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In vitro models for gluten toxicity: relevance for celiac disease
pathogenesis and development of novel treatment options
Katri Lindfors1, Tiina Rauhavirta1, Satumarja Stenman1, Markku Ma
¨ki1and Katri Kaukinen2,3
1
Pediatric Research Center, University of Tampere and Tampere University Hospital;
2
School of Medicine, University of Tampere, 33014
Tampere;
3
Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, 33521 Tampere, Finland
Corresponding author: Dr Katri Lindfors, Pediatric Research Center, School of Medicine, Finn-Medi 3, University of Tampere, Tampere
33014, Finland. Email: katri.lindfors@uta.fi
Abstract
In genetically predisposed individuals, dietary gluten in wheat, rye and barley triggers celiac disease, a systemic autoimmune
disorder hallmarked by an extensive small-bowel mucosal immune response. The current conception of celiac disease
pathogenesis is that it involves components of both innate and adaptive immunity whose activation typically leads to
small-bowel villous atrophy with crypt hyperplasia. Currently, the only effective treatment for celiac disease is a strict
lifelong gluten-free diet excluding all wheat-, rye- and barley-containing food products. During the diet, the clinical
symptoms improve and the small-bowel mucosal damage recovers, while re-introduction of gluten into the diet leads to
re-appearance of the symptoms and deterioration of the small-bowel mucosal architecture. In view of the restricted nature
of the diet, alternative treatment is warranted. Improved understanding of the molecular basis of celiac disease has
enabled researchers to suggest other therapeutic approaches. Although there is no animal model reproducing all features
of celiac disease, the use of in vitro approaches including a variety of cell lines and the celiac patient small-bowel mucosal
biopsy organ culture has generated knowledge about pathogenesis of celiac disease. In these culture systems, gluten
induces different effects that can be quantified, thus also enabling studies concerning the efficacy of candidate
therapeutic compounds for celiac disease. This review describes the intestinal epithelial cell models, celiac patient T-cell
lines and clones, as well as the small-bowel mucosal organ culture methods widely used in studies of celiac disease, and
summarizes the major findings obtained with these systems.
Keywords: celiac disease, gluten, epithelium, T-cells, organ culture
Experimental Biology and Medicine 2012; 237: 119 –125. DOI: 10.1258/ebm.2011.011294
Introduction
Dietary gluten in wheat, rye and barley is the etiological
agent in celiac disease. In a subgroup of individuals carry-
ing the human leukocyte antigen (HLA) DQ2 or DQ8 mol-
ecules, ingestion of gluten leads to a small-bowel mucosal
immune response and development of celiac disease. The
disorder often presents with small-bowel villous atrophy,
crypt hyperplasia and immune activation, including the
production of disease-specific antibodies against gluten-
derived gliadin peptides and an endogenous protein trans-
glutaminase 2 (TG2).
1
The current conception of celiac
disease pathogenesis is that it involves components of
both innate and adaptive immunity. Gluten contains a
high amount of repetitive glutamine- and proline-rich
sequences, making it highly resistant to proteolytic degra-
dation by gastrointestinal enzymes.
2,3
This results in the per-
sistence of relatively large gluten-derived gliadin peptides
which predispose to activation of the small-bowel mucosal
immune system. Some of the gliadin peptides (the so-called
toxic peptides, for example p31–43) are able to induce
harmful effects on the small-intestinal epithelium through
interleukin (IL)-15, whose overexpression eventually leads
to epithelial cell destruction.
4,5
Other gliadin peptides, the
immunodominant ones ( for example p57– 68 and the
33-mer) in turn activate T-cell-mediated adaptive immunity
in the mucosa. A key step in this process is the post-
translational modification of immunogenic gliadin peptides
by TG2. TG2 has the capacity to deamidate distinct gluta-
mine residues in gliadin peptides to glutamic acid, which
favors their interaction with the celiac-type HLA DQ2,
thereby promoting the activation of T-cells.
