PERSPECTIVES

Five checkpoints for fine discrimination

OPINION
Hereditary or acquired immunodeficiencies
often predispose to fatal infections by other­
Beyond pattern recognition: wise harmless microorganisms16,17. On the
other hand, overt and uncontrolled immune
five immune checkpoints for scaling responses can cause inflammatory tissue
damage and lead to debilitating diseases.
the microbial threat As immune homeostasis is a prerequisite for
species survival, it is imperative for the host
to invoke measured immune responses that
J. Magarian Blander and Leif E. Sander are appropriate for the infectious threat and
that cause minimal collateral tissue damage.
Abstract | Pattern recognition by the innate immune system enables the detection How does the immune system sense and
of microorganisms, but how the level of microbial threat is evaluated — a process scale microbial menace?
that is crucial for eliciting measured antimicrobial responses with minimal Janeway’s concept of pattern recogni-
inflammatory tissue damage — is less well understood. New evidence has shown tion has provided the key framework for
immune discrimination between host (self)
that features of microbial viability can be detected by the immune system and
and microorganism (non-self), but it has
thereby induce robust responses that are not warranted for dead microorganisms. not been fully sufficient in explaining the
Here, we propose five immune checkpoints that, as defined here, collectively finer distinctions that are made between
determine the gravity of microbial encounters. viable and dead or pathogenic and non-
pathogenic microorganisms, or between
infection and colonization. Furthermore,
In 1989, Charles A. Janeway Jr proposed the immune response have been shown correlations between the severity of infec-
that the detection of microbial patho- to be controlled by TLRs6. Besides TLRs, tion and the strength of the resultant
gens occurs through pattern-recognition other PRR families have been described11 immune response are frequently observed
receptors (PRRs) of the innate immune (BOX 1); these families include the cytosolic and even exploited as clinical biomarkers.
system, and that these receptors sense NOD-like receptors (NLRs)7,8 and RIG‑I- The main dilemma has been that PAMPs
invariant microbial components which he like receptors (RLRs)9, and the cell-surface are shared among microorganisms regard-
termed pathogen-associated molecular C‑type lectin receptors (CLRs)10. The struc- less of their viability or pathogenicity, but
patterns (PAMPs)1. In 1997, Janeway and tures and functions of PRRs are as diverse as the quality and magnitude of the immune
Ruslan Medzhitov reported the discovery those of the PAMPs that act as their ligands. response triggered following activation of
of Toll-like receptor 4 (TLR4), the first PAMPs range from lipids to proteins, carbo­ the corresponding PRRs varies according
mammalian homologue of the Drosophila hydrates and nucleic acids11. PRR signals to the nature of the microbial encounter,
melanogaster Toll protein2, which had converge on common intracellular path- as illustrated by the following examples.
been shown by Bruno Lemaitre and ways, including those mediated by nuclear First, expression of virulence factors, such
Jules Hoffman to invoke innate immune factor-κB (NF-κB), mitogen-activated as bacterial secretion systems or pore-
responses in D. melanogaster 3. Activation protein kinases (MAPKs) and interferon- forming toxins, imparts pathogenicity and
of TLR4 triggered the expression of co- regulatory factors (IRFs)11,12. Some NLRs strongly augments the resultant immune
stimulatory molecules for T cells and assemble in multimeric cytosolic complexes, response18. Second, live attenuated patho-
of pro-inflammatory cytokines, thereby termed inflammasomes, leading to the acti- gens that are used as vaccines generally
bridging innate immune recognition with vation of the cysteine protease caspase 1, induce superior immunity compared with
adaptive immune activation2. Bruce Beutler which processes pre­cursor forms of their dead counter­parts19,20. Third, microbial
and colleagues later provided formal evi- pro-inflammatory cytokines into their invasion evokes rapid and vigorous immune
dence that TLR4 was a PRR by showing biologically active forms13 (BOX 1). responses, whereas colonization of our body
that mutation or loss of the Tlr4 locus was Since the inception of the pattern surfaces by a plethora of microorganisms
responsible for the phenotypes of the recognition theory, over two decades of triggers no measurable inflammation21.
C3H/HeJ and C57BL/10ScCr strains of mice, investigations have proven its central role These examples clearly illustrate an inherent
which are known for their unresponsiveness in host defence and immunity. Indeed, ability of the innate immune system to make
to lipopolysaccharide (LPS) and suscep- defects in PRR pathways result in dangerous fine distinctions that enable the evalua-
tibility to infections with Gram-negative infections14,15. However, various functions tion of the severity of the infectious threat,
bacteria4. Various other TLRs have now of PRRs and their signalling pathways are such that dangerous microbial encounters
been identified5, and multiple aspects of still under intense investigation12. evoke more aggressive immune responses.

