Forest laws, Indian forest laws, why they are important
Seminars immunopathology2007
1. REVIEW
Toll like receptor-5: protecting the gut from enteric microbes
Matam Vijay-Kumar & Jesse D. Aitken &
Andrew T. Gewirtz
Received: 24 September 2007 /Accepted: 5 November 2007 / Published online: 7 December 2007
# Springer-Verlag 2007
Abstract The intestine is normally colonized by a large
and diverse commensal microbiota and is occasionally
exposed to a variety of potential pathogens. In recent years,
there has been substantial progress made in identifying
molecular mechanisms that normally serve to protect the
intestine from such enteric bacteria and which may go awry
in chronic idiopathic inflammatory diseases of the gut. One
specific molecular interaction that appears to play a key
role in governing bacterial–intestinal interactions is that of
the bacterial protein flagellin with toll-like receptor 5. This
article reviews studies performed in vitro, in mice, and in
humans that indicate an important role for the flagellin-
TLR5 interaction in regulating both the innate and adaptive
immune responses in the intestine.
Introduction: protecting against the needle
and the haystack
The human intestine is normally colonized by a large
(1013
–1015
) and diverse (over 7,500 different species)
population of commensal bacteria known collectively as
the microbiota [26]. The microbiota can be viewed as a
metabolic “organ” exquisitely tuned to our physiology and
capable of performing essential functions that we have not
had to evolve on our own. Microbial colonization of the
intestine confers both local and systemic benefits spanning
diverse processes that include host defense from infection,
energy and nutrient metabolism, and tissue development
and repair. In addition, the gut is sometimes exposed to
potential food and water-borne pathogens. The mucosal
immune system is charged with the task of protecting the
host against such pathogens. One might compare the task of
detecting such pathogens to that of “finding a needle in a
haystack” although, by the numbers, this likely greatly
understates how rare most pathogens would be after their
ingestion. Moreover, unlike hay and needles, which have
distinct appearances and composition, intestinal pathogens
are often highly similar to closely related commensal
species, sometimes differing in only a handful of genes
that make them pathogenic. In protecting against such
pathogens, it is important that the intestinal defenses do not
seek to indiscriminately eliminate all bacteria as this would
jeopardize the beneficial affects of the enteric microflora on
digestion and would likely result in severe chronic
intestinal inflammation. However, the intestinal defense
system should not completely ignore the commensal
microbiota as its overgrowth can result in a variety of
problems, and furthermore, some of these bacteria are
commensals in the intestine but may cause severe disease
upon attaining access to other tissues (e.g., some Escherichia
coli strains). Thus, the intestine requires a system to protect
itself against both pathogens and scenarios in which
commensal bacteria might potentially cause disease.
The complex system of intestinal defense system utilizes
physical barriers and soluble antibacterial mediators that, in
large part, are generated by the epithelial cells that line the
intestine [15]. Furthermore, the intestine is populated by a
number of immune cells and possesses mechanisms to
quickly increase the number/activation status of such cells
in response to a particular challenge. Given the complex-
ities of its challenge, it is not surprising that the intestinal
defense system relies on a variety of receptors and in
Semin Immunopathol (2008) 30:11–21
DOI 10.1007/s00281-007-0100-5
DO00100; No of Pages
M. Vijay-Kumar :J. D. Aitken :A. T. Gewirtz (*)
Department of Pathology, Emory University,
Atlanta, GA, USA
e-mail: agewirt@emory.edu
2. targeting a variety of bacterial molecules. Nonetheless,
work from ourselves and others suggests that bacterial
flagellin, the molecular subunit of bacterial flagella, is
particularly important in intestinal host defense in that it is
targeted by both the innate and adaptive immune systems in
the gut.
In vitro studies: how flagellin activates intestinal epithelial
pro-inflammatory gene expression
The original appreciation of flagellin’s role in innate
immunity came from in vitro studies. Furthermore, such
studies have been the source of most of what is known
regarding the host–bacterial interactions that result in TLR5
activation and the biology of the flagellin-TLR5 interaction.
This section gives a brief overview of what has been
learned from such studies emphasizing those aspects that
are unique to the flagellin–TLR5 interactions and only
briefly describes those mechanisms that are similar to that
used by other TLRs.
Discovery of flagellin as a pro-inflammatory agonist
in the gut
The notion that flagellin is a dominant innate immune
activator of the gut originated from studies that modeled the
interaction of Salmonella with polarized gut epithelia [14].
