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Despite indications that probiotic treatment can modulate ODN]), and an inactive control oligonucleotide (mutated oligodeoxyri-
some immune responses in the lung, there have been no reports bonucleotides [M-ODN]) were supplied to us by Dr. E. Raz, University
of the effects of oral treatment with a probiotic organism on of California, San Diego.
major characteristics of asthma, including allergic airway in-
Treatment Protocols
flammation and bronchial hyperresponsiveness. Here, we de-
scribe the ability of one probiotic organism, Lactobacillus reuteri, Commencing on Day 6 (i.e., at time of second OVA sensitization),
but not another, Lactobacillus salivarius, to attenuate antigen- animals received 1 ⫻ 109 live, heat-killed, or irradiated L. reuteri,
L. salivarius, or equivalent isolated L. reuteri DNA (50 g) in 200 l
induced eosinophil influx to the airway as well as local cytokine
of MRS broth via a gavaging needle for 9 consecutive days. Animals
responses and hyperresponsiveness to methacholine in an oval- gavaged with broth alone served as control animals. In other experi-
bumin (OVA)-sensitized mouse model of allergic airway disease. ments, isolated L. reuteri DNA, calf thymus DNA, or ISS-ODN
Portions of the data presented in this article have previously (50 g) was given intraperitoneally on Days 6, 8, 10, and 12.
been published in abstract form (13).
Airway Responsiveness
METHODS Airway responsiveness was assessed based on the response of total
respiratory system resistance (RRS) to saline and increasing intravenous
Animals doses of methacholine. RRS was measured as described previously (17)
Adult male BALB/c or Toll-like receptor 9–deficient (TLR9⫺/⫺) mice using the flow interrupter technique, modified for use in mice. The
(20–25 g) were maintained in an automatic light/dark cycle (light periods slope of the dose response was calculated by linear regression between
of 12 h) and provided water and chow ad libitum. Mice were acclima- the measured RRS and the log10-transformed methacholine dose, using
tized to the animal facility for 1 week before experimentation. Trans- data from the 10-, 33-, and 100-g doses.
genic TLR9⫺/⫺ mice on a balb/c background were obtained from Dr.
K. Rosenthal (McMaster University) with permission from Dr. S. Akira BAL
(Department of Host Defense, Osaka University, Osaka, Japan). Age- Two aliquots of 250 l PBS were injected and withdrawn through a
matched (8–9 wk old) animals were used in all experiments. These tracheal cannula. Airway inflammation was assessed by inflammatory
experiments were performed in accordance with guidelines of the Cana- cell counts in BAL fluid. Cells were removed from BAL fluid by centri-
dian Council for Animal Care. fugation at 200 ⫻ g for 15 minutes, and supernatants stored at ⫺80⬚C
until evaluation of cytokine content. Cells were resuspended in PBS
Experimental Asthma (1 ml). BAL cells were stained with Trypan blue, and viable cells
This study used an OVA-sensitized mouse model of allergic airway counted using a hemocytometer. Smears of BAL cells were prepared
inflammation. Briefly, mice were sensitized by intraperitoneal injection with a Cytospin (Thermo Shandon, Pittsburgh, PA) and stained with
of 20 g OVA adsorbed with 500 g alum in saline on Day 0 and Day HEMA 3 reagent (Biochemical Sciences, Swedesboro, NJ) for differen-
6. On Days 12 and 14, mice were challenged intranasally with 5 g tial cell counts. A total of 200 cells were counted for each lavage, and
OVA per mouse (14). Twenty-four hours after the last challenge (Day cells were classified, based on morphologic criteria, as macrophages,
15), mice were subjected to measurements of airway responsiveness neutrophils, lymphocytes, or eosinophils. An independent observer
followed by bronchoalveolar lavage (BAL). OVA/alum-sensitized, sa- blinded to the experimental conditions performed all cell counts.
line-challenged mice served as control animals.
