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Oral administration of Lactobacillus salivarius inhibits the allergic airway response in mice Chia-Yang Li, Hung-Chih Lin, Kai-Chung Hsueh, Shu-Fen Wu, and Shih-Hua Fang

Abstract: Asthma is recognized throughout the world as a chronic airway inflammatory disease. In this study, we investigated the effect of probiotics in response to antigen challenge in an ovalbumin (OVA)-sensitized asthma model in BALB/c mice. Lactobacillus salivarius PM-A0006 was orally administered to mice before antigen challenge. After antigen challenge, serum OVA-specific antibody levels, airway responsiveness to methacholine, influx of inflammatory cells to the lung, and cytokine levels in bronchoalveolar lavage (BAL) fluid and splenocytes were assessed. Oral treatment with live L. salivarius PM-A0006 significantly attenuated the influx of eosinophils to the airway lumen and reduced the levels of serum OVA-specific IgE and eotaxin in BAL fluid of antigen-challenged animals. Furthermore, L. salivarius PM-A0006 also decreased allergen-induced airway hyperresponsiveness and elevated the levels of IFN-g. These results showed that oral treatment with L. salivarius PM-A0006 could have therapeutic potential in the treatment of allergic airway disease. Key words: bronchoalveolar lavage fluid, cytokines, Lactobacillus salivarius, probiotics. Re´sume´ : L’asthme est une maladie inflammatoire chronique des voies respiratoires re´pandue a` travers le monde. Dans cette e´tude, nous avons examine´ l’effet de probiotiques dans la re´ponse a` une provocation antigeńique dans un mode`le d’asthme induit par l’OVA chez la souris BALB/c. Les souris ont reçu par voie orale Lactobacillus salivarius PM-A0006 preálablement a` la provocation antigeńique. Apre`s la provocation antigeńique, les niveaux se´riques d’anticorps spećifiques a` l’OVA, la re´ponse bronchique a` la me´thacoline, l’influx de cellules inflammatoires aux poumons et les niveaux de cytokines dans le liquide de lavage broncho-alveólaire et dans les spleńocytes, ont e´te´ e´value´s. Un traitement oral a` L. salivarius PM-A0006 vivant atteńuait de façon significative l’influx d’eósinophiles dans la lumie`re respiratoire, et re´duisait les niveaux d’IgE se´riques spećifiques a` l’OVA et d’eótaxine dans le liquide de lavage broncho-alveólaire des animaux provoque´s. De plus, L. salivarius PM-A0006 diminuait aussi l’hyperreáctivite´ bronchique induite par l’allerge`ne, ainsi que les niveaux e´leve´s d’IFN-g. Ces re´sultats montrent qu’un traitement oral a` L. salivarius PM-A0006 posse´derait un potentiel the´rapeutique dans le traitement de maladies respiratoires allergiques. Mots-cle´s : liquide delavage broncho-alveólaire, cytokines, Lactobacillus salivarius, probiotiques. [Traduit par la Re´daction]

Introduction Asthma is a chronic airway disorder that is characterized by reversible airway inflammation, obstruction, hyperresponsiveness, and elevated IgE production and mucus secretion (Djukanovic´ 2000). The degree of airway infiltration by inflammatory cells, such as eosinophils, lymphocytes, and mast cells, reflects the severity of asthma (Wills-Karp 1999). Several inflammatory mediators can promote the progression of asthma, such as PGE2, LTC4, and eotaxin (Cohn et al. 2004). Previous studies demonstrated that Th2 cells and their cytokines play a fundamental role in the pathogenesis of asthma (Robinson et al. 1992; Walker et al. 1992; Del Prete et al. 1993; Brusselle et al. 1995; Kips et al. 1995; Foster et al. 1996; Gru¨nig et al. 1998; Wills-Karp et

