Current Drug Targets - Inflammation & Allergy, 2003, 2, 145-154 145

Probiotics and Immune Regulation of Inflammatory Bowel Diseases

Yingzi Cong*, Astrid Konrad, Nuzhat Iqbal and Charles O. Elson

Division of Gastroenterology and Hepatology, The University of Alabama at Birmingham, Birmingham,

AL 35294, USA
Abstract: Intestinal microflora play an important role in the pathogenesis of inflammatory bowel diseases
(IBD). Certain probiotic bacteria provide beneficial effects for human health and intervention of IBD. Possible
mechanisms underlying these effects are diverse and include many aspects of the interaction of the host with
its commensal microflora, with immunological and non-immunological effects. This review paper will focus
on the recent progress in the use of probiotics in the treatment of IBD, and discuss the potential
immunological mechanisms underlying their effects, such as the modulation of mucosal T cell, B cell,
epithelial cell, dendrtic cell, macrophage, nature killer cell, antibody, and cytokine responses.
Keywords: Probiotics; Inflammatory Bowel Diseases; Immune Regulation; Microflora.; Mucosa.

1. INTRODUCTION The human body contains 10 times more microbes than

The importance of commensal microflora for human
health was first addressed by Elie Metchnikoff at the
beginning of the 20th century. He attributed beneficial
effects on human health to the consumption of Lactobacillus
present in yogurt [1]. The concept that currently groups a
number of bacterial strains as “probiotics” was introduced in
1965 by Lilly and Stillwell [2], and is defined as living
non-pathogenic microorganisms, which upon ingestion exert
a positive influence on the health or physiology of the host
beyond inherent basic nutrition [3]. Probiotics include
bacteria, such as Lactobacilli sp and Bifidobacteria sp,
some Escherichia coli, Enterococci, and certain yeast,
mainly Saccharomyces. Their fate in the gastrointestinal tract
and their effects differ among strains [4]. To date, therapeutic
and preventive effects of probiotics on infections, intestinal
disorders, and food allergy have been described [5]. Some
probiotics demonstrate immunostimulatory properties by
enhancing innate immune responses [6], and some others
may help eradicate pathogens in chronic carriers of
S a l m o n e l l a and provide a beneficial effect during
Clostridium difficile infection [7, 8]. These studies suggest
that probiotics may enhance immune response and positively
affect indigenous microflora. Although many health claims
have been made concerning the potential of probiotics to
prevent or treat intestinal disorders, only a few probiotic
strains have shown to be effective in randomized placebo-
controlled clinical trials. In this paper, we review some of
recent progress in the use of probiotics in the treatment of
inflammatory bowel diseases, and discuss the potential
immunological mechanisms underlying their effects.
2. INTESTINAL COMMENSAL FLORA
Gastrointestinal tract is colonized by a vast and diverse
community of microbes that are essential to its function.
*Address correspondence to this author at the Division of
Gastroenterology and Hepatology, The University of Alabama at
Birmingham, 633 Ziegler Research Building, 703 South 19th Street,
Birmingham, AL 35294-0007, USA; Tel: +1-205-934-6061; Fax: +1-205-
934-8493; E-mail: ycong@uab.edu
eukaryotic cells. These microbes have evolved in concert
with their host to occupy specific regions and niches in the
gastrointestinal tract. A balanced, complex microflora is
necessary for normal digestion and to maintain the
homeostasis of the intestinal ecosystem [9]. Bacteria are
present throughout the gastrointestinal tract, but their
distribution pattern and concentration vary greatly. The
microbial biota varies qualitatively and quantitatively along
the length of the intestine with an increasing gradient of
microbes from the stomach to the colon, reaching 1011-12
organisms/gram of stool in the colon. The viability of
ingested bacteria is reduced dramatically by contact with
gastric acid, and the stomach normally contains only very
few bacteria (< 103 c.f.u/mL) that are predominantly Gram-
positive and aerobic. The human small intestine represents a
transitional zone between the sparse population of aerobic
flora found in the stomach and the profuse bacterial flora of
the colon. The microflora of the proximal small bowel is
similar to that of the stomach. In the distal ileum, the
concentration of microorganisms increases, and Gram
negative bacteria begin to outnumber Gram-positive
organisms. A dramatic increase of bacterial concentration
occurs across the ileocecal valve; within the colon, the
bacterial concentration is between 1011 to 1012 and anaerobes
outnumber aerobes by a factor of 102-104 [10].
The enteric biota is complex with an estimate of many
hundreds of different cultivable organisms present [11].
However, not all microbes known to be present in the GI
tract can or have been cultured, and the majority of bacteria
remain uncultured [12]; so this is an underestimation.