A key initial observation was that while in response to the
enteric pathogen Salmonella typhimurium, only a small
percentage of epithelial cells had been colonized; nearly all
had been activated as assessed by both examining activa-
tion of the pro-inflammatory transcription factor NF-κB by
microscopy. This suggested the presence of a soluble
mediator whose existence was demonstrated by showing
that transfer of the media of infected epithelia to uninfected
cells also transferred the response (i.e., IL-8 secretion) to the
uninfected epithelia. Purification of the “active ingredient” of
such media conditioned by bacterial–epithelial interactions
followed by microscale protein sequencing revealed it to be
the protein flagellin, the primary structural component of
bacterial flagella. This identification was verified by demon-
strating that flagellin was necessary for S. typhimurium-
induced epithelial IL-8 secretion in that the response could
not be induced by flagellin-deficient mutants and sufficient
in that purified recombinant flagellin, made in E. coli or
eukaryotic cells, was a highly potent inducer of IL-8 secretion
[34]. Purified flagellin is a very potent inducer of IL-
8 expression displaying an ED50 in the picomolar range. In
contrast, human epithelial cells have been broadly observed
not to exhibit pro-inflammatory responses to lipopolysa-
charride, which is a very potent activator of most immune
cells [16]. The effects of flagellin on epithelial gene
expression are not restricted to IL-8, but rather, flagellin
was Salmonella’s dominant pro-inflammatory determinant
being necessary and sufficient for induction of nearly all
epithelial pro-inflammatory gene expression induced by S.
typhimurium [57].
The finding that Salmonella flagellin is such a potent
activator of epithelial pro-inflammatory gene expression
was rather surprising primarily because flagellin expression
is not restricted to pathogens but rather is expressed by all
motile bacteria, including a variety of commensal bacteria
that can be routinely colonized from healthy mucosal
biopsies. Furthermore, flagellin is a highly conserved
molecule, especially when in its monomeric form, and it is
flagellin monomers, rather than polymerized flagellin (i.e.,
flagella) that induce epithelial pro-inflammatory gene
expression. Consistent with its high conservation, it has
been demonstrated that most bacterial flagellins are potent
activators of pro-inflammatory gene expression in vitro
with, for example, E. coli and Salmonella flagellin having
similar pro-inflammatory potency in such assays [38]. Yet,
in contrast to Salmonella, the commensal E. coli strains
from which flagellin was purified do not, themselves,
substantially activate pro-inflammatory gene expression
when colonizing the apical (luminal) surface of polarized
model intestinal epithelia in vitro [14]. The reason for this
apparent discrepancy is that the flagellin receptor, TLR5, is
predominantly expressed on the basolateral surface of
polarized epithelia. This places the receptor in a position
to recognize flagellated pathogens that are invasive or have
opportunistically managed to breach the epithelium. Fur-
thermore, at least one pathogen, namely, S. typhimurium, is
able to transcytose its flagellin across model epithelia so it
can activate pro-inflammatory gene expression without
necessarily invading or crossing the epithelial surface
(Fig. 1). Such transcytosis of flagellin across model
epithelia is mediated by genes within the Salmonella
pathogenicity island 2, which are able to alter vesicular
trafficking in eukaryotic cells [29]. As flagellated com-
mensal bacteria would not have such ability, it seems likely
that their flagellin will not normally potently activate
epithelial cells in the gut. However, should there be
ulcerations in the epithelium, a context in which commensal
E. coli strains might be dangerous to the host, the
epithelium will be able to promote a localized inflammatory
response that should eliminate the bacteria. While this seems
an elegant way to protect the gut from a variety of bacteria, it
also presents scenarios which could be envisaged to
potentially result in aberrant inflammation. Specifically, upon
considering the large bacterial load in the gut, of which many
are flagellated, one can imagine that increases in gut
permeability, even in the absence of large ulcerations, might
result in significant activation of pro-inflammatory gene
expression and possibly drive some instances of chronic
inflammatory bowel disease.
12 Semin Immunopathol (2008) 30:11–21
3. Flagellin-induced signaling
The identification of toll-like receptor 5 (TLR5), as the
flagellin receptor, came from two reciprocal candidate-based
screens. One study screened all known TLRs for their ability,
upon expression in cells unresponsive to flagellin, to confer
ability to activate NF-kB in response to flagellin and
observed that only TLR5 had this ability [12]. Another
started with cells engineered to express TLR5 and sought
bacterial molecules that had the ability to activate the
receptor and, consequently, purified flagellin [22]. In
accordance with these, independent reciprocal screens
identified the same receptor–ligand interaction; the biological
importance of the TLR5-flagellin interaction was ultimately
verified by in vivo experiments (discussed below). In
contrast to some other TLRs that are able to heterodimerize,
TLR5 appears to only homodimerize in forming a functional
signaling complex [40]. Exogenously expressed TLR5 and
flagellin can be co-immunoprecipitated suggesting direct
interaction between flagellin and TLR5 [38].
