ARTICLE IN PRESS

Clinical Nutrition (2005) 24, 339–352

http://intl.elsevierhealth.com/journals/clnu

REVIEW

Aetiology of inflammatory bowel disease (IBD):

Role of intestinal microbiota and gut-associated
lymphoid tissue immune response
Oscar C. Thompson-Chagoyána, José Maldonadob, Angel Gilc,
’’
a
Department of Paediatrics, Los Venados’’ General Hospital, Mexican Institute of Social Security,
México City, México
b
Department of Paediatrics, Faculty of Medicine, University of Granada, Granada, Spain
c
Department of Biochemistry and Molecular Biology, School of Pharmacy, Faculty of Pharmacy,
University of Granada, Campus Cartuja, 18071 Granada, Spain

Received 18 February 2005; accepted 21 February 2005

KEYWORDS Summary The aetiology of inflammatory bowel disease (IBD) probably involves a
Inflammatory bowel combination of genetic predisposition and environmental factors that may be
disease; channelled through an abnormality in gut-barrier function, with a loss of antigen
Intestinal tolerance. Some genetic markers that predispose to inflammatory disease have been
microbiota; identified (alleles DR2, DRB1*0103, DRB1*12 and mutations in the NOD2/CARD15
Intestinal tolerance; gene on chromosome 16). Alterations in the pattern of cytokine production by T cell
Probiotics subclasses leading to loss of tolerance to oral antigens have been documented.
Moreover, a number of environmental factors (cigarette smoking, use of non-steroid
anti-inflammatory drugs, psychological stress and the presence of the caecal
appendix) have been postulated as a trigger of IBD. It has also been suggested that
the gut microbiota plays a major role in the development and persistence of IBD, and
numerous modifications of intestinal microbiota composition have been identified.
As a result, manipulation of the microbiota with antibiotics is a current therapeutic
strategy; more recently, however, a number of studies have reported promising
results when using probiotic organisms to manipulate gut microbiota composition in
order to restore tolerance to microbial antigens of the host’s own microbiota.
& 2005 Elsevier Ltd. All rights reserved.

Corresponding author. Tel.: +34 958 246139; fax: +34 958 248960.
E-mail address: agil@ugr.es (A. Gil).

0261-5614/$ - see front matter & 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.clnu.2005.02.009
 ARTICLE IN PRESS
340 O.C. Thompson-Chagoyán et al.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

Aetiological theories of IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Inappropriate reaction to a specific persistent pathogenic infection of the intestine . . . . . . . . . . . . . . . . 341
Subtle alterations in bacterial composition and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Overexposure to normal resident luminal bacterial products that induce an impairment of the mucosal
barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Aberrant host immune response including loss of tolerance to normal ubiquitous luminal antigens . . . . . . . 342

Intestinal microbiota in healthy and IBD subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

Functions of gut microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Metabolic functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Trophic functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Protective functions: the barrier effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Intestinal microbiota in human IBD patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
Immune response to antigens in the gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

Immune response and oral tolerance in IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

Mechanisms of oral tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Cellular mechanisms of oral tolerance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Active suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Clonal anergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Clonal deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

Potential roles of probiotics in the treatment of IBD: A way of inducing oral tolerance . . . . . . . . . . . . . . . . . 347
Probiotics in experimental models of colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Evidence supporting the successful response to probiotics for the prevention or treatment of IBD in
animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Probiotics in human inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Possible mechanisms of probiotic action in IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

Introduction respond in vitro to the antigens of their own flora

Inflammatory bowel disease (IBD) is a chronic
inflammatory condition of the gastrointestinal tract
that manifests as ulcerative colitis (UC) or Crohn’s
disease (CD). Notwithstanding intensive research,
the aetiology of this condition remains unknown;
however, it is thought to result from a combination
of genetic predisposition and environmental factors
that may be channelled through an abnormality in
gut-barrier function.1
Between 17% and 25% of patients with CD have
mutations in the NOD2/CARD15 gene on chromo-
some 16 that predispose to CD.2,3 The NOD2/
CARD15 gene codes for a protein that activates
monocyte NFkB in response to bacterial lipopoly-
saccharides, and it has been suggested that the
NOD2/CARD15 gene product confers susceptibility
to CD by altering the recognition of bacterial
components and/or altering the activation of host
response to bacteria.4 It has been demonstrated
that T cells from the lamina propria in CD patients
with a Th1 polarised response, indicating a loss of
tolerance to their intestinal microbiota.5 This Th1
response induces an increment in the production of
INF-g by CD4 and activated macrophages that
secrete TNFa and IL-126 (Fig. 1).
In UC a positive association has also been
reported with DR2, DRB1*0103 and DRB1*12 al-
leles,7,8 and genome-wide scanning studies have
shown linkage between UC and regions of chromo-
somes 3, 7, and 12.7 An increasing number of single
nucleotide polymorphisms and alleles associated
with IBD have been reported [186 for 1997, 261 for
1999 and 590 for 2003 for B HLA-1 polymorphism;
4235 and 70 for C HLA 1 polymorphism, and 83,125
and 282 for A HLA-1 polymorphism, respectively].
Similar increases have also been reported for HLA-
A*02 alleles in the same years (10, 22 and 65,
respectively). Moreover, it has very recently been
proposed that the differential expression of HLA-G
in UC and CD provides a potential way to distinguish
between these two entities.9
 ARTICLE IN PRESS
Aetiology of IBD 341

