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Innate Immunity Depends on
Toll-Like Receptors
From flies to mammals, these proteins provide a first-line defense and
are implicated in infectious and autoimmune diseases
William Check
omething about the invertebrate Toll
Center in Dallas and now at the Scripps Resystem and the mammalian toll-like
search Institute in La Jolla, Calif., and his colreceptors (TLRs) inspires scientific
laborators found similar receptors in mice. Early
insight and intellectual excitement. Colon, they identified a receptor, designated TLR4,
leagues recount how biologist Christhat specifically recognizes and binds lipopolytiane Nusslein-Volhard exclaimed “toll”—
saccharides (LPS) from gram-negative bacteria.
German slang, meaning “fantastic”—while
Subsequently, researchers from several laboramarveling in 1980 over a Drosophila mutant
tories revealed a family of (now) 10 mammalian
that proved fruitful for explaining fly embryoTLRs that appear to be involved exclusively in
genesis. The name stuck with that
the innate immune response but
mutant, and her efforts after that
not in development.
“Eureka” moment led NussleinSuch TLRs are “simply put, the
Efforts to
Volhard to a Nobel prize in 1995,
main
way that we [mammals] see
understand the
says Jean-Marc Reichhart of the
the
microbial
world,” Beutler says.
critical place of
Louis Pasteur University in Stras“They are the initial gateway to
TLRs in
bourg, France.
all— or nearly all—mammalian remammalian
The prototypic Toll-1 receptor
sponses to microbes.” TLRs can
host defense
identified by Nusslein-Volhard
activate innate defenses, mainly inand another eight Toll proteins
flammation, before adaptive imare giving rise
serve developmental functions in
mune responses come into play.
to hopes for
fruit flies. Additionally, Toll-1
Realizing that the TLR4 gene
novel
plays an important role in host despecifically recognizes LPS was “a
therapeutic
fenses against microorganisms, acshocking finding,” Beutler recalls.
applications.
cording to Reichhart and his colA mere few years ago, he and other
leagues, including Jules Hoffman.
biologists thought that mouse
“In invertebrates, Toll is one of the
TLRs served developmental funcmajor systems for protection and the first line of
tions. Moreover, he says, “This finding told us
defense against pathogens,” Reichhart says.
that in mice a Toll homolog is required for
“This is fascinating. How could these two funcresponse to gram-negative infection, while in
tions have evolved under different pressures at
flies Toll-1 is required for response to fungal
two stages of fly life? Which was first? These are
infection. We knew that this was more than a
difficult questions to answer.”
coincidence.” Other properties of the mouse
from which the mutant TLR4 gene was cloned
suggested that TLR4 senses LPS and mediates
Toll-Like Components Important for
host responses to this bacterial endotoxin.
Innate Immunity in Mammals
“That was really electrifying,” he exclaims. “I
In 1998, immunologist Bruce Beutler, then at
think it was the single most exciting moment of
the University of Texas Southwestern Medical
my scientific career.”
S
William Check is a
science writer in
Wilmette, Ill.
Volume 70, Number 7, 2004 / ASM News Y 317
Continuing efforts to understand the critical
place of TLRs in mammalian host defense are
giving rise to hopes for novel therapeutic applications. “I think the possibilities are very large,”
says Beutler. “And they will be even larger if it
can be demonstrated that endogenous types of
inflammatory disease, such as Crohn’s disease
or ankylosing spondylitis, that involve overproduction of tumor necrosis factor-alpha (TNF-␣)
are mediated primarily by activation of TLR
pathways—although this is not known yet.”
“TLRs will become important targets for
pharmaceutical development,” agrees David
Persing, vice president of Discovery Research at
Corixa Corporation, Seattle, Wash. “They are
relatively new targets in terms of recognition of
their potential importance, and are involved in
diseases ranging from infections to inflammatory diseases to autoimmune diseases and allergic disorders.” While scientists have been studying the adaptive immune response for several
decades, their appreciation of the importance of
innate immunity took shape only during the past
few years, Persing says. “The innate response
can in many ways dictate the tenor of the adaptive response.”
