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Seminars in Immunology 21 (2009) 208–214

Contents lists available at ScienceDirect

Seminars in Immunology

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he innate immune response to DNA

kosua Vilaysane, Daniel A. Muruve ∗

epartment of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada

r t i c l e i n f o

eywords:LR9nflammasome

a b s t r a c t

As a component of all living cells and microbes, it is not surprising that organisms have evolved mecha-nisms to detect foreign or aberrant DNA and trigger an innate immune response. TLR9 is an endosomal

ytosolic DNA sensornnate immunity

membrane bound receptor that is widely studied and the best understood DNA sensor. However, the exis-tence of TLR9-independent DNA sensing pathways have been recognized for many years. Recently, novelcytosolic DNA sensors have been uncovered that include ZBP1 or DNA-dependent activator of interferon-regulatory factors (DAI) and a DNA sensing inflammasome consisting of the HIN200 protein, absent inmelanoma 2 (AIM2). In combination with TLR9, these receptors provide a diverse repertoire of mecha-nisms to alert the cell to microbial DNA and possibly aberrant host DNA leading to the activation of the

innate immune system.

. Introduction

The innate immune system is an integral part of the hostesponse to invading pathogens and other danger signals. It offershe first line of defense against viral and bacterial organismshrough the recognition of pathogen associated molecular pat-erns (PAMPs) by a range of germline encoded pattern recognitioneceptors (PRRs). Activation of the innate immune system leadso diverse cellular responses including the induction of interferonegulatory factors (IRF) 3, 7 and NF�B that regulates type I inter-eron and pro-inflammatory cytokine gene expression respectively1]. The host recognition of nucleic acids is an essential compo-ent of the innate immune system. It was first suggested in the960s that nucleic acids were a stimulus for interferon produc-ion in cells [2]. In 1963, Rotem et al. found that non-viral foreign

urine nucleic acids, including DNA could induce an antiviral inter-eron response in chick embryo fibroblasts and mouse cells [3], anffect that was also observed using yeast nucleic acids [4]. Surpris-ngly, these intriguing studies were not actively pursued until thend of the century, when it was observed that pathogenic and self-NA upregulated the expression of host immune response genes

n various different cell types [5–7]. Coinciding with the discov-ry of Toll-like receptors (TLRs) and other PRRs, the understanding

f nucleic acid recognition by the host has since increased expo-entially. The sensing of microbial DNA and RNA is distinct andediated by different innate pathways and effector molecules. For-

ign DNA and RNA can arise directly from the genomes of invading

∗ Corresponding author at: University of Calgary, 3330 Hospital Dr. NW, Calgary,lberta, T2N 4N1 Canada. Tel.: +1 403 220 2418; fax: +1 403 210 3949.

E-mail address: dmuruve@ucalgary.ca (D.A. Muruve).

044-5323/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2009.05.006

© 2009 Elsevier Ltd. All rights reserved.

pathogens or from the transcription/replication of viral and bacte-rial (pathogenic) genes. Non-microbial (endogenous) DNA can alsohave a significant impact on the innate immune system sheddingnew light on several clinical syndromes such as systemic lupus ery-thematosus (SLE) that are typified by autoimmunity to self-DNA.Recent studies have advanced the understanding of DNA sensingand the host immune response. The discovery of ZBP1 (DLM-1 orDAI, DNA-dependent activator of interferon-regulatory factors), theAIM2 (absent in melanoma 2) inflammasome and the characteri-zation of nucleases such as TREX1 in the innate immune responseto DNA, in combination with the increased understanding of TLR9biology, provides clarity to a phenomenon first discovered over 40years ago [8–13].

2. TLR9: a membrane bound endosomal DNA sensor

The most extensively studied group of PRRs is the TLRs. Thesereceptors activate signaling events that result in the transcriptionand regulation of pro-inflammatory cytokines and type I inter-ferons. Since the discovery of the first human TLR [14], signalingpathways downstream of these receptors have been well delin-eated. TLRs contain leucine rich repeat (LRR) motifs, a Toll/IL-1Rhomology domain and are considered type I integral membraneglycoproteins. Once stimulated, the TLRs dimerize, change in con-figuration and recruit TIR domain containing adaptor molecules(MyD88, TIRAP/MAL, TRIF/TICAM1 and TRAM) that initiate signaltransduction [15].

