Download STING and the innate immune response to nucleic acids in the cytosol.

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STING and the innate immune response to
nucleic acids in the cytosol
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© 2013 Nature America, Inc. All rights reserved.
Dara L Burdette & Russell E Vance
Cytosolic detection of pathogen-derived nucleic acids is critical for the initiation of innate immune defense against diverse
bacterial, viral and eukaryotic pathogens. Conversely, inappropriate responses to cytosolic nucleic acids can produce severe
autoimmune pathology. The host protein STING has been identified as a central signaling molecule in the innate immune
response to cytosolic nucleic acids. STING seems to be especially critical for responses to cytosolic DNA and the unique bacterial
nucleic acids called ‘cyclic dinucleotides’. Here we discuss advances in the understanding of STING and highlight the many
unresolved issues in the field.
The detection of pathogen-derived nucleic acids is a central strategy
by which the innate immune system senses microbes to then initiate
protective responses1. Conversely, inappropriate recognition of self
nucleic acids can result in debilitating autoimmune diseases such as
systemic lupus erythematosus2. It is therefore important to understand the molecular basis of the detection of nucleic acids by the
innate immune system. Studies have established that nucleic acids
derived from extracellular sources are sensed mainly by endosomal
Toll-like receptors (TLRs), such as TLR3, TLR7 and TLR9, whereas
cytosolic nucleic acids are detected independently of TLRs by a variety
of less-well-characterized mechanisms1.
Studies have identified STING (‘stimulator of interferon genes’; also
known as TMEM173, MPYS, MITA and ERIS) as a critical signaling molecule in the innate response to cytosolic nucleic-acid ligands.
STING was first described as a protein that interacts with major histocompatibility complex class II molecules3, but the relevance of this
interaction remains unclear. Subsequent studies have instead focused
on the role of STING in the transcriptional induction of type I interferons and coregulated genes in response to nucleic acids in the cytosol.
Several groups have independently isolated STING by screening for
proteins able to induce interferon-B (IFN-B) when overexpressed4–6.
Studies of STING-deficient mice have subsequently confirmed the
essential role of STING in innate responses to cytosolic nucleic-acid
ligands, particularly double-stranded DNA (dsDNA) and unique bacterial nucleic acids called ‘cyclic dinucleotides’7–9. Several studies have
also linked STING to the interferon response to cytosolic RNA5–7, but
this has not been found consistently7,8,10,11; thus, we focus here on the
role of STING in response to DNA and cyclic dinucleotides.
STING has been shown to have critical roles in the innate immune
responses to many bacterial pathogens4,7,12–20, viral pathogens4,7,21–26
Department of Molecular and Cell Biology, Division of Immunology and
Pathogenesis, University of California, Berkeley, Berkeley, California, USA.
Correspondence should be addressed to R.E.V. ([email protected]).
Received 27 September; accepted 8 November; published online 14 December
2012; doi:10.1038/ni.2491
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and eukaryotic pathogens27. STING also seems to have a central role
in certain autoimmune diseases initiated by inappropriate recognition
of self DNA28, and STING-dependent signaling seems to be required
for the induction of adaptive immunity in response to DNA vaccines7.
STING has also been proposed to sense membrane-fusion events
associated with viral entry, in a manner independent of the sensing
of nucleic acids21. Thus, STING is clearly a central participant in a
variety of innate and adaptive immune responses.
Human and mouse STING are 81% similar and 68% identical
at the amino-acid level, and there are putative STING orthologs in
diverse species, including zebrafish and Xenopus. Several distinct
alleles encoding STING have been described in humans, including
a potentially nonfunctional allele found to be homozygous in ~3%
of two American cohorts29. STING seems to be expressed mainly
in the thymus, heart, spleen, placenta, lung and peripheral leukocytes
but is poorly expressed in the brain, skeletal muscle, colon, small
intestines, liver and kidney4–6, suggestive of a function in the immune
system. STING is expressed only in certain transformed cell lines,
including HEK293 human embryonic kidney cells, A549 adenocarcinomic human alveolar basal epithelial cells, THP-1 human monocytic cells and U937 human leukemic monocyte lymphoma cells, but
is undetectable in most cell lines, including HEK293T cells, HuH-7
human hepatocyte-derived cellular carcinoma cells or HeLa human
cervical cancer cells5,6.
The amino-terminal domain of STING, which encompasses
approximately the first 130 amino acids, seems to comprise four transmembrane domains, whereas the final 250 amino acids comprise a
globular carboxy-terminal domain (CTD; Fig. 1), of which several
crystal structures have been reported30–34. It is presumed that the
STING CTD resides in the cytosol, though this remains to be shown.