Early Progress in Search for Simple Host
Defenses in Studies of Fruit Flies
Contemporary investigators siezed on the centrality of Toll-1 and TLRs to host defense in part
because of efforts by their predecessors to answer an age-old question, one that is implicit in
a verse by 18th-century Irish cleric and satirist
Jonathan Swift. About the time that Antonie van
Leeuwenhoek was peering through his microscopes to see “very little living animalcules,”
Swift wrote:
So, naturalists observe, a flea
Hath smaller fleas that on him prey;
And these have smaller still to bite ’em
And so proceed ad infinitum.
Swift humorously framed a simple fact—pathogens are ubiquitous. But how do organisms recognize and combat infections?
When flies are infected by fungi, they effectively dose themselves with an antimicrobial
metabolite, called drosomycin. But flies do not
have diagnostic capabilities for determining
whether they are infected and when to produce
318 Y ASM News / Volume 70, Number 7, 2004
internal antimicrobial defenses. How does that
happen? And is anything similar about pathogen response mechanisms among other species?
Similarities between Toll-1 and TLRs establish that link, and they are part of a more complex system for discriminating nonself from self.
Indeed, the innate immune system uses genetically encoded receptors to respond to conserved
microbial structures. Toll-1 in flies and the 10
human TLRs alert their respective hosts to infections.
Reichhart and Hoffman recognized the antimicrobial function of Toll-1 while searching for
the control mechanisms of innate immunity in
Drosophila. “We asked very simple questions at
the beginning,” Reichhart says. “When you
challenge the insect with a microbe, it responds
by expressing antimicrobial peptides. So what
are those peptides?” They isolated the peptides,
then the genes encoding the peptides. Next they
asked, what is important about the promoters of
the peptide genes? Response elements in these
genes resemble those in mammals that bind the
transcription factor, called nuclear factor-␬ B
(NF␬B), following activation by interleukin-1
(IL-1). A sequence search turned up only one
NF␬B-like transcription factor in Drosophila, a
protein called dorsal that is involved in early
embryogenesis. A whole pathway was already
known that triggers dorsal activity in the embryo.
“The central key molecule in this signaling
pathway that leads to dorsal is the transmembrane receptor Toll,” Reichhart says. In 1996 he
and Hoffman and their colleagues reported that
“mutations in the Toll signaling pathway dramatically reduce survival after fungal infection.” They later showed that Toll also is involved in sensing gram-positive bacteria.
Comparable but More Complex
Components in Mammals
When Beutler in Dallas set out to find the mammalian gene responsible for sensing LPS, he soon
focused on two mutant mouse strains. “It was
known since 1965 that these mice were unable
to survive challenge with gram-negative bacteria, but that their responses to things other than
endotoxin were normal,” he explains. By positional mapping, he and his collaborators identified a critical region in which the best candidate
FIGURE
1
The TLRs (of which there are 10 in humans) signal via interactions with TIR adapter proteins, of which five are known. Four are shown here;
the function of the fifth (SARM) is not known yet. TLR4 (the LPS receptor) uses all four TIR adapters. TLR2 uses two; TLRs 3 and 9 each
use one. It is likely that they are dimeric as shown. The usual pathway used is via IRAK4/IRAK, TRAF6, TAK1, and numerous other kinases,
to activate NF-␬B. But Trif/Trif and Trif/Tram act to activate IRF-3 (Trif can also activate TRAF6). This is the basis of so-called
“MyD88-independent signaling.” Note that TLR3 has no requirement for MyD88. In a manner not yet understood, TLR9 can also activate
synthesis of type I interferons. Hence both TLRs 3 and 9 are very important for antiviral defense. MyD88 and Trif adapters are as well. It
is at the adapter level that antiviral and antibacterial pathways converge.
was the gene encoding mouse TLR4. Five other
mouse genes that are also similar to TLR had
been identified in EST databases. “However, no
one knew what any of them did until our work
was published,” Beutler says. “The fact that
TLR4 sensed LPS immediately suggested that
other TLRs might sense other molecules unique
to microbes.”
The following year, Shizuo Akira of Osaka
University and his collaborators reported that
TLR2 knockout mice are hyporesponsive to
gram-positive bacterial cell walls, including to
peptidoglycan from Staphylococcus aureus.
