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MINI REVIEW ARTICLE
published: 18 February 2014
doi: 10.3389/fimmu.2014.00060
Regulation of intestinal immune responses throughTLR
activation: implications for pro- and prebiotics
Sander de Kivit1*, Mary C. Tobin2, Christopher B. Forsyth1, Ali Keshavarzian1,3 and Alan L. Landay 2,3
1Division of Digestive Diseases and Nutrition, Rush University Medical Center, Chicago, IL, USA
2Department of Immunology/Microbiology, Rush University Medical Center, Chicago, IL, USA
3Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, Netherlands
Edited by:
Christophe M. Filippi, Genomics
Institute of the Novartis Research
Foundation, USA
Reviewed by:
Ari Waisman, University Medical
Center of Johannes Gutenberg
University Mainz, Germany
Muriel Moser, Université Libre de
Bruxelles, Belgium
*Correspondence:
Sander de Kivit, Division of Digestive
Diseases and Nutrition, Rush
University Medical Center, 1735 West
Harrison Street, Chicago, IL 60612,
USA
e-mail: sander_dekivit@rush.edu
The intestinal mucosa is constantly facing a high load of antigens including bacterial anti-
gens derived from the microbiota and food. Despite this, the immune cells present in the
gastrointestinal tract do not initiate a pro-inflammatory immune response.Toll-like receptors
(TLRs) are pattern recognition receptors expressed by various cells in the gastrointestinal
tract, including intestinal epithelial cells (IEC) and resident immune cells in the lamina pro-
pria. Many diseases, including chronic intestinal inflammation (e.g., inflammatory bowel
disease), irritable bowel syndrome (IBS), allergic gastroenteritis (e.g., eosinophilic gas-
troenteritis and allergic IBS), and infections are nowadays associated with a deregulated
microbiota.The microbiota may directly interact withTLR. In addition, differences in intesti-
nal TLR expression in health and disease may suggest that TLRs play an essential role in
disease pathogenesis and may be novel targets for therapy. TLR signaling in the gut is
involved in either maintaining intestinal homeostasis or the induction of an inflammatory
response. This mini review provides an overview of the current knowledge regarding the
contribution of intestinal epithelial TLR signaling in both tolerance induction or promoting
intestinal inflammation, with a focus on food allergy. We will also highlight a potential role
of the microbiota in regulating gut immune responses, especially through TLR activation.
Keywords: toll-like receptors, intestinal epithelial cells, food allergy, microbiota, probiotics, prebiotics, circadian
rhythm
THE MUCOSAL IMMUNE RESPONSE IN THE INTESTINE – AN
OVERVIEW
The mucosal tissue of the intestines contains the largest part of
the immune system present in the human body, and is constantly
exposed to many antigens, which are derived from amongst oth-
ers food and micro-organisms including the commensal micro-
biota or invading pathogens. Approximately, 70% of the cells of
the immune system are present in the gut and are continuously
discriminating between harmless and pathogenic antigens. Nev-
ertheless, the majority of oral foreign antigens do not result in
inflammatory responses in healthy individuals. This phenome-
non is known as oral tolerance. Local or systemic pathological
inflammation may occur when oral tolerance toward some harm-
less luminal antigens is lost. This is seen for instance in food allergy,
which is characterized by an inflammatory immune response
toward generally harmless food-derived antigens.
Intestinal epithelial cells (IEC) provide a physical and chemical
barrier between the intestinal lumen and the lamina propria. The
expression of tight junction proteins by IEC, production of mucus
by goblet cells and Paneth cell-derived antimicrobial peptides
prevent translocation of luminal antigens and micro-organisms
into the lamina propria (1,2). Nevertheless, antigens are actively
sampled into the gut-associated lymphoid tissue (GALT). Under-
standing of the GALT is essential to gain insight in both disease
pathogenesis and to design new therapeutic strategies to prevent or
cure inflammatory diseases of the intestine. As an antigen ends up
in the lumen of the intestine, it is generally recognized by dendritic
cells (DC) present in Peyer’s patches, after the antigen has been
transported into the Peyer’s patch via specialized IEC known as M
cells (3,4). Antigen sampling also occurs via dendrites of DC that
protrude between the IEC (5,6). Upon antigen recognition, DC
migrate toward the draining mesenteric lymph nodes (MLN) and
activate T cells, which migrate back toward the intestinal lamina
propria to carry out their effector functions (7).