3,6,7
Similar to other autoimmune disorders, celiac disease is
self-perpetuating if the specific exogenous trigger, gluten,
is not removed. Its removal by a strict gluten-free diet
ISSN: 1535-3702
Copyright #2012 by the Society for Experimental Biology and Medicine
Experimental Biology and Medicine 2012; 237: 119 – 125
leads to recovery of the small-bowel mucosal lesion and dis-
appearance of the clinical symptoms. Re-introduction of
gluten into the diet, on the other hand, results in the
re-appearance of clinical symptoms and deterioration of
the small-bowel mucosal architecture. Today, the only effec-
tive treatment for celiac disease is a lifelong gluten-free diet
excluding all wheat-, rye- and barley-containing food pro-
ducts. As the diet is restricted and often burdensome,
novel treatment options would be highly welcomed by
celiac patients. During recent years, several lines of research
have in fact aimed to develop alternative therapies.
Approaches include blocking of innate and adaptive
immune components as well as the function of TG2.
8
Another prominent strategy is to hydrolyze gluten by
exogenous enzyme supplements designed for this
purpose. This promising strategy is based on the finding
that distinct peptidases or their combinations from different
sources are able to hydrolyze gliadin and gliadin peptides
in vitro.
3,9
Indeed, data are available to indicate that distinct
peptidase is capable of accelerating the degradation of
gluten in the gastrointestinal system, closely mimicking
in vivo digestion and suggesting that gluten toxicity might
be eliminated by such an approach.
10
Regardless of the progress made in understanding the
pathogenesis of celiac disease, the most conspicuous limit-
ation in the field is the lack of a functional disease-specific
animal model. Such a model would also be highly appreci-
ated among researchers developing novel treatment strat-
egies for the disease, as the initial testing of novel drug
candidates could then be conducted in animals. A number
of attempts have been made to create an animal model for
celiac disease by various approaches. These include the gen-
eration of transgenic mice expressing the celiac-type human
HLA DQ2 or DQ8
11 – 14
and introduction of celiac-type anti-
bodies in mice,
15 – 17
but none of the animals used have
developed a full-blown villous atrophy together with
crypt hyperplasia that are characteristic for untreated
celiac disease. Gluten-dependent small-intestinal mucosal
damage can be induced in mice
18
and has been described
in rhesus macaques,
19
but these two models are not depen-
dent on celiac-type HLA and their applicability as models
for celiac disease has thus to be considered with caution.
Very recently, DePaolo et al.
20
reported that gliadin-fed
humanized HLA-DQ8 mice that overexpress IL-15 in the
lamina propria (DQ8-D
d
-IL-15 transgenic mice) present
with a condition resembling early developing celiac
disease, where intestinal inflammation is present but the
mucosal architecture remains normal. This promising
model may prove useful in the future, although its applica-
bility needs to be verified in further studies.
Regardless of numerous attempts, the animal model for
celiac disease that reproduces all the aspects of the disorder
still awaits development. However, the use of in vitro
approaches including a variety of cell lines and the celiac
patient small-bowel mucosal biopsy organ culture has gen-
erated knowledge about pathogenesis of celiac disease and
enabled the initial testing of novel treatment options for
celiac disease. This review describes the cell-based and
organ culture methods widely used in celiac disease and
summarizes the major findings obtained with these systems.
Epithelial cell culture models
After ingestion and progress to the small intestine, the
gluten-derived gliadin peptides come into contact with the
small-bowel mucosal epithelium. In untreated celiac
disease, small-bowel mucosal epithelial cells proliferate
more rapidly without differentiating to enterocytes, and
their turnover by apoptosis is augmented. Moreover, the
epithelial junctional integrity is compromised
21,22
and the
gliadin peptides therefore gain access to the lamina
propria. However, it is not well understood how gluten-
derived gliadin peptides modulate the biology of the epi-
thelium and how they cross the epithelial barrier prior to
substantial mucosal remodeling during the early stages of
the disorder. For all of the above-mentioned reasons,
study of the intestinal epithelium in the context of celiac
disease is thus manifestly relevant. There are several intesti-
nal epithelial cell lines available, but two of those mostly
used in celiac disease research are described here.
