CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:274 284
REVIEWS
Gut Microbiota: Mining for Therapeutic Potential
ANN M. OHARA* and FERGUS SHANAHAN*,
*Alimentary Pharmabiotic Centre, and Department of Medicine, University College Cork, National University of Ireland, Cork, Ireland
The resident microbiota of the human intestine exerts a
conditioning effect on intestinal homeostasis, delivering
regulatory signals to the epithelium and instructing muco-
sal immune responses. Pattern recognition receptors are
key mediators of innate host defense, and in healthy indi-
viduals, the mucosal immune system exhibits an exquisitely
regulated restrained response to the resident microbiota.
However, in genetically susceptible hosts, unrestrained mu-
cosal immune activation in response to local bacterial sig-
nals can contribute to the pathogenesis of inflammatory
bowel disease. Manipulation of the microbiota to enhance
its beneficial components thus represents a potential ther-
apeutic strategy for inflammatory bowel disease. Moreover,
the microbiota might be a rich repository of metabolites
that can be exploited for therapeutic benefit. Modern mo-
lecular techniques are facilitating improved understanding
of host-microbe dialogue in health and in several disease
processes, including inflammatory bowel disease. It follows
that elucidating the molecular mechanisms of host-micro-
bial interactions is now a prerequisite for a bugs to drugs
program of discovery.
T he chronic inflammatory bowel diseases (IBDs), Crohns
disease and ulcerative colitis, are characterized by bouts of
uncontrolled, chronic mucosal inflammation, followed by re-
modeling processes that occur during periods of remission.
Together, these result in tissue-damaging inflammatory re-
sponses in the intestine that might result in much personal
suffering and impaired quality of life. Crohns disease and
ulcerative colitis have a combined prevalence of 250/100,000 in
the Western world, and they represent a substantial economic
burden on health care resources. Although the precise etiology
of IBD remains to be elucidated, a complex interaction of
environmental, genetic, and immunoregulatory factors contrib-
utes to the initiation and perpetuation of the disease.1
Under normal circumstances, the mucosal immune system is
exquisitely regulated and exhibits a restrained response to the
resident microbiota while retaining an ability to mount appro-
priate immune responses to pathogenic bacteria. However, sev-
eral lines of evidence indicate that genetically influenced dys-
regulation of the mucosal immune response to antigens of the
indigenous microbiota can contribute to the pathogenesis of
IBD.2,3 In susceptible individuals, environmental triggers can
impact on the initiation or reactivation of disease, and tissue
damage might result from immune cell misperception of dan-
ger within the indigenous microbiota or from failure of normal
tolerance to enteric bacteria.2,4 Within the gastrointestinal tract,
the inflammatory capacity of commensal bacteria is varied.
Some resident bacteria are proinflammatory, whereas others
attenuate inflammatory responses.511
The 2005 Nobel prize awarded to Barry Marshall and Robin
Warren is a sobering reminder that the solution to some chronic
disorders cannot be unraveled by exclusive investigation of the
host response. The host response must be considered in terms of
bacterial residents and prokaryotic-eukaryotic interactions. Mod-
ern molecular techniques are facilitating our understanding of
microbe-microbe and host-microbe communications at mucosal
interfaces and are providing glimpses of tangible therapeutic strat-
egies. Therefore, the intent here is to present an overview of the
commensal microbiota, their interactions with the intestinal mu-
cosa, and clinical relevance to IBD.
Indigenous Gut Microbiota
Composition of the Normal Gut Microbiota
Dialogue between commensal bacteria and the host
occurs primarily along mucosal surfaces, and the largest inter-
face is the human gastrointestinal mucosa. The intestine is
habitat to a dynamic and diverse bacterial community that is
separated from the internal milieu by only a single layer of
epithelial cells. Intestinal bacteria outnumber human somatic
and germ cells 10-fold12 and represent a combined microbiome
well in excess of the human genome. Co-evolution of the host
and indigenous microbes has fostered mutually beneficial and
cooperative interactions mediated by bidirectional host-micro-
biota exchange.
Traditionally, studies of indigenous bacteria focused on the
characterization of fecal diversity. Detailed analysis was hin-
dered by conventional microbiology techniques, and most bac-
terial species still cannot be cultured. Modern molecular tech-
niques such as broad-range sequencing of 16S ribosomal RNA
(rRNA) from amplified bacterial nucleic acid extracted from
feces or biopsies indicate evolutionary divergence and can be
used to identify and classify bacteria. The availability of bacte-
rial sequence data has facilitated the development of molecular
probes for DNA microarrays, fluorescent in situ hybridization,
and gene chips that identify and enumerate specific microbial
species. These molecular approaches are being used to examine
the individuality and temporal stability of the microbiota and
Abbreviations used in this paper: AIEC, adherent-invasive Esche-
richia coli; CARD, caspase recruitment domain; DC, dendritic cell; IBD,
inammatory bowel disease; IL, interleukin; IFN, interferon; MAP,
Mycobacterium avium subspecies paratuberculosis; MDP, muramyl
dipeptide; NF, nuclear factor; NOD, nucleotide-binding oligomerization
domain; PPAR, peroxisome proliferator activated receptor; PRR, pat-
tern recognition receptor; rRNA, ribosomal RNA; Th1/Th2, T-helper cell
type 1/2; TLR, toll-like receptor; TNF, tumor necrosis factor.