As discussed below in greater detail, STING seems to respond to the
cytosolic presence of dsDNA and cyclic dinucleotides by relocalizing
to discrete foci in the cell cytoplasm. This relocalization is associated
with recruitment and activation of the kinase TBK1, which phosphorylates IRF3, a key transcription factor required for the induction
of Ifnb expression (Fig. 2). Other additional reported functions for
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DD
379
CTD
Crystallized
Figure 1 STING protein architecture. Amino acids 1–379 of human
STING include the transmembrane regions (TM1–TM4; black) and the
STING dimerization domain (DD; gray). The CTD (amino acids 138–379)
includes the dimerization domain and the CTT (amino acids 340–379);
published structures have crystallized the CTD lacking the CTT. Residues
Tyr167 and Glu260 (above) are proposed to be critical for c-di-GMP
binding.
Recognition of cyclic dinucleotides by STING
Cyclic dinucleotides are second-messenger signaling molecules produced by diverse bacterial species, but there is no evidence that they
are produced by mammalian cells. They consist of two ribonucleotides
that are circularized by canonical 5`-to-3` phosphodiester bonds.
Cyclic di-GMP (c-di-GMP) is the most well-studied cyclic dinucleotide and has been shown to regulate diverse aspects of bacterial physiology36, whereas cyclic di-AMP (c-di-AMP) and the hybrid cyclic
di-AMP-GMP are less well understood37,38. Notably, cyclic dinucleotides seem to be detected in the cytosol of mammalian cells; this
leads to activation of TBK1-IRF3 and the downstream production of
type I interferons39–41. Microarray analyses suggest that the transcriptional response to cytosolic c-di-GMP is essentially indistinguishable
from the response to cytosolic dsDNA40. STING is required for interferon production in response to cyclic dinucleotides8,9 by a mechanism that, surprisingly, seems to involve direct recognition of cyclic
dinucleotides by STING itself 42. Recombinant purified STING protein produced by bacteria has been found to
bind c-di-GMP and c-di-AMP (but not AMP,
GMP, cAMP, pGpG or other nucleic acids)
Cyclic
dinucleotides
DNA
?
ING
ST
ING
ST
ING
ING
ST
ST
G
IN
NG
STI
ST
TBK1
ER
Mitochondria?
Autophagy-like
responses
ST
ING
TBK1
TB
K1
P
MAM?
IRF3
P
P
P
P
P
Ifnb
Ccl2, Ccl20
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TBK1
STAT6
IRF3
P
STAT6
Nucleus
P
20
Structural analysis of STING
Five separate groups have published crystal structures of human
STING alone and in complex with c-di-GMP30–34. The structures are
in broad agreement with each other and show that STING adopts an
A-B fold of novel topology (Fig. 3a) that bears some distant similarity
to the nucleotide-binding domain of the kinase LRRK2 and to small
G proteins of the Ras family of GTPases31,34.
When not ligand bound, the STING CTD crystallizes as a symmetrical dimer (Fig. 3a). Gel-filtration experiments have confirmed
that STING that is not ligand bound exists mainly as a dimer30,31,33,34,
even in solution, and thus the dimers observed in the structures are
presumably not an artifact of crystallization. The observation of a
preformed dimer is an important result, because if endogenous
STING exists in this state in vivo, then dimerization is probably not
the mechanism of ligand-induced signaling, despite some suggestions
G
STIN
Figure 2 Overview of STING signaling.
In response to dsDNA and cyclic dinucleotides,
dimeric STING interacts with TBK1 and
relocalizes to an unknown compartment.
After that relocalization, TBK1 interacts
with and phosphorylates IRF3, which
induces its dimerization and translocation
to the nucleus. In the nucleus, IRF3 (along
with other transcription factors) binds to
promoter elements to induce transcription
of Ifnb and other coregulated genes. After
relocalization, STING can also interact with
STAT6. This interaction leads to TBK1dependent phosphorylation of STAT6, which
induces its dimerization and translocation to
the nucleus, where it induces transcription of
Ccl2 and Ccl20 (independently of IRF3 and
interferon). The STING-TBK1 complex can
also recruit autophagy factors, which leads to
autophagy-like responses independently of Ifnb
transcription. ER, endoplasmic reticulum;
MAM, mitochondria-associated membrane.
STAT6
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© 2013 Nature America, Inc. All rights reserved.