al. 1998; Larche´ et al. 2003; Saha et al. 2008). Consequently, the feasibility of down-regulating pathological Th2 responses by enhancing Th1 development has received much attention. Probiotics are defined by the Food and Agriculture Organization of the United Nations as ‘‘live microorganisms, which, when consumed in adequate numbers, confer a health benefit on the host’’ (Guarner and Schaafsma 1998). Of the many probiotic effects of lactobacilli (LAB), their immunomodulating effects have been the most extensively studied (Meydani and Ha 2000). Recent studies reported that LAB strains reduce allergic reactions in humans and mice (Kallioma¨ki and Isolauri 2003; Fujiwara et al. 2004; Ishida et al. 2005; Prescott et al. 2005). Probiotic microbes residing in the gastrointestinal tract of the host can induce Th1-based

Received 23 December 2009. Revision received 8 March 2010. Accepted 18 March 2010. Published on the NRC Research Press Web site at cjm.nrc.ca on 7 May 2010. C.-Y. Li.1 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan. H.-C. Lin,1 K.-C. Hsueh, and S.-F. Wu. Department of Pediatrics, China Medical University Hospital, School of Chinese Medicine, Taichung, Taiwan. S.-H. Fang.2 Institute of Athletics, National Taiwan Sport University, No. 16, Sec. 1, Shuan-Shih Road, Taichung 40404, Taiwan. 1These

authors contributed equally to this work. author (e-mail: [email protected]).

2Corresponding

Can. J. Microbiol. 56: 373–379 (2010)

doi:10.1139/W10-024

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antiallergic responses (Martinez and Holt 1999; Shida et al. 1998). In addition, Th2 cell-induced allergic inflammation could also be suppressed by probiotic-induced production of TGF-b (Fuller 1989; Hansen et al. 2000). Although many studies have demonstrated a beneficial role of probiotics in the experimental treatment of allergies (Gionchetti et al. 2003; Kim et al. 2005; O’Mahony et al. 2005), whether oral administration of probiotics has therapeutic potential in the treatment of allergic airway disease still remains elusive. In addition to effects in the gut, there is evidence indicating that orally delivered probiotics might regulate pulmonary immune responses. For example, the probiotic Lactobacillus rhamnosus GG was shown to reduce the frequency and severity of viral infections of the respiratory tract in children (Hatakka et al. 2001). Furthermore, Hori et al. (2002) demonstrated that oral administration of Lactobacillus casei can significantly enhance the pulmonary natural killer cell activity and promote the production of IFN-g and TNF by nasal lymphocytes in mice. However, action and efficacy depend on the probiotic in a strain-specific manner (Borchers et al. 2009). The purpose of this study was to determine whether Lactobacillus salivarius could affect the immune response in an ovalbumin (OVA)-sensitized asthma model. Experimental results demonstrated that L. salivarius PM-A0006 can attenuate antigen-induced eosinophil influx to the airway as well as local cytokine responses and hyperresponsiveness to methacholine in an OVA-sensitized mouse model of allergic airway disease.

Materials and methods Animals Female BALB/c mice, aged between 6 and 8 weeks, were obtained from the Laboratory Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan). All mice were kept in plastic cages in an environmentally controlled room with a 12 h light : 12 h dark cycle and were allowed free access to water. Mice were acclimatized to the animal facility before experimentation, and 8-weekold animals were used in all experiments. Mice were divided into 5 groups, and each group included 14 mice. All procedures were performed according to the Guide for the care and use of laboratory animals (National Research Council 1996). Experimental asthma In an OVA-sensitized mouse model of allergic airway inflammation, mice were sensitized by intraperitoneal injection of 50 mg of OVA mixed with 4 mg of alum in saline on day 2 and sequentially repeatedly sensitized with 25 mg of OVA emulsified in 4 mg of alum in saline on days 16, 30, and 44. On days 54 and 55, mice were challenged intranasally with 100 mg of OVA per mouse. Airway responsiveness of allergic sensitized mice was measured at 24 h after the last challenge (day 55) followed by collection of bronchoalveolar lavage (BAL) fluid (Fig. 1). The native mice were gavaged with distilled water without OVA sensitization as a negative control. Mice were sensitized with OVA without probiotic treatment as a positive control.

