Innate Immune Sensing of DNA
Shruti Sharma, Katherine A. Fitzgerald*
Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of
DNA Is a Potent Activator of Innate Immunity
When a pathogen attacks, the immune system rapidly mobilizes
host defenses in order to reduce the microbial burden and limit
damage to the host . Innate immunity is the first line of defense
and relies on germ line–encoded pattern recognition receptors
(PRRs) such as the Toll-like receptors (TLRs), which sense
microbial products that are not normally found on or in
mammalian cells. The considerable potency of nucleic acids as
triggers of the innate immune response has gained appreciation
over the last few years. In particular, nucleic acid sensing of viruses
is central to anti-viral defenses through recognition of viral
genomes or nucleic acids generated during viral replication.
Distinct classes of nucleic acid sensing molecules have been
uncovered that function in different cell types and subcellular
compartments to coordinate innate defenses (reviewed in ).
While recognition of RNA molecules is dependent on members
of the TLR family and cytosolic RNA helicases, the mechanisms
underlying the sensing of DNA have been less well defined. It has
been known for over a decade that DNA, the most recognizable
unit of life, is a potent trigger of inflammatory responses in cells.
The discovery of TLR-9, a receptor for hypomethylated CpG-rich
DNA, partially explained these findings . TLR9 is localized to
the endosomal compartment and in humans is expressed in B cells
as well as in plasmacytoid dendritic cells (pDCs). However, it
became clear that the immune stimulatory activity of microbial
DNA was not compromised in many cells lacking TLR9 .
These observations prompted new efforts to understand how DNA
triggers immune responses, an endeavor that has led to the
discovery of several new DNA recognition receptors and fresh
insights into infectious as well as autoimmune diseases.
There Are Multiple Receptors for Microbial DNA
A significant effort from many laboratories has highlighted the
importance of cytosolic DNA sensing in the innate immune
response. At least six intracellular receptors have been implicated
to some degree. These include DNA-dependent activator of
interferon (IFN)-regulatory factors (DAI) (also called Z-DNA-
binding protein 1, ZBP1) , absent in melanoma 2 (AIM2) [6–9],
RNA polymerase III (Pol III) [10,11], leucine-rich repeat (in
Flightless I) interacting protein-1 (Lrrfip1) , DExD/H box
helicases (DHX9 and DHX36) , and most recently, the IFN-
inducible protein IFI16 . DAI was the first to be implicated in
synthetic B- and Z-form dsDNA recognition ; however, the role
of DAI is still unclear, as DAI-deficient mice and cells coordinate
normal immune responses to DNA . Cytoplasmic dsDNA also
triggers IFN production via RNA Pol III, which transcribes the
DNA into 59-ppp RNA, a ligand for the RNA helicase RIG-I
[10,11]. In pDCs, DHX9 and DHX36 contribute to cytosolic
CpG-DNA and HSV-1-driven IFN responses , which likely
account for previously reported TLR9-independent cytokine
responses to some DNA viruses . Lrrfip1 appears to bind
both DNA and RNA; however, Lrrfip1 does not regulate the
transcription factors that drive IFN gene transcription, but rather
signals a co-activator pathway involving b-catenin and CBP/p300
histone modifying complexes to enhance the transcription of type I
IFNs in the nucleus . DNA from Listeria monocytogenes and RNA
from vesicular stomatitis viral (VSV) activate this Lrrfip1-b-catenin
pathway to mediate these effects.
Immune responses to DNA are not restricted to type I IFN-
inducing pathways: cytosolic DNA also activates caspase-1-
dependent maturation of the pro-inflammatory cytokines inter-
leukin (IL)-1b and IL-18. This pathway is mediated by AIM2, a
PYHIN (Pyrin- and HIN200-domain-containing) protein. Recent
evidence from knockout studies has revealed the importance of
AIM2 in host defense to cytosolic bacteria such as Fransicella spp.,
as well as DNA viruses like mouse cytomegalovirus (reviewed in
[17–20]). The newest receptor identified, IFI16, binds viral DNA
and is critical in the immune response to certain DNA viruses .