Intestinal epithelial cells have been described to suppress DC
activation as well and contribute to tolerance induction by secret-
ing amongst others TSLP and TGF-β, and metabolize vitamin A
into retinoic acid to induce the development of CD103+DC (8–
12). These CD103+DC induce antigen-specific regulatory T cells
(Treg) as well as the expression of the specific gut-homing mole-
cules α4β7 integrin and CCR9 on T cells in the MLN (13). Treg
cells suppress adaptive immune responses through cell–cell con-
tact dependent mechanisms or secretion of the anti-inflammatory
cytokines IL-10 or TGF-β. Indeed, induction of Treg cells results
in abrogation of food hypersensitivity responses (14,15). A
higher frequency of allergen-specific Treg cells is observed in chil-
dren that have outgrown cow’s milk allergy and allergen-specific
immunotherapy has been shown to induce Treg cells (16,17),
implicating that the induction of Treg cells is essential for mucosal
tolerance.
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de Kivit et al. TLR, microbiota, and gut health
REGULATION OF INTESTINAL IMMUNITY AND TOLERANCE
BY TLRs EXPRESSED BY IEC
Toll-like receptors (TLRs) recognize a wide range of microbial
fragments and therefore recognize both antigens derived from the
microbiota as well as invading pathogens. TLRs are expressed by
a variety of cells, including IEC. TLR2 can dimerize with TLR1 or
TLR6 to recognize bacterial cell wall lipoproteins. LPS produced
by Gram-negative bacteria is recognized by TLR4 in conjunc-
tion with CD14 and MD2, whereas unmethylated CpG motifs
of bacterial DNA are recognized by TLR9. In addition, flagellin is
recognized by TLR5, which is expressed at the basolateral mem-
brane by IEC. TLR2, 4, and 5 are generally expressed at the cell
membrane, whereas TLR9 is expressed intracellularly. However, in
IEC, TLR9 has been reported to be expressed at the cell membrane
as well (18,19).
Under homeostatic conditions, IEC show low expression of
TLR2 and TLR4 and are therefore unresponsive to TLR stimuli
(20,21). However, under inflammatory conditions,epithelial TLR
expression is increased, which contributes to both inflammation
as well as immune tolerance (19,22,23). Increased epithelial TLR2
and TLR4 expression is associated with inflammatory bowel dis-
ease (24). In contrast, apical TLR9 stimulation has been described
to contribute to intestinal homeostasis (18). Interestingly, TLR
activation of IEC appears to be important in regulating adap-
tive immune responses. Using an in vitro co-culture system, it was
shown that TLR4 and basolateral TLR9 activation on IEC is impor-
tant in driving an inflammatory response, whereas apical TLR9
activation supported the differentiation of an anti-inflammatory
response (25). The underlying mechanisms by which TLR9 pro-
motes tolerance are not well understood, but it has been described
that apical but not basolateral TLR9 ligation on IEC prevents
degradation of IκB-α, and therefore suppresses NF-κB-induced
pro-inflammatory cytokine production by IEC (18). In addition,
it has recently been indicated that apical TLR9 activation sup-
ports the expression and secretion of galectin-9, a soluble protein
of the lectin family, which supports the differentiation of Treg
cells potentially by supporting the development of tolerogenic
DC (26,27). Though IEC are important in driving the devel-
opment of tolerogenic CD103+DC and suppress DC activation
(8), it is not known whether TLR activation on IEC influences
the generation of CD103+DC. Recently, it has been shown that
gut bacteria stimulate the recruitment of CD103+DC into the
epithelium potentially via TLR-dependent mechanisms in both
IEC and hematopoietic cells (28). Altogether, TLR stimulation in
the intestinal epithelium plays an important role in regulating
mucosal immune responses in the intestine.