Lung Histology
Bacterial Preparations
Lungs were inflated with 10% formalin (to a pressure of 20 cm H2O),
L. reuteri were purchased originally from the American Type Culture fixed for 24 hours, and embedded in paraffin. Fixed and embedded
Collection (ATCC No. 23272; Manassas, VA). L. salivarius were a gift tissue was stained with hematoxylin and eosin for histologic assessment
from Dr. B. Kiely (Alimentary Health, Cork, Ireland). Both strains are using light microscopy.
of human intestinal origin and have demonstrated antiinflammatory
effects in animal models of colitis (15, 16). From frozen stocks (⫺80⬚C), Cytokine Measurement
bacteria were suspended in Man-Rogosa-Sharpe liquid medium (MRS
Cytokines (IL-6, IL-10, IL-12, monocyte chemoattractant protein
broth; Difco Laboratories, Detroit, MI) plated in MRS agar, cultured
[MCP]-1, TNF, IFN-␥) were assessed using the BD Cytometric Bead
anaerobically at 37⬚C for 24 hours, then inoculated in fresh MRS broth
Array System (BD Biosciences, San Jose, CA) or commercial ELISA
and grown at 37⬚C under anaerobic conditions for 48 hours in 50-ml
for individual cytokines (IL-5, IL-13, and eotaxin; R&D Systems, Min-
tubes. After 2 days, tubes were centrifuged at 2,000 rpm for 15 minutes
neapolis, MN).
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 Indoleamine 2,3-Dioxygenase Activity
colorimeter (bioMérieux, Hazelwood, MO). Bacterial suspensions were
centrifuged in 15-ml tubes at 2,000 rpm for 15 minutes at 20⬚C, superna- Maximal indoleamine 2,3-dioxygenase (IDO) activity was indirectly
tants discarded, and bacteria resuspended in MRS broth to give a determined as described previously (18). Briefly, lung tissue homoge-
concentration of 5 ⫻ 109/ml. Heat-killed L. reuteri were prepared by nates were incubated with an excess of exogenous tryptophan (Tryp;
heating aliquots of viable bacterial suspensions for 20 minutes at 80⬚C. 780 M) as a substrate in the reaction buffer for 30 minutes in vitro.
Suspensions of L. reuteri were killed by ␥-irradiation with Cobalt 60 The resulting kynurenine (Kyn) levels in the reaction homogenates
for 20 hours at 8.05 Gy/minute. The resulting viability was determined were measured by HPLC. IDO activity was expressed as nanograms
by plating on MRS agar plates under anaerobic conditions for 72 hours of Kyn per milligram of protein per 30-minute interval (14). Protein
at 37⬚C. No bacterial growth was detected in either the heat-killed or concentrations were measured using a BioRad protein assay (BioRad,
irradiated L. reuteri preparations after 72 hours of culture. Hercules, CA). To determine systemic IDO activity, plasma Kyn and
Tryp were simultaneously determined in HPLC and expressed as a
DNA Isolation ratio (19).
Genomic DNA was isolated from L. reuteri using the EndoFree DNA
isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s
Statistics
instructions. The purity of DNA was confirmed by measuring the ultra- Experimental results are expressed as means ⫾ SEM. Statistical analy-
violet 260/280 absorbance ratio (⬎ 1.8). Calf thymus DNA (Sigma ses were performed with unpaired two-tailed Student’s t tests or one-
Chemical Co., St Louis, MO) was used as a control in all experiments way analysis of variance, followed by Newman-Keuls test for comparing
using bacterial DNA. A synthetic immunostimulatory CpG-containing all pairs of groups (GraphPad Prism, version 4.0; GraphPad, San Diego,
oligodeoxyribonucleotide sequence, which acts as a ligand for TLR-9 CA). A p value of less than 0.05 was considered statistically significant,
(immunostimulatory CpG-containing oligodeoxyribonucleotides [ISS- and n represents the number of experiments performed.
Forsythe, Inman, and Bien: Probiotics and Airway Inflammation 563
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 and increased airway responsiveness compared with saline-
irradiated organisms did not modulate the Kyn:Tryp ratio (Fig- challenged animals. However, treatment with live L. reuteri did
ure 5A). Maximal IDO in lung tissue was not changed by treat- not result in attenuation of eosinophil influx (99.2 ⫾ 32.7 ⫻ 104),
ment with either live or killed organisms (Figure 5B). BAL cytokine levels (data not shown), or airway responsiveness
to methacholine (Figure 6).