Tolerance Inflammation

sIgA Antigens

sIgA

TGF-β
INF-γ, IL-2
Apoptosis TNF-α
Th3
APC
IL-10
IL-4 Peyer’s patches

Th2 Th1
CD-4

IL-10 IL-12
TNF-α
Thr Systemic recirculation

Figure 1 Tolerance vs. inflammation in gut-associated lymphoid tissue (GALT). APC: antigen-presenting cell; IL:
Interleukin; INF g: Interferon g; sIgA: secretory IgA; Th1, Th2, Th3 and Thr: T helper lymphocyte subclasses; TGF-b:
transforming growth factor b; and TNF-a: tumour necrosis factor a.

Several environmental factors associated with
IBD have been reported, and many researchers
believe that environmental factors are more
important than genetic factors, at least in UC;
the main argument adduced in support of this claim
is the low rate of concordance between mono-
zygotic twins with UC (6–14%) compared with twins
affected by CD (44–50%).8
Other factors frequently associated with IBD3,8
include cigarette smoking (protective to UC and
detrimental to CD), use of non-steroid anti-inflam-
matory drugs, psychological stress and the pre-
sence of caecal appendix (appendectomy at a
young age has a protective effect on development
of UC), but the microbial factor is the strongest
environmental factor studied until now. Although a
long search has so far failed to confirm a direct
pathogenic role for a specific infectious agent,
ample evidence suggests that commensal enteric
’’
bacteria and their products are the triggers’’ that
initiate and perpetuate IBD.10
The aim of this review is to examine some of the
main factors influencing the onset and develop-
ment of IBD focusing on the alteration of intestinal
microbiota and gut-associated lymphoid tissue
(GALT) immune responses leading to loss of oral
tolerance to antigens. Since IBD may result from
alterations in intestinal bacterial composition and
overexposure to luminal bacterial products, the
review will focus on how faecal microbial colonisa-
tion and intestinal microbiota composition may
affect the course of the disease. By contrast, some
microorganisms, mainly lactic acid bacteria (LAB),
could have a beneficial impact on IBD, influencing
both microbiota composition and GALT immune
responses. Current data on probiotics and IBD are
reviewed here.
Aetiological theories of IBD
IBD is thought to be due to an impairment of
functional immune responses. The increase in
mucosal B cell and plasma cell populations in IBD
suggests that the disease is antibody-mediated and
complement-dependent, but the main abnormality
driving the inflammation is an exaggerated T cell
response causing mucosal hyper-responsiveness to
commensal bacteria.11
Inappropriate reaction to a specific
persistent pathogenic infection of the
intestine
It has long been proposed that resident bacterial
flora play an essential role in the pathogenesis of
human IBD. Indeed, direct interaction of commen-
sal microbiota with the intestinal mucosa is
believed to stimulate inflammatory activity in gut
lesions. Because CD and UC closely mimic defined
intestinal infections, and occur in areas with the
highest luminal bacterial concentrations, many
microbial pathogens have been suggested as
causes of IBD. These include: Mycobacterium
paratuberculosis and M. kansaii, Escherichia coli,
 ARTICLE IN PRESS
342
Diplostreptococcus sp, Virus (measles, RNA virus),
Lysteria monocytogenes, Fusobacterium necro-
phorum, Chlamydia sp., Listeria monocytogenes,
Pseudomona maltophila and Helicobacter hepati-
cus.10,12 Moreover, faecal stream diversion has
been shown to prevent recurrence of CD, whereas
infusion of intestinal contents to the excluded ileal
segments reactivates mucosal lesions.12
Subtle alterations in bacterial composition
and function
The gut microbiota represents an extremely com-
plex ecosystem. Because the microbiota in healthy
humans is relatively stable when diet remains
unchanged, subtle alterations in bacterial composi-
tion and function may have profound implications
for mucosal barrier function and immune response.
Some modified E. coli strains have been reported in
patients with UC and CD; similarly, patients with
IBD have larger amounts of bacteria attached to
their epithelial surfaces than do healthy people.
Isolated bacteria belong to a wide range of genera;
some are identified within the epithelial layer,
while others are found in intracellular locations.
Nevertheless, the role of these altered bacteria in
IBD pathogenesis remains unclear.13,14
Overexposure to normal resident luminal
bacterial products that induce an
impairment of the mucosal barrier
An alternative mechanism by which enteric micro-
bial agents could stimulate chronic enterocolitis
and systemic inflammation is by means of an
impairment of the intestinal mucosal barrier,
allowing increased uptake of antigens and proin-
flammatory molecules, including endotoxin and
other bacterial products. It has been reported that
mucosal permeability is increased in most intestinal
inflammatory conditions. In such cases, bacterial
components can activate intestinal immune cells,
endothelial cells and epithelial cells to secrete
many substances (cytokines, eicosanoids, oxygen
metabolites, nitrogen oxide and proteases), indu-
cing local damage and inflammation that contri-
butes to disrupting mucosal barrier function.10
Aberrant host immune response including
loss of tolerance to normal ubiquitous
luminal antigens
The inflammatory bowel syndrome may be the
result of an abnormal host response to ubiquitous
antigens, including the normal resident microbiota,
O.C. Thompson-Chagoyán et al.
bacterial products, toxins, and food antigens.