TLR9 is the membrane bound receptor for DNA in the cell (Fig. 1).Hemmi et al. first proposed the function of human TLR9 by show-ing that it responded to bacterial unmethylated CpG DNA but notself-DNA, resulting in MyD88-dependent NF-�B activation [16].Studies also suggested the endosomal location of TLR9 with its lig-

A. Vilaysane, D.A. Muruve / Seminars in Immunology 21 (2009) 208–214 209

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ig. 1. Overview TLR9 interactions and signaling. TLR9 is localized in the ER and traroteins UNC93B1, gp96 and PRAT4A. Following endosomal acidification and liganctivate NF�B and induce the expression of type I interferons via IRF7.

nd binding sites within the vesicular space and away from theytoplasm [16,17]. In humans, TLR9 is found primarily in plasmacy-oid dendritic cells (pDCs), specialized cell types that are capable ofroducing large amounts of type I interferons during viral infection18]. The importance of TLR9 in host antiviral defense is confirmedy several studies that demonstrate a requirement for TLR9 inhe response to DNA viruses such as herpes simplex virus and

urine cytomegalovirus [19–21]. Ligands specific for TLR9 inducehe production of interferon-� in pDCs cells in a MyD88-dependent

anner that differs from signaling downstream of most other TLRs.pon TLR9 stimulation within the endosome, MyD88 generates aolecular complex with IRAK-1, IRAK-4 and the interferon reg-

latory factor, IRF-7 [22,23]. IRF-7 is phosphorylated by IRAK-1hich then translocates to the nucleus to mediate the transcrip-

ion of interferon-� genes [24]. In this regard, and unlike signalingo NF�B, IRAK-1 appears to be an essential component in the MyD88athway that results in IRF-7 activation. IRAK-1 deficient mice are

ncapable of activating IRF-7 although NF�B activation, which isependent primarily on IRAK-4 remains intact [23,24]. The activa-ion of NF�B downstream of TLR9 is also dependant on TRAF6 E3biquitin ligase activity [22].

TLR9 activation by DNA requires endosomal acidification andaturation consistent with the observation that the receptor is

ot constitutively active in resting cells [17,25]. TLR9 resides inhe endoplasmic reticulum, but not the Golgi of unstimulatedDCs. The internalization of CpG DNA induces the translocationf TLR9 to endolysosomes and subsequent activation [26,27]. Thendoplasmic reticulum UNC93B1 membrane spanning protein isecognized as a key player in TLR9 trafficking, through its abilityo bind strongly to the transmembrane regions of the nucleotideensing TLRs [28–30]. Mice that carry an H412R mutation in theNC93B1 gene (3d mice) are unable to interact with nucleotide

ensing TLRs and display impaired TLR3, TLR7 and TLR9 signaling.he biology is significant since 3d mice and patients with muta-ions in UNC93B1 have defects in the response to DNA viruses suchs herpes simplex virus and murine cytomegalovirus [29,31]. Thehaperone protein gp96 and PRAT4A (protein associated with TLR4)

lso interact with TLR9 (and TLR7) and are necessary for proper pro-ein folding and trafficking from the ER to endolysosomes [32,33].owever, the exact mechanism by which these proteins mediate

he effects needed for proper TLR9 signaling remain unknown. Oncen the endosome, binding between the ligand and receptor occurs

tes to DNA-containing endosomes to initiate signaling. Translocation requires ERraction, TLR9 is activated and binds MyD88, IRAK4 and IRAK1 that are essential to

and MyD88 is rapidly recruited to these areas to initiate the signal-ing pathway. Endosomal acidification and maturation is requiredto cleave the ectodomain of the TLR9 receptor to the functionalreceptor. Once cleaved, the truncated form of TLR9 is able to recruitMyD88 and initiate signaling [34,35]. The complex regulation ofTLR9 signaling illustrates an elegant mechanism to separate therecognition of microbial and self-nucleic acids and protect the hostfrom an inappropriate immune response.

