Molecular Basis of DNA Recognition in the Immune System

The innate immune system is the first line of defense against infectious agents. Germline-encoded pattern recognition receptors (PRRs), including TLRs, NOD-like receptors (NLRs), retinoic acid inducible-I (RIG-I)–like receptors, and C-type lectins, recognize a wide range of microbial products, often referred to as microbe-associated molecular patterns (1). Recognition of microbe-associated molecular patterns by these surveillance receptors turns on signaling pathways that coordinate transcription of hundreds of inflammatory genes, the products of which control infection directly and marshal the T and B cells of the adaptive immune system (2). In addition to classical microbial products, such as bacterial LPS or lipoproteins, microbial nucleic acids have emerged as major triggers of innate immune defenses.

The best-characterized nucleic acid sensors are a subset of TLRs, type I transmembrane receptors localized to the endosomal compartment that sense dsRNA (TLR3) (3), ssRNA (TLR7 and TLR8) (4–6), and hypomethylated CpG DNA (TLR9) (7, 8). TLRs induce type I IFN and other inflammatory 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 melanoma differentiation-associated protein 5, have also been identified (10, 11); they signal via a unique adaptor mitochondrial antiviral signaling (MAVS; also known as IFN-β promoter stimulator-1) to mediate NF-κB and IRF-dependent transcription of inflammatory genes (12).

The cellular machinery that senses cytosolic DNA is still being elucidated. Although significant progress has been made in understanding how DNA is recognized and how DNA signaling ensues and leads to inflammation, the molecular basis of cytosolic DNA recognition in innate immunity is still being worked out in detail. A number of new molecules (which will be described below) have been identified that contribute in various ways to recognition and signaling in response to DNA. TLR9 expressed in endosomal membranes was the first identified DNA receptor that recognizes hypomethylated CpG motifs (7). However, in humans, TLR9 expression is restricted to B cells (13) and plasmacytoid dendritic cells (pDCs) (14) and, therefore, does not account for DNA-induced immune responses in other cell types, such as macrophages. Two seminal studies demonstrated that the targeted delivery of synthetic dsDNA or a 45-nt immunostimulatory DNA into the cytosol of macrophages and dendritic cells (DCs) triggered Tank-binding kinase 1 (TBK1)/IRF3-dependent induction of type I IFN and other inflammatory genes in a TLR9-independent manner (15, 16). These findings led to a search in many laboratories for sensor(s) that could couple cytosolic DNA recognition to immune signaling. Two conceptually distinct signaling pathways have since emerged. The first of these leads to the proteolytic activation of the cysteine protease caspase-1 associated with maturation and secretion of the proinflammatory cytokines IL-1β and IL-18. A second pathway that is still being worked out leads to the transcriptional induction of type I IFN and proinflammatory genes. These two pathways are depicted in their simplest form in Fig. 1. In this article, we briefly summarize and discuss the state of play of DNA sensing and signaling and the functional significance of these event in antimicrobial immunity and autoimmune diseases.

Cytosolic DNA leads to the production of the proinflammatory cytokines IL-1β and IL-18. IL-1β is secreted by many cell types and is essential for both the innate and adaptive arms of the immune response (17). IL-1β is important in activating neutrophils, macrophages, DCs, and T cells, whereas IL-18 is crucial for IFN-γ production by NK cells and T cells (18, 19). In contrast to the majority of inflammatory mediators that are regulated transcriptionally, IL-1β and IL-18 are regulated at both the transcriptional and posttranslational levels. Upon transcriptional induction by TLRs and other sensor systems, IL-1β and IL-18 are synthesized as inactive precursor proteins, which are subsequently processed by the cysteine protease caspase-1 (IL-1β converting enzyme) (20). Conversion of procaspase-1 into an enzymatically active form, caspase-1, occurs upon formation of a multiprotein complex in the cytosol referred to as the “inflammasome” (21). The nucleotide binding and leucine-rich repeat containing receptor (NLR; aka NOD-like receptors) and pyrin and HIN200 domain-containing (PYHIN) family proteins (discussed further below) are known to form inflammasome complexes by recruiting procaspase-1 via an adaptor protein ASC (22). ASC is a bipartite molecule containing 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 (or PYHIN proteins) and procaspase-1 via homotypic PYD:PYD and CARD:CARD interactions, respectively (Fig. 2). Several inflammasome 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 to intracellular DNA, as well as bacterial or DNA virus infections.