Normal luminal bacteria may induce and perpe-
tuate intestinal inflammation in IBD patients by
initiating an aberrant host immune response,
although in most cases no specific pathogen can
be identified.
Progression or resolution of acute inflammation
after bacterial stimulation depends on the delicate
balance between proinflammatory and immunosup-
pressive forces.10,13 It has been shown that both
mice and humans are normally tolerant to auto-
logous microbiota, and that breakdown of this
tolerance is associated with the development of
chronic intestinal inflammation.5 It may be that
potentially pathological responses to components
of the intestinal flora do occur under physiological
conditions, but these are suppressed by immunor-
egulatory mechanisms.15
There is increasing evidence that the antigens
driving the tissue immune response are derived
from normal bacterial flora. Proof of this concept
has come from studies of at least 11 models of IBD,
in which inflammation is found to be dependent on
the presence of normal flora; the absence of
normal flora is associated with non-appearance of
the illness.13,16–18 This phenomenon is seen in
different species (mice, rats and guinea pigs), and
occurs in manipulated organisms such as transgenic
mice with targeted deletion of the T cell receptor
(TCRa), that spontaneously develop colitis in
response to the gut microbiota.19 It also has been
observed in rats and mice with IBD treated with
broad-spectrum antibiotics, which have been
shown to mitigate mucosal inflammation.20 More-
over; in some models, colonisation with normal
flora rapidly results in T-cell-mediated gut inflam-
mation, and disease can be transferred to other
animals using activated T cells against enteric
bacteria.21 Nevertheless, it appears that a constant
bacterial stimulus is needed for the induction and
continuation of inflammatory process21 and that
not all commensal bacteria have an equivalent
ability to induce mucosal inflammation, but instead
are influenced by the host genetic background.22
Intestinal microbiota in healthy and IBD
subjects
The alimentary tract is a specialised organ that
extends from the lips to the anus, and represents
the largest contact area between the body and the
external environment.23 It has been estimated that
the adult human body contains 1014 cells, of which
only 10% constitute the body proper; the remaining
 ARTICLE IN PRESS
Aetiology of IBD
90% are microbiota cells, mostly living in the lumen
of the large intestine.13,23–28 The fact that we are
90% bacteria is frequently overlooked, and the
significance of the relationship between prokar-
yotes and eukaryotes is only just beginning to be
appreciated.
The mammalian intestinal tract hosts a complex,
dynamic, and diverse society of bacteria. Although
the precise quantitative and qualitative composi-
tion of the indigenous digestive microbiota is not
yet known, more than 100 different bacterial
species have been identified in faecal specimens
from normal individuals, and it is thought that at
least 500 species are present in the digestive
tract.24,25,29 On a quantitative basis, approximately
10–20 genera probably predominate,25 but the real
magnitude of the intestinal microbiota is under-
lined by the fact that the number of genes it
contains is about 50–100 times that of our own
genome.28
The gastrointestinal microbiota can be consid-
ered a functionally and metabolically adaptable
and rapidly renewable organ of the body.4,13,30 Its
relationship with the host is so specific that any
alteration in the balance of organisms might result
in illness; some of its members are considered
potentially harmful in view of their involvement in
toxin production, mucosal invasion, or activation of
carcinogens and inflammatory responses. However,
more recently attention has turned to indigenous
non-pathogenic microorganisms and the ways in
which they benefit the host,13,25,31 since there is
evidence that they play a major role in host
nutrition, physiology, and control of the immune
system.23
Functions of gut microbiota
Evidence obtained with animals feed under germ-
free conditions suggests that gut microbiota have
important and highly specific metabolic, trophic,
and protective functions.29
Metabolic functions
One major metabolic function of the intestinal
microbiota is the fermentation of non-digestible
dietary residue and endogenous mucus produced by
the epithelia.29 Gut-bacteria metabolism is respon-
sible for the conversion of many substances into
metabolites that can be absorbed and used by the
host (60–70% of their energy requirements).29 The
overall outcomes of this complex metabolic activity
are the recovery of metabolic energy and absorb-
able substrates for the host, and the supply of
energy and nutritive products for bacterial growth
343
and proliferation. Fermentation of carbohydrates is
a major source of energy in the colon. Non-
digestible carbohydrates include large polysacchar-
ides (resistant starches, cellulose, hemicellulose,
pectins, and gums), some oligosaccharides that
escape digestion, and unabsorbed sugars and
alcohols.32 The metabolic endpoint is the genera-
tion of the short-chain fatty acids (SCFA) acetate,
propionate and butyrate. Anaerobic metabolism of
peptides and proteins (putrefaction) by the micro-
biota also produces SCFA but, at the same time, it
generates a series of potentially toxic substances
included ammonia, amines, phenols, thiols, and
indols.29
Intestinal microorganisms are also involved in
vitamin synthesis29 and in the absorption of
