Effects of bifidobacterial cytoplasm, cell wall and exopolysaccharide on mouse lymphocyte proliferation and cytokine production

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International Dairy Journal 16 (2006) 70–80 www.elsevier.com/locate/idairyj

Effects of bifidobacterial cytoplasm, cell wall and exopolysaccharide on mouse lymphocyte proliferation and cytokine production T. Amrouchea,b,c, Y. Boutinb,d, G. Prioulta, I. Flissa,b, a

Dairy Research Center STELA, Pavillon Paul-Comtois, Universite´ Laval, Que´., Canada G1K 7P4 Institute of Nutraceutical and Functional Foods INAF, Universite´ Laval, Que´., Canada G1K 7P4 c Department of Food Technology, Faculty of Agronomy and Biological Sciences, University of Tizi Wezzu, Algeria d TransBiotech, CEGEP Le´vis Lauzon, Le´vis (Qc), Canada G6V 9V6 b

Received 20 August 2004; accepted 12 January 2005

Abstract Probiotic bifidobacteria have been reported to stimulate the immune system and thus offer the possibility of improving host immune defense against pathogens. The aim of this work was to study the effects of bifidobacterial cytoplasm, cell wall, and exopolysaccharide (EPS) on splenocyte proliferation and production of -IFN-g and IL-10. Three bifidobacteria, RBL64, RBL81, and RBL82 isolated from newborn infant faeces were used in our study. Commercial strain Bifidobacterium lactis Bb12 was used as a positive control. Among different cell extracts, the cell wall components showed the most profound effects on cell proliferation and cytokine production. EPS neither stimulated lymphocyte proliferation nor induced cytokine secretion. B. lactis Bb12 exhibited a significant immunostimulating effect (stimulation index ¼ 16) compared with other bifidobacteria studied. More -IFN-g (44 ng mL1) was produced in response to cell wall and about 0.8 ng mL1 of IL-10 was detected in the cell culture supernatant. The results demonstrate that bifidobacterial extracts, mainly the cell walls, stimulate the proliferation of lymphocytes and suggest that such extracts could be used in controlling certain immune pathologies. r 2005 Elsevier Ltd. All rights reserved. Keywords: Bifidobacteria; Cell wall; Cytokines; Cell proliferation; Cytoplasm; Exopolysaccharides

1. Introduction Recent studies on human and animal infections suggest that the natural intestinal microflora plays a major role in resisting both viral infections and colonization of the gastrointestinal tract by pathogenic bacteria (Sherwood & Gorbach, 2000; Ibnou-Zekri, Blum, Schiffrin, & von der Weid, 2003; Asahara et al., 2004). It has been shown that protection by microflora can be improved by ingesting probiotics, which are micro-organisms that favourably influence host physiology by modifying the intestinal flora (Kirjavainen, Corresponding author. Tel.: +1 418 656 2131x6825; fax: +1 418 656 3353. E-mail address: ismail.fl[email protected] (I. Fliss).

0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.01.008

Arvola, Salminen, & Isolauri, 2002; Hattori et al., 2003). Bifidobacteria, discovered in 1899 by Tessier, are a major component of the gastrointestinal tract microflora (Mitsuoka, 1990) and a constituent of the gut mucosal barrier (Mullie et al., 2002). Like many other bacteria, bifidobacteria are known to produce capsular and extracellular polysaccharides (Roberts et al., 1995), which have a role in cell recognition, adhesion to surfaces, and formation of biofilms to facilitate colonization of various ecosystems. They also have a protective function in the natural environment, for example against phagocytosis, phages, and osmotic stress (Withfield & Valvano, 1993; Looijesteijn, Trapet, De vries, Abee, & Hugenholtz, 2001). Probiotic bifidobacteria, especially Bifidobacterium longum, B. infantis and B. breve, have been shown to

