Further studies on immunomodulatory effects of exopolysaccharide isolated from Lactobacillus rhamnosus KL37C

Introduction

Recently, an increasing number of studies have suggested that the immunoregulatory effect of probiotic bacteria can be achieved not only by using live or dead bacteria, but also by using components of the bacterial cell wall or their secretory products such as exopolysaccharides (EPS) excreted into the environment during growth of bacteria [1, 2]. Most lactic acid bacteria are capable of synthesizing and secreting EPS. Lactobacillus, Streptococcus and Lactococcus prevail among these genera but also it can be produced by some strains of Bifidobacterium [3-6]. Over 50 different exopolysaccharides derived from lactic acid bacteria have been described, and more than 30 of these bacteria belong to Lactobacillus [7].

EPSs are linear macromolecular carbohydrate biopolymers which potentially express biological activity [8]. Lactobacillus bacteria can both secrete EPS to the environment and synthesize its cell wall-bound type. EPS, which is the main component of biofilm, facilitates adhesion of bacteria to intestinal epithelium, enables bacterial cells to survive in bile salts [9] and prevents infection of bacteria by the bacteriophage [10]. Genes encoding enzymes and proteins required for EPS synthesis have been discovered and described [11]. However, involvement of EPS in an interaction between Lactobacillus bacteria and MALT system is not fully understood. It is not known either whether a receptor for EPS exists.

In our previous study we have shown immunomodulating properties of crude EPS isolated form Lactobacillus rhamnosus KL37C [12]. We have compared the effect of EPS on production of inflammatory mediators by mouse peritoneal macrophages with the effect of lipopolysaccharide (LPS). EPS could effectively stimulate production of cytokines by macrophages, especially tumor necrosis factor  (TNF-), interleukin 6 (IL-6) and IL-12. However, its stimulatory potential was significantly lower than that of LPS. Furthermore, our data demonstrated that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by a reduced production of TNF- after restimulation with either LPS or EPS (‘cross-tolerance’). Our results also indicated that inhibitors of both ERK and p38 MAPKs inhibit production of TNF- induced by LPS or EPS. Elsewhere, we have shown that Lactobacillus rhamnosus exopolysaccharide ameliorates arthritis induced by a systemic injection of collagen and lipopolysaccharide in DBA/1 mice [13]. However, further studies with highly purified EPS are necessary to find out whether EPS can be clinically useful as an immunomodulatory agent.

In the present study we have focused on analyzing in vitro immunoregulatory properties of a highly purified exopolysaccharide from L. rhamnosus KL37C (EPS₃₇). The immunostimulatory potential of EPS₃₇ was compared with that of lipoteichoic acid (LTA) isolated from the same bacteria and with LPS from Escherichia coli. We have examined the effect of EPS on some selected biological functions of major inflammatory cells, namely, neutrophils, macrophages and dendritic cells.

Material and methods

Mice



Inbred CBA/J mice and Balb/c mice (8-12 weeks of age, 18-22 g) were maintained in the Animal Breeding Unit, Chair of Immunology, Jagiellonian University Medical College, Krakow. All mice were housed in the laboratory room with water and standard diet ad libitum. The authors were granted permission by the Local Ethics Committee to use mice in this study.



EPS₃₇ isolation



Exopolysaccharide was obtained from L. rhamnosus KL37C strain as described before [14]. The strain was isolated from feces of human newborns and then stored at –70°C in MRS broth supplemented with 10% glycerol. Bacteria were cultivated in supplemented MRS liquid broth (Oxoid, UK) under anaerobic conditions at 37°C for 48 h. Cells were harvested by centrifugation at 7300 γ (4°C, 30 min) and washed twice with PBS. Freeze-dried bacterial mass was extracted with 10% TCA (25°C, 2 h) and then centrifuged at 14 500 γ for 20 min. The pellet was discarded and EPS₃₇ from the supernatant was precipitated with 5 volumes of cold 96% ethanol (4°C, 16 h) and collected by centrifugation at 23 500 γ (4°C, 50 min). The pellet was suspended in water, dialyzed for 48 h against water and then lyophilized.