calcium, magnesium and iron.33 Absorption of ions
in the caecum is improved by carbohydrate
fermentation and SCFA production. All of these
fatty acids have important functions in host
physiology.29
Trophic functions
Possibly the most important role of the intestinal
microbiota in colon physiology is its trophic effect
on the intestinal epithelium.29 Epithelial cell
differentiation is considerably influenced by inter-
action with resident microorganisms and their
metabolic products, mainly short chain fatty acids;
all of these stimulate epithelial cell proliferation
and differentiation of the small and large bowel.34
Protective functions: the barrier effect
Intestinal bacteria form a barrier against non-
indigenous microorganisms, including pathogens.35
All mechanisms that do not allow the installation of
pathogenic bacteria in the human gut are said to
exert a mucosal barrier effect. Intact indigenous
flora are known to represent a formidable barrier to
the establishment of pathogenic populations on
’’
host surfaces; this phenomenon is called bacterial
’’
antagonism’’, bacterial interference’’ or more
’’
commonly colonisation resistance’’. This barrier
effect includes many activities such as production
of bacteriocins by indigenous flora components,
production of metabolic products which will be
toxic for pathogens, conditions inhibitory to patho-
genic organisms, such as low pH and depletion of
nutrients required for their multiplication.36 Ad-
herent non-pathogenic bacteria can prevent at-
tachment and subsequent entry of pathogenic
enteroinvasive bacteria into epithelial cells.37
Furthermore, bacteria compete for nutrient avail-
ability in ecological niches, and maintain their
collective habitat by administering and consuming
all resources. The host actively provides a nutrient
 ARTICLE IN PRESS
344
that the bacterium needs, and the bacterium
actively indicates to the host how much it needs.
This symbiotic relationship prevents unwanted
overproduction of the nutrient, which would favour
intrusion of microbial competitors with potential
pathogenicity for the host.29 Finally, bacteria can
also inhibit the growth of their competitors by pH
modification, control of motility, nutrient depletion
and production of anti-microbial substances called
bacteriocins.30
Intestinal microbiota in human IBD patients
Major modifications of the intestinal microbiota
reported in patients with IBD include: high con-
centrations of mucosal bacteria in both inflamed
and non-inflamed bowel tissue14; increased num-
bers of coliforms and bacteroids, and a decrease in
LAB38; and increased E. coli numbers in the colon.39
Immune response to antigens in the gut
After reaching the lamina propria, the antigens
encounter various host defence mechanisms and
are internalised and processed by antigen-present-
ing cells (APC) for presentation to T cells. Recogni-
tion of antigen by T cells requires interaction
between the antigen, the major histocompatibility
antigen complex on the surface of the APC and the
T cell antigen–receptor complex (TCR) on the T
helper cell. An appropriate interaction leads to
activation of the T cell. T cells are classified as CD4+
and CD8+ T cells; CD8+ cells (suppressor cells) have
a downregulatory influence on the immune system
and can kill cells infected with cytosolic pathogens.
CD4+ cells (helper cells) are currently divided into
four types—Th1, Th2, Th3 and Tr—distinguishable
by their differential pattern of cytokine secretion:
Th1 cells release interleukin (IL)-2, interferon (INF)
g, and tumour necrosis factor (TNF)-a; Th2 cells
secrete IL-4, IL-5, IL-6 and IL-10; Th3 cells secrete
mainly transforming growth factor (TGF)-b; and T
Table 1 Alteration in cytokine production by CD4+ Th cells in IBD.
CD4+ T cell (Helper T cell)
mTh1 CD4+ kTh2 CD4+
mRelease kRelease
IL2 IL-4
IFN-g IL-5
TNF-a IL-6
IL-10
Mediate cellular immune responses Mediate humoral immune responses
O.C. Thompson-Chagoyán et al.
regulatory cells (Tr) are the main source of IL-
10.40,41 An altered pattern of cytokines is observed
in IBD compared with tolerant subjects (Fig. 1).
Increased release of IL-2, INF-g TNF-a, which
mediate cellular immune responses, and decreased
release of IL-4, IL-5, IL-6, IL-10, which are
implicated in humoral immune responses, in con-
junction with decreased secretion of TGF-b by Th3
cells and IL-10 by Thr, are well known to occur in
IBD (Table 1).
Lack of peripheral T cell response may be
achieved in three different ways: (1) the T cells
encounter the antigen in an irrelevant immunolo-
gical form and are thus unaware of it; (2) the T cells
may encounter the antigen in circumstances that
result in the subsequent functional or actual
elimination of the cell (anergy or deletion); (3)
regulatory cells or mediators can modify the
immune response.
Whether antigens induce peripheral immunity or
tolerance may be dependent on costimulatory
signals like CD40, CD44v7, CD80 and CD86, all of
them provided to the T cells by APC.15,40
Immune response and oral tolerance in
IBD
The gastrointestinal tract provides a protective
interface between the internal environment and
the constant challenge from food-derived antigens
and from microorganisms in the external environ-
ment. A mutual interdependence exists between
the gut microbiota and the host immune system.
Successful coexistence with a complex microbiota
presents a particular challenge to the immune
system of the host. On the one hand, the host needs
to avoid an overly aggressive response to this
