Download Pattern recognition receptors and control of adaptive immunity

Noah W. Palm
Ruslan Medzhitov
Pattern recognition receptors and
control of adaptive immunity
Authors’ address
Noah W. Palm1, Ruslan Medzhitov1
1
Howard Hughes Medical Institute and Department of
Immunobiology, Yale University, School of Medicine,
New Haven, CT, USA.
Summary: The mammalian immune system effectively fights infection
through the cooperation of two connected systems, innate and adaptive
immunity. Germ-line encoded pattern recognition receptors (PRRs) of
the innate immune system sense the presence of infection and activate
innate immunity. Some PRRs also induce signals that lead to the activation of adaptive immunity. Adaptive immunity is controlled by PRRinduced signals at multiple checkpoints dictating the initiation of a
response, the type of response, the magnitude and duration of the
response, and the production of long-term memory. PRRs thus instruct
the adaptive immune system on when and how to best respond to a
particular infection. In this review, we discuss the roles of various PRRs
in control of adaptive immunity.
Correspondence to:
Ruslan Medzhitov
Howard Hughes Medical Institute and
Department of Immunobiology
Yale University, School of Medicine
300 Cedar Street
New Haven, CT 06510, USA
Tel.: +1 203 785 7541
Fax: +1 203 785 4461
e-mail: [email protected]
Keywords: Toll-like receptors ⁄ pattern recognition receptors, T cells, inflammation, cell
activation
Introduction
Immunological Reviews 2009
Vol. 227: 221–233
Printed in Singapore. All rights reserved
2009 The Authors
Journal compilation 2009 Blackwell Munksgaard
Immunological Reviews
0105-2896
All living organisms, from bacteria through humans, have
evolved strategies to counter parasitic infections (1). In higher
organisms, the varied and numerous strategies involved in
defense from parasitic microbes are collectively referred to as
the immune system. The mammalian immune system consists
of two interrelated arms—the evolutionarily ancient and
immediate innate immune system, and the highly specific,
but temporally delayed, adaptive immune system. The combination of innate and adaptive immunity enables the mammalian immune system to recognize and eliminate invading
pathogens with maximal efficacy and minimal damage to self,
as well as to provide protection from re-infection with the
same pathogen. The innate and adaptive immune systems use
two fundamentally different strategies to recognize microbial
invaders—specifically, the innate immune system detects
infection using a limited number of germ-line encoded receptors that recognize molecular structures unique to classes of
infectious microbes, while the adaptive immune system uses
randomly generated, clonally expressed, highly specific receptors of seemingly limitless specificity (2, 3). It is the combination of these two strategies of recognition that makes the
mammalian immune system highly efficacious.
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
The mammalian immune system evolved to maximize host
defense while minimizing damage to the host tissues. The
innate immune system employs a variety of highly efficient
immune effectors to combat infection; however, the efficacy
of these effectors is limited by the collateral damage, or
immunopathology, they can cause. Thus, the efficacy of the
innate immune system is limited by the level of immunopathology that can be tolerated. The adaptive immune system
increases the potential efficacy of immunity partially by minimizing collateral damage by focusing immune defense in an
antigen-specific manner using highly specific antigen receptors. However, by using randomly generated receptors that
cannot reliably distinguish self from non-self, adaptive
immunity creates another set of opposing forces that must be
balanced: the ability of the adaptive immune system to
respond to pathogenic non-self must be maximized, while the
possibility of responses to self and ⁄ or innocuous non-self
must be minimized.
The innate and adaptive immune systems are ideal partners
because the differing strategies that they use to recognize
infection possess complementary strengths and weaknesses.
Because the innate immune system uses germ-line encoded
receptors to recognize invariant features typical of classes
of microbes, it is highly efficient at distinguishing self from
non-self (4). However, the non-clonal mechanism of activation of innate immune effectors has the potential to cause
significant collateral damage to self-tissues resulting in immunopathology. Adaptive immunity, because it uses random,
diverse, clonally expressed receptors produced via somatic
recombination, is highly effective in specifically targeting
immune responses towards the infection while sparing the
surrounding uninfected tissue. However, cells of the adaptive
immune system cannot by themselves reliably determine the
origin of the antigen they are specific for. By working together,
innate and adaptive immunity can both effectively target
immune effectors and reliably distinguish self from non-self.
A conceptual framework for the current understanding
of the functioning of the innate immune system and its control of adaptive immunity was proposed by the late Charles
Janeway Jr nearly 20 years ago (4). Janeway’s hypothesis was
essentially as follows: the adaptive immune system, because
of the use of randomly generated receptors for antigen recognition, cannot reliably distinguish between self and non-self.
Therefore, adaptive immune cells must be instructed as to the
origin of an antigen by a system that can determine, with high
fidelity, whether an antigen is derived from self, infectious
(i.e. microbial) non-self, or innocuous (i.e. non-infectious
and non-microbial) non-self. Janeway suggested that the
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evolutionarily ancient, and at that point severely understudied, innate immune system might be able to provide such
instruction. Furthermore, he proposed a concrete mechanism
by which the innate immune system could sense the presence
of infection and relay its conclusions to the adaptive immune
system. Janeway posited that the innate immune system
would sense the presence of infection via recognition of conserved microbial pathogen-associated molecular patterns
(PAMPs), by germ-line encoded receptors he dubbed pattern
recognition receptors (PRRs). These PAMPs would possess
certain qualities to be effective. First, they would have to be
unique to microbes and absent from eukaryotic cells so that
they would accurately signal infection. Second, they would be
common to a broad class of microbes so that a limited number
of germ-line encoded receptors could detect all infections.