2007 by the AGA Institute
1542-3565/07/$32.00
doi:10.1016/j.cgh.2006.12.009
March 2007
Figure 1. Changes in intestinal structure and function in germ-free animals compared with colonized animals raised under conventional conditions.
Reconstitution of germ-free mice with a microbiota restores the mucosal immune system, and commensal bacteria exert numerous protective,
structural, and metabolic effects on the intestinal epithelium.
the impact of weaning, antibiotics, or dietary changes on its
composition. It is now realized that the mucosa-associated
community is significantly different from the luminal and fecal
communities.13 Although every adult intestine harbors a par-
ticular combination of predominant species, the composition
of the intestinal microbiota might alter with lifestyle, diet, and
age.14,15 Nonetheless, a comparative study of the microbiota of
human adults with varying degrees of genetic relatedness, in-
cluding monozygotic twins, emphasized the prevailing influ-
ence of host genotype over diet in determining the microbial
composition of the gut.16
Acid, bile, and pancreatic secretions hinder the colonization
of the stomach and proximal small intestine by most bacteria.
However, bacterial density dramatically increases in the distal
small intestine and in the large intestine increases to approxi-
mately 10111012 bacteria per gram of colonic content, which
contribute to 60% of fecal mass.17,18 Although archaea and
eukarya are also represented,19 bacteria predominate, and co-
lon-residing bacteria achieve the highest cell densities recorded
for any ecosystem.20 The most common anaerobic genera in-
clude Bifidobacterium, Clostridium, Bacteroides, and Eubacterium,
and aerobic Escherichia, Enterococcus, Streptococcus, and Klebsiella
are also found. However, sequencing of 16S rRNA gene clone
libraries has indicated that a significant fraction of the bacteria
represent uncultivated species and novel microorganisms.13
Mammalian fetuses are born germ-free, but immediately
after birth, establishment of the resident microbiota is driven by
environmental factors such as mode of delivery, type of infant
diet, hygiene levels, and medication.21,22 Enterobacteria and
bifidobacteria species represent early colonizers, but differences
in gut microbial composition and incidence of infection occur
THERAPEUTIC POTENTIAL OF THE GUT MICROBIOTA 275
between breast-fed and formula-fed infants.21 These pioneer
bacteria can modulate gene expression in the host to create a
suitable environment for themselves and can prevent growth of
other bacteria introduced later in the ecosystem.23,24 Increased
credence is being given to the hypothesis that the modern
sanitized environment of developed societies has altered the
normal pattern of gut colonization during infancy. This might
result in a lack of tolerance to otherwise harmless food proteins
and other antigens, including those of the intestinal micro-
biota.25
Functions of the Normal Gut Microbiota
Enteric bacteria confer many benefits to intestinal phys-
iology including protective, structural, and metabolic effects.
Their influence on intestinal structure and function has been
demonstrated in comparative studies of germ-free and colo-
nized animals. Some of the differences between animals raised
under germ-free and conventional conditions are listed in Fig-
ure 1. Such comparisons, together with studies indicating that
reconstitution of gnotobiotic mice with a microbiota is suffi-
cient to restore the mucosal immune system,26 illustrate that
the microbiota provide regulatory signals that instruct intesti-
nal development and function. Indeed, colonization of germ-
free mice with a single species, Bacteroides thetaiotaomicron, af-
fects the expression of a variety of host genes influencing
nutrient uptake, metabolism, angiogenesis, mucosal barrier
function, and the development of the enteric nervous system.23
Furthermore, ligands from resident bacteria and commensal-
derived symbiosis factors influence the normal development
and function of mucosal immunity.27,28 Indigenous bacteria
educate the mucosal immune system and modulate the fine
276 OHARA AND SHANAHAN
tuning of T-cell repertoires and T-helper cell type 1 (Th1)/Th2
cytokine profiles.29 Taken together, the composition of the
colonizing microbiota might influence individual variations in
immunity.