microbial population that would lead to the
elimination of beneficial organisms and would
almost certainly result in inflammation and exten-
sive tissue damage. On the other, the ability to
kTh3 kThr
kRelease kRelease
TGF-b IL10
 ARTICLE IN PRESS
Aetiology of IBD
limit the spread of bacteria from the lumen into
underlying tissues and to mount an effective
response to intestinal pathogens needs to be
maintained.4 Mucosal immune response and non-
response are marked by their ability to distinguish
between the antigenic challenge posed by patho-
genic invaders, and not dangerous resident organ-
isms or proteins in the food stream.
The first line of host defence is directed towards
the exclusion of antigens, the elimination of foreign
antigens that have penetrated the mucosa, and the
regulation of the ensuing antigen-specific immune
response. As a result, the gastrointestinal barrier
controls antigen transport and the generation of
immunological phenomena in the gut; but even in
physiological conditions, an insignificant but im-
munologically important fraction of antigens bypass
the defence barrier4 and the intestinal immune
system is faced with the dilemma of discriminating
between antigens with no pathogenic potential and
antigens and microorganisms that are potentially
harmful. It has long been recognised that the
encounter of dietary antigens by the immune
system normally leads to abrogation of the immune ’’
response. This phenomenon has been termed oral
tolerance’’41 and is defined as the induction of a
state of systemic immune non-responsiveness to
orally administered antigen upon subsequent anti-
gen challenge. This mechanism presumably pre-
vents the development of an immune reaction or
allergy against intestinal intraluminal antigens.
Thymus
CD4+ CD8+
Peripheria
CD8+
CD8+ CD45hi CD4+
CD45RBhi
Th2
Autoimmunity and inflammation
IBD, experimental granulomatose
colitis, autoimmune thyroiditis
Figure 2 Mechanisms of central and peripheral tolerance. IBD: inflammatory bowel disease; IL: Interleukin; and TGF-b:
transforming growth factor b.
345
Mechanisms of oral tolerance
The presence of large numbers of immunogenic and
pro-inflammatory molecules in the intestinal lu-
men, together with evidence for the interaction of
the host’s immune system with this material, has
led to the concept that the gut mucosa is in a state
of restrained immune reactivity. This state has
’’ ’’
been termed controlled’’ or physiological’’
inflammation4; when the body comes into contact
with a specific antigen, it should be able to identify
whether this molecule is dangerous, whether it
must be eliminated or if it is possible to tolerate its
presence.
The best-understood mechanism of immune
tolerance is the central tolerance established in
thymus and bone marrow. However, not all antigens
are present in the lymphoid organs; therefore,
central tolerance must be complemented by addi-
tional mechanisms of peripheral tolerance. Figure 2
depicts some of the most important mechanisms
involved in central and peripheral tolerance. Most
data concerning this selective immune response
have been obtained from experimental animals
(ovalbumin-TCR transgenic mice). When an antigen
is tolerated, the site of tolerance induction and the
type of APC generating the tolerogenic immune
response are not clear, although T cells appear to
be the major target of tolerance, and the reduction
in antibody responses after antigen feeding is due
to the reduction in T helper activity.42
CD4+ CD8+
CD4+ CD8+
CD4+ +
CD4+
CD45RBlow
- +
TGF-β
IL- 4
IL-10 ?
 ARTICLE IN PRESS
346
Cellular mechanisms of oral tolerance
It is accepted that the major mechanisms of
tolerance induction are the development of sup-
pressor T cells and the induction of clonal deletion
and/or anergy clonal deletion, and it is believed
that different mechanisms are favoured at differ-
ent antigen doses40: low doses of antigen favour
active suppression, whereas high doses are
mediated principally by anergy or clonal dele-
tion28,40 (Fig. 3).
Active suppression
Active suppression occurs when T cells obtained
from an antigen-fed donor animal and transferred
to a recipient animal induce unresponsiveness to
the administration of any antigen. Without prior
cell transfer, the administration of the same
antigen induces a strong immune response.43 The
transferred cells are CD4+ T cells originating in
Peyer’s patches,15,43 and the primary mechanism of
active cellular suppression is via the secretion of
suppressive cytokines. The crucial contributor to
oral tolerance appears to be TGF-b, and the
response could be summarised as follows: after
the generation of antigen-specific regulatory cells
in GALT, these cells migrate to peripheral lymphoid
organs, and suppress the immune response by
secreting TGF-b, thereby inhibiting the generation
Oral administration of antigens
APC
Low doses
T cell
Th2 Th3
IL-4, IL-10 TGFβ
Inhibition of autoimmunity
and inflammation
Figure 3 Mechanisms of dose-dependent oral tolerance in the gut-associated lymphoid tissue (GALT). APC: antigen-
presenting cell; IL: Interleukin; TGF-b: transforming growth factor b; and Th2: T helper lymphocyte subclass 2.
O.C. Thompson-Chagoyán et al.
of antigen-specific effector cells. These suppressor
cells can cause cytokine suppression in other
lymphoid cells in the surrounding area; this
’’
phenomenon is known as bystander suppression’’
and a specific antigen is required for the activation
of suppressor cells. When these activated cells
reencounter the same antigen, they release TGF-b