EPS₃₇ purification



Freeze-dried preparation of crude EPS₃₇ was dissolved in buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2) and treated with DNase and RNase (37°C, 6 h) and with protease from Streptomyces griseus (37°C, 16 h) (both Sigma-Aldrich, Germany), then dialyzed against water at 4°C for 24 h. Resuspended polysaccharide was purified by ion-exchange chromatography on 1.6 × 20 cm DEAE-Sephadex A-25 column (Pharmacia Fine Chemicals, Sweden). EPS₃₇ was eluted with 20 mM Tris buffer pH 8.2. Fractions containing EPS₃₇ were pooled, desalted by dialysis against water at 4°C for 24 h and lyophilized.



LTA isolation



Defrosted aliquot of bacteria was mixed with equal volume of n-butanol (Merck, Germany) under stirring for 30 min at RT. After centrifugation at 13 000 γ for 20 min, the aquatic phase was collected and dialyzed against distilled water, lyophilized, resuspended with chromatography start buffer (15% n-propanol in 0.1 M ammonium acetate, pH 4.7) and centrifuged at 45 000 γ for 15 min. The supernatant was subjected to hydrophobic interaction chromatography on 1.6 × 40 cm octyl-Sepharose CL-4B column (Pharmacia Fine Chemicals, Sweden). Unbound material was washed out with start buffer. Afterwards, bound materials were eluted with 35% n-propanol in 0.1 M ammonium acetate buffer (pH 4.7). Column fractions containing LTA were pooled and dialyzed against distilled water.



Macrophages



Peritoneal mouse (CBA/J) macrophages (Mθ) were induced by an intraperitoneal injection of 2.0 ml of thioglycolate (Sigma, USA). Cells were collected 72 h later by washing out the peritoneal cavity with 5 ml of DPBS containing 5 U heparin/ml (Polfa, Poland). Cells were centrifuged and red blood cells were lysed. Osmolarity was restored by addition of PBS. At least three mice were used as donors of peritoneal macrophages for each experiment.



Dendritic cells



Bone marrow derived dendritic cells (DC) were cultured from Balb/c mice. Briefly, bone marrow cells were flushed out from femurs and grown at starting density of 2 × 106 cells/ml of IMDM (Cytogen, Germany). The medium was supplemented with HEPES (24 g/l), 50 nM of 2-mercaptoethanol (both Sigma-Aldrich, Germany), 10% FBS (PAA, USA), gentamicin (50 mg/ml, Krka, Slovenia) and 3 ng/ml GM-CSF (Invitrogen, USA). After 3 days, the medium containing most of non-adherent cells, was removed and replaced with the fresh one. None adherent DCs, released spontaneously from proliferating cell clusters, were recovered at 7th day of culture. Prepared cells were suspended in PBS containing 0.05% FBS and 2 mM EDTA and purified with Magnetic Advanced Cell Sorting system (MACS, Miltenyi Biotec, Germany). Firstly, cells were incubated on ice for 40 min with anti-CD11c antibodies. Then, cells were washed in PBS and passed through a column with iron wool placed in the magnetic field. First collected fractions of cells were CD11c negative. After removing the column from the magnetic field, CD11c-positive cells were collected. Thus, purified DC were suspended in the culture medium IMDM and used for further experiments.



Reactive oxygen species generation: luminol-dependent and lucigenine-dependent chemiluminescence assay



The effect of EPS₃₇ on induction production of reactive oxygen species (ROS) by neutrophils was evaluated in vitro using luminol-dependent chemiluminescence or lucigenine-dependent chemiluminescence. Chemiluminescence was counted at 37°C in temperature-stabilized luminometer Lucy 1 (Anthos, Austria). Briefly, 18-hour peritoneal cells induced by thioglycolate (5 × 105/well), were preincubated with tested agents in Hank’s balanced salt solution (60 min at 37°C in an atmosphere of 5% CO2) on a 96-well flat-bottom black plate (Nunc, Denmark). Then, the cells were mixed with luminol (0.8 mg/ml) or lucigenine (0.25 mM) in 1 : 1 volume ratio (both Sigma-Aldrich, Germany) and incubated at 37°C for another 30 min. After incubation, the cells were quickly stimulated with yeast zymosan (200 µg/ml, Sigma-Aldrich, Germany) and photon emission over 75 min with 3-min intervals was measured. Results are expressed as relative light units (RLU) where photons were counted every 5 seconds.