Molecular recognition functions have since been
assigned to other TLRs, including TLR1 to lipopeptide; TLR3 to double-stranded RNA
(dsRNA); TLR5 to flagellin; TLR6 to zymosan
and lipopeptide; and TLR9 to CpG (unmethylated DNA).
TLRs are part of the larger superfamily of
Toll-IL-1 receptors (TIRs). “Mammals have
two kinds of receptors with TIR domains,” Beutler says, “TLRs and receptors for the cytokines
IL-1 and IL-18, as well as some orphan receptors.”
All TIRs signal by way of TIR domain adapters,
such as MyD88 and Trif (see figure). These systems are very ancient. Thus, an ancestral (prevertebrate) TLR may have adopted a pro-inflammatory function as long ago as 500 million years,
Beutler and his colleagues speculate.
Drosophila and mammalian innate immune
sensors achieve specificity in different ways. In
Volume 70, Number 7, 2004 / ASM News Y 319
mice, LPS binds to a cell-surface molecule, CD14.
Direct exposure of TLR4 to LPS then activates
intracellular signaling. “In a CD14 knockout
mouse, you can bypass the need for CD14 by
increasing LPS concentration,” Beutler says.
“Based on this fact, together with genetic complementation studies showing that TLR4 confers species-dependent ligand recognition, we believe that
TLR4 has direct contact with LPS.”
However, in Drosophila, Toll does not directly engage microbial molecules, but is activated through a cleaved form of the cytokine
peptide called spaetzle. Specificity for gram-positive bacteria occurs prior to spaetzle being
cleaved, in a peptide that Reichhart and Hoffman named semmelweis.
Akira distinguishes two phylogenetically different groups of TLRs. The first includes TLR2
and TLR4, which are expressed on cell surfaces
and recognize microbial structures in the blood.
The second consists of TLR7, TLR8, and TLR9,
which are present in endosomes within the cytoplasm. “Activation of newer receptors takes
place after pathogens are phagocytosed and digested in the phago/lysosome,” Akira says.
Once Toll-1 or any of the TLRs is activated,
intracellular signaling pathways are very close between insects and mammals, according to Reichhart. In mammals, TLR signaling involves activation of one or more of the “TIR adapter proteins.”
The adapters relevant to TLR signaling are known
as MyD88, Tirap, Trif, and Tram. Most TLRs act
through MyD88 alone or through both MyD88
and Tirap, which leads to production of inflammatory cytokines, chiefly TNF-␣.
By screening thousands of mice with induced
mutations, Beutler’s group recently identified a
second, independent pathway for TLR4 that operates through Trif and Tram and leads to synthesis of type I interferons, upregulates costimulatory
molecules, and induces nitric oxide synthase
(iNOS). TLR3 is activated by dsRNA through Trif
alone, whereas mice with mutations in the Trif
gene fail to produce type I interferons in response
to viral infection. Because dsRNA serves as an
adjuvant in Trif mutants, there may be a separate
pathway for dsRNA sensing.
Links Being Found between Adaptive and
Innate Immune Systems
The Trif/Tram pathway provides a direct link
between TLR4 activation and adaptive immu-
320 Y ASM News / Volume 70, Number 7, 2004
nity. Although purified LPS is a strong adjuvant,
its effects are abolished in the mutant mouse
strains that Beutler worked with, suggesting that
both the inflammatory and the adjuvant effects
of LPS flow through TLR4. Finding that mice
with mutations in Trif/Tram lack the adjuvant
effect of LPS provides a specific post-TLR4 route
for this effect. “As one would expect, adjuvants
are sensed through TLRs,” Beutler says.
Combining the MyD88/Tirap and Trif/Tram
pathways provides a biochemical basis for how
adjuvants work. Activating the adaptive immune system requires antigen-presenting cells—
macrophages and dendritic cells—to express costimulatory molecules such as CD80, CD86,
and CD40, and proinflammatory cytokines.
When TLR4 recognizes LPS on the surface of
macrophages or dendritic cells, it leads to production of cytokines via MyD88/Tirap and costimulatory molecules via Trif/Tram, providing
both components for activating T helper lymphocytes of the adaptive immune system.
Seeing that the innate and adaptive immune
responses are so tightly linked answers a longstanding question, according to Reichhart.