Along the epithelium, enteric bacteria form a natural defense
barrier against exogenous microbes. Colonization resistance
involves several mechanisms including displacement, competi-
tion for nutrients and epithelial binding sites by the resident
microbiota, and production of antimicrobial factors such as
lactic acids and bacteriocins.30 Commensal bacteria also help
fortify the epithelial barrier by various mechanisms that include
the induction of the complement inhibitor decay-accelerating
factor or increasing barrier function.31,32 Exposure of colonic
epithelial cell lines to bacterial ligands also results in apical
tightening and sealing of the tight junctional protein ZO-1 and
increased transepithelial resistance.33
The metabolic activity of the microbiota is equivalent to that
of an organ within an organ.34 Through the production of short
chain fatty acids, resident microorganisms positively influence
the differentiation and proliferation of intestinal enterocytes.35
Resident bacteria can break down dietary carcinogens; synthe-
size biotin, folate, and vitamin K; ferment nondigestible dietary
residue, particularly carbohydrates and endogenous epithelial-
derived mucus; and assist in the absorption of calcium, mag-
nesium, and iron.36 38 Together, this complex metabolic activity
recovers valuable energy and absorbable substrates for the host
and provides energy and nutrients for bacterial growth and
proliferation. Colonization increases glucose uptake in the gut,
and compared with colonized mice, germ-free mice require a
greater caloric intake to sustain a normal body weight.39 This
implicates intestinal bacteria as modulators of fat deposition in
the host. It has been proposed that an individuals gut micro-
biota has a specific metabolic efficiency, and differences in gut
microbial composition between individuals might regulate en-
ergy storage and predispose to obesity.19 Therefore, exploration
of the metabolic activity of the microbiota offers strong poten-
tial for exploitation of the microbiota as biomarkers for im-
pending disease and the development of novel therapies.
Host-Microbiota Dialogue at the Mucosal
Surface
Host defense demands precise interpretation of the
microenvironment to distinguish commensal from pathogenic
microorganism and must entail exquisite regulation of re-
sponses. Disruption of these processes might lead to inappro-
priate responses. Thus, although the microbiota are generally a
health asset, they might become a liability in certain circum-
stances. These include syndromes of bacterial overgrowth
and/or translocation, metabolite-mediated conversion of pro-
carcinogens to carcinogens, and IBD.29,40,41
Along the epithelium, active sampling of commensal bacte-
ria, pathogens, and other antigens is mediated by various types
of immunosensory cells. These include surface enterocytes, M
cells, and dendritic cells (DCs). Surface enterocytes are inter-
connected with tight junctions and overlain with mucus. They
serve as afferent sensors of danger signals within the luminal
microenvironment by secreting defensins, immunoglobulin A,
chemokines, and cytokines that alert and direct innate and
adaptive immune responses.42 45 M cells, specialized epithelial
cells, that overlie lymphoid follicles sample the environment
and transport luminal antigens to subadjacent DCs and other
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 3
antigen-presenting cells. Intestinal DCs themselves participate
in immune surveillance and can directly sample gut contents by
either entering or extending dendrites between surface entero-
cytes without disrupting tight junctions.46 DCs can ingest and
retain live commensal bacteria and transit to the mesenteric
lymph node where immune responses to these bacteria are
induced locally. This prevents access of commensal bacteria to
the internal milieu.47 In addition, enterocyte-derived mediators
such as thymic stromal lymphopoietin can induce noninflam-
matory DCs, suggesting that physiologic interactions between
DCs and surface enterocytes contribute to intestinal homeosta-
sis.48
Mucosal Recognition of Bacteria
The ability of immunosensory cells to discriminate
pathogen from commensal is mediated, in part, by 2 major host
pattern recognition receptor (PRR) systems. These are the fam-
ily of toll-like receptors (TLRs) and the nucleotide-binding
oligomerization domain/caspase recruitment domain (NOD/
CARD) molecules.49 Several of the signal transduction path-
ways induced by these receptors have been elucidated and their
effector responses characterized.49 53 Both TLRs and NOD pro-
teins trigger innate and adaptive immune responses, including
the synthesis of proinflammatory cytokines and chemokines,
and TLR signaling also impacts on subsequent T-cell responses
by activating DCs that overcome the suppressive effects of T
regulatory cells.54 Together, these PRRs play a fundamental role
in immune cell activation in response to specific microbial-
associated molecular patterns (Table 1).