Despite the many levels of regulation, under the proper cir-cumstances, TLR9 is also capable of recognizing self-DNA andis associated with autoimmunity [36,37]. The specialized local-ization of TLR9 prevents it from encountering self-DNA in thecytosol or present in the extracellular space. Unlike viruses andbacteria that have evolved specialized mechanisms to gain entryinto cells, self-DNA has a limited ability to be internalized intoendosomal compartments [25,38]. The importance of this compart-mentalization in preventing recognition of self-DNA is illustratedby several studies. LL37 is an antimicrobial peptide producedin the skin of patients with psoriasis [39,40]. LL37 complexedwith human DNA is retained in early endosomes and facilitatesthe activation of TLR9 in dendritic cells. Similarly, TLR9 activa-tion is increased by DNA–cationic lipid (DOTAP) complexes orimmunoglobulin–chromatin immune complexes that also enhanceinternalization and DNA-targeting to endosomes [25,37,38]. TLR9targeting to the endosome is regulated by its transmembrane andcytoplasmic domains [41,42]. Using a TLR9/TLR4 chimeric protein,Barton et al. showed that TLR9 expressed on the cell surface couldbe activated by mammalian and vertebral DNA [41]. Together, thesestudies strongly suggest that it is the localization of TLR9, andnot the sequence specificity of the DNA that determine receptoractivation. Wagner and co-workers, in a series of studies, haveprovided compelling evidence that support this view [38,43,44].Using a panel of oligonucleotides, the determinants of TLR9 acti-vation were shown to depend primarily on targeting and retentionof DNA to early and late endosomes. Oligonucleotides complexedwith cationic lipids (DOTAP) or containing 3′-polyguanosine tails(to increase endosomal translocation and nuclease resistance) acti-

vated dendritic cells in a CpG-independent manner. Studies bythis group identified the phosphodiester 2′ deoxyribose backboneof single-stranded DNA, and not sequence as the primary deter-minant of TLR9 activation. However, CpG-containing DNA stilldisplayed a higher binding affinity for TLR9 than non-CpG contain-

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ng sequences lending some capacity to differentiate self-DNA andicrobial DNA at a molecular level [38,43].

. Cytosolic DNA sensors

The introduction of TLR9 antagonists and TLR9 deficient miceelped uncover the presence of a TLR9-independent cytosolic DNAensor (s) [44–53]. For example, several studies showed that theesponse to DNA vaccination remained intact in TLR9 deficient

ice [49,50]. Consistent with this, Cortez-Gonzalez et al. foundhat bacterial plasmid DNA could be taken up and activate Bymphocytes in a TLR9-independent manner [54]. The existencef a TLR-independent type I interferon pathway was also sug-ested following L. monocytogenes infection that triggered a potentRF-3/interferon-�-dependent immune response in macrophagesacking TRIF, MyD88, TRAM and TLR9 [55]. More recently, sev-ral groups have characterized the TLR-independent response toNA, also referred to as the interferon-stimulatory DNA (ISD)athway [6,44,45,47,56]. Unlike TLR9 activation that occurs inndosomes and influenced by DNA sequence and methylation, theLR-independent response to DNA is believed to be cytosolic andriggered by double-stranded DNA in a size but not sequence-estricted manner [6,7,44]. Furthermore, the sensing of DNA islearly distinct from the TLR system in that it does not require TRIFr MyD88 and it is separate from the cytosolic RNA-sensing RIG-

ike receptors (RLRs) that signal via the adaptor IPS-1 (also knowns Cardif/VISA/MAVS) [6,44,45,47,56]. Several new molecules haveow been described that play a role in the TLR-independent sensingf DNA.

.1. ZBP1

In 2007, Takaoka et al. described a receptor capable of sens-ng cytosolic DNA and regulating the type I interferon response8]. Renaming the molecule DAI, it was previously known underwo names: DLM-1 and Z-DNA binding protein-1 (ZBP1) (Fig. 2)

57,58]. ZBP1 was originally found to be up-regulated in tumortromal cells [58] but was later shown to be highly expressed inumerous tissues, lymphocytes and macrophages [57,59,60]. ZBP1inds uncommon left handed Z-form DNA with high affinity and it

ig. 2. ZBP-1 and signaling. ZBP-1 binds DNA via its Z�, Z� and D3 domains. NF�Bctivation is mediated via a homotypic RHIM interaction with RIP1. RIP3 is alsoequired and interacts with the ZBP-1 RHIM domain. Signaling to type I interferonathways is mediated through the carboxy-terminal of the protein and involvesBK-1 and IRF3.

