Download Plasmacytoid dendritic cells: sensing nucleic acids in viral infection

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Plasmacytoid dendritic cells: sensing nucleic acids in viral infection
and autoimmune diseases
Michel Gilliet*, Wei Cao* and Yong-Jun Liu
Abstract | Plasmacytoid dendritic cells (pDCs) are important mediators of antiviral immunity
through their ability to produce large amounts of type I interferons (IFNs) on viral infection.
This function of pDCs is linked to their expression of Toll-like receptor 7 (TLR7) and TLR9,
which sense viral nucleic acids within the early endosomes. Exclusion of self nucleic acids from
TLR-containing early endosomes normally prevents pDC responses to them. However, in some
autoimmune diseases, self nucleic acids can be modified by host factors and gain entrance to
pDC endosomes, where they activate TLR signalling. Several pDC receptors negatively
regulate type I IFN responses by pDCs during viral infection and for normal homeostasis.
Type I interferons
Molecules produced by cells of
the immune system that are
rapidly induced by infection
with viruses and bacteria.
They immediately limit viral
replication and increase
subsequent antigen-specific
immune responses.
CpG-containing DNA
DNA oligodeoxynucleotide
sequences that include a
cytidine–guanosine sequence
and certain flanking
nucleotides. These sequences
have been found to induce
innate immune responses
through interaction with
Toll-like receptor 9.
Department of Immunology
and Center of Cancer
Immunology Research,
The University of Texas M. D.
Anderson Cancer Center,
Houston, Texas 77030, USA.
Correspondence to Y.-J.L.
e-mail: [email protected]
*These authors contributed
equally to this manuscript.
doi:10.1038/nri2358
Published online 18 July 2008
Plasmacytoid dendritic cells (pDCs) were first described
by pathologists in the 1950s as plasmacytoid T cells or
plasmacytoid monocytes on the basis of their plasma-cell
morphology and expression of the T‑cell marker CD4 or
the myeloid-cell markers MHC class II, CD36 and CD68
(Ref. 1). In the 1980s, virologists and immunologists were
intrigued by an ill-defined human blood cell type, which
was referred to as a natural type-I-interferon-producing
cell (IPC) owing to its capacity to produce large amounts
of type I interferons (IFNs) following culture with viruses2.
This mysterious cell type was found to correspond to
pDCs3–9; therefore, in this Review, we use the term pDCs
to refer to this cell population.
One important question raised following the identification of pDCs was how they sense viral infection to
rapidly produce a large amount of type I IFNs. At that
time, it was known that two classes of nucleic acids,
namely microbial-derived oligonucleotides containing
unmethylated CpG dinucleotides10 and double-stranded
RNA (dsRNA) synthesized by various viruses, could
strongly induce type I IFN production by mammalian
cells11. In 2001, Kadowaki et al. reported that pDCs
produced type I IFNs in response to CpG-containing DNA
(as found in DNA viruses and bacteria) but not to the
viral dsRNA mimic polyinosinic–polycytidylic acid
(polyI:C). By contrast, myeloid DCs (mDCs) produced
type I IFNs and interleukin‑12 (IL‑12) in response to
polyI:C but not to CpG-containing DNA12. Although the
receptors for CpG-containing DNA and polyI:C were
unknown at the time of these observations, the different
responses of pDCs and mDCs to these microbial-associated
nucleic acids suggested that pDCs and mDCs may
express different pattern-recognition receptors and that
pDCs may detect viral infection by recognizing viral
nucleic acids12.
A second intriguing observation in this study was
that not all of the known CpG-containing oligodeoxynucleotides (CpG ODNs) could induce pDCs to produce
type I IFNs12. Interestingly, at the same time, Klinman
and colleagues classified CpG ODNs into two main
groups: D‑type (now known as A‑type) CpG ODNs,
which stimulate IFNγ production by natural killer (NK)
cells, and K‑type (now known as B‑type) CpG ODNs,
which stimulate B-cell proliferation and B-cell IL‑6 and
antibody production13,14. It is now clearly established that
the A‑type CpG ODNs can efficiently stimulate IFNα production by pDCs that then activate NK cells15–17, whereas
the B‑type CpG ODNs do not12,14,18. Instead, B‑type CpG
ODNs stimulate pDCs to produce IL‑6 and tumournecrosis factor (TNF) and induce B‑cell proliferation and
antibody production12,14. These studies suggest that pDCs
may have developed two different signalling mechanisms
to respond to different types of CpG ODN.
In this article, we review the remarkable progress
made over the past 7 years in understanding the molecular mechanisms by which the innate immune system,
in particular pDCs, sense and respond to microbial
nucleic acids during infection. Furthermore, we discuss
the mechanism by which pDCs sense and respond to
self nucleic acids — a mechanism that can potentially
trigger autoimmune diseases — and the way that these
responses might be regulated.
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Pattern-recognition
receptors
Host receptors that can sense
pathogen-associated molecular
patterns and initiate signalling
cascades that lead to an innate
immune response. These can
be membrane bound (for
example, Toll-like receptors) or
soluble cytoplasmic receptors
(for example, RIG‑I, MDA5 and
NLRs).
A-type CpG ODNs
(Also known as D‑type CpG
ODNs). Synthetic oligodeoxynucleotides (ODNs) with the
following three features: polyG
sequences at the 3′ end;
a central palindromic
sequence; and cytosineguanine dinucleotides within
the palindrome. The polyG tails
on A‑type CpG ODNs can
interact with each other,
resulting in the formation of
guanine tetrads and clusters.
B-type CpG ODNs
(Also known as K‑type CpG
ODNs). Synthetic oligodeoxynucleotides that contain a CpG
motif(s) on a phosphorothioate
backbone.
Nucleic-acid sensing by the immune system
Multiple receptors have evolved in mammals for detecting non-self nucleic acids during microbial infection,
with several receptor systems often existing within a
single cell. Interestingly, however, pDCs are unique in
that they only use one such receptor system to sense
nucleic acids and produce type I IFNs.
Toll-like receptors. Toll-like receptors (TLRs) are a family of conserved membrane-spanning molecules that
contain an ectodomain of leucine-rich repeats, a transmembrane domain and a cytoplasmic domain known
as the TIR (Toll/IL‑1 receptor) domain. In Drosophila
melanogaster, the Toll receptor controls a main pathway
that is triggered in response to bacterial and fungal infections, induces strong and rapid expression of genes, such
as the nuclear factor-κB (NF-κB) family of transcription
factors, and ultimately induces antimicrobial peptide
production19. Since the first mammalian TLR (now
known as TLR4) was identified by Janeway, Medzhitov
and Preston-Hurlburt20, 10 TLRs have been identified in
humans (FIG. 1) and 12 in mice. The leucine-rich repeats
of the TLR ectodomains bind to pathogen-associated
molecular patterns (PAMPs), such as the bacterial
cell-wall component lipopolysaccharide (a ligand of
TLR4)20,21, bacterial flagellin (a ligand of TLR5)22, and
lipoprotein and peptidoglycan (which are recognized by
TLR2 complexed with TLR1 or TLR6)23–25. Therefore,
these TLRs, which are expressed on the surface of host
cells, facilitate the immediate detection of and response
to microorganisms in their environment26. Other TLRs,
including TLR3 (ref. 27), TLR7 (refs 28–31), TLR8
(refs 29,30) and TLR9 (ref. 32), are located in intracellular
endosomal-lysosomal compartments26,33,34, where they
are involved in the detection of microbial nucleic acids.
