Molecular Basis of DNA Recognition in the Immune System

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    Immune SystemMolecular Basis of DNA Recognition in the

    Maninjay K. Atianand and Katherine A. Fitzgerald 10.4049/jimmunol.1203162

    2013; 190:1911-1918; ;J Immunol


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    Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright 2013 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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  • Molecular Basis of DNA Recognition in the ImmuneSystemManinjay K. Atianand and Katherine A. Fitzgerald

    Recognition of microbial nucleic acids is one strategy bywhich mammalian hosts respond to infectious agents.Intracellular DNA that is introduced into cells duringinfection elicits potent inflammatory responses by trig-gering the induction of antiviral type I IFNs and thematuration and secretion of inflammatory cytokines,such as TNF-a, IL-1b, and IL-18. In addition, if nu-cleases, such as DNase II or DNase III (Trex1), fail toclear self-DNA, accumulated DNA gains access to in-tracellular compartments where it drives inflammatoryresponses leading to autoimmune disease. In this re-view, we discuss a rapidly evolving view of how cyto-solic DNA-sensing machineries coordinate antimicrobialimmunity and, if unchecked, lead to autoimmune dis-ease. The Journal of Immunology, 2013, 190: 19111918.

    The innate immune system is the first line of defenseagainst infectious agents. Germline-encoded patternrecognition receptors (PRRs), including TLRs, NOD-

    like receptors (NLRs), retinoic acid inducible-I (RIG-I)likereceptors, and C-type lectins, recognize a wide range of mi-crobial products, often referred to as microbe-associated mo-lecular patterns (1). Recognition of microbe-associated molecularpatterns by these surveillance receptors turns on signaling path-ways that coordinate transcription of hundreds of inflammatorygenes, the products of which control infection directly andmarshal the T and B cells of the adaptive immune system (2).In addition to classical microbial products, such as bacterialLPS or lipoproteins, microbial nucleic acids have emerged asmajor triggers of innate immune defenses.The best-characterized nucleic acid sensors are a subset of

    TLRs, type I transmembrane receptors localized to the endo-somal compartment that sense dsRNA (TLR3) (3), ssRNA(TLR7 and TLR8) (46), and hypomethylated CpG DNA(TLR9) (7, 8). TLRs induce type I IFN and other inflam-matory genes via Toll/IL-1R domain containing adaptor

    molecules, such as MyD88 (TLR7/8/9) or TRIF for TLR3(9). Cytosolic RNA sensors, such as RIG-I and melanomadifferentiation-associated protein 5, have also been identified(10, 11); they signal via a unique adaptor mitochondrial an-tiviral signaling (MAVS; also known as IFN-b promoterstimulator-1) to mediate NF-kB and IRF-dependent tran-scription of inflammatory genes (12).The cellular machinery that senses cytosolic DNA is still

    being elucidated. Although significant progress has been madein understanding how DNA is recognized and how DNAsignaling ensues and leads to inflammation, the molecular basisof cytosolic DNA recognition in innate immunity is still beingworked out in detail. A number of newmolecules (which will bedescribed below) have been identified that contribute in variousways to recognition and signaling in response to DNA. TLR9expressed in endosomal membranes was the first identifiedDNA receptor that recognizes hypomethylated CpG motifs(7). However, in humans, TLR9 expression is restricted toB cells (13) and plasmacytoid dendritic cells (pDCs) (14)and, therefore, does not account for DNA-induced immuneresponses in other cell types, such as macrophages. Twoseminal studies demonstrated that the targeted delivery ofsynthetic dsDNA or a 45-nt immunostimulatory DNA intothe cytosol of macrophages and dendritic cells (DCs) trig-gered Tank-binding kinase 1 (TBK1)/IRF3-dependent in-duction of type I IFN and other inflammatory genes in aTLR9-independent manner (15, 16). These findings led toa search in many laboratories for sensor(s) that could couplecytosolic DNA recognition to immune signaling. Two con-ceptually distinct signaling pathways have since emerged. Thefirst of these leads to the proteolytic activation of the cysteineprotease caspase-1 associated with maturation and secretion ofthe proinflammatory cytokines IL-1b and IL-18. A secondpathway that is still being worked out leads to the transcrip-tional induction of type I IFN and proinflammatory genes.These two pathways are depicted in their simplest form inFig. 1. In this article, we briefly summarize and discuss thestate of play of DNA sensing and signaling and the functional

    Division of Infectious Diseases and Immunology, Department of Medicine, Universityof Massachusetts Medical School, Worcester, MA 01605

    Received for publication November 15, 2012. Accepted for publication January 7, 2013.