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PERSPECTIVES

Although pattern recognition alone cannot the immune system distinguishes between: First checkpoint
explain how these distinctions are made, first, soluble and particulate PAMPs; second, Soluble or particulate PAMPs — the virtues
signal transduction via PRRs is absolutely viable and dead microorganisms; third, of a phagocytic synapse. Most studies on
crucial for protective immunity 6. invasive and non-invasive microorganisms; PRR activation have focused on delineating
Based on recent evidence, we define five and, fourth, pathogenic and non-pathogenic the response to individual microbial ligands,
immune checkpoints that collectively inte- microorganisms. Last, the immune system or synthetic mimics thereof, in soluble form.
grate information about the class of PAMP integrates tissue-specific signals to elicit the Although this system proved valuable in
and the context of pattern recognition in appropriate response. These five checkpoints, delineating the signalling events and biologi-
order to evaluate the level of threat that a discussed below in no particular hierarchical cal responses following pattern recognition, it
microorganism poses to the host organism order, extend the role of pattern recognition has not been able to reflect the complexity of
(FIG. 1). The result of this evaluation critically beyond host versus microorganism discrimi- PAMP recognition in the context of an intact
determines the magnitude and quality of the nation, and may critically aid in the process microorganism. Individual PAMPs may trig-
ensuing immune response. We propose that of immunological decision-making. ger distinct responses when sensed as part of
whole microorganisms. Soluble PAMPs that
are shed from viable microorganisms or leak
Box 1 | Pattern-recognition receptors from killed microorganisms can be sensed at
Pattern-recognition receptors (PRRs) are evolutionarily conserved germline-encoded receptors, a distance from the local site of infection, and
which, although structurally different, share a common feature of detecting pathogen-associated this leads to cytokine and chemokine pro-
molecular patterns (PAMPs)11. duction. By contrast, the detection of PAMPs
Toll-like receptors (TLRs) in the context of a microbial cell indicates the
Ten TLRs have been identified in humans and twelve in mice. TLRs are type I transmembrane presence of a microorganism and necessitates
glycoproteins that localize to either the plasma membrane (in the case of TLR1–TLR6, TLR10 and direct microbicidal responses. Indeed, such
TLR11, for example) or the endosomal membrane (in the case of TLR3, TLR7 and TLR9, for example). responses downstream of the CLR dectin 1
Ligands for TLRs include bacterial lipoproteins and lipopeptides (for TLR2), double-stranded RNA (also known as CLEC7A) require the forma-
(for TLR3), lipopolysaccharide (for TLR4), flagellin (for TLR5), single-stranded RNA (for TLR7), CpG tion of a phagocytic synapse22. Although
DNA (for TLR9) and profilin (for TLR11)11. Following ligand binding, the receptors form heterodimers both soluble and particulate β‑glucans
or homodimers and recruit signalling adaptor molecules through their Toll/IL‑1R (TIR) domain. With derived from Saccharomyces cerevisiae bind
the exception of TLR3, all TLRs signal through myeloid differentiation primary-response protein 88
to dectin 1 on macrophages, only particles
(MYD88). TLR3 signals solely through TIR-domain-containing adaptor protein inducing IFNβ (TRIF)12,
whereas TLR4 signals through both MYD88 and TRIF.
0.5 μm or larger in diameter trigger synapse
formation and downstream signalling events
NOD-like receptors (NLRs)
that result in the generation of reactive
NLRs constitute a large family of cytosolic proteins. The first family members to be discovered —
nucleotide-binding oligomerization domain protein 1 (NOD1) and NOD2 — recognize bacterial
oxygen species (ROS) in addition to pro-
peptidoglycan fragments and activate nuclear factor-κB (NF‑κB) signalling7. Several NLRs — including inflammatory cytokine production22. This
NOD‑, LRR- and pyrin domain-containing (NLRP) proteins, NOD‑, LRR- and CARD-containing was shown to be a direct result of excluding
(NLRC) proteins and neuronal apoptosis inhibitory proteins (NAIPs) — form multimeric cytosolic phosphatases, such as CD45 and CD148,
complexes termed inflammasomes, which serve as platforms for the activation of caspase 1 and from dectin 1 clusters at β‑glucan particle
the release of interleukin‑1β (IL‑1β) and IL‑18 (REF. 91). More recent data show that some NLR contact sites, thereby allowing unimpeded
family members carry out important inflammasome-independent functions, including regulating signal transduction through SRC and SYK
type I interferon (IFN) production, and may even have roles aside from pathogen recognition92. family kinases.
RIG‑I-like receptors (RLRs) Therefore, for some PRRs (such as CLR
There are three known RLRs: retinoic acid-inducible gene I (RIG‑I), melanoma differentiation- family members that double up as both
associated gene 5 (MDA5) and LGP2. RLRs are expressed in the cytosol and sense nucleic acids, phagocytic and inflammatory signalling
such as viral RNA. RIG‑I and MDA5 contain an amino‑terminal caspase-recruitment domain (CARD), receptors10), coupling phagocytosis with full
which binds to IFNB-promoter stimulator 1 (IPS1; also known as MAVS and VISA) at the mitochondria
signalling potential ensures the mobilization
to induce type I IFN production. Whereas RIG‑I binds to 3ʹ-triphosphate single-stranded RNA, MDA5
is thought to bind to double-stranded RNA. LGP2 is thought to act as a regulator of RLR activity. of appropriate antimicrobial responses (such
as ROS production) that can only be effec-
Other cytosolic nucleic acid sensors
tive when immune cells are in direct contact
DNA-dependent activator of IFN-regulatory factors (DAI; also known as DLM1 and ZBP1) is
thought to be involved directly or indirectly in sensing cytosolic DNA and inducing type I IFNs. with microorganisms. Although hitherto
Another more recently identified DNA sensor is IFNγ-inducible protein 16 (IFI16; known as p204 in demonstrated only for antifungal responses,
mice)93, which is a member of the PYHIN protein family. Absent in melanoma 2 (AIM2) is another the novel concept of discriminating particu-
PYHIN family protein that recognizes cytosolic DNA and forms inflammasomes with the adaptor late from soluble PAMPs could generally aid
protein ASC39,94. It has been proposed that the PYHIN proteins AIM2 and IFI16 form a new family of the risk-assessment process during infections
PRRs called AIM2‑like receptors (ALRs)93. A recent study reported that the helicases DDX1, DDX21 with extracellular microorganisms, effective
and DHX36 sense cytosolic double-stranded RNA and form a complex with TRIF to induce the clearance of which would require the
transcription of IFNB 95. mobilization of both highly inflammatory
C-type lectin receptors (CLRs) and microbicidal responses.
The CLRs are a large family of proteins that possess one or more C‑type lectin domains and one or
more immunoreceptor tyrosine-based activation motifs (ITAMs). They recognize a wide range of Microorganisms present multiple PAMPs.
carbohydrate ligands (and probably also non-carbohydrate ligands). Following activation, they
Microorganisms not only present individual
signal through the kinase SYK and CARD9 to promote the production of pro-inflammatory
PAMPs in particulate rather than soluble
cytokines and reactive oxygen species10.
form to the immune system, they also

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 PERSPECTIVES

present more than one PAMP at once. This
begins at the phagocytic synapse, where
multiple microbial cell wall components
(such as LPS and flagellin from flagellated
Gram-negative bacteria) trigger more than
one PRR. It continues with microbial deg-
radation within phagolysosomes, where
new PAMPs (such as DNA and RNA) are
released and engage intracellular PRRs.
Triggering specific combinations of TLRs
in dendritic cells (DCs) induces synergistic
cytokine production23,24, and targeting both
TLR4 at the plasma membrane and TLR7 in
endosomes mediates vaccine efficacy, pre-
sumably via combinations of as yet poorly
defined signals for protective immunity 25.
Cooperation of TLR and NLR signals is best
exemplified by TLR-mediated activation
of interleukin‑1β (IL‑1β) gene transcrip-
tion and NLR-mediated activation of the
inflammasome complex, which initiates
the processing and secretion of IL‑1β13.
TLR and CLR synergy occurs during the
recognition of fungi such as Aspergillus
fumigatus and Coccidioides posadasii, when
TLR2 synergizes with dectin 1 to induce
pro-inflammatory cytokines, such as tumour
necrosis factor (TNF)24. Integrating signal-
ling from multiple PRRs during phagocyto-
sis ensures a robust response that is tailored
to meet the increased level of microbial
threat, with minimal room for evasion by
potentially dangerous microorganisms24.
Second checkpoint
Dead or alive — detecting microbial viabil-
ity. Although PAMPs convey the nature of
their microbial origin (for example, LPS is a
major cell wall component of Gram-negative
bacteria, whereas lipoteichoic acid is pre-
sent in Gram-positive bacterial cell walls5),
their detection does not necessarily indicate
an active infection with a viable micro­
organism. However, the immune system
responds more vigorously to viable micro­
organisms than to dead ones, a phenomenon
that has been attributed to the ability of live
microorganisms to replicate and express
virulence factors that facilitate the invasion
and infection of their hosts19,26. However,
live vaccines usually comprise attenuated
variants of pathogenic microorganisms, and
successfully induce long-term protective
immunity despite their low virulence and
invasiveness26. Therefore, we reasoned that
viability itself, being the quintessential basis
of infectivity, might alert the immune system
independently of virulence factors.
In support of this, we found that viable
bacteria contain a special class of PAMPs
that we have termed ‘vita-PAMPs’, recog-
nition of which signifies microbial life to
the innate immune system27. We identi-
fied bacterial mRNA, which is present in
viable bacteria but not in killed bacteria,
as one such vita-PAMP. Following phago-
cytosis of viable bacteria, but not dead
bacteria, bacterial mRNA is released, and
this leads to pronounced production of
interferon‑β (IFNβ) in a manner dependent
on TIR-domain-containing adaptor protein
inducing IFNβ (TRIF)27. By imaging the
translocation of fluorescein–dextran from
acidic phagosomes, where its fluorescence
is quenched, to the pH-neutral cytosol,
where it regains fluorescence, we could
demonstrate low-level leakage from phago-
somes carrying avirulent Escherichia coli.