TLRs and NOD proteins are expressed by surface enterocytes
and DCs,51 and in the gut, TLRs and NOD proteins appear to
be crucial for bacterial-host communication. Decreased entero-
cyte proliferation and levels of cytoprotective factors have been
observed in TLR-defective mice compared with wild-type
mice.27 TLR signals mediated by commensal bacteria or their
ligands are essential for intestinal barrier function and repair of
the gut.55 Many PRR ligands are expressed by commensal bac-
teria, yet the healthy gut does not evoke inflammatory re-
sponses to these bacteria.11 PRRs participate in several recently
described host molecular immune mechanisms that mediate
intestinal homeostasis (Table 2), and these are reviewed else-
where.56 In the healthy gut, regulatory T cells and tolerance-
inducing DCs contribute to the control of excessive Th1 re-
sponses to the indigenous microbiota also.57,58 Phenotypic
alterations in regulatory T-cell populations have been observed
in IBD patients,59 and several lines of evidence indicate that
peripheral tolerance, as well as local tolerance to commensal
bacteria, is mediated by the suppressive effects of regulatory T
cells.59 61
Commensal bacteria have been shown to suppress inflam-
matory responses and inhibit specific intracellular signal trans-
duction pathways, thereby contributing to the maintenance of
mucosal homeostasis. The transcription factor, nuclear factor
(NF)-B, is a master coordinator of immune responses to
pathogenic bacteria and other stress signals. However, most
commensal bacteria do not activate NF-B8,11; instead, some
commensal strains antagonize NF-B within epithelial cells by
a variety of mechanisms. These include inhibition of epithelial
proteasome function or degradation of the NF-B counter-
regulatory factor IB- or by the nuclear export of the p65
subunit of NF-B in a peroxisome proliferator activated recep-
March 2007 THERAPEUTIC POTENTIAL OF THE GUT MICROBIOTA 277

Table 1. List of PRRs and Their Ligands

TLR Ligand Source

TLR1 Triacyl lipopeptides Bacteria & mycobacteria

TLR2 Lipoprotein Most bacteria
Peptidoglycan Gram-positive bacteria
Lipoteichoic acid Gram-negative bacteria
Lipoarabinomannan Mycobacteria
TLR3 Double-stranded RNA Viruses
TLR4 Lipopolysaccharide Gram-negative bacteria
TLR5 Flagellin Flagellated bacteria
TLR6 Diacyl lipopeptides Mycoplasma
Lipoteichoic acids Gram-positive bacteria
Zymosan Fungi
TLR7 Single-stranded RNA Viruses
TLR8 Single-stranded RNA Viruses
TLR9 Unmethylated CpG-containing DNA Bacteria & viruses
TLR10 Undetermined Undetermined
TLR11 Profilin-like ligands Protozoa
NOD
NOD1/CARD4 -D-glutamyl-meso-diamino-pimelic acid of peptidoglycan Gram-negative bacteria
NOD2/CARD15 MDPs of peptidoglycan Most bacteria

tor (PPAR)-dependent manner.7,8,62 Some commensal bacte-
ria can attenuate colonic inflammation through TLR4 by ele-
vating PPAR- expression in intestinal enterocytes and
uncoupling NF-B dependent target genes in a negative feed-
back loop.8,63 It is clear that many of these mechanisms are
strain-specific, and not all commensal bacteria with immuno-
modulatory effects use these mechanisms; other signal trans-
duction pathways are likely to account for their anti-inflamma-
tory effects.
Pathogenesis of Inflammatory Bowel
Disease
Both forms of IBD represent the clinical outcome of a
complex interaction of environmental, genetic, and immune
factors. The normal physiologic response to the indigenous
microbiota is one of immunologic quiescence. Deviations from
this and, in particular, genetically influenced aberrant immune
responses to luminal antigens can lead to the development of
IBD. Crohns disease bears the immunologic signature of an
exaggerated Th1 response, with excess interleukin (IL)-12, IL-
18, interferon (IFN)-, and tumor necrosis factor (TNF).64
Conversely, ulcerative colitis is associated with a dominant
atypical Th2 response that is probably driven by the production
of IL-13.65
An environmental contribution to the pathogenesis of IBD
is clear from studies of monozygotic twins in which an incom-
plete accordance rate has been observed for both Crohns dis-
ease (50%) and ulcerative colitis (10%).66 Smoking, dietary

Table 2. Host Systems That Limit PRR Signaling and Inflammation

Mechanism Example Reference

Decreased ligand recognition due to TLR Low expression of functional TLRs (eg, TLR2 and TLR4) 107, 130, 131
expression profiles TLR localization (surface vs cytoplasmic expression) 108, 132, 133
State of cell differentiation (apical vs basolateral 132, 134
expression)
Limited repertoire of TLR co-receptor Low expression of TLR co-receptors (eg, MD-2) 130
expression Lack of expression of TLR co-receptors (eg, CD14) 108
Inhibitors of TLR signaling High expression of TLR signaling suppressors (eg, toll- 133, 135137
interacting protein/Tollip, toll IL-1 receptor 8/Tir8)
Sequestration of the TLR signaling adaptor proteins 138
MyD88 and Mal by ST-2 (toll IL-1 receptor family
member)a
Enzyme-mediated inhibition of TLR Proteolytic degradation of TLR4 and TLR9 by TRIAD3A, 139
signaling a ubiquitin ligasea
Deubiquitination of TLR adaptor proteins by the zinc 140, 141
finger protein A20a
Cross-regulation by other PRRs Negative regulation of TLR signaling by NOD2 103, 104, 142, 143
Ligand neutralization Secretion of immunoglobulin A by intestinal 47
enterocytes
aThe regulatory effects of ST2, TRIAD3A, and A20 on TLR signals have been identified in other systems, but it remains to be determined whether
these suppressors mediate TLR signaling in the gut.