Third, they would be essential for the life of the microbe so
that their detection could not be easily eliminated via mutation. Maybe most importantly, Janeway also predicted that
recognition of infection by PRRs on cells of the innate
immune system would lead to the induction of signals
involved in activation of the adaptive immune system and thus
would result in initiation of adaptive immunity. This conceptual framework has now been proven to be correct, although
new advances naturally require further development of the
theory.
Adaptive immunity: advantages and dangers
Antigen receptors of the adaptive immune system offer
a number of unique advantages because of their ability to
recognize nearly any antigen and their clonal expression (3).
First, adaptive immune responses possess exquisite specificity
and, thus, can specifically target the immune response; therefore,
adaptive immunity can maximize the efficacy of the immune
response while minimizing unnecessary collateral damage.
Second, because of clonal expression and selection, adaptive
immunity endows the immune system with a mechanism by
which to remember previous infections and, thus, provide
protection from future infection with the same pathogen.
Because randomly generated antigen receptors cannot determine the origin of the antigen, they can potentially activate
the immune response against self-antigens, leading to autoimmunity, or innocuous non-self antigens, leading to allergy.
Negative selection of autoreactive lymphocytes in the thymus
provides an important, albeit incomplete, solution to the
self ⁄ non-self discrimination problem. However, negative
selection is incomplete in the sense that it does not eliminate
all self-reactive specificities and it cannot account for the
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
discrimination between different sources of non-self antigens
(for example, microbial versus food antigens). Thus, despite
central tolerance, opportunities for inappropriate adaptive
immune responses abound, and other mechanisms to prevent
inappropriate responses also exist (5). Peripheral tolerance
consists of a variety of mechanisms that prevent inappropriate
responses by autoreactive lymphocytes that escape negative
selection and lymphocytes specific for innocuous non-self
antigens. One aspect of peripheral tolerance is the inactivation
of T cells that see their antigen in the absence of signals indicating the presence of infection; these cells are thus rendered
permanently unresponsive or anergic (6). Another aspect of
peripheral tolerance is enforced by regulatory T cells, which
suppress, via various mechanisms, inappropriate adaptive
immune responses to self or innocuous non-self (7).
Checkpoints in control of adaptive immunity
The adaptive immune response is a multistep process where
different types of decisions are made before the onset of every
subsequent stage. These checkpoints control different aspects
of the adaptive immune responses and integrate information
provided by various signals induced by infection through
PRRs (Fig. 1). The major checkpoints determine the origin of
the antigen, the type of infection (pathogen class), the extent
and duration of infection, and finally, the requirement for
immediate defense or future defense.
The first checkpoint is controlled by the signals that indicate
the origin of the antigen. This is presumably a binary, yes or no,
decision that determines whether to initiate the response.
Dendritic cells (DCs), as well as other cell types of the innate
immune system, upon activation by PRRs, produce a variety of
signals that couple the identity of the antigen with its microbial
origin (8). The coupling mechanisms often rely on physical
association between an antigen and a PAMP. For example,
attachment of the C3dg fragment to an antigen ‘flags’ the antigen
as foreign, or microbial, and directs efficient antibody responses
against the antigen (9). Similarly, co-occurrence of antigens and
Toll-like receptor (TLR) ligands in the same phagosome (which
normally only happens if the two are physically associated, for
example as parts of the same bacterial cell), signals the microbial
origin of the antigen and promotes selection of microbial antigens for major histocompatibility complex class II presentation
by DCs (10–12). In general, however, the exact mechanisms
involved in coupling antigens with PRR ligands are not entirely
understood. Signals produced by PRRs that indicate the origin of
the antigen to T cells are also not well defined. Although the
costimulatory signals, CD80 and CD86, are commonly thought
to be critical, by themselves they are clearly not sufficient to
induce T-cell responses (13, 14).
The second checkpoint controls the type of adaptive
immune response induced. To be effective in combating
infection, adaptive responses are tailored to the class of infection; for example, T-helper 1 (Th1) responses are induced
PRR-Induced Signals
Fig. 1. PRR-mediated control of checkpoints of adaptive immunity. Pattern recognition receptors (PRRs) detect the presence and features of an
infection and PRR-induced signals control adaptive immunity accordingly at various checkpoints to insure a productive adaptive response. PRR-mediated determination of whether an antigen is self, innocuous non-self, or non-innocuous non-self leads to control of activation of an adaptive response.
PRR-mediated determination of the class of infection leads to control of the type of adaptive response induced. PRR-mediated determination of the
level of infection and persistence of an infection leads to control of the magnitude and duration of the adaptive response. Finally, PRR-mediated determination that immediate defense is needed to combat an ongoing infection leads to effector cell generation, while determination that an infection has
been cleared and resources can be shuttled towards future defense leads to memory cell generation. A failure to properly regulate immunity at each
checkpoint can lead to various immune pathologies including autoimmunity, allergy, failure to protect from infection, and immunopathology.
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in response to intracellular bacteria, Th2 responses are
induced in response to helminths, and Th17 responses are
induced in response to extracellular bacteria and fungi. PRRs
sense the presence of different classes of infection and control the induction of appropriate effector responses through
the production of specialized instructive signals. Thus,
bacterial infections can induce interleukin-12 (IL-12) production, viral infections trigger type I interferon (IFN)
production, and helminthes induce thymic stromal lymphopoietin, IL-4, and IL-13 production. These signals can in
turn direct T-cell differentiation into the appropriate effector
classes (15).