Soluble microbial components Dead microorganisms Viable microorganisms Pathogens

Virulence
factors Vita-PAMP
PAMP
PAMP Vita-PAMP PAMP

Pathogenic
Dead
Level of inflammatory response

microorganism
microorganism Viable
microorganism
PAMP PAMP Pattern recognition
• Recognition of PAMPs and
vita-PAMPs
• Production of pro-inflammatory
cytokines (such as IL-6,
IL-12 and TNF)
Pattern recognition • NLRP3 inflammasome activation
• Recognition of PAMPs and (which induces IL-1β production
vita-PAMPs and pyroptosis)
• Production of pro-inflammatory • Increased IFNβ production
cytokines (such as IL-6, Direct and indirect effects of
IL-12 and TNF) virulence
Pattern recognition • NLRP3 inflammasome activation • Virulence factors (such as toxins,
• Recognition of PAMPs (which induces IL-1β production effector proteins and secretion
• Production of pro-inflammatory and pyroptosis) systems)
cytokines (such as IL-6, IL-12 and TNF) • Increased IFNβ production • Cell death and barrier disruption
Level of microbial threat
Figure 1 | Correlation of the microbial threat with inflammatory interferon-regulatory factors or NOD‑, LRR- and pyrin domain-containing 3
responses. Soluble pathogen-associated molecular patterns (PAMPs) and (NLRP3) inflammasomes. Activation of the NLRP3 inflammasome results in
dead microorganisms pose a relatively low-level threat to the host. They are the production of IL‑1β and pyroptotic cell Nature
death.Reviews | Immunology
Classical pathogens
recognized by pattern-recognition receptors (PRRs), which induce the pro- (defined as microorganisms that express genes encoding virulence factors
duction of pro-inflammatory cytokines — such as interleukin‑6 (IL‑6), IL‑12 that actively disrupt or alter host tissue homeostasis) are extremely danger-
and tumour necrosis factor (TNF) — as well as cellular responses with the ous for the host organism and usually trigger immediate and strong inflam-
goal of removing PAMPs and microbial debris from the tissue. Viable micro- matory responses. In addition to PRR activation, direct and indirect effects
organisms, which are by definition infectious, pose a significant threat and of virulence are sensed by the immune system, triggering the rapid mobili-
elicit robust inflammatory responses. Bacterial viability is sensed through zation of multiple PRR systems, including Toll-like receptors (TLRs) and
the detection of vita-PAMPs, and possibly through other mechanisms, lead- NOD-like receptors (NLRs). Thus, the inflammatory response is scaled to the
ing to the activation of additional PRR pathways, including those involving microbial threat. IFNβ, interferon‑β.

NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | MARCH 2012 | 217

© 2012 Macmillan Publishers Limited. All rights reserved

PERSPECTIVES

Bacteria
Surface TLRs

TLR4
Plasma membrane Phagocyte

TIRAP TIRAP
MYD88 MYD88
Internalization Late
phagosome Sensing of cytosolic
bacterial mRNA
Bacterial
Early Viable triggers inflammasome
mRNA
phagosome bacteria assembly

TLR4

Inflammasome
MYD88
TRAM TRIF Endosomal TRIF Pyroptosis
TLR TRIF promotes
NLRP3
inflammasome
activation

Pro-caspase 1

Activation of Active
MAPK signalling TRIF activates IRF3 caspase 1
pathways

IL-1β
pro-IL-1β IL-1β production

IRF3 Type I IFN

IFN production

NF-κB
NF-κB
activation Production
p50 p65 IL-6, IL-12, TNF, IL-1β of IL-6, IL-12
and TNF
Cytosol Nucleus

Figure 2 | Sensing vita-PAMPs. Viable bacteria trigger unique responses
(highlighted in orange boxes), namely: the induction of high levels of
interferon‑β (IFNβ); and the activation of the NOD‑, LRR- and pyrin domain-
containing 3 (NLRP3) inflammasome with subsequent interleukin-1β (IL‑1β)
production and pyroptosis. Cell wall components of non-pathogenic bacteria
are sensed through surface Toll-like receptors (TLRs), which trigger signalling
cascades that lead to the transcription of genes encoding pro-inflammatory
cytokines such as IL‑6 and pro-IL‑1β. Following the phagocytosis of bacteria,
phagolysosomal degradation exposes additional ligands, such as bacterial
DNA and, in the case of viable bacteria, bacterial mRNA. The presence of
bacterial mRNA signifies bacterial viability because it is not found in dead
bacteria. Bacterial mRNA probably binds to endosomal pattern-recognition
receptors (PRRs) to induce signalling. Small amounts of the phagosomal
contents gain access to the cytosol, where they can be sensed by cytosolic
PRRs. Translocation of bacterial mRNA to the cytosol triggers the formation
of the NLRP3 inflammasome and subsequent activation of caspase 1. Pro-
IL‑1β, which is equally induced by viable and dead bacteria, is specifically
Nature Reviews | Immunology
processed into its bioactive mature form by active caspase 1 only in response
to viable bacteria. NLRP3 activation also triggers caspase 1‑dependent inflam-
matory cell death (pyroptosis). Interestingly, although the transcription of the
gene encoding pro-IL‑1β strongly depends on TLR signalling through myeloid
differentiation primary-response protein 88 (MYD88), the activation of NLRP3
and the production of active IL‑1β and IFNβ are completely dependent on the
TLR signalling adaptor protein TIR-domain-containing adaptor protein induc-
ing IFNβ (TRIF). Although the link between TRIF and interferon-regulatory
factor 3 (IRF3)-dependent IFNβ production is well established, TRIF-mediated
NLRP3 activation occurs through an as yet unidentified mechanism.
MAPK, mitogen-activated protein kinase; NF‑κB, nuclear factor-κB;
TIRAP, TIR-domain-containing adaptor protein; TNF, tumour necrosis factor;
TRAM, TRIF-related adaptor molecule.

This leakage could presumably allow the
translocation of mRNA from viable bacteria
into the cytosol, where it triggers cytosolic
PRRs. Indeed, using various avirulent
bacteria, we found that live forms of these
bacteria, but not killed forms, triggered the
activation of the NOD‑, LRR- and pyrin
domain-containing 3 (NLRP3) inflamma-
some and subsequent caspase 1‑depend-
ent IL‑1β production and pyroptosis (an
inflammatory form of cell death). mRNA
alone or together with LPS could not
recapitulate these responses, suggesting
a requirement for phagocytosis. Notably,
NLRP3‑dependent pyroptosis and IL‑1β
production were undetectable in the
absence of TRIF. Thus, TRIF has a crucial
and previously uncharacterized role in link-
ing the TLR pathway to NLRP3, through
a mechanism that is independent of
transcriptional regulation of NLRP3 inflam-
masome components. Although commu-
nication with the cytosol occurs regardless
of the viability of the microbial contents of
phagosomes27, it is possible that TRIF
hastens this process by recruiting channel
proteins that facilitate mRNA translocation.
TRIF may alternatively have a different, so
far unknown, role in mediating crosstalk
between the TLR and NLRP3 pathways.