The Nlrp3 inflammasome is the best-studied inflammasome and is associated with bacterial, viral, parasitic, and fungal infections (23). NLRP3 is also activated in response to endogenous danger signals (24). The Nlrp3 inflammasome in macrophages responds to modified vaccinia virus Ankara strain (25) and adenovirus (26) to facilitate caspase-1 activation and IL-1β secretion. For adenovirus, intracellular viral DNA, but not viral capsids, activate the Nlrp3 inflammasome. However, caspase-1 activation and IL-1β secretion in response to transfected nonviral cytosolic DNA (synthetic, bacterial, or mammalian origin) was Nlrp3 independent, but ASC dependent, indicating that cytosolic DNA-driven inflammasome activation requires an ASC-dependent sensor (26). Whether adenoviral DNA is the ligand required to trigger assembly of the Nlrp3 inflammasome is unclear, but an indirect mode is more likely, as suggested by a recent study that perturbations in cell membrane associated with adenoviral entry are the trigger for Nlrp3 activation (27). The Nlrp3 inflammasome also recognizes influenza A infection, a negative stranded RNA virus (28). Therefore, the Nlrp3 inflammasome is involved in mediating the inflammatory responses to both DNA and RNA viruses. It is likely that the Nlrp3 inflammasome senses cytosolic nucleic acids indirectly.

Several groups independently identified AIM2 as the receptor for cytosolic DNA that leads to caspase-1 activation and IL-1β secretion (29–31). Contrary to other cytosolic DNA sensors, which are primarily involved in the induction of type I IFN, AIM2 triggers the activation of the inflammasome. Notably, the AIM2 inflammasome is the first among all inflammasome-activating proteins identified in which a direct receptor–ligand interaction is demonstrated. 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 HIN200 domain, AIM2 undergoes oligomerization and, thereby, recruits caspase-1 via ASC. In vivo studies in Aim2-deficient mice clearly indicate that the Aim2 inflammasome is essential and functions in a nonredundant manner for innate defense responses to DNA viruses and intracellular bacterial infections (32, 33). Caspase-1 activation and IL-1β/IL-18 secretion in response to mouse CMV and vaccinia virus infection in murine macrophages is dependent upon Aim2 inflammasome (32). Consistently, Aim2-dependent IL-18 secretion and NK cell activation are essential for an early control of mouse CMV infection in vivo. Similarly, Aim2 plays a crucial role in controlling Francisella tularensis infection in vivo, because Aim2-deficient mice are more susceptible to infection and show higher bacterial counts in infected organs (33, 34). Activation of the Aim2 inflammasome is also reported in other bacterial infections including Listeria monocytogenes (35, 36) and Mycobacterium tuberculosis (37). How Aim2 is exposed to bacterial DNA during infection is not well understood. There is some evidence that DNA released into the cytosol following lysosomal bacteriolysis is the trigger for Aim2 activation (38). Recent evidence indicates that the AIM2-related protein IFI16 also forms an inflammasome complex following Kaposi sarcoma–associated herpes virus infection of endothelial cells (39).

Type I IFN production is another major consequence of cytosolic DNA sensing and is essential for antiviral immunity and immunity to many classes of infectious agents. Although we understand in detail how DNA via AIM2 leads to IL-1β production, the molecular bases to DNA-induced type I IFN gene transcription is less clear. A core signaling module consisting of stimulator of type I IFN gene (STING)–TBK1–IRF3 is engaged and is absolutely essential for type I IFN responses to DNA. TBK1, an IKK-related kinase originally characterized as the kinase responsible for the phosphorylation-induced activation of IRF3 in TLR and then RIG-I–like receptor signaling, is also central for DNA-signaling pathways (40, 41). Therefore, TBK1 activation acts as a point of convergence of multiple PRR-driven pathways that results in IRF3 phosphorylation and transcription of type I IFN genes and related IFN-stimulated genes.