Two related checkpoints in the adaptive immune response
control the magnitude and duration of the response, which
should correlate with the extent and persistence of infection,
respectively. Unlike the origin of the antigen or the class of
pathogen, the magnitude and duration of a response are
graded functions. They can be ‘read-out’ by PRRs that can
sense the extent and the persistence of infection because
infection is the source of PRR ligands. Aside from a few model
systems, little is known about how the magnitude of the
adaptive immune response is controlled. Since the magnitude
of an immune response is dependent upon the extent of
infection, presumably it is controlled by a signal that is proportional to the level of infection. The duration of response
may be controlled by similar signals that must be produced
only in the presence of infection. Indeed, in the case of
Listeria infection, the initial infectious dose and antigen dose
are critical for determining the magnitude of the CD8+ T-cell
response, whereas the persistence of infection determines the
duration of the response (16). Thus, both antigens and PAMPs
may be sensed to co-ordinately control the magnitude and
duration of the adaptive immune response. In addition, magnitude and duration are also controlled by negative regulators
of the response, such as the negative costimulatory molecule
cytotoxic T-lymphocyte antigen-4.
In addition to the checkpoints that control the primary
immune response, there is a checkpoint that controls the
generation and activation of a memory response. This checkpoint is also regulated by PRRs. Recent data on TLR-induced
adaptive responses have demonstrated that, under certain
conditions, a primary CD4+ T-cell response can be induced
that does not lead to T-cell memory (13). These data
suggested the existence of a PRR-induced signal that is necessary for memory generation, but dispensable (at least under
certain experimental conditions) for induction of a primary
response. The identity of this signal is currently unknown. In
another study, TLR-mediated activation of memory B cells
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was shown to elicit a memory antibody response, indicating
that the recall response is also regulated by PRRs (17).
Memory generation is also controlled by the perceived need
for immediate defense (effector cells) during ongoing
infection versus future defense (memory cells) after the
infection has been cleared; this also is controlled by PRRs.
PRRs and control of adaptive immunity
All PRRs can detect the presence and type of microbial infection and activate the appropriate innate immune response
(18). Some PRRs can also control adaptive immunity accordingly, particularly at the various checkpoints outlined above.
This instruction of adaptive immunity occurs largely through
triggering the maturation of DCs from highly phagocytic,
weakly immunogenic, tissue-resident cells into weakly phagocytic, highly immunogenic, lymph node-homing cells that are
competent to induce tailored T-cell responses to non-self
antigens acquired in the periphery (8, 19).
Not all PRRs are equal in terms of their ability to trigger the
adaptive immune response. Indeed, while some PRRs (e.g.
TLRs) are sufficient to induce both T- and B-cell responses,
other PRRs (e.g. the mannose receptor and scavenger receptors) are not competent to induce adaptive immunity by
themselves (8). Presumably, the ability and sufficiency of a
given PRR to control adaptive immunity at each of the checkpoints discussed above should correlate with the ability of that
particular PRR to accurately detect the presence, extent, and
duration of infection, and the microbial origin of the antigens,
as well as to effectively relay this information to the adaptive
immune system.
In the next sections we discuss what is currently known
about control of adaptive immunity by different classes of
PRRs (Table 1). For this purpose, two classes of PRRs,
transmembrane and cytosolic, are discussed separately.
Transmembrane PRRs
Transmembrane PRRs recognize PAMPs in the extracellular
space and ⁄ or in phagosomes or endosomes. Here, we discuss
members of two families of transmembrane PRRs—the TLRs
and the C-type lectin Dectin-1.
TLRs
TLRs are the canonical PRRs that fulfill all of the predicted
qualities of a PRR that links innate and adaptive immunity—that is, TLRs sense infection through the recognition of
PAMPs and induce appropriately tailored innate and adaptive
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Table 1. Transmembrane and cytosolic PRRs and their connection to adaptive immunity
PRR
Ligands recognized
Microbes recognized
Adaptive response induced
References
Gram-positive and
Gram-negative
bacteria, DNA and RNA
viruses, fungi, protozoa
Fungi
Sufficient to induce Th1,
antibody (particularly IgG2),
and CD8+ T-cell responses
(Reviewed
in 8, 20)
Sufficient to induce Th17 and
antibody responses
(31, 32)
Cytosolic bacterial cell wall
components, peptidoglycans
Intracellular bacteria
(48, 50)
Pathogenic bacteria
ISD sensor
Potassium efflux, LPS plus ATP,
pore-forming toxins,
bacterial secretion systems
Cytosolic DNA
RIG-I ⁄ MDA5
Cytosolic RNA
RNA viruses
Sufficient to induce Th2 and antibody
responses; potentiates Th1, Th2, Th17,
and antibody responses initiated by TLR;
may favor Th17 responses
Required for robust T cell-dependent
hypersensitivity; may potentiate Th2
driven antibody responses
Sufficient to induce CD4+ T-cell and
antibody responses (in hematopoietic cells);
sufficient to induce CD8+ T-cell responses
(in non-hematopoietic cells)
Sufficient to induce CD8+ T-cell responses;
insufficient to induce CD4+ T-cell and
antibody responses
Transmembrane PRRs
TLRs
Bacterial cell wall components,
viral nucleic acids in endosomes, etc.
Dectin-1
Cytosolic PRRs
Nod1, Nod2
NALP3
Fungal cell wall components, b-glucan
DNA viruses, retroviruses
(53–58)
(64)
(69)
PRRs, pattern recognition receptors; TLR, Toll-like receptor; Nod, nucleotide-binding oligomerization domain; NALP3, Nacht domain-, LRR-, and PYDcontaining protein 3; ISD, interferon stimulatory DNA; RIG-I, retinoic acid-inducible gene I; MDA5, melanoma differentiation-associated gene 5; LPS,
lipopolysaccharide; ATP, adenosine triphosphate; Th, T-helper; IgG2, immunoglobulin G2.
immune responses (8, 20). TLRs have been shown to be sufficient to control adaptive immunity at all checkpoints leading
to a tailored adaptive response, characterized by Th1 induction, immunoglobulin G2c (IgG2c) production, CD8+ T-cell
induction, and protection from re-infection (13, 21, 22).