    This work was supported by National Institutes of Health Grants AI093752, AI067497,and AI083713 (to K.A.F.).

    Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald, Division ofInfectious Diseases and Immunology, Department of Medicine, University of Massa-chusetts Medical School, LRB, Room 309, 364 Plantation Street, Worcester, MA01605. E-mail address:

    Abbreviations used in this article: AIM2, absent in melanoma 2; CARD, caspase-1activation and recruitment domain; c-di-AMP, cyclic di-AMP; c-di-GMP, cyclic di-

    GMP; cGAMP, cyclic GMP-AMP; CTD, C-terminal domain; DAI, DNA-dependentactivator of IRF; DC, dendritic cell; IFI16, IFN inducible protein 16; LRRFIP1,leucine-rich repeat in flightless-I interacting protein 1; MAVS, mitochondrial antiviralsignaling; NLR, NOD-like receptor; pDC, plasmacytoid dendritic cell; PRR, patternrecognition receptor; PYD, pyrin domain; PYHIN, pyrin and HIN200 domain-con-taining; RIG-I, retinoic acid inducible-I; SLE, systemic lupus erythematosus; STING,stimulator of type I IFN gene; TBK1, Tank-binding kinase 1.

    Copyright 2013 by TheAmerican Association of Immunologists, Inc. 0022-1767/13/$16.00

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  • significance of these event in antimicrobial immunity andautoimmune diseases.

    Inflammasome-activating cytosolic DNA sensors

    Cytosolic DNA leads to the production of the proin-flammatory cytokines IL-1b and IL-18. IL-1b is secreted bymany cell types and is essential for both the innate andadaptive arms of the immune response (17). IL-1b is im-portant in activating neutrophils, macrophages, DCs, andT cells, whereas IL-18 is crucial for IFN-g production by NKcells and T cells (18, 19). In contrast to the majority of in-flammatory mediators that are regulated transcriptionally, IL-1b and IL-18 are regulated at both the transcriptional andposttranslational levels. Upon transcriptional induction byTLRs and other sensor systems, IL-1b and IL-18 are syn-thesized as inactive precursor proteins, which are subsequentlyprocessed by the cysteine protease caspase-1 (IL-1b convert-ing enzyme) (20). Conversion of procaspase-1 into an enzy-matically active form, caspase-1, occurs upon formation ofa multiprotein complex in the cytosol referred to as theinflammasome (21). The nucleotide binding and leucine-rich repeat containing receptor (NLR; aka NOD-like recep-tors) and pyrin and HIN200 domain-containing (PYHIN)family proteins (discussed further below) are known to forminflammasome complexes by recruiting procaspase-1 via anadaptor protein ASC (22). ASC is a bipartite molecule con-taining an N-terminal pyrin domain (PYD) and the C-terminal caspase-1 activation/recruitment domain (CARD);therefore, it acts as an adaptor to bring together NLRs (orPYHIN proteins) and procaspase-1 via homotypic PYD:PYDand CARD:CARD interactions, respectively (Fig. 2). Severalinflammasome complexes have been identified in recent years.Of the known inflammasomes, Nlrp3, absent in melanoma 2(AIM2), and, most recently, IFN inducible protein 16 (IFI16)

    inflammasomes, have been linked to immune responses tointracellular DNA, as well as bacterial or DNA virus infec-tions.

    The Nlrp3 inflammasome. The Nlrp3 inflammasome is the best-studied inflammasome and is associated with bacterial, viral,parasitic, and fungal infections (23). NLRP3 is also activatedin response to endogenous danger signals (24). The Nlrp3inflammasome in macrophages responds to modified vacciniavirus Ankara strain (25) and adenovirus (26) to facilitatecaspase-1 activation and IL-1b secretion. For adenovirus,intracellular viral DNA, but not viral capsids, activate theNlrp3 inflammasome. However, caspase-1 activation and IL-1b secretion in response to transfected nonviral cytosolic DNA(synthetic, bacterial, or mammalian origin) was Nlrp3 inde-pendent, but ASC dependent, indicating that cytosolic DNA-driven inflammasome activation requires an ASC-dependentsensor (26). Whether adenoviral DNA is the ligand requiredto trigger assembly of the Nlrp3 inflammasome is unclear, butan indirect mode is more likely, as suggested by a recent studythat perturbations in cell membrane associated with adenoviralentry are the trigger for Nlrp3 activation (27). The Nlrp3inflammasome also recognizes influenza A infection, a negativestranded RNA virus (28). Therefore, the Nlrp3 inflammasomeis involved in mediating the inflammatory responses to bothDNA and RNA viruses. It is likely that the Nlrp3 inflammasomesenses cytosolic nucleic acids indirectly.