Nucleic acid
TLR7 (ssRNA)
TLR8 (ssRNA)
Lipid
TLR9
(CpG-containing DNA)
TLR4 (LPS)
TLR2
(Lipoprotein or PGN)
TLR3 (dsRNA)
TLR10 (?)
TLR6
(Lipoprotein)
TLR1
(Lipoprotein)
TLR5 (Flagellin)
Protein
Figure 1 | Overview of the human TLR family. The molecular tree of the human
Toll‑like receptors (TLRs) is grouped based on the sequence homology and specificity
Reviews | Immunology
for certain pathogen-associated molecular patterns. dsDNA,Nature
double-stranded
DNA;
LPS, lipopolysaccharide; PGN, peptidoglycan; ssRNA, single-stranded RNA.
TLR activation by their cognate agonists leads to the
recruitment of cellular adaptor molecules that contain
TIR domains and the formation of multi-component
signal transduction complexes in the cytoplasm. Four
key adaptor proteins, namely, myeloid differentiation
primary-response gene 88 (MyD88), TIR-domaincontaining adaptor protein (TIRAP; also known as
MAL), TIRAP inducing IFNβ (TRIF) and TRIF-related
adaptor molecule (TRAM), directly bind to TLRs
in various combinations (reviewed in refs 26,35 ).
Adaptor‑molecule binding to TLRs then activates multiple signalling pathways, such as those involving NF‑κB,
mitogen-activated protein kinases (MAPKs) and IFNregulatory factors (IRFs), and initiates the transcription of genes encoding cytokines, chemokines and
co-stimulatory molecules.
The endosomal TLR7/TLR8/TLR9–MyD88 signalling
pathway. Following the discovery by Akira and colleagues that DNA from bacteria has stimulatory effects
on cells of the mammalian immune system through
TLR9 (ref. 32), the function of a group of closely related
endosomal TLRs has been revealed. TLR7, TLR8 and
TLR9, which have high sequence homology36,37, recognize a range of RNA and DNA molecules. TLR7 and
TLR8 respond to guanosine- or uridine-rich singlestranded RNA (ssRNA) from viruses and the synthetic
imidazoquinoline compounds imiquimod and R‑848,
as well as guanosine analogues28,29,38. TLR9 seems to
detect ssDNA molecules that contain unmethylated
CpG-containing motifs, which are commonly found
in the genomes of viruses and bacteria32,39. Synthetic
ODNs containing CpG dinucleotides can interact
directly with the ectodomain of TLR9 and induce
conformational changes 40 that lead to downstream
signalling activation.
After ligand engagement, activated TLR7, TLR8
and TLR9 interact with MyD88, which contains a TIR
domain and a death domain (FIG. 2a). MyD88 serves as a key
adaptor molecule that functions to recruit IL‑1-receptorassociated kinases (IRAKs) to almost all TLRs (except
TLR3) and to the IL‑1 receptor26,41. MyD88‑deficient
mice therefore fail to produce TNF or IL‑6 when exposed
to IL‑1 or microbial components that are recognized by
TLR2, TLR4, TLR5, TLR7 or TLR9 (refs 26,42). Among
the TLRs that associate with MyD88, TLR7 and TLR9
strictly depend on the function of MyD88; TLR4 signals
through MyD88, TIRAP, TRIF and TRAM; and TLR2
uses both MyD88 and TIRAP26,41.
The endosomal TLR3–TRIF signalling pathway. TLR3,
which is localized in endosomes, recognizes viral dsRNA
and its synthetic mimic polyI:C. However, unlike the
other endosomal TLRs (TLR7, TLR8 and TLR9),
TLR3 seems to transduce its signals mainly through
an MyD88-independent pathway, as stimulation with
polyI:C in MyD88-deficient cells does not affect the
production of pro-inflammatory cytokines and co‑
stimulatory molecules26,43. Indeed, TLR3 was shown to
associate with TRIF and signal through IRF3, a key factor
involved in IFNβ production43,44 (FIG. 2b).
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a TLR7/TLR8/TLR9–MyD88 signalling pathway
c RLR signalling pathway
b TLR3–TRIF signalling pathway
MDA5
Endosome
TLR7 or
TLR8
RIG-I
Endosome
TLR9
TLR3
TRIF
TRAF6
p50 p65
MAPKKs
IRF7
p50 p65
MAPKs
IκB
Nucleus
(PAMPs). Molecular patterns
that are found in pathogens
but not mammalian cells.
Examples include terminally
mannosylated and
polymannosylated compounds
(which bind the mannose
receptor) and various microbial
components, such as
bacterial lipopolysaccharide,
hypomethylated DNA, flagellin
and double-stranded RNA
(all of which bind Toll-like
receptors).
Death domain
A protein–protein interaction
domain found in many proteins
that are involved in signalling
and apoptosis.
DExD/H box RNA helicases
A group of enzymes that can
unwind double-stranded RNA
using energy derived from
ATP hydrolysis. The DExD/H
box is a characteristic
amino‑acid signature motif of
many RNA-binding proteins.
Caspase-recruitment
domain
(CARD). A protein domain
that is found in certain
initiator caspases (for example,
mammalian caspase‑9) and
their adaptor proteins (for
example, apoptotic-proteaseactivating factor 1, APAF1).
This domain mediates
protein–protein interactions.
Mitochondrion
TRAF3
P
IRAK1
IRAK4
TRAF3
TBK1
FADD
Caspases
IKKε
IRF3
TBK1
IKKε
IRF3
p50 p65
IRF7
NF-κB
Cytosol
Pathogen-associated
molecular patterns
IPS1?
MyD88
BTK
IPS1
dsDNA
sensor ?
TIR domain
MyD88
LGP2
IFNα,
IFNβ,
IFNλ,
IFNω
IL-6,
TNF
CD80,
CD86
IFNβ
IL-6,
TNF
IFNα,
IFNβ
Figure 2 | Intracellular nucleic-acid sensors and signalling pathways. a | Endosomal Toll-like receptor 7 (TLR7),
TLR8 and TLR9 recognize RNA and DNA ligands, and signal through a key adaptor MyD88 (myeloid differentiation
primary-response gene 88) in the cytosol. MyD88 in turn associates with a signal complex comprised
of TRAF6
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| Immunology
(tumour-necrosis factor (TNF)-receptor-associated factor 6), BTK (Bruton’s tyrosine kinase), IRAK4 (interleukin‑1receptor (IL-1R)-associated kinase 4) and IRAK1, which leads to the activation of IRF7 (interferon-regulatory factor 7),
NF‑κB (nuclear factor‑κB) and MAPKs (mitogen-activated protein kinases). Overall, this pathway potently induces
the production of high levels of type I IFNs (such as IFNα, IFNβ, IFNλ and IFNω), pro-inflammatory cytokines and
expression of co-stimulatory molecules. b | Alternatively, the binding of double-stranded RNA (dsRNA) to endosomal
TLR3 triggers a signalling pathway mediated by TRIF (Toll/IL-1R (TIR) domain-containing adaptor protein inducing
IFNβ), which involves TRAF3, TBK1 (TANK-binding kinase 1) and IKKε (inhibitor of NF‑κB kinase ε). Subsequent IRF3
activation leads to IFNβ production. The TLR3–TRIF signalling pathway also activates NF‑κB and MAPKs and results
in the production of pro-inflammatory cytokines and the expression of co-stimulatory molecules (CD80 and CD86).
