Oral Treatment with Live Lactobacillus reuteri Inhibits
the Allergic Airway Response in Mice
Paul Forsythe1, Mark D. Inman2, and John Bienenstock1
1
The Brain–Body Institute and Department of Pathology and Molecular Medicine and 2Firestone Institute for Respiratory Health,
McMaster University, and St. Joseph’s Healthcare, Hamilton, Ontario, Canada
Rationale: Clinical trials have demonstrated that probiotics may be
effective in the treatment and prevention of atopic disease in chil-
dren but there have been few reports of therapeutic effects of oral
probiotics outside the gastrointestinal tract.
Objectives: We investigated the effect of two probiotic organisms
on the response to antigen challenge in a mouse model of allergic
airway inflammation.
Methods: We used an ovalbumin-sensitized asthma model in BALB/c
and Toll-like receptor 9–deficient mice. Animals were treated with
probiotic organisms via gavaging needle before antigen challenge.
After antigen challenge, airway responsiveness to methacholine,
influx of inflammatory cells to the lung, and cytokine levels in bron-
choalveolar lavage fluid were assessed.
Results: Oral treatment with live Lactobacillus reuteri but not Lactoba-
cillus salivarius significantly attenuated the influx of eosinophils to
the airway lumen and parenchyma and reduced the levels of tumor
necrosis factor, monocyte chemoattractant protein-1, IL-5, and
IL-13 in bronchoalveolar lavage fluid of antigen-challenged animals,
but there was no change in eotaxin or IL-10. L. reuteri but not
L. salivarius also decreased allergen-induced airway hyperrespon-
siveness. These responses were dependent on Toll-like receptor 9
and were associated with increased activity of indoleamine 2,3-
dioxygenase. Killed organisms did not mimic the ability of the live
L. reuteri to attenuate inflammation or airway hyperresponsiveness.
Conclusion: Oral treatment with live L. reuteri can attenuate major
characteristics of an asthmatic response in a mouse model of allergic
airway inflammation. These results suggest that oral treatment with
specific live probiotic strains may have therapeutic potential in the
treatment of allergic airway disease.
Keywords: airway inflammation; bronchial hyperresponsiveness;
probiotics; Toll-like receptor 9; mouse model
Probiotics are defined as live microorganisms, which, when con-
sumed in adequate numbers, confer a health benefit on the host
(1). Organisms used as probiotics are most frequently of the
Lactobacillus or Bifidobacterium species and are generally com-
mensals, occurring naturally as part of the gut microbiota. There
is increasing evidence to support a therapeutic role for probiotics
in the treatment of various inflammatory conditions. Random-
ized controlled trials of probiotics have proved successful in
patients with chronic pouchitis and irritable bowel syndrome (2,
3). A multispecies probiotic (VSL#3; VSL, Gaithersburg, MD)
given to IL-10–deficient mice with established colitis normalized
gut barrier function, reduced proinflammatory cytokines IL-1
and tumor necrosis factor (TNF)-␣, and diminished histologic
evidence of disease (4). Although there is support for the efficacy
(Received in original form June 19, 2006; accepted in final form December 20, 2006 )
Correspondence and requests for reprints should be addressed to John Bienens-
tock, M.D., Departments of Pathology and Molecular Medicine, McMaster Univer-
sity, Brain–Body Institute, St. Joseph’s Healthcare, 50 Charlton Avenue East, T3304
Hamilton, ON, L8N 4A6 Canada. E-mail: bienens@mcmaster.ca
Am J Respir Crit Care Med Vol 175. pp 561–569, 2007
Originally Published in Press as DOI: 10.1164/rccm.200606-821OC on January 4, 2007
Internet address: www.atsjournals.org
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Despite indications that probiotics can modulate immune
responses in the lung, there have been no reports of the
effects of oral treatment with a probiotic on major charac-
teristics of asthma, including airway inflammation and hyp-
erresponsiveness.
What This Study Adds to the Field
Oral treatment with a probiotic organism can attenuate
major characteristics of an asthmatic response, including
airway eosinophilia, local cytokine responses, and hyperres-
ponsiveness.
of probiotics in the treatment of experimental colitis and food
allergy (2, 3, 5) and several studies have characterized the ability
of various strains of probiotics to alter the activity and cytokine
production of the gut and associated lymphoid tissue, clarifica-
tion of the underlying mechanisms of these antiinflammatory
effects is still obscure.
In addition to effects in the gut, there is evidence that pro-
biotics, given perinatally, may be protective against manifesta-
tions of atopic disease. Clinical trials have demonstrated that
Lactobacillus rhamnosus GG may be effective in treatment and
prevention of early atopic disease in children (6, 7). Lactobacillus
fermentum was shown to be beneficial in improving the extent
and severity of atopic dermatitis in young children (8). Such
findings have lead to an increased interest in the role of commen-
sal organisms in the regulation of systemic immune responses.
Although long-term follow-up studies of children at high risk
for developing allergy have not shown significant beneficial ef-
fects of probiotics on the incidence of asthma (7), there is evi-
dence indicating that oral administration of certain microbial
organisms can modulate immune responses in the lung. Rats
orally immunized with killed Pseudomonas aeruginosa exhibited
enhanced bacterial clearance from the airways compared with
nonimmunized donors after intratracheal challenge with live
P. aeruginosa (9). This response was associated with increases in
bronchoalveolar neutrophils and in recruitment and phagocytic
activity of alveolar macrophages. Furthermore, infection with
the gut-restricted bacterium Citrobacter rodentium attenuated
the airway eosinophilia that results from pulmonary Cryptococ-
cus neoformans infection (10). A double-blind randomized trial
found that oral treatment with the probiotic L. rhamnosus GG
reduced the rate and severity of respiratory virus infection in
children (11). Oral treatment of mice with Lactobacillus casei
increased pulmonary natural killer cell activity and enhanced
IFN-␥ and TNF production by nasal lymphocytes. The probiotic-
treated animals also had reduced viral titers in nasal washings
after influenza infection (12).