    The AIM2 inflammasome. Several groups independently iden-tified AIM2 as the receptor for cytosolic DNA that leads tocaspase-1 activation and IL-1b secretion (2931). Contrary toother cytosolic DNA sensors, which are primarily involved inthe induction of type I IFN, AIM2 triggers the activation ofthe inflammasome. Notably, the AIM2 inflammasome is thefirst among all inflammasome-activating proteins identified inwhich a direct receptorligand interaction is demonstrated.

    FIGURE 1. DNA in cytosol triggers transcription

    of inflammatory genes and inflammasome-depen-

    dent proteolytic activation of caspase-1. Cytosolic

    DNA leads to the engagement of two conceptually

    distinct signaling pathways involved in host immune

    responses. (A) Activation of IRF3, IRF7, and NF-kB

    leads to the transcriptional induction of type I IFN orproinflammatory genes, such as IL-6 and TNFa. (B)Cytosolic DNA-driven inflammasome assembly via

    homotypic PYD:PYD and CARD:CARD interactions

    leads to caspase-1 activation and subsequent proteo-

    lytic cleavage of proIL-1b and proIL-18 into their

    biologically active, mature forms IL-1b and IL-18. In

    addition to cytokine processing, caspase-1 also medi-

    ates cell death under certain biological contexts.


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  • AIM2 binds cytosolic DNA of self and nonself origin,including bacterial, viral, and mammalian DNA, in a sequence-independent manner. Upon DNA binding via its HIN200domain, AIM2 undergoes oligomerization and, thereby,recruits caspase-1 via ASC. In vivo studies in Aim2-deficientmice clearly indicate that the Aim2 inflammasome is essentialand functions in a nonredundant manner for innate defenseresponses to DNA viruses and intracellular bacterial infections(32, 33). Caspase-1 activation and IL-1b/IL-18 secretion inresponse to mouse CMV and vaccinia virus infection in murinemacrophages is dependent upon Aim2 inflammasome (32).Consistently, Aim2-dependent IL-18 secretion and NK cellactivation are essential for an early control of mouse CMVinfection in vivo. Similarly, Aim2 plays a crucial role incontrolling Francisella tularensis infection in vivo, becauseAim2-deficient mice are more susceptible to infection andshow higher bacterial counts in infected organs (33, 34).Activation of the Aim2 inflammasome is also reported inother bacterial infections including Listeria monocytogenes(35, 36) and Mycobacterium tuberculosis (37). How Aim2 isexposed to bacterial DNA during infection is not wellunderstood. There is some evidence that DNA released intothe cytosol following lysosomal bacteriolysis is the trigger forAim2 activation (38). Recent evidence indicates that the AIM2-related protein IFI16 also forms an inflammasome complex

    following Kaposi sarcomaassociated herpes virus infectionof endothelial cells (39).

    Type I IFNproducing cytosolic DNA sensors

    Type I IFN production is another major consequence of cy-tosolic DNA sensing and is essential for antiviral immunity andimmunity to many classes of infectious agents. Although weunderstand in detail how DNA via AIM2 leads to IL-1bproduction, the molecular bases to DNA-induced type I IFNgene transcription is less clear. A core signaling moduleconsisting of stimulator of type I IFN gene (STING)TBK1IRF3 is engaged and is absolutely essential for type I IFNresponses to DNA. TBK1, an IKK-related kinase originallycharacterized as the kinase responsible for the phosphorylation-induced activation of IRF3 in TLR and then RIG-Ilike re-ceptor signaling, is also central for DNA-signaling pathways(40, 41). Therefore, TBK1 activation acts as a point of con-vergence of multiple PRR-driven pathways that results inIRF3 phosphorylation and transcription of type I IFN genesand related IFN-stimulated genes.Several groups independently identified STING (also

    known as MPYS, TMEM173, ERIS, and MITA) as a keycomponent of the DNA-sensing pathway (4244). STINGwas shown to localize to the endoplasmic reticulum and outermitochondrial membranes via five transmembrane-spanningregions. In response to DNA, STING translocates to peri-nuclear regions where it interacts with TBK1 to relay down-stream signals to IRF3. STING deficiency in macrophages orDCs leads to a markedly impaired type I IFN response to B-DNA and immunostimulatory DNA or to infection withDNA viruses, including HSV-1, human CMV, and vacciniavirus (42, 45). STING is also essential for type I IFN in-duction in response to intracellular bacteria, including F.tularensis (46, 47), L. monocytogenes (46, 48), and Brucellaabortus (49), and in response to extracellular bacteria, such asStreptococcus pneumoniae (50) and Streptococcus pyogenes (51).Initial studies showed that STING also interacted with...


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