c | The dsRNA receptors RIG‑I (retinoic-acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5)
and LGP2 (laboratory of genetics and physiology 2) constitute the third nucleic-acid sensor system, exclusively
located in the cytoplasm of most cells. RIG‑I, MDA5 and LGP2 signal through a common adaptor molecule IPS1
(IFNB-promoter stimulator 1), which is associated with the mitochondria. By coupling with TRAF3, TBK1 and IKKε,
IPS1 directly activates IRF3 and NF‑κB through FADD (FAS-associated via death domain), caspase‑8 and caspase‑10.
This pathway induces the production of type I IFNs and other cytokines. dsDNA-induced IFN production involves a
yet-to-be defined sensor that signals through certain components of the RLR (RIG-I-like receptor) pathway, such as
TBK1 and IRF3. IkB, inhibitor of NF-kB; MAPKK, MAPK kinase.
The RIG‑I-like-receptor signalling pathway. The
observation that nearly all nucleated cells can produce type I IFNs after viral infection or in response
to dsRNA in the cytosol, even in the absence of
endosomal TLRs and their adaptors, suggested that
there is an independent sensory system that detects
nucleic acids in the cytosol (FIG. 2c) . Indeed, three
genes have been identified in humans and mice that
encode a group of proteins known as the RIG‑I-like
receptors (RLRs) — retinoic-acid-inducible gene I
(RIG‑I), melanoma differentiation-associated antigen 5 (MDA5) and laboratory of genetics and physio­
logy 2 (LGP2) 45,46. The RLRs are DExD/H box RNA
helicases , and RIG‑I and MDA5 also contain an
N‑terminal caspase-recruitment domain (CARD). These
receptors bind to dsRNA poly(rI:rC), poly(rA:rU)47
and, in the case of RIG‑I, 5′-triphosphate RNA48,49,
which are present in viral genomes or replication intermediates. RLRs interact with a central adaptor IFNBpromoter stimulator 1 (IPS1; also known as MAVS,
VISA or CARDIF)50–52, which assembles a signalling
complex that contains TNF-receptor-associated factor 3
(TRAF3), TRAF-family-member-associated NF-κB
activator-binding kinase 1 (TBK1) and inhibitor of
NF‑κB kinase ε (IKKε; also known as ΙΚΚi), and signals
through IRF3 to induce IFN production45,53.
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Other nucleic-acid sensors. Nucleic acids are also able
to engage several other intracellular components. First,
a recently described molecule termed DNA-dependent
activator of IRFs (DAI) has been shown in vitro to act as a
cytosolic DNA sensor by binding dsDNA and signalling
through TBK1 and IRF3 to activate NF‑κB and induce
type I IFN production54. However, the role of DAI in sensing dsDNA in vivo has been questioned in a study using
DAI-deficient mice55. Second, in macrophages, cytoplasmic DNA has been shown to trigger pro-inflammatory
responses through components of the inflammasome, a
process that is independent of TLRs and IRFs56. Third,
type I IFNs induce the expression of a variety of genes
with antiviral functions57,58, and several such molecules
seem able to sense certain nucleic‑acid structures. For
example, RNAs with short stem-loops can activate IFNinducible dsRNA-dependent protein kinase R (PKR) in a
5′‑triphosphate-dependent manner, independent of RIG‑I
(REF. 59). In addition, activation of the ribonuclease L by
2′,5′-linked oligoadenylate leads to the production of
small RNA cleavage products from self RNA, which then
function to amplify antiviral IFN responses54.
Inflammasome
A molecular complex of several
proteins that, on assembly,
cleaves pro-interleukin‑1,
thereby producing active
interleukin‑1.
Ubiquitin E3 ligase
An enzyme that is required
to attach the molecular
tag ubiquitin to proteins.
Depending on the position
and number of ubiquitin
molecules that are attached,
the ubiquitin tag can target
proteins for degradation in
the proteasomal complex,
sort them to specific
subcellular compartments or
modify their biological activity.
Osteopontin
An extracellular matrix
protein that supports
adhesion and migration of
inflammatory cells. It has
recently been recognized
as an immunoregulatory
T‑helper‑1-type cytokine.
Nucleic-acid sensing by pDCs
pDCs sense nucleic acids through TLR7 and TLR9. In
humans, pDCs selectively express TLR7 and TLR9, but
not other TLRs. This is in contrast to mDCs, which
preferentially express TLR1, TLR2, TLR3 and TLR8,
and monocytes, which express TLR1, TLR2, TLR4,
TLR5 and TLR8 (refs 8,9,60,61). Mouse pDCs that lack
MyD88 or TLR9 completely lose their ability to produce
IFNα when challenged with the TLR9 ligand CpG
ODNs62. Importantly, TLR9 was found to be required
for pDCs to respond to DNA viruses, such as herpes
simplex virus 1 (HSV‑1), HSV‑2 and murine cytomegalovirus63–66. In addition, TLR7 is required by pDCs
to respond to ssRNA viruses, such as influenza virus,
respiratory syncytial virus, Sendai virus and vesicular
stomatitis virus (VSV)28,29,38,67. Given that MyD88 functions as an adaptor protein for both TLR7 and TLR9,
MyD88-deficient pDCs have defective responses to a
wide range of viruses9,29,68,69. However, several recent studies have suggested that pDCs from certain tissues70,71 or
in response to particular viral infections68 might activate
MyD88-dependent pathways independently of TLR7 and
TLR9, but the molecular components of these pathways
are currently unclear. Consistent with a lack of TLR3
expression by pDCs60,61,72, pDCs from Tlr3–/– mice were
found to secrete normal levels of IFNα in response to
polyI:C, whereas Tlr3–/– conventional DCs and B cells had
defective responses to the TLR3 ligand polyI:C28,29.
In fibroblasts and conventional DCs, RLRs and their
adaptor IPS1 are essential for the induction of IFNs after
infection with RNA viruses45,46,69. By contrast, RIG‑Ideficient or IPS1-deficient pDCs retain the ability to
produce large amounts of IFNα in response to the RNA
viruses Newcastle disease virus and Sendai virus, by using
the TLR–MyD88 pathway69,73. Consistent with these findings, TBK1 and IKKε, two essential components of the
RLR signalling pathway, are dispensable in pDCs during
antiviral responses53.
However, it is clear that pDCs also respond to viruses
that enter the cytoplasm. Recently, autophagy — a conserved cell-autonomous process involving lysosomal
degradation of cellular organelles74,75 — has been implicated in the innate type I IFN responses of pDCs to RNA
viruses67 (FIG. 3). Autophagy may facilitate the delivery of
cytoplasmic viral RNA to TLR7-containing endosomal
compartments, thereby bypassing a need for cytoplasmic
sensors. In summary, the exclusive dependence on the
endosomal TLR–MyD88 pathway for nucleic-acid sensing and activation by pDCs implies a unique function of
these cells and probably a non-redundant role of these
cells in the immune system.