Mutagenesis studies indicate that the key portion of the
flagellin molecule that is recognized by TLR5 is a region of
the flagellin molecule that is exposed on free flagellin
monomers, but that is buried within the assembled flagella
that mediates bacterial motility [46]. In accordance, TLR5
recognizes flagellin monomers rather than whole flagella.
The region of the monomer that is recognized by TLR5 is
highly conserved, and thus, TLR5 has the ability to detect
flagellin monomers from a wide variety of gut microbes.
Most flagellated bacteria readily shed some flagellin
monomers possibly as inherent result of flagella polymer-
izing at the distal end at a substantial distance from the
bacteria [29]. Some bacteria, notably Helicobacter and
Vibrio species encase their flagella in a sheath and thus
avoid shedding flagellin monomers possibly to reduce
immune detection. Alternatively, Salmonella species are
reported to have a distinct mechanism that allows them to
release flagellin monomers, not physically associated with
the flagellum, upon detection of the eukayotic metabolite
lyso-phosphatidytic acid [48]. The in vivo importance of
this pathway is not yet determined, but it may be a means
of activating an inflammatory response to aid dissemination
within the host and/or between new hosts.
The basic mechanisms by which TLR5 signals appear to
be relatively similar to that used by other TLRs. Briefly, as
outlined in Fig. 2, ligation of TLR5 results in rapid
activation of interleukin-1 receptor-associated kinase 4
(IRAK-4) leading to activation of MAP kinases (p38 and
ERK) and IkB kinase, which results in activation of NF-kB
thus driving the expression of a number of pro-inflammatory
genes such as the neutrophil chemoattractant IL-8 [54].
TLR5-mediated p38 activation is not needed for initial
transcriptional activation, at least of the IL-8 gene, but rather
was required for efficient translation of mRNAs induced by
flagellin. While p38 and NF-kB activation are common to
epithelial cells stimulated with flagellin or the pro-inflam-
matory cytokine TNFα, only flagellin was able to induce
significant activation of tyrosine phosphorylation of STAT-1
and STAT-3 thus explaining why flagellin, but not TNFα,
can induce iNOS activation in gut epithelial cells [55].
Consequences of TLR5 activation
The overall downstream outcome of such signaling is to
result in the transcriptional activation of an extensive panel
of at least 500 genes whose overall function seems to be to
Y
IRAK-4
NF-kB
p38
Pro-inflammatory
Survival
TLR5
IKK
PI3K
MyD88
Casapases
Transcription
Apoptosis
Fig. 2 Summary cartoon of TLR5 signaling as described in text and
references
Fig. 1 TLR5 can be activated by pathogens or loss of epithelial
barrier. Pathogens such as Salmonella typhimurium can breach the
epithelium or transcytose their flagellin across the epithelium to
activate TLR5. Alternatively, TLR5 may become activated by
commensal flagellin upon epithelial barrier dysfunction
Semin Immunopathol (2008) 30:11–21 13
4. protect the cells against various challenges [57]. Conse-
quently, TLR5-activated genes include those with direct
antibacterial function (e.g., defensins), immune cell chemo-
attractants, and a number of more general stress-induced
genes such as heat-shock proteins. In addition, TLR5 ligation
results in the activation of a number of genes with anti-
apoptotic function [58]. Activation of such anti-apoptotic
gene expression allows cells to stay alive in response to
challenges that would otherwise result in cell death. In spite
of its cytoprotective attributes, the ability of TLR5 signaling
to drive inflammation is likely potentially dangerous to its
host. Consequently, there appear to be multiple mechanisms
of down-regulating TLR5 signaling. One of these is that
flagellin ligation of epithelial TLR5 also results in a rapid
activation of phosphoinositide 3-kinase (PI3K) that serves to
limit pro-inflammatory gene expression largely via activation
of the phosphatases that are responsible for down-regulating
p38 [56]. Another mechanism of down-regulating TLR5-
induced signaling may be via flagellin’s interaction with the
mucin Muc1, whose expression dampens or attenuates
flagellin-induced TLR5 signaling [28]. Such means of
counter-regulating TLR5 signaling may prevent excessive
activation of pro-inflammatory gene expression.
A unique feature of epithelial TLR5 signaling is that its
expression is highly polarized to the basolateral surface,
and thus, both model epithelium and human colon can only
signal in response to flagellin reaching the basolateral
surface [13]. While polarization of TLR5 to the basolateral
surface of the intestinal epithelium should normally
minimize TLR5 activation in response to commensal
flagellated microbes, it can also be envisaged to provide a
safety net in that it would become activated if such
commensal microbes breech the epithelium via an oppor-
tunistic mechanism. However, there are also a variety of
scenarios by which activation of TLR5 might occur in
response to flagellin derived from commensal microbes,
and such TLR5-mediated pro-inflammatory gene expres-
sion might play a role in triggering acute flares of
inflammation in IBD. For example, multiple studies have
observed bacteria attaining seemingly inappropriate locales
in IBD either adherent, within, or beyond the epithelium
that might result in increased TLR5 activation [4, 49].