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stimulate the immune system and thus may increase the capacity of the host to fight against gastrointestinal infections (Mullie et al., 2004) and help reduce allergic inflammation (Von der Weid, Ibnou-Zekri, & Pfeifer, 2002). Some evidence suggests that the effects of probiotic bacteria on the immune system may be utilized to treat immunologically exacerbated pathologies in humans (Matsuzaki & Chin, 2000; Perdigon, Fuller, & Raya, 2001; Cross, Stevenson, & Gill, 2001; Prioult, Fliss, & Pecquet, 2003). Probiotic modulation of humoral, cellular and non-specific immunity has been studied in disease models (Yasui, Shida, Matsuzaki, & Yokokura, 1999; Qiao et al., 2002; Hart et al., 2004), and new species and strains of probiotic bacteria are continually being identified and evaluated for their capacity to modulate immune functions. The mode of action of bifidobacteria and lactic acid bacteria (LAB) in the gastrointestinal tract appears to be non-specific, increasing immune responsiveness to a wide variety of antigens (De Roos & Katan, 2000). Ouwehand, Kirjavainen, Gronlund, Isolauri, and Salminen (1999) reported that probiotics exert effects through interactions between lymphoid tissue and intact micro-organisms, fragments thereof or metabolites produced in situ. Bacterial cell wall breakdown products may play an important role in a number of homeostatic mechanisms as well as non-specific immunity (Erickson & Hubbard, 2000; Kankaanpa, Sutasb, Salminena, & Isolaurib, 2003). It has also been reported that probiotic bacteria can preferentially promote Th1-type responses interferon gamma (IFN-g) secretion) (He et al., 2002; Morita et al., 2002). Regular ingestion of several Lactobacillus species has been shown to enhance the capacity of murine splenic leukocytes to produce IFN-g following mitogenic stimulation, while IL-4 or IL-5 production is unaffected (Gill, 1998; Gill, Rutherfurd, Prasad, & Gopal, 2000). Preliminary results reported for lactic acid bacteria suggest that EPS may be useful not only for their rheological properties but also for health-promoting properties, which may include anti-tumor and immunostimulatory actions (Ricciardi & Clementi, 2000; Chabot et al., 2001). Ruas-Madiedo, Hugenholtz, and Zoon (2002) reported that EPS from LAB may influence the immune system by enhancing lymphocyte proliferation, macrophage activation and cytokine production. At present, there is no clear understanding of the molecular or cellular basis for immunostimulation by bifidobacteria. To address the lack of data that convincingly show immunomodulatory effects of bifidobacteria, we have attempted to elucidate a mechanism by studying the action of four bifidobacteria. Three isolates of Bifidobacterium spp. from newborn infant faeces were compared with the commercial strain Bifidobacterium lactis Bb12. The abilities of cytoplasm, cell wall and EPS from bifidobacteria to stimulate mouse splenocyte

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proliferation and production of cytokines IFN-g and interleukin 10 (IL-10) were measured.

2. Material and methods 2.1. Bacterial strains and culture conditions Isolates from newborn infant faeces were identified as bifidobacteria by fructose-6-phosphate phosphoketolase assay and by the PCR method (Toure´, Kheadr, Lacroix, Moroni, & Fliss, 2003). Isolates RBL64, RBL81, and RBL82 were selected on their basis of exopolysaccharide production. B. lactis Bb12 (Chr. Hansen, Moersolm, Denmark) was used as positive control. Bacteria were first cultured for 18 h at 37 1C in De Man–Rogosa–Sharpe broth (MRS) (Rosell Institute Inc., Montreal, Canada) supplemented with 1% Tween 80 and 0.05% cysteine-HCl (v/v) to lower the oxidation–reduction potential of the medium (Screekumar & Hosono, 1998). After incubation, 100 mL of this pre-culture was transferred into 900 mL MRS broth and incubated under anaerobic conditions for 24 h at 37 1C. All incubations were stationary. 2.2. EPS isolation, purification and fractionation EPS was isolated and purified by the method of Cerning et al. (1994). After incubation, cultures were heated at 100 1C for 15 min to inactivate potential EPS hydrolases and improve detachment of EPS from the microbial cell walls (Tuinier et al., 2001). The cultures were then centrifuged at 16,000 g for 20 min at 4 1C to separate bacteria and cell debris. EPS were precipitated with three volumes of chilled 95% ethanol and were made to stand overnight at 4 1C. The precipitate, collected by centrifugation at 13,000 g for 20 min at 4 1C, was re-suspended in 25 mL of deionized water and then freeze-dried. The resulting powder was dissolved in 15 mL of 10% trichloroacetic acid (TCA) and the solution was centrifuged at 13,000 g for 15 min at 4 1C to remove proteins. The protein content of the supernatant was determined by the Lowry method using bovine serum albumin (BSA) as standard. As required, TCA extraction was repeated to remove the remaining proteins. EPS solutions were dialysed (1000 Da-cut off) at 4 1C against deionized water. After dialysis, EPS solutions were fractionated by successive filtrations through Centricon (Millipore) 5000 and 100,000 Da-cut off filters. Three fractions were obtained: F1:4100,000 Da; F2: 5000–100,000 Da; and F3:o5000 Da. The total sugar content of the EPS suspensions was estimated by the phenol/sulphuric method (Dubois, Gilles, Hamilton, & Roberts, 1956). All samples were aliquoted and kept at 20 1C until use.