Beneficial metabolic activities of microflora include
synthesis of vitamin B and K, production of epithelial
nutrients such as short-chain fatty acids, metabolism of
dietary carcinogens to inactive compounds and the
conversion of pro-drugs into active drugs. Direct
involvement of the flora in host defense is demonstrated
when disturbed by broad-spectrum antibiotics therapy that is
occasionally complicated by overgrowth of Clostridium
difficile [13]. The indigenous flora also promotes host
defense indirectly by influencing the development and

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146 Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2
function of mucosal immune response. This is shown in
adult germ-free animals that have little or no mucosal
lymphoid tissue or S-IgA, despite exposure to a variety of
food antigens. When germ-free animals are colonized by an
intestinal biota, their mucosal lymphoid tissue greatly
expands along the length of the intestine and S-IgA is
produced in quantity [14]. In segmented filamentous bacteria
(SFB)-monoassociated mice, TCRαβ + IELs expand and
express cytotoxic activity from 2-3 weeks after
administration [15]. The capacity to generate IgA-producing
cells progressively increases in response to intestinal
antigenic stimulation, particularly with the establishment of
the gut microflora [16, 17]. The intestinal IgA fully
develops by the age of 6 weeks in conventionally raised
mice [18]. Upon colonization, some bacteria have been
shown to translocate to the mesenteric lymph node (MLN),
but the number of translocating bacteria begins to decrease
with the onset of specific IgA response, reflecting maturation
of the intestinal immunologic defense mechanisms. Among
the many bacterial strains that comprise the normal enteric
biota, not all are equally able to stimulate the mucosal
immune system [19]. When given to germ-free mice, some
bacteria are more effective than others in inducing germinal
center reactions in Peyer’s patches [20, 21], stimulating IgA
plasma cells in the gut lamina propria [17, 22], and specific
antibodies in the circulation [23-25]. Bacterial colonization
can also alter gene expression of the epithelium, such as
production of fucosylated glycoconjugates and α , 2-
fucosyltransferase mRNA in the small-intestinal epithelium
[26-28], which may have secondary effects on the mucosal
immune system. The selective antibody response to bacterial
species found in colitic mice suggests that variability in
immuno-stimulatory capability of gut bacteria extends to
pathologic states [29]. Consistent with these observations,
reconstitution of germ-free HLA-B27/ß2m transgenic rats
and IL-10 deficient mice with certain bacterial species, but
not others, result in the induction of intestinal inflammation
[30, 31].
During neo-natal colonization of mice with the enteric
bacterial biota, there are large swings in bacterial strains that
occur in a sequential manner [32-35]. This occurs in mice
from about the 10th to the 18th day after birth. This period is
a time of profound rearrangement of bacterial populations in
mice, which eventually reaches the “adult” pattern by 3 to 4
weeks and then is thought to remain stable throughout adult
life. This period of colonization with bacterial biota
corresponds to a time of immaturity of the systemic immune
system and to a rapid development of the mucosal immune
system, and thus this encounter of the host with the rich
assortment of bacterial antigens could have profound
consequences later in life. Neo-natal antigen exposure has
been shown to alter immune responses to the same antigen
when re-encountered again as an adult [36]. The intestinal
flora from 1-23 days old mice is inefficient, whereas that
from 25 days old mice can induce intestinal IgA
plasmocytes [18]. In humans, microbial colonization begins
after birth. The maternal intestinal flora is the source of
bacteria colonizing the newborn's intestine. Colonization is
also determined by contact with surroundings. Initially,
facultative anaerobic strains dominate. Later, the lactic acid
bacteria and coliforms become the predominant part of the
microflora [37]. Breast-feeding encourages the growth of
Cong et al.
Bifidobacteria in the gut, whereas formula-fed infants have a
more complex microflora. After weaning, the composition of
the microflora resembles that of the adult flora [38].
One potentially important immunoregulatory function of
the intestinal microflora is their involvement in the
generation of the immunocompetent cells during
development and maintenance of homeostasis of the gut-
associated immune system [22, 39]. Bacterial colonization is
required for newborns to make serum antibodies to T-
dependent and type 2 T-independent antigens [40]. Neo-natal
immune response is initially characterized by a Th2 cell
cytokine profile, and later the bacterial stimuli emerging
from the post-natal gut seems to play a major role in driving
this Th2-skewed immune response toward a more finely
balanced Th1 and Th2 immune responses. Although no
bacterial species has been found to be uniquely involved in
these processes, Lactobacilli are potential candidates because
many species of Lactobacillus have been demonstrated to be
immunomodulatory both in vitro and in vivo [41, 42].
3. MICROFLORA IN PATHOGENESIS OF
INFLAMMATORY BOWEL DISEASE
Inflammatory bowel diseases (IBD), consisting of
Crohn’s disease (CD) and ulcerative colitis (UC), are chronic
immune-mediated diseases of the intestinal tract of unknown
etiology [43]. Similar to other chronic inflammatory and
autoimmune disorders, initiation of IBD appears to involve
interactions among genetic, environmental and immune
factors. Although the pathogenesis of IBD is poorly
understood, the characteristic histopathology of the lesion as
well as an abundance of immunologic abnormalities
indicates that it involves a state of local immune
hyperreactivity. Much of this immune reactivity appears to
be directed at the enteric bacterial biota. The commensal
flora conditions the level of activation of mucosal immune
response and appears to be a key factor in driving mucosal
inflammation in genetically susceptible individuals.
