Exopolysaccharides Produced by Leuconostoc mesenteroides Strain

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Exopolysaccharides Produced by Leuconostoc mesenteroides Strain NTM048 as an Immunostimulant To Enhance the Mucosal Barrier and Influence the Systemic Immune Response Chiaki Matsuzaki,*,† Asuka Hayakawa,† Kenji Matsumoto,‡ Toshihiko Katoh,† Kenji Yamamoto,† and Keiko Hisa§ †

Research Institute for Bioresources and Biotechnology and ‡Department of Food Science, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836, Japan § Nitto Pharmaceutical Industries, Ltd., Kamiueno, Muko, Kyoto 617-0006, Japan ABSTRACT: Leuconostoc mesenteroides strain NTM048 has been shown to have intestinal IgA-inducing ability. In this study, we investigated the immunostimulant potency of an exopolysaccharide secreted from strain NTM048 (NTM048 EPS) in vitro and in vivo in a murine model. NTM048 EPS ranges in size from 10 to 40 kDa and is speculated to be mainly composed of glucose and fructose. The in vitro study revealed that NTM048 EPS induced total and antigen-specific IgA production by Peyer′s patch cells and influenced Th1 and Th2 cell-mediated response in splenocytes. Oral administration of NTM048 EPS dose-dependently induced fecal IgA production accompanied by the up-regulation of retinoic acid synthase and transforming growth factor-β receptor genes in Peyer′s patch cells. Flow cytometric analysis of the splenocytes revealed an increase of the CD3+ T-cell population and the ratio of CD4+ T-cells/CD8+ T-cells. These results indicate that NTM048 EPS could enhance the mucosal barrier and influence the systemic immune response. KEYWORDS: exopolysaccharide, immunostimulant, antigen-specific IgA, systemic immune response, Leuconostoc mesenteroides

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INTRODUCTION Lactic acid bacteria (LAB) have been used around the world to produce a great variety of fermented food. Some LAB species contributes to the improvement of the texture and viscosity of fermented products owing to the exopolysaccharides (EPS) that they synthesize. Therefore, they can be considered as natural biothickeners because they are produced in situ by the LAB-starters that are permitted to be used under their General Recognized As Safe status.1 The biological properties of the EPS are also of interest for their antitumor, antiulcer, cholesterol-lowering, and immunomodulating activities, the last of which includes IgA-inducing activity.1,2 IgA is the most abundant immunoglobulin isotype in the epithelial mucus and can entrap antigens and pathogens, thus preventing them from binding to cell surface receptors, as exemplified by the IgA-mediated neutralization of cholera toxin and the motility reduction of Salmonella spp.3,4 Generating massive amounts of IgA is indispensable for mucosal protection.5,6 Additionally, because of the insufficient efficacy of the systemic IgG response against infection by influenza viruses, IgA has alternatively gained attention because of its ability to act in the airway mucosal epithelium and its broad spectrum heterosubtypic protective immunity.7 EPSs produced from LAB, e.g. Lactobacillus delbrueckii subsp. bulgaricus OLL 1073R-1, and Lactobacillus kef iranofaciens, have been demonstrated to modulate immune cells to produce intestinal IgA.8,9 Nevertheless the enhanced effect of the EPSs on intestinal IgA secretion has not been reported. In spite of the demand for immunostimulants to strengthen the mucosal barrier, EPSs have not been applied yet as immunostimulants for human health and disease.1,9 © XXXX American Chemical Society

Our previous study demonstrated that oral administration of Leuconostoc mesenteroides subsp. mesenteroides strain NTM048 to BALB/cA mice induced a significant increase in the fecal IgA content, and the EPS produced by this strain (NTM048 EPS) significantly induced IgA production in vitro.10 However, it has not been shown that NTM048 EPS can act as an immunostimulant and influence the systemic immune response. In this study, we examined the effect of NTM048 EPS on mucosal and systemic immune systems in vitro and in vivo and found that NTM048 EPS could enhance the mucosal barrier and influence the systemic immune response.

