMFG-E8-deficient female mice that develop aSLE-like autoimmune disease with ANA andglomerular nephritis (165). Mice deficient in

MFG-E8Gas6Protein S

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Figure 3Multiple mechanisms reinforce self/nonself discrimination by innate receptors that recognize nucleic acids. Self nucleic acid ligands forinnate immune receptors are released from necrotic cells or from apoptotic cells that undergo secondary necrosis. Receptors that bindand clear apoptotic or necrotic cells reduce the likelihood that self ligands will encounter innate receptors. DNaseI can also destroyextracellular DNA ligands prior to internalization; DNaseII plays a similar role within lysosomes after ligand internalization. Selfnucleic acids that bypass these clearance or degradation mechanisms may associate with proteins that facilitate uptake and delivery tointracellular compartments. Antibodies (either on the B cell surface or as immune complexes), antimicrobial peptides (LL37), orHMGB1 can all mediate nucleic acid uptake. DNA that escapes the phagosome or otherwise gains access to the cytosol (e.g., reversetranscribed retroelements) may activate cytosolic sensors. Trex1 (DNaseIII) can degrade these cytosolic ligands. Whether self-RNAcan in some instances activate RLRs is not yet known.

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all three TAM receptors develop splenomegalyand hyperresponsive APCs (166). TAM recep-tors have been implicated in negative regula-tion of TLR signaling, so the phenotype ofTAM receptor–deficient mice cannot be solelyattributed to defective apoptotic cell clearance.Nevertheless, failure to clear apoptotic cells inthese mice likely leads to release of self nucleicacid ligands and contributes to disease. In addi-tion to the TAM receptors, members of the Tcell immunoglobulin and mucin domain (TIM)family have been shown to bind PS and to fa-cilitate apoptotic cell uptake. In fact, severalputative PS receptors have been identified, al-though the importance of individual receptorsfor apoptotic cell clearance and whether recep-tors bind PS directly has remained controversial(164, 167–169).

An important issue that remains poorly un-derstood is why macrophages are not activatedby nucleic acid ligands that are contained withinengulfed apoptotic cells. One potential expla-nation is that these ligands are degraded bylysosomal hydrolases such as DNaseII, therebypreventing TLR recognition. DNaseII is aubiquitously expressed endonuclease that lo-calizes in lysosomal compartments and de-grades chromosomal DNA from apoptotic cellsand expelled nuclei from erythroid precursors(123, 170). DNaseII-deficient mice die fromsevere anemia due to an inability to breakdown expelled erythrocyte nuclei in the fe-tal liver. Although the severe anemia and em-bryonic lethality could be rescued by type IIFN receptor (IFNAR) deficiency (123), themice later develop arthritis and produce in-flammatory cytokines such as TNF, IL-1β, andIL-6 (171). The sensor responsible for thetype I IFN production has not been identi-fied, yet the observation that additional de-ficiency in MyD88 and TRIF did not res-cue the embryonic lethality suggests that acytosolic sensor is involved (123). AlthoughTLR9 has been excluded, whether other TLRsare responsible for the later production of in-flammatory cytokines observed in DNaseII-/IFNAR-double-deficient mice is not yet clear(124).

In addition to clearance of apoptotic cells,mechanisms have evolved to facilitate removalof necrotic cells. One proposed mechanismby which necrotic cells may be cleared isthrough the serum complement protein C1q(Figure 3). C1q can clear apoptotic andnecrotic cells in an IgM-dependent manner(172). Another protein involved in avoiding selfligand recognition is serum amyloid P (SAP).SAP is believed to bind chromatin and preventDNA:protein complexes from becoming im-munogenic (173). Interestingly, C1q- and SAP-deficient mice develop ANA. Moreover, mosthumans deficient in C1q develop severe SLE(174). More recently, receptors for necrotic cellclearance, such as CLEC9A and Mincle, havebeen identified (175, 176). Whether deficiencyin these receptors increases the likelihood ofresponses to self nucleic acids has not beenexamined.