HOCl-specific UV absorption spectra measurement



HOCl concentration was determined before each reaction by UV absorption spectra (l = 200 to 400 nm) measured at 292 nm using extinction coefficient factor 350 M–1cm–1. HOCl and EPS₃₇ were mixed in concentration 1 mM HOCl and 100 µg/ml EPS₃₇ and incubated for 5 min in RT. Each reaction of HOCl with EPS₃₇ was monitored by UV absorption spectra (l = 200 to 400 nm) to estimate the influence of EPS₃₇ on UV spectra of HOCl.



Macrophage culture and treatment



Mθ were cultured in 24-well flat-bottom cell culture plates (5 × 105/well) IMDM (Cytogen, Germany) supplemented with 5% FBS, and gentamicin at 37°C in atmosphere of 5% CO2. After 1 h, non-adherent cells were removed, whereas the adherent cells were stimulated with indicated concentrations of EPS₃₇, LTA and LPS (E. coli 001:B4; Sigma-Aldrich, Germany) After 24 h, culture supernatants were collected and frozen at –80°C until used. All groups were investigated in duplicates.



Dendritic cell culture and treatment



Dendritic cell prepared as described above, were cultured on 96-well plates in IMDM supplemented with 5% FBS, HEPES and gentamicin at 37°C in atmosphere of 5% CO2. Cells were stimulated with selected concentrations of examined bacterial structures, and after 24 h supernatants were collected and level of cytokines IL-6, IL-10 and IL-12p40 was estimated.



Cytokines determination



The concentration of cytokines in supernatants was determined using the ELISA method with some modifications, as described before [12]. 96-well plates (Costar, USA) were coated with antibody against the selected cytokine (TNF-α, IL-2, IL-6 and IL-12p40 – Bioscience, USA) and incubated for 24 h at 4°C. After blocking the plate with a solution of 3% skimmed milk (2 h), tested samples and standards for each of the cytokines were applied. After further 24 h of incubation at 4°C, biotinylated antibodies against the selected cytokine were added for 1 h. The ELISA was developed using horseradish peroxidase conjugated with streptavidin (Vector, USA) followed by o-phenylenediamine and H₂O2 (both Sigma-Aldrich, Germany). After 30 min, reaction was stopped by addition of 3M H2SO4. The optical density of each sample was measured at 492 nm in microplate reader PowerWavec (Bio-Tek Instruments, USA). 0.05% Tween-20 in phosphate buffer was used as a washing solution. Exception to the above procedure was the measurement of IL-10, which was performed according to the manufacturer's instructions (BD Biosciences, USA).



Phagocytosis



To evaluate the effect of EPS on phagocytosis of dextran by macrophages, cells were resuspended in 1% FBS in PBS and then incubated with EPS₃₇ or LPS and RITC-dextran (Sigma-Aldrich, Germany) for 30 min at 37°C. The reaction was stopped by addition of cold 1% FBS in PBS. Cells were washed three times using the same buffer, and then macrophages were prepared for analysis by flow cytometry. Cells were stained with allophycocyanin-conjugated anti-mouse F4/80 monoclonal antibody (eBioscience, USA). Nonspecific binding was blocked using monoclonal 2.4G2 antibody (BD Pharmingen, USA). Cells were incubated with antibodies for 40 min, at 4°C in the dark, then washed twice with 1% FBS in PBS and suspended for analysis in the same buffer. To exclude dead cells, propidium iodide was added just before analysis (Sigma-Aldrich, Germany). Cells were analyzed using FACSCalibur flow cytometer (Beckton Dickinson, USA) and CellQuest Pro software (Beckton Dickinson, USA).



PGE2 determination



PGE₂ concentration in supernatants was determined by Prostaglandin E₂ Monoclonal EIA kit (AssayDesigns, USA) according to the manufacturer’s instructions.