STING include the activation of autophagy pathways 11,14,35 and activation of the STAT6 transcription factor10 (Fig. 2). Here we review
the present understanding of STING in innate immune responses to
nucleic acids in the cytosol.
G
TM4
ST
IN
TM3
STAT6
TM2
CTT
IR
F3
TM1
Glu260
IRF3
1
with a dissociation constant of 2.5–5 MM (refs. 30,31,33,34,42), consistent with the reported dose-responsiveness of cells41. Whereas
wild-type STING restores the ability of STING-deficient 293T cells
to respond to cyclic dinucleotides, STING mutants defective in the
binding of cyclic dinucleotides do not34,42. Interestingly, expression of
wild-type STING does not restore the ability of 293T cells to respond
to dsDNA, which suggests that additional factors upstream of STING
are required for the detection of dsDNA (discussed below). Indeed,
there is at present no evidence for the direct binding of dsDNA by
STING42. Consistent with the idea that STING has distinct roles
in responses to cyclic dinucleotides and DNA, mutational analysis
has identified a point substitution in STING (R231A) that abolishes
the cytosolic response to cyclic dinucleotides but not to dsDNA 42.
Surprisingly, the R231A substitution does not affect binding of c-diGMP to STING but instead seems to affect the ability of STING to
transmit signals in response to cyclic dinucleotides. Although such
studies strongly suggest that STING functions as a direct sensor of
cyclic dinucleotides, but as a signaling adaptor in the response to
dsDNA, it remains possible that STING participates in the direct recognition of DNA and also that additional host factors are required for
the detection of cyclic dinucleotides.
340
180
IRF3
Tyr167
STAT6
155
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Figure 3 STING structure. (a) Structure of the
STING dimer not bound to ligand. The loop
region between B2 and B3 (not shown here) is
indicated by dotted lines connecting B2 and B3
on each STING protomer (Protein Data Bank
(PDB), 4EMU)34. (b) Change in the structure
of STING after ligand binding, indicated by the
crystal structures of STING not bound to ligand
(blue; PDB, 4EMU) and STING in complex
(magenta) with c-di-GMP (cyan; PDB, 4EMT),
aligned with the PyMOL molecular graphics
system; dotted lines indicate the loop region
between B2 and B3 (as in a)34. (c) Four crystal
structures of the STING dimer (magenta) bound
to c-di-GMP (cyan), aligned with PyMOL (PDB,
4EF4, 4F9G, 4F54 and 4EMU); yellow, Arg232
(refs. 30,31,33,34). (d) Overlay of a fifth
structure (yellow; PDB, 4F5D) that differs from
those in c, aligned as in c (magenta).
a
b
Ligand-free STING
STING–c-di-GMP complex (4EMT)
STING protomer 1
STING protomer 2
Ligand-free STING (4EMU)
c
Arg232
Arg232
d
to the contrary6,9,43–45. The STING dimer
resembles a pair of wings, or a butterfly,
with a deep cleft between the two dimers
STING–c-di-GMP complex
STING–c-di-GMP complex
(4EF4, 4F9G, 4F54, 4EMU)
(4EF4, 4F9G, 4F54, 4EMU, 4F5D)
(Fig. 3a). Dimer formation is mediated by a
hydrophobic A-helix (A1) that encompasses
approximately residues 153–190, a region that some structure- of c-di-GMP to STING or the interferon response to dsDNA. The
prediction algorithms (apparently incorrectly) model as a trans- common human allele encoding STING also produces a protein with
membrane segment3,4. The hydrophobic residues from one STING arginine at the corresponding codon (Arg232)29. Interestingly, howprotomer form intermolecular interactions with the other STING pro- ever, three of the five crystal structures30,31,34 are of a rare His232
tomer, forming a substantial hydrophobic core that buries ~1,800Å2 human isoform that is unresponsive to cyclic dinucleotides (D.L.B.
and R.E.V., unpublished observations), although it is still responsive
of combined surface area.