Immunology 21 (2009) 208–214

shares similar DNA-binding domains (Z� and Z�) to the RNA editingenzyme adenosine deaminase acting on RNase1 (ADAR1) and thevaccinia virus protein E3L (Z�). The presence of multiple C-terminalserine/threonine phosphorylation sites and the observation thatZBP1 is interferon inducible hinted at a role in signal transduc-tion and in the immune response to pathogens [8,58]. Indeed, theoverexpression of ZBP1 mediates an IRF-3-dependent type I inter-feron response to transfected B-DNA and forms a complex withTBK1 and IRF3 through its C-terminal domain [8]. ZBP1 also inter-acts with RIP1 and RIP3 via a RHIM (RIP homotypic interactionmotif) domain that results in NF�B activation [61]. ZBP1-targetingsiRNA reduced the B-DNA and alpha-herpesvirus (HSV-1) activa-tion of IRF-3 and the expression of type I interferons suggestingthat this molecule played a role in the TLR-independent DNA sens-ing mechanism. While the interaction between ZBP1 and certainforms of DNA was known previously, it was not studied exten-sively. Using deletion mutant vectors, Takaoka et al. identifieda new region (D3) that participated in DNA binding in addi-tion to the two previously discovered Z-DNA binding domains(Z� and Z�). Consistent with the previous observations regardingthe TLR-independent recognition of dsDNA, DNA binding to ZBP1occurred irrespective of its sequence but the activation of inter-feron responses was DNA-length restricted with little activationin response to DNA less than 100 bp. From the length studies itwas suggested that DNA was needed to maintain a ZBP1 multi-meric complex required for activation of type I interferon genes[62,63].

While ZBP1 was the first cytosolic DNA sensor identified, Wanget al. found evidence that other redundant DNA sensing pathwaysexisted, though evidence suggested this response to be depen-dent on cell type [62]. In vivo, Ishii et al. showed that ZBP1 andMyD88/TRIF pathways were dispensible for the innate response toB-DNA since the induction of type I interferons and the responseto DNA vaccination was intact in mice genetically deficient in thesemolecules. The innate response to DNA and DNA vaccination reliedon TBK-1 which is downstream of both ZBP1 and MyD88/TRIF (i.e.TLR9) pathways. However since a double knockout approach wasnot utilized the possibility remains that ZBP1 and TLR9 are redun-dant to each other and thus, the biological significance of ZBP1remains to be determined [64].

3.2. AIM2 and the inflammasome

The nucleotide-binding domain leucine rich repeat (NLR) fam-ily of proteins represent a group of intracellular PRRs that mediateNF�B signaling and caspase 1 activation. These cytosolic receptorscontain a NOD domain, LRRs, and a signaling domain (CARD, PYD,or BIR). They have been connected to the recognition of microbialcomponents such as iE-DAP (NOD1, NLRC1) and muramyl dipeptide(NOD2, NLRC2). In the case of these two receptors, stimulation bytheir respective ligands causes them to oligomerize and recruit aserine/threonine kinase (RIP2/RICK) to their CARD domains, result-ing in activation of NF-�B [65]. The majority of NLRs however playa role in the formation of proteolytic complexes termed inflam-masomes [66,67]. The NLRP3 (NALP3) inflammasome is the bestunderstood and consists of NLRP3, the adaptor protein ASC andthe protease caspase 1. Upon activation NLRP3 proteins oligomerizeand recruit via homotypic PYD interactions, the PYD-CARD adap-tor protein ASC. The formation of this complex then recruits andactivates caspase 1 via its CARD domain. Caspase 1 is an inflam-matory caspase that does not participate in apoptosis. Instead,

activated caspase 1 processes numerous diverse cellular substratesincluding the cytokines IL-1� and IL-18. In the case of IL-1�, cas-pase 1 cleaves the 35 kDa pro-IL-1� to the mature and secreted17 kDa cytokine. The potential targets of the inflammasome andcaspase 1 go beyond cytokines such as IL-1� and IL-18. For exam-

A. Vilaysane, D.A. Muruve / Seminars in

Fig. 3. AIM2 inflammasome. The HIN200 family member AIM2 binds DNA via itsHIN200 domain. The oligomerization of the protein with the adaptor ASC recruitspAa

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AIM2 and p202 fall within susceptibility loci for SLE in humans

ro-caspase1 via a homotypic CARD interaction to form the AIM2 inflammasome.ctivated caspase1 mediates processing and secretion of pro-IL-1� and pro-IL-18 inddition to triggering pyroptosis.

le, the inflammasome has been implicated in glycolysis and inn inflammatory form of cell death termed “pyroptosis” [68,69].yroptosis is only recently described in macrophages and con-ists of programmed cell death (associated with loss of membranentegrity, unlike apoptosis) in association with IL-1� and IL-18ecretion. Other NLRs proteins that are known to form inflamma-omes include NLRP1 (NALP1) and NLRP2 (NALP2) that also dependn ASC to link to caspase 1. In contrast, NLRC4 (Ipaf-1) is an NLR

hat forms a caspase 1 inflammasome in the absence of ASC via

direct CARD–CARD interaction. Although caspase 1 appears toe the dominant caspase associated with the inflammasome, other

nflammatory caspases such as caspase 5 can also be recruited to

Immunology 21 (2009) 208–214 211

this platform, however the biological significance remains obscure[66,67].

