SUPPLEMENT ARTICLE

Mechanisms of Action of Probiotics

W. Allan Walker
Division of Nutrition and Department of Pediatrics, Harvard Medical School, Boston, and Mucosal Immunology Laboratory, Massachusetts General
Hospital for Children, Charlestown, Massachusetts

At birth, the newborn leaves the germ-free intrauterine environment and enters a highly contaminated ex-
trauterine world, which requires potent host defenses to prevent disease. Intestinal defenses develop during
gestation and have the capacity to respond but first must be exposed to colonizing bacteria. I review the
importance of bacterial colonization for the appearance of normal mucosal immune function and the clinical
consequences of inadequate colonization with regard to development of disease. For example, we now know

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that an imbalance in T-helper (Th) cells (e.g., Th2 levels greater than Th1 levels) can predispose to autoimmune
disease and gut inflammation or disease, such as necrotizing enterocolitis. As we determine the role of bacterial
colonization in the gut (bacterial-epithelial “cross talk”), we should have more-appropriate ways to modulate
the gut immune responses—for example, by use of probiotics to prevent the expression of these gastrointestinal
diseases.

If we define probiotics as “live microorganisms that
when ingested have a positive effect on health” [1], we
can begin to include as probiotics certain commensal
bacteria found in the human gut. During the past de-
cade, microbiologists, immunologists, and gastroenter-
ologists have actively studied the mechanism by which
commensal bacteria improve mucosal defenses of the
gastrointestinal tract. This article will review selected
aspects of these observations.
BACTERIAL-EPITHELIAL “CROSS TALK”
It has been known that colonizing bacteria that interact
with the gastrointestinal mucosa can communicate with
the underlying epithelial and mucosal lymphoid ele-
ments and that such interaction stimulates host de-
fenses in the gut [2]. However, it was not until recently
that investigators began to understand the so-called
bacterial-epithelial cross talk at the cellular level. With
the discovery of toll-like receptors (TLRs) on eukaryotic
epithelial, endothelial, and lymphoid cells, which could
Reprints or correspondence: Dr. W. Allan Walker, Mucosal Immunology
Laboratory, Massachusetts General Hospital for Children, 114 16th St. (114-3503),
Charlestown, MA 02129 (wwalker@partners.org).
Clinical Infectious Diseases 2008; 46:S87–91
 2008 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2008/4603S2-0008$15.00
DOI: 10.1086/523335
interact with molecular patterns on both pathogens and
commensal bacteria [3], a molecular and cellular basis
for communication could be appreciated. The TLR that
has been studied the most is TLR-4, whose primary
ligand is gram-negative lipopolysaccharide (LPS) (i.e.,
endotoxin). This receptor interacts with LPS as an LPS-
binding protein complex after being anchored to the
cell surface by a surface molecule, CD14. With this
interaction, a series of signaling molecules is activated
in the cell to release the transcription factor nuclear
factor kB (NFkB) into the nucleus, which in turn tran-
scribes inflammatory cytokines—for example, IL-8 and
IL-6—to provide the basis for an acute innate inflam-
matory response to an invading pathogen. Since the
initial discovery of TLR-4, ten additional TLRs have
been cloned, and a variety of microbial molecular pat-
terns have been identified as ligands, including patterns
on commensal bacteria [4]. The appreciation of mi-
crobial patterns that interact with pattern-recognition
receptors on eukaryotic cells has been the basis of our
understanding of bacterial-epithelial cross talk and its
role in both innate and adaptive mucosal immunity.
DEVELOPMENT OF HOST DEFENSE
A vital practical application of bacterial-epithelial cross
talk is the communication between colonizing bacteria
and the germ-free neonatal gastrointestinal tract im-

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mediately after birth. The nature and composition of these
colonizing bacteria are important contributors to the devel-
opment of gastrointestinal host defenses against infection and
allergic reaction. Table 1 lists several important components of
host defenses affected by this initial colonization. At birth, the
full-term neonate has the developmental capacity to mount an
adequate host defense. However, for the efferent (i.e., active)
component of that defense to occur, the gut lymphoid tissue
must first be stimulated by colonizing bacteria [5]. This in-
cludes expression of follicular epithelial (i.e, microfold) cells
over Peyer’s patches in the ileum and colon. These cells facilitate
interaction between microbes and lymphoid cells [6]. In ad-
dition, intraepithelial and lamina propria sites are activated to
produce protective cytokines [7], and mesenteric lymph nodes
secrete polymeric IgA (pIgA) [8]. Recently, it has been dem-
onstrated that dendritic cells in the lamina propria can extend
their appendices between epithelial cells, and, via TLR-2 and
Table 1. Probiotic effects on the development of host defense.
Generalized mucosal immune response
Balanced T-helper cell response
Self-limited inflammatory response
Polymeric IgA secretion
gut is the capacity of epithelial and lymphoid cells, after an
acute innate immune response, to turn off the inflammatory
response in the absence of a self-limited manner. In the absence
of a self-limited and innate inflammatory response, chronic
clinical conditions such as inflammatory bowel disease and
necrotizing enterocolitis (NEC) can occur. Accordingly, several
mechanisms have evolved to control chronic inflammation of
the gut. These include the interaction between CpG DNA of
bacteria and the intracellular receptor TLR-9, to which the
bacteria bind and thus activate T regulatory cells by the pro-