TLRs have also been shown to be critical for the induction of
adaptive immunity in response to various immunizations and
infections (20, 23).
The critical roles of TLRs in activation of adaptive immune
responses have been well documented and extensively
reviewed elsewhere (8, 20). TLRs have been shown to control
adaptive immune responses at multiple levels, including control of antigen uptake (24) and antigen selection for presentation in DCs (12), control of DC maturation and cytokine
production (8, 13), control of naive T-cell susceptibility to
suppression by regulatory T cells (Tregs) (25), and control of
survival of activated T cells (26). TLRs can also control B-cell
responses to T-dependent and T-independent antigens (21,
27, 28), as well as self-antigens (29). As mentioned above,
TLRs can also directly activate memory B cells for antibody
production (17).
Dectin-1
Dectin-1 is a member of the C-type lectin family of PRRs and
recognizes b-glucans from fungal pathogens such as Candida
albicans (30). Aside from the TLRs, Dectin-1 may be the PRR
whose role in induction of adaptive immunity is best understood. Recent work has shown convincingly that Dectin-1
stimulation alone is sufficient to induce adaptive immunity
(31). However, unlike TLRs, which induce Th1 responses,
Dectin-1-induced adaptive immunity was shown to favor the
differentiation of T cells into the Th17 phenotype (31, 32).
Dectin-1 stimulation led to the preferential secretion of the
Th17-supporting cytokine IL-23 in place of the Th1-skewing
cytokine IL-12 (32). Notably, Th17 cells are specialized for
defense against extracellular pathogens (33, 34). Thus, Dectin-1 triggering by extracellular fungi leads to a Th17 response
resulting in recruitment of neutrophils, which engulf and kill
the extracellular fungi leading to clearance of the infection.
Dectin-1 is thus sufficient to induce an adaptive immune
response that is tailored to the offending infection and is a perfect example of PRR-mediated tailoring of adaptive responses
for the class of infection that is sensed. Furthermore, Dectin-1
signaling is critical for the clearance of fungal infections (35,
36). Interestingly, the signaling pathways used by Dectin-1
are very similar to those engaged by the antigen receptors in
lymphocytes and are distinct from those used by TLRs (35,
37). This observation suggests that there are multiple signaling
pathways that can lead to control of adaptive immunity.
Dectin-1 recognizes b-glucans that are unique, to microbial
non-self (30). Thus, barring discovery of a self-ligand for
Dectin-1, Dectin-1 can reliably distinguish between self and
non-self and control induction of adaptive immunity. Dectin-1,
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
like TLRs, may also control the selective processing and
presentation of contents from phagosomes containing fungal
particles, although this has yet to be examined. Dectin-1,
unlike the TLRs, is also a phagocytic receptor and can influence phagosome trafficking (38). This property may enhance
the ability of Dectin-1 to induce selective presentation of
non-self antigens. Finally, Dectin-1 engagement appears to be
largely fungal specific. Thus, Dectin-1 can determine the class
of infection and control the type of adaptive response induced
accordingly (39).
Cytosolic PRRs
The cytosolic PRRs can be separated into two classes based on
their mechanism of activation. The first class of cytosolic
PRRs, which includes the founding nucleotide-binding oligomerization domain containing (Nod)-like receptor (NLR)
family members Nod1 and Nod2, the retinoic acid-inducible
gene I (RIG-I)-like helicases (RLHs), and presumably the IFN
stimulatory DNA (ISD) sensor, directly detect cytosolic PAMPs
and activate various signaling cascades (40, 41). The second
class consists of members of the NLR family that are involved
in the formation and activation of large, multimeric protein
complexes referred to as inflammasomes, which control the
activation of caspase-1, and the secretion of caspase-1-dependent cytokines such as IL-1b (42). At least some of these
NLRs, such as Nacht domain-, LRR-, and PYD-containing protein 3 (NALP3), which can be activated by potassium efflux,
sense activities that result from microbial infection rather than
cytosolic PAMPs. However, other inflammasome NLRs, such
as ICE protease-activating factor (Ipaf), are activated in
response to cytosolic PAMPs.
The role of cytosolic PRRs in control of adaptive immunity
is just beginning to emerge. Cytosolic PRRs, unlike transmembrane PRRs, can distinguish between intracellular infections
and extracellular infections, between cell-intrinsic infections
and cell-extrinsic infections, and may even distinguish pathogenic microorganisms from harmless, commensal microorganisms. However, it is not clear whether or how cytosolic
PRRs relay information about antigen origin to the adaptive
immune system. We next discuss recent data on the role of
cytosolic PRRs in control of adaptive immunity.
NLRs
Nod1 and Nod2
Nod1 and Nod2 sense bacterial infection through the detection of cytosolic peptidoglycan fragments from bacterial cell
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walls. They activate an innate immune response that is critical for protection from bacteria that are able to escape the
endolysosomal compartment and for production of antimicrobial peptides in the intestinal crypts (43–46). Notably,
mutations in Nod2 have been linked to Crohn’s disease,
causing great interest in the role of this PRR in immunity
and pathology (45). In one study, Nod2-deficient animals
had defects in antibody responses to protein immunization
when the Nod2 ligand muramyldipeptide was used as adjuvant (47). Recent experiments have shown that specific activation of Nod1 in the absence of ligands for other PRRs is
sufficient to induce a type 2 T- and B-cell response (48).