NLRP3 is known to be activated by numerous microbialand non-microbial stimuli including bacterial peptidoglycan, ATP,monosodium urate and silica crystals suggesting that this inflam-masome senses danger signals through a common but indirectmechanism. Recently, we demonstrated that the adenovirusactivated the NLRP3 inflammasome in macrophages that wasdependent on its dsDNA genome [9]. In the same cellular system,the transfection of naked DNA surprisingly triggered NLRP3-independent but ASC dependent caspase 1 activation and IL-1�maturation identifying a novel DNA-sensing inflammasome. Theactivation of this inflammasome was restricted to dsDNA in asize but not sequence-dependent manner consistent with previousobservations regarding TLR9-independent DNA sensing. As such,the source of DNA was unimportant as viral, bacterial, mammalianand synthetic dsDNA could all activate caspase-1. These results alsohighlight that the innate response to DNA is regulated not onlyby the specificity of the respective innate receptors, but also bythe localization of DNA in the cell. The internalization of viral DNAby adenovirus in macrophages proceeds through phagocytosis andtriggers phagosome-linked PRRs such as the NLRP3 inflammasomeand TLR9 [46,70] whereas cytosolic DNA triggers a completely dif-ferent set of pathways.

Four studies identify the HIN-200 family member, AIM2, as thecytoplasmic sensor that mediates caspase 1 activation in responseto cytoplasmic dsDNA [10–13] (Fig. 3). AIM2 is expressed in thecytosol and contains an HIN200 and a PYD domain. AIM2 bindsdirectly to dsDNA, but not ssDNA through the C-terminal HIN200domain resulting in AIM2 oligomerization and the recruitment ofASC through homotypic PYD interactions. The subsequent recruit-ment and activation of caspase 1 result in the formation of anAIM2 inflammasome that mediates IL-1� maturation and pyrop-tosis. Interestingly, these results document the assembly of aninflammasome by a protein outside of the NLR family and showthe first evidence of a direct interaction between an inflammasomereceptor and its ligand. DNA sensing and inflammasome forma-tion is not a general feature of the other human HIN-200 familymembers, IFIX, IFI16 and MNDA that are primarily localized to thenucleus. Stacey and co-workers reported that the mouse HIN200protein, p202 negatively regulated the AIM2 inflammasome. p202contains two HIN domains but no PYD domain. p202 binds specif-ically to dsDNA in a sequence-independent but length-dependentmanner, similar to AIM2 but cannot bind ASC and therefore appearsto function as a dominant negative inhibitor. However, since p202has no known human ortholog, its relevance remains unclear[13].

Importantly, the AIM2 inflammasome was critical for caspase-1activation and cell death but completely dispensable for interferon-� induction in response to cytoplasmic DNA and thus distinct fromthe TLR-independent DNA sensing or ISD pathway. The discreteredundancy in innate immune pathways eliminates the potentialfor an “Achilles heel” that could be targeted by evolving pathogenssuch as viruses and provides the host several layers of antimicrobialdefense. The physiological situations in which the AIM2 inflam-masome pathway might be engaged and its relative place amongthe other pathogen- or danger-recognition systems remain unclear.The ability to activate cytosolic DNA sensors such as AIM2 withnon-microbial or self-DNA sheds some light on the possible mech-anism that drives the inflammatory response in patients with theautoimmune disease, systemic lupus erythematosus (SLE). Both

and mice, and p202 is differentially expressed between lupus-susceptible and lupus-resistant mice. Further research is requiredto clarify the involvement of the HIN-200 family in this and otherdiseases.