that inhibits the response of other neighbouring T
cells that are sensitive to antigens other than those
initiating an antigen-mediated response.44,45
Clonal anergy
The administration of high doses of antigen
produces clonal anergy. Clonal anergy exists when
T cells are unable to secret IL-2 or to proliferate;
this process is not passive, and seems to be
preceded by activation and proliferation of anti-
gen-specific T cells in lymph nodes.46 The tolerance
induced after administration of high doses of
antigen resembles the tolerance induced by admin-
istering antigen intravenously, but it is now
accepted that high-dose oral tolerance occurs due
to the absence of adequate costimulation, since T-
cell activation requires two signals, one via TCR
interaction with the antigen–MHC complex and a
second via the B7–CD28 interaction. If the costi-
mulatory signal is missing, the T cell will undergo
deletion or become anergic. Likewise, B7 interacts
Macrophages, dendritic cells,
B cells, enterocytes
APC-T cell Interaction
High doses
T cell
Th1 Th2
Anergy, deletion
Oral tolerance
 ARTICLE IN PRESS
Aetiology of IBD
APC
B7s B7s
MHC II
TCR
CD3 CD28
CTLA-4
T cell
IL-2
TGF-β
- Th2
Figure 4 Function of TGF-b in the suppression of T cell response mediated by CTLA-4 receptor. APC: antigen-presenting
cell; MHCII: major hystocompatibility complex II; TGF-b: transforming growth factor b; and Th1, Th2, Th3 and Thr: T
helper lymphocyte subclasses.
with CTLA-4 leading to TGF-b secretion and im-
portant molecule in the induction of oral tolerance
since it inhibits T cell activation41 (Fig. 4).
Clonal deletion
This mechanism has been observed under non-
physiological conditions in transgenic animals, and
occurs following the activation of T cells by strange
proteins.46
Potential roles of probiotics in the
treatment of IBD: A way of inducing oral
tolerance
In the absence of an aetiological therapy, the
inflammatory process itself has become the current
target for the treatment of IBD. A large number of
chemical compounds have been used in an attempt
to revert to the non-inflammatory state, as well as
for the maintenance of the remission state. The
manipulation of intestinal microbiota has received
little attention, and the few studies addressing this
option have been carried out with antibiotics.
Although corticoid drugs remain the most effective
treatment for active IBD, other therapeutic agents
such as mesalazine, azathioprine, 6-mercaptopur-
ine, methotrexate and cyclosporine have also been
tested.47 With the knowledge that activated T cells
can produce pro-inflammatory and anti-inflamma-
tory cytokines, physicians have begun to use
347
TGF-β
Th3
-
Th1
IL-12
T cell
Activation
IL-4
immunological strategies, including administration
of neutralising monoclonal antibodies or anti-
inflammatory cytokines with the intention of
modulating the immune alterations causing IBD.47
Nevertheless, for various reasons long-term
therapy is often the fate of patients suffering from
IBD; unfortunately, however, unpleasant side ef-
fects may result from drug use. Accordingly,
researchers have sought to develop methods which
allow for a more localised delivery of therapeutics.
The most widely used techniques are: colonic
instillation of conventional chemical therapy or
interleukins (IL-4 and IL-10), administration of SCFA
enemas,48 intranasal instillation of DNA delivery
systems,47 and injection of IL-4 using human
adenovirus as a vector.49 Finally, due to the possible
role of indigenous microbiota in the pathogenesis of
IBD,11 the external manipulation of its composition
using probiotic organisms seems to be a promising
therapeutic form.
It appears that normal humans develop tolerance
to their own microbiota and do not mount an
immune response to these bacteria. However, IBD
patients seem to lose this tolerance and their
immune response is upregulated50; for this reason,
and with the support of findings in experimental
models, it has been postulated that IBD is the result
of genetically determined defective immunosup-
pression resulting in an overly aggressive response
to ubiquitous luminal bacteria constituents.14,17,38
It has been suggested that manipulation of
 ARTICLE IN PRESS
348
Table 2 Criteria for considering bacteria as
probiotics.
Human origin
Non pathogenic for the host
Resistant to destruction by gastric acid, bile or
technical processing
Be able to adhere to gut epithelial tissue
Capacity to colonize the gastrointestinal tract
Produce antimicrobial substances
Modulate immune responses
Influence host metabolic activities
Source: Annu. Rev. Nutr. 2002;22:107–38.
intestinal bacteria may be a useful approach in the
treatment of these patients, and that probiotics
offer a promising option.51
Probiotics are live microbial food supplements
that beneficially affect the host by improving its
intestinal microbial balance; when administered in
adequate amounts, they confer a health benefit.52
Recent research has expanded the definition of
probiotics, as it has been shown that genetically
engineered microbes and non-viable microbes may
equally possess such potential.53 Current criteria
for defining probiotics are shown in Table 2.
There is some evidence that administration of
probiotics could be effective in the prevention and
treatment of infant acute infectious diarrhoea,
mainly of viral origin, and in the prevention of
antibiotic-associated diarrhoea, as well as in the
treatment of Helicobacter pylori infection and in
the prevention and management of atopic diseases.
One of the mechanisms of action is the modification