Interestingly, this response was dependent upon Nod signaling in both hematopoietic and non-hematopoietic compartments. Furthermore, Nod1 stimulation was able to
potentiate Th1, Th2, and Th17 responses initiated in
conjunction with TLR ligands either during immunization
or infection with Helicobacter pylori. It is thus suggested that,
when given with TLR ligands, Nod1 potentiates and may
shape adaptive immunity; however, because Nod1 stimulation potentiates all Th responses, it is unclear whether Nods
are involved in controlling the type of response induced.
However, because Nods can detect the presence of cell
intrinsic, intracellular infections, it is tempting to speculate
that they might be critically involved in shifting immunity
in the intestine from tolerogenic to immunogenic (49). Furthermore, work in the human system has suggested that
Nod stimulation specifically favors anti-bacterial Th17
responses through the selective induction of IL-23 and IL-1;
this response was abrogated in cells from Crohn’s disease
patients that carried a mutation in Nod2 (50). A role for
the Nods in distinguishing pathogens from commensal
organisms would fit well with the linkage between Nod2
mutations and Crohn’s disease as well as the ability of Nods
to potentiate anti-microbial Th cell responses. However,
further work will be required to solidify this hypothesis.
NALP3
NALP3 inflammasomes can be activated in multiple ways.
Experimentally, NALP3 inflammasomes are most often
activated by pretreatment with lipopolysaccharide (LPS)
followed by induction of potassium efflux via high dose adenosine triphosphate (51). Inflammasomes can also be activated
by some intracellular Gram-positive pathogens by pore-forming
exotoxins, as well as by the type III secretion system used by
Gram-negative pathogens to inject virulence effectors into the
host cell (49, 52). In each of these cases, in vitro activation of
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
the NALP3 inflammasome in macrophages requires two
signals- signal one (e.g. LPS) primes for activation and induces
IL-1b production while signal two (e.g. potassium efflux)
activates the inflammasome and leads to Caspase-1 activation
and IL-1b processing and secretion.
Although NALP3 is so far the best-studied NALP involved in
inflammasome activation and IL-1b secretion, little is known
about the involvement of NALP3 in control of adaptive immunity. The most compelling evidence for the involvement of
NALP3 in activation of adaptive immunity comes from studies
showing that NALP3 is required for priming of T-cell-dependent contact hypersensitivity responses to skin painting
of haptens (53, 54). Recent studies have revealed that the
common adjuvant Alum can also activate the NALP3 inflammasome, and one study suggests that NALP3 is required for
optimal antibody responses following immunization using
Alum as an adjuvant (55–58). It remains unclear whether
inflammasome activation in vivo also always requires priming
by TLRs, as it does in vitro.
While the requirement for two separate signals to induce
inflammasome activation may complicate experimental
analysis, it may also provide the immune system with a
powerful tool for sensing the insult responsible for disruption of homeostasis. As noted above, inflammasomes are
specifically activated in response to bacteria that attempt to
manipulate the host response through virulence factors,
potentially providing a mechanism to distinguish between
pathogens and commensal organisms. Thus, PAMP sensing
alone by a transmembrane PRR might indicate the presence
of microorganisms (commensal or pathogenic), while PAMP
plus inflammasome activation might specifically indicate the
presence of pathogenic microbes. One problem with this
hypothesis is that inflammasomes can be activated under
non-infectious conditions (e.g. in gout) in addition to their
activation by pathogens (59). However, the inflammasomes
that are activated by endogenous and/or non-infectious
stimuli (e.g. uric acid crystals) may be largely specialized
to deal with non-infectious stressors, and thus may not be
connected to adaptive immunity. For example, ultraviolet
treatment of keratinocytes also induces inflammasome
activation (60). However, this activation may serve a very
different purpose—it may induce mainly tissue repair rather
than immunity. The triggering of the inflammasome by
endogenous and/or non-infectious conditions could also be
unintentional. Indeed, the endogenous triggers of NLRs are
notable not for their normal role in maintaining homeostasis
but instead for their role in pathological conditions such as
gout (59).
Cytosolic DNA sensor
An innate immune response to cytosolic DNA leading to the
production of large amounts of type I IFNs has recently been
described (61, 62). Subsequently, the role of the cystolic DNA
sensor, or interferon stimulatory DNA (ISD) sensor, in control
of adaptive immunity has also been examined. DNA vaccines
can induce both T-cell and antibody responses and activate
both TLR9 and the yet-to-be-identified ISD sensor (63, 64).
However, these two pathways can be distinguished via their
signaling adapter usage. TLR9 signals through the TLR adapter
myeloid differentiation factor 88 (MyD88), while the ISD
sensor signals through TRAF family member-associated NF-jB
activator (TANK)-binding kinase 1 (TBK1) to IFN regulatory
factor 3 (IRF3) (65). Surprisingly, adaptive immune responses
to DNA vaccines are largely TLR9 independent (64, 66).
Instead, adaptive responses to DNA vaccines are TBK1- and
type I IFN-dependent, implicating the ISD sensor (64). In
these experiments, TBK1 was required in hematopoietic cells
for CD4+ T-cell and B-cell responses, while TBK1 was
required in non-hematopoietic cells for CD8+ T-cell
responses. While the requirement for TBK1 for induction of
adaptive immunity to DNA vaccines is compelling, the role of
the ISD sensor in controlling adaptive immunity to infectious
stimuli under physiological conditions and its ability to
induce memory remain to be studied. Nonetheless, these
experiments with DNA vaccines suggest that, in principle, the
ISD sensor can control induction of antiviral T- and B-cell
responses. It is particularly interesting to consider the mechanism by which the ISD sensor instructs the adaptive immune
system on the origin of an antigen. Considering its cytosolic
location, it is difficult to imagine a mechanism by which the
coincidence of antigen and PAMP could be detected by the
ISD sensor in such a way as to selectively induce responses to
foreign antigens. However, it is certain that future studies will
reveal more about the mechanisms of ISD-induced adaptive
immunity.