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Probiotic treatment Live lyophilized L. salivarius PM-A0006 used in this study was provided by ProMD Biotech Co., Ltd. (Southern Taiwan Science Park, Tainan, Taiwan). Commencing on day 1, mice received live 2.6 106 colony-forming units (CFU) (low dose, 0.5), 5.2 106 CFU (middle dose, 1), or 2.6 107 CFU (high dose, 5) lyophilized powder of L. salivarius PM-A0006 in 500 mL of cell-free distilled water via a gavaging needle for 8 consecutive weeks prior to intranasal antigen challenge. Measurement of serum OVA-specific IgE, IgG1, and IgG2a To determine serum antibody responses, blood samples were collected from the ophthalmic veins after sacrifice. Anti-OVA specific antibodies were measured by enzymelinked immunosorbent assay (ELISA). Briefly, microplates (Nunc, Roskilde, Denmark) were coated with 0.5 mg of OVA in coating buffer (0.1 mol/L of NaHCO3, pH 9.6) overnight at 4 8C. Plates were blocked with 1% bovine serum albumin in saline and then washed. Samples were added to the plates and incubated overnight at 4 8C. The quantities of OVA-specific IgE, IgG1, and IgG2a were detected by incubation (1 h at room temperature) with biotinylated rat anti-mouse IgE, IgG1, and IgG2a monoclonal antibodies, respectively. Avidin-HRP-conjugated streptavidin was used for detection, and plates were developed with the ABTS substrate for 30 min at room temperature. The color reactions were stopped with 5% SDS and read at 450 nm. The level of IgE, IgG1, or IgG2a was calculated by reference to a standard curve generated using purified mouse IgE, IgG1, or IgG2a, respectively. Results were presented as ELISA units, which were calculated with the following equation: ELISA unit = (Asample – Ablank) / (Apositive control – Ablank), where A is absorbance. Airway responsiveness On day 56, in vivo pulmonary function was assessed by whole-body plethysmography (Buxco system; Biosystem XA, Buxco Electronics Inc. Sharon, Conn., USA), as previously reported (Hamelmann et al. 1997). Mice were exposed to aerosolized PBS to establish the base line and then to incremental concentrations of methacholine (6.25, 12.5, 25, and 50 mg/mL). After each nebulization, enhanced pause (Penh) values were measured for 3 min and averaged. The mean Penh represented the airway tone. BAL fluid BAL and airway hyperresponsiveness measurements were carried out in the same animals. Pilot experiments revealed that the type and number of cells recovered from BAL fluid were not affected by the combination of these 2 techniques. After completion of the methacholine dose-response curve, the animals were lavaged 3 times through the tracheal cannula with 1 mL aliquots of pyrogen-free Hank’s buffered salt solution. BAL cells were prepared by using Cytospin (Shadon Scientific Ltd., Cheshire, UK) and stained with Liu’s stain. After they were coded, differential BAL cell counts were assessed by one observer using oil immersion microscopy (magnification: 1000) on all cytospin preparations to avoid observer bias. Cells were identified and differPublished by NRC Research Press

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Fig. 1. Experimental protocol: probiotic administration and intraperitoneal OVA sensitization. Mice were sensitized on days 2, 16, 30, and 44 with OVA and alum. Mice were fed lyophilized Lactobacillus salivarius PM-A0006 via a gavaging needle for 8 consecutive weeks prior to intranasal antigen challenge. The native mice were gavaged with distilled water without OVA sensitization as a negative control. Mice were sensitized with OVA without probiotic treatment as a positive control. Twenty-four hours after the last challenge (day 55), mice were subjected to measurements of airway hyperresponsiveness.

entiated into macrophages, lymphocytes, neutrophils, and eosinophils by standard morphology. At least 300 cells were counted for each cytospin preparation, and the absolute number of each cell type was calculated. Levels of inflammatory mediator in BAL fluid Cells were removed from BAL fluid, and the supernatants were stored at –80 8C until analyzed for inflammatory mediators. Inflammatory mediators (eotaxin, LTC4, and PGE2) were assessed using commercial ELISA kits (R&D Systems Ltd., Oxford, UK) according to the manufacturer’s protocols.