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MATERIALS AND METHODS

Animals. Six-week-old male BALB/cA mice were purchased from CLEA Japan (Tokyo, Japan) and were fed an AIN-76A diet purchased from Research Diets (New Brunswick, NJ). The composition of AIN76A was as follows (w/w): 20.0% milk casein, 0.3% DL-methionine, 5.0% corn oil, 50.0% sucrose, 15.0% corn starch, 5.0% cellulose powder, 1.0% AIN-76 vitamin mix, 3.5% AIN-76 mineral mix, and 0.2% choline hydrogen tartrate. Animals were handled in accordance with the guidelines for the proper conduct of animal experiments issued by the Science Council of Japan (2006). The animal experimentation ethics committee of Ishikawa prefectural University approved this study (No. 26-14-11). Isolation and Purification of Exopolysaccharide. The EPS produced by strain NTM048 was extracted and purified as details in our previous study.10 The sugar concentration was measured by the Received: April 27, 2015 Revised: July 24, 2015 Accepted: July 24, 2015

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DOI: 10.1021/acs.jafc.5b01960 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

splenocytes. The prepared splenocytes were cultured (5 × 105 cells/ well) on 96-well T-cell activation plates (Becton Dickinson) with sizeexclusion chromatography-purified NTM048 EPS (20 μg/mL) for 66 h, followed by fluorescence-augmented cell sorter (FACS) analysis to examine the frequency of T cells (CD4+ and CD8+). Aliquots of 1 × 106 splenocytes were incubated with 10 μg/mL of Purified Rat AntiMouse CD16/32 (BD Biosciences, San Diego, CA) for 15 min on ice and washed twice in stain buffer (PBS with 2% FBS). Cells were then stained with PE-Cy7 conjugated antimouse CD3e (clone 145-2C11), PE conjugated antimouse CD4 (clone RM4-5), and APC conjugated antimouse CD8a (clone 53-6.7) using Mouse T Lymphocyte Subset Antibody Cocktail (BD Biosciences) for 30 min at 4 °C. Flow cytometry was performed on 20,000 cells by using FACSAria and FACSDiVa4.1 software (Becton Dickinson). The frequency of CD4+ cells producing IFN-γ, IL-4, and IL-17 in splenocytes cultured with NTM048 EPS was examined by intracellular cytokine staining (ICCS) using the BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences) according to the manufacturer′s instructions. In brief, the NTM048 EPS-stimulated splenocytes were resuspended in 1 mL of stimulation medium [RPMI 1640 medium containing 10 ng/mL of phorbol 12-myristate 13 acetate (Sigma-Aldrich), 1 ng/mL of ionomycin calcium salt (Sigma-Aldrich), 0.6 μL/ml of BD Golgistop] and incubated at 37 °C in 5% CO2 for 5 h. Cells were washed twice with staining buffer and incubated with fixation/permeabilization solution for 20 min in the dark. Cells were washed twice again and stained by CD4 PERCP-Cy5.5, IL-17 PE, IFNγ FITC, and IL-4 APC antibodies using a Mouse Th1/Th2/Th17 Phenotyping Kit (BD Biosciences) for 30 min on ice in the dark. Cells were washed twice and resuspended in staining buffer. Staining was assessed by FACSAria (BD Biosciences) and analyzed using BD FACSDiVa 4.1 software (BD Biosciences). A total of 30,000 events/ sample were analyzed. In Vivo Analysis of Immune Responses for NTM048 EPS. After 2 weeks of acclimation, 20 mice were assigned to 5 groups of four each based on body weights and fecal IgA levels. Each group was administered ad labium the NTM048 EPS-containing experimental water at a concentration of 0, 0.05, 0.1, 0.5 or 1% w/v. The mice were individually housed and administered experimental water for up to 42 days. Feces were collected every 7 days, and the IgA content in the feces was analyzed according to our previous study.10 On day 42, the mice were euthanized with excessive somnopentyl, and Peyer′s patch cells, spleen, and blood were harvested. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed using the Transaminase CII kits (Wako Pure Chemical Industries, Osaka, Japan) to detect liver damage. Plasma creatinine (CRE) content was analyzed by the LabAssay Creatinine kit (Wako Pure Chemical Industries) to detect kidney disease. Measurements of plasma IgA and IgG levels were performed using a mouse IgA ELISA kit (Bethyl Laboratories) and a mouse IgG ELISA kit (Bethyl Laboratories), respectively. Gene Expression Analysis of Peyer′s Patch cells. Peyer′s patch cells collected from the small intestine of mice were used for gene expression analysis. Total RNA samples were isolated using a QuickPrep total RNA extraction kit (GE Healthcare, Piscataway, NJ, USA). For preparation of cDNA, 1 μg of each total RNA sample was reverse-transcribed using Super Script III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo (dT) primers (Invitrogen), and the obtained cDNA was then purified using a PCR Purification kit (Qiagen, Cambridge, MA, USA). Quantitative real-time RT-PCR was performed in a 7300 Fast Real-Time PCR system (Life Technologies Japan, Tokyo, Japan), using a Power SYBR Green Master Mix (Life Technologies) according to the manufacturer′s instructions. A housekeeping transcript, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used as an internal control to standardize the efficiency of each reaction. The sequences of the primers used were as follows: GAPDH (Gapdh), 5′-ctacactgaggaccaggttgtct-3′ and 5′attgtcataccaggaaatgagctt-3′; IL-5 (Il5), 5′-tgaggcttcctgtccctactcataa-3′ and 5′-ttggaatagcatttccacagtaccc-3′; IL-6 (Il6), 5′-aatagtccttcctaccccaatttc-3′ and 5′-atttcaagatgaattggatggtct-3′; IL-21 (Il21),13 5′-gcagcacaggctaagagcttgta-3′ and 5′-tggctagtggagaagccttca-3′; BCL6 (Bcl6),13