The Role of TLR-9 in L. reuteri Treatment
As in wild-type animals, OVA sensitization and challenge Effect of Isolated L. reuteri DNA
of TLR9⫺/⫺ mice led to eosinophil influx to the airway (76.2 ⫾ Given the requirement for TLR-9 in the effects of L. reuteri,
12.4 ⫻ 104 vs. 0.62 ⫾ 0.1 ⫻ 104, n ⫽ 6, p ⬍ 0.001) (Figure 6) we assessed the ability of DNA isolated from the organism to
reduce allergic airway inflammation. As previously described by viable L. reuteri, before intranasal antigen challenge, resulted
Hayashi and colleagues (20), intraperitoneal administration of in significant attenuation of inflammatory cell influx to the lung
the synthetic TLR-9 ligand ISS-ODN significantly reduced eosin- and airway responsiveness to methacholine. The decreases in
ophil influx to the airway (110.6 ⫾ 17.0 vs. 45.2 ⫾ 3.5 ⫻ 104, cellular influx and airway hyperresponsiveness (AHR) were as-
n ⫽ 10, p ⬍ 0.05); however, neither treatment with L. reuteri sociated with reduced levels of inflammatory cytokines (MCP-1,
nor calf thymus DNA had any effect on the allergic airway TNF, IL-5, IL-13) in the BAL fluid. However, oral consumption
response when administered either orally or through intraperito- of live L. reuteri did not result in an increase in the Th1-type
neal injection (data not shown). cytokines IL-12 and IFN-␥ or the antiinflammatory cytokine
IL-10.
DISCUSSION Strain-specific characteristics of this probiotic appear to be re-
sponsible for the antiinflammatory action we observed. Gavage
Studies have demonstrated strong immunomodulatory proper- with an alternative probiotic Lactobacillus strain, L. salivarius,
ties of lactobacilli, which include antiinflammatory and antitu- did not modulate the allergic airway response in our model.
mor activity, modulation of autoimmune diseases, and preven- Strain-specific effects of probiotics have been demonstrated in
tion of infections (4, 21–23). There is clinical evidence suggesting other systems, and the distinct immune responses between
that probiotic treatment is protective against atopic dermatitis strains may be due to differing inherent characteristics of the
and studies indicating that certain probiotic strains, when com- organisms, which include persistence in the gut, colonization,
bined with allergens, are candidates for mucosal vaccination and intrinsic immunogenicity. In this regard, the cytokine profile
against allergy. Kruisselbrink and colleagues constructed an elicited by a probiotic organism clearly plays an important role
L. plantarum strain that expressed an immunodominant T-cell in determining the immunologic outcome.
epitope of the Der p1 allergen of the house dust mite (24). In a detailed investigation, Maassen and colleagues (26) ana-
Intranasal administration of this organism suppressed antigen- lyzed eight common Lactobacillus strains with respect to induc-
induced Th1 and Th2 immune responses in Der p1–sensitized tion of cytokines by the murine gut mucosa in response to a
animals. Coapplication of Lactococcus lactis and L. plantarum parenterally administered antigen and found distinct cytokine
with the major birch pollen allergen (Bet v 1) caused suppression profiles elicited by different strains. L. reuteri induced several
of allergen-induced basophil degranulation (25). In a mouse proinflammatory and/or Th1 cytokines, such as IL-1, IL-2, and
model of food allergy, feeding mice with L. casei was shown to TNF, but not antiinflammatory or Th2 cytokines, such as IL-10
prevent responses to antigen challenge (5). and IL-4. L. casei tended to induce production of IL-10 and
Here, we have studied the effect of L. reuteri on an allergic IL-4. In this system, L. reuteri enhanced the systemic antibody
airway response in OVA-sensitized mice. Oral treatment with response to antigen. In contrast, in a study of the capacity of
566 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007
may indicate that the antiinflammatory effects of oral L. reuteri CD8⫹Treg cells) (46). Indeed, DCs expressing IDO contribute
treatment in our model are not solely mediated by bacterial to the generation and maintenance of peripheral tolerance by
DNA acting on TLR-9, and it is possible that metabolic products depleting autoreactive T cells and by inducing Treg responses
of L. reuteri act in concert with TLR-9 to mediate the antiin- (47). In other studies, L. reuteri and L. casei, but not L. plan-
flammatory response. Another potential explanation for our re- tarum, have been demonstrated to prime monocyte-derived DCs
sults is that intact, live organisms are required for effective pre- to drive the development of Treg cells (48) In vivo, oral treatment
sentation of bacterial DNA to TLR-9. TLR-9 is an intracellular with L. rhamnosus induced T-cell hyporesponsiveness in healthy
receptor, localized to late endosomes or lysosomes (42). Results human volunteers and patients with Crohn’s disease with corre-
from recent studies suggest that the intracellular localization of sponding in vitro studies, suggesting this occurred via modulation
TLR-9 controls access of the receptor to different sources of of DC function (49). The capacity of lactobacilli to variably
DNA (43). In mammals, the extracellular environment contains induce maturation and the cytokine profile expressed by DCs
DNase I, which promotes degradation or extracellular DNA. indicate that different species of probiotics may differentially
Therefore, the isolated DNA from L. reuteri, whether adminis-
determine whether a DC drives Th1, Th2, or a Treg response,
tered orally or intraperitoneally, was presumably degraded ex-
and subsequently whether DCs have therapeutic potential in the
tracellularly before it could interact with TLR-9. This is consis-
treatment of allergic disorders.