562 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007
Despite indications that probiotic treatment can modulate
some immune responses in the lung, there have been no reports
of the effects of oral treatment with a probiotic organism on
major characteristics of asthma, including allergic airway in-
flammation and bronchial hyperresponsiveness. Here, we de-
scribe the ability of one probiotic organism, Lactobacillus reuteri,
but not another, Lactobacillus salivarius, to attenuate antigen-
induced eosinophil influx to the airway as well as local cytokine
responses and hyperresponsiveness to methacholine in an oval-
bumin (OVA)-sensitized mouse model of allergic airway disease.
Portions of the data presented in this article have previously
been published in abstract form (13).
METHODS
Animals
Adult male BALB/c or Toll-like receptor 9–deficient (TLR9⫺/⫺) mice
(20–25 g) were maintained in an automatic light/dark cycle (light periods
of 12 h) and provided water and chow ad libitum. Mice were acclima-
tized to the animal facility for 1 week before experimentation. Trans-
genic TLR9⫺/⫺ mice on a balb/c background were obtained from Dr.
K. Rosenthal (McMaster University) with permission from Dr. S. Akira
(Department of Host Defense, Osaka University, Osaka, Japan). Age-
matched (8–9 wk old) animals were used in all experiments. These
experiments were performed in accordance with guidelines of the Cana-
dian Council for Animal Care.
Experimental Asthma
This study used an OVA-sensitized mouse model of allergic airway
inflammation. Briefly, mice were sensitized by intraperitoneal injection
of 20 ␮g OVA adsorbed with 500 ␮g alum in saline on Day 0 and Day
6. On Days 12 and 14, mice were challenged intranasally with 5 ␮g
OVA per mouse (14). Twenty-four hours after the last challenge (Day
15), mice were subjected to measurements of airway responsiveness
followed by bronchoalveolar lavage (BAL). OVA/alum-sensitized, sa-
line-challenged mice served as control animals.
Bacterial Preparations
L. reuteri were purchased originally from the American Type Culture
Collection (ATCC No. 23272; Manassas, VA). L. salivarius were a gift
from Dr. B. Kiely (Alimentary Health, Cork, Ireland). Both strains are
of human intestinal origin and have demonstrated antiinflammatory
effects in animal models of colitis (15, 16). From frozen stocks (⫺80⬚C),
bacteria were suspended in Man-Rogosa-Sharpe liquid medium (MRS
broth; Difco Laboratories, Detroit, MI) plated in MRS agar, cultured
anaerobically at 37⬚C for 24 hours, then inoculated in fresh MRS broth
and grown at 37⬚C under anaerobic conditions for 48 hours in 50-ml
tubes. After 2 days, tubes were centrifuged at 2,000 rpm for 15 minutes
at 20⬚C and washed twice with sterile phosphate-buffered saline (PBS)
to a concentration of 6 ⫻ 108 bacteria/ml as determined by a Vitek
colorimeter (bioMérieux, Hazelwood, MO). Bacterial suspensions were
centrifuged in 15-ml tubes at 2,000 rpm for 15 minutes at 20⬚C, superna-
tants discarded, and bacteria resuspended in MRS broth to give a
concentration of 5 ⫻ 109/ml. Heat-killed L. reuteri were prepared by
heating aliquots of viable bacterial suspensions for 20 minutes at 80⬚C.
Suspensions of L. reuteri were killed by ␥-irradiation with Cobalt 60
for 20 hours at 8.05 Gy/minute. The resulting viability was determined
by plating on MRS agar plates under anaerobic conditions for 72 hours
at 37⬚C. No bacterial growth was detected in either the heat-killed or
irradiated L. reuteri preparations after 72 hours of culture.
DNA Isolation
Genomic DNA was isolated from L. reuteri using the EndoFree DNA
isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s
instructions. The purity of DNA was confirmed by measuring the ultra-
violet 260/280 absorbance ratio (⬎ 1.8). Calf thymus DNA (Sigma
Chemical Co., St Louis, MO) was used as a control in all experiments
using bacterial DNA. A synthetic immunostimulatory CpG-containing
oligodeoxyribonucleotide sequence, which acts as a ligand for TLR-9
(immunostimulatory CpG-containing oligodeoxyribonucleotides [ISS-
ODN]), and an inactive control oligonucleotide (mutated oligodeoxyri-
bonucleotides [M-ODN]) were supplied to us by Dr. E. Raz, University
of California, San Diego.
Treatment Protocols
Commencing on Day 6 (i.e., at time of second OVA sensitization),
animals received 1 ⫻ 109 live, heat-killed, or irradiated L. reuteri,
L. salivarius, or equivalent isolated L. reuteri DNA (50 ␮g) in 200 ␮l
of MRS broth via a gavaging needle for 9 consecutive days. Animals
gavaged with broth alone served as control animals. In other experi-
ments, isolated L. reuteri DNA, calf thymus DNA, or ISS-ODN
(50 ␮g) was given intraperitoneally on Days 6, 8, 10, and 12.
Airway Responsiveness
Airway responsiveness was assessed based on the response of total
respiratory system resistance (RRS) to saline and increasing intravenous
doses of methacholine. RRS was measured as described previously (17)
using the flow interrupter technique, modified for use in mice. The
slope of the dose response was calculated by linear regression between
the measured RRS and the log10-transformed methacholine dose, using
data from the 10-, 33-, and 100-␮g doses.
BAL
Two aliquots of 250 ␮l PBS were injected and withdrawn through a
tracheal cannula. Airway inflammation was assessed by inflammatory
cell counts in BAL fluid. Cells were removed from BAL fluid by centri-
fugation at 200 ⫻ g for 15 minutes, and supernatants stored at ⫺80⬚C
until evaluation of cytokine content. Cells were resuspended in PBS
(1 ml). BAL cells were stained with Trypan blue, and viable cells
counted using a hemocytometer. Smears of BAL cells were prepared
with a Cytospin (Thermo Shandon, Pittsburgh, PA) and stained with
HEMA 3 reagent (Biochemical Sciences, Swedesboro, NJ) for differen-
tial cell counts. A total of 200 cells were counted for each lavage, and
cells were classified, based on morphologic criteria, as macrophages,
neutrophils, lymphocytes, or eosinophils. An independent observer
blinded to the experimental conditions performed all cell counts.