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. DNA sensing and the distinction of self

The recognition of nucleic acids by the innate immune systems regulated at various levels. One level of regulation involves lig-nd specificity of the different PRRs. Due to the ability of virusesnd bacteria to rapidly evolve new strategies to avoid detection,t is logical to use a sensing system that targets critical microbialomponents that are present during all stages of their life cycle,ndispensable for pathogen survival but absent from the host. Thus,ighly conserved distinct pathogenic RNA and DNA sequences andtructures are excellent targets for host recognition of foreign mate-ial. RLRs and TLRs detect specific patterns common in foreign RNAnd DNA that are absent in host material such as long dsRNA, 5′-riphosphate RNA and unmethylated CpG DNA [1,16,71]. In the casef DNA viruses, which often use the host machinery to replicate,he distinction between self-DNA and viral DNA becomes less obvi-us. Regulation can therefore also occur based on PRR localizations described in the previous section. In this regard, the regulationnd endosomal localization of receptors such as TLR9 prevents thenappropriate activation by endogenous DNA and focuses the innateesponse in compartments commonly used by pathogens [41,44].he innate immune system has also been observed to respondo DAMPs (danger associated molecular patterns) released dur-ng infection and cellular injury. These endogenous signals includeTP, ROS (reactive oxygen species), uric acid and lysosomal stress

72–75]. DAMPs signal to the host that cells are injured or dyingn an uncontrolled manner. These danger signals are important asying cells may very well contain viral or microbial components.hus DAMPs may act as an adjuvant to sensitize the cell to less dis-inct pathogenic constituents such as DNA and elicit an immuneesponse. This process ensures that the immune system is primedt sites of potential infection and suggests that the response to DNAay also depend on the context and cellular conditions in whichNA is presented.

.1. DNA regulating mechanisms and autoimmunity

The obvious problem with the use of nucleic acids as a targets the fact that nucleic acids are also present in the host. Whileystems have evolved to separate and delineate between host andathogenic nucleic acids, they have also been known to fail, con-ributing to the pathogenesis of autoimmune diseases such asLE [36,37]. SLE is a multifactorial disease caused by a combina-ion of genetic and environmental factors. In SLE, the presence ofntinuclear antibodies against DNA and DNA structures such asucleosomes [76,77] causes a wide range of pathology includingrthritis, glomerulonephritis and vasculitis [78]. In addition to theegulatory mechanisms described above, the body must maintainonstant vigilance in its clearance of endogenous unsequesteredNA to avoid the inappropriate activation of the innate immune

ystem and the development of autoimmunity. Mammalian DNAs usually sequestered in the nucleus or mitochondria of cellsxcept during replication or cell death when the nuclear enve-ope is compromised. Host nucleases such as DNaseI, DNaseII andREX1 (DNaseIII) degrade endogenous DNA found in the extracellu-

ar space, endosomes and the cytosol. The presence of nucleases inndolysosomal compartments also limits the activation of innateeceptors such as TLR9 by rapidly degrading internalized DNA.his is exemplified by the observation that DNA modificationshat create nuclease resistant phosphothiorate-stabilized or 3′-olyguanosine oligonucleotides are potent TLR9 activators [38,43].

herefore, it is not surprising that the failure to effectively clearndogenous DNA can lead to an inappropriate immune responseith serious consequences.

Numerous studies show that nuclease deficiency or functionalefects results in inappropriate immune activation. DNaseI defi-

Immunology 21 (2009) 208–214

ciency, along with other environmental factors in both humans andmice, can lead to SLE [79]. DNaseI gene mutations have been iden-tified in a subgroup of patients with SLE, an observation that issupported by the SLE-like phenotype observed in DNaseI-deficientmice. In both cases, the lack of DNaseI activity is associated with thedevelopment of autoantibodies directed against dsDNA and nucle-osomes that are strongly associated with glomerulonephritis inSLE patients [79,80]. DNaseI is an essential endonuclease that, inconjunction with the proteolytic activity of plasmin degrades chro-matin released from dying or necrotic cells to low molecular weightforms [81]. In the absence of DNaseI, higher molecular weight DNAstructures persist that are believed to be immunostimulatory. Sim-ilarly, the DNaseII endonuclease plays a major role in the clearanceof apoptotic DNA generated primarily during erythropoiesis. DNa-seII deficiency results in the accumulation of phagocytosed dsDNAin macrophages that induces TLR-independent interferon-� pro-duction that is lethal in mice [48,82]. On an interferon receptor 1(IFNR1) deficient background or using a conditional knockout, thelethality of DNaseII deficiency is rescued confirming the importanceof type I interferons in mediating the observed murine pheno-type. However the inability to process endogenous DNA persistsand results in an autoimmune polyarthritis that mimics rheumatoidarthritis within 2 months of birth [83].