As predicted by binding studies30–34,42, the crystal structures show to DNA4,7. The R232H substitution may account for the lack of subthat each STING dimer binds one molecule of c-di-GMP, which stantial structural changes observed after the binding of c-di-GMP in
is accommodated in the deep cleft between the two wings of the these structures. The other two crystal structures32,33 are of STING
dimer (Fig. 3b). Notably, c-di-GMP is itself a symmetric dimeric proteins with arginine at position 232. Human STING with Arg232
molecule and is thus accommodated naturally in the symmetrical may be responsive to cyclic dinucleotides, although it is important
STING dimer. When bound to STING, c-di-GMP adopts a bent to emphasize this has not yet been demonstrated. Residue 232 lies
U-shape with the phosphates deep in the cleft and the guanine in the loop region between B-sheet 2 (B2) and B-sheet 3 (B3) that
rings pointing upward. In all of the structures, the aromatic ring is predicted to cover the binding pocket. In the His232 structures,
of Tyr167 mediates critical stacking interactions with the guanine these loops are considerably disordered, such that the position of
ring of c-di-GMP. These stacking interactions would probably not residue 232 cannot be confirmed. In the Arg232 structures, the B2-B3
form with smaller pyrimidine bases, so c-di-TMP and c-di-CMP loop is modeled, although the two structures differ in the position
are not predicted to bind strongly to STING30, although this has not of Arg232. One published structure shows Arg232 oriented down
been addressed experimentally. Interestingly, many of the interac- into the binding pocket32, whereas the other shows Arg232 pointing
tions between STING and c-di-GMP are mediated by solvent, and out and away from the binding pocket33. Interestingly, the former
there is little change in the residues lining the pocket after c-di-GMP STING–c-di-GMP structure32 differs the most from the other four
binding (Fig. 3b). This raises the important question of how STING structures (Fig. 3c,d). One possible interpretation of these varying
induces signals after ligand binding (addressed below).
results is that the loop between B2 and B3 is highly flexible and that
Although all five structures are of human STING bound to the binding of c-di-GMP induces changes in the conformation of
c-di-GMP, STING also seems to detect c-di-AMP, although with per- the critical arginine that are important for downstream signaling.
haps lower affinity than its affinity for c-di-GMP31. The lower affinity Unfortunately, the structures do not provide insight into why the
may be explained by a lack of an amine at position 2 of the purine ring arginine in the B2-B3 loop is required for signaling only in response
of adenine (in contrast to guanine), which would prevent the forma- to cyclic dinucleotides but not in response to DNA.
tion of some key hydrogen bonds30. The structures also suggest that
cyclic dideoxyribonucleotides might not bind as strongly to STING, How does STING transduce signals?
as the 2` hydroxyl of ribose forms a hydrogen bond with Thr263 of As the crystal structures reported thus far do not show consistent
STING. The structures also do not suggest an obvious mechanism by structural rearrangement after c-di-GMP binding (Fig. 3b), these
which dsDNA might bind directly to STING, although it is certainly structures do not indicate an obvious conformational change that
possible that the deep cleft in the STING dimer could accommodate could be responsible for inducing downstream signaling. Thus, structures of STING in association with downstream signaling molecules
diverse ligands.
As mentioned above, replacement of the arginine at position 231 may be critical for identifying the mechanism of signal transduction
in mouse STING with alanine (R231A) renders STING unresponsive after ligand binding. In addition, the published structures are of only
to cyclic dinucleotides but, interestingly, does not affect the binding a fragment of STING and do not, for example, provide structural
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DNA sensor
DNA sensor
N
CTT
N
CTT
N
N
N
CTT
DNA-bound
(active)
N
DNA-bound
(active)
Arg232
Dimerized
(resting or inactive)
STING
STING relocalization and autophagy
One report has suggested that STING localizes to mitochondria5,
but a subsequent consensus is that in resting cells, STING localizes
TBK1
CTT
CTT
N
Cyclic dinucleotide
information about the carboxy-terminal tail
(CTT) of STING (residues 340–379; Fig. 1),
which has been proposed by one group to be critical for STING to
transduce signals30,44. In this model, the CTT is proposed to bind
the STING CTD and, in the absence of ligand, maintain STING in
an inactive state. In the presence of ligand, the CTT is then proposed
to disengage from the CTD and become available to recruit TBK1
and IRF3 (Fig. 4). Although some mutant analysis and biochemical
evidence supports this model30,44, it will be important to obtain direct
biochemical information about the arrangement of the CTT in the
presence and absence of ligand.
In addition to interacting with TBK1 and IRF3, STING has been
proposed to interact with other signaling proteins. For example,
STING contains putative binding sites for the adaptor TRAF2 (ref. 29)
and has been suggested to interact with the ubiquitin ligase TRAF3
(ref. 5), but a requirement for TRAF proteins in STING signaling
is not well established. STING has also been shown to be phosphorylated on several residues, including Ser358 (ref. 45). However, the
importance of phosphorylation for signaling is not fully resolved34,44.