Lung Histology
Lungs were inflated with 10% formalin (to a pressure of 20 cm H2O),
fixed for 24 hours, and embedded in paraffin. Fixed and embedded
tissue was stained with hematoxylin and eosin for histologic assessment
using light microscopy.
Cytokine Measurement
Cytokines (IL-6, IL-10, IL-12, monocyte chemoattractant protein
[MCP]-1, TNF, IFN-␥) were assessed using the BD Cytometric Bead
Array System (BD Biosciences, San Jose, CA) or commercial ELISA
for individual cytokines (IL-5, IL-13, and eotaxin; R&D Systems, Min-
neapolis, MN).
Indoleamine 2,3-Dioxygenase Activity
Maximal indoleamine 2,3-dioxygenase (IDO) activity was indirectly
determined as described previously (18). Briefly, lung tissue homoge-
nates were incubated with an excess of exogenous tryptophan (Tryp;
780 ␮M) as a substrate in the reaction buffer for 30 minutes in vitro.
The resulting kynurenine (Kyn) levels in the reaction homogenates
were measured by HPLC. IDO activity was expressed as nanograms
of Kyn per milligram of protein per 30-minute interval (14). Protein
concentrations were measured using a BioRad protein assay (BioRad,
Hercules, CA). To determine systemic IDO activity, plasma Kyn and
Tryp were simultaneously determined in HPLC and expressed as a
ratio (19).
Statistics
Experimental results are expressed as means ⫾ SEM. Statistical analy-
ses were performed with unpaired two-tailed Student’s t tests or one-
way analysis of variance, followed by Newman-Keuls test for comparing
all pairs of groups (GraphPad Prism, version 4.0; GraphPad, San Diego,
CA). A p value of less than 0.05 was considered statistically significant,
and n represents the number of experiments performed.
Forsythe, Inman, and Bien: Probiotics and Airway Inflammation
RESULTS
Inflammatory Cell Influx to the Airway
Total cell numbers in BAL fluids were significantly increased
24 hours after the final antigen challenge in OVA-sensitized mice
compared with OVA-sensitized, saline-challenged mice (155.9 ⫾
30.1 ⫻ 104 vs. 18.08 ⫾ 0.8 ⫻ 104, respectively; n ⫽ 12, p ⬍
0.001) (Figure 1A), thus confirming that challenge with OVA
was effective. Although the cell population in BAL fluid from
saline-challenged mice consisted almost exclusively of alveolar
macrophages, OVA challenge caused a dramatic increase in the
proportion of eosinophils (Figure 1B). OVA-challenged mice
gavaged with L. reuteri had a significant reduction in cells recov-
ered in BAL fluid compared with MRS broth–fed animals (59.2
⫾ 11.8 ⫻ 104 vs. 155.9 ⫾ 30.1 ⫻ 104, respectively; n ⫽ 12, p ⫽
0.004) (Figure 1A). This corresponded to a significant decrease
in both eosinophil (38.2 ⫾ 2.7 ⫻ 104 vs. 114.6 ⫾ 19.0 ⫻ 104) and
macrophage numbers (19.1 ⫾ 3.6 ⫻ 104 vs. 38.6 ⫾ 4.5 ⫻ 104)
(Figure 1C). Histologic analysis demonstrated that the increase
in eosinophils in the lung parenchyma of OVA-sensitized and
-challenged mice was also reduced by treatment with live
L. reuteri (Figure 2). Treatment of animals with either heat-
killed or ␥-irradiated organisms did not attenuate eosinophil
influx to the airway (Figure 1). In contrast to L. reuteri,
live L. salivarius did not modulate cellular influx to the airway
(Figure 1D).
Blood Eosinophils
To determine if the reduction in eosinophil influx to the airway
was entirely due to a decrease in migration from the blood, or
also a result of decreased production/release from bone marrow,
we measured circulating eosinophils. In OVA-challenged ani-
mals, treatment with live L. reuteri resulted in a significant reduc-
tion in the population of circulating eosinophils compared with
MRS broth–fed animals (4.7 ⫾ 0.54 vs. 1.6 ⫾ 0.27% of total
563
white blood cells, respectively; n ⫽ 6, p ⬍ 0.001) (data not
shown).
Cytokine Levels in BAL Fluid
After OVA challenge, levels of MCP-1, TNF, IL-5, IL-13, and
eotaxin were significantly increased in BAL fluid compared with
saline-challenged animals (Figure 3). Levels of IFN-␥, IL-12,
and IL-10 were not significantly changed compared with saline
controls. Treatment with L. reuteri significantly attenuated the
increase in MCP-1, TNF, IL-5, and IL-13 but did not alter eotaxin
levels. There was no effect of L. reuteri on IL-10, whereas IFN-␥
and IL-12 were below the limit of detection in all groups assessed.
Treatment with either heat-killed or irradiated L. reuteri also
significantly attenuated the increase in MCP-1 TNF and IL-5
after antigen challenge; however, in contrast to treatment with
live bacteria, killed organisms did not significantly alter levels
of IL-13 (Figure 3). L. salivarius had no effect on cytokine levels
in BAL fluid (data not shown).
Airways Response to Methacholine
Challenge with OVA in sensitized animals produced an increase
in airway responsiveness to methacholine, as determined by an
increase in the maximum resistance from 3.9 ⫾ 0.9 to 7.0 ⫾
0.8 cm H2O/ml/second (n ⫽ 15, p ⬍ 0.01) and the slope of
dose–response curve from 1.9 ⫾ 0.7 to 4.8 ⫾ 0.7 (p ⬍ 0.01). This
hyperresponsiveness was significantly reduced by treatment with
live L. reuteri (maximum resistance, 4.5 ⫾ 0.7 cm H2O/ml/s;
slope, 2.9 ⫾ 0.6) but not with heat-killed or irradiated organisms
(Figure 4).