Increasing number of both clinical and laboratory-derived
observations implicate the normal flora in the pathogenesis
of IBD. It has been recognized for a long time that the
anatomic sites of the highest bacterial concentration (distal
ileum and colon) are the sites most frequently affected by
inflammation in patients with IBD [44]. There is growing
evidence that the fecal flora, including the mucosal-
associated flora, differs in patients with active IBD compared
with quiescent disease or healthy volunteers [45]. Several
studies demonstrate decreased luminal concentrations of
lactobacilli and bifidobacteria concentrations in patients with
active Crohn's disease or ulcerative colitis (UC) [46-48].
High concentrations of bacteria adherent to the mucosa have
been found in patients with IBD, but not in control subjects,
and the concentrations of adherent mucosal bacteria increase
progressively with the severity of disease [49]. There is
persuasive evidence for increased immune reactivity to
components of the commensal flora in experimental models
and IBD patients [50, 51, 56]. The most compelling
evidence for the role of normal flora in pathogenesis of IBD
comes from the experimental animal models which have
significant advantages in studying interactions among
environmental, immune, and genetic components that lead
to chronic intestinal inflammation. While a diversity of
Probiotics and Immune Regulation of Inflammatory Bowel Diseases
sporadic and engineered genetic defects have been described
which a predisposed to chronic inflammatory bowel diseases
in mice, with varying immunologic mechanisms mediating
inflammation, colonization with normal enteric flora is
required for full expression of the disease. In a number of
models, such as IL-2 deficient mice, IL-10 deficient mice and
HLA-B27/ß2m transgenic rats, no disease occurs when the
animals are raised under germ-free conditions [53-55], or the
disease is dramatically ameliorated when the animals are
raised under specific pathogen-free conditions [54]. Most
importantly, colitis has been induced in SCID mice by
adoptive transfer of CD4+ T cells reactive to enteric bacterial
antigens, providing direct evidence that bacterial-reactive T
cells can mediate inflammatory bowel disease [56].
4. THERAPEUTIC APPLICATION OF PROBIOTICS
IN IBD
Conventional and experimental therapy of IBD mainly
targets the inflammatory process by suppressing the host
response. Because of the key role of the intestinal microflora
in the development of IBD, manipulating this component
provides a very intriguing novel therapeutic approach. Not
all members of the gastrointestinal flora are considered
proinflammatory. HLA-B27/ß2m transgenic rats mono-
associated with Bacteroides vulgatus developed colitis,
whereas those mono-associated with E. coli remained
healthy [30]. Administration of probiotic bacteria to IL-10
deficient mice ameliorated their colitis [57]. Multiple reports
have documented that strains of lactobacilli and
bifidobacteria, which may contain strains with probiotic
activities, are decreased in patients with active Crohn's
disease or ulcerative colitis [46, 47, 58]. However, many
reports include vague or imprecise data. Only recently have
the effects of probiotics on human and animal health been
evaluated using elegant scientific tools.
4.1 Selection of Probiotics
Probiotic strains are often selected empirically from
bacterial pools, isolated from normal humans or from
traditional fermented foods. A number of criteria have been
proposed to allow more efficient and rational selection.
Attractive properties of potentially effective probiotic
bacteria include human origin, non-pathogenic behavior,
resistance to technologic processing and bile toxicity,
tolerance to gastric acid, adhesion to intestinal epithelial
surfaces, ability to persist within the gastrointestinal tract,
production of anti-microbial substances, ability to modulate
immune responses, and antagonism of potentially
pathogenic micro-organisms [59-61].
4.2 Therapeutic Application of Probiotics in Animal
Model of IBD
Recent studies have suggested a potential therapeutic role
of probiotics in experimental inflammatory bowel disease, in
that different strains of lactobacilli have been shown to
prevent the development or attenuate active inflammation in
methotrexate [62] and in acid acetic-induced colitis [58], and
to prevent colitis in IL-10 deficient mice [57, 63, 64].
In methotrexate-induced enterocolitis in rats,
administration of Lactobacillus reuteri and Lactobacillus
Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 147
plantarum decreased weight loss, intestinal permeability and
myeloperoxidase level, re-established intestinal
microecology, and reduced bacterial translocation to
extraintestinal sites [62], with L. plantarum more effective
than L. reuteri. Feeding of Lactobacillus salivarius to IL-10
knockout mice consistently decreased mucosal inflammation
and significantly reduced fecal coliform and enterococci
levels [64]. At 2 weeks of age, IL-10 deficient mice showed
no colonic injury but displayed abnormal colonic bacterial
colonization with reduced Lactobacillus sp. levels and
increased number of bacteria adherent to mucosa and of
translocated bacteria. In association with this abnormal
colonic bacterial flora, colitis developed by 4 weeks of age.