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EPS solutions were passed through a 0.22 mm filter prior to addition to cell cultures. 2.3. EPS molecular mass determination EPS molecular mass was determined by gel permeation chromatography (GPC) on a Waters HPLC system (Milford, MA, USA). Two columns, TOSOHAS G 4000 PWXL (30 cm  7:8 mm) and BECKMAN TSK 4000 SW (60 cm  7:5 mm) were connected in series. The mobile phase was 0.1 M ammonium acetate (pH 7.2) at a flow rate of 0.5 mL min1. Pullulan standards (Shodex Standards, Japan) of 100,000 Da and 1.6  106 Da and purified EPS were adjusted to 1 mg L1 and 50 mL volumes were injected. A light-scattering detector (SEDX Scientific Products & Equipment, Concord, Canada) was used at 45 1C to detect the presence of exopolysaccharides resulting in variation of scattered light intensity.

a concentration of 5  105 cells mL1. Various concentrations of bifidobacterial extracts were added alone or with phytohemagglutinin (PHA) (10 mg mL1) to the cells. They were incubated for 48 h in a humidified 5% CO2 atmosphere at 37 1C.

2.5.1. Cell proliferation After incubation, the plates were centrifuged (300 g for 10 min at 41C) and the supernatants were kept for cytokine analysis while the cells were used to measure proliferation. Cell proliferation was measured by adding 20 mL of 5-bromo-20 -deoxyuridine (BrdU) for the last 24 h of culture and determining BrdU incorporation using a cell proliferation ELISA kit (Roche Diagnostics Gmbh, Mannheim, Germany). Proliferation responses were expressed as a stimulation index calculated as the ratio of mean OD (450 nm) obtained for stimulated culture to the mean OD (450 nm) obtained for unstimulated control cell cultures.

2.4. Preparation of cytoplasm and cell wall extracts of bifidobacteria Fourteen-hour MRS broth cultures were centrifuged at 5000 g for 10 min at 4 1C and washed with 0.01 M potassium phosphate buffer (PBS) at pH 7.0. Pelleted cells were disrupted manually for 30 min at 4 1C with three or four volumes of alumina micro-beads (Sigma Co., Saint Louis, MO, USA) in a mortar laid in ice. PBS (1 mL) was added to the mixture to facilitate extraction. The alumina beads were separated from the suspension by centrifugation at 1000 g for 15 min at 4 1C. The homogenate was re-centrifuged at 30,000 g for 30 min at 4 1C to pellet cell walls and obtain cytoplasmic extract in the supernatant fraction. Cell wall material was resuspended in 2 mL of bi-distilled water. The protein content of the cytoplasmic and cell wall extracts were determined by the Lowry method. The extracts were diluted in RPMI 1640 medium to a final protein concentration of 2 mg mL1 and passed through a 0.22 mm-filter.