L. salivarius did not modulate airway responsiveness to meth-
acholine (data not shown).
IDO Activity
The plasma ratio of Kyn to Tryp was significantly increased after
treatment with live L. reuteri versus MRS broth (0.24 ⫾ 0.05 vs.
Figure 1. Effect of oral treatment with live
(LR), heat-killed (HK), and ␥-irradiated (IR)
L. reuteri (1 ⫻ 109 organisms daily for 9
consecutive days) on (A ) total and (B )
differential cell counts (macrophages, eo-
sinophils, neutrophils, and lymphocytes)
and (C ) absolute numbers of macro-
phages and eosinophils in bronchoal-
veolar lavage (BAL) fluid from ovalbumin
(OVA)-sensitized male mice 24 hours after
challenge with intranasal OVA or saline.
(D ) The effect of live L. salivarius treat-
ment (1 ⫻ 109 organisms daily for 9 con-
secutive days) on eosinophil numbers in
BAL fluid is also shown. Each column rep-
resents the mean ⫾ SEM (n ⫽ 10). *p ⬍
0.05; **p ⬍ 0.01 compared with Man-
Rogosa-Sharpe broth–treated control.
564 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007

Figure 2. Representative sections of lung tissue from L. reuteri–treated (A ) and untreated (B ) OVA-sensitized mice after antigen challenge. A section
from a saline-challenged control animal is shown for comparison (C ). Arrows indicate inflammatory cell influx to the parenchyma. This influx was
markedly reduced by treatment with live L. reuteri.

0.09 ⫾ 0.03, respectively; n ⫽ 10, p ⬍ 0.05). Heat-killed and
irradiated organisms did not modulate the Kyn:Tryp ratio (Fig-
ure 5A). Maximal IDO in lung tissue was not changed by treat-
ment with either live or killed organisms (Figure 5B).
The Role of TLR-9 in L. reuteri Treatment
As in wild-type animals, OVA sensitization and challenge
of TLR9⫺/⫺ mice led to eosinophil influx to the airway (76.2 ⫾
12.4 ⫻ 104 vs. 0.62 ⫾ 0.1 ⫻ 104, n ⫽ 6, p ⬍ 0.001) (Figure 6)
and increased airway responsiveness compared with saline-
challenged animals. However, treatment with live L. reuteri did
not result in attenuation of eosinophil influx (99.2 ⫾ 32.7 ⫻ 104),
BAL cytokine levels (data not shown), or airway responsiveness
to methacholine (Figure 6).
Effect of Isolated L. reuteri DNA
Given the requirement for TLR-9 in the effects of L. reuteri,
we assessed the ability of DNA isolated from the organism to

Figure 3. Effect of oral treatment with live

(LR), heat-killed (HK), and ␥-irradiated (IR)
L. reuteri (1 ⫻ 109 organisms daily for 9 con-
secutive days) on cytokine levels in BAL fluid
from OVA-sensitized male mice 24 hours
after challenge with intranasal OVA or saline.
Each column represents the mean ⫾ SEM (n
⫽ 10). *p ⬍ 0.05 compared with OVA chal-
lenged, Man-Rogosa-Sharpe broth–treated
control.
Forsythe, Inman, and Bien: Probiotics and Airway Inflammation
reduce allergic airway inflammation. As previously described by
Hayashi and colleagues (20), intraperitoneal administration of
the synthetic TLR-9 ligand ISS-ODN significantly reduced eosin-
ophil influx to the airway (110.6 ⫾ 17.0 vs. 45.2 ⫾ 3.5 ⫻ 104,
n ⫽ 10, p ⬍ 0.05); however, neither treatment with L. reuteri
nor calf thymus DNA had any effect on the allergic airway
response when administered either orally or through intraperito-
neal injection (data not shown).
DISCUSSION
Studies have demonstrated strong immunomodulatory proper-
ties of lactobacilli, which include antiinflammatory and antitu-
mor activity, modulation of autoimmune diseases, and preven-
tion of infections (4, 21–23). There is clinical evidence suggesting
that probiotic treatment is protective against atopic dermatitis
and studies indicating that certain probiotic strains, when com-
bined with allergens, are candidates for mucosal vaccination
against allergy. Kruisselbrink and colleagues constructed an
L. plantarum strain that expressed an immunodominant T-cell
epitope of the Der p1 allergen of the house dust mite (24).
Intranasal administration of this organism suppressed antigen-
induced Th1 and Th2 immune responses in Der p1–sensitized
animals. Coapplication of Lactococcus lactis and L. plantarum
with the major birch pollen allergen (Bet v 1) caused suppression
of allergen-induced basophil degranulation (25). In a mouse
model of food allergy, feeding mice with L. casei was shown to
prevent responses to antigen challenge (5).
Here, we have studied the effect of L. reuteri on an allergic
airway response in OVA-sensitized mice. Oral treatment with
565
Figure 4. The effect of oral treatment with live
(LR) L. reuteri (A ) and heat-killed (HK) and ␥-
irradiated (IR) organisms (B ) on airway reactivity
(slope RRS) and maximum resistance (Max RRS) in
OVA-sensitized male mice 24 hours after chal-
lenge with intranasal OVA or saline. Airway re-
sponsiveness was measured in response to in-
creasing doses of intravenous methacholine.
Using the resulting dose–response curve, indices
of airway reactivity and maximal degree of bron-
choconstriction (Max RRS) were determined.
Each column represents the mean ⫾ SEM (n ⫽
10). *p ⬍ 0.05; **p ⬍ 0.01 compared OVA chal-
lenged, Man-Rogosa-Sharpe broth–treated con-
trol. MCh ⫽ methacholine.
viable L. reuteri, before intranasal antigen challenge, resulted
in significant attenuation of inflammatory cell influx to the lung
and airway responsiveness to methacholine. The decreases in
cellular influx and airway hyperresponsiveness (AHR) were as-
sociated with reduced levels of inflammatory cytokines (MCP-1,
TNF, IL-5, IL-13) in the BAL fluid. However, oral consumption
of live L. reuteri did not result in an increase in the Th1-type
cytokines IL-12 and IFN-␥ or the antiinflammatory cytokine
IL-10.