Several groups independently identified STING (also known as MPYS, TMEM173, ERIS, and MITA) as a key component of the DNA-sensing pathway (42–44). STING was shown to localize to the endoplasmic reticulum and outer mitochondrial membranes via five transmembrane-spanning regions. In response to DNA, STING translocates to perinuclear regions where it interacts with TBK1 to relay downstream signals to IRF3. STING deficiency in macrophages or DCs leads to a markedly impaired type I IFN response to B-DNA and immunostimulatory DNA or to infection with DNA viruses, including HSV-1, human CMV, and vaccinia virus (42, 45). STING is also essential for type I IFN induction in response to intracellular bacteria, including F. tularensis (46, 47), L. monocytogenes (46, 48), and Brucella abortus (49), and in response to extracellular bacteria, such as Streptococcus pneumoniae (50) and Streptococcus pyogenes (51). Initial studies showed that STING also interacted with components of the RNA-recognition machinery, such as RIG-I, where it was linked to type I IFN induction in response to vesicular stomatitis virus, a negative-strand RNA virus (42). However, subsequent studies in STING-deficient cells suggested that viruses or ligands that engaged RIG-I have normal IFN responses in STING-deficient murine macrophages (48, 52). However, additional studies linked STING to DNA- and RNA-recognition pathways by uncovering a role for it in the activation of STAT6 and induction of STAT6-dependent chemokines (52).

The assumption from these earlier studies is that, analogous to MyD88 or MAVS, STING functions as an adaptor molecule. This assumption implies that DNA-binding proteins that recognize microbial DNA engage STING and activate TBK1 and downstream signaling. Several candidate sensors have been discovered and implicated to varying degrees in this DNA-dependent TBK1–IRF3 and type I IFN production pathway (Fig. 3).

DNA-dependent activator of IRFs (DAI), also known as Z-DNA–binding protein, ZBP, or DLM1, is an IFN-stimulated gene and the first cytosolic DNA sensor identified (53, 54). DAI binds Z-form DNA via two N-terminal Z-DNA–binding domains and an adjacent protein region carrying B-DNA–binding potential. DAI was shown to mediate DNA-induced type I IFN production in cell-based studies. However, subsequent studies in DAI ^−/− mice revealed that DAI-deficient cells and mice retain normal type I IFN responses to DNA viruses and various types of synthetic DNA (55), indicating that DAI is either dispensable or a redundant sensor of cytosolic DNA. Whether DAI impacts other aspects of DNA-induced signaling is still unclear.

Following the discovery of DAI, RNA polymerase III was linked to DNA recognition, adding another layer of complexity to the cytosolic DNA-recognition pathway (56, 57). RNA polymerase III present in the cytosol transcribes AT-rich DNA into immunostimulatory dsRNA transcripts characterized by uncapped 5′-triphosphate moieties, which act as a ligand for RIG-I (58). Subsequently, RIG-I signals via MAVS to induce the expression of type I IFN and other cytokines. Therefore, RNA polymerase III does not sense cytosolic DNA directly, but it generates a ligand to engage the RIG-I pathway. Involvement of the RNA polymerase III/RIG-I pathway in response to AT-rich DNA explains the partial dependence upon MAVS for DNA-induced induction of type I IFN in certain cell types. The RNA polymerase III pathway is functional in both human and mouse cells and is engaged in response to the intracellular dsDNA mimetic poly(dA:dT), as well as with certain DNA viruses, like adenovirus and EBV. Additional studies also linked RNA polymerase III and type I IFN responses during Legionella pneumophila and HSV-1 infections; however, there is some controversy over these latter data (59, 60).