Daily intrarectal inoculation of Lactobacillus sp or oral
lactulose therapy which stimulates Lactobacilli resulted in
Lactobacillus sp repopulation, reduced numbers of bacteria
adherent to mucosa and of translocated bacteria and
prevented the onset of colitis [63]. Administration of
combinations of Lactobacillus salivarius subsp. salivarius
UCC118 and Bifidobacterium longum infantis 35624 to IL-
10 deficient mice also ameliorated colitis [52]. More recent
studies demonstrated prevention and treatment of the colitis
in IL-10 deficient mice by continuous feeding of L .
plantarum. This attenuated established colitis as manifested
by decreased mucosal IL-12, IFNγ, and IgG2a levels.
Gnotobiotic IL-10 deficient mice mono-associated with L.
plantarum exhibited mild immune system activation but no
colitis. Germ-free mice did not develop colitis. Colonization
of IL-10 deficient germ free mice with L. plantarum, and
subsequent exposure to a complex SPF flora and continued
probiotic therapy significantly decreased the severity of
inflammation [57].
4.3 Clinical Application of Probiotics to IBD
Acceptance of benefit to human health from putative
probiotic bacteria requires demonstration of effect in formal
clinical studies. Some recent clinical trials have provided
evidence of positive effects. Treatment with non-pathogenic
E. coli Nissle 1917 has almost an equivalent effect of
mesalazine in maintaining remission of ulcerative colitis
[65, 66]. Lactobacillus GG is a safe probiotic bacterium
known to transiently colonize the human intestine. In an
open-label pilot trial of children with stable Crohn's disease,
administration of Lactobacillus GG in enteric coated tablets
significantly improved gut barrier function and clinical
activity [67]. Saccharomyces boulardii, non-pathogenic
yeast with beneficial effects on the human intestine, may
also be beneficial in the maintenance treatment of Crohn's
disease when combined with mesalamine [68]. Recently,
more promising effects of probiotics on IBD have been
observed with a new combined probiotic preparation, VSL #
3, which contains 5x10 1 1 cells/g of 3 strains of
Bifidobacteria (B. longum, B breve, and B. infantis), 4
strains of L a c t o b a c i l l i (L. casei, L. plantarum, L.
acidophilus, and L. delbruekii subsp. bulgaricus), and 1
strain of Streptococcus salivarius subsp. thermssophilus. In
a small pilot trial, VSL#3 was effective in maintaining
remission in mesalazine intolerant patients with ulcerative
colitis [69]. In a double-blind, placebo-controlled trial, oral
administration of VSL#3 was effective in the treatment of
chronic pouchitis, an inflammatory complication that can
148 Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2
arise after ileal pouch-anal anastomosis surgery for the
treatment of ulcerative colitis [70, 71].
4.4 Possible Mechanisms
The possible mechanisms underlying the beneficial
effects of probiotics in IBD are rather diverse and include
numerous aspects of the interactions of the host with its
commensal microflora. They can be divided into
immunological and non-immunological effects. For non-
immunological effects, most lactobacilli species are able to
adhere to colonic epithelial cells, thereby interfering with the
adherence of other potentially pathogenic bacteria [72] and
possibly competing for nutrients while they temporarily
colonize the intestine. Recent in vitro data suggest that
inhibition of the adherence of attaching and effacing E. coli
to intestinal epithelial cells by lactobacilli is mediated
through increased expression of intestinal mucin genes [73].
Some probiotic bacterial species are able to restore damaged
intestinal permeability [63, 74, 75], which is deranged in
IBD and experimental colitis. An additional mechanism of
protection is the direct inhibition of the growth of potential
pathogenic bacterial species. For example, Lactobacillus GG
produces a bacteriocin [76], and both lactobacilli and
bifidobacteria lower the luminal pH, which can be inhibitory
for certain microbes [38, 69, 77]. More detailed information
regarding non-immunological effects of probiotics is
described in an excellent review by Steidler [78].
Because IBD involves abnormal immune responses to
enteric flora, the modulation of the mucosal immune system
by probiotics may play a key role in their therapeutic
mechanisms. Probiotic organisms can directly or indirectly
influence mucosal and systemic immune function by altering
cytokine gene expression, antibody production, and by
modulating cellular immune responses [71, 79-81]. Some
reports have demonstrated that probiotic bacteria promote
intestinal immunologic barrier by enhancing humoral
immune responses [82-84], stimulate nonspecific host
resistance to microbial pathogens [85-87], and modulate the
host immune responses to potentially harmful antigens with
a potential to down-regulate hypersensitivity reactions [88].
4.4.1 Effects of Probiotics on Intestinal IgA
The increase in local intestinal IgA levels resulting from
ingestion of probiotics may contribute to enhancement of the
mucosal resistance against gastrointestinal infections.