“There was always a question of how an adaptive immune system could defend us if it were
alone, because adaptive immunity depends on
the multiplication of host cells with a generation
time of least 12 hours, whereas microbes can
divide every 20 minutes,” he says. To cover this
lag, the rapidly reactive innate system responds
nearly immediately to infectious agents, protecting the host until the slower adaptive system
kicks in and eventually also makes memory cells
for long-term responses.
Some evidence suggests that the initial innate
immune process influences the type of adaptive
immune response that is generated. “When naive T helper cells are presented with antigens by
antigen-presenting cells, they differentiate into
two subsets, T helper 1 (Th1) and T helper 2
(Th2),” Akira says. “Th1 cells secrete interferon-␥, which promote mainly cellular immunity,
whereas Th2 cells produce IL-4, IL-5, IL-10, and
IL-13, and promote mainly humoral immunity.” Akira says that autoimmune diseases such
as Crohn’s disease and multiple sclerosis are
associated with an abnormally strong Th1 response, whereas allergic diseases seem to involve
an abnormally strong Th2 response.
Which limb of adaptive imunity predominates may be modulated by the innate immune
response. For instance, Akira says, MyD88-deficient mice are skewed toward a Th2 response.
Beutler suggests that perhaps this pattern means
that the default pathway for adaptive immune
development, absent MyD88 signaling, is the
Th2 pathway. Reichhart notes that activation of
TLR4 by LPS stimulates Th1 activity, while
activating TLR2 through schistosomal egg antigen activates Th2 cells.
Therapeutic Possibilities for Infectious
and Autoimmune Diseases
A number of therapeutic possibilities arise from
these basic findings. It seems likely that the TLR
system links diseases from infection to arthritis
to allergy, promising a broad spectrum of therapeutic benefits.
For example, Beutler points to two novel approaches for combating infectious diseases.
First, he says, blocking TLR signaling with LPS
antagonists might prevent sepsis syndrome. Second, people with particular mutations in TLR
signaling pathways who develop severe infections might benefit from drugs that would enable them to bypass those defective steps.
For instance, one patient with recurrent bacterial infections and whose leukocytes were hyporesponsive to LPS and IL-1 in vitro carries a
defective gene for IRAK4 (interleukin-1 receptor-associated kinase). As well, three children
from Saudi Arabia with an inherited IRAK4
deficiency are unusually susceptible to pyogenic
bacterial infections. And among several patients
with systemic meningococcal disease, the TLR4
locus has a highly significant excess of rare coding changes, according to Beutler and his collaborators. Potentially, such patients could be
treated with specific agents to help them overcome these deficiencies.
Beutler also sees “huge therapeutic possibilities” if endogenous stimuli of TLRs are part of
the etiology of autoimmune or inflammatory
diseases. “There may be both endogenous and
exogenous sources of ligands for TLRs,” Persing
of Corixa says. “We are just beginning to understand the full range of those ligands.” He cites
work by Akira suggesting that TLR4 may play
an important role in inflammatory bowel disease. “The idea is that a change in gut permeability increases access of microbial components
such as LPS, which further drives the inflammatory process,” he says. Blocking the proinflam-
matory effects of those components at the level
of the TLR might reduce inflammation.
Additionally, Persing and his colleagues are
working with a detoxified lipid A derivative,
monophosphoryl lipid A (MLA), which has
been tested in more than 40,000 human volunteers as a vaccine adjuvant, and several synthetic
lipid A mimetics called aminoalkyl glucosaminide 4-phosphates (AGPs). All appear to operate
through TLR4, and changes in structure of the
AGPs lead to drastic differences in TLR4 signaling capacity. When mixed with a vaccine protein, these compounds dramatically accelerate
and augment the immune response to that protein.
“Both MLA and AGPs, when delivered to the
airways via aerosol, produce a rather profound
level of resistance against infectious challenge
with influenza virus and Listeria,” Persing says.
“We could essentially protect animals against 5
to 6 LD50’s of both infectious agents. We think
the intranasal approach works with LPS-type
compounds because TLR4 is one of the few
TLRs that appear to be expressed throughout
the respiratory tract.” Certain structures of
AGPs appear to be a good fit for human TLR4;
in mice, these compounds promote protective
innate immune responses in the airways
against infectious challenge for up to a week.