278 OHARA AND SHANAHAN
factors, nonsteroidal anti-inflammatory drugs, socioeconomic
conditions, and the more hygienic lifestyles of developed coun-
tries have all been associated with IBD.67 Contributing environ-
mental factors are probably multiple and complex. Numerous
studies have pursued a specific infectious cause for IBD. The
most notable inconclusively incriminate either Mycobacterium
avium subspecies paratuberculosis (MAP) or adherent-invasive
Escherichia coli (AIEC) in the development of Crohns disease.68,69
Viable MAP have occasionally been detected in peripheral
blood and intestinal tissue in a higher proportion of individuals
with Crohns disease than in controls, and antibodies to the
organism have been disease-associated.68,70,71 A recent study
identified 2 sets of cross-reactive MAP and human peptides that
are specific for Crohns disease.72 Although these peptide pairs
might represent structural mimics whereby an anti-MAP im-
mune response could trigger a host autoimmune response,
definite proof for a role of molecular mimicry in Crohns
diseasespecific autoimmunity has not been established. This
study did not test the existence of cross-reactive autoantibodies
or T-cell immunity with pathogenic activity. Nonetheless, there
is limited evidence for T-cell immunity to MAP in association
with Crohns disease, and a cause and effect relationship re-
mains unproved.73 Among several arguments against persistent
MAP infection as a cause of Crohns disease, the most persua-
sive is the clinical experience with anti-TNF therapy. Therapeu-
tic blockade of TNF- greatly impairs host resistance to myco-
bacterial infection, but this is not associated with disseminated
MAP in patients with IBD.73 Furthermore, controlled trials have
failed to show a therapeutic effect of anti-tuberculosis therapy
in Crohns disease.74
A number of studies have reported abnormally high num-
bers of virulent AIEC and hemagglutinin-expressing Esche-
richia coli adjacent to the ileal or colonic mucosa in Crohns
disease.69,75 AIEC can adhere to epithelial cells, invade mac-
rophages without inducing apoptosis, and persist and repli-
cate within phagocytes.76 Nonetheless, these reports do not
fulfil Kochs postulates, and several lines of available evi-
dence that are reviewed elsewhere stand against an etiologic
role for any single pathogenic microorganism in the patho-
genesis of IBD.67 Moreover, it is difficult to reconcile the
concept of an infectious etiology with the beneficial effects of
host immunosuppression; any infection putatively stimulat-
ing such an intense inflammatory reaction as seen in Crohns
disease is unlikely to respond to long-term suppression of
host immune defenses. However, after the lesson of Helico-
bacter pylori and peptic ulcer disease, it would be unwise to
completely dismiss an infectious contribution to the patho-
genesis of IBD. It is interesting to note that both MAP DNA
and E coli DNA have been detected in the granulomas of
resected tissues from some Crohns disease patients.77,78
However, other forms of bacterial DNA were also detected,67
and rather than reflect a specific causal relationship, it might
reflect impaired innate handling of luminal bacteria in
Crohns disease. It is possible that in a subset of genetically
susceptible Crohns disease patients, particularly those with
a defect in their ability to sense bacterial ligands or clear
intracellular infections, persistent infection with MAP, AIEC,
or an as yet unidentified pathogen might underlie their
disease.
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 3
Microbial Contribution to Inflammatory
Bowel Disease
Although a specific infectious cause for IBDs has not
been established, current clinical and experimental data incrim-
inate a loss of tolerance to commensal bacteria in the develop-
ment of chronic intestinal inflammation.67 Animal models of
IBD provide persuasive evidence implicating the resident mi-
crobiota as a causative factor of IBD. Irrespective of the under-
lying genetic defect(s) in various animal models of the disease,
colonization with commensal bacteria is required for an inflam-
matory phenotype, and germ-free animals do not develop IBD-
like lesions.29,79 82 Of note, antibiotics are beneficial in a num-
ber of animal models. It has been shown in some models that
the disease can be adoptively transferred to immune-deficient
recipients when reconstituted with T cells from a diseased
donor that was sensitized to enteric bacteria.83 Moreover, mod-
ification of the microbiota by the administration of probiotic
bacteria, which are commensal organisms that can be harnessed
for therapeutic benefits, has been shown to delay the onset of
disease and attenuate the inflammatory process.84 86 Although
these animal models do not reveal whether commensal bacteria
passively or directly elicit the inflammatory response, they
clearly demonstrate that exposure to the microbiota is essential
for expression of the disease.