Fig. 2. Effect of probiotic administration on production of OVAspecific IgE in serum from OVA-sensitized mice. Sera from all groups of mice were obtained after sacrifice. IgE levels were determined by ELISA. Data are shown as means ± SE (n = 10–14 animals per group). An asterisk indicates a significant difference between the positive control group and the probiotic-treated groups (*, p < 0.05).

In vitro cytokine measurement The levels of IFN-g and IL-5 in the culture supernatants were measured upon restimulation with allergens. Whole spleens were removed to isolate splenocytes and cultured in 24-well flat-bottom plates at 37 8C in a 5% CO2 humidified atmosphere. Samples of 5 106 cells were stimulated with concanavalin A in a total volume of 1 mL. After 48 h, cellfree supernatants were harvested and the levels of IFN-g and IL-5 were assessed by ELISA. Statistics Experimental data were analyzed using SPSS software version 10 (SPSS Inc., Chicago, Ill., USA). The intergroup variation was assessed by one-way analysis of variance followed by Fisher’s least significant difference test. Differences were regarded as statistically significant for p values under 5% (p < 0.05) and 1% (p < 0.01).

Results Level of OVA-specific antibodies To examine whether oral administration of L. salivarius PM-A0006 affected the production of OVA-specific antibodies after OVA sensitization, the levels of OVA-specific IgE, IgG1, and IgG2a were determined by ELISA. Experimental results revealed that oral administration of L. salivarius PM-A0006 inhibited the production of OVA-specific IgE (Fig. 2). No significant differences were found in serum OVA-specific IgG1 and IgG2a levels between the positive control group and the probiotic-treated groups (data not shown). Airway response to methacholine Challenge with OVA in sensitized animals resulted in an increased airway responsiveness to methacholine. To examine whether oral administration of L. salivarius PM-A0006 affected the methacholine-induced airway hyperresponsiveness in OVA-sensitized mice, the pulmonary function was

assessed by whole-body plethysmography. Experimental results indicated that oral administration with L. salivarius PM-A0006 suppressed the methacholine-induced airway hyperresponsiveness of OVA-sensitized mice compared with that of the positive control. This suppressive effect was substantially more pronounced in the group fed the low dose (Fig. 3). Inflammatory cell influx to the airway Challenge with OVA in sensitized animals resulted in an increased total cell number in BAL fluid (Fig. 4A). Experimental results showed that animals that were orally administrated with L. salivarius PM-A0006 had a slightly lower total cell number in BAL fluid compared with that in the positive control but were not significant (Fig. 4A). 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 (Fig. 4B). In animals that were fed different doses of L. salivarius PM-A0006 before and during OVAsensitization and airway challenges, a significant suppression of the eosinophil influx was observed (Fig. 4B). Inflammatory mediator levels in BAL fluid Several inflammatory mediators affect the progression of Published by NRC Research Press

376 Fig. 3. Effect of oral treatment with Lactobacillus salivarius PMA0006 on airway responsiveness to methacholine. In vivo airway reactivity in response to increasing doses of aerosolized methacholine was calculated by whole-body plethysmography. Data are shown as the means ± SE (n = 10–14 animals per group) of Penh values for each concentration of methacholine. An asterisk indicates a significant difference between the positive control group and the probiotic-treated groups (*, p < 0.05; **, p < 0.01).