Strain-specific characteristics of this probiotic appear to be re-
sponsible for the antiinflammatory action we observed. Gavage
with an alternative probiotic Lactobacillus strain, L. salivarius,
did not modulate the allergic airway response in our model.
Strain-specific effects of probiotics have been demonstrated in
other systems, and the distinct immune responses between
strains may be due to differing inherent characteristics of the
organisms, which include persistence in the gut, colonization,
and intrinsic immunogenicity. In this regard, the cytokine profile
elicited by a probiotic organism clearly plays an important role
in determining the immunologic outcome.
In a detailed investigation, Maassen and colleagues (26) ana-
lyzed eight common Lactobacillus strains with respect to induc-
tion of cytokines by the murine gut mucosa in response to a
parenterally administered antigen and found distinct cytokine
profiles elicited by different strains. L. reuteri induced several
proinflammatory and/or Th1 cytokines, such as IL-1␤, IL-2, and
TNF, but not antiinflammatory or Th2 cytokines, such as IL-10
and IL-4. L. casei tended to induce production of IL-10 and
IL-4. In this system, L. reuteri enhanced the systemic antibody
response to antigen. In contrast, in a study of the capacity of
566 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007
Figure 5. Effect of oral treatment with live (LR), heat-killed (HK), and
␥-irradiated (IR) L. reuteri (1 ⫻ 109 organisms daily for 9 consecutive
days) on activity of indoleamine 2,3-dioxygenase (IDO) in plasma and
lung tissue as assessed by kynurenine (Kyn) to tryptophan (Tryp) ratio
and maximum IDO activity per milligram of protein, respectively. IDO
activity was assessed in OVA-sensitized mice 24 hours after challenge
with intranasal OVA or saline. Each column represents the mean ⫾ SEM
(n ⫽ 10). *p ⬍ 0.05 compared with OVA-challenged, Man-Rogosa-
Sharpe broth–treated control.
Lactobacillus strains to induce cytokine production from bone
marrow–derived dendritic cells (DCs), L. reuteri proved to be
a poor IL-12 inducer, whereas L. casei strongly induced IL-12,
IL-6, and TNF production (27). Therefore, it is clear that changes
in cytokine profile induced by probiotic strains may be site spe-
cific and dependent on the experimental system used.
Furthermore, Pelto and colleagues (28) demonstrated that
probiotic bacteria modulate phagocytosis differently in healthy
and allergic subjects. In healthy people, there was a stimulatory
effect, whereas in subjects with milk hypersensitivity, there was
down-regulation of the immune response (28). Therefore, the
outcome of probiotic treatment may depend on the immunologic
state of the host; in this regard, it will be of interest to determine
the impact of specific probiotics on allergic inflammation in a
range of mouse strains with distinct immune responses.
Neither heat-killed nor ␥-irradiated organisms mimicked the
ability of the live L. reuteri to protect against antigen-induced
eosinophil influx or AHR. However, killed organisms did retain
some immunomodulatory capacity in that they significantly at-
tenuated antigen-induced increases in TNF, MCP-1, and IL-5.
IL-13 is critical to the development of experimental asthma after
acute allergen exposure (29, 30). Delivery of IL-13 to the airway
induces mucus production, AHR, and eosinophilia, whereas
blockade of IL-13 in animal models of allergic asthma leads to
inhibition of all of these effects (29). Therefore, the observation
that live but not killed L. reuteri inhibit IL-13 may be central
to the distinct ability of live organisms to modulate cell influx
and AHR.
It is clear that certain physiologic effects of probiotics are
dependent on live organisms, whereas other effects can be medi-
ated by killed bacteria (31). Furthermore, the dependency on
live organisms for efficacy appears to be strain specific. In vitro,
L. reuteri can reduce TNF-induced IL-8 production by human
intestinal epithelial cells (32). This effect is dependent on live
organisms that are in contact with the epithelial cells. However,
the same modulation of IL-8 release has been reported with
both live and heat-killed L. rhamnosus GG (33). In contrast to
the inability of killed L. reuteri to modulate the allergic response
in the airway, Hunt and colleagues demonstrated that intragas-
tric administration of heat-killed Mycobacterium vaccae signifi-
cantly reduced pulmonary inflammation after antigen challenge
in OVA-sensitized mice (34). However, treatment with M. vac-
cae resulted in a markedly different cytokine profile in BAL
fluid than that observed after L. reuteri feeding, with significantly
increased IL-10 levels and no change in IL-5. These differences
in cytokine profile and requirement for live organisms suggest
that killed M. vaccae and live L. reuteri exert their protective
effects via distinct mechanisms. Importantly, L. reuteri reduces
both inflammation and AHR, whereas the impact of intragastric
M. vaccae treatment on AHR has not been reported. It does
not necessarily follow that reduction in airway inflammation
leads to reduced airway responsiveness, and a causal relationship
between eosinophilic inflammation and AHR is far from
established.
Despite clear evidence for strong immunomodulatory proper-
ties of lactobacilli, the mechanisms underlying these effects are
poorly understood. Our results indicate that feeding with live
L. reuteri increased systemic IDO activity as assessed by the
ratio of Tryp to Kyn in plasma. There is evidence, in animal
models of colitis, that a mixture of eight different probiotic
organisms (VSL#3) mediates their antiinflammatory effect
through activation of TLR-9 by unmethylated CpG motifs de-
rived from the organism’s own DNA (35). Furthermore, Hayashi
and coworkers recently demonstrated that parenterally adminis-
tered synthetic immunostimulatory DNA sequences (ISS-ODN)
that act as TLR-9 ligands significantly reduced allergen-induced
airway inflammation in mice (20). In this system, the antiin-
flammatory response was dependent on TLR-9–mediated activa-
tion of the tolerogenic enzyme IDO. However, unlike intraperi-
toneal treatment with ISS-ODN described by Hayashi and
colleagues, we did not see an increase in maximum IDO activity
in the lung after oral administration of L. reuteri (20). The ISS-
ODN–induced increase in IDO activity in pulmonary epithelial
cells is dependent on IFN-␥, whereas no changes in IFN-␥ were
detectable in the airway after L. reuteri treatment. Pulmonary
IDO activity is essential to the antiinflammatory effects of ISS-
ODN in the airway; therefore, any role for IDO in the attenua-
tion of the allergic response by L. reuteri will be distinct from
that induced by the synthetic TLR-9 ligand.