Ubiquitination may also have an important role in regulating STING
signaling. The ubiquitin ligase RNF5 seems to negatively regulate
STING signaling46, whereas the ubiquitin ligases TRIM56 and
TRIM32 have been suggested to positively regulate STING signaling
by catalyzing the addition of Lys63 (K63)-linked ubiquitin chains to
Lys150 (and perhaps other residues) of STING45. Ubiquitination of
Lys150 is suggested to be important for inducing the dimerization
of STING and/or recruitment of TBK1 (refs. 45,47). However, that
model conflicts with a structural analysis suggesting that Lys150 is not
involved in dimerization or activation31. K150A, K150L and K150R
mutants form dimers equally well, as assessed by size-exclusion
chromatography, and, in addition, STING-deficient mouse embryonic fibroblasts reconstituted with K150R mutant of STING induce
interferon in response to B-form DNA31.
Finally, in addition to its role in the recruitment of TBK1 and IRF3,
the carboxyl terminus of STING has also been proposed to recruit
STAT6, which is then phosphorylated on Ser407 by TBK1 (ref. 10).
The activation of STAT6 by this pathway is independent of the kinase
Jak and is thus distinct from canonical STAT6 activation downstream
of cytokine receptors. Interestingly, STING-dependent STAT6 activity
does not seem to be important for the induction of type I interferons
but is instead required for the transcription of a subset of STINGdependent genes, including those encoding the chemokines CCL2
and CCL20 (ref. 10).
22
DNA sensor
CTT
CTT
CTT
IRF3
P
TBK1
TBK1
CTT
CTT
N
N
P
IRF3
Figure 4 Potential model of STING signaling.
In the inactive and autoinhibited state (left),
STING exists as a constitutive dimer. After
recognition of cytosolic dsDNA by a DNA
sensor (middle and right), autoinhibition is
relieved and the CTT is exposed to facilitate
interaction with TBK1 and phosphorylation
of IRF3. Cyclic dinucleotides are recognized
directly by STING, but this similarly leads
to relief of autoinhibition, interaction with
TBK1 and phosphorylation of IRF3. Signaling
downstream of cyclic dinucleotides requires
Arg232 (red dots in the loop regions between
B2 and B3), whereas signaling in response to
dsDNA does not30,42.
N
Cyclic dinucleotide bound
(active)
Cyclic dinucleotide bound
(active)
to the endoplasmic reticulum4,6,35 or perhaps to the mitochondriaassociated membrane7, a compartment that transiently tethers the
endoplasmic reticulum to mitochondria48. After activation by DNA or
cyclic dinucleotides, STING relocalizes to concentrated foci (puncta)
in the cell14,35. The nature of this intracellular compartment is not
entirely clear. However, several reports have suggested that the relocalization of STING is associated with the activation of autophagy.
Activated STING localizes together with several autophagy-associated
proteins, including Atg9a, p62 and LC3 (ref. 35), and Atg7-deficient
cells that are defective in autophagy are also reported to be defective
in the STING-dependent induction of type I interferons11. However,
a different study has reported normal relocalization of STING and
induction of interferon in both Atg7-deficient cells and Atg16Ldeficient cells35. Moreover, the localization of STING together with
other autophagy-associated proteins, such as ULK1, Atg14L or Atg5,
was not observed in that study, and electron microscopy did not show
localization of STING to the classic double-membraned structures
considered definitive markers of autophagy35. In fact, Atg9a−/− cells
that are also defective in autophagy actually have a heightened interferon response35, which suggests that autophagy negatively regulates
interferon induction. Therefore, the extent to which STING puncta
are to be considered ‘true’ autophagosomes is uncertain. Nevertheless,
some important work has demonstrated a requirement for STINGinduced autophagy-like responses for innate defense during infection. For example, the intracellular bacterial pathogen Mycobacterium
tuberculosis has been shown to produce cytosolic DNA ligands during
infection13, and these ligands have been found to activate STING,
resulting in ubiquitination of bacteria and the subsequent recruitment of TBK1, p62 and NDP52, all of which are required for ‘selective’
autophagic targeting and innate defense against M. tuberculosis14.
A STING-dependent autophagy-like response has also been observed
in response to infection with A-herpesvirus11. Thus, STING seems to
coordinate multiple immunological defense responses to infection,
including the induction of interferons and STAT6-dependent chemokines and selective induction of autophagy (Fig. 2).