IFI16 was identified as a DNA-binding protein in human monocytes in affinity-purification studies (61). Like AIM2, IFI16 is a member of the PYHIN (aka p200 protein) family. IFI16 is primarily localized in the nucleus. In some cell types, such as macrophages, a small pool of cytoplasmic IFI16 colocalizes with transfected DNA or viral DNA that gains access to the cytosolic compartment during HSV-1 infection. In other cell types, such as fibroblasts, which are permissive to HSV1 infection, IFI16 engages viral DNA that accumulates during productive infection in the nucleus (62). IFI16 does not leave the nucleus in HSV-1–infected human foreskin fibroblast cells; however, this nuclear sensing of HSV-1 requires cytoplasmic STING signaling. Therefore, HSV-1 DNA recognition may differ in permissive versus nonpermissive cells, but, in both cases, IFI16-dependent recognition of HSV-1 DNA is coupled to STING signaling. It is important to understand the role of nuclear and cytoplasmic IFI16 function during HSV infection in these contexts and to better understand how signaling from these compartments is initiated and regulated. IFI16 has two C-terminal DNA-binding HIN200 domains and, like AIM2, it binds DNA via nonsequence-specific DNA recognition accomplished through electrostatic attractions between the positively charged HIN200 domain residues and the negatively charged dsDNA sugar–phosphate backbone (63). IFI16 then interacts with STING to activate TBK1 to trigger IFN-β induction. It is unknown whether IFI16 binds STING directly or indirectly. Humans have 4 PYHIN proteins (IFI16, AIM2, MNDA, and IFIX), whereas mice have 13 (64). The murine PYHIN protein Ifi204 (p204) is proposed to function in an analogous manner to IFI16. Knockdown of IFI16 (or Ifi204) compromise DNA-induced, as well as HSV-1–induced, IRF3 activation and IFN induction (61). Evidence is also accumulating for a functional role of IFI16/Ifi204 in controlling HSV infection, because p204 plays a role in resistance to HSV-1 infection in the corneal epithelium (65). Interestingly, the HSV viral nuclear ICP0 protein can target IFI16 for degradation, thereby documenting a mechanism by which HSV evades IFI16-mediated detection (62).

Aspartate–glutamate–any amino acid–aspartate/histidine box–containing helicases are also beginning to emerge as important players in the recognition of cytosolic nucleic acids. DHX36 and DHX9 have been identified as TLR9-independent, but MyD88-dependent, sensors of CpG-A and CpG-B DNA, respectively, in human pDCs (66). These two distinct oligodeoxynucleotides are known inducers of either type I IFN (CpG-A) or proinflammatory cytokines, such as TNF-α and IL-6 (CpG-B), in pDCs (67). Consistently, DHX36 is involved in the production of IFN-α, whereas DHX9 mediates TNFα/IL-6 production in HSV-1–infected (or CpG-treated) human pDCs. DHX36 and DHX9 interact directly with the Toll/IL-1R domain of MyD88 to trigger downstream signaling to activate IRF7 and NF-κB p50, respectively. Subsequent studies also ascribed a role for DHX36 and DHX9 in the recognition of cytosolic RNA. DHX36, together with DDX1 and DDX21, forms a complex with the adaptor TRIF to mediate type I IFN induction in response to synthetic dsRNA mimic polyinosinic-polycytidylic acid (68). Similarly, DHX9 interacts with MAVS to induce a type I IFN in response to dsRNA in myeloid DCs (69). It is unclear whether DHX36 and DHX9 are involved in the recognition of RNA viruses. In a comprehensive shRNA screen for all 59 members of the aspartate–glutamate–any amino acid–aspartate/histidine box family, Zhang et al. (70) identified DDX41 as a cytosolic DNA receptor in both mouse and human DCs. DDX41 binds to dsDNA through its helicase domain and triggers the activation of IRF3, NF-κB, and MAPK signaling. Knockdown of DDX41 in DCs led to impaired type I IFN and proinflammatory cytokine production in response to transfected dsDNA or DNA viruses, including HSV-1 and adenovirus, but not against polyinosinic-polycytidylic acid or influenza virus infection. Moreover, DDX41 binds STING in DCs in response to transfected poly(dA:dT) or HSV-1 DNA, suggesting that STING is involved in signaling downstream of DDX41 recognition of the cytosolic DNA. Unlike IFI16, which is a type I IFN inducible gene, DDX41 is expressed at relatively high levels in immune cells. Therefore, DDX41 was proposed to recognize DNA in the early stages of infection, whereas IFI16, a type I IFN inducible gene, is induced at later stages (as a result of DDX41-dependent IFN production), where these two sensors together coordinate DNA-induced IFN responses.

Several additional DNA-binding proteins have been linked to DNA recognition and innate immunity. A recent study by Zhang et al. (71) identified Ku70, a component of the DNA repair and telomere maintenance pathway, as the newest member of the cytosolic DNA-sensing machinery. Ku70 was identified as a DNA-binding protein in HEK293 cells by DNA-affinity purification followed by mass spectrometry. Notably, Ku70 is involved in the production of type III IFN (IFN-λ1; also known as IL29), but not type I IFN (IFN-α or IFN-β), in response to a variety of transfected DNA (>500 bp) in HEK293, murine macrophages, and DCs. Subsequent studies indicated that the DNA-induced IFN-λ1 induction required IRF1 and IRF7 binding to the IFN-λ1 promoter, which implicates Ku70 in a signaling pathway involving IRF1/IRF7. Although Ku70 was shown to mediate HSV-induced IFN-λ1 induction in HEK293 cells, it remains to be fully elucidated whether Ku70 recognizes DNA viruses and bacterial infections in primary immune cells. Because the biological activity and the signaling pathway used by both types I and III IFN are very similar, it is also unclear how the engagement of Ku70-mediated IFN-λ1 production contributes to the overall DNA-induced innate immune responses.