Several reports demonstrate that probiotic strains enhance
intestinal IgA production. An increase in systemic and
mucosal IgA response to dietary antigens was shown after
oral administration of lactobacilli [74, 89]. When formula
containing viable bifidobacteria was given to children, the
administered strain was detected in feces together with
higher fecal levels of total IgA and anti-poliovirus IgA [90,
91]. Oral administration of Lactobacillus casei strain GG to
patients with Chrohn's disease greatly increased the
intestinal IgA response and thus promoted the gut-
immunological barrier [82]. In a mouse model,
administration of a diet with yogurt containing various
lactobacilli, the number of IgA secreting cells in small
intestine increased, while the values for cells secreting IgM
and IgG and for T lymphocytes remained similar to those of
the controls. The effect of giving yogurt with probiotics on
Cong et al.
colonic lymphoid cells was mainly on the numbers of IgA-
secreting B cells and T cells, with a marked increase in T
cells. Interestingly, although the number of macrophages
was increased, there was no enhancement in their activity
that might have harmed the host by producing an
inflammatory response. Different probiotic strains have
different effects on intestinal IgA levels [83, 84, 92].
4.4.2 Effects of Probiotics on Circulating Antibody
Responses
Kaila et al. reported that when patients with diarrhea
were given Lactobacillus GG, the duration of diarrhea was
significantly shorter than in the placebo group. This effect
was associated with a significantly enhanced nonspecific
humoral response during the acute phase of the infection, in
that total peripheral blood IgG-, IgA-, and IgM-secreting
cells, as well as IgA anti-rotavirus, were greatly increased
[89]. Oral administration of Wistar and Brown Norway rats
with viable Lactobacillus casei Shirota strain YIT9029
significantly enhanced the T. spiralis-specific delayed-type
hypersensitivity (DTH) response together with increased
serum T. spiralis-specific IgG2b antibody production.
Importantly, no significant effects of L. casei on larval
counts or inflammatory reactions in the tongue musculature,
body weights, or lymphoid organ weights were observed
[93]. In another study, eight different common Lactobacilli
strains were assessed for their ability to induce pro- and anti-
inflammatory cytokines, IgA-producing plasma cells in the
gut, as well as systemic antibody responses against a
parenterally administered antigen. Oral administration of L.
r e u t e r i and L. brevis induced expression of the
proinflammatory Th1 cytokines IFNγ, IL-2 and IL-1. Oral
administration of these two strains and L. fermentum also
significantly enhanced the IgG response against parenterally
administered haptenated chicken gamma globulin. L. reuteri
induced relatively high levels of IgG2a against haptenated
chicken gamma globulin compared to L. murine. The five
other strains did not show any mucosal adjuvanticity [85].
4.4.3 Effects of Probiotics on T Cells
T cells have been shown to play a key role in immune
responses against foreign pathogens and in control of
immune homeostasis. Oral administration of mice with
probiotic-supplemented yogurts containing Streptococcus
thermophilus, Lactobacillus bulgaricus, Bifidobacterium,
and Lactobacillus acidophilus significantly increased
percentage of CD4+ T cells in spleen [94]. Oral ingestion of
lactobacilli greatly increased T cell proliferative responses to
bacteria in Peyer's patches, peripheral blood and spleen, but
did not affect macrophage and T cell proportions and
lymphocyte subset composition in these three sites [95],
suggesting that live lactobacilli may interact with the
intestinal immune system and influence the systemic
immune system. Different probiotic strains vary in their
ability to modulate T cell and B cell responses. Some
promote Th1 type responses, and some others enhance both
Th1 and Th2 type responses. In vitro stimulation with heat-
killed Lactobacillus casei strongly enhanced murine
splenocyte IL-12 and IFN-γ production, but inhibited IL-4
and IL-5 secretion, and markedly suppressed total and
antigen-specific IgE secretion by antigen (OVA)-stimulated
splenocytes, indicating that in this situation L. casei
preferentially induce Th1 development [96]. Lactobacillus
Probiotics and Immune Regulation of Inflammatory Bowel Diseases
acidophilus treatment enhanced T cell proliferation to Con A
and B-cell response to LPS, whereas Lactobacillus casei,
Lactobacillus gasseri, and Lactobacillus rhamnosus
inhibited T cell and B cell proliferation, indicating that
Lactobacillus strains can have strain-specific effects on B-
and T-cell responses [97, 98].
4.4.4 Induction of Regulatory T Cells
The inhibitory activity of regulatory T cells is believed
to be central to the prevention of autoimmune diseases,
allergies, transplant rejection, immune-deficiency disorders
and inflammation. Multiple types of regulatory T cells have
been identified, including CD4+ CD25 + T cells, Tr1 cells,
and Th3 cells [99-105]. Recently, bacterial specific-T
regulatory cells have been identified that inhibit pathogenic
responses to enteric flora and appear to play an important
role in the maintenance of normal intestinal immune
homeostasis [106]. Possibly related to these observations are
the data regarding effects of certain strains of lactic acid
bacteria, including L. johnsonii, L. gasseri, L. paracasei,
L. acidophilus, L. casei strain Shirota, and L. casei strain
GG, to modulate specific T cell responses. All strains tested
in this study induced various levels of both IL-12 and IL-10
production in murine splenic cells in vitro. Among these
strains, L. paracasei induced the highest levels of IL-12 and
IL-10. Interestingly, when L. paracasei bacteria were added
to mixed lymphocyte cultures in which BALB/c CD4+ T
cells were stimulated weekly in the presence of irradiated
allogeneic splenocytes, the L. paracasei strongly inhibited
the proliferative response of CD4+ T cells in a dose-
dependent fashion. This was accompanied by a marked
decrease of both Th1 and Th2 cytokines, including IFNγ,
IL-4, and IL-5, but maintenance of IL-10 and induction of
TGF-β in a dose-dependent manner [107]. It seems possible
that this probiotic is stimulating regulatory T cells similar
to the Tr1 cells that are involved in the maintenance of
intestinal immune homeostasis [106, 108].