TLR-mediated activation of pDCs. In pDCs, the engagement between TLR and MyD88 leads to the assembly
of a multiprotein signal-transducing complex in the
cytoplasm, which contains IRAK4 (Refs 76–78), TRAF6
(refs 79,80), Bruton’s tyrosine kinase (BTK)81–83 and
IRF7 (refs 84,85) (FIG. 3). IRF7 is activated through
ubiquitylation by the ubiquitin E3 ligase activity of
TRAF6 (ref. 86) and phosphorylation by IRAK4.
Activated IRF7 interacts with TRAF3, IKKα, IRAK1,
osteopontin and probably phosphatidylinositide
3‑kinase (PI3K), and then translocates to the nucleus
where it has a central role in initiating type I IFN gene
transcription53,84,86–89. Simultaneously, TRAF6 in the
signal-transducing complex ubiquitylates the protein
kinase transforming growth factor-β (TGFβ)-activated
kinase 1 (TAK1), a signal transducer that activates
NF‑κB and MAPKs90,91 for the induction of transcription of pro-inflammatory cytokines, chemokines and
co-stimulatory molecules92.
In most cells, the production of IFNα depends on
type I IFN receptor (IFNAR) triggering by IFNβ, which
results in IRF7 induction93. Remarkably, however, pDCs
constitutively express high levels of IRF4, IRF7 and IRF8
(ref. 18), and the high levels of endogenous IRF7 facilitate
a rapid type I IFN response that is independent of IFNARmediated feedback signalling94. The recent observation
that pDCs express low levels of the translational repressors, known as 4E-BPs, that are responsible for regulating
IRF7 translation, might explain the constitutive expression of IRF7 in these cells95. IRF8 can further amplify the
IFN response during the second phase of type I IFN gene
transcription in pDCs96,97. Aside from IRF7, IRF5 directly
interacts with MyD88 and mediates the TLR-dependent
induction of type I IFNs and pro-inflammatory cytokines,
including IL‑6 and TNF98–100. However, IRF4 competes
with IRF5, but not IRF7, to interact with MyD88, thereby
negatively regulating cytokine production induced
by TLR stimulation100.
Additional cellular factors involved in TLR-mediated
activation of pDCs. In the absence of PAMPs, TLR9
resides in the endoplasmic reticulum (ER) of resting
pDCs, and only translocates to the endosomal compartment on exposure to TLR agonists 101. The delivery
of TLRs to endolysosomes and their subsequent proper
activation requires UNC93B, an ER‑resident transmembrane protein that can specifically interact with TLR3,
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TLR7 and TLR9 (refs 102,103). The ER chaperone gp96
can also interact with TLR3, TLR7, TLR9 and several
other TLRs, and is also thought to be involved in TLR
function104. The transcription factor X‑box-binding
protein 1 (XBP1), which is highly expressed by pDCs, is
Virus or self nucleic acid
Plasmacytoid DC
Plasma membrane
Endosome
DNA
RNA
ATG5
TLR9
Autophagosome
TLR7
MyD88
UNC93B
MyD88
TLR7 or
TLR9
gp96
TRAF3
XBP1
TRAF6
IRAK1
IKKα
OPN
PI3K
BTK IRAK4
ER
IRF7
TAK1
IRF5
IRF8
P
P
p50 p65
IRF7
IRF4
MAPKs
NF-κB
Cytosol
Nucleus
IFNα, IFNβ,
IFNλ, IFNω
IL-6,
TNF
CD80,
CD86,
CD40
Reviews | Immunology
Figure 3 | Activation pathway in plasmacytoid dendriticNature
cells responding
to
nucleic acids. Resting plasmacytoid dendritic cells (pDCs) predominantly express
Toll-like receptor 7 (TLR7) and TLR9, which reside in the endoplasmic reticulum (ER) in
association with UNC93B and gp96. Following exposure to virus or nucleic acids, TLR7
and TLR9 relocate from the ER to the endosomes to engage with their RNA or DNA
agonists. Conformational changes in the TLRs lead to the activation of MyD88 (myeloid
differentiation primary-response gene 88) and its further association with TRAF6
(tumour-necrosis factor (TNF) receptor-associated factor 6), BTK (Bruton’s tyrosine
kinase) and IRAK4 (interleukin‑1-receptor-associated kinase 4). The MyD88–TRAF6–
IRAK4 complex then activates IRF7 (interferon-regulatory factor 7), TAK1
(transforming‑growth-factor-β-activated kinase 1), nuclear factor-kB (NF-kB) and IRF5 to
propagate the downstream signals. Most importantly, IRF7 is activated through TRAF3,
IRAK1, IKKα (inhibitor of NF-κB kinase α), osteopontin (OPN) and phosphoinositide
3‑kinase (PI3K). Following ubiquitylation and phosphorylation, IRF7 translocates to the
nucleus and initiates the transcription of type I interferons (such as IFNα, IFNβ, IFNλ and
IFNω). TAK1 triggers the activation of NF‑κB and MAPKs (mitogen-activated protein
kinases) and, together with IRF5, leads to the production of pro-inflammatory cytokines
and the expression of co-stimulatory molecules. IRF8, although not involved in the initial
induction of IFN, magnifies IFN production through a feedback mechanism. By contrast,
IRF4 inhibits the function of IRF5 through direct competition. Autophagosomes, which
are constitutively formed in pDCs via ATG5 (autophagy-related gene 5), are probably
involved in transferring the nucleic-acid agonists to endosomal TLRs for the production
of IFNs. IL-6, interleukin-6; XBP1, X‑box-binding protein 1.
involved in maintaining normal IFN production through
its role in maintaining ER homeostasis105. So, the unique
ability of pDCs to mount a rigorous type I IFN response
relies on the function of a range of other cellular factors
that are now being revealed.
pDC responses to nucleic acids
pDCs are professional type I IFN-producing cells
in viral infections. Within 6 hours of activation by a
virus, human pDCs dedicate 60% of the newly induced
transcriptome to type I IFN genes18, producing a large
amount of type I IFNs (1–2 units per cell or 3–10 pg per
cell) within 24 hours, which is 200 to 1,000 times more
than the amount produced by any other blood cell type7.
Human pDCs express all the subtypes of type I IFNs,
namely IFNα, IFNβ, IFNλ, IFNω and IFNτ (ref. 18).