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Nonetheless, phagosomal communication
with the cytosol, be it intrinsic or facilitated,
is likely to have a general role in mobilizing
both phagocytic and cytosolic pathways
of pattern recognition when a heightened
level of microbial threat is detected (FIG. 2).
Given the well-known superior protection
afforded by viable vaccines, elevated threat
levels are also likely to influence adaptive
immune responses. Indeed, we have shown
that vita-PAMPs enhance humoral immu-
nity 27, and we predict they also positively
regulate CD4+ and CD8+ T cell responses.
Other viability-associated molecules.
Besides bacterial mRNA, other vita-PAMPs
or viability-associated molecules are likely
to exist that could signal bacterial life to
immune cells. Indeed, Vance, Isberg and
Portnoy have previously proposed that
molecules associated with growth could
signify life, and they suggested several
potential candidates for what they called
PAMP-PV (PAMP per vita), including
tracheal cytotoxin (a monomeric unit of
peptidoglycan produced by growing Gram-
negative bacteria), quorum-sensing mol-
ecules, bacterial pyrophosphates and second
messengers (such as cyclic di-GMP)18. The
sensing of active processes such as bacterial
replication is also possible18, although experi-
mental evidence for this does not yet exist.
The same authors further proposed that
certain PAMPs, such as muramyl dipeptide,
may be associated with dead bacteria, and
they termed these molecules PAMP-PM
(PAMP postmortem)18, although the impact
of the recognition of such PAMPs on immu-
nity remains unclear. Nevertheless, the general
concept of different classes of PAMPs inform-
ing the immune system about microbial
viability, and thus the microbial threat level,
is intriguing and may potentially add a new
layer to the concept of pattern recognition.
The detection of vita-PAMPs that act in
concert with conventional PAMPs to syner-
gistically engage several PRR systems may
have a crucial role in antimicrobial immu-
nity 27. We are only beginning to understand
how this translates into the generation
of protective immunity 28. Clearly more
work is required to elucidate the intricate
relationships between various PAMPs and
vita-PAMPs, and how they affect immune
responses for optimal protection against
infectious microorganisms. Determining the
unique molecular properties of vita-PAMPs
and the identity of their PRRs could facilitate
the development of new vaccines that
combine the safety profile of dead vaccines
with the higher efficiency of live ones.
NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | MARCH 2012 | 219
Third checkpoint
Pathogen or non-pathogen — detecting the
activity of virulence factors. Microorganisms
are traditionally classified as ‘pathogens’ or
‘non-pathogens’ based on their ability to
cause disease. This useful clinical classification
of virulence usually reflects the expression of
gene products known as virulence factors,
which facilitate the disruption or invasion of
host physical barriers and entry into sterile
tissues with the potential for systemic spread.
Thus, infection with virulent bacteria gravely
threatens host survival and necessitates
serious responses from the host (FIG. 1).
If we consider mucosal pathogens, many
of these — such as Legionella, Yersinia,
Salmonella and Helicobacter spp. — use
type II, type III or type IV secretion systems
(T2SS, T3SS and T4SS, respectively). These
secretion systems are large protein com-
plexes that transport virulence-associated
proteins (pathogen effectors) across epithe-
lial cell membranes and into the cytosol,
where they interfere with normal cellular
functions29. For example, the T4SS of
virulent Helicobacter pylori translocates the
CagA effector protein into gastric epithelial
cells, where it becomes phosphorylated and
binds to various host signalling proteins,
thereby disrupting their functions in actin
cytoskeleton rearrangements, tight junc-
tion formation, polarity, proliferation and
inflammation30. Yersinia spp. use a T3SS
to transport effector proteins called YOPs
(Yersinia outer-membrane proteins), which
also exert various immunomodulatory
effects on host cell function by inhibiting
phagocytic, MAPK and NF‑κB signalling
pathways31. Legionella pneumophila uses
its T2SS and T4SS for host cell attachment
and entry, and the T4SS for the secretion of
effector proteins32. The Dot/Icm T4SS facili-
tates the establishment of the L. pneumophila
vacuole, which provides an intracellular niche
for bacterial replication32. The Salmonella
enterica effectors SopE and SptP — which
are secreted via the Salmonella pathogen­
icity island 1 (SPI1) T3SS — have guanine
nucleotide-exchange factor (GEF) and
GTPase-activating protein (GAP) activities,
respectively. Thus, by controlling the
activity of RHO family GTPases, these
effector proteins can modulate the actin
cytoskeleton to facilitate bacterial invasion29.
Pathogen-mediated disruption of
host cell functions, however, does not go
unnoticed by the innate immune system,
and leads to the activation of various PRRs,
most notably NLRs. During infection by
H. pylori, peptidoglycan-derived muramyl
dipeptides that are delivered into the cytosol
© 2012 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES
via the T4SS are detected by nucleotide-
binding oligomerization domain protein 1
(NOD1) and trigger the activation of NF‑κB
and pro-inflammatory responses in epithe-
lial cells33. The Yersinia spp. T3SS triggers
caspase 1‑dependent pyroptosis and IL‑1β
secretion via both NLRP3 and NOD‑, LRR-
and CARD-containing 4 (NLRC4), and
the adaptor protein ASC34. Cytosolic trans­
location of flagellin derived from Salmonella
or Legionella spp. leads to its recognition
by neuronal apoptosis inhibitory protein 5
(NAIP5), which associates with NLRC4
to initiate inflammasome formation and
caspase 1 activation35,36. Besides detecting
PAMPs that are translocated directly into
the cytosol, NLRs have been suggested
to sense cytoskeletal rearrangements that
occur as a result of cytosolic bacterial
invasion. Reports have demonstrated the
recruitment of NLRs such as NOD1 to
actin-rich entry sites of Shigella flexneri, and
of the signalling adaptor ASC and caspase 1
to actin tails of motile intracellular Listeria
monocytogenes, although it remains poss­
ible that this recruitment is secondary to
the translocation of PAMPs at those sites
(reviewed in REF. 18).
Unlike TLRs, NLRs are exquisitely sensi-
tive in detecting the ‘activity’ of virulence
factors such as toxins or secretion systems,
although it has been difficult to ascertain
whether NLR activation is a result of direct
binding to toxins, the secretion machinery,
translocated effectors, or PAMPs. In the
absence of evidence demonstrating direct
binding of any of these components to NLRs,
indirect sensing of membrane damage (an
outcome commonly induced by virulence
factors) was thought to explain how various
toxins or secretion system effectors from dif-
ferent bacteria could all converge on the acti-
vation of a few NLRs37,38 (see also the ‘guard
hypothesis’ below). However, oligomeriza-
tion of NLRC4 with NAIP5 or NAIP2 was
recently shown to occur in direct response to
S. enterica flagellin and PrgJ (a rod protein of
the T3SS), respectively 35,36. Yeast two-hybrid
assays and co-immunoprecipitation demon-
strated interactions of NAIP5 with different
bacterial flagellins and of NAIP2 with PrgJ
and another T3SS rod protein, BsaK from
Burkholderia thailandensis 36, thus providing
evidence for direct interactions of NLRs with
PAMPs. Similarly, fluorescence resonance
energy transfer (FRET) and binding studies
have demonstrated direct recognition of
transfected double-stranded DNA (dsDNA)
by the cytosolic non-NLR DNA sensor
AIM2 (absent in melanoma 2), leading to
ASC-dependent caspase 1 cleavage, IL‑1β
PERSPECTIVES