Western blot analysis: evaluation of COX-2 expression



The level of induction of COX-2 protein was determined by the Western blot technique. Mθ were lysed in lysis buffer containing mixture of protease inhibitors (Sigma-Aldrich, Germany). The total protein concentration in resulting lysates was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich, Germany). Samples containing equal amounts of protein (14 mg) were suspended in loading buffer (reagents Sigma, USA), in a 2 : 1 ratio and denatured for 4 min at 100°C. Samples were applied to polyacrylamide gel with 10% SDS and were separated electrophoretically in the Laemmli system using Mighty Small II apparatus (Amersham Biosciences, USA) [15]. Separated proteins were transferred to the nitrocellulose membrane (Bio-Rad, USA) using Hoefer TE22 transfer equipment (Amersham Biosciences, USA). After overnight incubation with protein blocking solution at 4°C (3% skimmed milk), membranes were incubated for 2 h at RT with rabbit polyclonal anti-COX-2 antibodies (Cayman, USA) and mouse monoclonal anti-b-actin antibodies (Sigma-Aldrich, Germany). Then, membranes were incubated for 2 h with secondary antibodies, anti-rabbit IgG (Sigma-Aldrich, Germany) and goat IgG conjugated with phosphatase alkaline (Sigma-Aldrich, Germany) at RT. The bands were detected with phosphatase alkaline substrate BCIP/NBT (Sigma, USA). Protein bands were scanned and analyzed using freeware Scion Image for Windows (Scion, USA). Results are presented as a ratio of optical density of protein to b-actin, which is constitutively expressed in cells.



Mice immunization with OVA



Mice were immunized intraperitoneally with OVA (200 µg/mouse) in the presence of LPS (1 µg/mouse; both Sigma-Aldrich, Germany), or/and of EPS₃₇ (50 µg/mouse) or/and of LTA (50 µg/mouse) twice, on days 0 and 14. Seven days after the second immunization mice were bled and serum was processed further.



Measurement of serum OVA-specific antibody titers



The level of antibodies specific to OVA in mouse serum was determined using the ELISA test as described before [13]. Briefly, plates were coated with OVA (5 µg/ml) overnight. Serial dilutions of mouse serum in PBS were applied to antigen-coated wells for 1 h at RT. Levels of IgGs were determined using biotin-conjugated antibodies against IgG (Sigma-Aldrich, Germany), IgG1 (MP Biomedicals, USA), or IgG2a (Southern Biotech, USA). The amount of antibody was expressed in arbitrary ELISA units calculated from anti-OVA titer: 1 Unit = 1/100 titers of antibodies specific to OVA.



Statistical analysis



Statistical significance between two groups were tested using Student's t test. For more groups, comparison one-way ANOVA and Tukey post hoc tests were used. Results are expressed as a mean ± SEM values. A p value < 0.05 was considered statistically significant. Analysis was performed using GraphPad Prism ver. 5.01 program (GraphPad Software, USA).

Results

Influence of EPS₃₇ on the production of ROS by neutrophils



To answer the question whether exopolysaccharides released from bacteria may affect ROS production by inflammatory cells, non-stimulated and zymosan-stimulated neutrophils were incubated with EPS₃₇. The effect was compared with that of LTA and LPS (Fig. 1). In order to determine the effect of tested substances on an intracellular and extracellular level of ROS, luminol-dependent (Fig. 1A) or lucigenine-dependent (Fig. 1B) chemiluminescence was measured, respectively. The cells generated and secreted significant amounts of ROS in response to zymosan. In contrast, EPS₃₇, LTA and LPS did not induce ROS production when added alone to non-stimulated neutrophils. Interestingly, EPS₃₇ and LTA added to neutrophils before stimulation with zymosan inhibited formation of ROS by about 40%. The effect was observed only in the case of luminol-dependent chemiluminescence (Fig. 1A). What is more, incubation of EPS₃₇ with HOCl resulted in scavenging of HOCl as indicated from alteration of the HOCl-specific UV absorption curve (Fig. 1C).