An additional mechanism that reduces thepotential for recognition of self nucleic acidsis the degradation of extracellular nucleicacids prior to recognition by innate receptors.DNaseI is the major endonuclease foundin serum and urine, where it functions todegrade extracellular dsDNA into tri- ortetraoligonucleotides (177). DNaseI-deficientmice develop features of SLE, including ANA,glomerulonephritis, and eventual death (178).In humans, mutations in dnaseI are associatedwith SLE, and low DNaseI activity correlateswith glomerulonephritis in patients (179).These studies point to DNaseI as having a crit-ical role in degrading free DNA and resultingin destruction of an otherwise stimulatory selfligand.

Compartmentalization ofInnate Receptors

Failure of the clearance mechanisms discussedin the previous section does not a priori result inactivation of nucleic acid–sensing TLRs. As dis-cussed in other sections of this review, TLR3,TLR7, TLR8, and TLR9 are localized intra-cellularly within endolysosomal compartments.For reasons that are not well understood, free


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self nucleic acids are not efficiently delivered tothese compartments. Instead, multiple mecha-nisms facilitate uptake of nucleic acid ligandsand short-circuit the compartmentalization ofTLRs. Four main examples illustrate how de-livery of self-DNA to intracellular compart-ments can lead to immune cell activation byself-DNA: Fc receptors (FcRs), B cell receptor(BCR) cross-linking, LL37, and HMGB1.

Binding to FcRs or BCRs can lead to inter-nalization of self ligands followed by traffickingto compartments containing TLRs. The pro-duction of ANA by autoreactive B cells is a keycomponent in the pathogenesis of SLE, andthe initial break in tolerance is thought to in-volve activation of B cells by ICs containing nu-cleic acid. B cells primarily utilize the BCR as amethod to internalize these ICs. Elegant studiesinvolving autoreactive B cells have shown thatthe BCR can facilitate internalization of ICs,which leads to synergistic activation throughthe BCR and TLR9 or TLR7 (150, 151). In-terestingly, there is recent evidence that BCRsignals are sufficient to cause relocalization ofTLR9 to autophagosomes, where it colocalizeswith the BCR after internalization of ICs (180).Although this strategy may allow TLRs to sam-ple the contents of antigens internalized by theBCR, this relocalization of TLR9 may be re-sponsible for the increased responsiveness ofautoreactive B cells to mammalian DNA ligandsafter BCR stimulation (181). These studies il-lustrate that the uptake of ICs by the BCR canlead to nucleic acid sensor and autoreactive Bcell activation, which can potentially tilt the bal-ance toward autoimmunity. Another method ofinternalization of self ligands is through FcRs.FcRs are expressed on a wide range of im-mune cell types, where they bind Fc regionsof antibodies and provide inhibitory or activat-ing signals. In addition to the contribution ofFcRs to activating or inhibitory signals, furtherstudies using serum from SLE patients haveshown that FcRs can deliver DNA-containingICs into TLR9-containing compartments inhuman pDCs (162). Thus, BCRs and FcRs al-low internalization of ICs that potentially carryself ligands.

Another protein that facilitates entry of selfnucleic acids into immune cells is HMGB1.HMGB1 is a ubiquitous, highly conservedDNA-binding protein that recognizes theminor groove of DNA with low sequencespecificity to enable bending during transcrip-tion initiation. During apoptosis, chromatindeacetylation enhances HMGB1-DNA associ-ation, which results in nuclear retention of theprotein; however, during necrotic cell deaththe deacetylation program is not engaged,and HMGB1 is passively released from dyingcells (182). Interestingly, monocytes and DCsalso secrete HMGB1 in response to proin-flammatory cytokines or TLR ligands (183).These studies along with the observation thatadministering HMGB1-blocking antibodiesduring sepsis is sufficient to delay lethality inmice have led to the suggestion that HMGB1 isa mediator of inflammation (183). Since theseinitial reports, investigators have shown thatextracellular HMGB1 associates with DNA re-leased from necrotic cells or DNA-containingICs, thereby inducing TLR9 responses toself-DNA, rather than HMGB1 directlystimulating immune cells (184, 185). Some re-ports have indicated that HMGB1 associationwith receptor for advanced glycosylation endproducts (RAGE) is required to engage TLR9responses. In this model, HMGB1 facilitatesuptake of self nucleic acids by engaging RAGEat the cell surface and mediating internaliza-tion. Recently, HMGB1 and the related familymembers HMGB2 and HMGB3 have all beenshown to bind model DNA and RNA ligandsas well as to mediate viral recognition (186).Simultaneous siRNA knockdown of all threefamily members blocked sensing by TLRsas well as the cytosolic nucleic acid sensors,suggesting that these proteins play a universalrole in nucleic acid sensing (186). These resultsindicate that the presence of HMGB proteinsduring infection may increase the probabilityof detecting rare viral ligands; however, thisenhanced sensitivity may come at the potentialcost of recognizing self nucleic acids thatmay be present in the local environment aswell. Understanding whether HMGB family