T84 cells
T84 cells are derived from a lung metastasis of a colon
cancer, and the cell line was first established as a model
for studying intestinal epithelial chloride transport. When
grown on permeable supports, the cells form confluent
polarized layers with high transepithelial resistance, and
they evince a chloride secretory response to different
chemicals.
23
In addition, the polarized T84 cell layers are
characterized with well-delineated intracellular junctions
including circumferential tight junctions.
24
For the above-
mentioned reason, the cell line, although of colonic origin,
is widely used in studying intestinal epithelial permeability
to macromolecules and ions even though the cells do not
present with features of intestinal enterocyte-type differen-
tiation in such cultures. However, when T84 cells are cul-
tured three-dimensionally in collagen in the presence of
either fibroblasts or transforming growth factor
b
, they
differentiate to intestinal enterocytes as assessed by mor-
phological and biochemical criteria.
25
T84 cells have been applied in research into celiac disease
pathogenesis, for instance in studying the innate immune
reactions elicited by the toxic gliadin peptides. It has been
shown that the toxic gliadin peptide p31– 43 creates cellular
stress by inducing the production of reactive oxygen
species
26
and Fas-dependent apoptosis.
27
Furthermore, it
has been shown that untreated celiac disease patients’
serum antibodies induce proliferation and reduce differen-
tiation of the three-dimensionally cultured T84 cells
28
and
increase the permeability of the T84 cell epithelial mono-
layer.
29
Moreover, Bethune et al.
30
have demonstrated that
interferon (IFN)-
g
derived from gluten-stimulated celiac
patient–specific intestinal T-cells enhances the passage of
gluten through the T84 epithelial layer.
Caco-2 cells
The Caco-2 cell line was originally obtained from a relatively
well-differentiated human colon adenocarcinoma. When
reaching confluence, the Caco-2 cells differentiate
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120 Experimental Biology and Medicine Volume 237 February 2012
spontaneously.
31
In such cultures, the cells are polarized
and possess an ultrastructural morphology of differentiated
enterocytes with an apical surface covered by microvilli.
32
In
the monolayer, the cells are joined together through apical
junctional complexes which include tight junctions and des-
mosomes.
33
Postconfluent cultures are hallmarked by the
emergence of domes in which cells express sucrase isomal-
tase, alkaline phosphatase and aminopeptidase N,
enzymes typical for differentiated enterocytes.
33
Owing to
the above-mentioned characteristics, the Caco-2 cell line is
widely used as a model for the intestinal epithelial barrier.
Caco-2 cells probably comprise the most extensively used
epithelial cell line in studies related to celiac disease.
Gliadin or distinct gliadin peptides are reported to induce
actin rearrangement,
34
proliferation,
34,35
intracellular oxi-
dative imbalance
36
and apoptosis in Caco-2 cells.
37,38
Gliadin also inhibits the spontaneous differentiation of
these cells.
39
Such findings suggest that the celiac
disease-inducing agent gliadin itself could at least partly
account for the behavioral abnormalities of the small
bowel epithelial cells in celiac disease.
In view of the importance of the compromised small-
bowel epithelial barrier function in celiac disease pathogen-
esis, researchers have used the Caco-2 cells in studies
seeking to establish which factors could account for this.
At least gliadin and two proinflammatory cytokines abun-
dantly expressed in celiac disease, IFN-
g
and tumor necrosis
factor
a
, have been reported to affect the expression of
several epithelial junctional proteins (for example occludin
and ZO-1) and permeability in Caco-2 cells.
40 – 48
Caco-2 cells have also been used in studies clarifying how
individual gliadin peptides are processed in intestinal epi-
thelial cells. Using Caco-2 cells it has been found that both
the toxic p31– 43 and the immunodominant p57–68
gliadin peptides are taken up by the cells and interact
with the endocytosis compartment.
49
However, the toxic
and the immunodominant gliadin peptides seem to be dif-
ferentially processed in Caco-2 cells,
49
but at least the immu-
nodominant 33-mer gliadin peptide eventually crosses the
epithelial barrier to the luminal side.