IDO is expressed in various cell types, including fibroblasts
(36), trophoblasts (37), macrophages, epithelial cells, DCs (38),
and nonimmune cells of the lung (20). IDO has been implicated
in T-cell tolerance to tumors and dysfunctional self-tolerance in
nonobese diabetic mice, as a protective negative regulator in
autoimmune disorders (39), and has shown a protective role
in a mouse model of colitis (40). Perhaps most notably, the
maintenance of a clinically unresponsive state after aeroallergen
exposure in atopic individuals has been associated with increased
systemic IDO activity and IL-10 production (41). Thus, while
we did not see an overt increase in IL-10 in BAL fluid, the
enhancement of systemic IDO activity by an oral probiotic may
be significant to the therapeutic potential of these organisms.
The effect of L. reuteri on the allergic airway response was
dependent on TLR-9. However, neither ␥-irradiated organisms
that retain intact DNA nor isolated DNA, whether given system-
ically or orally, altered inflammatory cell influx or AHR. This
Forsythe, Inman, and Bien: Probiotics and Airway Inflammation
may indicate that the antiinflammatory effects of oral L. reuteri
treatment in our model are not solely mediated by bacterial
DNA acting on TLR-9, and it is possible that metabolic products
of L. reuteri act in concert with TLR-9 to mediate the antiin-
flammatory response. Another potential explanation for our re-
sults is that intact, live organisms are required for effective pre-
sentation of bacterial DNA to TLR-9. TLR-9 is an intracellular
receptor, localized to late endosomes or lysosomes (42). Results
from recent studies suggest that the intracellular localization of
TLR-9 controls access of the receptor to different sources of
DNA (43). In mammals, the extracellular environment contains
DNase I, which promotes degradation or extracellular DNA.
Therefore, the isolated DNA from L. reuteri, whether adminis-
tered orally or intraperitoneally, was presumably degraded ex-
tracellularly before it could interact with TLR-9. This is consis-
tent with the finding that purified viral DNA is a poor inducer
of TLR-9 when compared with the intact virus (44). Similarly,
Rachmilewitz and colleagues found that, although oral treatment
with intact probiotic organisms could attenuate disease severity
in a mouse model of colitis via TLR-9, DNA isolated from this
same mixture of probiotic organisms was ineffective via the oral
route (35).
Although, to date, little is known about the fate of probiotic
organisms in the gastrointestinal tract, Macpherson and Uhr (45)
demonstrated that intestinal DCs can ingest and retain small
numbers of live commensals for several days. In this state, they
drive DCs to a tolerogenic phenotype. Furthermore, these DCs
did not move beyond the draining mesenteric lymph node.
Therefore, if this mechanism is in play in our model system,
local effects at or before the mesenteric lymph node (MLN) must
be assumed to be responsible for the systemic down-regulation of
the allergic response observed in the lung. Given that DCs are
pivotal in early bacterial recognition and are essential in pre-
venting asthmatic reactions to inhaled antigens, these cells may
be central in mediating the beneficial effects of probiotics on the
allergic airway response. DCs can induce a range of regulatory
T-cell subtypes (CD4⫹ CD25⫹, IL-10–producing Treg, and
567
Figure 6. Effect of oral treatment with live
(LR) L. reuteri (1 ⫻ 109 organisms daily
for 9 consecutive days) on (A ) eosinophil
numbers in BAL fluid and (B ) airway re-
sponsiveness to methacholine in OVA-
sensitized TLR-9–deficient mice. Cell
counts and airway responsiveness were
assessed 24 hours after challenge with intra-
nasal OVA or saline. Airway responsiveness
was measured in response to increasing
doses of intravenous methacholine. Using
the resulting dose–response curve, indices
of airway reactivity (slope RRS) and maxi-
mal degree of bronchoconstriction (Max
RRS) were determined. Each column repre-
sents the mean ⫾ SEM (n ⫽ 6). WT ⫽
wild type. *p ⬍ 0.05 compared with OVA-
challenged, Man-Rogosa-Sharpe broth–
treated control.
CD8⫹Treg cells) (46). Indeed, DCs expressing IDO contribute
to the generation and maintenance of peripheral tolerance by
depleting autoreactive T cells and by inducing Treg responses
(47). In other studies, L. reuteri and L. casei, but not L. plan-
tarum, have been demonstrated to prime monocyte-derived DCs
to drive the development of Treg cells (48) In vivo, oral treatment
with L. rhamnosus induced T-cell hyporesponsiveness in healthy
human volunteers and patients with Crohn’s disease with corre-
sponding in vitro studies, suggesting this occurred via modulation
of DC function (49). The capacity of lactobacilli to variably
induce maturation and the cytokine profile expressed by DCs
indicate that different species of probiotics may differentially
determine whether a DC drives Th1, Th2, or a Treg response,
and subsequently whether DCs have therapeutic potential in the
treatment of allergic disorders.
In conclusion, we report that oral administration of live L.
reuteri but not L. salivarius resulted in significant attenuation of
the allergic airway response. This effect depends on both viable
organisms and TLR-9 and is associated with systemic evidence
reflecting IDO activation. Our results indicate that the immuno-
modulatory effects of oral treatment with L. reuteri are not
limited to the gastrointestinal tract and we present the first report
that oral treatment with a probiotic organism can attenuate
major characteristics of an asthmatic response, including airway
eosinophilia, local cytokine responses, and hyperresponsiveness
to methacholine. These beneficial effects appear to be strain
specific, and it is clear that realizing the true potential of pro-
biotics as regulators of allergic airway disease will require a
better understanding of the mechanisms behind the quantitative
and qualitative differences in immune regulation that exist
among candidate organisms.