In patient-related studies, increased numbers of bacteria
within the mucosa of IBD patients compared with that of
noninflamed and inflammatory disease controls have been re-
ported.87,88 Furthermore, the distribution of lesions and inci-
dence of inflammation in either Crohns disease or ulcerative
colitis is greatest in the area with the highest concentrations of
luminal bacteria.29 Diversion of the fecal stream is associated
with an improvement in disease severity in the distal bowel, and
cellular and humoral immune reactivity against components of
the bacterial microbiota is evident in patients with IBD.2,29,89
Flagellin derived from commensal bacteria has been identified
as a dominant antigen in Crohns disease, suggesting that
TLR5-dependent recognition of flagellin might play a role in
the immune responses to the commensal bacteria observed in
IBD.90 In addition, therapeutic efficacy from the administration of
antibiotics and probiotics in IBD patients has been reported.3,9193
Together, these and other circumstantial observations in humans
that are reviewed in more detail elsewhere29,94 substantiate the
current dogma that IBD is a heterogeneous syndrome caused by
dysregulated immunity to normal microbiota. However, the model
must allow for heterogeneity within both Crohns disease and
ulcerative colitis. It remains unclear whether the inflammatory
responses in IBD, both within the gut and at extraintestinal sites,
are elicited in response to a specific subset of intestinal microbes,
or whether tolerance to commensal bacteria in general is affected.
Genetic Influences on Microbial Perception in
Inflammatory Bowel Disease
The pathophysiologic impact of bacterial-mucosal in-
teractions has been emphasized by an increased awareness of
the association of genetic aberrations with increased suscepti-
bility to IBD. In particular, mutations of CARD15, which en-
codes NOD2, and certain TLR polymorphisms have been
strongly associated with Crohns disease. This implicates a role
of PRR dysfunction and impaired bacterial sensing in the
pathogenesis of IBD.
March 2007
Three major polymorphisms in the CARD15 gene on chro-
mosome 16q12 have been specifically associated with 15% of
Crohns disease patients, and individuals who carry 2 copies of
the risk alleles have a 20-fold to 40-fold increase in their risk of
developing Crohns disease.9597 However, this risk factor is
neither sufficient nor necessary for development of the dis-
ease.98 Moreover, NOD2-deficient mice do not develop sponta-
neous gut inflammation, and NOD2 knockout mice that lack
full-length NOD2 protein do not demonstrate significantly
enhanced susceptibility to experiment-induced colitis.99,100
The ligands for NOD2 are muramyl dipeptides (MDP) of
bacterial peptidoglycan (Table 1). All 3 Crohns diseaseassoci-
ated mutations occur in the ligand-binding domain of NOD2.
Intracellular triggering of NOD2 leads to NF-B activation.
Thus, mutations in the NOD2 protein compromise MDP-in-
duced signaling and result in aberrant or defective NF-B re-
sponses to gut bacteria.101,102 However, stimulation of NOD2
with MDP has been shown to inhibit TLR2-driven NF-B acti-
vation and Th1 cytokine responses. This suggests that in the
absence of NOD2, stimulation with MDP results in NF-B
dysregulation and imbalanced TLR2-mediated cytokine pro-
duction (elevated IL-12, decreased IL-10).103,104 Together these
might contribute to the pathogenesis of Crohns disease (Figure
2A). On the other hand, in a study of knock-in mice expressing
the most common Crohns diseaseassociated NOD2/CARD15
mutation, it was shown that MDP triggers increased NF-B
activation, IL-1 secretion, and apoptosis105 (Figure 2B). More-
over, NOD2-deficient mice are unusually susceptible to infec-
tion with Listeria monocytogenes and have reduced production of
defensin-like antimicrobial peptides termed cryptidins.100 Con-
sidering that reduced expression of -defensin has been ob-
served in Crohns disease patients with NOD2 mutations,106
this has led to the proposal that NOD2 mutations might
predispose to Crohns disease by reducing -defensinmediated
innate antimicrobial activity100 (Figure 2C). To date, these dif-
ferent experimental models have implicated conflicting loss of
function, loss of regulation, or gain of function phenotypes for
NOD2 mutations, and the precise biologic role of NOD2 in
normal intestinal physiology and in the pathogenesis of
Crohns disease remains elusive.
Expression of some TLRs, including TLR4, is differentially
altered in Crohns disease and ulcerative colitis.107,108 It has been
proposed that increased TLR4 expression in patients with IBD
confers lipopolysaccharide hyperresponsiveness.107 Alterna-
tively, it might reflect a loss of response. The TLR4 gene is
localized on chromosome 9q32-33, a region harboring a
Crohns disease susceptibility gene,109 and in selective popula-
tions, a TLR4 polymorphism has been associated with IBD.49,110
It is plausible that variant alleles in the TLR4 gene could induce
functional dysregulation in response to lipopolysaccharide.