4.4.5 Effects of Probiotics on Natural Killer Cells
Natural killer (NK) cells have been implicated in immune
surveillance in innate immune responses [109]. Activated
NK cells circulate through blood and lymph and accumulate
at sites of injury, infections, and tumors [110], and quickly
respond to immune activation signals, in particular for
inducing the early production of IFNγ that is necessary to
control microbial pathogen infections [111, 112]. The rapid
activation of NK cells is characteristic of innate immunity
and constitutes a first line of defense against invading
pathogens. Signals generated during the early host response
have an additional role in the shaping of subsequent adaptive
immune response. Production of IFN-γ ensures activation of
phagocytosis by monocytes and T cell differentiation to the
Th1 phenotype, thus directing the adaptive immune response
towards an appropriate effector pathway to clear the
infection. Administration of a dietary supplement with
probiotic lactic acid bacteria promoted activity of circulating
NK cells in the elderly [113]. Several studies have shown
that commensal lactobacilli isolated from the human
gastrointestinal tract activate human mononuclear cells and
are potent inducers of IFN-γ and monocyte-derived IL-12
[79, 114]. The combination of IL-12 and IL-18 is a potent
inducer of IFN-γ in human CD56bright NK cells, whereas the
combination of IL-12 and IL-15 stimulates NK cells to
Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 149
secrete IL-10 [115]. Recently, it was shown that treatment of
human peripheral blood mononuclear cells (PBMC) with
Lactobacillus johnsonii selectively activated CD3-CD56 +
NK cells, detected by enhanced expression of the activating
antigens-CD69 and CD25. Purified NK cells upregulated
CD25 and proliferated when stimulated with Lactobacillus
johnsonii and Lactobacillus sakei in vitro [81]. Stimulation
of PBMC with Staphylococcus aureus and Lactobacillius
johnsonii resulted in the activation of CD3- CD16+ CD56+
human peripheral blood NK cells to express activation
antigen CD69 and the secretion of IL-12 and IFNγ. These
effects required cell contact-dependent costimulation by
autologous monocytes [116], suggesting that phagocytosis
of such probiotic strains provided additional co-activation
signals on accessory cells that may differ from those induced
by cytokines. This was not observed when PBMC were
stimulated with non-pathogenic E. coli or lipopolysaccharide
(LPS) [81].
4.4.6 Effects of Probiotics on Epithelial Cells
Epithelial cells that line the intestinal tract participate in
the initiation and regulation of the mucosal immune
response to bacteria by interacting with immune cells of the
gut-associated lymphoid tissue, such as lamina propria
lymphocytes (LPL) and intra-epithelial lymphocytes (IEL)
[117-121]. Intestinal epithelial cells (IEC) may change
phenotype as a consequence of stimulation by IEL derived
soluble mediators, such as IFNγ and regulated production of
IFN-inducible T-cell chemoattractants [120, 122, 123]. In
the case of the colonized intestinal mucosa, however,
protection against microbial pathogens is mediated in part
by the microflora [124, 125]. IEC may be implicated in the
recognition of components of the intestinal microflora and
the transduction of bacterial-derived signals to resident
mucosal immune cells [126-129]. Changes in IEC, LPL,
and IEL have been described in response to modifications of
the intestinal microflora [6, 130-132]. It has been shown that
treatment of IL-10 deficient mice with VSL #3 resulted in an
enhancement of epithelial barrier function and a reduction of
TNFα production. Direct application of VSL #3 on to the
apical surface of gut epithelial cell T84 monolayers greatly
increased their barrier function and resistance to Salmonella
invasion [133]. L. casei or Clostridium butyricum greatly
enhanced gut epithelial cell proliferation in rats and thus
possibly promoted tissue repair [134]. In a CaCo-2 epithelial
cell-leucocyte co-culture system, probiotic bacteria elicited a
differential cytokine response by human IEC in the presence
of human blood leucocytes. Leucocyte-sensitized CaCo-2
cells in vitro distinguished between signals delivered by
different probiotic bacteria and in turn transduced a
discriminative signal to underlying PBMC. L. sakei
stimulated IEC TNFα and IL-1 production, similar to non-
pathogenic E. coli stimulation. In these co-cultures,
differences in the regulation of inflammatory immune
response by IEC were observed between enteropathogenic E.