The role of STING in responses to cytosolic DNA
Two important papers published in 2006 demonstrated that cells
can respond to the cytosolic presence of dsDNA by inducing the
expression of genes encoding type I interferon and other coregulated
genes49,50. It was later shown that in certain cells (notably HEK293T
cells), this response is induced only by highly AT-rich dsDNA (for
example, poly(dAT:dTA)) that is transcribed by RNA polymerase III
into RNA that activates the cytosolic RNA sensor RIG-I and its downstream signaling adaptor MAVS51,52. As the RNA polymerase III
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pathway is selective only for very highly AT-rich DNA51,52, it does
not seem to be of major relevance to the response to most pathogens.
Notably, however, many cell types (including mouse and human cells)
are also able to respond to ‘normal’ non-AT-rich dsDNA50,53. This
response requires STING and is independent of RIG-I–MAVS7. A key
unresolved issue for the field is how STING is activated in response to
DNA. One possibility is that STING senses DNA directly, but because
STING expression in 293T cells restores responses to cyclic dinucleotides but not to dsDNA, it seems that additional host factors may be
required for cytosolic responses to DNA42. DNA also does not compete for the binding of cyclic dinucleotides to STING42. Although that
last result does not rule out the possibility that STING binds DNA and
cyclic dinucleotides at distinct sites, there is at present no published
evidence of a direct STING-DNA interaction.
Thus, it is widely presumed that at least one additional protein is
required for STING-dependent responses to cytosolic DNA. Indeed,
as is discussed below, several proteins have been proposed to function as DNA ‘sensors’. None of these has yet been met with universal
acceptance, as it has been surprisingly challenging to provide conclusive experimental evidence that a given protein is a sensor of cytosolic
DNA. At least 2,600 proteins with identifiable DNA-binding domains
are encoded by the human genome54, and presumably few if any of
those proteins function as DNA sensors in the innate immune system.
Thus, binding to DNA is not itself sufficient evidence that a given
protein is a sensor of DNA. Therefore, in addition, any putative sensor should be shown to act upstream of STING, and the mechanism
by which the sensor activates STING should be identified. In many
cases, this is proposed to occur via a direct interaction with STING.
However, a major issue with many studies so far is that they have
relied on assays such as immunoprecipitation and immunofluorescence, which may detect nonspecific or indirect interactions between
STING and a putative sensor of DNA. For this reason, it is critical that
immunoprecipitation and immunofluorescence data be supported
by genetic evidence that demonstrates a requirement for the sensor
and its ability to interact with STING. Unfortunately, redundancy
among multiple sensors may make this condition difficult to fulfill.
In addition, in most studies so far, knockdown of the putative sensor by small interfering RNA or short hairpin RNA, rather than the
more conclusive knockout of the gene encoding the sensor, has been
used to demonstrate the role of the sensor in responses to DNA. In
some existing studies, such knockdown affects interferon induction
by as little as twofold, which is very modest, given that Ifnb can be
induced ~1,000-fold or more. Indeed, the large induction window
of Ifnb means that this gene is particularly sensitive to off-target or
indirect effects of small interfering or short hairpin RNA, a problem
compounded by the fact that the DNA-binding proteins proposed
to act as ‘sensors’ might have central roles in the biochemistry and
metabolism of nucleic acids and thus might be expected to exert indirect effects on the interferon response. Such confounding indirect
effects could be controlled for by the demonstration that knockdown
of a putative DNA sensor does not affect direct activation of STING
by cyclic dinucleotides, but this control is in general lacking in most
studies at present. Below we summarize the present state of knowledge
of several putative sensors of cytosolic DNA.
The DNA-binding protein ZBP1 (also called DLM1 or DAI) was
the first protein to be proposed to be a sensor of cytosolic DNA on
the basis of several lines of evidence55,56. First, knockdown of ZBP1
in mouse L929 fibroblasts results in a modest (approximately threefold) defect in the induction of Ifnb in response to cytosolic DNA. In
addition, ZBP1 contains DNA-binding domains and thus (perhaps
unsurprisingly) binds DNA. Finally, overexpressed ZBP1 associates
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with overexpressed IRF3 and TBK1, as shown by coimmunoprecipitation. However, although ZBP1 was initially characterized before
the discovery of STING, subsequent studies have not established a
physical or genetic link to STING signaling. Moreover, knockdown of
ZBP1 in mouse embryonic fibroblasts has almost no effect on interferon responses to DNA56, and targeted deletion of Zbp1 in mice57
does not produce any interferon-related altered phenotype. In the 5
years since the initial report on ZBP1, few additional studies have
confirmed a role for ZBP1 in interferon responses to DNA. One report
described a modest (approximately twofold) lower interferon induction in response to pneumococcal DNA15, and another found a role
for ZBP1 in responses to human cytomegalovirus22, but several other
studies have observed no effect of knockdown of ZBP1 in a variety
of cell types13,25,27,58. Some studies have suggested a role for ZBP1 in
activation of the transcription factor NF-KB rather than induction of
interferon59–61. Interestingly, a report has linked ZBP1 to a very different host response to infection: induction of programmed necrosis
via recruitment of the kinase RIP3 (ref. 62). At most, such studies lead
us to conclude that ZBP1 has a very minor role in STING-dependent
interferon responses to dsDNA.