In addition to regulating intestinal immunity, TLR activation
on IEC is also known to modulate the expression of tight junction
proteins. In many inflammatory disorders, including food allergy,
epithelial tight junctions are impaired and increased bacterial
translocation occurs (29). This increased bacterial translocation
into the lamina propria may sustain the inflammatory response.
In particular, epithelial TLR2 activation has been described to
protect against barrier disruption by enhancing zonula occludens
(ZO)-1 expression in IEC in a protein kinase C-dependent manner
(30). In contrast, activation of TLR4 increases intestinal perme-
ability and results in enhances bacterial translocation (31). NF-κB
signaling as a result of TLR4 activation by LPS appears to play
a major role in LPS-mediated barrier disruption (32,33). Simi-
larly, apical Campylobacter jejuni infection of T84 cell monolayers
results in a rapid decrease in the transepithelial resistance of the
monolayer involving NF-κB signaling (34). Activation of TLR9
apically on IEC prevents TLR4-induced gut leakiness and infec-
tion of IEC monolayers with Campylobacter jejuni disrupts the
intestinal epithelial barrier function by reducing TLR9 expression
at the surface membrane of IEC (33). In this similar study, the
authors also indicate an increase in the intestinal barrier func-
tion upon apical, but not basolateral TLR9 stimulation with a
synthetic CpG DNA (35). Preliminary data from our group also
report a potential protective effect of apical TLR9 activation in T84
cell monolayers co-culturedw ith CD3/28-activated PBMC. Hence,
paracellular transport of antigens as well as bacterial translocation
under pathological conditions may be affected by TLR activation
on IEC.
With respect to food and environmental allergens, the contri-
bution of TLR activation on IEC is not well studied. Recently,
TLR4 activation by wheat α-amylase trypsin inhibitors, a rec-
ognized plant-derived allergen (36), has been described to drive
intestinal inflammation (37). The percentage of α-amylase trypsin
inhibitors is markedly higher in genetically modified grain seeds
that are more resistant to infection than traditional seeds (38–40),
which might explain why a wheat-free diet could be beneficial
in a wide range of inflammatory and allergic disorders. Simi-
larly, the house dust mite allergen Der p 2 as well as the major
cat allergen Fel d 1 enhance signaling through TLR2 and TLR4
(41). Although these studies were carried out on innate immune
cells, this does not exclude that these allergens may interact with
TLR expressed by IEC as well. Especially, since TLR activation on
IEC affects the mucosal barrier function and potentially shapes
mucosal immune responses in the intestine, interactions of aller-
gens with TLR expressed by IEC may facilitate their entry into the
gut mucosa and sustain the allergic inflammatory response. Inter-
estingly, treatment with CpG oligodeoxynucleotides improved the
intestinal barrier function and increased the percentage of Treg
cells in the spleen and MLN (42). Since epithelial TLR may interact
with the gut microbiota and luminal antigens, further under-
standing of the role of epithelial TLR activation in food allergy
is necessary.
INTERACTIONS BETWEEN THE MICROBIOTA AND TLRs
The microbiota is the largest source of microbial stimulation in the
gut. Furthermore, the microbiota is necessary for development of
the intestinal immune system (43). The “hygiene hypothesis,” cur-
rently the most popular theory of deregulation of the microbiota,
theorizes that specific microbial stimulation is necessary for gut
health. Originally, it states that microbial stimulation polarizes the
immune response toward Th1,while lack of microbial stimulation
maintains a Th2 polarized immune response, which is character-
istic for atopy (44). Recently, a specific microbiota signature was
linked to oral allergic sensitization in mice exhibiting a gain-of-
function mutation in the IL-4 receptor αchain, which rendered
these animals more prone to developing food allergy. This micro-
biota signature was characterized by a reduction in Firmicutes spp.