Another possibility is that the alterations in mucosal
permeability that have long been associated with IBD [23]
might result in greater access of flagellin to basolateral
TLR5. As flagellin is readily released by most flagellated
microbes, such activation of epithelial TLR5 in states of
barrier dysfunction need not require abnormal localization
of bacteria themselves, and thus, in this case, might be
promoting an “inappropriate” inflammatory response in that
it is not serving a necessary role in host defense. In vivo
studies described below are shedding light on these
possibilities.
Mouse studies
Mouse models are generally regarded as the gold standard
in validating receptor–ligand interactions that were identi-
fied in vitro and, moreover, have played a key role in
understanding basic mechanisms that regulate host–bacte-
rial interactions in the gut both with regard to bacterial
pathogenesis and intestinal inflammation. Such models
make it possible to study mechanisms that govern both
early and late events in the enteric inflammatory diseases
and, consequently, should eventuate in the development and
evaluation of new therapeutic strategies to treat such
diseases.
Mice lacking TLR5 lack innate immune response to purified
flagellin
Mice injected with purified flagellin exhibit robust
increases in a panel of serum cytokines. This response is
absent in mice lacking the TLR5 gene [50]. This result
confirms the notion that TLR5 is, in fact, the host’s primary,
if not only, receptor for detecting free flagellin monomers.
Generation of TLR5 bone marrow chimeras indicated that
TLR5 in both hemopoietic and non-hemopoietic cells was
important for generation of cytokines consistent with a
significant biologic role for TLR5 in both epithelial and
hemopoietic cells [8]. While murine macrophages and
dendritic cells have generally been observed to lack TLR5
and not respond to soluble flagellin [35], intestinal DC
appear to be an exception in that they express TLR5 and
respond to flagellin [50] thus suggesting the possibility that
these cells mediate a portion of the in vivo hemopoietic cell
responsiveness to flagellin. In any case, that TLR5KO
lacked innate immune responses to flagellin provides a nice
model to study the biological importance of the TLR5–
flagellin interaction.
Loss of TLR5 results in spontaneous colitis
Inflammatory bowel disease is thought to result from a
breach in physiological immune homeostasis between
commensal microflora and the host immune system. There
are several experimental mouse models in which induced
mutations of host genes have resulted in inflammatory
bowel disease (IBD). In most instances, genetically mod-
ified mice do not get IBD if they are raised in gnotobiotic
conditions, i.e., without a commensal flora sometimes
referred to as “germ-free” underscoring the importance of
the microbiota in this disorder [47]. While the enteric
microbiota does not normally cause inflammation in normal
mice (or healthy humans), neither should it be viewed as an
inert bystander. Rather, the microbiota exerts profound
effects on the intestine, via interactions with the epithelial
14 Semin Immunopathol (2008) 30:11–21
5. layer, enteric nervous system, and mucosal immune system.
In light of in vitro findings that indicate flagellin is a
“dominant” activator of the epithelial innate immune
response, we reasoned that mice lacking TLR5 might be
resistant to models of IBD. In contrast, we observed that
mice lacking TLR5 had a tendency to develop spontaneous
colitis [52].
The most severe manifestation of spontaneous colitis was
rectal prolapse, which was exhibited by about 10–12% of
mice lacking TLR5, but not their wild-type littermates, both
of which had been backcrossed onto the C57BL/6 back-
ground. Furthermore, an additional 25% of TLR5KO mice
had gross, histopathologic, and serologic evidence of colitis.
In addition, TLR5KO mice develop other typical features of
murine colitis including splenomegaly, anemia, and enlarged
mesenteric and sublingual lymph nodes. This phenotype is
specific to TLR5 in that there was no evidence of colitis
observed in mice lacking other TLRs [2–4, 6, 7, 9, 11] or the
global TLR signaling adapter MyD88 maintained in our
facility. While, based on gross or histopathologic analysis,
about 60% of TLR5KO appeared relatively normal, analysis
of their intestinal gene expression via microarray indicated
they had substantial elevation in pro-inflammatory gene
expression and thus may be poised to develop colitis. In
accordance, when bred onto an IL10-deficient background,
loss of TLR5 resulted in severe colitis with 100% penetrance
in the double knockout mice (about 60% of IL10-deficient
littermates get colitis).