coli and probiotic non-pathogenic bacteria, suggesting that
IEC discriminated between enteropathogens and commensal
bacteria. L. johnsonii had a low potential to induce
proinflammatory response but rather favored induction of
TGFβ expression in CaCo-2 cells [80]. TGFβ is a key factor
implicated in the regulation of intestinal barrier function
[135-137], and is thought to mediate tolerance to the
indigenous microflora via bystander suppression [138-142],
150 Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2
as well as mediate the inhibitory function of T regulatory
cells [143]. These results support the hypothesis that
bacterial signaling at the mucosal surface requires a network
of cellular interactions. According to these results,
sensitization of CaCo-2 cells by neighboring
immunocompetent cells constitutes a crucial step for the
recognition of non-pathogenic bacteria because CaCo-2 cells
alone remained hyporesponsive to probiotic bacterial
stimuli. Interestingly, L sakei which induced biosynthesis of
TNF-α and IL-1 in CaCo-2-leucocyte co-cultures also
strongly stimulated IL-10 secretion from underlying PBMC,
indicating that a negative feedback loop might be activated
[80]. IL-10 is crucial in the control of gut homeostasis and
immunity [144-152], and downregulates chemokine response
in activated IEC [153]. Interestingly, Lactobacilli and
Bifidobacteria strains from VSL#3 and Lactobacillus GG
did not induce IL-8 of epithelial cell HT29/19A monolayers,
whereas both cell debris and cell extracts from E. coli Nissle
1917 induced IL-8 production in a dose-dependent way,
suggesting that probiotic VSL#3 and Lactobacillus GG and
E. coli Nissle 1917 may exert their beneficial effects on the
host by a different mechanism [154]. It is noteworthy that
certain bacteria can actively interfere with the
proinflammatory I-κB/NF-κB signaling pathway in IEC by
blockade of I-κBα degradation [155]. In addition to the
effects on IEC cytokine production, L a c t o b a c i l l u s
rhamnosus promoted survival of IEC by inhibition of
apoptosis in TNFα stimulated mouse IEC line YAMC and
human epithelial cell line HT29. Live Lactobacillus
rhamnosus GG or its supernatant stimulated anti-apoptotic
Akt activation, but did not alter ERK, NF-κ B or
SAPK/JNK activities [156].
4.4.7 Effects of Probiotics on Dendritic Cells
Dendritic cells (DC) are highly specialized APC of the
immune system [157]. Depending on their maturational
state, they possess different functional properties. Fully
mature DCs are potent activators of naïve T cells and are
regarded as important initiators of primary T cell responses.
Immature DCs or “tolerant DC” are poor stimulator of T cell
responses and have been shown to induce anergic or
regulatory T cells [158-163]. DCs are present throughout the
gastrointestinal tract [164, 165], and are in close proximity
to the intestinal microbiota. Thus, DC may be the targets for
immune modulation by probiotics. In that regard, a recent
study has shown that species of Lactobacilli differentially
activated DC by modulating expression of cytokines and
surface costimulatory molecules [166]. Mouse DC IL-12 and
TNFα production was substantially different when treated
with various strains of Lactobacillus. Similar but less
pronounced differences were observed among lactobacilli in
the induction of IL-6 and IL-10. The cytokine induction was
bacterial dose-dependent. Low concentrations of L. casei
yielded maximal levels of IL-6, IL-12, and TNFα but
virtually no IL-10 induction. At higher concentrations of L.
casei, IL-10 production increased radically with no change in
the level of other cytokines. This observation indicates that
the threshold of bacterial concentration necessary to induce
cytokine production varies among cytokines. All strains up-
regulated surface MHC class II and CD86, which is
indicative of DC maturation. Interestingly, Lactobacillus
reuteri DSM12246 inhibited IL-12, IL-6, and TNFα
induction by L. casei CHCC3139, whereas IL-10 production
Cong et al.
remained unaltered. In analogous fashion, L. reuteri reduced
L. casei-induced up-regulation of CD86. These results
suggest that different strains of Lactobacilli exert very
different DC activation patterns, and some strains may be
capable of inhibiting activities of other strains in the species.
IL-12 and IL-10 produced by APCs exert largely opposite
immune effects. IL-12 is critical for the induction of Th1
cells and elicits IFNγ production by T cells and by NK cells
[167]. IL-10 is an anti-inflammatory cytokine that
suppresses IL-12 production, and acts on macrophages to
prevent their activation and elaboration of proinflammatory
molecules and chemokines, thus inhibiting T cell
recruitment into the intestine [148, 149, 168]. Thus, by
inhibiting DC IL-12 production and promoting IL-10
production some probiotics may enhance regulatory T cell
induction through modulation of DC maturation [160-163].