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members are absolutely required for nucleicacid sensing as well as the role these proteinsmay play in self/nonself discrimination areimportant areas of future investigation.

Recently, a role for the antimicrobial pep-tide LL37 was also defined in mediating TLR9and TLR7 responses to self nucleic acids dur-ing psoriasis. When extracts derived from psori-atic lesions were compared with healthy skin intheir ability to elicit type I IFN responses frompDCs, LL37 was identified as the immuno-logically active component in psoriatic samples(187). This cationic, amphipathic antimicrobialpeptide is highly induced in psoriatic lesionsand bears structural similarity to α-helical pep-tides used to transfect DNA (188). LL37 bindsand protects DNA and RNA from nucleases andgreatly enhances uptake into early endosomes(187, 189). Interestingly, LL37 complexed withclass B CpG ODN induces increased type I IFNproduction, suggesting that the cationic peptidecan alter uptake and/or trafficking (190).

Thus, the four mechanisms discussed in thissection (uptake through the BCR and FcRs orbinding to HMGB1 and LL37) share the prop-erty of facilitating delivery of self nucleic acidligands to intracellular, TLR-containing com-partments. In this way, the sequestration of thereceptors that appears central to self/nonselfdiscrimination is carefully regulated. In addi-tion, these complexes appear to protect nu-cleic acids from nucleases. Whether LL37 andHMGB simply enhance general uptake of nu-cleic acids to endosomal compartments or ac-tively influence the delivery of these complexesto compartments specialized for IFN produc-tion is yet to be shown. These proteins mayplay a role in directly interacting with the TLRitself, as has been suggested for HMGB1 (184).

Regulation of Cytosolic Sensors

Although extensive studies have been per-formed to address the role of TLRs inautoimmune disease, much less is known aboutthe role of cytosolic nucleic acid sensors in thepathogenesis of autoimmunity. For most cy-tosolic RNA and DNA sensors, the mechanisms

of self/nonself discrimination remain incom-pletely defined. The specificity of certaincytosolic sensors may provide the basis forself/nonself discrimination. For example, RIG-I’s specificity for uncapped 5′-triphosphateRNAs favors recognition of foreign RNAs(101). However, not all host RNAs are capped,suggesting that additional regulatory mech-anisms may aid self/nonself discrimination.Determination of the origin of DNA may belargely based on localization, as DNA outsideof the nucleus is clearly an anomaly, although,as discussed below, mechanisms regulatecytosolic DNA recognition as well.

One of the first studies to highlight thecontribution of other DNA sensors to autoim-mune pathology was the analysis of DNaseII-deficient mice. As mentioned above, DNaseIIdegrades DNA from apoptotic cells and DNAfrom expelled nuclei from erythroid cells.DNaseII-knockout mice are embryonic lethaldue to severe anemia, but DNaseII-/IFNAR-double-deficient mice that are rescued from theanemia phenotype later develop polyarthritis,which is dependent on TNF-α (171). It is notyet clear which innate sensors are activatedin these animals, but IFN-β appears to beresponsible for the anemia phenotype, asIFNAR deficiency rescues the embryoniclethality (123). The type I IFN production cor-relates with an increase in macrophage numberin the fetal liver and thymus. Moreover,these macrophages contain undigested DNA,suggesting that macrophages, not pDCs, areresponsible for the type I IFN that is produced.