and increase in Proteobacteria spp. (45). Another example that
Frontiers in Immunology | Immunological Tolerance February 2014 | Volume 5 | Article 60 | 2
de Kivit et al. TLR, microbiota, and gut health
indicates the importance of the gut microbiota composition in the
development of food allergy is a recent study showing that colo-
nization of germ-free mice with the fecal microbiota of a healthy
infant rich in Bifidobacterium spp. and Bacteroides spp. protected
against the development of cow’s milk allergy following sensi-
tization to β-lactoglobulin (46). This was associated with lower
T cell reactivity toward the allergen, an increase in Foxp3+Treg
and lower bacterial translocation into the lamina propria. Bifi-
dobacterium breve potentially activates CD103+intestinal DC to
produce IL-10 and IL-27 in a TLR2-dependent fashion to induce
IL-10-producing Tr1 cells (47), whereas colonization of germ-free
mice with Bacteroides fragilis restores the Th1/Th2 balance and
prevents intestinal inflammation through induction of IL-10 pro-
ducing CD4+T cells. This was dependent on recognition of B.
fragilis polysaccharide A by gut DC (48,49).
Disturbances in the commensal bacterial composition in the
gut, reflected by increased colonization with Escherichia coli or
Clostridium difficile, is associated with an increased risk in the
development of allergic disease and IBD in humans (50,51). The
fecal microbiota of allergic infants shows a higher prevalence of
Clostridium spp. and Staphylococcus aureus. In parallel, lower lev-
els of Bifidobacteria,Enterococci, and Bacteroides were found in
the stool of allergic infants compared to healthy individuals (52,
53). Bacterial colonization early in life has been shown to affect
cytokine production by T helper cell subsets, implicating that dys-
biosis at an early age may increase the risk of developing food
allergy (54). Likewise, infants that have developed eczema by the
age of 12 months show a lower diversity in the gut microbiota
during the early postnatal period (55). Thus, it appears that low
abundance of Bifidobacteria,Enterococci, and Bacteroides and a
higher abundance of Clostridium spp. and Staphylococcus are asso-
ciated with loss of tolerance and an exaggerated allergic response
toward food-derived antigens. However,it was recently shown that
Clostridium butyricum can induce IL-10 producing macrophages
in the gut in a TLR2-dependent manner and suppresses TLR4
expression by colonic IEC (56,57). Hence, host–microbiome
interactions not only promote a normal Th1/Th2 balance, but sup-
port the development of Treg responses as well. Whether changes
in microbiota composition are a factor to promote an allergic
response to food or are a consequence of food allergy remains to
be studied.
It is important to note that not only changes in the microbiota
are present in individuals with food allergy, but the response of
immune cells toward the microbiota has also been described to
be different. The so-called beneficial bacteria are not necessarily
associated with anti-inflammatory responses in allergic patients.
For example, although an increased prevalence of Bifidobacteria
is rendered as beneficial, specific Bifidobacterium strains isolated
from the feces of allergic infants were shown to induce increased
production of the pro-inflammatory cytokines IL-1β, IL-6, and
TNF-α(58). This is supported by the observation that the aller-
gic infants showed an increased IL-6 and TNF-αresponse toward
TLR2, TLR4, and TLR5 stimuli (59).
Using in vitro models it was shown that IEC play an impor-
tant role in discrimination between different bacterial strains at
the apical membrane (60,61). In addition, commensal bacteria
have the capacity to enhance TLR expression by IEC (62–66).