RIG-I ⁄ melanoma differentiation-associated gene 5
RIG-I and melanoma differentiation-associated gene 5
(MDA5) (which are both members of the RLH family) recognize RNA viruses in all cells, except for the specialized plasmacytoid DCs, and activate an antiviral innate immune response
that is typified by the production of type I IFNs (67). The
TLRs also play an important role in viral detection and
defense, specifically in the IFN-producing plasmacytoid DC.
Recent experiments examining the adaptive immune response
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
to lymphocytic choriomeningitis virus (LCMV) and influenza
infection in PRR-signaling adapter-deficient mice have begun
to examine the relative importance of TLR versus RLH signaling in control of adaptive immunity.
LCMV triggers type I IFN production through both
TLR- and RLH-dependent pathways. CD8+ T cells mediate
protective–adaptive immune responses to LCMV. Recent
experiments demonstrated that the CD8+ T-cell response to
LCMV was entirely TLR signaling dependent and that RLH
signaling was dispensable for this response (68). Thus, in
response to LCMV, TLRs are responsible for induction of
protective CD8+ T-cell responses, and RIG-I ⁄ MDA5 signaling
is insufficient to induce adaptive immunity.
Both MyD88-dependent (TLR7) and IFN-b promoter stimulator 1 (IPS-1)-dependent (RIG-I ⁄ MDA5-dependent) pathways are induced in response to influenza A virus infection
and mediate the innate response to the virus. Recently, the
adaptive immune responses to influenza in MyD88-deficient
and IPS-1-deficient mice were compared, revealing a role for
RIG-I ⁄ MDA5 in control of adaptive immunity (69). In
response to influenza A virus, IPS-1-dependent signaling (in
the absence of TLR-dependent signaling) was sufficient to
induce a CD8+ T-cell response. However, TLR-signaling was
required for induction of CD4+ T-cell and antibody responses
to influenza A; TLR signaling was required to provide protection from re-infection. Therefore, while RIG-I ⁄ MDA5 can
induce CD8+ T-cell effector responses to certain viral infections, under these conditions they are insufficient to induce
helper T-cell activation, antibody production, and protection
from re-infection. It thus appears that adaptive immune
responses to influenza A and LCMV are controlled largely by
the TLRs. However, further research using additional viral
infections must be performed before a final conclusion on the
role of RIG-I ⁄ MDA5 in control of adaptive immunity can be
reached.
Coincident triggering of transmembrane and cytosolic
PRRs
Transmembrane and cytosolic PRRs differ notably in the types
of microbes and infections that they detect. Transmembrane
PRRs can sense the presence of both cell-intrinsic and non-cell
intrinsic infections and are triggered by both pathogens and
commensal organisms (70). Certain cytokines produced in
response to infection, such as type I IFNs and TNF-a, also
signal the presence of infection, and do not distinguish
between cell-intrinsic and non-cell intrinsic infections, or
pathogens and commensal organisms. In contrast, cytosolic
PRRs are activated mainly by cell-intrinsic infections, and
228
some cytosolic PRRs may be specifically activated in response
to pathogens and not commensal organisms (49). Thus, triggering of cytosolic PRRs may tag a cell as infected and may
indicate pathogenicity. Because transmembrane PRRs, inflammatory cytokine receptors, and cytosolic PRRs are differentially activated in response to different types and classes of
infections, these different signals probably serve distinct purposes. Isolated triggering versus coincident triggering of these
different classes of receptors may lead to differing immune
outcomes. Further studies into the role of different PRRs in
immunity and the coincident triggering of intracellular PRRs,
cytokine receptors, and cytosolic PRRs will certainly lead to a
deeper understanding of the control of innate and adaptive
immunity.
PRRs in non-DCs and control of adaptive immunity
The current view of innate instruction of adaptive immunity
involves a linear progression of signals specifying to adaptive
immune cells when and how to respond to a pathogenic
insult. In this model, DCs are activated by the presence of
infection through PRRs. Activated DCs presenting pathogenderived antigens migrate to the draining lymph node where
they activate and instruct naive T cells. Finally, activated helper
T cells migrate to the B-cell zone and provide antigen-specific
help to B cells (8). However, a role for PRR signaling in
non-DCs in control of adaptive immunity has been documented in a number of systems. For example, a role for PRRs
on non-hematopoietic cells in control of adaptive immunity
has been described for several systems (48, 64, 71, 72). Here,
we focus on the role of TLRs on B cells in T-cell-dependent
antibody responses.
B-cell-intrinsic TLR signaling and control of T-dependent
antibody responses
Mice deficient in TLR signaling show severe defects in antibody responses to protein plus LPS in simple depot adjuvants.