2.5.2. Measurement of IFN-g and IL-10 IFN-g and IL-10, in supernatants of splenocyte cultures, were determined by cytokine-specific sandwich ELISAs. Antibodies R 4-6A2 and biotinylated XMG1.2 were used for IFN-g while MM-011 and biotinylated MM-16E3 were used for IL-10 (all from Endogen, Woburn, MA, USA). Coating antibodies were diluted to 2 mg mL1 and conjugate antibodies were diluted to 0.5 mg mL1. Streptavidin-horseradish peroxydase (Endogen, Woburn, MA, USA) and 3, 3, 5, 5-tetramethylbenzidine (KPL, Maryland, USA) were used in these assays. The reaction was stopped with 1 M H2SO4 and optical density at 450 nm was determined using an ELISA plate reader (Molecular Device, Sunnyvale, USA). Cytokine concentrations were derived from linear dose-response standard curves obtained using dilutions of recombinant mouse IFN-g (BD Pharmigen, San Diego, CA, USA) or recombinant mouse IL-10 (Endogen). Fresh cell-free complete RPMI 1640 was used as negative control.

2.5. Stimulation of mouse splenocytes by bifidobacterial extracts 2.6. Statistical analysis Spleens were removed aseptically from euthanized Balb/C mice and single cell suspensions were prepared by mechanically disrupting the tissues through a cell strainer into RPMI 1640 medium (Gibco BRL Inc., Paisley, Scotland). Red blood cells were removed from the cell suspension by lysis with 0.87% NH4Cl solution. Cells were then washed, suspended in RPMI 1640 complete medium (with 10% fetal calf serum (FSC), 0.1 mL mL1 50 mM mercaptoethanol, 100 U mL1 penicillin, and 100 mg mL1 streptomycin, all from Gibco). Cells were transferred to 96-well round bottom plates at

Splenocytes proliferation assay and cytokines evaluation were carried out in triplicate for each bacterial extract, and the analyses were done in duplicate. Statistical analyses were carried out with STATGRAPHICS plus 4.1 (Manugistics Inc., Rockville, MD, USA). Significant differences between treatments were tested by analysis of variance (ANOVA) followed by a comparison between treatments performed by Fisher’s least significant difference (LSD) method, with a level of significance of Po0:05:

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3. Results and discussion

73

100

% Total EPS

76.1

50 22.5 2.1 0 F1

(A)

F2

F3

EPS fraction 100 79.3

% Total EPS

Certain studies suggest that EPS produced by certain lactic acid bacteria may have immunostimulatory (Hosono et al., 1997), and antitumoral (Oda, Hasegawa, Komatsu, Kambe, & Tsuchiya, 1983; Ebina, Ogata, & Murata, 1995) activities. Induction of cytokine (IFN-g and IL-1a) production has been reported (Kitazawa, Itoh, Tomioka, Mizugaki, & Yamaguchi, 1996) and phosphate groups in these polysaccharides are believed to play an important role in macrophage and lymphocyte activation (Kitazawa et al., 1998, 2000; NishimuraUemura et al., 2003). We therefore sought to evaluate the effect of crude and fractioned bifidobacterial EPS as well as cytoplasm and cell wall extracts on splenocytes proliferation and production of IFN-g and IL-10. Since similar results were obtained with all fractions of the three isolates RBL81, RBL82, and RBL64, we present herein only the comparison of RBL64 and B. lactis Bb12.