More recently, Trex1 (also known asDNaseIII) was identified as a negative regu-lator of cytosolic DNA sensors. Trex1 is themost abundant 3′ to 5′ exonuclease in the cell.It localizes to the ER and cytosol and acts onboth ssDNA and dsDNA (191). Furthermore,Trex1 is the only 3′ to 5′ exonuclease known tohave type I IFN–inducible expression. Trex1-deficient mice succumb to autoimmune inflam-matory myocarditis that is rescued by additionaldeficiency in IRF3, IFNAR, or RAG2. How-ever, Trex1-/RAG2-double-deficient mice stillinduce IFN-β, which implies that disease is


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initiated by innate immune activation. Remark-ably, Trex1 deficiency leads to an increasein cytosolic DNA derived from endogenousretroelements, indicating that one of the func-tions of Trex1 is to degrade cytosolic DNA de-rived from reverse transcribed retroelements.This work suggests that cytosolic DNA sen-sors need to be negatively regulated to pre-vent autoimmunity in a cell-intrinsic manner,as opposed to TLRs that work in a non–cell au-tonomous manner. Moreover, these data pro-vide mechanistic insight for earlier findings thatTrex1 is a susceptibility locus for patients withthe neurological inflammatory disease Aicardi-Goutieres Syndrome (AGS). Heterozygousmutations in Trex1 cause familial chilbain lu-pus, which is considered a SLE subtype and hasalso been linked to SLE (192). Both AGS andchilbain lupus can show evidence of ANA.

CONCLUSIONS ANDPERSPECTIVES

In the sections above, we have detailed howmultiple innate immune receptors utilize nu-cleic acids as a signature of microbial infec-tion. These receptors have evolved to samplethe lumenal contents of endolysosomal com-partments as well as the cytosol. This strategy

enables recognition of diverse microbes with alimited set of receptors. The past decade hasseen rapid progress in the identification of thereceptors involved in nucleic acid sensing aswell as their downstream signaling pathways.Future research will certainly add new com-ponents to these networks, especially in thenewer field of cytosolic innate immune path-ways. These discoveries are likely to shed lighton some of the remaining puzzles in the field,such as how differential gene induction is me-diated by individual innate receptors (e.g., typeI IFN by TLR7 and TLR9).

A trade-off associated with innate immunerecognition of nucleic acids is the potentialfor self recognition. As discussed above, inap-propriate recognition of self nucleic acids hasbeen implicated in several autoimmune disor-ders. The risk stems from the inherent simi-larity between self versus foreign nucleic acids.Accordingly, various mechanisms have evolvedto ensure reliable self/nonself discrimination.Although some of these mechanisms have beendefined, additional mechanisms likely remainundiscovered. The fact that failure of thesemechanisms can result in quite distinct diseases(e.g., SLE versus inflammatory myocarditis)underscores the nonoverlapping roles of the in-nate immune pathways discussed in this review.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

The authors are supported by funding from the NIH (R01AI072429, R21AI079358, P01AI063302to G.M.B.; Ruth L. Kirschstein Predoctoral Fellowship to M.L.M.), the Lupus Research Institute,and the University of California Cancer Research Coordinating Committee.

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IY29-Frontmatter ARI 4 February 2011 21:56

Annual Review ofImmunology

Volume 29, 2011Contents

Innate Antifungal Immunity: The Key Role of PhagocytesGordon D. Brown � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Stromal Cell–Immune Cell InteractionsRamon Roozendaal and Reina E. Mebius � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �23