release and pyroptosis39. Recognition of
cytosolic dsDNA by AIM2 was shown to be
essential for inflammasome activation dur-
ing infections with Francisella tularensis 40.
In contrast to these examples, mutant
strains of S. enterica lacking or expressing a
dysfunctional T3SS are not able to rapidly
induce high levels of IL‑1β secretion27,41,42.
The guard hypothesis and patterns of patho-
genesis. The ‘guard hypothesis’, which was
put forward by Dangl and Jones to describe
plant resistance to pathogens, suggests that
rather than binding to bacterial effectors
(or PAMPs) directly, plant R gene-encoded
NB–LRR (nucleotide-binding site plus
leucine-rich repeat) proteins sense patho-
gens indirectly by monitoring the integrity
of certain cellular proteins or processes
(guardees)96. When pathogen effectors target
the guardees, NB–LRR proteins are activated,
and this triggers the expression of plant
resistance genes. Two recent examples of this
type of mechanism have been demonstrated
in mammalian cells. Inhibition of protein
synthesis by L. pneumophila effectors that are
secreted through the Dot/Icm T4SS induces
prolonged activation of NF‑κB, presum-
ably owing to an inability to resynthesize
NF‑κB inhibitor (IκB; the guard), leading
to a unique transcriptional response that is
not elicited by avirulent Legionella spp.43. In
another example, modification of the host
RHO family GTPase RAC2 (the guardee) by
the E. coli effector protein cytotoxic necrotiz-
ing factor 1 (CNF1) specifically triggers the
activation of the immune deficiency (IMD)
pathway in flies and the receptor-interacting
protein (RIP) kinase pathway in mammalian
cells44. Together, these studies show that an
immunological distinction between patho-
gen and non-pathogen is made through
the detection of pathogen activities that are
designed to alter various aspects of normal
host cell physiology. Collectively, these
data, and those above, demonstrate that
pathogenic bacteria generally trigger more
robust immune responses than avirulent
strains, despite the fact that both contain very
similar or even identical PAMPs. Besides
the examples above, pathogens use numer-
ous other invasion strategies, many of which
share common themes. These themes can be
viewed as ‘patterns’, as proposed by Vance,
Isberg and Portnoy 18, by analogy to Janeway’s
microbial patterns (PAMPs)1. The authors
coined the term ‘patterns of pathogenesis’ to
propose that pattern recognition in the con-
text of an altered cellular physiology conveys
the pathogenicity status of microorganisms
to the host immune system18.
220 | MARCH 2012 | VOLUME 12 www.nature.com/reviews/immunol
Although primarily sensed through vita-
PAMPs27, bacterial viability as a prerequisite
for infectivity and pathogenicity could in itself
be viewed as a pattern of pathogenesis — but
only when it is detected in sterile tissues. The
reason for this clause is obvious: commensals
and non-pathogenic bacteria, although viable,
do not trigger pro-inflammatory responses as
long as they are confined to body surfaces or
specialized niches (more on this point under
the fourth and fifth checkpoints). These bac-
teria, which are classically viewed as harmless,
can cause severe infections following trans­
location to sterile tissues and should therefore
be regarded as a serious infectious threat.
Consequently, the immune system may have
developed a strategy to sense bacterial viabil-
ity, and thus infectivity, independently of
classical hallmarks of virulence, through the
detection of vita-PAMPs27.
Detecting microbial viability in sterile tis-
sues as a pattern of pathogenesis may also be
important during distinct stages of infections
with pathogenic bacteria such as S. enterica
subsp. enterica serovar Typhimurium.
Pathogenic S. Typhimurium represses SPI1
T3SS and flagellin expression after breaching
the intestinal epithelium and reaching mes-
enteric lymph nodes and the spleen45. Given
the potential for temporal and site-specific
variations in virulence factor expression, the
detection of vita-PAMPs and the simultane-
ous induction of multiple viability-associated
responses could potentially override
pathogen-mediated blockade of specific host
defence pathways. A redundancy in the roles
of NLRC4 and NLRP3 during infections
with S. Typhimurium has been proposed to
reflect an adaptation of the host to respond
to changes in the expression of bacterial
molecules such as flagellin46. Interestingly,
NLRP3 — which mediates the detection of
bacterial mRNA27 — is necessary for the host
to respond to mutants of S. Typhimurium
that lack a functional T3SS46. In light of our
findings summarized under the second
checkpoint 27, perhaps the recognition of
S. Typhimurium by NLRP3 during certain
phases of infection, when flagellin and T3SS
expression is repressed, centres around the
recognition of S. Typhimurium-derived
mRNA. In such a scenario, the detection of
vita-PAMPs, as invariant molecules essential
for pathogen viability, would be a fail-safe
strategy for sensing pathogens. Although
this idea requires rigorous testing, it is
notable that TRIF signalling — which is
crucial for the responses to bacterial mRNA
(FIG. 2) — is important in the response to
S. Typhimurium when the intestinal barrier
is bypassed by intraperitoneal injection27.
© 2012 Macmillan Publishers Limited. All rights reserved
Fourth checkpoint
Invasion or colonization — detecting fea-
tures of invasiveness. Viable microorganisms
that have gained access to sterile tissues
trigger robust immune responses, whereas
commensal microorganisms coexist with the
host in an energetically and metabolically
beneficial relationship. Host–microbiota
mutualism relies on fine immunological
equilibriums that allow commensal coloni-
zation while strictly avoiding the invasion
and infection of host tissues. The risk is that
many commensals (such as Enterococcus
faecalis and Streptococcus viridans) can
cause endocarditis, sepsis and other serious
diseases47. Others (including Staphylococcus
spp.) colonize body surfaces, such as the
skin, without causing overt pathologies
— as long as they remain restricted to the
surface47. Thus, the immune system must
rapidly sense and respond to the presence
of microorganisms in sterile tissues, while
tolerating harmless colonization of epithelial
surfaces.
How this delicate distinction is made on
a cellular level remains largely unknown.
Strict compartmentalization — through
epithelial tight junctions, mucus layers and
secreted antimicrobial peptides, which
together create a firm border between
commensals and the host — is thought to
be a main mechanism. Microbial breach
of these barriers is regarded as invasion.
There is evidence, however, for low-level
translocation of intestinal bacteria48 and for
the sampling of luminal contents by spe-
cialized mucosal phagocytes49, challenging
the concept of absolute separation of host
tissues from the microbiota. Moreover, the
presence of the commensal microbiota is
clearly sensed by the immune system and
shapes lymphogenesis and other key aspects
of mucosal and systemic immunity, as well
as tolerance mechanisms50,51. Accordingly,
although compartmentalization is likely to
be a major mechanism of protection, it does
not fully explain the discrimination between
microbial colonization and infection.
The detection of microbial features asso-
ciated with invasiveness may be one mecha-
nism by which an increased threat level is
detected and an antimicrobial response is
mobilized (FIG. 3). Some invasive forms of
microorganisms differ structurally from their
innocuous commensal counterparts, a case
best illustrated in fungi. Airborne A. fumiga-
tus conidia are covered with a ‘rodlet’ layer
consisting of the immunologically inert
hydrophobin RodA52. This allows the fungus
to remain dormant and undet­ected by host
defence mechanisms, while simultaneously
 PERSPECTIVES