phenol-sulfuric acid method using glucose as a standard. Samples were tested for the absence of free sugars by thin layer chromatography on silica gel G type 60 plates (MERCK, Tokyo, Japan) using chloroform− methanol−water (3:3:1 v/v/v) as the mobile phase. The ethanolprecipitated and dried EPS was then finely milled and moderately sonicated to dissolve in distilled water (for the in vivo experiment) or saline (for the in vitro cell assay). Characterization of NTM048 EPS. To determine the monosaccharide composition of purified NTM048 EPS, 3 mg of EPS was hydrolyzed by addition of 750 μL of perchloric acid (1.75 M) and incubated at 80 °C for 16 h. After incubation, 250 μL of KOH (5 M) was added to precipitate the potassium perchlorate. The solution was centrifuged, and then the supernatant was filtered through a 0.45 μm pore size filter and the monosaccharide composition was analyzed by high performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) using a 4 × 250 mm Dionex CarboPac PA1 column. A gradient of sodium acetate from 0 to 500 mM in 100 mM sodium hydroxide was used for elution over 40 min at 1 mL/min. Detection was performed using a Dionex ICS-3000 module. D-Glucose and D-fructose (Nacalai Tesque, Tokyo, Japan) were used as standards. The average molecular mass of NTM048 EPS was determined by size exclusion chromatography using Sepharose CL6B (Sigma-Aldrich, Missouri, USA). Two types of commercially purchased dextrans (Sigma-Aldrich, average MW 36,000−45,000 and 9,000−11,000) were used as standards. In Vitro Analysis of IgA-Inducing Activity of EPS on Peyer′s Patch Cells. To evaluate the IgA-inducing activity of NTM048 EPS, mouse Peyer′s patch (PP) cells were prepared according to our previous study10 and resuspended at 2.5 × 106 cells/mL in RPMI 10 medium [RPMI 1640 (Gibco BRL, CA, USA) containing 100 U/mL penicillin, 100 μg/mL streptomycin, 55 μmol/L 2-mercaptoethanol, and 10% fetal bovine serum (FBS; Gibco BRL)]. Various concentrations of size-exclusion chromatography-purified NTM048 EPS were added to PP cells (1.25 × 106 cells/mL final concentration) and incubated in 96-well T-cell activation plates (Becton Dickinson, Franklin Lakes, NJ) at 37 °C in a humidified atmosphere of 5% CO2 in air. After 5 days, the IgA levels in the supernatants were measured by ELISA using a mouse IgA ELISA kit (Bethyl Laboratories, Montgomery, TX). Assay for Specific IgA-Inducing Activity of EPS. The specific IgA-inducing ability of NTM048 EPS to the H1N1 antigen was measured according to the method of Yasui et al.11 with some modifications. Mouse PP cells (4 × 106 cells/mL) in Eagle′s minimum essential medium (Nissui, Tokyo, Japan) were cultured with size exclusion chromatography-purified NTM048 EPS (250 μg/mL) and influenza A (H1N1) virus antigen (0.34 μg/mL) on 96-well T-cell activation plates (Becton Dickinson) at 37 °C in a humidified atmosphere of 5% CO2 in air to which a 10% v/v aliquot of a nutrient mixture was added daily.12 After 2 days of incubation, the medium containing H1N1 antigen was removed by centrifugation, and then the PP cells were further incubated with NTM048 EPS (250 μg/mL) for an additional 6 days. Specific IgA levels against H1N1 antigen in the cultured medium were measured by ELISA as follows. H1N1 antigens (3 μg/mL) were coated on the wells of a 96-well ELISA plate (Thermo Scientific, MA, USA), and the culture supernatant was added to each well. Horseradish peroxidase-labeled mouse IgA detection antibody (Bethyl Laboratories) was added to the wells, and then an aliquot of 3,3′,5,5′-tetramethylbenzidine substrate solution was added to each well. After 15 min, the reaction was stopped by addition of 1 M HCl, and the absorbance of the contents of the wells was measured at 450 nm. In Vitro Analysis of Immune Responses on Splenocytes. To evaluate the response of NTM048 EPS stimulation on splenocytes, mice were euthanized by CO2 gas, and their spleens were harvested. Their spleens were gently teased and dispersed through a 70-μm cell strainer to obtain single-cell suspensions. After centrifugation (100 × g, 8 min), red blood cell lysis buffer (NH4Cl 0.83%, KHCO3 0.1%, EDTA-2Na 0.00372%) was added to the pellet and incubated for 5 min. After washing with RPMI 10 medium twice, the cells were used as B