Like AIM2, IFI16 is a PYHIN protein that binds viral DNA via
HIN domains; however, IFI16 does not appear to associate with
ASC to regulate IL-1b maturation. Rather, IFI16 activation
induces IFN-b and inflammatory cytokine production in response
to cytosolically administered viral DNA or HSV1 infection.
Distinct Classes of DNA Sensors Engage Distinct
Most of these DNA sensors utilize a subset of adapter molecules,
which relay signals to NF-kB and members of the interferon
regulatory factor (IRF) family. TLR9 as well as DHX9 and
DHX36 recruit MyD88 to activate IFN production in pDCs in
response to DNA. In contrast, recognition of DNA by RNA-Pol
III generates an RNA intermediate, which signals via RIG-I and
MAVS. In the case of IFI16, the endoplasmic reticulum–resident
protein stimulator of interferon genes (STING) relays signaling
downstream . Whether STING binds IFI16 directly or merely
acts as a signaling intermediate for this pathway is unclear. AIM2
triggers caspase-1 activation via the PYD domain containing
adapter molecule ASC. Although IFI16 also contains a PYD
domain, it does not appear to utilize ASC for IFN production. It is
likely that the DAI pathway also involves STING, although this
Citation: Sharma S, Fitzgerald KA (2011) Innate Immune Sensing of DNA. PLoS
Pathog 7(4): e1001310. doi:10.1371/journal.ppat.1001310
Editor: Hiten D. Madhani, University of California San Francisco, United States of
Published April 21, 2011
Copyright: ? 2011 Sharma, Fitzgerald. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This work is supported by grants from the NIH (AI067497, AI64349,
AI083713, and AI079293) awarded to KAF. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests
* E-mail: firstname.lastname@example.org
PLoS Pathogens | www.plospathogens.org1 April 2011 | Volume 7 | Issue 4 | e1001310
has not been formally demonstrated. Downstream of STING,
MAVS, or MyD88, the nucleic acid sensing pathways converge on
different IKK kinases to phosphorylate and activate IRFs
(reviewed in ). In the case of the TLRs and possibly DHX
helicases, IKKa is involved in phosphorylating IRF7, while
downstream of MAVS and STING, TANK-binding kinase 1
(TBK-1), an IKK-related kinase, phosphorylates and activates
IRF3. There is no evidence for the involvement of adaptor
proteins in the Lrrfip1-b-catenin pathway, although intermediary-
signaling molecules may be required for Lrrfip1-dependent b-
Cytosolic DNA Recognition Pathways Also
Contribute to the Pathogenesis of Autoimmune
While DNA recognition receptors and associated signaling
pathways are part of the normal immune response to infection, self
DNA that gains access to compartments where these sensors are
localized can also trigger inflammation, with deleterious conse-
quences for the host (reviewed in ). Systemic lupus erythema-
tosis (SLE) is one of the first autoimmune diseases where aberrant
self-DNA recognition and type I IFNs play a role in disease
pathogenesis. DNA and RNA complexed with autoantibodies
trigger immune activation, leading to autoantibody production
and significant cell death. Here, TLR7- and TLR9-sensing
pathways in autoreactive B cells and pDCs appear to be central
to disease pathogenesis . Mutations in enzymes that normally
degrade DNA have been linked to SLE and other diseases. For
example, defective clearance of extracellular nucleic acids from
dying cells due to deficiency or mutation of DNAse I causes a
lupus-like syndrome in mice and humans [22,23]. The sensing of
accumulated DNAse I substrates is unclear but likely involves
TLRs as well as other DNA sensors.
DNases regulate the accumulation of DNA in more than one
compartment. For instance, DNase II is localized to lysosomes
where it normally degrades DNA from engulfed apoptotic and
necrotic cells. Interestingly, DNAse II–deficient mice are embry-
onic lethal due to overproduction of type I IFNs [24,25]. However,
mice deficient in both DNAse II and the type I IFN receptor are
viable. The DNA sensing mechanism triggering IFN in this case is
known to be TLR independent but dependent on IRF3 and IRF7.