Nonredundant Roles of Basophils in ImmunityHajime Karasuyama, Kaori Mukai, Kazushige Obata, Yusuke Tsujimura,

and Takeshi Wada � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Regulation and Functions of IL-10 Family of Cytokinesin Inflammation and DiseaseWenjun Ouyang, Sascha Rutz, Natasha K. Crellin, Patricia A. Valdez,

and Sarah G. Hymowitz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Prevention and Treatment of Papillomavirus-Related CancersThrough ImmunizationIan H. Frazer, Graham R. Leggatt, and Stephen R. Mattarollo � � � � � � � � � � � � � � � � � � � � � � � � 111

HMGB1 Is a Therapeutic Target for Sterile Inflammation and InfectionUlf Andersson and Kevin J. Tracey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 139

Plasmacytoid Dendritic Cells: Recent Progress and Open QuestionsBoris Reizis, Anna Bunin, Hiyaa S. Ghosh, Kanako L. Lewis, and Vanja Sisirak � � � � � 163

Nucleic Acid Recognition by the Innate Immune SystemRoman Barbalat, Sarah E. Ewald, Maria L. Mouchess, and Gregory M. Barton � � � � � � 185

Trafficking of B Cell Antigen in Lymph NodesSantiago F. Gonzalez, Søren E. Degn, Lisa A. Pitcher, Matthew Woodruff,

Balthasar A. Heesters, and Michael C. Carroll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 215

Natural Innate and Adaptive Immunity to CancerMatthew D. Vesely, Michael H. Kershaw, Robert D. Schreiber,

and Mark J. Smyth � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 235

Immunoglobulin Responses at the Mucosal InterfaceAndrea Cerutti, Kang Chen, and Alejo Chorny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

HLA/KIR Restraint of HIV: Surviving the FittestArman A. Bashirova, Rasmi Thomas, and Mary Carrington � � � � � � � � � � � � � � � � � � � � � � � � � � � 295

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IY29-Frontmatter ARI 4 February 2011 21:56

Mechanisms that Promote and Suppress Chromosomal Translocationsin LymphocytesMonica Gostissa, Frederick W. Alt, and Roberto Chiarle � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 319

Pathogenesis and Host Control of Gammaherpesviruses: Lessons fromthe MouseErik Barton, Pratyusha Mandal, and Samuel H. Speck � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 351

Genetic Defects in Severe Congenital Neutropenia: Emerging Insights intoLife and Death of Human Neutrophil GranulocytesChristoph Klein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 399

Inflammatory Mechanisms in ObesityMargaret F. Gregor and Gokhan S. Hotamisligil � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 415

Human TLRs and IL-1Rs in Host Defense: Natural Insightsfrom Evolutionary, Epidemiological, and Clinical GeneticsJean-Laurent Casanova, Laurent Abel, and Lluis Quintana-Murci � � � � � � � � � � � � � � � � � � � � 447

Integration of Genetic and Immunological Insights into a Model of CeliacDisease PathogenesisValerie Abadie, Ludvig M. Sollid, Luis B. Barreiro, and Bana Jabri � � � � � � � � � � � � � � � � � � � 493

Systems Biology in Immunology: A Computational Modeling PerspectiveRonald N. Germain, Martin Meier-Schellersheim, Aleksandra Nita-Lazar,

and Iain D.C. Fraser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 527

Immune Response to Dengue Virus and Prospects for a VaccineBrian R. Murphy and Stephen S. Whitehead � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

Follicular Helper CD4 T Cells (TFH)Shane Crotty � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 621

SLAM Family Receptors and SAP Adaptors in ImmunityJennifer L. Cannons, Stuart G. Tangye, and Pamela L. Schwartzberg � � � � � � � � � � � � � � � � � 665

The Inflammasome NLRs in Immunity, Inflammation,and Associated DiseasesBeckley K. Davis, Haitao Wen, and Jenny P.-Y. Ting � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 707

Indexes

Cumulative Index of Contributing Authors, Volumes 19–29 � � � � � � � � � � � � � � � � � � � � � � � � � � � 737

Cumulative Index of Chapter Titles, Volumes 19–29 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 744

Errata

An online log of corrections to Annual Review of Immunology articles may be found athttp://immunol.annualreviews.org/errata.shtml

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Documents

Nucleic Acid Recognition by the Innate Immune System