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TLR-4 on their surface, they can sample commensal-bacterial
molecular patterns (figure 1) [9]. The interaction leads to mat-
uration of the dendritic cells and to the release of cytokines,
which orchestrate the conversion of naive T-helper cells (Th0)
into a mature, balanced response of T-helper cells (Th1, Th2,
and Th3/Tr1), an important component in the prevention of
disease. Furthermore, commensal bacteria can cross microfold
cells and interact with antigen-presenting cells in mesenteric
lymph nodes to activate naive plasma cells into becoming pIgA-
producing B cells [10]. pIgA, in turn, coats the mucosal surface
to control subsequent microbial and antigen penetration.
An important component of mucosal host defense in the
duction of anti-inflammatory cytokines (e.g., IL-10) (figure 2)
[11]. Commensal bacteria can also secrete small molecules that
can enter intestinal epithelial cells to inhibit activation of NFkB
[12]. In addition, it has been reported recently that prolonged
exposure to molecular patterns (of peptidoglycans and LPS)
that interact with TLR-2 and TLR-4 can activate cell surface
and intracellular negative regulators, respectively, which, in
turn, can turn off transcription factors, leading to a reduction
in the production of inflammatory cytokines and chemokines
[13]. These cellular mechanisms are critical to both intestinal
host defense and the prevention of chronic disease in the gas-
trointestinal tract. With an inadequate initial bacterial coloni-

Figure 1. The direct effect of commensal bacteria on a balanced T-helper cell response. Immature dendritic cells in the lamina propria extend an
appendage between enterocytes into the intestinal lumen. Through toll-like receptor 2 (TLR-2) and TLR-4 on this appendage, they sample luminal
commensal bacteria, and, through this interaction, the dentritic cell matures and releases cytokines, which create a microenvironment for naive T-
helper cells (Th0) to mature into balanced Th1 and Th2/Tr1 helper subsets. TREG, T regulatory cells. Data are from [9].

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Figure 2. The interaction of commensal bacteria CpG DNA with the intracellular toll-like receptor 9 (TLR-9) in dendritic cells, which stimulates the
production of Th3/Tr1 cells. Commensal bacterial penetrate microfold cells to enter Peyer’s patches and interact with dendritic cells, or they interact
with dendritic cells directly. CpG DNA from these bacteria interacts with the TLR-9 intracellular receptor in dendritic cells and triggers a cytokine
response that facilitates the maturation of T regulatory (TREG) cells that down-regulate acute inflammatory responses to prevent chronic intestinal
inflammation. TGF, transforming growth factor. Data are from [11].

zation, probiotics can be used to accomplish the same balanced was directly correlated with the inhibition of pathogenic Esch-
immune response [14]. erichia coli attachment and of damage to the intestinal tract.

OTHER PROTECTIVE EFFECTS OF PROBIOTICS PROBIOTIC MECHANISMS OF ACTION THAT

Probiotics have been demonstrated to have an adjuvant effect
on immunologic responses. As mentioned above, their inter-
action with mesenteric lymph nodes can result in an up-reg-
ulation of pIgA against intestinal pathogens and food antigens
[10]. In addition, Saccharomyces boulardii, a probiotic fungal
organism, has been demonstrated to enhance the specific IgG
and IgA antibody response to Clostridium difficile toxin A after
Clostridium infection [15]. Infants with rotaviral gastroenteritis
who were given Lactobacillus rhamnosus strain GG had an in-
creased IgA response to the virus [16]. Finally, a pilot study
involving healthy adults about to receive typhoid vaccine for
foreign travel showed that those given Lactobacillus GG for 10
days before vaccination had significantly higher levels of anti-
typhoid antibodies than did those who received a placebo [17].
Furthermore, the effect of Lactobacillus GG was specific because
levels of antibodies to antigens in other vaccines received pre-
viously were not increased.
The use of probiotics has also been effective in enhancing
the mucosal barrier to pathogens and antigen presentation. A
recent study reported that known probiotics, Lactobacillus
strains, could stimulate up-regulation of mucous genes in in-
testinal goblet cells [18]. Furthermore, the effect of these pro-
biotics on the activation and secretion of mucus in the intestine
ARE DISEASE SPECIFIC
Oral tolerance, allergy, and immunity. The capacity to de-
velop oral tolerance is an important deterrent to the expression
of allergy and autoimmune disease. Oral tolerance relates to
commensal bacteria and innocuous antigens in the gut and
prevents excessive immunologic responses to increased
amounts of antigen stimulation of the gut. Oral tolerance oc-
curs when innocuous antigens and commensal bacteria cross
the mucosal surface and interact with mucosal regulatory T
cells that release a cytokine (i.e., transforming growth factor
b), which is thought to mediate the down-regulation of humeral
and cellular immune responses to those antigens. A recent ob-
servation suggests that oral tolerance cannot occur in the ab-
sence of colonized bacteria. In a study that compared oral
tolerance between germ-free and colonized animals exposed
orally to ovalbumin and that measured specific IgE levels to
quantify the humeral systemic response, it was noted that oral
tolerance, seen as a down-regulation of the IgE anti-ova re-
sponse, could not be achieved in the germ-free state [19]. In
subsequent studies, both humeral and cellular systemic toler-
ance to oral antigens could be achieved in germ-free animals
if they were conventionalized during the newborn period but
not if conventionalized as adults, which suggests that early ex-