preventing the host from mounting an Microbial invasion is coupled to the biology niches into sterile tissues, may all serve to
unnecessary response to ubiquitous inhaled of the host. Factors other than loss of com- trigger invasive infections by commensals.
fungal particles. Following germination, partmentalization can also trigger the poten- In the case of Staphylococcus spp., which
RodA is presumably degraded, exposing tial for aggressive behaviour by commensals. are colonizing bacteria with the potential
β‑1,3‑glucans (particularly at sites of hyphal Microenvironmental changes brought about to cause severe disease, the host actively
extension), which signal a potentially threat- by pregnancy, haematological malignancy, antagonizes the switch from the colonizing
ening scenario and trigger the activation of immunodeficiency, antibiotic treatment to the invasive form by inhibiting the agr
PRRs such as dectin 1 (REF. 53). Notably, live or chemotherapy, or the accidental reloca- quorum-sensing system, which is required
A. fumigatus conidia, but not killed conidia, tion of microorganisms from commensal for the induction of invasion-promoting
induce the production of pro-inflammatory
cytokines by phagocytes, and this correlates
with the ability of live conidia (but not dead Environmental factors
conidia) to undergo ‘swelling’, a process that Non-sterile (for example, immuno-
precedes hyphal formation53. suppression or
antibiotics) promote Pseudohyphal
Similarly, Candida albicans can switch C. albicans C. albicans transition to form of C. albicans
between colonizing yeast and invasive yeast invasive hyphal form β-1,3-glucans
hyphal forms54. Oral epithelial cells distin- Mucus
guish between hyphal and yeast forms of
C. albicans through an unidentified PRR,
causing a biphasic MAPK response. Only
hyphal forms induce the second phase of Sterile Mucosal Injured Hyphal form
MAPK activation and the subsequent pro- epithelium epithelium of C. albicans
duction of cytokines and antimicrobial
Unknown PRR TLR2 Dectin 1
peptides55. DCs — which are frequently
found in high numbers at mucosal sites
where C. albicans resides — can also
discriminate between the two forms, Conversion of yeast Hyphal
even in vitro in the absence of epithelial to hyphal form form
allows C. albicans to
barriers and compartmentalization56. They escape from the
produce different cytokines in response phagosome and Production of ROS
to hyphal versus yeast forms56, and hyphal promote inflamma- Phagosome and pro-inflammatory
some activation cytokines
forms but not yeast forms induce subse-
quent T helper 17 (TH17) cell responses57. Inflammasome
Interestingly, live C. albicans, but not killed activation
C. albicans, activates the NLRP3 inflamma- Phagocyte
some58,59, a property attributed to the ability
of live C. albicans to form filaments that IL-1β,
enable its escape from the phagolysosome IL-6 TNF,
CXCL2
into the cytosol, where NLRP3 resides.
The examples involving A. fumigatus
IL-1β, IL-6 and TGFβ Tissue inflammation
and C. albicans demonstrate that the TGFβ TH17 cell promote TH17 cell and leukocyte
responses to these facultative pathogens development recruitment
follow the same logic: an aggressive
response is generated only to the invasive
hyphal forms and not to the innocuous Figure 3 | Detecting features of invasiveness. Candida albicans yeast colonize mucosal epithelial
Nature Reviews | Immunology
surfaces, causing no pathology to the host organism. Germination — which is triggered by micro­
commensal forms. But does the detection
environmental factors such as immunosuppression or antibiotic treatment — leads to the transition
of microbial viability in the case of fungi of C. albicans from the innocuous yeast form to the invasive hyphal form. This transition is associated
have a role in measuring the threat level? with the exposure of β‑1,3‑glucans specifically at sites of hyphal extension, leading to recognition of
The recognition mechanism used for the C. albicans by the C-type lectin receptor dectin 1. Dectin 1 clustering at the phagocytic synapse
detection of labile prokaryotic mRNA obvi- induces the production not only of pro-inflammatory cytokines, but also of microbicidal reactive
ously cannot be used. Instead, the immune oxygen species (ROS). Signalling from dectin 1 and Toll-like receptor 2 (TLR2) synergistically leads to
system may rely on the detection of a differ- the production of pro-inflammatory cytokines, such as tumour necrosis factor (TNF) and CXC-
ent proxy of viability, such as a morphology chemokine ligand 2 (CXCL2; also known as MIP2). C. albicans yeast may also translocate across an
associated with the invasive hyphal form, as injured epithelium into the underlying tissue, where they encounter phagocytic cells and engage
the switch to this form can only be carried an undefined pattern-recognition receptor (PRR). Phagocytes promptly internalize these yeast into
phagosomes, from which only viable yeast can escape by transitioning into the hyphal form. This process
out by fungi that are alive. Indeed, RodA-
might lead to phagosomal membrane destabilization and subsequent activation of the NOD‑, LRR- and
deficient A. fumigatus mutants induce pyrin domain-containing 3 (NLRP3) inflammasome, resulting in caspase 1‑dependent processing of
immune activation52 and, conversely, pro-interleukin‑1β (pro-IL‑1β) into its mature secreted form. The translocation of yeast into sterile
engineered C. albicans mutants that are tissues may itself be associated with germination. The cytokines that are produced in response to the
‘locked’ in the yeast form fail to activate recognition of hyphal forms, but not yeast forms, of C. albicans include IL‑6 and IL‑1β, which together
the inflammasome57 and are avirulent 60. with transforming growth factor-β (TGFβ) can induce the development of T helper 17 (TH17) cells.

NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | MARCH 2012 | 221

© 2012 Macmillan Publishers Limited. All rights reserved

PERSPECTIVES
genes61. Thus, although intact physical bar-
riers are essential to keep the microbiota
at bay, the immune system is crucial for
border protection and the maintenance
of host–commensal mutualism. Indeed,
immunodeficiencies often predispose to
rare infections with harmless colonizers16,47.
These examples illustrate how the potential
for some commensals to become invasive
is tightly coupled to the biology of the host
organism. Such behaviour of commensals
also illustrates the nature of the ‘facultative’
or ‘opportunistic’ pathogens, categories that
exist between ‘pathogen’ and ‘commensal’.
Fifth checkpoint
Regulatory or inflammatory responses —
a matter of location. Although the check-
points mentioned above help to gauge the
microbial threat on a cellular level, microbial
infections must ultimately be viewed in the
broader context of the specific physiologies
of the infected tissue or organ. Consequently,
correlations between the microbial threat
and the resultant immune response, as
simplified in FIG. 1, are subject to strong
tissue-specific regulation (FIG. 4). The levels
of exposure to microorganisms and micro-
bial products, as well as the numbers and
compositions of resident immune cells, vary
greatly among different tissues. Although
some tissues, such as the brain or the lung,
must remain sterile to ensure proper organ
function, the intestinal mucosa relies on
a harmonious relationship with the com-
mensal microbiota21. Accordingly, immune
responses to microorganisms vary greatly
depending on the tissue and its local micro-
environment; this is best exemplified by the
differential responses of DCs from different
anatomical sites. For example, Peyer’s patch
DCs produce higher levels of IL‑10 than
splenic DCs and skew CD4+ T cell differenti-
ation towards the TH2 cell lineage (reviewed
in REF. 62). Similarly, small intestinal lamina
propria DCs are better than splenic DCs at
instructing the differentiation of forkhead
box P3 (FOXP3)-expressing regulatory T
(TReg) cells62. Furthermore, a recent study has
described how microenvironments in the
spleen and the intestine differentially deter-
mine the requirements for IL‑6 in TH17 cell
differentiation63. The nature of the immune
response to microorganisms is also affected
by the simultaneous recognition of molecules
signifying tissue damage. For example, micro-
bial pathogens that induce the apoptosis of
infected target cells preferentially trigger
a TH17 cell response. In this case, innate
recog­nition of apoptotic cell ligands (such as
phosphatidylserine), together with PAMPs,
222 | MARCH 2012 | VOLUME 12 www.nature.com/reviews/immunol
induces the synthesis of pro-inflammatory
IL‑6 and IL‑23 as well as anti-inflammatory
transforming growth factor-β (TGFβ),
leading to TH17 cell differentiation64.
Within the intestinal tract, phago-
cytes express molecules that negatively
regulate TLR signalling 65. This may explain
their hyporesponsiveness to PAMPs66,67,
a phenomenon that has been referred to
as inflammatory anergy 67 and that may
contribute to intestinal homeostasis given
the high amounts of microbial ligands at
this site. Tissue-derived factors such as
IL‑10, TGFβ, thymic stromal lympho­
poietin and retinoic acid further shape this
tolerogenic environment, favouring the
development of FOXP3+ TReg cells62,68. In
addition, some commensals induce regula-
tory immune responses. For example, com-
mensals such as E. coli strain Nissle 1917
have anti-inflammatory properties that are
exploited to maintain remission of ulcera-
tive colitis69. Moreover, various species of
Lactobacillus 70,71, Clostridium clusters IV and
XIVa72 and Bacteroides fragilis 73 induce TReg
cell development in the intestinal lamina
propria. Although the underlying mecha-
nisms remain poorly understood, in one
study B. fragilis polysaccharide A (PSA)
increased the frequency and immuno­
suppressive functions of FOXP3+ TReg cells
in a TLR2‑dependent manner 73. In another
example, colonization of the mouse small
intestine by segmented filamentous bacteria
(SFB; members of the order Clostridiales74)
induced the development of TH17 cells,
which promote resistance to the rodent
pathogen Citrobacter rodentium75. Therefore,
despite the fact that commensal micro­
organisms are alive, their sequestration
behind epithelial barriers and their recogni-
tion within the context of tissue-specific
cues, combined with the possible expression
of host-adapted microbial structures (such as
PSA) that engender symbiosis73, collectively
promote tolerance rather than immunity to
these viable microorganisms.
The importance of cell specificity. Cell
specificity is another element that greatly
affects the response to commensal micro-
organisms. Commensals such as SFB and
B. fragilis are intimately associated with
intestinal epithelial cells73,75, and SFB trig-
ger an increase in the expression of genes
associated with immune system pathways
and antimicrobial defence75. Paneth cells
(a population of specialized intestinal
epithelial cells) respond to bacteria by
making antimicrobial peptides that help
to maintain the species balance in the
© 2012 Macmillan Publishers Limited. All rights reserved
steady-state gut bacterial communities
(reviewed in REF. 76). Subsets of intestinal
DCs mediate immune tolerance towards
commensals by inducing tightly controlled
immune responses that are both protective
against the gut microbiota and regulatory
in nature. Unlike mucosal macrophages,
which readily kill phagocytosed commen-
sals, Peyer’s patch DCs retain commensals
such as Enterobacter cloacae for several
days and carry them to mesenteric lymph
nodes, where they induce IgA secretion
from B cells77,78. In humans, intestinal IgA is
not only directed against pathogens (such
as S. Typhimurium), but is also specific for
an extensive range of commensal bacteria,
showing that the recognition of com-
mensals in the intestinal mucosa induces
protective responses79. Recent evidence
demonstrates that specific colonic TReg cells
develop in response to various commensal
bacteria, including a previously unchar-
acterized Clostridiales species as well as
species in the genuses Parabacteroides and
Bacteroides 80. Collectively, these studies
demonstrate that, like pathogens, intestinal
commensals are recognized by the immune
system but, instead of evoking an inflam-
matory response, they induce a regulatory
immune response that maintains intestinal
homeostasis. In contrast to this regulatory
response that occurs under steady-state
conditions, newly recruited phagocytes that
migrate to the intestinal mucosa during
infections or other inflammatory condi-
tions display a pro-inflammatory pheno-
type and respond robustly to PAMPs81,
indicating that tissue-specific immune
regulation can be overcome by certain sig-
nals. This pathway is important for host
defence against intestinal pathogens; how-
ever, chronic activation is dangerous and can
trigger the development of inflammatory
bowel disease.
The host microenvironment shapes bacterial
gene expression. The host immune system
responds to specific microbial signals and,
similarly, gene expression in bacteria is
tightly coupled to host microenviron­mental
cues. This is best illustrated by the tight
regulation of virulence factor expression
in various pathogenic bacteria to ensure
timely deployment 82. For S. Typhimurium,
the SPI1 T3SS is activated following contact
with an epithelial cell to ensure that effec-
tors are secreted into the cytosol, whereas
the SPI2 T3SS is activated in intracellular
compartments to facilitate S. Typhimurium
replication29. During the systemic phase of
salmonellosis, the expression of flagellin is
 PERSPECTIVES

Microbial
colonization Facultative
External Commensal Pathogen
Commensal pathogen
environment
Sampling

Epithelium

Low
security
Epithelial Immuno- risk
Tissue Expression of
surface suppression or
injury virulence factors
antibiotic
treatment
Secondary
Low risk translocation Invasion Invasion
Recognition of PAMPs
and vita-PAMPs from
commensals promotes Pathogen may Medium
regulatory immune repress virulence security
responses (such as IgA factors but can risk
and TReg cell induction) be detected (level
Sterile via PAMPs and influenced
tissue vita-PAMPs by location)
Detection of PAMPs
and vita-PAMPs

Medium risk Immune system detects Medium risk

Inflammatory response to features of invasiveness, Inflammatory
restore host–microorganism activity of virulence factors, responses to
mutualism and tissue PAMPs and vita-PAMPs clear pathogen
homeostasis

Systemic Blood endothelium High risk

circulation Heightened
Detection of PAMPs or High
inflammatory security
vita-PAMPs signals systemic response, which can
spread of infection risk
lead to septic shock