Approaches to decipher the mechanism by which loss of
TLR5 results in colitis (or leaves them in a pre-colitic state)
focused on studying TLR5KO mice that lacked overt
colitis. Such studies indicate that TLR5KO mice develop
colitis due to an inability to manage their enteric micro-
biota. Specifically, non-colitic TLR5KO mice exhibit
moderately elevated (about fivefold) numbers of total
culturable bacteria in their feces and, moreover, have many
bacteria that are tightly associated with the colonic surface,
which contrasts to WT mice that have most bacteria in the
intestinal lumen. TLR5KO mice are also permissive of
bacterial translocation to both liver and spleen on the order
of approximately 100–200 bacteria per organ. Because
TLR5KO mice and their WT littermates begin life with the
same enteric microbiota, which they largely acquire from
their mother during suckling, these differences likely reflect
an inability to control their commensal microbiota rather
than a pathogenic organism per se. TLR5KO housed in a
separate colony in Japan have not elicited the most severe
manifestations of colitis and, thus, the specific components
of the microbiota may dictate the extent of disease severity.
A variety of approaches, including much of the work
discussed above suggests that it is probably loss of TLR5
function on epithelial cells that underlies TLR5KO colitis.
Loss of epithelial TLR5 may reduce epithelial secretion of
antibacterial mediators and/or reduce recruitment of
immune cells. In any case, the resulting failure to manage
the enteric microbiota likely results in colitis via activation
of other pathways of TLR signaling. Ex vivo cultures of
colons from TLR5KO showed significantly elevated levels
of proinflammatory, TH1, and TH17 cytokines. Microarray
analysis of colonic tissue from TLR5KO mice reveals the
upregulation of classical proinflammatory genes, specifi-
cally TLR4, and its co-receptors CD14 and LBP suggest-
ing a potential role for TLR4 pathway. Interestingly, in the
TLR4/5 double KO, there is no incidence of rectal
prolapse or evidence of inflammation despite an increased
bacterial burden in the feces and colons. In summary, as
outlined in Fig. 3, TLR5KO mice show a decreased
induction of flagellin-specific gene expression and are
thus unable to efficiently regulate their intestinal micro-
biota. This state of dysregulation necessitates the activa-
tion of TLR4 in hemopoietic cells and results in the
induction of the proinflammatory cytokines that drive
colitis.
Consequences of TLR5 loss upon infection by Salmonella
In humans, Salmonella species causes a continuum of
diseases ranging from a largely gut-restricted enteritis to
typhoid fever in which the gastrointestinal manifestations
are minimal, but the bacteria disseminates throughout the
host. These diseases states can be modeled in mice. In
general, oral infection of mice with Salmonella causes
systemic and generally lethal typhoid-like illness. It
seemed reasonable to predict that mice lacking TLR5
might be more susceptible to such illness. However,
surprisingly, Uematsu et al. have found that TLR5KO
mice display significant resistance to infection and a
decreased extraintestinal bacterial burden compared to
their WT littermates [50]. This resistance to Salmonella-
induced mortality is lost when mice are challenged
intraperitoneally. The resistance of TLR5KO to Salmonella
dissemination has been suggested to result from impaired
transport of Salmonella from the intestinal tract to the
mesenteric lymph nodes possibly due to a role for TLR5 in
regulating dendritic cell movement. However, as outlined in
Fig. 3, we propose that TLR5KO resistance to Salmonella
may reflect the significant basal alterations in intestinal gene
expression.
If, before exposure to Salmonella, mice are treated with a
large bolus of antibiotic, they develop disease reminiscent
of the enterocolitis commonly seen in humans after human
ingestion of food-borne Salmonella [2]. The role of
flagellin in this disease model was studied in mice by
infecting them with a aflagellate salmonella mutants or
isogenic control strains [51]. When examined within hours
of colonization, aflagellate strains elicited an attenuated
Semin Immunopathol (2008) 30:11–21 15
6. initial inflammatory response relative to flagellated salmo-
nella. This result is consistent with in vitro data that
implicates flagellin as a dominant proinflammatory effector
of the response to enteropathogenic salmonella. In contrast,
when examined at a later time (24–48 h), aflagellate
salmonella appeared substantially more virulent by 48 h
after infection and had induced peritonitis and intussuscep-
tion of bowel segments. Furthermore, aflagellate-infected
mice show gross cecal contraction, reduced weight, and
pallor, which are macroscopic hallmarks of this Salmonel-
losis model. In addition, infection with aflagellate Salmo-
nella results in increased apoptosis of epithelial cells. These
observations suggest that TLR5-mediated gene expression
protects the intestine against Salmonella. Such protection
could reflect a role for the early pro-inflammatory gene
expression in retarding bacteria and/or a role for antiapop-
totic/cytoprotective gene expression in protecting against
Salmonella-induced cytotoxicity.