Dosing at weekly intervals maintains protection, but protection for a week at a time theoretically allows the compounds to be used on an
“as-needed” basis.
Sponsored by a U.S. Army grant, Persing and
his colleagues are now preparing one of the
AGPs for a dose-escalation study in humans in
2005. “Even before the anthrax scare, the military was interested in broad measures for protection of the airways against infectious challenge,” Persing says. “Military planners believe
that the main portal of entry for bioweapons
will be the airways and mucosal route.” Such a
threat could come from weaponized Bacillus
anthracis or other pathogens such as Francisella
or Yersinia pestis. Meanwhile, perhaps more
imminent threats arise from natural “bioweapons,” including the severe acute respiratory syndrome (SARS) coronavirus and avian strains of
influenza.
Persing recently was awarded a substantial
five-year contract by the National Institute of
Allergy and Infectious Diseases to develop agonists of TLRs that can be used to develop rapidacting vaccines. He and his colleagues found
Volume 70, Number 7, 2004 / ASM News Y 321
that delivering an AGP along with an influenza
virus antigen intranasally produces a strong adjuvant effect along with a mucosal IgA immune
response and a systemic IgG response. In the
meantime, while the vaccine is taking effect, the
AGP component of the vaccine provides shortterm protection against viral infection. By exploiting the protective features of the innate and
adaptive responses, the researchers designed a
dosing schedule to protect against an influenza
viral challenge within a few hours of the first dose.
In separate studies, Persing’s group is using
these compounds to suppress allergic responses
to ragweed. “By manipulating the innate immune response in the context of an allergen
challenge,” he says, “we can dramatically reduce the response to the allergen so it is no
longer characterized by the allergic phenotype.”
Broad Effects, Ancient Lineage
The toll-like receptors link a broad spectrum of
species. When 17th-century poet and clergyman
John Donne wrote, “Therefore never send to ask
for whom the bell tolls, it tolls for thee,” he was
emphasizing kinship within the human species.
Donne could not have imagined a protective
system that tolls not only for humans, but for all
mammals and invertebrates as well. The Toll/
TLR system offers yet another proof of relatedness across the animal kingdom, as well as providing insights into the workings of evolution.
“It is amazing when you understand this homology for the first time,” Reichhart says. “As
biologists, however, we are not so surprised that
things are going so far back, 600 million years,
to the Cambrian explosion. All systems were
already present at that time.”
Beutler raises an even more extreme possibility—that the Toll/TLR family goes back to
plants. “Even plants have TIR domain proteins,” he says. “And wherever you have TIR
domains, those domains have defensive functions.” That pushes the origin of these protective
proteins back to 1 or 2 billion years ago.
Moving from the remote past to the near
future, Beutler sees one of the major challenges
in the TLR field as discovering how TLRs, the
“eyes” of the innate immune system, “see” their
microbial quarry. “TLRs engage ligands according to the same rules of protein chemistry that
apply to all other receptors,” he says. “In the
years to come, we will understand exactly how
LPS is bound by TLR4, how DNA is bound by
TLR9, and how all of the other TLRs recognize
their specific ligands. Then we will understand
how such interactions might be mimicked,
encouraged, or effectively blocked.”
SUGGESTED READING
Hoffmann, J. A., and J. M. Reichhart. 2002. Drosophila innate immunity: an evolutionary perspective. Nature Immunol.
3:121–126.
Lemaitre, B., E. Nicolas, L. Michaut, J. M. Reichhart, and J. A. Hoffmann. 1996. The dorsoventral regulatory gene cassette
spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973–983.
Persing, D. H., R. N. Coler, M. J. Lacy, D. A. Johnson, J. R. Baldridge, R. M. Hershberg, and S. G. Reed. 2002. Taking toll:
lipid A mimetics as adjuvants and immunomodulators. Trends Microbiol. 10(Suppl.):32–37.
Poltorak, A., X. He, I. Smirnova, et al. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4
gene. Science 282:2085–2088.
Reichhart, J.-M. 2003. TLR5 takes aim at bacterial propeller. Nature Immunol. 4:1159 –1160.
Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, and S. Akira. 1999. Differential roles of
TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443– 451.
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