However, the functional phenotypic consequences of TLR4
polymorphisms in IBD remain unresolved. A preliminary report
from a single German cohort study has suggested that Crohns
disease might also be associated with TLR9 promoter polymor-
phisms.111 Recent reports demonstrating a hyperreactivity to
flagellins in sera from Crohns disease patients90,112 suggest that
these TLR or other PRR polymorphisms could lead to impaired
bacterial clearance and thus increased load of bacterial antigens,
including flagellin, in the lumen. This would be consistent with
evidence for defective handling of bacteria and increased bacterial
numbers in the mucosa of patients with Crohns disease.90,112
THERAPEUTIC POTENTIAL OF THE GUT MICROBIOTA 279
Therefore, it is probable that additional IBD susceptibility genes
that affect the interpretation of the microbial microenvironment
and/or regulate the host response to it remain to be identified.
Modifying the Ecosystem as
Therapeutic Strategy
Traditional drug therapies for IBD address only one
aspect of the pathogenesis by targeting the mucosal inflamma-
tory response, but this strategy remains suboptimal in terms of
disease management and efficacy. Optimal disease management
might require the microbial contribution to be addressed. Ma-
nipulating the microbiota and exploiting host-microbial signal-
ing pathways could provide benefit for the treatment of both
acute and chronic intestinal diseases. This underpins the ratio-
nale for therapeutic manipulation of the microbiota with pro-
biotic bacteria or other pharmabiotics.
Lactobacilli and bifidobacteria have traditionally been the most
common probiotic candidates, but multi-strain cocktails (eg,
VSL#3), nonpathogenic E coli, and nonbacterial organisms such as
Saccharomyces boulardii and nematode parasites have been used for
probiotic effect.113,114 Pharmabiotic is an umbrella term to encom-
pass any form of therapeutic exploitation of the gut microbiota. In
addition to live probiotic bacteria, pharmabiotics might include
bacterial constituents such as DNA, probiotic-derived biologically
active metabolites, food ingredients that modulate the composi-
tion of the microbiota (prebiotics), probiotic/prebiotic combina-
tions (synbiotics), or genetically modified commensal bacteria.
Recently, in the first human trial with genetically engineered ther-
apeutic bacteria, modified Lactococcus lactis was used to deliver IL-10
locally to the gut in 10 Crohns disease patients. The treatment was
safe, disease activity was reduced, and the modified bacteria were
biologically contained.115 Therefore, bacterial-based topical deliv-
ery of biologically active proteins represents a highly promising
and safe therapeutic strategy for combating mucosal diseases.
Increasing evidence supports a therapeutic role for probiotic
strategies for treating enteric infections, post-antibiotic syn-
dromes, necrotizing enterocolitis, and irritable bowel syn-
drome.116 119 In murine models of colitis, probiotics have also
demonstrated prophylactic effects that are associated with a
reduction in proinflammatory cytokines and an induction of
regulatory cytokines.84,120 However, the role of probiotics in
human IBD is more complex. The best evidence for probiotic
efficacy in IBD is in the prevention of pouchitis,121 but results
in clinical practice appear to be inconsistent. This might relate
to variability in patient populations or the quality or choice of
probiotic preparation. In ulcerative colitis, E coli Nissle 1917,
Lactobacillus rhamnosus GG, and VSL#3 have shown efficacy sim-
ilar to the drug mesalazine in maintaining remission.93,122,123
Probiotics have induced remission of acute ulcerative colitis
also.93,124 However, in a randomized, double-blind, placebo-
controlled trial involving 157 patients, neither Bifidobacterium
infantis 35624 nor Lactobacillus salivarius subspecies salivarius
UCC118 demonstrated a significant benefit over placebo in the
maintenance of steroid-induced remission of ulcerative coli-
tis.125 These strains have attenuated disease severity in animal
models of IBD.85,126 It might be that the differences in efficacy
between animal and human IBD might reflect the timing of
administration, differences in disease severity, or effective pro-
biotic dose/body weight.
The evidence for probiotics in Crohns disease is even weaker.
Controlled studies have not established efficacy for L rhamnosus
280 OHARA AND SHANAHAN CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 3

Figure 2. Possible mechanisms contributing to the association of mutations in the CARD15 gene with Crohns disease. CARD15 encodes the NOD
protein, NOD2, which is expressed in the cytosol of cells of the macrophage/monocyte lineage, DCs, and enterocytes. Stimulation of NOD2 by MDP
of bacterial peptidoglycan leads to the activation of the transcription factor NF-B, and peptidoglycan can signal also to antigen-presenting cells
through TLR2. (A) In normal mucosa, TLR2-driven activation of NF-B is negatively regulated by non-mutated NOD2, thereby preventing excessive
production of the proinflammatory Th1 cytokines IL-12 and IFN-. Conversely, in NOD2-deficient cells, IL-10 secretion is decreased, and Th1-cell
mediated inflammation occurs through IL-12 as a result of NOD2 to negatively regulate TLR2 signaling. (B) Alternatively, it has been postulated that
in antigen-presenting cells expressing mutated NOD2, MDP elevates NF-B activation and results in excessive IL-1 production and uncontrolled
apoptosis, which together might mediate the pathogenesis of Crohns disease. (C) Deficient expression of NOD2 by intestinal enterocytes,
particularly by Paneths cells at the base of the small intestinal crypts, results in decreased secretion of antimicrobial peptides known as -defensins.