In mice, pDCs are mainly responsible for type I IFN
production in vivo during infection with murine cytomegalovirus and VSV, as depletion of pDCs abrogates the
systemic production of IFN94,106,107.
pDC-derived type I IFNs link innate and adaptive
antiviral immunity. Type I IFNs derived from pDCs
not only directly inhibit viral infection, but also activate the antiviral functions of NK cells, mDCs, B cells
and T cells, and therefore initiate and orchestrate
innate and adaptive antiviral immunity. In response to
HIV infection, human pDCs induce type I IFN‑dependent
maturation of mDCs into potent antigen-presenting
cells108. During HSV infection, pDC-derived type I
IFNs and CD40 ligand induce maturation of bystander
mDCs to potently stimulate antiviral T‑cell-mediated
immunity109. Type I IFNs derived from pDCs have been
shown to trigger IL‑12, IL‑15, IL‑18 and IL‑23 production by mDCs, and to induce monocytes to differentiate
into DCs110,111. They also increase the ability of mDCs
to cross-present exogenous antigens to CD8+ T cells and
promote their clonal expansion112,113 and their ability to
induce the differentiation of naive T cells into T helper 1
(TH1) cells114. Type I IFNs derived from pDCs also
stimulate NK cells16 and, in conjunction with IL‑6, drive
B cells to differentiate into mature antibody-secreting
plasma cells115. These findings point to a crucial role
for innate pDC activation in the initiation of a network
of cellular and molecular events that lead to protective
immune responses against viruses. Furthermore, a
recent study suggests that pDCs are specialized to crosspresent viral antigens to memory CD8+ T cells, owing to
their unique ability to rapidly load viral peptides onto
pre-synthesized MHC class I molecules in recycling
early endocytic compartments116.
Two classes of CpG-containing DNA sequences induce
distinct responses in pDCs. pDCs and, to a lesser extent,
B cells express TLR9, the sensor for DNA. However,
pDCs mount distinctive responses to at least two classes
of synthetic ODNs with hypomethylated CpG motifs117.
On the one hand, A‑type CpG ODNs do not activate
B cells but induce robust production of type I IFNs
by pDCs. On the other hand, B‑type CpG ODNs can
activate B cells but only trigger pDCs to produce the
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Plasmacytoid DC
Cytosol
Nucleus
TfR
Early endosome
Type A
ODN
P
TLR9
EEA1
TRAF6
MyD88
IRAK4
P
IRF7
TRAF3
IRAK1
OPN IRF7
IFNα,
IFNβ,
IFNλ,
IFNω
Acidification
LAMP1
Late endosome
or lysosome
p50 p65
NF-κB
Type B
ODN
TLR9
LysoTracker
MyD88
TRAF6
TAK1
MAPKs
BTK IRAK4
IRF5
IL-6,
TNF
CD80,
CD86,
CD40
Chemokines
Figure 4 | Signalling of CpG ODN classes in different endosomal compartments.
In plasmacytoid dendritic cells (pDCs), the binding of aggregated A‑type CpG oligodeoxynucleotides (ODNs) to Toll-like receptor 9 (TLR9) occurs in the early endosomes,
Nature Reviews | Immunology
which express markers such as transferrin receptor (TfR) and early endosomal antigen
1 (EEA1). Prolonged TLR9 signalling from the early endosomes activates MyD88
(myeloid differentiation primary-response gene 88) and other signal mediators,
importantly IRF7 (interferon-regulatory factor 7), which promote strong type I
interferon (IFN) production. By contrast, monomeric B‑type CpG ODNs that bind to
the TLR9 complex quickly traffic through the early endosomes and into the more
acidic late endosomes or lysosomes, which are marked by the presence of LAMP1
(lysosomal-associated membrane protein 1) and LysoTracker. This presumably
activates a different set of signal mediators, particularly NF‑κB (nuclear factor-κB)
and probably MAPKs (mitogen-activated protein kinases) and IRF5, and thereby
leads to distinct outcomes of pDC activation without high levels of IFN production.
BTK, Bruton's tyrosine kinase; IRAK, interleukin-1-receptor-associated kinase;
OPN, osteopontin; TAK1, transforming-growth-factor-β-activated kinase 1;
TRAF, tumour-necrosis factor (TNF) receptor-associated factor.
Systemic lupus
erythematosus
(SLE). An autoimmune disease
in which autoantibodies
specific for DNA, RNA or
proteins associated with
nucleic acids form immune
complexes that damage small
blood vessels, especially in the
kidneys. Patients with SLE
generally have abnormal
B‑ and T‑cell function.
CpG islands
Sequences of 0.5–2
kilobases that are rich in
CpG dinucleotides. They are
mostly located upstream of
housekeeping genes and are
also present in some tissuespecific genes. They are
constitutively non-methylated
in all animal cell types.
pro-inflammatory cytokines TNF and IL‑6, and small
amounts of IFN, as well as inducing the upregulation
of CD80, CD86 and the expression of MHC class II
molecules12–14,60,61,118.
Two important features distinguish A‑type versus
B‑type CpG ODNs, and these account for their differing
ability to trigger type I IFN production by pDCs. First,
A‑type CpG ODNs are characterized by a polyG tail that
mediates the spontaneous formation of large multimeric
aggregates with a diameter of about 50 nm119. By contrast,
B‑type CpG ODNs are monomeric and do not form such
higher order structures. Second, monomeric B‑type CpG
ODNs rapidly traffic through early endosomes into late
endosomes or lysosomes, whereas multimeric A‑type
CpG ODNs are retained for longer periods of time in
the early endosomes of pDCs120,121 (FIG. 4). The prolonged
retention of multimeric A‑type CpG ODNs provides a
platform for extended activation of the signal-transducing
complex, consisting of MyD88 and IRF7, which leads
to robust type I IFN production120 (FIG. 4). Interestingly,
when A‑type CpG ODNs are disrupted into monomeric
structures, they fail to be retained in the early endosome
and to induce high amounts of type I IFN production,
but instead rapidly traffic to late endosomes and induce
maturation and production of IL‑6 and TNF. Conversely,
liposome or particle encapsidation of B-type CpG ODNs
allows their retention in early endosomes and the subsequent induction of much higher levels of type I IFNs and
lower levels of IL‑6 and TNF and impaired pDC maturation119–122, which supports the important link between
the physical size of TLR9 ligands and their stimulatory
capacity. Therefore, pDCs seem to detect aggregated
DNA structures in the early endosomes through TLR9,
and this is coupled with IRF7 activation and type I IFN
responses. By contrast, pDCs sense linear DNA structures in the late endosomes through TLR9, and this
seems to be coupled with NF‑κB activation, which leads
to TNF and IL‑6 production and pDC maturation.
pDCs sense self DNA in autoimmunity
Mechanisms for innate tolerance to self DNA. The
release of host-derived (self) DNA into the extracellular
environment is a common feature of both necrotic and
apoptotic cell death123. To preserve the integrity of the
organism, it is of pivotal importance that the immune
system avoids the recognition of extracellular self DNA,
while retaining the ability to sense pathogen-derived
nucleic acids. The discrimination between pathogenderived DNA and self DNA seems to be controlled
at three distinct levels. First, the subcellular localization of TLR9 allows immune responses to DNA from
pathogens that invade the cells by endocytosis, whereas
self DNA fails to spontaneously access this compartment124,125. Second, the high concentration of DNases
in the extracellular environment ensures a rapid degradation of self DNA that is released by dying cells but
not DNA that is contained in viruses or microorganisms. The importance of this pathway in preventing
autoimmune responses is shown by the fact that mice
deficient in DNase 1 develop a systemic lupus erythematosus (SLE)-like syndrome126, and that some patients
with SLE have mutations in DNase 1 (ref. 127). Finally,
viral or bacterial DNA contains multiple unmethylated
CpG motifs that can bind and activate TLR9 (ref. 128),
whereas mammalian self DNA contains fewer such
motifs and these are mostly masked by methylation129.