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Figure 1. Characterization of NTM048 EPS. A: Molecular mass distribution of NTM048 EPS by size exclusion chromatography. Two kinds of industrial dextrans (average MW 36,000−45,000 and 9,000−11,000) were used as standards. B: HPAEC-PAD analysis of the monosaccharide composition of NTM048 EPS hydrolysate. Glucose and fructose were used as standards. 5′-gcactgggcaaacacaacat-3′ and 5′-agcgtgccgggtaaactg-3′; BAFF (Tnfsf13b),14 5′-tgctatgggtcatgtcatcca-3′ and 5′-ggcagtgttttgggcatattc3′; APRIL (Tnfsf13),14 5′-tcacaatgggtcaggtggtatc-3′ and 5′-tgtaaatgaaagacacctgcactgt-3′; RALDH1 (Aldh1al),15 5′-atggtttagcagcaggactcttc-3′ and 5′-ccagacatcttgaatccaccgaa-3′; CD40L (Cd40l), 5′-tttatccttgctgaactgtgagga-3′ and 5′-gatcctcatcacctctttgcattt-3′; TGF-β1 (Tgf b1),15 5′-tggagcaacatgtggaactc-3′ and 5′-tgccgtacaactccagtgac-3′; TGF-βR2 (Tgf br2),16 5′-cctactctgtctgtggatga-3′ and 5′-gctcgtaatccttcacttctc-3′; and AID (Aicda),17 5′-aaattctgtccggctaacca-3′ and 5′cattccaggaggttgctttc-3′. To confirm whether or not PCR correctly amplified the aimed genes, the PCR products were sequenced and identified by means of BLAST search (DNA Data Bank of Japan), and we analyzed their melting curves (data not shown). All experiments were performed in triplicate. Flow Cytometric Analysis of Splenocytes. Single-cell suspensions of splenocytes were prepared, and the frequencies of T cells (CD4+ and CD8+) were examined as described above using a FACSAria (BD Biosciences) and analyzed by BD FACSDiVa4.1 software (BD Biosciences). A total of 20,000 events/sample were analyzed. Statistical Analysis. Each result is expressed as the mean ± standard error (SE). Dunnett′s test or two-way repeated-measures ANOVA with a posthoc analysis followed by Dunnett′s test was used to compare multiple groups. Statistical analyses were conducted using Ekuseru-Toukei software version 2010 (SSRI, Tokyo, Japan). P values less than 0.05 were considered statistically significant.