Another putative sensor of cytosolic DNA is DDX41. The cytosolic
RNA sensors RIG-I (DDX58) and its paralog Mda5 are members of a
large superfamily of DExD/H-box RNA helicases. This protein superfamily might also include DNA sensors; however, most DExD/H-box
proteins seem to have critical roles in RNA biogenesis63. Thus, knockdown or knockout of these proteins may have nonspecific effects on
the expression of interferon-encoding genes. Nevertheless, in one
published study, a small interfering RNA screen was done in which
individual DExD/H-box mRNAs were knocked down in a mouse
dendritic cell line (D2SC cells)64. In that study, knockdown of DDX41
strongly affected the interferon response to the synthetic B-form
dsDNA poly(dAT:dTA) and poly(dGC:cCG) and a DNA virus (herpes
simplex virus type 1), producing a decrease of >90%, but did not affect
the interferon response to poly(I:C) RNA or an RNA virus (influenza
A virus). Similar specific effects of DDX41 knockdown were also seen
in primary bone marrow–derived DCs, as well as human THP-1 cells.
Despite its homology to RNA helicases, DDX41 did not bind poly(I:C)
or poly(U) RNA but, unexpectedly, instead bound both AT-rich and
GC-rich dsDNA64. DDX41 also associated with STING, as assessed
by coimmunoprecipitation64. Together these results indicate involvement of DDX41 in the cytosolic response to DNA but do not formally
prove that the role of DDX41 in the interferon response to DNA is
achieved via an ability to activate STING. It will be important to determine how DDX41 associates with and activates STING. It will also
be important for the knockdown experiments to be confirmed with
mice in which the gene encoding DDX41 is targeted.
Interestingly, knockdown of DDX41 also affected the interferon
response of dendritic cells to Listeria monocytogenes64, a bacterial
pathogen that induces interferon via the secretion of c-di-AMP41.
Indeed, another report has proposed that, in addition to its role
in sensing DNA, DDX41 is also the “main” direct sensor of cyclic
dinucleotides65. This new model is at odds with the proposal that
STING is the direct sensor of cyclic dinucleotides42. The authors
of the new study have determined that c-di-GMP binds to recombinant DDX41 with a dissociation constant of 5.7 MM (ref. 65).
Interestingly, they also confirm that STING binds c-di-GMP but
report a dissociation constant of ~15 MM (ref. 65), which is a lower
measured affinity than that of the consensus of five other studies
(~2.5–5 MM)30,31,33,34,42. Nevertheless, because in their studies,
DDX41 has a slightly higher affinity for c-di-GMP than does STING,
the authors propose that DDX41 is the “main” sensor of cyclic
23
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REVIEW
dinucleotides65. However, if DDX41 is the direct sensor of both cyclic
dinucleotides and DNA, then it is difficult to explain why 293T cells
expressing STING selectively respond to cyclic dinucleotides and not
to DNA42. The authors of the new study suggest STING may merely
act as a “secondary receptor or coactivator” for c-di-GMP65. A clear
picture of how this might work, and confirmation of the association
of DDX41 with c-di-GMP and/or STING, awaits crystallographic and
biochemical analysis of a DDX41–c-di-GMP complex (with or without
STING), analogous to the crystallographic and biochemical studies
that seem to confirm STING as a sufficient and direct sensor of cyclic
dinucleotides30,31,33,34,42.
Two more putative sensors of cytosolic DNA are IFI16 and IFI204.
IFI16 is a member of the PYHIN family of proteins that contain at
least one carboxy-terminal HIN-200 DNA-binding domain and
(except for mouse p200) an amino-terminal PYRIN domain. The
PYHIN family also includes AIM2, a protein that has been shown
to activate a caspase-1 inflammasome in response to cytosolic DNA.
Thus, members of this family have also been dubbed ‘ALRs’ (‘AIM2like receptors’)53.