Role of TLR5 in humans
A large portion of studies on TLRs have focused on
macrophages, which are relatively amenable to being
isolated from humans and studied ex vivo. However,
macrophages are relatively unresponsive to flagellin mak-
ing human studies rather difficult and limited. Yet, one ex
vivo study of human gut has been informative as it has
studies examining the consequences of a genetic alteration
in TLR5.
Basolaterally restricted in vivo response to flagellin
The initial discovery and characterization of flagellin as
potent activator of epithelial cells involved two well-studied
models of polarized epithelial cells (MDCK and T84).
These studies found that responses to flagellin required this
protein to reach the basolateral surface of the epithelium
Loss of Toll-like Receptor 5 (TLR5)
Failure to Manage Commensal Microflora
Increased Activation of Hemopoietic TLR4
Increased Expression of Colitogenic Cytokines
(IL-23. IL-17, IL-12, IFNγ, TNFα)
Loss of Epithelial Barrier
Function
Systemic Dissemination of
Bacteria and their Products
Persistent Inflammation of the
Gut and Colitis
Rectal Prolapse
Flagellated Bacteria
Flagellin
ICE Protease Activation Factor
(Cytosolic Flagellin Receptor, IPAF)
Caspase 1/ ICE
Mature IL-1β, IL-18
Acute Phase Response
(SAA, Lipocalin-2, Haptoglobin, Hepcidin)
Anemia, Splenomegaly,
Leukocytosis
Reduced Epithelial Expression of Host-Defense Genes
Fig. 3 Potential mechanism un-
derlying spontaneous colitis
TLR5KO mice
16 Semin Immunopathol (2008) 30:11–21
7. [12, 41]. However, subsequent findings with other cell
types have not uniformly replicated this polarity. Thus, it
was not until Rhee et al set up an elegant system to study
responses to flagellin in human colon ex vivo that the
significance of in vitro observations of polarity could be
determined. These studies found that native human colonic
mucosa responded ex vivo to basolateral but not apically
applied flagellin by inducing chemokine IL-8 thus validat-
ing the original in vitro studies [43]. This system also found
that flagellin activation of human colon occurred without
alterations in transepithelial electrical resistance indicating
that in contrast to some bacterial activators of the
epithelium, flagellin does not have properties of a toxin.
In accordance, mice lack detectable innate responses to
luminal flagellin unless given under a state of epithelial
barrier dysfunction [44].
Dominant-negative TLR5 Polymorphism associates
with disease
Four single nucleotide polymorphisms have been identified
in the human TLR5 gene [20]. One of these has clear
functional consequences and thus has proved very helpful
in understanding the role/importance of TLR5 in humans.
Specifically, the polymorphism referred to as TLR5-stop is
a cytosine-thymidine transition at base pair 1,174 that
replaces the arginine at amino acid 392 with a stop codon.
This change results in deletion of a large portion of the
cytoplasmic region of TLR5, including loss of the toll-IL-1
receptor (TIR)-domain that mediates TLR signaling. TLR5-
stop is thus unable to signal and, furthermore, results as a
dominant-negative allele. Approximately 10% of the
world’s population across a variety of ethnicities carry
TLR5-stop as heterozygotes and appear to have a 75%
reduction in TLR5 function [21]. Persons who are
homozygous for TLR5 are presumed to have complete loss
of TLR5 function and would be predicted by Mendelian
distribution to be about 1% of the population. Studies to
date have not been sufficiently large to determine the actual
frequency of TLR5-stop homozygotes, but such persons do
indeed exist and have not as yet manifested with any
particular health problems. Yet, heterozygous carriage of
TLR5-stop is associated with increased likelihood of
developing clinical disease upon exposure to Legionella
[20] suggesting that TLR5 function is important for
protecting the lung against this infection and, more
generally, supports the concept that TLR5 protects against
pathogens that first colonize mucosal surfaces. In contrast,
study of a cohort in Vietnam found that TLR5-stop
polymorphism has no significant impact on the susceptibil-
ity to typhoid fever caused by Salmonella typhi suggesting
TLR5 may not offer protection to pathogens that breech
mucosal surfaces to cause systemic disease [7].
In light of the above-described observations that
TLR5KO exhibit spontaneous colitis, one might expect
that reduced TLR5 function might predispose one to
developing inflammatory bowel disease. However, in fact,
TLR5-stop negatively associates with Crohn’s disease
(CD), although the association was only statistically
significant in Jewish cohorts [18]. The association was
specific for Crohn’s disease as it did not extend to
ulcerative colitis. These seemingly disparate observations
may reflect an inherent difference between mice and
humans or may reflect the possibility that reduced TLR5
function can indeed protect against some instances of gut
inflammation, but total loss of TLR5 function, which
occurs in TLR5KO mice, results in inability to manage
commensal microbiota. The concept that loss of TLR5 can
protect against seemingly inappropriate immune responses
is supported by the observation that the TLR5-stop poly-
morphism, but not other TLR5 alleles, is associated with
protection from developing SLE, and the protection was
most pronounced in individuals who are seronegative for
anti-dsDNA autoantibodies. As will be discussed below,
TLR5 can also regulate adaptive immunity, and thus, the
ability of TLR5-stop to protect against these diseases may
ultimately prove to result from effects on adaptive im-
munity as a result of reduced TLR5 function.