Figure 4 | The class and context of pattern recognition indicates the
microbial threat level and dictates the nature and magnitude of the
immune response. This schematic model is composed of multiple parts
identified by colour. The figure denotes different host tissues, with surface
epithelia at the top (yellow), sterile tissue in the middle (orange) and the
systemic circulation at the bottom (red). Blue denotes the microbial world
and green boxes show the immune responses triggered. The colours were
inspired by the US Department of Homeland Security’s former terrorism
threat level system, in which yellow meant elevated significant risk, orange
meant high risk and red meant severe risk. At each level, several check-
points must be bypassed before the microorganism can access the sys-
temic circulation. Note the inverse correlation between the number of
immune checkpoints that need to be bypassed and the security level of the
host tissue. Commensal-derived pathogen-associated molecular patterns
(PAMPs) and vita-PAMPs drive regulatory effector immune functions that
restore host–commensal mutualism in the intestinal mucosa. PAMPs from
commensals that accidentally translocate into sterile tissues trigger
inflammatory responses that promote microbial clearance.
Nature ReviewsSimultaneous
| Immunology
detection of vita-PAMPs signifies viability and warrants a heightened
response. Detection of PAMPs within the context of invasion or virulence
factor activity only occurs in response to viable pathogens (or viable facul-
tative pathogens) and leads to a heightened inflammatory response. Once
in sterile tissues, pathogens might repress their expression of virulence
factors, and their detection as a significant microbial threat might rely pri-
marily on vita-PAMPs. Detection of PAMPs alone, without vita-PAMPs,
might signify a successful response. Detection of either PAMPs or vita-
PAMPs in the circulation would signal systemic spread, which poses a
severe threat to the host. A heightened systemic inflammatory response
would be warranted, although such a response, if uncontrolled, could lead
to septic shock. Mobilization of adaptive immune responses is not
depicted, but their magnitude and longevity is expected to increase with
heightened innate responses. TReg, regulatory T.

repressed in an effort to evade recognition
by NAIP5–NLRC4 (REF. 45). Engineered
S. Typhimurium mutants rendered incapable
of switching off flagellin expression are
severely attenuated owing to prompt acti-
vation of the NLRC4 inflammasome in
infected macrophages83. In another example,
expression of the Yersinia pseudotubercu-
losis effector protein YopJ — which blocks
TNF and IL‑10 production and induces
the apoptosis of macrophages — is crucial
during the intestinal stage of an infection
with Y. pseudotuberculosis 84, but dispensable
during the systemic phase of infection85,86.
Instead, other effectors from pathogenic
Yersinia spp. (such as YopK, which has been
shown to suppress inflammasome activa-
tion) could be induced during systemic
infection34. Pathogens and commensals alike
receive host signals and respond to them in
a way that enables them to survive in their
respective niches. In the case of pathogens,
modulating virulence factor expression in
accordance with cues received from host tis-
sues facilitates either the invasion of physical
barriers or the evasion of immune detection
depending on the location within the host.
This in turn directly influences the ability of
the immune system to accurately assess the
level of microbial threat.

NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | MARCH 2012 | 223

© 2012 Macmillan Publishers Limited. All rights reserved

PERSPECTIVES
Local versus systemic infection. Besides
the tissue-specific regulation of immune
responses to local infections, it is crucial to
respond aggressively to the systemic spread
of microorganisms, as this could quickly
kill the host. If one defines the detection of
microorganisms or microbial components in
the blood circulation as a definite indicator
of systemic spread, one would expect that
blood-derived phagocytes would respond
more vigorously to microbial stimulation
than their tissue-resident counterparts.
Indeed, monocytes readily release the
highly pro-inflammatory cytokine IL‑1β in
response to soluble LPS, even in the absence
of secondary signals such as ATP, owing
to their expression of constitutively active
caspase 1 (REF. 87). Given the serious con-
sequences of IL‑1β release88, its activation
is generally reserved for situations of acute
threat. By contrast, inflammasome activa-
tion and IL‑1β release by tissue-resident
macrophages occurs only in response to an
active infection (that is, following detection
of vita-PAMPs or virulence factors), and not
to LPS alone. Thus, tissue-specific (mucosal
versus non-mucosal, sterile versus non-
sterile) and compartment-specific (local
versus systemic) regulations are as important
as pathogen-specific factors in dictating
antimicrobial responses (FIG. 4).
Concluding remarks
In delineating pathways and checkpoints
that enable the immune system to measure
the microbial threat in order to invoke the
appropriate responses, we cannot help but
be reminded of the danger theory, proposed
by Polly Matzinger 89, which stood in stark
contrast with Janeway’s theory of pattern
recognition in self–non-self discrimination1.
The danger theory posited that the primary
function of the immune system is to sense
and respond to danger in the form of signals
derived from damaged tissue, regardless of
whether this damage is caused by an infec-
tious challenge or a sterile, non-infectious
insult 89. Certain forms of cell death that
are associated with pathological situations
(resulting from tumour chemotherapy or
necrosis, for example) are highly inflamma-
tory in the apparent absence of microbial
components90. Apoptosis, on the other hand,
induces an immune response geared towards
tissue repair 90. In the context of microbial
recognition, the presence of live micro­
organisms, pathogenic or non-pathogenic,
in sterile tissues or of microorganisms that
cause damage via the activity of virulence
factors would, as we have discussed, repre-
sent greater dangers to the immune system
224 | MARCH 2012 | VOLUME 12 www.nature.com/reviews/immunol
than PAMPs alone. Perhaps the checkpoints
we define here bring together the views of
both Janeway and Matzinger in not only
determining that a microorganism is present
(Janeway), but also assessing the level of
danger it poses (Matzinger). It is important
to emphasize that, in the absence of dan-
ger, PAMPs derived from commensals or
death by apoptosis still induce an immune
response, but of a nature that fosters tissue
homeostasis and regeneration rather than
inflammation.
One final note: because immune
responses and pathogens often co-evolve,
we predict that a major strategy for micro-
bial immune evasion might be subversion
of some of the proposed checkpoints (which
by no means constitute an exhaustive list),
leading the host to underestimate the infec-
tious threat. Many viruses and pathogenic
bacteria are known to specifically target
host innate immune pathways to avoid
inflammatory responses34, especially in the
establishment phase of infection. In addi-
tion, dysregulation of this system caused by
inadequate risk assessments at any of the
checkpoints could lead to chronic inflam-
matory diseases, such as Crohn’s disease.
Understanding the exact mechanisms that
govern these decision-making processes
will require further efforts that are well
worth their while, as they are sure to yield
valuable new targets for improving vaccines
or treating inflammatory diseases.
J. Magarian Blander is at the Mount Sinai School of
Medicine, Immunology Institute, Department of
Medicine, 1425 Madison Avenue, New York,
New York 10029, USA.
Leif E. Sander is at the Charité University Hospital
Berlin, Department of Infectious Diseases and
Pulmonary Medicine, Charitéplatz 1,
10117 Berlin, Germany.
e‑mails: Julie.blander@mssm.edu;
leif-erik.sander@charite.de
doi:10.1038/nri3167
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Acknowledgements
We thank R. Medzhitov for critical reading of the manuscript.
J.M.B. is a Burroughs Wellcome Investigator in the pathogen-
esis of infectious disease, and is also supported by grants
from the US National Institute of Allergy and Infectious
Diseases, the US National Institute of Diabetes and Digestive
and Kidney Diseases, the American Cancer Society and the
Hirschl Trust Fund. L.E.S. is supported by the German
Research Foundation (DFG).
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
J. Magarian Blander’s homepage: http://www.mssm.edu/
research/programs/innate-immunity-research-program
Leif E. Sander’s homepage: http://www.charite.de/inflab/
index.php?id=170&L=0%252C
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