Leucine-rich repeat in flightless-I interacting protein 1 (LRRFIP1) was originally identified by Wilson et al. (72) using a cDNA library to screen for cellular interacting proteins of HIV-1 RNA; it was shown to bind RNA, as well as DNA with a lower affinity. Subsequent studies indicated that LRRFIP1 recognizes both RNA and DNA in the cytosol and enhances the transcription of the Ifnβ gene through a novel β-catenin–dependent–signaling pathway (73). In a subsequent RNA interference screen against leucine-rich repeat–containing and leucine-rich repeat–interacting proteins in primary murine macrophages, Yang et al. (73) identified LRRFIP1 as a cytosolic sensor involved in the production of IFN-β in response to L. monocytogenes or vesicular stomatitis virus infection. LRRFIP1 is similarly involved in IFN-β production in response to transfected dsRNA or dsDNA. Interestingly, LRRFIP1 uses β-catenin, an integral component of the Wnt-signaling pathway, to turn on Ifnβ gene transcription. Upon recruitment by LRRFIP1 by unidentified mechanisms, β-catenin is phosphorylated at serine 552, which then translocates to the nucleus and binds to IRF3, leading to enhanced recruitment of the histone acetyltransferase p300, thereby enhancing transcription of the Ifnβ gene.

In addition to its role as a signaling intermediate in DDX41- and IFI16-signaling pathways, STING acts as a receptor for the bacterial second messenger molecules cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) (74). These secondary messenger molecules are potent inducers of type I IFNs and, in the case of L. monocytogenes, are proposed to represent the major trigger of IFN production in macrophages (75). Forward genetic studies in N-ethyl-N-nitrosourea–mutagenized mice revealed that STING was essential for the type I IFN response to c-di-AMP/c-di-GMP and following Listeria infection (48). STING binds these small molecules directly through its C-terminal domain (CTD), which leads to activation of the TBK1–IRF3 axis for the induction of Ifnβ genes. Recently, several groups resolved the crystal structures of human STING-CTD bound to c-di-AMP/c-di-GMP (76–78). Collectively, these studies impart dual functions to STING: it either serves as an adaptor for DNA sensing or as a direct sensor of bacterial second-messenger molecules, like c-di-AMP. Whether c-di-AMP or DNA is the major microbial trigger involved in the IFN-β response during Listeria infection remains to be determined. Because genes encoding c-di-AMP are also predicted to exist in several other pathogenic bacteria, including staphylococci, streptococci, Mycobacterium, and Chlamydia (79), it will be interesting to define the importance of STING/c-di-AMP interactions in host type I IFN responses to these pathogens. Very recently, Chen and colleagues (80) showed that STING directly binds cyclic GMP-AMP (cGAMP), a novel endogenous second messenger generated in response to DNA, to trigger IRF3 activation and the induction of IFN-β in response to transfected DNA or DNA viruses. They further identified cGAMP synthase, a mammalian nucleotidyltransferase family member, which binds to cytosolic DNA and catalyzes the generation of cGAMP to trigger STING-dependent induction of the type I IFN response (81). Therefore, STING recognition of cGAMP represents a new avenue for mounting host immune responses to cytosolic DNA.

Surprising recent evidence indicates that DDX41 also serves as the PRR involved in detecting c-di-GMP and c-di-AMP to trigger TBK1–IRF3 signaling via STING (82). Surprisingly, the interaction of c-di-GMP with DDX41 was shown to be greater than that observed for STING binding. These data indicate that DDX41 is required to facilitate c-di-GMP signaling via STING. The solved crystal structure of the STING-CTD in complex with c-di-GMP showed that one molecule of c-di-GMP binds one dimer of STING (76–78). Cheng and colleagues proposed that the detection of c-di-GMP via DDX41 promotes enhanced DDX41–STING interactions, leading to an increase in the binding affinity of STING for c-di-GMP, which ultimately drives downstream signaling events (82). This model is consistent with the possibility that STING functions as a secondary receptor or coactivator in the cyclic dinucleotide–signaling pathway.