NTM048 EPS was eluted between the two dextran standards whose sizes ranged from 36,000−45 000 Da and 9,000−11 000 Da, respectively, indicating the NTM048 EPS molecular mass range is approximately 10−40 kDa (Figure 1A). Next, we attempted to determine the monosaccharide composition of NTM048 EPS. HPAEC-PAD analysis was used to monitor the partial hydrolysis of the EPS to its monosaccharides. The chromatogram showed two monosaccharide peaks, which corresponded to the retention time of D-glucose and D-fructose, respectively (Figure 1B). This result suggests that NTM048 EPS contains glucose and fructose. Effect of NTM048 EPS on Immune Cells in Vitro. Isolated PP cells and splenocytes were used to examine the effect of NTM048 EPS on immune cells. As shown in Figure 2A, total IgA production of PP cells was dose-dependently stimulated by NTM048 EPS (1.5-fold and 1.7-fold increases compared with that of the saline control in the presence of 25 and 250 μg/mL EPS, respectively). Moreover, costimulation of NTM048 EPS with H1N1 antigen significantly induced antiH1N1 IgA production by 1.5-fold (Figure 2B). These results indicate that NTM048 EPS can stimulate PP cells to produce total and antigen-specific IgA (see Discussion). FACS analysis on the NTM048 EPS-stimulated splenocytes was also carried out. The isolated splenocytes were treated 20 μg/mL of NTM048 EPS. The number of CD3+ cells and the ratio of CD4+ T cells to CD8+ T cells from the NTM048 EPSstimulated splenocytes were significantly higher (P < 0.05) than those of nonstimulated splenocytes (Figure 3A and B). Additionally, NTM048 EPS stimulation significantly expanded the CD4+ T cell population (P < 0.01) (Figure 3C upper

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RESULTS Characterization of NTM048 EPS. We obtained 1.46 g of NTM048 EPS from a 50-mL culture in the EPS-production medium. To estimate the molecular mass of the NTM048 EPS, size exclusion chromatography was performed. The majority of C


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concentration of 20 μg/mL. No change was observed in the frequency of CD4+ T cells producing IL-17 (nontreated, 1.7 ± 0.1%; NTM048 EPS treated, 1.7 ± 0.1% [data not shown]). These results suggest that EPS stimulation affects the Th1 and Th2 cell-mediated response in splenocytes. Effect of NTM048 EPS on the Mouse Intestinal Immune System. Having established the in vitro effect of NTM048 on mucosal and systemic immune responses as described above, we next performed in vivo experiments using BALB/cA mice. Drinking water supplemented with NTM048 EPS at 0, 0.05, 0.1, 0.5, or 1.0% (w/v) was given to mice ad libitum for 42 days. All animals were in good health throughout the experimental period, and no side effects, such as diarrhea, occurred. There were no significant differences in food and water intake, body weight gain, and plasma AST, ALT, and CRE levels among the groups (Table 1). In agreement with the in vitro study, oral administration of NTM048 EPS increased the fecal IgA concentration (Figure 4). While the fecal IgA content of the 0 and 0.05% groups were minimally changed through the experimental period, those of the 0.1, 0.5, and 1% groups were significantly increased after 35 days compared with their initial IgA content. Peyer′s patches are the main inductive sites for intestinal IgA responses.18,19 To determine the mechanism responsible for the mucosal IgA production upon administration of NTM048 EPS, we analyzed the expression levels of genes involved in intestinal immunostimulation in isolated PP cells (Figure 5). The gene expression of the TGF-β receptor 2 (TGF-βR2) found on B cells was significantly increased in the 0.5 and 1% groups (P < 0.05), suggesting the occurrence of TGF-β1 signaling.5 The gene expression of retinal dehydrogenase (RALDH), which is required for retinoic acid (RA) production from dendritic cells (DCs), was also increased in the 1% group (P < 0.05). The gene expression of B cell lymphoma 6 (BCL6), which is abundantly expressed by follicular helper T (TFH) cells, tended to increase in the 0.5 and 1% groups (P = 0.14 and P = 0.17, respectively). On the other hand, the expression of IL-5, IL-6, IL-21, B cell-activating factor (BAFF), a proliferationinducing ligand (APRIL), CD40 ligand (CD40L), TGF-β1, and activation-induced cytidine deaminase (AID) genes remained unchanged. Effect of NTM048 EPS on the Systemic Immune System in Mice. To investigate the effect of NTM048 EPS administration on the systemic immune system, we examined the plasma IgA and IgG levels and carried out flow cytometric analysis on the isolated splenocytes of NTM048 administratedmice. Oral administration of NTM048 EPS did not affect plasma IgA and IgG levels (Table 1), but a significant effect on T cell population in the spleen was observed. The CD3 positive T-cell population in total lymphocytes of the 0.5% and 1% groups were significantly higher than that of the control group (Figure 6A) (P < 0.05). Additionally, the CD4+ T cells/CD8+ T cells of the 1% group were significantly higher than that of the control group (Figure 6B) (P < 0.01), which was consistent with the in vitro result (Figure 3A and B).