This suggests that TLR responses toward microflora constituents
may be important. However, not all bacterial strains are equally
effective in suppressing food allergy. This is reflected by the selec-
tive capacity of bacterial strains to induce Foxp3+Treg cells in a
murine model for OVA-induced asthma and OVA-induced food
hypersensitivity (67). Similarly, only specific Lactobacillus strains
attenuate Th2 responses by inducing CD103+tolerogenic DC
(68). Both Lactobacillus and Bifidobacterium strains have been
shown to induce Treg type immune responses, thereby suppressing
allergy (47,69–72). Recently, it has been shown that the bacter-
ial DNA from Lactobacillus spp. or probiotics contain a higher
frequency of immunoregulatory CpG motifs – potentially stimu-
lating TLR9 – when compared to pathogenic bacteria like E. coli,
which is important for Treg conversion in the intestinal mucosa
(73). Exposure of IEC to DNA derived from E. coli or S. dublin
induces high IL-8 production by IEC (19,74), whereas DNA from
Lactobacillus rhamnosus GG prevents NF-κB-induced IL-8 pro-
duction by IEC (66). Similarly,apical exposure of IEC to genomic
DNA from B. breve M-16V was found to enhance IFN-γand IL-10
secretion by PBMC in an HT-29/PBMC co-culture model (26). In
line with this study, it was shown that DC cultured in the condi-
tioned medium of IEC apically exposed to S.Dublin DNA, but not
from B. breve, produced increased amounts of pro-inflammatory
cytokines (75). This suggests that not all probiotic bacterial strains
are potentially effective in treating allergic diseases. Selection of
probiotic bacterial strains should possibly be based on their rich-
ness in CpG motifs, targeting TLR9, and bacterial strains high in
these motifs may be considered for clinical trials.
PREBIOTICS SHAPE THE INTESTINAL MICROBIOTA
Breast feeding also affects the microbiota composition by increas-
ing the amount of Bifidobacteria as shown by higher fecal Bifi-
dobacteria counts (76). Human milk contains a high amount of
non-digestible oligosaccharides with over 1000 different oligosac-
charide structures and it has been shown that human milk, as well
as specific dietary fibers like chicory-derived inulin and lactose-
derived short-chain galacto-oligosaccharides (scGOS), selectively
support the growth of Lactobacillus and Bifidobacterium strains
(77). Therefore, these oligosaccharides have prebiotic effects in
the intestine. Based on the basic structure and size of neutral
non-digestible oligosaccharides present in human milk, a specific
prebiotic mixture consisting of scGOS and long-chain fructo-
oligosaccharides (lcFOS) in a 9:1 ratio has been developed. Oral
supplementation of scGOS/lcFOS has been shown to reduce aller-
gic symptoms in mice and humans (78–80). Especially dietary
supplementation with a combination of scGOS/lcFOS and B. breve
M-16V (GF/Bb) is effective in reducing allergic symptoms (81,
82). In a colitis model in rats, inulin, and FOS reduced coli-
tis, which was associated with increased Bifidobacterium species
and reduced Enterobacteriaceae and C. difficile in the feces (83).
The underlying mechanisms are not known. However, exposure
of IEC to GF/Bb may result in the generation of tolerogenic DC
and consequently Treg polarization in the GALT. In addition to
supporting Treg conversion, stimulation of the growth of Lacto-
bacillus and Bifidobacterium strains may also improve the intesti-
nal barrier function in a TLR2 and potentially TLR9 dependent
manner (84,85).
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de Kivit et al. TLR, microbiota, and gut health
CIRCADIAN CLOCK AND TLR
Although the type of microbiota composition is a critical fac-
tor for the state of TLR activation in the gut of patients with
allergic disorders, other environmental factors can also influence
TLR activation. It has recently been shown that the expression
of TLRs is under regulation of the circadian clock. This impli-
cates that the expression of TLRs is not temporally fixed in a 24-h
day and night cycle. Recently, the expression of TLR9 as well as
other TLRs were shown to be regulated by the circadian clock
(86,87). Interestingly, the severity of TLR9-mediated induction of
sepsis is associated with the time-dependent expression of TLR9
(86). Moreover, further studies have indicated that the interaction
between the microbiota and TLRs expressed by the gut epithelium
is dependent on the circadian rhythm as well (88). Besides the
observation that the expression of TLRs is under circadian con-
trol, cytokine production by macrophages and CD4+T cells, the
suppressor function of Foxp3+Treg cells,leukocyte trafficking ,and
antibody production also show a circadian pattern (89–97). Fur-
thermore, it was recently shown that the circadian clock is critical
for regulation of intestinal permeability as well, as disruption of the
circadian rhythm led to increased microbial translocation and dis-
ruption of the epithelial tight junctions (98). Hence, interactions
between the microbiota and the intestinal mucosal immune system
may not only be dependent on the type of bacterial species present
in the microbiome, but are also temporally regulated, which may
contribute to regulation of immune responses in the intestine.