Can. J. Microbiol. Vol. 56, 2010 Fig. 4. Effect of cell distribution in BAL fluid by oral treatment with Lactobacillus salivarius PM-A0006. Effect of oral treatment with L. salivarius daily for 8 consecutive weeks on (A) the total cell number and (B) the differential cell counts (macrophages, eosinophils, neutrophils, and lymphocytes) in BAL fluid from OVAsensitized mice 24 h after challenge with intranasal OVA or saline. Data are presented as means ± SE (n = 10–14 animals per group). An asterisk indicates a significant difference between the positive control group and the probiotic-treated groups (*, p < 0.05; **, p < 0.01).

asthma, such as PGE2, LTC4, and eotaxin (Cohn et al. 2004). We further assayed the production of inflammatory mediators in BAL fluid by ELISA. As shown in Fig. 5, OVA-challenged mice gavaged with L. salivarius PMA0006 had a reduction in eotaxin production. Moreover, an even greater reduction was observed in the group fed a low dose (Fig. 5). No significant differences were found in the production of PGE2 and LTC4 between the positive control group and the probiotic-treated groups (Fig. 5). Cytokine production in splenocyte cultures It is generally believed that allergic diseases such as asthma occur when the Th1/Th2 balance is shifted in favor of Th2. As a result, raising the Th1 response is a therapeutic strategy to counter asthma symptoms (Suzuki et al. 2001). We further examined whether treating OVA-sensitized mice with L. salivarius PM-A0006 affected the Th1/Th2 balance. Experimental results indicated that treatment with low doses of L. salivarius PM-A0006 significantly elevated the levels of IFN-g in OVA-stimulated splenocytes (Fig. 6). No significant differences were found in the level of IL-5 between the positive control group and the probiotic-treated groups (data not shown).

Discussion An increasing body of evidence is accumulating to support the notion that probiotics, like lactobacilli, exert profound stimulatory effects on the intestinal immune system (Vaarala 2003; Isolauri et al. 2004; Boyle and Tang 2006). Therefore, it has been suggested that lactobacilli and other probiotic microorganisms could be effective in the management of allergic disorders, such as rhinitis, atopic dermatitis, and asthma (Noverr and Huffnagle 2004; Vanderhoof and Young 2004; Feleszko et al. 2006). Furthermore, clinical evidence provided by Kruisselbrink et al. (2001) and others indicates that probiotic treatment is effective in the prevention of allergen-induced atopic dermatitis. In the present study, an OVA-sensitized mouse model of

allergic airway inflammation was used to investigate the effects of oral probiotic administration on the allergen-induced asthmatic airway responses. Our results demonstrate that oral administration with L. salivarius PM-A0006 prior to and during allergen sensitization and airway challenges leads to a suppression of various features of the asthmatic phenotypes, including specific IgE production, airway inflammation, and development of airway hyperresponsiveness (Figs. 2, 3, 4). The decreases in cellular influx and airway hyperresponsiveness were associated with a reduced level of inflammatory cytokine (eotaxin) in the BAL fluid (Fig. 5). Additionally, oral consumption of L. salivarius PM-A0006 resulted in an increase in the Th1-type cytokine IFN-g (Fig. 6). Other studies have reported similar antiinflammatory effects of lactobacilli, where oral administration of L. rhamnosus GG suppressed airway reactivity Published by NRC Research Press

Li et al. Fig. 5. Effect of oral treatment with Lactobacillus salivarius PMA0006 on inflammatory mediator levels in BAL fluid in OVAsensitized mice. Inflammatory mediators (eotaxin, LTC4, and PGE2) were assessed by ELISA. Data are presented as means ± SE (n = 10–14 animals per group). An asterisk indicates a significant difference between the positive control group and the probiotictreated groups (*, p < 0.05).

Fig. 6. The oral administration of Lactobacillus salivarius PMA0006 augmented the production of IFN-g by splenocytes. BALB/c mice were orally administered distilled water or L. salivarius PMA0006 for 8 consecutive weeks. In the eighth week, single-cell suspensions of spleen cells were prepared. Spleen cells were cultured with concanavalin A, and then the supernatant was collected. Cytokine levels were determined by ELISA. Data are presented as means ± SE (n = 10–14 animals per group). An asterisk indicates a significant difference between the positive control group and the probiotic-treated groups (*, p < 0.05).