Conflict of Interest Statement : None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgment : The authors thank Ursula Kadela, Gudrun Goettsche, and
Jennifer Wattie for their invaluable technical support.
568 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007
References
1. Guarner F, Schaafsma GJ. Probiotics. Int J Food Microbiol 1998;39:237–
238.
2. Gionchetti P, Rizzello F, Helwig U, Venturi A, Lammers KM, Brigidi
P, Vitali B, Poggioli G, Miglioli M, Campieri M. Prophylaxis of pou-
chitis onset with probiotic therapy: a double-blind, placebo-controlled
trial. Gastroenterology 2003;124:1202–1209.
3. O’Mahony L, McCarthy J, Kelly P, Hurley G, Luo F, Chen K, O’Sullivan
GC, Kiely B, Collins JK, Shanahan F, et al. Lactobacillus and bifido-
bacterium in irritable bowel syndrome: symptom responses and rela-
tionship to cytokine profiles. Gastroenterology 2005;128:541–551.
4. Madsen K, Cornish A, Soper P, McKaigney C, Jijon H, Yachimec C,
Doyle J, Jewell L, De Simone C. Probiotic bacteria enhance murine
and human intestinal epithelial barrier function. Gastroenterology
2001;121:580–591.
5. Kim H, Kwack K, Kim DY, Ji GE. Oral probiotic bacterial administration
suppressed allergic responses in an ovalbumin-induced allergy mouse
model. FEMS Immunol Med Microbiol 2005;45:259–267.
6. Kalliomaki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics
and prevention of atopic disease: 4-year follow-up of a randomised
placebo-controlled trial. Lancet 2003;361:1869–1871.
7. Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri
E. Probiotics in primary prevention of atopic disease: a randomised
placebo-controlled trial. Lancet 2001;357:1076–1079.
8. Weston S, Halbert A, Richmond P, Prescott SL. Effects of probiotics
on atopic dermatitis: a randomised controlled trial. Arch Dis Child
2005;90:892–897.
9. Cripps AW, Dunkley ML, Clancy RL. Mucosal and systemic immuniza-
tions with killed Pseudomonas aeruginosa protect against acute respi-
ratory infection in rats. Infect Immun 1994;62:1427–1436.
10. Williams AE, Edwards L, Hussell T. Colonic bacterial infection abrogates
eosinophilic pulmonary disease. J Infect Dis 2006;193:223–230.
11. Hatakka K, Savilahti E, Ponka A, Meurman JH, Poussa T, Nase L,
Saxelin M, Korpela R. Effect of long term consumption of probiotic
milk on infections in children attending day care centres: double blind,
randomized trial. BMJ 2001;322:1–5.
12. Hori T, Kiyoshima J, Shida K, Yasui H. Augmentation of cellular immu-
nity and reduction of influenza virus titer in aged mice fed Lactobacillus
casei strain Shirota. Clin Diagn Lab Immunol 2002;9:105–108.
13. Forsythe P, Wattie J, Inman M, Bienenstock J. Oral treatment with live
lactobacillus reuteri attenuates the allergic airway response in mice.
J Allergy Clin Immunol 2006;117:S315.
14. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN. Lactoba-
cillus species prevents colitis in interleukin 10 gene-deficient mice.
Gastroenterology 1999;116:1107–1114.
15. Sheil B, McCarthy J, O’Mahony L, Bennett MW, Ryan P, Fitzgibbon JJ,
Kiely B, Collins JK, Shanahan F. Is the mucosal route of administration
essential for probiotic function? Subcutaneous administration is associ-
ated with attenuation of murine colitis and arthritis. Gut 2004;53:694–
700.
16. Ebeling C, Forsythe P, Ng J, Gordon JR, Hollenberg M, Vliagoftis H.
Proteinase-activated receptor 2 activation in the airways enhances
antigen-mediated airway inflammation and airway hyperresponsive-
ness through different pathways. J Allergy Clin Immunol 2005;115:
623–630.
17. Leigh R, Southam DS, Ellis R, Wattie JN, Sehmi R, Wan Y, Inman MD.
T-cell-mediated inflammation does not contribute to the maintenance
of airway dysfunction in mice. J Appl Physiol 2004;97:2258–2265.
18. Takikawa O, Kuroiwa T, Yamazaki F, Kido R. Mechanism of interferon-
gamma action: characterization of indoleamine 2,3-dioxygenase in cul-
tured human cells induced by interferon-gamma and evaluation of the
enzyme-mediated tryptophan degradation in its anticellular activity.
J Biol Chem 1988;263:2041–2048.
19. Laich A, Neurauter G, Widner B, Fuchs D. More rapid method for
simultaneous measurement of tryptophan and kynurenine by HPLC.
Clin Chem 2002;48:579–581.
20. Hayashi T, Beck L, Rossetto C, Gong X, Takikawa O, Takabayashi K,
Broide DH, Carson DA, Raz E. Inhibition of experimental asthma
by indoleamine 2,3-dioxygenase. J Clin Invest 2004;114:270–279.
21. Reid G, Jass J, Sebulsky MT, McCormick JK. Potential uses of probiotics
in clinical practice. Clin Microbiol Rev 2003;16:658–672.
22. Moudgil KD, Kim E, Yun OJ, Chi HH, Brahn E, Sercarz EE. Environ-
mental modulation of autoimmune arthritis involves the spontaneous
microbial induction of T cell responses to regulatory determinants
within heat shock protein 65. J Immunol 2001;166:4237–4243.
23. Matsuzaki T, Hashimoto S, Yokokura T. Effects on antitumor activity
and cytokine production in the thoracic cavity by intrapleural adminis-
tration of Lactobacillus casei in tumor-bearing mice. Med Microbiol
Immunol (Berl) 1996;185:157–161.
24. Kruisselbrink A, Heijne Den Bak-Glashouwer MJ, Havenith CE, Thole
JE, Janssen R. Recombinant Lactobacillus plantarum inhibits house
dust mite-specific T-cell responses. Clin Exp Immunol 2001;126:2–8.