50 18.1 1.6 0 F1

3.1. EPS production by bifidobacteria (B)

F3

EPS fraction

100

% Total EPS

The average yields of crude (total) EPS obtained from 24-h cultures of isolates RBL81, RBL82, RBL64, and B. lactis Bb12 were, respectively, 0.53, 0.42, 0.32, and 0.33 mg mL1 of supernatant. Concentrations of EPS were in the same range as those obtained by Roberts et al. (1995) from B. longum BB-79 (0.47 g L1). EPS yield has been reported to vary widely depending on species and culture conditions. For example, Streptococcus thermophilus has been shown to produce 20–100 mg EPS L1 in fermented milk medium (Vaningelgem et al., 2004), while Lactobacillus rhamnosus RW 9595 yielded from 931 to 1275 mg L1 of purified EPS when grown on glucose or lactose at 32 or 37 1C (Van Calsteren, Pau-Roblot, Begin, & Roy, 2002). Lactococcus lactis subsp. cremoris SBT 0495 has been reported to produce 600 mg L1 in a lactose-containing medium (Higashimura, Mulder-Bosman, Reich, Iwasaki, & Robin, 2000). Bifidobacterial EPS were fractionated into F1 (4100,000 Da), F2 (5000–100,000 Da) or F3 (o 5000 Da) portions. Proportions of each fraction of the isolate are shown in Fig. 1. Fraction F1 of all isolates was found to have a higher sugar content (ranging from 63.5% to 79.3%) than fraction F2 (18.1–30.9%) or F3 (1.6–5.6%). Isolate RBL82 produced more EPS in the molecular mass range 4100,000 Da than did RBL64 or RBL81. The comparison of the EPS molecular mass profiles of isolate RBL82 and L. rhamnosus is shown in Fig. 2. With the aid of Fig. 3, which shows three peaks corresponding to the EPS of fractions F1, F2, and F3, it can be seen that most of the EPS produced by bifidobacteria appears to be of lower molecular mass.

F2

63.5 50 30.9 5.6 0 F1

(C)

F2

F3

EPS fraction

Fig. 1. EPS content of polysaccharide fractions from three bifidobacterial strains isolated from newborns faeces: (A) strain RBL81; (B) strain RBL82; and (C) strain RBL64. The fractions were separated by centricon based on their molecular mass: F14100,000 Da; F2: 5000–100,000 Da; F3:o5000 Da.

Fraction F1 appears to contain some of fraction F2, which may have been retained by the 105 Da cut-off filter or may be an artefact of fractionation. ToneShimokawa, Toida, and Kawashima (1996), who analysed cell wall polysaccharides of B. infantis Reuter ATCC 15697 by gel filtration chromatography using pullulan standards, reported that polysaccharides were affected by the ionic strength of the eluting solution and may interact with the column matrix. The mass range of F1 appears consistent with the findings of Roberts et al. (1995), who estimated by chromatography the average mass EPS from B. longum BB-79 to be greater than 200,000 Da.

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Fig. 2. Gel permeation chromatography analysis of crude bacterial exopolysaccharides: (A1) 1600  103 Da; (A2) 100  103 Da; (B) L. rhamnosus; and (C) strain RBL 82.

Fig. 3. Gel permeation chromatography analysis of fractioned EPS from bifidobacterial strain RBL82 using pullulan standard: (A1) 1600  103 Da; (A2) 100  103 Da; (D) F 1 4100; 000 Da; (E) F2: 5000–100,000 Da; and (F) F 3 o5000 Da:

3.2. Splenocyte proliferation induced by bifidobacterial extracts The crude EPS preparation had only a weak stimulating effect on cell proliferation (Fig. 4). Tzianabos, Wang, and Kasper (2003) reported that different bacterial capsular polysaccharides with molecular weights greater than 17 kDa stimulated CD4+ cell proliferation in vitro. Our results suggest that molecular weight does not likely influence any possible stimulatory effect of bifidobacterial EPS on the proliferation of mouse splenocytes. Several factors influencing the effects of EPS on lymphocyte proliferation have been reported. Kitazawa et al. (1998), who fractionated EPS from Lactobacillus delbrueckii ssp. bulgaricus 1073R-1 into neutral and acidic fractions, demonstrated that the acidic polysaccharide stimulated mitogenic responses of murine splenocytes and Peyer’s patches but not thymocytes. They also reported that de-phosphorylation of EPS reduced their mitogenic activity in lymphocytes. Obviously, polysaccharide structure may be modified during extraction and purification procedures, causing losses of their properties to stimulate the cell prolifera-