It was thought that T-cell help alone was sufficient to induce
optimal T-dependent B-cell responses. Therefore, the defect in
B-cell responses observed in mice deficient in TLR signaling
could be expected to be entirely caused by the failure to
induce a T-cell response. However, we recently demonstrated
that TLR-dependent signals are required not only in DCs (to
induce a T-cell response) but also in B cells themselves
for induction of a robust T-dependent antibody response
(21). This requirement for a B-cell-intrinsic TLR signal
varied depending on the antibody isotype. IgM and IgG1
responses were largely TLR-dependent, IgG2c responses
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
were nearly entirely TLR-dependent, and IgE responses were
TLR-independent. Thus, it appears that TLRs on B cells themselves act as an additional checkpoint in the regulation of
adaptive immune responses. Similar results were obtained in
human B cells (27). It is interesting to speculate that one
purpose of this additional checkpoint may be to regulate the
magnitude and duration of the B-cell response based on the
availability of TLR ligands in the lymph node. Presumably,
the relative concentration of PRR ligands in the lymph node
correlates with the extent of infection, and the absence of
PRR ligands in the lymph node correlates with clearance of
infection. In addition, this checkpoint may control effector
versus memory cell differentiation; indeed, TLRs stimulation
on B cells, which would represent persistence of infection and
necessity for effector (plasma cell) generation, increases the
expression of the critical regulator of plasma cell differentiation B-lymphocyte induced maturation protein-1 (Blimp-1)
(21). Interestingly, TLR signaling in B cells is also critically
involved in antibody-dependent autoimmunity (73–75).
A number of investigators have since found similar requirements for TLRs on B cells in the induction of optimal antibody
responses to various infections and immunizations. Notably,
the link between TLR signaling on B cells and IgG2c production is maintained in every system examined, suggesting that
this isotype is intimately connected to B-cell-intrinsic TLR
signaling (73, 76–79). The reasons for this intimate connection and the need for TLR ligands in addition to T-cell help
and the IgG2c switch factor IFN-c are unclear. However, it is
tempting to hypothesize that the presence of B-cell stimulating
TLR ligands in the lymph node might signal either the type or
severity of the infection, and that this would shape the
immune response to deal with the specific type of infection
through production of IgG2c. Notably, IgG2 is highly efficient
for both viral and bacterial clearance and efficiently fixes
complement (80); therefore, TLRs on B cells may also shape
the adaptive response to maximize resistance to the particular
offending infection.
Although many studies have found a role for TLRs in
antibody responses, two reports have failed to find any
role for TLRs in general, or on B cells in particular, in
induction of antibody responses to common immunizations (79, 81, 82). Because the reason for this discrepancy
in data sets was unclear, the role of B-cell-intrinsic TLR
signals became controversial. However, we have recently
found that this discrepancy is due to an experimental
difference—experiments that found a role for TLRs were
done using either native proteins in adjuvant or natural
infections, while experiments finding little or no role for
TLRs were done using proteins chemically modified by
haptenation (haptenated proteins). We found that haptenated proteins, unlike native proteins, were uniquely
immunogenic and thus could induce robust T- and B-cell
responses in a TLR-independent manner (NP and RM,
unpublished results). The reason for the unique immunogenicity of haptenated proteins is unclear, but it may be
related to the antigen specificity of the adaptive responses
that are induced - haptenated induce strong anti-hapten
responses yet induce very weak responses to the carrier
protein to which the haptens are conjugated. However,
regardless of the reason for the immunogenicity of haptenated proteins, it is clear that haptenated proteins display
altered innate requirements for induction of adaptive
immunity that can affect the outcome of experiments
testing adjuvant effects and often obscure the pathways
used by common adjuvants.
PRRs and adjuvanticity
Common adjuvants contain at least two different types of
activities that are critical for inducing adaptive immune
responses to immunizations with soluble antigens. One activity is a depot activity that prevents the dispersion of soluble
antigen and promotes the concentrated delivery of antigen to
the draining lymph node where priming of the adaptive
immune response can occur (83, 84). (Notably, infectious
organisms do not require this activity, as they are naturally
concentrated at the site of infection.) The other activity is
based on activation of the innate immune system through
triggering of appropriate PRRs, such as the TLRs, leading to
activation of antigen-presenting cells and immunogenic
presentation of antigen (85). The best adjuvants provide both
activities and can convey immunogenicity on an otherwise
non-immunogenic antigen. In the case of complete Freund’s
adjuvant (CFA), emulsification in mineral oil acts largely to
provide a depot, while heat killed mycobacteria act to trigger
the innate immune system (83).
Both depot and innate immune-stimulating activities are
necessary for induction of robust adaptive immune
responses to soluble proteins (83). Some common adjuvants, such as CFA, contain components that have both
activities, while other common adjuvants have only one of
the two necessary activities. These adjuvants are, by themselves, insufficient to induce robust adaptive responses. For
example, TLR ligands, which efficiently stimulate the innate
immune system, are insufficient to induce adaptive immunity when used alone as adjuvant for soluble proteins (86).
2009 The Authors • Journal compilation 2009 Blackwell Munksgaard • Immunological Reviews 227/2009
229
Palm & Medzhitov Æ Innate instruction of adaptive immunity
Fig. 2. Proteins commonly used for immunizations are highly
contaminated with TLR ligands. Bone marrow-derived DCs (BMDCs)
from wildtype or myeloid differentiation factor 88 (MyD88) ⁄ TIRdomain-containing adapter-inducing interferon-b (TRIF)-deficient mice
were stimulated with lipopolysaccharide (LPS) (100 ng ⁄ ml), ovalbumin
(OVA), endotoxin-free human serum albumin (HSA), keyhole limpet
hemocyanin (KLH), or chicken c-globulin (CGG) for 20 h before measuring interleukin-6 (IL-6) secretion into the supernatant by enzymelinked immunosorbent assay. All proteins were used at 300 lg ⁄ ml.