Figure 2. In vitro response of Peyer′s patch cells stimulated by NTM048 EPS. A: IgA-inducing ability of NTM048 EPS. Each value is presented as the mean ± SE (n = 5; ***P < 0.001). B: Augmentation of anti-influenza virus H1N1 IgA antibody production by NTM048 EPS. Each value is presented as the mean ± SE (n = 8; ***P < 0.001).

Figure 3. Flow cytometric analysis of splenocytes in vitro. Splenocytes were cultured with 20 μg/mL of NTM048 EPS or saline (control). Each value is presented as the mean ± SE (n = 5). A: Ratio of CD3+ cells/lymphocytes. B: Ratio of CD4+ cells/CD8+ cells (*P < 0.05). C: Ratio of CD4+ cells/lymphocytes, IFN-γ+ cells/CD4+ cells, and IL-4+ cells/CD4+ cells. Statistical results are shown in the panels.

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panel). The ratio of a subset of CD4+ T cells producing IFN-γ was significantly increased by NTM048 EPS stimulation (14.5% vs 22.4%, P < 0.001) with a concomitant decrease in the ratio of a subset of CD4+ T cells producing IL-4 (6.1% vs 2.7%, P < 0.001) (Figure 3C lower panel). We also examined the effects of 100 μg/mL of EPS treatment on splenocytes, which results were almost the same as those of 20 μg/mL of EPS treatment (data not shown). This indicates that the immune stimulation of splenocytes by NTM048 EPS was induced enough at the

DISCUSSION One of the immunomodulating properties induced by LAB is the promotion of host mucosal IgA secretion to strengthen the first lines of defense, such as the mucosal barrier.20−22 It has been reported that several types of LAB are effective against pathogenic infection via their IgA-inducing ability. Lactobacillus pentosus strain b24023 and Lactobacillus plantarum strain AYA24 D


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Journal of Agricultural and Food Chemistry Table 1. Physiological Parameters of Mice Administered NTM048 EPS at Doses of 0, 0.05, 0.1, 0.5, and 1%a 0% group water intake (g/d) initial body weight (g) final body weight (g) body weight gain (g) blood chemistry AST (Karmen unit) ALT (Karmen unit) CRE (μg/mL) IgA (μg/mL) IgG (μg/mL) a

0.05% group

0.1% group

0.5% group

1% group

3.8 30.4 37.1 6.7

± ± ± ±

0.1 0.9 1.5 0.7

4.4 30.8 37.1 6.3

± ± ± ±

0.3 1.4 2.0 1.4

3.9 30.9 37.3 6.4

± ± ± ±

0.1 0.8 1.7 1.0

4.3 31.1 36.1 5.1

± ± ± ±

0.4 0.4 1.3 1.3

4.2 30.0 35.9 5.9

± ± ± ±

0.2 1.3 2.0 0.9

32.9 28.1 3.1 227.7 87.8

± ± ± ± ±

6.3 5.5 0.2 9.9 4.8

25.9 19.4 4.4 220.9 92.5

± ± ± ± ±

3.4 1.6 0.4 16.9 4.5

24.1 18.8 4.3 235.8 94.3

± ± ± ± ±

3.8 3.0 0.3 14.1 6.3

21.5 14.4 4.9 259.7 93.5

± ± ± ± ±

1.3 1.3 0.5 42.5 13.3

22.2 14.8 4.4 266.3 82.0

± ± ± ± ±

3.1 3.0 0.3 10.0 4.3

Results are shown as the mean ± SE (n = 4).

Figure 4. Fecal IgA content induced by NTM048 EPS intake. Mice were administered 0, 0.05, 0.1, 0.5, and 1% of EPS-containing water and the IgA content in feces were compared. Differences were assessed by a two-way repeated-measure ANOVA with a post hoc analysis by Dunnett′s test. Each value is presented as the mean ± SE (n = 4). *P < 0.05, **P < 0.01 vs each initial IgA content (day 0).