However, hypomethylated CpG islands that show reactivity to TLR9 have also been found in mammalian
DNA. These CpG islands are preferentially enriched
in DNA fragments that are released by apoptotic or
necrotic cells and are found in the DNA of immune
complexes associated with SLE130.
A recent study however challenges the concept of self
versus non-self discrimination based on the presence of
unmethylated CpG motifs131. Although synthetic phosphothioate DNA depends on unmethylated CpG motifs
to induce TLR9 activation, phosphodiester DNA, which
is commonly found in pathogen DNA and self DNA,
depends on the 2′ deoxyribose backbone131. In addition,
the ability of phosphodiester DNA to activate TLR9
depends on the formation of multimeric aggregates,
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REVIEWS
indicated by the findings that self DNA activates TLR9
following the formation of aggregates with the antimicrobial peptide LL37 (detailed in the next section),
and synthetic phosphodiesteric DNA activates TLR9
through a 3′ polyG extension, which promotes DNA
aggregation. These data suggest that activation of TLR9
by DNA depends on both the 2′ deoxyribose backbone
for TLR9 binding and the aggregate forms for extended
retention in the early endosomes.
Immune complexes
Complexes of antigen
bound to antibody and,
sometimes, components of
the complement system.
The levels of immune
complexes are increased in
many autoimmune disorders,
in which they become
deposited in tissues and
cause tissue damage.
Breakdown of innate tolerance to self DNA in autoimmunity. The barriers that prevent pDCs from sensing
self DNA are not infallible, and responses to self DNA
do occur in some autoimmune diseases. In patients with
SLE, pDCs are continuously activated by circulating
immune complexes comprising self DNA and antibodies
to DNA or nucleoproteins132–134. The ongoing production
Damaged
epithelial cell
Autoreactive
B cell
LL37
LL37
LL37
HMGB1
Self DNA
DNA-specific IgG
Neutrophil
LL3
7
LL37
FcγRIIA
Lipid raft
ITAM
Plasmacytoid DC
RAGE
TLR9
LL37
LL
37
Early endosome
TLR9
Figure 5 | A model for sensing self DNA. The antimicrobial peptide LL37 is secreted by
damaged epithelial cells or deposited by tissue-infiltrating neutrophils. It binds self-DNA
fragments released by dying cells to form aggregated and condensed
structures that are
Nature Reviews | Immunology
protected from extracellular nuclease degradation and delivered via lipid rafts from the
extracellular environment into the early endosomes of plasmacytoid dendritic cells (pDCs).
Dying cells also release high-mobility group box 1 protein (HMGB1), which binds
aggregated self-DNA–LL37 complexes and promotes their association with Toll-like
receptor 9 (TLR9) in early endosomes by binding to RAGE (receptor for advanced glycation
end-products). In systemic lupus erythematosus, DNA-specific IgG autoantibodies
produced by autoreactive B cells bind self-DNA–LL37–HMGB1 complexes and potently
increase their translocation into TLR9-containing endosomes in pDCs through FcγRIIA
(low-affinity Fc receptor for IgG). ITAM, immunoreceptor tyrosine-based activation motif.
of type I IFNs by pDCs in these patients induces an
un­abated activation and maturation of mDCs that stimulate autoreactive T cells135. Furthermore, pDC-derived
type I IFNs, together with IL‑6, stimulate the differentiation of autoreactive B cells into autoantibody-secreting
plasma cells115. In psoriasis, a common autoimmune
disease of the skin, pDCs sense self DNA that is released
locally following skin injury136. The resulting activation of
pDCs triggers autoreactive T‑cell activation and initiates
the development of skin lesions through type I IFNs137.
Several host factors have been implicated in converting self DNA into triggers of pDC activation — these are
LL37 (also known as CAMP), autoantibodies and highmobility group box 1 protein (HMGB1) (FIG. 5). LL37 is
an endogenous antimicrobial peptide that is produced
by keratinocytes and neutrophils in wounded skin138 and
has been found to be overexpressed in psoriatic skin136. A
direct link between LL37 and pDC activation in psoriasis
was recently revealed136: through its unique cationic and
α‑helical properties, LL37 was found to bind self-DNA
fragments released by dying cells, forming large aggregated structures that are resistant to extracellular nuclease
degradation136. Moreover, self-DNA–LL37 complexes can
enter pDCs, a process that seems to involve lipid-raftand proteoglycan-dependent endocytosis139. Aggregated
self-DNA–LL37 complexes are retained in the early
endosomes of pDCs, and, similar to A‑type CpG ODNs,
they trigger the TLR9–MyD88–IRF7 pathway of type I
IFN production without inducing pDC maturation.
Therefore, LL37 overexpression in psoriasis might be
responsible for breaking tolerance to self DNA in psoriatic skin, leading to a sustained activation of pDCs and
type I IFN production (TABLE 1).
In SLE, the breakdown of innate tolerance to self
DNA has been classically attributed to complex formation between self DNA and DNA-specific antibodies140.
DNA-containing immune complexes isolated from the
sera of patients with SLE trigger type I IFN production
by pDCs through the binding of the DNA-specific auto­
antibody to low-affinity Fc receptor for IgG (FcγRIIA;
also known as CD32). Self-DNA-containing immune
complexes can then be internalized by FcγRIIA and translocated to TLR9-containing endosomal compartments141.
Interestingly, antibodies to DNA and nucleoprotein alone
seem to be unable to protect extracellular DNA fragments
from nuclease activity (TABLE 1). We found that immune
complexes isolated from patients with SLE contained
LL37 and that the presence of LL37 is a prerequisite for the
ability of DNA-specific antibodies to promote an FcγRIIAmediated uptake of self DNA into pDCs to trigger an early
endosomal TLR9 response (R. Lande et al., unpublished
observations). In line with these findings, Pascual and
colleagues have found that the gene encoding LL37 was
among the most upregulated genes in blood cells from
patients with SLE and correlated with high levels of expession of type I IFNs and IFN-induced genes (referred to as
the ‘IFN signature’) and disease activity142. Although the
exact origin of LL37 in SLE-associated immune complexes
remains to be determined, LL37 expression was linked to
the presence of immature granulocytes in the peripheral
blood of patients with SLE142.
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Table 1 | Breaking innate tolerance to self DNA in plasmacytoid dendritic cells
LL37
Antibody/autoantibody
HMGB1
Aggregation and condensation of DNA
++
–
–
DNA protection from extracellular degradation
++
–
–
Intracellular uptake of DNA
+ (via lipid rafts)
++ (mediated by FcγRIIA)
–
Sustained TLR9 activation in early endosomes
++
–
++ (via RAGE)
Role in SLE
+
+
+
Role in psoriasis
+
–
?
FcγRIIA, low-affinity Fc receptor for IgG; HMGB1, high-mobility group box 1 protein; RAGE, receptor for advanced glycation endproducts; SLE, systemic lupus erythematosus; TLR9, Toll-like receptor 9.