50
These findings in
Caco-2 cells, together with others made in celiac patient
biopsies or mucosal organ cultures, have considerably
increased our understanding of gliadin processing in intes-
tinal epithelial cells, this presumably being crucial for our
understanding of celiac disease pathogenesis.
The epithelial modulating properties of yet another factor
possibly participating in celiac disease pathogenesis,
namely celiac disease antibodies, have also been studied
in Caco-2 cells. It has been shown that celiac patient anti-
bodies induce actin remodeling,
51
interfere with the
uptake of p31–43
52
and modify its intracellular transport,
53
and further induce proliferation in Caco-2 cells.
51,54
Moreover, celiac patient IgA has been reported to increase
the epithelial permeability of both toxic ( p31 –43) and
immunodominant ( p57–68) gliadin peptides.
55
The Caco-2 cell line has also been used fairly extensively
in initial testing of novel treatment options for celiac
disease. The cell line has been used in studying the
potency of at least a tight junction modulator, zonulin
antagonist, Bifidobacteria, germinating cereal enzymes
and polymeric binders of gliadin as putative future treat-
ment options.
41 – 43,45,56 – 59
All the studies in question
demonstrated protective effects of the tested compounds
against gluten-induced effects in Caco-2 cells, but their effi-
cacy obviously need to be tested in other systems as well as
in patients. Taken together, the use of Caco-2 cells in celiac
disease research has shed significant light on the disease
pathogenesis and the proof-of-principle efficacy of novel
treatment options.
Celiac patient-derived T-cell lines and clones
The extensive evidence that CD4þT-cells, associated with
recognition of HLA DQ2 and DQ8 molecules, are markedly
involved in the pathogenesis of celiac disease has inspired
researchers to develop in vitro T-cell models. The celiac
disease-specific T-cells, raised against gluten, have been
obtained both from the small-intestinal mucosa
60 – 62
and
the peripheral blood
63 – 65
of patients suffering from celiac
disease. The HLA-DQ2 or DQ8 restricted T-cells isolated
from small-bowel mucosal biopsies are typically cultured
in vitro in the presence of stimulating gluten peptides
(T-cell lines), whereafter single, gluten-reactive monoclonal
cells (T-cell clones) can be further isolated and maintained
in the culture conditions. In contrast, peripheral blood
mononuclear cells are isolated from treated celiac disease
patient’s serum after a short-term in vivo gluten challenge.
66
In both cases, activation of T-cells is typically presented as
an increase in cell proliferation and IFN-
g
-predominant
cytokine production.
62,64,67
Most of the gut-derived T-cells do not recognize native
gluten peptides unless they are deamidated by tissue trans-
glutaminase,
6,7
a phenomenon which is thought to occur
also in in vivo conditions. The majority of studies conducted
with celiac patient T-cells have concentrated on identifi-
cation of gliadin epitopes such as diverse epitopes found
in immunodominant
a
-gliadin 33-mer;
3,68
however, intesti-
nal T-cell lines and clones have also been shown to recog-
nize deamidated rye secalin and barley hordein
peptides.
69,70
T-cell lines and clones have been especially
useful in the systematic characterization of T-cell reactive
gluten epitopes in wheat, rye and barley.
71 – 73
However,
patients seem to respond to distinct gluten peptides,
74,75
resulting in limitations when investigating the overall tox-
icity of gluten peptides. Application of several simultaneous
T-cell lines from different patients is thus probably needed
to overcome these challenges.
Similarly to intestinal epithelial cells, celiac patient T-cell
lines and clones have been used in testing newly developed
treatment forms. For instance, testing of different types of
modified gliadin or gluten peptides
76 – 79
and enzyme
therapy
3,42,80 – 83
as a potential novel treatment form has
been carried out in celiac patient T-cell lines or clones.
Small-bowel mucosal biopsy organ culture
The culturing of small-intestinal mucosal biopsies derived
ex vivo from celiac disease patients was originally intro-
duced by Browning and Trier.