of the host gut microbiota.54
In view of these preliminary successes, attempts
have been made to modify the microbiota in
patients with IBD: The scientific rationale for
therapeutic manipulation of enteric microbiota in
IBD can be divided into experimental and human
evidence.
Probiotics in experimental models of colitis
The findings of several studies suggest that some
bacterial products influence epithelial proinflam-
matory responses and exchange regulatory signals
with subepithelial cells and the mucosal immune
system51; as a result, it is difficult to induce
intestinal inflammation in germ-free animals.55
Similarly, while transgenic rats with normal intest-
inal flora spontaneously develop colitis, in those
bred in germ-free conditions, the inflammation is
either attenuated or non-existent.16 Moreover, IL-
2, IL-10, and T-cell receptor knockout mice17 do not
O.C. Thompson-Chagoyán et al.
develop disease when they are reared in a germ-
free environment. However, once the normal
intestinal microbiota is restored, inflammatory
disease occurs.56
It has likewise been demonstrated that among
the commensal gut microbes colonising both IBD
patients and experimental animals, anaerobic
bacteria are thought to be responsible for the
development of immune response and inflamma-
tion.57,58
Finally, in mice with IBD, the tolerance to
commensal microorganisms is lost while colitis is
active.5
Evidence supporting the successful response to
probiotics for the prevention or treatment of IBD
in animals
Several studies in animal IBD models have yielded
encouraging results, although they have also shown
that a single microorganism may not be ideally
suited to all patients; use of probiotic cocktails has
been tested with promising results in patients with
UC,59 but more rigorous scientific study is required
before they can be recommended for routine use.
The most promising results obtained to date are as
follows:
1. Administration of Lactobacillus reuteri prevents
acetic acid colitis in a rat model.60
2. Ingestion of Lactobacillus, and especially of L.
plantarum, improves colitis induced by intraper-
itoneal methotrexate administration in rats.61
3. In the neonatal period, IL-10 gene-deficient
mice have decreased levels of Lactobacillus sp.
in the colon. Daily anal administration of
lactobacilli reduces colonic mucosal adherent
and translocated bacteria and prevents colitis.18
In the same model, Lactobacillus salivarius
inhibits the development of both colitis and
colon cancer.62
4. Administration of a non-pathogenic E. coli
(serotype O6:K5:H1) prevents relapses of UC.63
5. Genetically engineered Lactobacillus lactis se-
creting active IL-10 has been used successfully in
the treatment and prevention of IBD in murine
models.64
Probiotics in human inflammatory bowel
disease
The main evidence supporting the involvement of
the gut microbiota in IBD is as follows:
1. High mucosal bacteria counts are reported in
patients with bowel inflammation, and counts
 ARTICLE IN PRESS
Aetiology of IBD
increase progressively with the severity of
disease, in both inflamed and non-inflamed
colon tissue.14
2. It has been demonstrated that T cells from the
lamina propria of CD patients respond in vitro
to antigens of their own microbiota with a Th1
polarised response, indicating a loss of toler-
ance of their normal microbiota.4
3. Among the commensal intestinal microbes
colonising both IBD patients and experimental
animals, anaerobic Gram positive bacteria,
particularly those of the Bacteroides genus,
are thought to be responsible for the develop-
ment of inflammation and immune response,65
and evidence suggests that bifidobacteria
might be used as a probiotic to ameliorate
IBD through the suppression of Bacteroides in
the intestinal microbiota.58
4. Coliforms and Bacteroides are potentially
pathogenic to the gut mucosa, and an increase
in both bacteria, together with a decrease in
LAB, has been reported in patients with IBD.38
5. Normal gut microorganisms have a considerable
influence on the integrity and development of
the intestinal mucosa.36,66
6. UC shares certain histological features with
infective colitis.67
7. Lymphocytes in contact with an extract of E.
coli lipopolysaccharides exhibit cytotoxicity to
colon cells; patients with UC are more likely to
carry E. coli strains with these lipopolysacchar-
ides.68
8. Tobramycin, an antibiotic with activity against
Gram negative bacteria, when administrated
orally together with corticosteroids, prompts
an improvement in patients with active UC.69
9. An exaggerated antibody response against the
intestinal microbiota has been reported in IBD
patients.57
10. Surgical bypass leads to remission in patients
with IBD and its reconnection produces a
relapse.39
11. High serum E. coli antibody levels are reported
in IBD patients; this species has been isolated in
65% of patients with CD.39
A number of trials in patients with IBD report
successful use of probiotics:
1. The administration of Lactobacillus GG may be
an effective therapeutic agent for pouchitis, a
complication of ileal reservoir surgery occurring
in 10–20% of patients undergoing surgical treat-
ment for chronic UC.70
2. Bifidobacteria ameliorates IBD through suppres-
sion of Bacteroides in the gut.58
349
3. Short-term administration of Lactobacillus GG in
children with CD increases the intestinal IgA
immune response71; long-term administration of
this probiotic reduces intestinal permeability
and improves clinical status.72
4. Saccharomyces boulardii, in association with