Effect of EPS₃₇ and LTA on release of cytokines from macrophages



In order to determine immunostimulatory properties of pure EPS₃₇ and LTA, peritoneal macrophages were cultured for 24 h with selected concentrations of examined factors. The results clearly indicate strong stimulatory properties of LTA. LTA at a concentration of 30 µg/ml and 100 µg/ml LTA significantly induced the production of all tested cytokines (TNF-α, IL-6, IL-12p40 and IL-10), while EPS₃₇ had no effect, as it is shown in Fig. 2.



Effect of EPS₃₇ on phagocytic activity of macrophages



To answer the question whether EPS₃₇ can affect the phagocytic activity of macrophages, the cells were incubated with EPS₃₇ and then with RITC-Dextran. The effect was compared with that of LPS. As shown in Fig. 3, neither EPS₃₇ nor LPS altered phagocytosis of dextran molecules by macrophages.



Effect of EPS₃₇ on COX-2 expression and PGE2 synthesis by macrophages



To examine the relative potency of EPS₃₇, LTA and LPS in induction of eicosanoids production by macrophages, the cells were cultured with the tested agents. The expression of COX-2 was estimated in the cell cytosol and the level of PGE2 was determined in supernatants, as described in Material and methods. Non-stimulated macrophages neither expressed COX-2 protein nor released PGE2, the major eicosanoid of macrophages, as shown in Fig. 4. LTA and LPS-stimulated macrophages expressed high levels of COX-2 and produced a significant amount of PGE2. In contrast, the pure EPS₃₇ did not induce expression of COX-2 protein and the level of secreted PGE2 was comparable to that observed in non-stimulated macrophages.



Effect of EPS₃₇ and LTA on release of cytokines from dendritic cells



Dendritic cells were stimulated with selected concentrations of EPS₃₇ to determine whether EPS₃₇ affects cytokines production by antigen presentation cells. The effect was compared with that of LTA and LPS (Fig. 5). LPS was the strongest inducer of all tested cytokines. There was also a massive release of IL-12p40 in response to LTA, while the influence of LTA on IL-6 and IL-10 production was negligible. In contrast to LPS and LTA, EPS₃₇ did not stimulate the production of any of the tested cytokines.



Effect of EPS₃₇ on humoral immune response in mice



Previously we have shown that pure EPS inhibited the in vivo production of total IgG specific to OVA. In the present study we have extended the observation to other IgG isotypes to examine the hypothesis considering polarization of the immune response by bacterial products. The results shown in Fig. 6 clearly indicate that EPS₃₇ is able to inhibit humoral response to OVA in mice. EPS₃₇ injected simultaneously with the antigen decreased markedly the production of anti-OVA IgG (65% of inhibition), IgG1 (90%) and IgG2a (82%) antibodies. Interestingly, EPS₃₇ given together with LPS diminished its adjuvant properties by inhibiting OVA-specific IgG production, especially anti-OVA IgG2a (87%) (Fig. 6C).

Discussion

Lactobacilli represent bacteria of human microbiome and are most commonly used for probiotic therapy [16, 17]. Our previous study indicates that immunoregulatory properties of lactobacilli are strain specific [18]. In addition, other authors have shown that this effect may be achieved using bacterial components such as LTA or peptidoglycan [19, 20]. Much less is known about the immunoregulatory effect of EPS. However, we have previously shown that crude EPS from Lactobacillus rhamnosus KL37C exerts an immunostimulatory effect on mouse macrophages in vitro [12]. It is of great interest to explain a contribution of EPS in bacteria – immune system cross-talk, especially in bacteria which synthesize EPS as a major component of biofilm matrix [21].