84
The biopsies can be kept
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Lindfors et al. In vitro models for gluten toxicity 121
viable in the organ culture system for 24 – 48 h
85
or even
longer, although there is no guarantee of the quality of
mucosal morphology after a long-term culture.
86
In prelimi-
nary studies, the organ culture method was used in an
opposite manner to the current practice. Culture conditions
were thought to mimic a gluten-free diet, so that the small-
bowel mucosal morphology improved after gluten was
excluded.
85 – 88
In contrast, the present organ culture
system is generated to illustrate the harmful effects of
gluten, gliadin or distinct gliadin peptides added to the
culture supernatant (Figure 1). Since the ex vivo biopsy
culture contains a wide variety of cell types expressed in
the intestinal mucosa in vivo, the method allows researchers
to investigate an extensive range of markers for gluten tox-
icity, all the way from the epithelium to activation of
immune components in the mucosal lamina propria. One of
the versatile benefits of the method lies in the fact that
various features characteristic for the disorder can be repro-
duced in biopsies from treated celiac disease patients, thus
allowing researchers to find mechanisms related to the
development of celiac disease from a healthy mucosa into
the active phase of the disease. In addition, some researchers
have suggested the organ culture method even as a poten-
tial tool for celiac disease diagnosis.
89,90
As the small-bowel mucosal biopsy organ culture system
has been used extensively in studies aiming to clarify the
pathogenesis of celiac disease, it would be impossible to
summarize the entire literature in this review. However,
since biopsies contain a number of different cell types, the
issues open to investigation in the system are numerous.
For instance, celiac disease researchers have studied the
immune mechanisms
4
as well as the contribution of different
cytokines
91
in this context. As an example, one of the major
findings related to celiac disease pathogenesis, namely the
importance of IL-15 during the disease process, was obtained
from studies conducted with the organ culture.
5,92 – 96
Considering that the small-bowel organ culture method
has been widely used in studies aiming to clarify the patho-
genesis of celiac disease, it is surprising that the method has
only been used in a few studies related to novel treatment
forms. To our knowledge, it has only been applied in study-
ing the therapeutic potential of a particular zonulin
antagonist
45
and gliadin-degrading proteolytic
enzymes,
42,81,97
with promising results.
Conclusions
In the absence of an animal model for celiac disease, research-
ers have used different cell lines and the celiac patient small-
bowel mucosal organ culture method in studying different
aspects of celiac disease pathogenesis and proof-of-concept
testing of novel treatment options which could in the future
replace the only effective treatment for celiac disease, the
gluten-free diet. Although widely used, all of the models pre-
sented in this review have their limitations and thus do not
always correlate with the circumstances in the human small
intestine in vivo. For example, the epithelial cell lines are not
derived from celiac disease patients but instead are of cancer-
ous origin. Moreover, the epithelial cells and celiac patient
T-cell lines and clones lack connection to other cell types
present in the intestine. In addition, the small-bowel organ
culture system lacks circulation, nervous system and connec-
tion to lymphatic organs. Furthermore, the organ culture
system is not a high-throughput method. In the future it
would thus be worthwhile to develop novel and improved
culture systems and to promote celiac disease-specific
models. However, even regardless of the above-mentioned
shortcomings, researchers have gained important infor-
mation and been able to addpieces to the celiac disease patho-
genesis puzzle using the models described in this review and
also other available model systems. Moreover, the studies
clarifying the efficacy of novel treatment options carried out
with the models described here have yielded encouraging
results and some potentially therapeutic compounds have
already entered phase II clinical trials.
Author contributions: KL, TR, SS, MM and KK all partici-
pated in the writing of the manuscript, and TR made the
figure.
ACKNOWLEDGEMENTS
The Celiac Disease Study Group have been financially sup-
ported by the Academy of Finland, the Sigrid Juselius
Foundation, the Foundation for Pediatric Research, the
Pirkanmaa Cultural Foundation, the Competitive Research
Funding of Tampere University Hospital, the Research
Fund of the Finnish Celiac Society and the European
Commission IAPP grant TRANSCOM (contract number
PIA-GA-2010-251506).
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(Received September 1, 2011, Accepted October 19, 2011)
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