mesalazine, increases the time without relapses
in patients with stable CD.73
5. In 120 patients with UC, 12-week administration
of a non-pathogenic E. coli strain was compared
with mesalazine; no differences were found in
the proportion of remission at the end of the
study63; when the E. coli strain was admini-
strated over 1 year, it prevented relapses.68
6. In patients with pouchitis, treatment with a
combined preparation of probiotics (four strains
of Lactobacilli, three strains of Bifidobacteria
and Streptococcus salivarius) containing 5  1011
bacteria per gram dramatically reduced relapses
(15% in patients with probiotics vs. 100% of the
placebo group); 4 months after the end of study,
all patients with bacteriotherapy were in remis-
sion.74 The same mixture was successful as a
maintenance treatment of UC in an open trial.75
7. Oral administration of Lactobacillus casei strain
GG leads to a significant increase in mucosa IgA
levels, suggesting an improvement of the im-
munological barrier in patients with CD.71
8. Treatment with E. coli strain Nissle 1917 reduces
the risk of relapse and decreases corticoid
requirements in patients with CD.76
Possible mechanisms of probiotic action in
IBD
There are a number of possible mechanisms by
which probiotics produce beneficial effects in IBD.
These mechanisms, which influence most of the
interactions between the host and his/her intest-
inal microbiota, include:
Production of inhibitory substances: including
the modification of pH and the production of
organic acids, hydrogen peroxide and bacteriocins.
These compounds reduce the number of viable cells
and affect bacterial metabolism or toxin produc-
tion.70
Blocking of adhesion sites: displacement of
harmful microorganisms may be a consequence of
physical competition. The ability of probiotics to
adhere to epithelial cells produces competitive
inhibition of bacterial adhesion sites, thus impeding
invasion by foreign organisms.29,47,77 Also, some
bacteria are able to block the intestinal mucosal
receptor, thus preventing the establishment of
adhesive pathogenic bacteria.68
 ARTICLE IN PRESS
350
Competition for essential nutrients: probiotics
utilise nutrients that would otherwise be consumed
by potentially harmful microorganisms, thus inhi-
biting their growth.70
Degradation of toxin receptor: this mechanism
protects against Clostridium difficile intestinal
disease.78
Stimulation of immunity: there is strong evi-
dence to suggest that probiotics modulate both
innate and acquired immunity. Probiotics could
interact with indigenous bacteria and/or host
mucosal cells to induce or modulate the immune
response, modifying the number of CD4 T cells; it is
well known that probiotics can actively interfere
with anti-inflammatory and proinflammatory sig-
nalling pathways, inducing production of IL-10 and
reducing INF and TNF release.39 Finally, the
increase in local IgA levels and IgA-secreting plasma
cell numbers found in IBD patients results from the
ingestion of probiotics.47,71
Promotion of gut integrity: Nitric oxide (NO) at
high concentrations is a very potent pro-inflamma-
tory stimulator. At low levels however, NO is a
promoter of gut integrity. It has been proven that
Lactobacillus plantarum has the ability to produce
NO in low doses with beneficial effects on the
intestinal mucosa.47 It has also been observed that
disruption of the epithelial barrier leads to a large
influx of antigen into the mucosa and therefore to
an enhanced intestinal immune response. In addi-
tion, the administration of L. casei or Clostridium
butyricum markedly enhances gut epithelial cell
proliferation (up to 200% in rat colon), thus
enhancing tissue repair.79
It has been suggested that SCFA (acetate,
propionate and butyrate) deficiency promotes
colitis,80 and that probiotic bacteria have the
capacity to produce SCFA,77 which in turn promote
mucosal intestinal restitution.47
Finally, probiotic bacteria can increase the gut
epithelial mucus that provides a barrier shielding
intestinal immune cells from the antigen-rich
luminal matrix by inducing genetic expression.81
Conclusions
Both genetic and environmental factors are in-
volved in the pathogenesis of IBD.
Most of the proposed aetiological theories for IBD
include a pathogenic role for the normal intestinal
resident bacteria. These theories include: an
inappropriate reaction to a specific infection of
the intestine, subtle alterations of bacterial com-
position and function, and overexposure to resident
O.C. Thompson-Chagoyán et al.
luminal bacterial products. Moreover, it has been
observed that patients with UC and CD have
abnormal intestinal microbiota and this alteration
induces the release of pro-inflammatory mediators.
Loss of tolerance to indigenous bacteria could be
implicated in the appearance of IBD; mechanisms
involved include the breakdown of active suppres-
sion, clonal anergy, clonal deletion and the release
of pro-inflammatory cytokines.
Treatment with probiotics represents an alter-
native to current drug treatment in IBD patients. In
these cases, previous intestinal ecology can be
restored by manipulating the microbiota through
administration of probiotic bacteria. Probiotics
could also be genetically modified to release
specific anti-inflammatory cytokines.
It must be remembered that, despite the
potential for application of probiotics in the
treatment and prevention of IBD, more controlled
studies are required before their routine use can be
recommended.
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