In this study we have investigated the effect of pure EPS₃₇ on the selected functions of major cells of acute inflammation, namely: neutrophils, macrophages and dendritic cells. We have found that EPS₃₇, and LTA significantly decrease the level of ROS in mouse neutrophils stimulated by zymosan. This can indicate an antioxidant capacity of pure EPS₃₇ similar to that of LTA. Antioxidant properties of EPS have been also described by Kodali et al. They have demonstrated that EPS from Bacillus coagulans RK-02 has antioxidant properties and may act as a scavenger of free radicals. These properties were comparable with those of standard antioxidants such as vitamin C and E [22]. Further, we investigated the influence of EPS₃₇ on the phagocytic function of macrophages. The ability of cells to phagocyte dextran molecules in the presence of pure EPS₃₇ was not inhibited. In addition, pure EPS₃₇, in contrast to the crude form described before [12], did not stimulate murine peritoneal macrophages to produce cytokines. Furthermore, there was no effect of EPS₃₇ on PGE2 synthesis and COX-2 protein expression in macrophages. This suggests that

pure EPS₃₇ is not a key component of Lactobacillus rhamnosus responsible for its immunostimulating properties. Therefore, we compared the effect of EPS₃₇ with LTA isolated from the same bacteria. LTA showed a strong stimulatory effect. Macrophages stimulated with high concentrations of LTA produced a significant level of both pro-and anti-inflammatory cytokines. LTA also stimulated macrophages to synthesize PGE2 and COX-2 protein. These data are consistent with other studies where LTA, derived from Gram (+) bacteria, exerts also immunostimulatory effects [23-25]. LTA can strongly induce synthesis of PGE2 and COX-2 not only in immune cells. For example, PGE2 secretion was observed in human lung epithelial cells and neurons of the cerebral cortex in rats after stimulation with LTA [26, 27]. As immunostimulatory effects depend on both, bacterial cell-wall components and on target cells, we have also examined effects of LTA and EPS₃₇ on cytokine production by dendritic cells. We have observed a significant production of IL-12p40 and small amounts of IL-6 in 24-hour DC cultures stimulated with tested agents. There are very few data showing effects of probiotic bacteria components on DC functions and they mainly concern whole bacteria. Weiss et al. have shown that probiotic bacteria can interact with DC in two ways. Such strains as Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus casei and Lactobacillus plantarum induced murine myeloid DC to synthesize IL-12 and TNF-α. They observed the opposite effect in the case of use of Bifidobacterium bacteria [28]. Evrard et al. showed that L. rhamnosus Lcr35 increased synthesis of the Th1/Th17 cytokine profile, but had no significant effect on the level of IL-10 [29]. Previously, we have demonstrated the impact of EPS₃₇ on humoral immune response in mice. We have shown that EPS₃₇ inhibited synthesis of IgG specific to OVA. The effect of EPS was opposite to the effect of LPS. LPS was a strong adjuvant and significantly enhanced OVA-specific response [30]. In this study, we expand our investigations to other OVA-specific IgG isotypes to examine the hypothesis considering polarization of the immune response by the bacterial product. We have found that EPS₃₇ diminished the serum level of OVA-specific IgG1 and IgG2a antibodies to the same degree. Moreover, EPS₃₇ also inhibited the adjuvant effect of LPS in the same way. Therefore, our present results did not show polarizing properties of EPS₃₇, while confirmed its suppressor effect on antigen-specific humoral response in mice.

In conclusion, the present study extends our knowledge concerning biological properties of bacterial EPSs. However, some fundamental questions are still unanswered. We can suggest that pure EPS, at least EPS from L. rhamnosus KL37C, shows a very weak, immunostimulatory action if compared with that of LTA (major component of lactobacilli cell wall). One may suggest that it is the effect of lack of EPS-specific PRR receptor (not yet found). On the other hand, EPS is the main component of biofilm matrix. It may suggest its protecting role against immune attack on bacteria hidden in biofilm. However, further studies are necessary to investigate the role of EPS in the interaction between bacteria and the immune system cells. Proper understanding of these interactions may open new strategies for probiotic therapies. Importantly, due to a large heterogeneity of bacterial EPSs, biological properties of each individual bacterial strain must be taken into account. Indeed, we have shown that potential of EPS is strain-dependent. EPSs isolated from three different strains of Lactobacillus (L. reuteri, L. johnsonii and L. animalis/murinus) stimulated the murine macrophage to produce both pro- and anti-inflammatory cytokines in a different manner [31].



The authors declare no conflict of interests.



This study was support by the Ministry of Science and Higher Education (Poland) grants No. NN401 048838 and No. NN401 042438.

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