tion. Recent studies suggest that the cell proliferation stimulation induced by bacterial EPS are indeed structure-dependent and that the active structures are in turn charge dependent. Kalka-Moll et al. (2002) emphasized that bacterial polysaccharides with a zwitterionic charge spatial motif, such as capsular polysaccharides, elicit potent CD4 T-cell responses both in vivo and in vitro. Recently, Stingele et al. (2004) demonstrated that zwitterionic polysaccharides interact directly with T cells with rapid association/dissociation kinetics. The proliferative response of T cells depends on free amino (positively charged) and carboxyl or phosphate groups (negatively charged) that are part of the repeating unit structure. Chemical neutralization of either of the charged group results in loss of the ability of the polysaccharide to activate T cells (Tzianabos et al., 2000; Tzianabos, Wang, & Kasper, 2001). Using gas–liquid chromatography and GLC-mass spectrometry, Roberts et al. (1995) found that EPS from bifidobacteria appear to be composed of galactose and an unidentified hexose (possibly glucose) with a lactic acid substituent. Splenocyte proliferation stimulated by bifidobacterial cytoplasm and cell wall from strains RBL64 and

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Strain RBL64

16 Stimulation index

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mL-1)

Fig. 4. Effect of (A) cytoplasmic content, (B) cell wall, and (C) EPS extracts from the strains RBL64 and B. lactis Bb12 on splenocyte proliferation when the preparations were normalized on the basis of their protein content. Columns with different letters are significantly different.

B. lactis Bb12 can be clearly observed after 48 h of incubation. Fig. 4 shows that splenocyte proliferation is dose dependent and the SI is significantly increased at protein concentrations ranging from 20 to 40 mg mL1. For both strains, cell wall extract was a more efficient stimulator of splenocyte proliferation than the cytoplasm fraction. However, B. lactis Bb12 extracts were more effective than those from RBL64. Prioult et al. (2003) reported that B. lactis Bb12 (NCC 362) was more effective than Lactobacillus paracasei (NCC 2461) or Lactobacillus johnsonii (NCC 533) at inducing and maintaining oral tolerance to bovine b-lactoglobulin in mice. Other studies have reported that Gram-positive

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bacteria, which do not contain lipopolysaccharide (LPS) but carry surface teichoic acids, lipoteichoic acids and peptidoglycan, can stimulate immune cells. However, whole peptidoglycan is much less active than LPS on a dry weight basis (Weintraub, 2003; Moreillon & Majcherczyk, 2003). The mechanisms by which Grampositive bacteria stimulate cellular response thus require further study. The stimulation of cell proliferation by cytoplasm may be due to DNA and bioactive peptides. Indeed, Hemmi et al. (2000) reported that bacterial DNA has stimulatory effects on mammalian immune cells, which depend on the presence of unmethylated CpG (deoxycytidylate-phosphate-deoxyguanylate) dinucleotides in the bacterial DNA. They postulated that cellular response to CpG DNA is mediated by a Toll-like receptor (TLR9), which consists of phylogenetically conserved transmembrane proteins recognizing bacteriaderived ligands. They demonstrated that TLR9-deficient mice express a non-responsive phenotype to CpG-DNA, suggesting that murine TLR9 is a CpG-DNA receptor. Bacterial DNA and synthetic oligodeoxynucleotides expressing unmethylated CpG motifs stimulate the immune system, inducing maturation, differentiation, and proliferation of multiple immune cells, including B and T lymphocytes, NK cells, monocytes, macrophages, and dendritic cells (Roman et al., 1997; Sun, Zhang, Tough, & Sprent, 1998; Klinman, Currie, Gursel, & Verthelyi, 2004). Other sources of immunomodulators from bifidobacteria may include peptidases, which could generate bioactive peptides with mitogenic effects on sple´nocytes (Gobbetti, Corsetti, Smacchi, Zocchetti, & De Angelis, 1998; Samartsev, Astapovich, & Novik, 2000). Differences in the level of stimulation may arise from the proportions of immuno-active components in specific probiotics. While our results demonstrate that cell extracts, mainly cell wall extract, stimulate splenocyte proliferation and suggest that such extracts could be used to enhance host immune responses or in controlling certain immunopathologies. Pessi, Sutas, Saxelin, Kalliooinen, and Isolauri (1999) reported that extracts from probiotics such as L. rhamnosus GG, B. lactis, L. acidophilus, L. delbrueckii subsp. bulgaricus, and S. thermophilus suppress immune response in vitro. Dijkstra, Alber, and Keck (1997) have suggested that bacterial surface constituents, being readily accessible to detection, have been biologically selected by the immune system as indicators of bacterial presence and are thus potent inducers of host responses. Since peptidoglycan is the major constituent of Gram-positive bacterial cell wall and is located at the cell surface, this glycoprotein is a suitable target for such immune responses. Moreover, the basic architecture of peptidoglycan appears to be highly conserved among bacteria, with minor variations in exact chemical composition while an additional layer