4.4.8 Effects of Probiotics on Macrophages and
Monocytes
Macrophages and monocytes have a central role in
initiating the innate immune response, which leads to
activation of the adaptive response. Macrophages
phagocytose infected cells present antigens to T and B cells,
and produce cytokines and chemokines that modulate
immune responses [169, 170]. These cytokines include
TNFα and IL-6, which are among the first produced during
the innate immune response toward bacteria. Some probiotic
bacteria have the potential to influence macrophages and
monocytes in a very specific way. Strain-dependent
induction of cytokines has been described after oral
Lactobacillus administration and may provide a way to
modulate local immune responses in any desired direction
[85, 88, 114, 171]. In vitro stimulation with heat-killed
Lactobacillus casei strongly enhanced IL-12 by antigen
OVA-stimulated splenocytes, and such IL-12 augmentation
was macrophage-dependent [96]. Lactic acid bacteria
stimulate human peripheral blood mononuclear cell TNFα,
IL-6, and IL-10 production [79]. Lactobacillus rhamnosus
GG induce NF-κB and STAT DNA-binding activity in
human primary macrophages. NF-κB activation was rapid
and was not inhibited by a protein synthesis inhibitor
cycloheximide, suggesting that these bacteria could directly
activate NF-κB [172]. Feeding of Lactobacillus rhamnosus,
Lactobacillus acidophilus and Bifidobacterium lactis
significantly enhanced natural and acquired immunity in
healthy mice, in that the phagocytic activity of peripheral
blood leucocytes and peritoneal macrophages was greatly
increased compared to the control mice [173]. An interesting
recent report showed that increased intestinal mucosal TNFα
production in Crohn’s disease was downregulated ex vivo by
the probiotic bacteria L. casei or L. bulgaricus. However,
these bacteria did not induce changes in non-inflamed
mucosa [174]. Treatment of patients with pouchitis with
VSL#3 increased tissue levels of IL-10 and decreased TNFα
and IL-1α, inducible nitric oxide synthase, and matrix
metalloproteinase [71]. These effects may favor the
generation of regulatory T cells and down-regulation of
inflammation.
As described above, different probiotic strains exert very
different effects on DC and macrophage cytokine induction.
DC and macrophage recognition and response to molecular
structures on bacteria (pathogen-associated molecular
Probiotics and Immune Regulation of Inflammatory Bowel Diseases
patterns) occur through Toll-like receptors (TLRs).
Activation of these surface receptors by bacterial components
is a key factor for regulation of the immune response and a
link between innate and adaptive immune responses [175].
Differential activation of TLR by multi-type pathogen-
associated molecular patterns could induce different cytokine
patterns. This might explain the observed differences in
cytokine induction found by various probiotics. TLR2 has
been found to be a receptor for peptidoglycan, lipoteichoic
acid, and bacterial lipoprotein in addition to LPS [176,
177]. LPS also binds to TLR4 and CD14, thus these
receptors constitute alternative activation pathways for LPS
[178-180]. Recent evidence suggests that there are structural
and functional differences in the LPS produced by different
bacteria [181, 182]. It has been proposed that the shape of
the lipid A component determines the bioactivity of LPS
[183], with lipid A that adopts a conical conformation being
more active than lipid A that adopts a cylindrical shape.
Some of the cylindrical LPS molecules may be antagonists.
Variation in the molecular composition and the three-
dimensional conformation of the lipid A component of
different types of LPS may explain the activation of different
TLR signaling pathways. Conical-shaped LPS stimulates
cells through TLR4, whereas cylindrical-shaped LPS
activates via TLR2 [184]. Therefore, it could be speculated
that the inhibitory effect of some probiotics is mediated
through interference with TLRs, perhaps via alterations in
the shape of LPS, or other TLR ligands. The involvement of
TLR in the differential activation capacities of species of
probiotic bacteria on DC and macrophages provides an
intriguing target for future mechanistic studies.
5. SUMMARY
The immune regulation of IBD by probiotics has been
shown to be promising and paradoxical. Proinflammatory
cytokines, IL-1, TNFα, and IFNγ, play a pivotal role in
inflammation. Cytokine transgenic or knockout mice have
been shown that a harmless immune response to commensal
intestinal microflora becomes a harmful inflammatory state
in the absence of certain cytokines [30, 53-55]. These
observations indicate that inflammation is induced by an
unbalanced local or systemic cytokine milieu. As described
above, treatment with some probiotic bacteria can modulate
TNFα and IFNγ production. Thus, ingestion of probiotic
bacteria may stabilize the immunologic barrier of the
intestinal mucosa by reducing the generation of local
proinflammatory cytokines. Specific probiotic strains can
normalize aberrant antigen-induced production of the
proinflammatory cytokines [185]. These data suggest that
the immunomodulating effect of probiotic bacteria may
depend on the immunologic state of the host, and there may
be differences between specific probiotic strains.
6. FUTURE TARGETS
Probiotics allow modulation of the intestinal immune
system and the commensal flora. However, the promise of
probiotics has often been overstated. Rigorously designed,
controlled clinical trials of efficacy in well-categorized
patients are required for future progress. It is now clear that
different probiotic bacteria have different effects on the host
Current Drug Targets - Inflammation & Allergy, 2003, Vol. 2, No. 2 151
and claims made for one probiotic strain cannot readily be
applied to another even within the same species. Thus,
probiotic bacterial strains must be evaluated individually.
Because many of the probiotic effects are mediated via
immune regulation, in particular by control of the balance of
proinflammatory and anti-inflammatory cytokines, the future
studies should focus on defining the specific
immunomodulatory properties of probiotic bacteria.
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