It is likely that one or more of the DNA sensors described above
account for these responses. Another type of deoxyribonuclease,
DNAse III, also called 39 repair exonuclease 1 (TREX1), is found
on the endoplasmic reticulum and has been shown to digest
Figure 1. Pathways of innate immune sensing of DNA. (A) Cytosolic DNA from invading viruses and bacteria engage and activate AIM2
binding to the adaptor ASC. ASC mediates caspase-1-dependent pro-IL-1b/pro-IL-18 cleavage and secretion of their bioactive forms, IL-1b and IL-18.
IL-1b and IL-18 are significant mediators of inflammatory responses to infection. (B) Four known cytosolic sensors are represented here. Lrrfip1
recognized viral DNA as well as RNA to induce IFNb via a b-catenin-IRF3 transactivator pathway independently of the kinase TBK1. DAI can bind
double-stranded B-form and atypical Z-form DNA to induce TBK1-IRF3-dependent IFNb production. Evidence for the role of adaptors MAVS/STING in
these pathways is lacking. IFI16 can directly bind viral DNA via its HIN200 domains and initiate IFNb induction in a STING-TBK1- and IRF3-dependent
manner. RNA polymerase III (Pol III) generates 59 tri-phosphate RNA that is a ligand for RIG-I. RIG-I signals via the adaptor MAVS, subsequently
activating ubiquitin ligase TRAF3 and subsequently TBK1 and IRF3. The ubiquitin binding protein RNF5 inhibits STING activation by targeting it to the
proteasome, while TREX1 inhibits/prevents IFNb production by degrading DNA substrate. (C) The receptor for advanced glycated end products
(RAGE) and HMGB1 can bind extracellular CpG-rich DNA and transport it to a TLR9-positive compartment. Here, it is recognized by TLR9 and signals
via MyD88 and the IKK kinase, IKKa, and IRF7 in pDCs to induce IFNa production. The cytosolic DExD/H box helicases DHX9/DHX36 can recognize
cytosolic CpG DNA and initiate signaling to IRF7 via MyD88.
PLoS Pathogens | www.plospathogens.org2 April 2011 | Volume 7 | Issue 4 | e1001310
cell-intrinisic DNA generated as a result of reverse transcription
from endogenous retroelements. Under normal circumstances
TREX1 prevents the accumulation of this reverse transcribed
DNA . However, in situations where TREX1 is non-
functional, DNA accumulates and can lead to activation of
cytosolic sensing pathways. Mutations in TREX1 are found in
patients with Aicardi-Goutie `res syndrome (AGS) and chilblain
lupus, diseases that clinically resemble congenital viral infections
[26,27]. Mutations in the sterile a motif (SAM domain) and HD
domain-containing protein 1 (SAMHD1) are also linked to this
disease [27,28]. Although there is no direct evidence linking
SAMHD1 to cytosolic DNA sensing per se, it is likely that
SAMHD1 also acts to counterbalance cytosolic DNA sensing and/
or signaling, perhaps by interfering with one or more of the sensors
There Are Still Major Unknowns in the World of
Fresh new insights into infectious as well as autoimmune
diseases have been gained as a result of the studies on DNA
sensing and signaling pathways. While there has been great
progress in this area, many important questions arise from these
discoveries. How these different sensors coordinate cell type–
specific and or species-specific responses to DNA is still a major
question and undoubtedly the focus of future research efforts in
this area. Another key issue to be resolved is how DNA ligands,
which are often enclosed in membrane-bound compartments (e.g.,
DNA viruses replicating in the nucleus), meet these cytosolic
receptors. The identification of TREX1 as well as SAMHD1
suggests that in healthy cells, tightly controlled DNA levels prevent
engagement of these pathways. It is likely that additional counter
regulatory mechanisms that dampen these responses will be
uncovered. Moreover, it is also likely that future discoveries will
unveil mechanisms by which pathogens inactivate these defenses
to prevent the immune response from sampling their genomes and
turning on anti-viral defenses. Further characterization of these
DNA sensing and counter regulatory mechanisms is likely to
impact our understanding of common autoimmune and autoin-
flammatory diseases as well as build a framework for our
understanding of infectious diseases. Future discoveries in this
area will no doubt unveil new opportunities for therapeutic
interventions in infectious and autoimmune disease.
We apologize to those in the field whose work was not cited here owing to
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