This failure to induce adaptive immunity is largely caused
by the lack of depot adjuvant, and thus the rapid dispersion
of the soluble antigen such that the required local concentration of antigen and PAMP is never reached (83, 86). By
contrast, Alum and incomplete Freund’s adjuvant (IFA) are
efficient depot adjuvants but contain minimal amounts of
PRR (84). For this reason, immunizations with purified proteins, such as endotoxin-free human serum albumin (which
is free of contaminating TLR ligands or other PRR ligands)
(Fig. 2) or subunit vaccine formulations, in either Alum or
IFA fail to induce robust adaptive responses because of the
lack of a sufficient PRR stimulating activity (13, 21, 87,
88). However, the addition of a single TLR ligand, such as
LPS, to a mixture of depot adjuvant and soluble protein
allows robust induction of adaptive immunity (13, 21).
Most proteins that are commonly used for immunization
studies- including keyhole limpet hemocyanin, bovine serum
albumin, ovalbumin, and chicken c-globulin- are highly contaminated with PRR ligands (Fig. 2). These contaminating
PAMPs provide the necessary PRR stimulating activity, which
accounts for the induction of adaptive immune response by
contaminated proteins in depot adjuvants, such as IFA and
Alum. The most common contaminants are LPS and bacterial
lipoproteins; thus, the immunogenic effects of these contaminants are dependent upon TLR signaling. However, PAMP
contaminants are not necessarily restricted to TLR ligands. For
example, recombinant proteins may also be contaminated
with TLR-independent PAMPs, such as Nod ligands. Also, we
230
have recently observed that, in contrast to native, PAMP-free
proteins, PAMP-free haptenated proteins in IFA or Alum
induce robust adaptive immune responses (NP and RM,
unpublished results). Thus, certain chemical modifications of
antigens can also influence the perceived requirements for
innate immunostimulatory adjuvants. Failure to consider these
possibilities when performing immunization-based experiments can greatly confuse the interpretation of data and lead
to incorrect conclusions on the requirements for innate
instruction of adaptive immunity.
Ideally, experiments testing the role of a particular PAMP or
PRR should be performed in a system where the role of a
given PRR can be isolated from the effects of other PRRs. This
separation can be achieved via targeting of a particular PRR
using its corresponding PAMP in conjunction with PRR-free
protein antigens in PRR-free depot adjuvants. In particular, it
is critical that experiments testing the sufficiency of a PRR for
induction of adaptive immunity be performed in a manner
where the role of a single PRR can be sufficiently isolated. In
the absence of this type of experiment, it is impossible to
know whether a PRR is sufficient for activation of adaptive
immunity.
An additional activity that adjuvants may provide relates to
the requirement for antigen and PRR to be delivered concomitantly and, ideally, to be delivered to the same phagosome, to
induce a maximal response. Adjuvants, such as Alum and IFA,
may thus also act to deliver antigen and PAMP in a complex
that results in delivery to the same phagosome. Indeed, the
co-delivery of antigen and PAMP is critical for induction of
adaptive immunity; separate immunizations with PAMP and
antigen emulsified separately fail to induce robust adaptive
immune responses despite the presence of both necessary
adjuvant activities (11). Furthermore, the immunogenicity of
a protein can be increased via conjugation with a PAMP, as is
achieved in the case of flagellin-fusion proteins (89). It is
interesting to consider how the ability of a protein to complex
with PAMPs may contribute to the immunogenicity and ⁄ or
immunodominance of certain proteins over others.
Common adjuvants also probably have other, yet to be
discovered activities, which may or may not be dependent
upon classical PRRs. For example, IFA can induce lymph node
swelling in a TLR-independent manner (NP and RM, unpublished observations); the mechanism responsible for this
innate response to IFA has yet to be defined. It is also of interest to note that Alum has recently been shown to cause IL-1b
secretion through activation of the NALP3 inflammasome,
which may account for Alum’s adjuvanticity in addition to its
depot effect (55–58).
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Palm & Medzhitov Æ Innate instruction of adaptive immunity
When is adaptive immunity induced?
While much has been learned over the past decade about
innate instruction of adaptive immunity, a number of
fundamental questions still remain. For example, it is not
entirely clear what determines whether an adaptive immune
response will be induced during a given infection. A textbook
notion is that during an infection an innate immune response
is first induced and attempts to eliminate the pathogen, and if
it is not sufficient to clear the infection, then the adaptive
immune response follows. This notion however is incompatible with the current understanding of the mechanisms of
initiation of adaptive immune responses. Namely, tissueresident DCs function as sensors of microbial invaders, and
upon contact with pathogens, they mature and migrate to the
lymph nodes where they activate naive T cells. According to
this view, regardless of the ability of the innate immune system to contain infection, DCs that have encountered microbes
should induce an adaptive immune response. However, lowgrade asymptomatic infections are commonplace, and they are
unlikely to always result in the activation of adaptive immune
responses. It is not clear whether activation of the adaptive
immune response is simply a matter of pathogen load, which
would imply the existence of a threshold for activation of
adaptive immunity, or whether additional mechanisms exist
that determine when an adaptive immune response is induced
based on other, currently unknown, parameters of infection.
Conclusions
Many recent studies have highlighted and clarified the role of
PRRs in induction and instruction of adaptive immunity and
have revealed roles for new PRRs, and new roles for old PRRs,
in control of adaptive immunity. We now know that PRRinduced signals control the adaptive immune system at various
checkpoints controlling the activation, effector class, magnitude, duration, and memory of an adaptive immune response,
and that the ability of a PRR to control adaptive immunity at
these checkpoints depends upon the ability of that PRR to
define the ongoing infection and relay that information to the
adaptive immune system. While the roles of various PRRs in
control of adaptive immunity are becoming clearer, it is also
certain that we are far from a complete understanding of how
innate immunity shapes adaptive responses to maximize
defense and the specific role that each class of PRRs plays in
this process. Future studies will undoubtedly reveal new
mechanisms of innate control of adaptive immunity.
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