Oral Treatment with Live Lactobacillus reuteri Inhibits ... - ATS Journals

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 children 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 bronchoalveolar lavage fluid were assessed. Results: Oral treatment with live Lactobacillus reuteri but not Lactobacillus 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 hyperresponsiveness. These responses were dependent on Toll-like receptor 9 and were associated with increased activity of indoleamine 2,3dioxygenase. 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 consumed 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 commensals, 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. Randomized 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 Bienenstock, M.D., Departments of Pathology and Molecular Medicine, McMaster University, Brain–Body Institute, St. Joseph’s Healthcare, 50 Charlton Avenue East, T3304 Hamilton, ON, L8N 4A6 Canada. E-mail: [email protected] 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 characteristics of asthma, including airway inflammation and hyperresponsiveness. 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 hyperresponsiveness.

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, clarification of the underlying mechanisms of these antiinflammatory effects is still obscure. In addition to effects in the gut, there is evidence that probiotics, given perinatally, may be protective against manifestations 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 commensal 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 effects of probiotics on the incidence of asthma (7), there is evidence 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 Cryptococcus 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 probiotictreated animals also had reduced viral titers in nasal washings after influenza infection (12).

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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 inflammation and bronchial hyperresponsiveness. Here, we describe the ability of one probiotic organism, Lactobacillus reuteri, but not another, Lactobacillus salivarius, to attenuate antigeninduced eosinophil influx to the airway as well as local cytokine responses and hyperresponsiveness to methacholine in an ovalbumin (OVA)-sensitized mouse model of allergic airway disease. Portions of the data presented in this article have previously been published in abstract form (13).

ODN]), and an inactive control oligonucleotide (mutated oligodeoxyribonucleotides [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 experiments, isolated L. reuteri DNA, calf thymus DNA, or ISS-ODN (50 ␮g) was given intraperitoneally on Days 6, 8, 10, and 12.

Airway Responsiveness

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 acclimatized to the animal facility for 1 week before experimentation. Transgenic 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). Agematched (8–9 wk old) animals were used in all experiments. These experiments were performed in accordance with guidelines of the Canadian 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, saline-challenged mice served as control animals.

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 centrifugation 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 differential 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 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 (bioMe´rieux, Hazelwood, MO). Bacterial suspensions were centrifuged in 15-ml tubes at 2,000 rpm for 15 minutes at 20⬚C, supernatants 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 ultraviolet 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-

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, Minneapolis, MN).

Indoleamine 2,3-Dioxygenase Activity Maximal indoleamine 2,3-dioxygenase (IDO) activity was indirectly determined as described previously (18). Briefly, lung tissue homogenates 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 analyses were performed with unpaired two-tailed Student’s t tests or oneway 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 recovered 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 heatkilled 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 animals, treatment with live L. reuteri resulted in a significant reduction in the population of circulating eosinophils compared with MRS broth–fed animals (4.7 ⫾ 0.54 vs. 1.6 ⫾ 0.27% of total

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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 methacholine (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, eosinophils, neutrophils, and lymphocytes) and (C ) absolute numbers of macrophages and eosinophils in bronchoalveolar 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 treatment (1 ⫻ 109 organisms daily for 9 consecutive days) on eosinophil numbers in BAL fluid is also shown. Each column represents the mean ⫾ SEM (n ⫽ 10). *p ⬍ 0.05; **p ⬍ 0.01 compared with ManRogosa-Sharpe broth–treated control.

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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 (Figure 5A). Maximal IDO in lung tissue was not changed by treatment 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 salinechallenged 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 consecutive 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 challenged, Man-Rogosa-Sharpe broth–treated control.


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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 challenge with intranasal 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 and maximal degree of bronchoconstriction (Max RRS) were determined. Each column represents the mean ⫾ SEM (n ⫽ 10). *p ⬍ 0.05; **p ⬍ 0.01 compared OVA challenged, Man-Rogosa-Sharpe broth–treated control. MCh ⫽ methacholine.

reduce allergic airway inflammation. As previously described by Hayashi and colleagues (20), intraperitoneal administration of the synthetic TLR-9 ligand ISS-ODN significantly reduced eosinophil 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 intraperitoneal injection (data not shown).

DISCUSSION Studies have demonstrated strong immunomodulatory properties of lactobacilli, which include antiinflammatory and antitumor activity, modulation of autoimmune diseases, and prevention 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 combined 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 antigeninduced 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

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 associated 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 responsible 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) analyzed eight common Lactobacillus strains with respect to induction 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

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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-RogosaSharpe 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 specific 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 attenuated 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 intragastric administration of heat-killed Mycobacterium vaccae significantly reduced pulmonary inflammation after antigen challenge in OVA-sensitized mice (34). However, treatment with M. vaccae 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 properties 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 derived from the organism’s own DNA (35). Furthermore, Hayashi and coworkers recently demonstrated that parenterally administered 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 antiinflammatory response was dependent on TLR-9–mediated activation of the tolerogenic enzyme IDO. However, unlike intraperitoneal 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 ISSODN–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 ISSODN in the airway; therefore, any role for IDO in the attenuation 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 systemically or orally, altered inflammatory cell influx or AHR. This


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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 responsiveness to methacholine in OVAsensitized TLR-9–deficient mice. Cell counts and airway responsiveness were assessed 24 hours after challenge with intranasal 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 maximal degree of bronchoconstriction (Max RRS) were determined. Each column represents the mean ⫾ SEM (n ⫽ 6). WT ⫽ wild type. *p ⬍ 0.05 compared with OVAchallenged, Man-Rogosa-Sharpe broth– treated control.

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 antiinflammatory response. Another potential explanation for our results is that intact, live organisms are required for effective presentation 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 administered orally or intraperitoneally, was presumably degraded extracellularly before it could interact with TLR-9. This is consistent 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 preventing 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

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. plantarum, 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 corresponding 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 immunomodulatory 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 probiotics 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.

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