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of S-proteins may provide additional variation (Beveridge & Graham, 1991; Labischinski & Maidhof, 1994). IFN-γ (ng mL-1)

3

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cd bcd bc

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bcd ab a

a a a 2

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IFN-γ (ng mL-1)

IFN-g is secreted by T-cell helper-type 1 cells (Th1), which are associated with antibody responses (Knopf, 2000). Although it has antiviral and antiparasitic activities in mice, the main biological activity of IFN-g appears to be immunomodulatory. IL-10 produced by T-cell helper type 2 cells (Th2) inhibits the synthesis of a number of cytokines, including IFN-g: IL-10 is also produced by regulatory T cells and lead to both suppression of Th2 responses and a switch from IgE to IgG4 antibody production preventing allergic disease (Robinson, Larche, & Durham, 2004). Induction or enhancement of cytokine production could be major mechanisms by which bifidobacteria exert immunomodulatory activities (Marin, Lee, Murtha, Ustunol, & Pestika, 1997). Since no cytokines were detected after adding bifidobacterial extracts alone, we checked for synergic effects with PHA. Levels of IFN-g and IL-10 produced by splenocytes in the presence of bifidobacterial extracts with PHA are shown in Figs. 5 and 6. A significant increase in IFN-g production was obtained in splenocyte cultures with bacterial cell wall at concentrations of 20–40 mg mL1. More than 4 ng mL1 of IFN-g was obtained by adding cell wall from B. lactis Bb12 while little or none was produced by adding EPS (Fig. 5c). IL-10 was not detected in response to EPS. These results are consistent with those obtained in the proliferation assay. In addition to the proliferation, immune cells also react to conserved bacterial molecules such as peptidoglycan by secreting cytokines (Moreillon & Majcherczyk, 2003). Increased IL-10 production by splenocytes (0.8 ng mL1) was obtained in response to isolate RBL64 cell wall and PHA. These results suggest a strain or species-dependent effect of bifidobacterial cell wall extracts on splenocytes, since they may induce proor anti-inflammatory cytokines. Indeed, IFN-g is known to be a major macrophage-activating lymphokine and regulates the induction of other cytokines, particularly Th2 cytokines such as IL-4, IL-5 and IL-10. Because of its role in mediating macrophage and NK cell activation, IFN-g is important in host defense against intracellular pathogens such as Mycobacterium tuberculosis and viruses and against tumours (Meydani & Ha, 2000). However, over-expression of cytokines may lead to inflammation in the intestine and is thus considered undesirable, especially for allergic infants and the elderly. He et al. (2002), in finding that bifidobacterial induction of cytokine secretion is strain and species dependant, demonstrated that Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory

Strain RBL64 B. lactis Bb12

* e cd

bcd

3

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a bc

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0 2

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-1

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IFN-γ (ng mL-1)

3.3. Cytokine production

4

2

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0 0.1

(C)

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Exopolysaccharides (µg mL -1)

Fig. 5. Synergistic effect of PHA and cellular preparations (A) cytoplasmic content, (B) cell wall, and (C) exopolysaccharides from bifidobacterial strain RBL64 and B. lactis Bb12 on IFN-g splenocyte production when cells were cultured in complete RPMI medium added with mitogen agent (PHA) (10 mg mL1). No IFN-g was detected in PHA-free media and there was no significant difference between treatments with exopolysaccharides. *Higher than 4 mg mL1.

cytokine secretion (IL-12 and TNF-a) by a murine macrophage-like cell line than did the infant-type bifidobacteria B. bifidum, B. breve and B. infantis. In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10.