Probiotic Mechanisms • CID 2008:46 (Suppl 2) • S89

posure to colonizing bacteria is necessary to achieve oral tol-
erance [20]. Finally, recent reports have suggested that the ex-
pression of TLR-4 on enterocytes is necessary to achieve
tolerance [21] and that extensive use of antibiotics would re-
duce levels of colonizing bacteria and thereby cause a loss of
oral tolerance [22]. Finally, probiotics can be used to regain
tolerance after extensive use of antibiotics [23].
NEC. A major condition that affects premature infants is
NEC. It is thought that necrotic inflammation of the distal
small intestine is due to an inadequate, immature, excessive
inflammatory response to both pathogenic and commensal bac-
teria [24, 25]. A recent clinical study in Taiwan showed that
the introduction of probiotics (Lactobacillus lactis and Bifido-
bacteria infantum) a few days after birth in premature babies
with body weight !1500 g can prevent the expression of NEC
[26]. A meta-analysis of different organisms used as probiotics
in this situation has shown that results are generally positive
[27]. We reported elsewhere that IkB, the inhibitor of NFkB,
is underdeveloped in premature enterocytes, which in part ac-
counts for the NFkB-mediated, excessive, immature increase in
IL-8 production and for the inflammation in NEC [28]. In
subsequent studies using a known enterocyte-maturation fac-
tor, corticosteroids, we showed that pretreatment with this
trophic factor in fetal enterocytes can reduce the IL-8 response
to inflammatory stimuli by increased IkB expression, suggesting
that developmental reduction in the expression of genes that
regulate the innate immune response may account for the ex-
pression of NEC in premature infants [29]. Most recently, using
DNA microarrays of fetal enterocyte RNA and biopsy of in-
testinal specimens from older children, colleagues and I have
shown that TLRs and their signaling-molecule genes are up-
regulated, and the negative regulators of innate inflammatory
response are down-regulated, again suggesting the develop-
mental basis for the extensive inflammatory response (N. Nan-
thakumar and A. Walker, personal communication). We have
also shown that culture media from the same probiotics shown
to prevent clinical NEC (i.e., L. lactis and B. infantum [26])
can modify expression of genes of the innate immune response
(e.g., TLRs and signaling molecules were decreased and negative
regulators were increased) and, at the same time, can strikingly
reduce the IL-8 response (N. Nanthakumar and A. Walker,
personal communication), which further suggests a mechanism
for protection against NEC.
CONCLUSIONS
In this review, I have suggested that, on the basis of the strict
definition of probiotics, certain human commensal bacteria
may qualify as probiotics. The demonstration of pattern-rec-
ognition receptors (e.g., TLRs) on eukaryotic cells, which in-
teract with molecular patterns on pathogens and commensal
bacteria, can provide the molecular basis for bacterial-epithelial
S90 • CID 2008:46 (Suppl 2) • Walker
cross talk, which results in both innate and acquired immune
responses. Different probiotics can function differently in the
development of intestinal host defenses, as an adjuvant of im-
mune responses or to strengthen the mucosal barrier. There-
fore, one needs to be careful when selecting a probiotic for a
specific function in the gastrointestinal tract.
Acknowledgments
Financial support. National Institutes of Health (R37 HD12437, RO1
DK70260, P30 DK40561, and PO1 DK33506).
Supplement sponsorship. This article was published as part of a sup-
plement entitled “Developing Probiotics as Foods and Drugs: Scientific and
Regulatory Challenges,” sponsored by the Drug Information Association,
the National Institutes of Health National Center for Complementary and
Alternative Medicine (1R13AT003805-01 to Patricia L. Hibberd), the Cal-
ifornia Dairy Research Foundation, Chr. Hansen, the Dannon Company,
General Mills, Institut Rosell, and Yakult International.
Potential conflicts of interest. W.A.W. is part of the Dannon/Yakult
Scientific Advisory Board and is a consultant for the Dannon Company.
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