Evasion of TLR5
TLR5 does not appear to recognize a simple linear epitope
but rather recognizes a nonlinear epitope on a region
thought to mediate polymerization of flagellin monomers.
An eight amino acid region (89–96), which comprises a
portion of the D1 α-helix is essential, but not sufficient, for
TLR5 activation [1, 46]. This region of the flagellin mole-
cule is highly conserved and is required for filament forma-
tion and essential for motility in most gut microbes. Thus, it
has been proposed that mutations in the flagellin gene that
would eliminate its being recognized by TLR5 would also
hinder function of flagellin in motility. Nonetheless, a
number of human pathogens have evolved novel methods
of evading TLR5 recognition. The oncogenic pathogen
Helicobacter pylori, for example, exhibits an alternate
amino acid sequence on the convex binding surface of the
flagellin monomer which prevents normal TLR5-mediated
recognition while compensatory alterations to the concave
binding surface preserve the ability to form flagella and,
thus, ensure motility. Purified monomers of H. pylori
flagellin have been shown to induce much less IL-8 and
to only weakly activate p38 signaling [17, 25]. Further-
more, in contrast to most flagellated microbes, H. pylori
envelopes its flagella in an extension of its cellular
membrane [11] referred to as a sheath and thus fails to
release flagellin monomers. That H. pylori is motile and yet
Semin Immunopathol (2008) 30:11–21 17
8. is hypostimulatory with regard to TLR5 likely contributes
to the human asymptomatic carrier phenotype, necessitates
antibiotic treatment to clear what would otherwise be a
lifelong infection, and has made H. pylori a highly
successful global pathogen with well over half of the
world’s population having been colonized. In addition to H.
pylori, Campylobacter jejuni, Bartonella bacilloformis, and
a number of other Proteobacteria also produce altered and
unrecognizable flagellins [1, 53]. In contrast, there is
evidence to suggest that some Salmonella species may
“deliberately” activate TLR5. Specifically, S. typhimurium
releases flagellin monomers upon extracellular detection of
host lysophospholipids. Such release of flagellin monomers
would likely result in robust activation of TLR5 and has
been suggested to be a possible attempt to manipulate the
local innate inflammatory response and alter the host’s
adaptive response towards one dominated by TH2 cells
[48].
Alternate flagellin receptors
Most populations of macrophages that have been studied
lack functional responses to extracellular flagellin presum-
ably due to insufficient expression of TLR5. However,
macrophages can recognize intracellular flagellin. Such
intracellular detection of flagellin is mediated by members
of the nucleotide-binding oligomerization domain-leucine-
rich repeats (NOD-LRR; also known as the NLR) family of
intracellular receptors. Many studies have utilized Legionella
pneumophila, the causative agent in Legionnaire’s Disease,
to deduce the mechanistic aspects of cytosolic flagellin
detection and the subsequent host immune response. L.
pneumophila is especially suited to this task in that its
propagation requires that it replicates inside phagocytic cells,
specifically macrophages but also monocytes. This strategy
of infection requires the bacterial type IV secretion system
Dot/Icm, which injects a number of effector proteins directly
into the cytosol to promote the formation of permissive
replicative vacuoles, prevent lysosomal processing of those
vacuoles, and suppress the host response to infection. Such
intracellular bacteria can be recognized by nod-like receptors
(NLRs). Flagellin has been recently shown to be recognized
by the NLR ICE-protease-activation factor (IPAF), in which
ICE stand for IL-1-converting enzyme [9, 36, 37]. Stimula-
tion of Ipaf, a member of the NOD-LRR family, by flagellin
monomers has been shown to disrupt intracellular replication
of L. pneumophila and restore the endogenous mechanisms
of lysosomal degradation by exposing the infectious vacuole
to LAMP-1 via an enigmatic caspase-1-dependent pathway
which may depend upon the adaptor protein apoptosis-
associated speck protein (ASC). Caspase 1 is known to
process a number of proinflammitory cytokines including IL-
1β and IL-18 and to be a major component of the pyroptotic
response to infection while ASC, an adaptor protein which
also interacts with Nalps, enhances, but is not necessary for,
Ipaf-mediated caspase 1 activation [10]. Another NOD-LRR
protein that plays a role in recognition of flagellin is Naip5,
also known as Birc1e, whose absence has been shown to
correlate with the severity of L. pneumophila infection both
in vitro and in vivo, particularly in A/J mice, which have
been shown to be permissive for Legionella replication due
to their having a mutant Naip5 protein [39, 42]. Although
conclusive mechanistic evidence is lacking, Naip5 has been
shown to associate with both caspase 1 and Ipaf and appears
to have a distinct but possibly semi-redundant role in
detecting cytosolic flagellin or may alternatively function in
a regulatory role as part of a higher order complex required
for activation of or interaction with the inflammasome. At
present, it is clear that Naip5 is necessary for macrophages to
mount a full strength flagellin-induced response to L.
pneumophila and that aflagellate bacteria are capable of
increased virulence on both the cellular and systemic levels
(27).