Although DNA-induced immune responses are central to immunity, inappropriate recognition of self-DNA can lead to deleterious consequences to the host. Systemic lupus erythematosus (SLE) is one such disease in which type I IFN and autoantibodies directed against dsDNA, RNA, and nucleosomes are implicated in disease pathogenesis (83). Multiple fail-safe mechanisms deployed by the host subvert endogenous DNA–induced immune responses. One level of regulation is provided by cellular endonucleases, such as DNase I, DNase II, and DNase III (also known as Trex1), which are involved in the clearance of extracellular, lysosomal, and cytosolic DNA, respectively. Recent evidence clearly indicates that the functional defects in these enzymes are associated with SLE and other human diseases. For example, DNase I gene mutations have been identified in subgroups of SLE patients (84), a clinical association further supported by lupus-like syndrome in DNase I–deficient mice (85). Defects in DNase I lead to the accumulation of extracellular DNA released by apoptotic/necrotic cells, which is highly immunostimulatory (86). DNase II is expressed in lysosomes, where it degrades DNA from phagocytosed apoptotic and necrotic cells. Interestingly, DNase II–knockout mice are embryonically lethal; however, they are viable on the IFNR1-knockout background, indicating that type I IFNs mediate the lethality of DNase II genetic deficiency (87). In this case, type I IFN responses are mediated in a TLR-independent, but IRF3/7-dependent manner. Recent evidence indicates that DNase II/STING–deficient mice are also viable, indicating the importance of STING in this model (88). The DNase II/IFNR1 knockout mice develop autoimmune polyarthritis by 2 mo of age with features reminiscent of human rheumatoid arthritis, further highlighting a central role for DNA-induced immune responses in autoimmune diseases. The sensors of DNase I and DNase II substrates are unclear, but it is likely that one or more of the aforementioned cytosolic DNA sensor(s) recognize and respond to this accumulated dsDNA. DNase III/Trex1 is another nuclease that is normally involved in the clearance of cell-intrinsic ssDNA (89). Trex1 is the most abundant 3′–5′ exonuclease and is localized to the endoplasmic reticulum. Recent studies in Trex1-knockout mice provide great insight into the regulation of endogenous DNA and its role in autoimmunity. Trex1^−/− mice are viable; however, they exhibit a shortened life span (2–4 mo) and manifest inflammatory myocarditis (90). In the absence of Trex1, there is an accumulation ∼60-bp ssDNA, believed to be produced during replication, which leads to the activation of DNA-damage associated signaling pathways. Further work by Stetson et al. (91) revealed a role for Trex1 in preventing cell-intrinsic initiation of autoimmunity. Trex1 substrates are ssDNA, which are either the by-products of replication and/or reverse transcribed from endogenous retroelements. As was observed in DNase II–deficient mice, inflammatory myocarditis associated with Trex1 deficiency is rescued by crossing Trex1-deficient mice to STING-deficient background. Together, these studies highlight the important role of strict regulation of the DNA-sensing pathway and underscore the importance of DNA sensing in both protective and pathological immune responses.

In recent years, there has been tremendous progress in understanding how cells recognize and respond to microbial threats via cytosolic DNA recognition. Studies from several groups clearly indicated that multiple sensors exist in the cytosol to trigger inflammatory responses to DNA. These responses are central to antimicrobial immunity; therefore, it is not surprising that multiple DNA sensors exist and operate in different cell types. However, as with all areas of progress, many new questions arise, and key aspects of DNA recognition remain to be better understood. The potential functional overlap and redundancy of key sensors and signaling intermediates, as well as a better clarification of the relative importance of DDX41 versus STING in sensing cyclic dinucleotides, likely will be assisted following the generation and characterization of DDX41-deficient cells and mice. A better understanding of the molecular mechanisms by which cells generate inflammation in response to DNA may provide new targets that could be manipulated for the treatment of infectious, as well as autoimmune, disease.

We apologize to colleagues whose work could not be cited because of space limitations.

The authors have no financial conflicts of interest.