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IL-10 (ng mL-1)

1 Strain RBL64 B. lactis Bb12

0.8 0.6 0.4 0.2

N.D

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Cytoplasmic content (µg protein mL-1)

(A) 1

IL-10 (ng mL-1)

against most viruses and intracellular bacteria, whereas, the coinduction of Th2-type immunity leads to the maintainance of immune homeostasis during Th1mediated responses (Bot, Smith, & Von Herrath, 2004). In addition, IL-10 detected in the culture media could be produced by regulatory T cells, distinct in function and phenotype from the Th1 and Th2 populations (McGuirk & Mills, 2002). CD4(+)CD25(+) regulatory T-cell, secreting high levels of IL-10, were recently shown to be re-directed against pathologic T-lymphocytes, facilitating the treatment of autoimmune disease (Mekala & Geiger, 2004).

4. Conclusions c

0.8

b

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b

0.4

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0 2

(B)

77

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Cell wall (µg protein mL -1)

Fig. 6. Synergistic effect of PHA and cellular preparations (A) cytoplasmic content and (B) cell wall from bifidobacterial strain RBL64 and B. lactis Bb12 on IL-10 splenocyte production when cells were cultured in complete RPMI medium added with mitogen agent (PHA) (10 mg mL1). No IL-10 was detected in PHA-free media and there is no significant difference between treatments with or with exopolysaccharides cytoplasmic content. ND—not detected.

Splenocyte proliferation and cytokine secretion in mice probably reflect stimulation of lymphocyte-governed inherent immunity to bacterial immunogens. It thus appears that the immunoregulatory action of bifidobacteria may be mediated primarily by enhancement of interferon production by spleen cells. In theory, this mechanism could serve to regulate over-expression of the Th2-type immune response and thus help fight against allergic response. Increased lymphocyte proliferation and IFN-g production in response to lactic acid bacteria in rat spleen has been previously reported (De Simone et al., 1989; De Simone, Vesely, Bianchi Salvadori, & Jirillo, 1993; Attouri, Bouras, Tome, Marcos, & Lemonnier, 2002). Decreased IL-10 secretion could in effect stimulate pro-Th1-type responses, since IL-10 is a potent deactivating signal for cytokine production and can suppress IFN-g production and Th1 phenotype expression (De Wall Malefyt, Abrams, Bennet, Figdor, & De Vries, 1993; D’Andrea et al., 1993). Establishment of a robust Th1-type immunity in the site of infection is a prerequisite for effective defense

The results demonstrate overall that cell components from bifidobacteria promote in vitro immune responses in mouse splenocytes. Cell wall and cytoplasm were found to stimulate lymphocyte proliferation and increase IFN-g; and IL-10 while bifidobacterial EPS recovered from culture broth did not produce a significant response. It is therefore suggested that live probiotic bacteria may not be required to influence the immune system. Components such as cell wall from autolysed bifidobacteria may stimulate lymphatic cell proliferation and cytokine production. The use of nonviable probiotics in foods should have the advantage of allowing a longer product shelf life and easier storage. Since the composition of bacterial cell wall and cytoplasm is very complex, additional research to identify specific immunoregulatory agents, particularly the components favouring pro-Th1 activity, should provide valuable insight into probiotic immunostimulation. Understanding these modulatory functions could provide a unique opportunity to prevent or treat intestinal disorders associated with food allergy, intestinal infections, inflammatory bowel disease and autoimmunity. Probiotic bifidobacteria could be used as nutritional supplements to improve the immune function of neonate or the elderly, for whom these functions are diminished. The use of probiotic homogenates as components of functional foods should also be studied clinically. Since bifidobacteria and lactic acid bacteria are indigenous inhabitants of the healthy intestine and have been used safely for the production of food all over the world since ancient times, they are particularly suited to be used as probiotics or immunomodulators for both the prevention and therapeutic treatment of diseases mediated by immune responses.

Acknowledgments This work was supported by Grants from FCAR, NOVALAIT, MAPAQ and NSERC. T. Amrouche was

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recipient of scholarships from the World Bank. Dr. Stephen Davids is thanked for reading the manuscript.

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