25. Repa A, Grangette C, Daniel C, Hochreiter R, Hoffmann-Sommergruber
K, Thalhamer J, Kraft D, Breiteneder H, Mercenier A, Wiedermann
U. Mucosal co-application of lactic acid bacteria and allergen induces
counter-regulatory immune responses in a murine model of birch
pollen allergy. Vaccine 2003;22:87–95.
26. Maassen CB, van Holten-Neelen C, Balk F, den Bak-Glashouwer MJ,
Leer RJ, Laman JD, Boersma WJ, Claassen E. Strain-dependent in-
duction of cytokine profiles in the gut by orally administered Lactoba-
cillus strains. Vaccine 2000;18:2613–2623.
27. Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli differentially modu-
late expression of cytokines and maturation surface markers in murine
dendritic cells. J Immunol 2002;168:171–178.
28. Pelto L, Isolauri E, Lilius EM, Nuutila J, Salminen S. Probiotic bacteria
down-regulate the milk-induced inflammatory response in milk-hyper-
sensitive subjects but have an immunostimulatory effect in healthy
subjects. Clin Exp Allergy 1998;28:1474–1479.
29. Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z,
Elias JA, Sheppard D, Erle DJ. Direct effects of interleukin-13 on
epithelial cells cause airway hyperreactivity and mucus overproduction
in asthma. Nat Med 2002;8:885–889.
30. Kamiya T, Wang L, Forsythe P, Goettsche G, Mao Y, Wang Y, Tougas
G, Bienenstock J. Inhibitory effects of Lactobacillus reuteri on visceral
pain induced by colorectal distension in Sprague-Dawley rats. Gut
2006;55:191–196.
31. Leigh R, Ellis R, Wattie J, Donaldson DD, Inman MD. Is interleukin-
13 critical in maintaining airway hyperresponsiveness in allergen-chal-
lenged mice? Am J Respir Crit Care Med 2004;170:851–856.
32. Ma D, Forsythe P, Bienenstock J. Live Lactobacillus reuteri is essential
for the inhibitory effect on tumor necrosis factor alpha-induced
interleukin-8 expression. Infect Immun 2004;72:5308–5314.
33. Zhang L, Li N, Caicedo R, Neu J. Alive and dead Lactobacillus rhamno-
sus GG decrease tumor necrosis factor-␣-induced interleukin-8 pro-
duction in Caco-2 cells. J Nutr 2005;135:1752–1756.
34. Hunt JR, Martinelli R, Adams VC, Rook GA, Brunet LR. Intragastric
administration of mycobacterium vaccae inhibits severe pulmonary
allergic inflammation in a mouse model. Clin Exp Allergy 2005;35:685–
690.
35. Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, Rudensky
B, Akira S, Takeda K, Lee J, Takabayashi K, et al. Toll-like receptor
9 signaling mediates the anti-inflammatory effects of probiotics in
murine experimental colitis. Gastroenterology 2004;126:520–528.
36. Yuan W, Collado-Hidalgo A, Yufit T, Taylor M, Varga J. Modulation of
cellular tryptophan metabolism in human fibroblasts by transforming
growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase
and tryptophanyl-tRNA synthetase gene expression. J Cell Physiol
1998;177:174–186.
37. Kudo Y, Boyd CA, Spyropoulou I, Redman CW, Takikawa O, Katsuki
T, Hara T, Ohama K, Sargent IL. Indoleamine 2,3-dioxygenase: distri-
bution and function in the developing human placenta. J Reprod Im-
munol 2004;61:87–98.
38. Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and
tryptophan catabolism. Nat Rev Immunol 2004;4:762–774.
39. Grohmann U, Fallarino F, Puccetti P. Tolerance, DCs and tryptophan:
much ado about IDO. Trends Immunol 2003;24:242–248.
40. Gurtner GJ, Newberry RD, Schloemann SR, McDonald KG, Stenson
WF. Inhibition of indoleamine 2,3-dioxygenase augments trinitroben-
zene sulfonic acid colitis in mice. Gastroenterology 2003;125:1762–
1773.
41. von Bubnoff D, Fimmers R, Bogdanow M, Matz H, Koch S, Bieber T.
Asymptomatic atopy is associated with increased indoleamine 2,3-
dioxygenase activity and interleukin-10 production during seasonal
allergen exposure. Clin Exp Allergy 2004;34:1056–1063.
42. Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol
2004;25:381–386.
43. Barton GM, Kagan JC, Medzhitov R. Intracellular localization of Toll-
like receptor 9 prevents recognition of self DNA but facilitates access
to viral DNA. Nat Immunol 2006;7:49–56.
44. Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A. Toll-like receptor 9-
mediated recognition of herpes simplex virus-2 by plasmacytoid den-
dritic cells. J Exp Med 2003;198:513–520.
Forsythe, Inman, and Bien: Probiotics and Airway Inflammation
45. Macpherson AJ, Uhr T. Induction of protective IgA by intestinal den-
dritic cells carrying commensal bacteria. Science 2004;303:1662–1665.
46. Groux H, Fournier N, Cottrez F. Role of dendritic cells in the generation
of regulatory T cells. Semin Immunol 2004;16:99–106.
47. Baban B, Hansen AM, Chandler PR, Manlapat A, Bingaman A, Kahler
DJ, Munn DH, Mellor AL. Minor population of splenic dendritic cells
expressing CD19 mediates IDO-dependent T cell suppression via type
I IFN signaling following B7 ligation. Int Immunol 2005;17:909–919.
569
48. Hart AL, Lammers K,. Brigidi P, Vitali B, Rizzello F, Gionchetti P,
Campieri M, Kamm MA, Knight SC, Stagg AJ. Modulation of human
dendritic cell phenotype and function by probiotic bacteria. Gut
2004;53:1602–1609.
49. Braat H, van den Brande J, van Tol E, Hommes D, Peppelenbosch M,
van Deventer S. Lactobacillus rhamnosus induces peripheral hypore-
sponsiveness in stimulated CD4⫹ T cells via modulation of dendritic
cell function. Am J Clin Nutr 2004;80:1618–1625.