tent with the finding that purified viral DNA is a poor inducer
In conclusion, we report that oral administration of live L.
of TLR-9 when compared with the intact virus (44). Similarly,
reuteri but not L. salivarius resulted in significant attenuation of
Rachmilewitz and colleagues found that, although oral treatment
with intact probiotic organisms could attenuate disease severity the allergic airway response. This effect depends on both viable
in a mouse model of colitis via TLR-9, DNA isolated from this organisms and TLR-9 and is associated with systemic evidence
same mixture of probiotic organisms was ineffective via the oral reflecting IDO activation. Our results indicate that the immuno-
route (35). modulatory effects of oral treatment with L. reuteri are not
Although, to date, little is known about the fate of probiotic limited to the gastrointestinal tract and we present the first report
organisms in the gastrointestinal tract, Macpherson and Uhr (45) that oral treatment with a probiotic organism can attenuate
demonstrated that intestinal DCs can ingest and retain small major characteristics of an asthmatic response, including airway
numbers of live commensals for several days. In this state, they eosinophilia, local cytokine responses, and hyperresponsiveness
drive DCs to a tolerogenic phenotype. Furthermore, these DCs to methacholine. These beneficial effects appear to be strain
did not move beyond the draining mesenteric lymph node. specific, and it is clear that realizing the true potential of pro-
Therefore, if this mechanism is in play in our model system, biotics as regulators of allergic airway disease will require a
local effects at or before the mesenteric lymph node (MLN) must better understanding of the mechanisms behind the quantitative
be assumed to be responsible for the systemic down-regulation of and qualitative differences in immune regulation that exist
the allergic response observed in the lung. Given that DCs are among candidate organisms.
pivotal in early bacterial recognition and are essential in pre-
venting asthmatic reactions to inhaled antigens, these cells may 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.
be central in mediating the beneficial effects of probiotics on the
allergic airway response. DCs can induce a range of regulatory Acknowledgment : The authors thank Ursula Kadela, Gudrun Goettsche, and
T-cell subtypes (CD4⫹ CD25⫹, IL-10–producing Treg, and Jennifer Wattie for their invaluable technical support.
568 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 175 2007
45. Macpherson AJ, Uhr T. Induction of protective IgA by intestinal den- 48. Hart AL, Lammers K,. Brigidi P, Vitali B, Rizzello F, Gionchetti P,
dritic cells carrying commensal bacteria. Science 2004;303:1662–1665. Campieri M, Kamm MA, Knight SC, Stagg AJ. Modulation of human
46. Groux H, Fournier N, Cottrez F. Role of dendritic cells in the generation dendritic cell phenotype and function by probiotic bacteria. Gut
of regulatory T cells. Semin Immunol 2004;16:99–106. 2004;53:1602–1609.
47. Baban B, Hansen AM, Chandler PR, Manlapat A, Bingaman A, Kahler 49. Braat H, van den Brande J, van Tol E, Hommes D, Peppelenbosch M,
DJ, Munn DH, Mellor AL. Minor population of splenic dendritic cells van Deventer S. Lactobacillus rhamnosus induces peripheral hypore-
expressing CD19 mediates IDO-dependent T cell suppression via type sponsiveness in stimulated CD4⫹ T cells via modulation of dendritic
I IFN signaling following B7 ligation. Int Immunol 2005;17:909–919. cell function. Am J Clin Nutr 2004;80:1618–1625.