(Blu¨mer et al. 2007; Feleszko et al. 2007; Karimi et al. 2009; Hougee et al. 2010), reduced the production of antigen-specific IgE (Feleszko et al. 2007; Hougee et al. 2010), decreased the recruitment of eosinophil (Blu¨mer et al. 2007; Karimi et al. 2009), and attenuated the development of airway hyperresponsiveness (Karimi et al. 2009). Furthermore, regulatory T cells play an important role in regulating Th2 responses to allergen, maintaining functional tolerance, and suppressing allergen-induced airway hyperresponsiveness

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(Strickland et al. 2006). Previous studies indicated that regulatory T cells induced by L. rhamnosus GG protected against an allergic airway response by attenuating the development of airway hyperresponsiveness, reducing the production of specific IgE, and decreasing the recruitment of eosinophil (Karimi et al. 2009; Lyons et al. 2010). We speculate that inhibition of allergic airway response by L. salivarius PMA0006 administration could be associated with the activity of regulatory T cells. Forsythe et al. (2007) analyzed the effects of oral administration with probiotic L. salivarius on allergic airway inflammation in a mouse model of antiallergic airway inflammation. Strain-dependent characteristics of this probiotic were observed, and the results revealed that oral treatment with L. salivarius did not modulate the allergic airway response. The antiallergic effects of LAB are strain dependent and mediated by effects on both Th1/Th2 cytokine expression and balance. Fujiwara et al. (2004) analyzed more than 100 LAB strains with respect to induction of cytokines by splenocytes from OVA-sensitized mice and found that distinct cytokine profiles were elicited depending on the strain of LAB, not depending on the species. Other studies have revealed that elevated allergen-induced Th-2 type cytokine production in airway leads to increased IgE formation and eosinophilic airway inflammation (Shi et al. 1997). These allergic airway responses can be inhibited by Th1 cytokines, such as IFN-g and IL-18, by tolerance induction (Wohlleben and Erb 2002). In our study, oral administration of L. salivarius PMA0006 prior to and during OVA sensitization resulted in a shift from a predominant Th2 towards a predominant Th1 immune response. In addition, the effects of different doses of probiotics on the response to antigen challenge in an OVA-sensitized asthma model were assessed here. Although dose-dependent effects were not observed, based on the major characteristics of asthma, oral treatment with a low dose (0.5, 2.6 106 CFU/day) of L. salivarius PM-A0006 might bring about repression. The precise dosage of probiotics must be further determined. Two relevant studies reported that administrating L. salivarius (1~2 109 CFU/ day) did not alter the production of IgE and recruitment of eosinophil following OVA challenge (Karimi et al. 2009; Lyons et al. 2010). We speculate that administrating a low dosage of L. salivarius might result in anti-inflammatory effects. Moreover, Drago et al. (2010) indicated that strains of the same species of L. salivarius have different modulatory effects on the Th1 or Th2 type of immune response. For these reasons, the selection of proper probiotic stains and precise dosage seems to be a notable issue for basic research and clinical application. In conclusion, we demonstrated that oral administration of live L. salivarius PM-A0006 resulted in significant attenuation of the allergic airway response. Our results indicate that oral treatment with L. salivarius PM-A0006 can attenuate allergen-induced asthmatic airway responses, including reduction in airway eosinophilia, shifting allergen-induced Th2 to a Th1 response, as well as blocking hyperresponsiveness to methacholine. On the basis of these beneficial effects, we thus conclude that L. salivarius PM-A0006 is a promising candidate for further investigation in the management of allergic airway disorders. Published by NRC Research Press

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Acknowledgements The authors thank Pei-Yu Shih for her expert technical assistance. This work was supported by the grants NSC 952320-B-028-001-MY2 from the National Science Council, R.O.C., and DMR-95-010 from the China Medical University and Hospital in Taiwan.

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