Several lines of evidence support the proposal that IFI16 is a
cytosolic sensor of DNA53. As expected, given the presence of a
DNA-binding domain, IFI16 binds DNA. Interestingly, IFI16 binds
both single-stranded DNA and dsDNA, even though only dsDNA is
stimulatory27,53. IFI16 also immunoprecipitates together with STING
in dsDNA-stimulated THP-1 cells, as well as when overexpressed in
293 cells. Several groups have observed modest (typically twofold)
decreases in the interferon response in cells in which IFI16 has been
knocked down13,66,67. This suggests that the effect of IFI16 deficiency,
although modest, is reproducible. The lack of a more substantial effect
of IFI16 deficiency may be due to compensation by other sensors of
DNA. It has also been difficult to show that expression of IFI16 and
STING is sufficient to restore a robust response to DNA by 293T cells,
which indicates other proteins may be required, although improper
nuclear localization of IFI16 in 293T cells may also be a complicating
factor53,68,69. Despite those studies, there is still no clear mechanism for
how IFI16 associates with and activates STING. PYRIN domains typically mediate homotypic interactions with other PYRIN-containing
adaptor proteins. For example, the PYRIN domain in AIM2 mediates
interactions with the PYRIN-containing adaptor ASC. STING lacks a
PYRIN domain, so it is not clear how it interacts with IFI16 or whether
the PYRIN domain of IFI16 is involved in the activation of STING.
Interestingly, IFI16 has been proposed to function in activation of the
inflammasome in response to Kaposi’s sarcoma–associated herpesvirus70 and to restrict the replication of human cytomegalovirus via
a mechanism independent of type I interferon71. Thus, considerable
work is still needed to disentangle the many potential roles of IFI16
and other ALRs in innate immune responses.
Several other putative DNA sensors have been described in the
literature. For example, knockdown of LRRFIP1 is reported to
result in modestly lower interferon response to dsDNA72, but activation of IRF3, NF-KB and mitogen-activated protein kinases is
unaffected, so the role of LRRFIP1 is probably not upstream of
STING. In addition, DHX9 and DHX36 have been linked to the
response to DNA73, but this was reported to occur via MyD88dependent signaling rather than STING-dependent signaling, and
subsequent studies have indicated roles for DHX9 and DHX36
instead in the response to RNA74,75. Ku70 has also been proposed
to be a cytosolic DNA sensor but is not proposed to signal via STING
or to activate type I interferons76.
Although the requirement for STING in responses to cytosolic
DNA is well established, the role of STING in responses to RNA
24
is much less clear. The initial characterization of STING-deficient
mouse embryonic fibroblasts suggested that these cells have a modest defect in the interferon response to RNA viruses such as Sendai
virus and vesicular stomatitis virus4. Indeed, additional studies have
shown that STING interacts with key components of the RNA-sensing
pathway, such as RIG-I and MAVS5,6,10,23,24,46. Although STING may
interact with those components, an absolute requirement for STING
in the interferon response to RNA, RNA viruses or their mimics (such
as poly(I:C)) is not always observed4,7,8,10,11. One study has shown
that STING is selectively required for STAT6-dependent responses
to RNA viruses, such as induction of CCL2, but is dispensable for the
IRF3-dependent interferon response to RNA viruses10, presumably
because these viruses can activate IRF3 via the MAVS pathway. Thus,
although STING may participate in a complex containing RIG-I or
MAVS and may also be essential for induction of certain STAT6 target
genes, it may not be required for interferon induction in response to
RNA or RNA viruses.
Concluding remarks
Although it is clear that STING has a central role in immune responses
to cytosolic nucleic acids, there are still major unresolved questions
about the underlying mechanisms that control STING activation and
signaling. Despite five crystal structures of ligand-free STING and
ligand-bound STING, it remains unclear how STING transitions from
an inactive state to a signaling-competent state. Moreover, the cellular
biological events that control the dynamic localization of STING in
cells are not well characterized. The role of STING in responses to
cytosolic RNA or RNA viruses is another area in which the literature
is inconsistent. It is also not clear whether cyclic dinucleotides are
the only physiological ligands for STING or whether perhaps other
ligands may also be able to activate STING. Finally, and perhaps most
importantly, it remains unclear how DNA is sensed in the cytosol and
how such sensing leads to the activation of STING. Resolving these
issues will constitute a major chapter in the understanding of innate
immune defense.
ACKNOWLEDGMENTS
We thank our colleagues in the field of STING biology and members of the Vance
and Barton laboratories for discussions. Supported by the US National Institutes
of Health (AI091100 to D.L.B., and AI063302, AI075039 and AI082357 to R.E.V.)
and the Burroughs Wellcome Fund (R.E.V.).
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/ni.2491.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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