HMGB1 is a nuclear DNA-binding protein that is
released by dying cells and has the ability to enhance type I
IFN production by pDCs in response to DNA‑containing
immune complexes143. Interestingly, in contrast to LL37,
HMGB1 can only bind multimeric aggregated DNA
structures, such as A‑type CpG ODNs, and not monomeric DNA sequences, such as B‑type CpG ODNs. The
DNA–HMGB1 complex binds to a surface receptor on
pDCs known as RAGE (receptor for advanced glycation
end-products). The interaction between RAGE and the
DNA–HMGB1 complex is not involved in the uptake of
the complex, but potently enhances type I IFN production by facilitating the association of the DNA with TLR9
in early endosomes143 (TABLE 1).
Based on the observation that LL37, DNA-specific
antibodies and HMGB1 have a role in the breakdown
of innate tolerance to self DNA in SLE, we propose the
following model. LL37 initially binds to self-DNA fragments released by apoptotic cells and forms aggregated
structures that are protected from nuclease degradation.
The aggregated self-DNA–LL37 complexes are then
bound by HMGB1. After internalization, the self-DNA–
LL37–HMGB1 complex associates with TLR9 in early
endosomes by engaging RAGE (FIG. 5). DNA‑specific
IgG antibodies can bind self-DNA–LL37–HMGB1
complexes and potently enhance the internalization of
the complex through FcγRIIA (FIG. 5).
The hypothesis that TLR9 activation by self DNA
may result in the development of autoimmunity has been
further supported by an experimental arthritis model.
More specifically, it was recently shown that cathepsin K,
a potent collagenase expressed by osteoclasts, is required
for TLR9 activation and the development of autoimmunity in an experimental arthritis model144. Although
the mechanism involved has not been elucidated, this
finding suggests that DNA-mediated TLR9 activation may be regulated by a variety of tissue- and/or
disease-specific cofactors.
pDCs sense self RNA in autoimmunity
Similar to self DNA, host-derived self RNA that is released
by damaged cells normally fails to activate pDCs, owing
to its rapid extracellular degradation by abundant RNases,
which thereby limits its accessi­bility to the TLR7‑containing
endosomes. Furthermore, vertebrate-specific RNA
modifications, including polyA tails145 and nucleotide
methylation146, contribute to the low immunogenicity
of self RNA. However, self-RNA molecules, in particular
those rich in uridine or uridine and guanosine (U/UGRNA) and those in small nuclear ribonucleoprotein
(snRNP), were shown to trigger pDCs to produce type I
IFNs through TLR7 when delivered to endosomes
by autoantibodies or liposomes147. These self-RNA and
autoantibody complexes were also shown to activate
autoreactive B cells through the B‑cell receptor (BCR)
and endosomal TLR7 (refs 148,147). SLE is characterized by the presence of autoantibodies to both self DNA/
chromatin and snRNPs that contain U/UG-RNA140,149.
The contribution of self-RNA‑triggered TLR7 signalling
to the development of autoimmune diseases such as SLE
is supported by the finding that Tlr7 gene duplication or
multiplication can promote the development of an SLElike disease in mice, which was associated with increased
production of autoantibodies and enhanced DC activation
and type I IFN production81,150–153. Accordingly, TLR7deficient lupus-prone mice have ameliorated autoimmune diseases with decreased lymphocyte activation
and serum autoantibodies, as well as reduced severity of
nephritis154,155. Paradoxically, TLR9-deficient lupus-prone
mice develop exacerbated diseases with increased levels
of serum IgG and lymphocyte numbers in the skin and
lymphoid tissues, enhanced pDC activation and IFNα
production, and increased severity of nephritis. However,
the level of autoantibody specific for DNA was lower
in the TLR9-deficient than the wild-type lupus-prone
mice. The underlying mechanism behind the opposing
effects of TLR7 and TLR9 on the development of disease
in this mouse model is not known. In addition, the relative contributions of TLR7 and TLR9 to the development
of SLE in humans require further investigation.
Negative regulation of pDC responses
Given the significance of type I IFNs in activating a
wide range of cells of the innate and adaptive immune
system, the IFN response has to be under tight control
to prevent aberrant immune responses that could harm
the host. Indeed, pDCs have an array of surface receptors and use a novel signalling pathway to modulate their
responses (FIG. 6).
pDC regulatory receptors. BDCA2 (blood DC antigen 2)
is a C‑type lectin that is uniquely expressed on the surface of human pDCs and was the first receptor shown
to potently suppress the ability of pDCs to produce
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type I IFNs in response to TLR ligands156. Recently, we
discovered that another human pDC-specific receptor,
immunoglobulin-like transcript 7 (ILT7; also known
as LILRA4), inhibits type I IFN production by pDCs
in response to both TLR7 and TLR9 agonists157. Both
BDCA2 and ILT7 associate with the γ‑chain of the highaffinity Fc receptor for IgE (FcεRIγ) and activate pDCs
through an immunoreceptor-based tyrosine activation motif
(ITAM)-mediated signalling pathway157,158. IgE engagement with FcεRIγ also downregulates CpG-induced
type I IFN production by human pDCs159,160.
In addition, several other receptors that signal through
ITAMs were found to inhibit IFNα responses by pDCs.
IgG binding to FcγRIIA, a receptor that contains a cytoplasmic ITAM, decreases the level of IFNα produced
by pDCs161,162. Crosslinking of NKp44, a receptor that
signals through the ITAM-bearing adaptor DAP12,
inhibits IFNα production induced by CpG ODNs163.
In mice, antibody crosslinking of Siglec‑H (sialicacid-binding immunoglobulin-like lectin H), another
DAP12‑associated receptor expressed by pDCs, reduces
type I IFN production by pDCs in vitro and in vivo164.
Moreover, pDCs from DAP12-deficient mice produce
BDCA2
ILT7
increased levels of IFNα in response to TLR9 activation.
These results support an emerging concept that signalling
through ITAM-associated activating receptors inhibits
innate immune responses.
However, PDC-TREM (triggering receptor expressed
on myeloid cells), a newly discovered pDC-specific
transmembrane receptor that forms a complex with
plexin A1, is activated by its endogenous ligand semaphorin 6D to induce DAP12-mediated activation of
PI3K and extracellular-signal-regulated kinase 1 (ERK1)
and ERK2, which leads to enhanced levels of type I IFN
production165. The reasons behind the different effects
of DAP12 signalling on the ability of pDCs to produce
type I IFNs require further investigation.
Last, P2Y receptors, a family of G‑protein-coupled
receptors, downregulate IFNα secretion by human pDCs
following stimulation by nucleotides such as ATP, ADP
and UDP-glucose166.
ITAM-mediated BCR-like signalling pathway suppresses
TLR activation. Many receptors on haematopoietic cells,
such as BCRs, T‑cell receptors, NK‑cell receptors and Fc
receptors, respond to extracellular stimuli by signalling
NKp44
FcεRIα
Siglec-H
C-type lectin
domain
FcγRIIA
Immunoglobulin
domain
γ γ
γ
γ
γ γ
DAP12
DAP12
Plasma
membrane
ITAM
SRC family PTKs
Plasmacytoid DC
SYK
ITAM pathway
BLNK
BCAP
Endosome
DNA
TLR7
IFNα, IFNβ, IFNγ, IFNω
RNA
TLR9
Immunoreceptor-based
tyrosine activation motif
(ITAM). A structural motif
containing tyrosine residues,
found in the cytoplasmic tails
of several signalling molecules.