The spleen contains various immune cells including T and B cells, DCs, and macrophages and regulates the systemic immune system to protect the body against blood-borne bacterial, viral, and fungal infections.26 NTM048 EPS influenced the T cell population of splenocytes in vitro and in vivo. In both experiments, NTM048 EPS increased the T cell (CD3+ cell) population and the ratio of CD4+ cells/CD8+ cells. Given the results of its IgA-inducing ability, intake of NTM048 EPS influenced not only the mucosal immune system but also the systemic immune system. Medrano et al. reported that oral treatment of EPS produced by microorganisms present in kefier grains modulated the balance of immune cells in intestinal mucosa including the increase in IgA+ cells but did not affect the cells in the spleen.27 Therefore, immunomodulation ability on the systemic immune system is considered as a marked characteristic of NTM048 EPS. Additionally, NTM048 EPS treatment of isolated splenocytes increased IFN-γproducing CD4+ cells and decreased IL-4-producing CD4+ cells. IFN-γ is a cytokine associated with the Th1 immune response rather than the allergic Th2 response, and IL-4 is a cytokine required for Th2 cell differentiation and Th1 development suppression.28 Hence, NTM048 EPS might influence the Th1 and Th2 cell-mediated response of systemic immune system. IgA induction through a T cell-dependent pathway leads to the production of high-affinity IgA because of the occurrence of somatic hypermutation (SHM), which is responsible for affinity

were selected for their IgA-inducing potency on mouse PP cells, and oral administration of the cells could facilitate protection against influenza virus in mice. However, the IgA-inducible components of these LABs remain unclear. In our previous study, we identified Leuc. mesenteroides strain NTM048 having IgA-inducing potency from among 173 LABs.10 NTM048 cells were tolerant to gastric and intestinal digestion in vitro, and oral administration of this strain induced intestinal IgA secretion in mice. Moreover, the NTM048 EPS dose-dependently induced IgA secretion from PP cells, suggesting that NTM048 EPS plays a pivotal role as an immunostimulant of strain NTM048. To confirm that NTM048 EPS is an immunostimulant, we investigated the effect of NTM048 EPS on the intestinal and systemic immune system in the present study. In agreement with the results from our previous study, NTM048 EPS dose-dependently enhanced total IgA secretion by isolated PP cells. Furthermore, we revealed that NTM048 EPS could induce specific IgA secretion by isolated PP cells. To effectively prevent mucosal infection, the ability to induce a specific IgA response is also important.25 In particular, a successful influenza virus vaccine containing an adjuvant which stimulates mucosal antigen-specific IgA production is attractive because of the insufficient efficacy of the systemic IgG response. Therefore, NTM048 EPS might be useful as an effective adjuvant of mucosal vaccination. E


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immunogenicity. Natural NTM048 EPS stimulated immune responses in mice without prominent side effects, suggesting its potency as a nontoxic mucosal adjuvant.34 Some strains of Leuc. mesenteroides produce fructose-containing dextrans,35 but their immunomodulating activity has not been reported yet. The precise determination of the NTM048 EPS structure is currently underway in our laboratory. In summary, we showed that Leuc. mesenteroides NTM048secreted EPS was a specific IgA-inducible immunostimulant and influenced systemic immunity in mice. These properties of NTM048 EPS indicate that it may be an effective immunostimulant useful for protection from mucosal pathogens.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 81-76-227-7513. Fax: 81-76-227-7557. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 5. Gene expression analysis of Peyer′s patch cells from mice treated with 0, 0.05, 0.1, 0.5, and 1% of NTM048 EPS. Gene expression was analyzed by SYBR Green-Based Real-Time RT-PCR. GAPDH was used as a control to standardize the efficiency of each reaction. Each value is presented as the mean ± SE (n = 4). Statistical analysis was performed with Dunnett′s multiple comparison test. *P < 0.05 vs the control group.

ACKNOWLEDGMENTS We thank Tetsuro Kitao, Kohei Kitao, and Dr. Yasunori Yonejima, Nitto Pharmaceutical Industries, Ltd., for helpful discussion and comments. We also thank Dr. Seiya Makino, Meiji Co., Ltd., for technical advice.

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REFERENCES

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Figure 6. T cell stimulation in the spleen of mice fed water containing 0, 0.05, 0.1, 0.5 and 1% of NTM048 EPS. A: Ratio of CD3+ cells/ lymphocytes. B: Ratio of CD4+ cells/CD8+ cells. Each values is presented as the mean ± SE (n = 4). *P < 0.05, **P < 0.01 vs the control group.

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Exopolysaccharides Produced by Leuconostoc mesenteroides Strain

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