Flagellin and adaptive immunity
Flagellin has long been known to be a major target of
adaptive immunity. Flagellar antigens are the basis of H-
serotyping that are used to classify isolates of Salmonella
and E. coli. Such serotypes are encoded by the flagellin
gene consistent with the fact that flagellin comprises about
97% of the flagella by mass. Bacterial flagellins are also a
dominant target of the elevated antibody response to
commensal microflora that has long been associated with
Crohn’s disease. Specifically, serologic expression cloning
of a large panel of bacterial proteins from spontaneously
colitic found that 25% of the antibody response associated
with colitic mice was directed at flagellin of various
commensal bacteria [27]. Flagellin’s serologic immunodo-
minance was subsequently confirmed in Crohn’s disease
patients [27, 45]. While the use of H-serotyping to classify
Salmonella isolates reflects the fact that antibodies to
flagella are rather specific for particular Salmonellae; this
specificity is somewhat lost when assaying responses to
flagellin monomers [44]. Antibodies from Crohn’s disease
patients are reactive to flagellin monomers from a variety of
commensal microbes, suggesting that these responses are
likely directed against flagellin monomers rather than
polymerized flagella [6]. While there is a commonly held
view that flagellin is a T-independent antigen, studies in
knockout mice indicate that T cells are absolutely required
for generation of flagellin-specific antibodies and, thus,
levels of flagellin-specific antibodies likely reflect levels/
activation of flagellin-specific T cells [44]. In accordance,
flagellin is the dominant T-cell epitope in murine Salmonella
infections [3, 33]. Thus, in serving as antigenic target of
18 Semin Immunopathol (2008) 30:11–21
9. adaptive immunity, flagellin plays a role in the host–bacterial
interactions in the gut.
Several lines of research indicate that flagellin’s immu-
nodominance as an antigen likely results, at least in part,
from its ability to activate innate immunity. Flagellin from
H. pylori, which does not activate TLR5, failed to elicit
antibodies but, when administered with a potent adjuvant,
was as immunogenic as Salmonella flagellin [44]. Mice
lacking ability to signal through TLR5 because of
deficiency in MyD88 lack antibody responses to both
purified flagellin and lack the acquisition of flagellin
antibodies that normally occurs in murine colitis [44].
Purified flagellin is also able to promote generation of
antibodies to coadministered antigens [5]. Furthermore,
flagellin serves as a potent T-cell adjuvant [34], and
flagellin–viral fusion protein was able to protect against
West Nile virus challenge in a TLR5-dependent manner
[32]. Thus, TLR5-mediation of innate immunity in the gut
likely has profound effects on adaptive immunity. In normal
mice, immune responses to intestinal flagellin are highly
restricted to intestinal IgA where one might presume they play
an important role in protecting against a variety of bacteria
[24]. How and why such responses may spread and become
systemic in Crohn’s disease and their pathophysiologic role
therein remain under investigation [19].
Concluding thoughts
As described herein, bacterial flagellin appears to be a
major target/effector of the immune response in the gut with
much of its bioactivity attributed, directly or indirectly, to
its ability to activate TLR5. It seems likely that such focus
of the immune system on a single molecule must reflect a
central role for that molecule’s function. In general, motility
seems a key aspect of so many living creatures, and thus, it
seems likely that the ability to move from place to place in
a directed manner would be a great asset to any form of life.
For bacteria, the cost is not only an immunologic one but
also a metabolic one as generation and use of flagella
consume 10–30% of a bacteria energy generation [31]. The
benefit of motility in experimental systems is often difficult
to appreciate. Removing flagellin genes increases growth
rates and does not reduce virulence of many pathogens in
lab settings. Nonetheless, most clinical isolates of Salmonella,
Pseudomonas, pathogenic E. coli are flagellated. It seems
reasonable to speculate that motility must be quite useful to
these organisms but that simply currently used lab models
may not be appropriate to appreciate its role. Perhaps
development of additional models combined with future
study of bacterial gene expression in their real world
reservoirs and actual physiologic hosts will shed light on
this area in the coming years.
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