These data may explain why many allergic reactions like asthma
attacks occur in the early morning (99,100). Recently, it was shown
that the expression of the FcεRI by mast cells and IgE-mediated
mast cell degranulation is temporally regulated by the circadian
clock (101,102). Also, it might, at least partially, explain the rapid
rise of incidence of (food) allergies in western societies where dis-
ruption of normal circadian patterns and stress is a consequence
of modern day society (103).
IMPLICATIONS FOR THE USE OF PRO- AND PREBIOTICS
There is still controversy about the effectiveness of probiotic and
prebiotic treatment in food allergy (104). However, given the
data that alteration of the gut microbiota influences mucosal
immune responses in the gut indicates that treatment using
FIGURE 1 | Schematic overview of potential interactions between the
gut microbiota and the intestinal mucosal immune system. A healthy
gut microbiota composition is high in the frequency of Bacteroides spp.,
Lactobacillus spp., and Bifidobacterium spp. (1) In particular, Bacteroides
fragilis supports Th1 and Treg polarization in a TLR2-dependent manner
through recognition of polysaccharide A by gut DC. Genomic DNA of
Bifidobacterium spp. and Lactobacillus spp. – rich in unmethylated CpG
motifs – potentially interact with TLR2 and/orTLR9 to enhance the
intestinal epithelial barrier function (2) and to support Treg conversion via
CD103+DC (3). Furthermore, apical TLR9 activation by IEC suppresses
NF-κB activation (3). In food allergy, the microbiota composition shifts
toward a higher frequency in Proteobacteria spp., Clostridium spp., and
Enterobacteriaceae. This may favor TLR4 mediated barrier disruption
facilitating allergen translocation in the gut mucosa (4) and
pro-inflammatory cytokine production (5) in a NF-κB-dependent fashion,
sustaining an allergic inflammation. Specific non-digestible
oligosaccharides (prebiotics) support the growth of Bifidobacterium spp.
and Lactobacillus spp. and suppresses the growth of Clostridium spp. and
Enterobacteriaceae, which may contribute to induction of tolerance toward
allergens in the intestines.
Frontiers in Immunology | Immunological Tolerance February 2014 | Volume 5 | Article 60 | 4
de Kivit et al. TLR, microbiota, and gut health
specific probiotic bacterial strains as well as prebiotics may be
useful in treatment for food allergy (Figure 1). Selection of the
right bacterial strains appears key to the effect of treatment using
probiotics. Especially,characterization of specific probiotics based
on CpG rich motifs in the DNA may improve the selection of
potential beneficial strains. Hence, studies aimed at the interaction
between probiotic bacteria and epithelial expressed TLRs may be
warranted. In addition, timing of treatment may play an essential
factor in the effectiveness of treatment using pro- and prebiotics
as expression of TLRs and immune cell functions appears to be
regulated by the circadian clock. In conclusion, more studies are
necessary focusing on interaction between the gut epithelium and
gut bacteria, both in terms of selecting potential beneficial bacterial
strains as well as appropriate timing of intervention.
AUTHOR CONTRIBUTIONS
Sander de Kivit wrote the manuscript; Mary C. Tobin, Christopher
B. Forsyth carefully reviewed the manuscript; Ali Keshavarzian
and Alan L. Landay reviewed the manuscript and provided overall
supervision.
ACKNOWLEDGMENT
This work is financially supported by a generous gift from Mr. and
Mrs. Burridge.
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Conflict of Interest Statement: The authors declare that the researchwas conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 02 December 2013; accepted: 03 February 2014; published online: 18 February
2014.
Citation: de Kivit S, Tobin MC, Forsyth CB, Keshavarzian A and Landay AL (2014)
Regulation of intestinal immune responses through TLR activation: implications for
pro- and prebiotics. Front. Immunol. 5:60. doi: 10.3389/fimmu.2014.00060
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