Recently, several studies have identified DNaseIII/TREX1 as aregulator of DNA homeostasis in the cell linked to autoimmuneand inflammatory diseases. TREX1 is the most abundant 3′–5′ DNAexonuclease [84,85]. Mutations in TREX1 have been identified insmall groups of patients with SLE [86] and in combination withmutations in the RNaseH2 complex contributes to the pathogenesisof Aicardi-Goutières Syndrome (AGS), an inherited inflammatorydisorder that targets the neurological system in children [87–90].Patients with AGS suffer from a syndrome that mimics congenitalinfection with cytomegalovirus or rubella and consists of a progres-sive inflammatory encephalopathy with high levels of interferon-�in the cerebrospinal fluid. Studies in TREX1 deficient mice havebeen enlightening and provide some insight into the disease, theregulation of cellular DNA and autoimmunity. First, mice lackingTREX1 do not develop a syndrome that mimics human AGS empha-sizing the multigenic and complex nature of this condition. Instead,TREX1 deficient mice have a reduced lifespan and succumb to aninflammatory myocarditis and cardiomyopathy within 2–4 months[91]. Yang et al. showed that TREX1 localized to the endoplasmicreticulum and translocated to BrdU positive foci at the nucleus pri-marily in S-phase cells following DNA damage. In the absence ofTREX1, 60–65 bp ssDNA polynucleotides accumulated in the ER thatwere believed to be produced during DNA replication. The accu-mulation of ssDNA in TREX1 deficient cells was associated withchronic ATM-dependent DNA damage checkpoint signaling char-acterized by persistent p53 activation, elevated levels of the cyclinD/E dependent kinase inhibitor, p21 and defective G1/S transition[92]. However, at this time there is no known crosstalk betweenthe DNA-damage response and the innate response to DNA. Furtherstudies by Medzhitov and co-workers added clarity to the role ofTREX1 in regulating the innate response to DNA. The inflammatorymyocarditis observed in TREX1 deficient mice was substantiallyreduced or eliminated in animals bred onto IRF3 or IFNR1 deficientbackgrounds. These data confirmed that an IRF3-dependent type Iinterferon response typical of TLR-independent DNA sensing or theISD pathway was essential to initiate the heart disease. TREX1 itselfis not a DNA sensor but instead regulates the accumulation of ssDNAthat can trigger an innate immune response. Interestingly, cloning

of accumulated ssDNA TREX1 substrates revealed a disproportion-ate amount of endogenous retrovirus elements in TREX1-deficientcompared to wild type cells leading the researchers to conclude thatTREX1 specifically targets reversed transcribed DNA [93]. Given thathuman AGS is also associated with mutations in the RNaseH2 com-

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lex that degrades RNA:DNA hybrids [94] occurring during reverseranscription, a potential link now exists between endogenousetroelements and autoimmunity via DNA sensing pathways in theell. Future studies will hopefully provide clarity to this intriguingossibility. Together, these results highlight the critical importancef DNA regulating pathways in preventing self-activation of the

nnate immune system that results in autoimmunity.

. Conclusions

Recent findings regarding the identity of cytosolic DNA sensorsave provided a few answers to questions posed regarding the exis-ence of TLR9 independent DNA sensing pathways. Similar to RNAensing mechanisms that are mediated via TLRs in endosomal com-artments and RLRs in the cytosol [95], a parallel system of cytosolicnd endosomal DNA receptors are present in the cell that providehe host several layers of innate defense mechanisms to deal withathogens that utilize different entry pathways during infection.dditional research into the biological significance of ZBP1 and

he AIM2 inflammasome is required to further define their rolesithin the innate immune system. In addition, other cytosolic DNA

ensors that mediate TLR-independent responses exist in the cellnd remain to be identified. More importantly, it will be particu-arly interesting to determine the role of nucleic acid sensors in theathogenesis of autoimmune and inflammatory diseases.

cknowledgements

D.A.M. is the recipient of an Alberta Heritage Foundation foredical Research Scholar award. Research funding is provided by

IHR operating and group grants.

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[3] Rotem Z, Cox RA, Isaacs A. Inhibition of virus multiplication by foreign nucleicacid. Nature 1963;197:564–6.

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