The motif has the form Tyr-XaaXaa-Leu/Ile, and the tyrosine is
a target for phosphorylation
by SRC tyrosine kinases and
subsequent binding of proteins
containing SRC-homology-2
domains.
TLR pathway
IL-6, TNF
Figure 6 | Dendritic-cell regulatory receptors and the ITAM pathway in the regulation of TLR responses.
Three cell-surface receptors, BDCA2 (blood dendritic cell (DC) antigen 2), ILT7 (immunoglobulin-like
transcript
7)
Nature Reviews
| Immunology
and FcεRIα (high-affinity Fc receptor for IgE), associate with the γ‑chain of FcεRI, which contains an ITAM
(immunoreceptor tyrosine-based activation motif), on human plasmacytoid DCs (pDCs). Human NKp44 and mouse
Siglec‑H (sialic-acid-binding immunoglobulin-like lectin H) bind to another ITAM-containing adaptor protein, DAP12.
These receptor complexes or receptors with intrinsic ITAMs, such as FcγRIIA, signal through their ITAMs and activate
a B‑cell receptor (BCR)-like pathway that involves SRC family protein tyrosine kinases (PTKs), SYK (spleen tyrosine
kinase) and the B‑cell-specific adaptors BLNK (B-cell linker) and BCAP (B-cell adaptor protein). This ITAM-mediated
pathway potently inhibits type I interferon (IFN) and cytokine production by pDCs in response to Toll-like receptor
(TLR) activation. IL-6, interleukin-6; TNF, tumour-necrosis factor.
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REVIEWS
through a common signalling pathway that relies on
the conserved ITAM sequence. Strikingly, most pDC
regulatory receptor complexes signal through a BCR-like
signalling cascade — that is, they signal through the SRC
family protein tyrosine kinases, such as LYN and SYK
(spleen tyrosine kinase), and two key B‑cell-specific signalling adaptors, B‑cell linker (BLNK) and B‑cell adaptor
protein (BCAP)158,167 (FIG. 6). By contrast, ITAM-bearing
receptors on mDCs or monocytes signal through a cascade involving non-BCR adaptors, such as SLP76 (SRChomology‑2-domain-containing leukocyte protein of 76
kDa)158.Activation of the BCR-like signalling pathway in
pDCs potently suppresses the activation of both TLR7
and TLR9, inhibiting the production of all type I IFNs,
as well as TNF and IL‑6 (refs 157,158) (FIG. 6). ITAMmediated signalling also suppresses the response to TLR
ligands by mouse macrophages in vitro and in vivo168,169.
So, pDCs use a powerful ITAM-mediated, BCR-like
regulatory pathway to counter-regulate the prominent
TLR signalling pathway. pDCs are constantly exposed
to self DNA or self RNA in the process of normal tissue
renewal or cell death following tissue injury. The nucleo­
tides that bind to P2Y receptors are also known to be
released and to accumulate at sites of cell stress and
tissue damage166. Therefore, the pDC regulatory receptors and the pathways they activate might function to
maintain a normal threshold of TLR responsiveness;
failure to do so might result in dysregulated type I IFN
production, a state that might predispose individuals to
autoimmune disease.
Future perspectives
Although the wealth of recent studies has greatly enhanced
our understanding of how pDCs sense DNA and RNA in
microbial infection and autoimmune diseases, several key
questions remain to be further investigated.
First, why are DNA aggregates, such as A‑type CpG
ODNs and genomic DNA complexed with LL37, selectively
retained in early endosomes and not in late endosomes
in pDCs?
1.
2.
3.
4.
5.
6.
7.
Facchetti, F. & Vergoni, F. The plasmacytoid monocyte: from morphology to function. Adv. Clin. Path. 4, 187–190 (2000).
Fitzgerald-Bocarsly, P. Human natural interferon-α
producing cells. Pharmacol. Ther. 60, 39–62 (1993).
Grouard, G. et al. The enigmatic plasmacytoid T cells
develop into dendritic cells with interleukin (IL)‑3
and CD40-ligand. J. Exp. Med. 185, 1101–1112
(1997).
This paper describes the first purification
and characterization of pDCs from human
tonsils and blood.
Rissoan, M. C. et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283, 1183–1186 (1999).
O’Doherty, U. et al. Human blood contains two
subsets of dendritic cells, one immunologically mature
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(1994).
Olweus, J. et al. Dendritic cell ontogeny: a human
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This paper was the first to show that pDCs
correspond to the long-sought natural type-IIFN-producing cells.
8.
9.
10.
11.
12.
13.
14.
15.
Second, the molecular mechanisms behind the
IRF7-mediated IFN responses associated with TLR7
and TLR9 activation in early endosomes and the
NF‑κB-mediated TNF and IL‑6 responses associated
with TLR7 and TLR9 activation in late endosomes need
to be identified.
Also, why does phosphothioate DNA but not phosphodiester DNA require unmethylated CpG motifs to
activate TLR9? And why does phosphothioate DNA
without the unmethylated CpG motifs become an
antagonist of TLR9 activation?
Another question concerns relocalization of TLRs
from the ER to the endosomal compartment, which
occurs in pDCs shortly after exposure to TLR agonists101.
This process is presumably facilitated by the ER protein
UNC93B and also perhaps by the molecular chaperone
gp96 through a novel process that bypasses the Golgi
apparatus33,40,102,103. If this is indeed the case, how do
pDCs detect DNA or RNA before TLR engagement with
ligands? And how are the functional TLRs assembled
and transported from one intracellular compartment to
the other?
Furthermore, the components of the TLR7- and
TLR9-independent and MyD88-dependent pathway
used by pDCs to respond to viral infection still need to
be identified68,70,71.
In addition, SLE and psoriasis are the best-studied
autoimmune diseases with a known link with dysregulated pDC activation and IFNα production; however,
various other autoimmune and inflammatory diseases
are also associated with such dysregulation, including
Sjögren’s syndrome, type 1 diabetes, Hashimoto’s disease
and dermatomyositis170. Are there other host-derived
factors that break innate tolerance to self nucleic acids
in these autoimmune diseases?
Finally, the natural ligands for the pDC-specific receptors, such as ILT7 and BDCA2, need to be identified. Are
they the putative endogenous factors that negatively regulate the type I IFN responses when pDCs are exposed to
DNA or RNA, in particular that of self-origin?
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Verthelyi, D., Ishii, K. J., Gursel, M., Takeshita, F. &
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Acknowledgements
We thank the former and current members of the laboratory for their critical contributions, the M.D. Anderson
Cancer Foundation and The Dana Foundation for financial
support, M. Haject for editorial assistance, T. Kim, M. Bao
and K. Arima for critical reading of the manuscript. This
Review is dedicated to Dr Ralph Steinman for his support
and encouragement.
DATABASES
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
HMGB1 | IFNs | IL-6 | IRF7 | LL37 | MyD88 | NF-κB | TIRAP |
TLR3 | TLR4 | TLR7 | TLR9 | TNF
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