Impaired NOD-2 dependent -defensin production might lead to increased bacterial colonization and a type of bacterial overgrowth that triggers an
immunologic response to the microbiota.

GG or Lactobacillus johnsonii LA1 as maintenance therapies for
Crohns disease.127,128 Crohns disease is a complex condition, vari-
able in its location as well as its manifestation. Variability in the
composition and diversity of the microbiota along and over the
cross-sectional axis of the gastrointestinal tract suggests that de-
pending on the topographic distribution of lesions in Crohns
disease, a single probiotic might not be equally suited to different
subsets of patients. Colonic location of disease seems to respond
better to antibiotics and might, as a result, be more susceptible to
pharmabiotic therapy. It might be that multi-strain probiotic com-
binations are required, and strain selection, dosing, and coloniza-
tion issues need more intensive investigation. Moreover, there
might be a role for probiotics in the enhancement of epithelial
barrier function in the very early stages of Crohns disease.
Several unresolved issues impede the clinical evaluation of
probiotics.67,129 These include determination of optimal dose
and vehicle of delivery, development of reliable predictors of in
vivo performance, regulation and verification of product stabil-
March 2007
ity, determination of which combinations of probiotics or other
pharmabiotics are synergistic or antagonistic, and strain-strain
comparisons of probiotic performance in different indications.
Furthermore, the microbial, immunologic, and functional char-
acteristics of individual probiotic strains and their mechanisms
of action in different clinical settings require clarification. In-
dividual variability in composition of the enteric microbiota
might also be a determining factor for optimal strain selection.
To protect consumers, there is also a pressing need for more
stringent regulation of unsubstantiated health claims.
Bacterial-mediated host immune responses involve several
pathways and signal transduction systems, and several modes
of action by which commensal and probiotic bacteria dampen
NF-B signaling have already been elucidated. More detailed
clarification of probiotic mechanisms will be facilitated by the
identification of the molecular basis of prokaryotic-eukaryotic
dialogue. Furthermore, the increasing availability of commen-
sal/probiotic genomes should facilitate the identification of
commensal effector molecules or other components with
pharmabiotic potential. The possibility of using these mole-
cules to specifically target distinct points of intracellular signal-
ing cascades might alleviate inflammation in a target area and
overcome the global immunosuppressive effects associated with
current therapies. In patients with severe IBD, the use of genet-
ically engineered or designer probiotics might offer a new
strategy for more targeted delivery of anti-inflammatory mole-
cules to the inflamed mucosa.
Conclusions and Future Perspectives
The rationale of pharmabiotic therapies appears to be
justified, and gastroenterologists need to be aware of an ex-
panding scope for the microbiota in clinical practice. The ap-
peal of probiotic strategies in IBD is their safety in comparison
with immunosuppressive drugs and corticosteroids. However,
safety concerns surrounding other pharmabiotic strategies in-
cluding the modification of food-grade bacteria to achieve spe-
cific functional activity in vivo need to be resolved. Clinical
evidence of efficacy also requires substantiation, and scientifi-
cally accredited medical evidence should underlie a clinicians
choice of pharmabiotic strategy. Moreover, mining the meta-
bolic activity of the indigenous microbiota might yield novel
therapeutic agents. Nevertheless, therapeutic manipulation of
the indigenous microbiota with any form of pharmabiotic re-
mains suboptimal as a result of incomplete understanding of
commensal bacteria, their anti-inflammatory properties, and
host-microbial interactions. The importance of PRRs in the
regulation of intestinal innate immunity and homeostatic in-
teractions with the commensal microbiota has only begun to be
appreciated, but it is clear that engagement with host immune
cells is central to pharmabiotic action. Further studies of phys-
iologic interactions within the complex network of host cell
commensalPRRligand signaling in gut health and disease
should lead to the optimal exploitation of pharmabiotic ap-
proaches to alleviate mucosal inflammation in IBD and possi-
bly other intestinal diseases.
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Address requests for reprints to: Professor Fergus Shanahan, Ali-
mentary Pharmabiotic Centre, Clinical Science Building, Cork Univer-
sity Hospital, Cork, Ireland. e-mail: f.shanahan@ucc.ie; fax: 353-21-
4345300.
Supported in part by Science Foundation Ireland. F.S. is also sup-
ported by the Irish Health Research Board, the Higher Education
Authority of Ireland, and the European Union (PROGID QLK-2000-
00563) and has been afliated with a campus-based company (Ali-
mentary Health Ltd).