Entête

Chapitre II: Effets du contenu cytoplasmique, de la paroi cellulaire et des exopolysaccharides de bifidobactéries sur la prolifération de lymphocytes de souris et la production de cytokines. Effects of bifidobacterial cytoplasm, cell wall and exopolysaccharides on mouse lymphocyte proliferation and cytokine production.

Table des matières

T. Amrouche a,b,c, Y. Boutin b,d, G. Prioult a, and I. Fliss a,b*

a Dairy Research Center STELA, Pavillon Paul-Comtois, Université Laval, Québec (Qc) G1K 7P4

b Institute of Nutraceutical and Functional Foods INAF, Université Laval, Québec (Qc) G1K 7P4

cDepartment of Food Technology, Faculty of Agronomy and Biological Sciences, University of Tizi-Wezzu, Algeria

d TransBiotech, CEGEP Lévis Lauzon, Lévis (Qc) G6V 9V6

International Dairy Journal : sous presse

Les bifidobactéries, candidates aux probiotiques, sont des bactéries exerçant des effets bénéfiques sur la santé de l’être humain comme le rétablissement de l’équilibre de la flore colique, l’amélioration de l’immunité, le traitement des diarrhées, etc. Des études récentes ont rapporté que les bactéries probiotiques ont la capacité de stimuler certaines fonctions immunitaires et offrent ainsi des possibilités d’augmenter les capacités de défense de l’organisme hôte contre les infections gastro-intestinales. Cependant, les mécanismes impliqués dans cet effet immunomodulateur demeurent mal connus. Le but de ce projet est d’étudier les effets de certains constituants des cellules de bifidobactérie notamment le contenu cytoplasmique, la paroi bactérienne et les exopolysaccharides (EPS) sur la réponse immunitaire. L’effet immunomodulateur a été étudié par une mesure de la prolifération lymphocytaire ainsi que par le dosage de certaines cytokines notamment IFN-γ et IL-10. Trois souches de bifidobactéries, B. thermoacidophilum RBL81, RBL82 et RBL64, isolées à partir de féces de bébés et sélectionnées pour leur capacité à produire des EPS, ont été étudiées. Une souche commerciale, Bifidobacterium lactis Bb12 a été utilisée comme souche de référence. Les résultats obtenus montrent qu’une meilleure stimulation de la prolifération cellulaire est obtenue avec la paroi cellulaire. Un effet immunostimulant important a été également obtenu avec le contenu cytoplasmique de B. lactis Bb12. Cependant, l’effet observé est dépendant de la souche microbienne et de la dose utilisée. Par contre, les EPS bruts ou fractionnés n’induisent aucun effet. La paroi cellulaire de B. lactis Bb12 s’est avérée plus immunostimulante (Indice de stimulation =16) que celle des autres souches testées. Le dosage des cytokines a confirmé les résultats de la prolifération cellulaire et a montré une forte production de IFN-γ (> 4 µg/ml) générée par la paroi cellulaire, et une augmentation de la sécrétion de IL-10 dans le milieu ( < 1 µg/ml ). Les résultats obtenus démontrent que les extraits cellulaires de bifidobactéries, notamment les parois, stimulent la prolifération des lymphocytes et laissent suggérer la possibilité d’utilisation de ces derniers dans le contrôle de certaines pathologies immunitaires.

Mots clés: Bifidobactéries, Cytokines, Cytoplasme, Exopolysaccharides, Paroi cellulaire, Prolifération cellulaire. 

Probiotic bifidobacteria have been reported to stimulate the immune system and thus offer the possibility of improving host immune defence 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-γ and IL-10. Three bifidobacteria, RBL64, RBL81, and RBL82 isolated from newborn infant feces 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 stimulate lymphocyte proliferation nor induced cytokine secretion. B. lactis Bb12 exhibited a significant immunostimulating effect (stimulation index = 16) compared to other bifidobacteria studied. More IFN-γ (> 4 ng mL-1) was produced in response to cell wall and about 0.8 ng mL-1 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.

Keywords: Bifidobacteria, Cell wall, Cytokines, Cell proliferation, Cytoplasm, Exopolysaccharides. 

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, Shimizu, Nomoto, Hamabata, Ozawa & Takeda, 2004). It has been shown that protection by microflora can be improved by ingesting probiotics, which are microorganisms that favourably influence host physiology by modifying the intestinal flora (Kirjavainen, 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 (EPS) (Roberts, Fett, Osman, Wijey, O’Connor & Hoover, 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 (Whitfield & Valvano, 1993; Looijesteijn, Trapet, De Vries, Abee & Hugenholtz, 2001).

Probiotic bifidobacteria, especially B. longum , B. infantis and B. breve , have been shown to stimulate the immune system and thus may increase the capacity of the host to fight against gastrointestinal infections (Mullie et al., 2004) and help to 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 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 microorganisms, 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 (IFN-γ 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-γ 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). Van Calsteren, Pau-Roblot, Begin and Roy (2002), and Ruas-Madiedo, Hugenholtz and Zoon (2002) reported that EPS from lactic acid bacteria may influence the immune system by enhancing lymphocyte proliferation, macrophage activation and cytokine production.

At the present time, 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 acidophilum from newborn infant feces were compared to the commercial strain Bifidobacterium lactis Bb12. The abilities of cytoplasm, cell wall and EPS from bifidobacteria to stimulate mouse splenocyte proliferation and production of cytokines IFN-γ and IL-10 were measured.

EPS was isolated and purified by the method of Cerning et al. (1994). After incubation, cultures were heated at 100°C for 15 min to inactivate potential EPS hydrolases and to improve detachment of EPS from the microbial cell walls (Tuinier, Van Casteren, Looijesteijn, Schols, Voragen & Zoon, 2001). The cultures were then centrifuged at 10,000 rpm for 20 min at 4°C to separate bacteria and cell debris. EPS were precipitated with three volumes of chilled 95% ethanol and standing overnight at 4°C. The precipitate, collected by centrifugation at 9,000 rpm for 20 min at 4°C, was re-suspended in 25 mL of deionised water and then freeze-dried. The resulting powder was dissolved in 15 mL of 10 % trichloroacetic acid (TCA) and the solution was centrifuged at 9,000 rpm for 15 min at 4°C 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 MW-cut off) at 4 °C for 2 days against deionised water. After dialysis, EPS solutions were fractionated by successive filtrations through Centricon (Millipore) 5,000 and 100,000 MW-cut off filters. Three fractions were obtained: F1: > 100,000 Da; F2: 5,000 to 100,000 Da; and F3: < 5 000 Da. The total sugar content of the EPS suspensions was estimated by the phenol/sulfuric method (Dubois, Gilles, Hamilton & Roberts, 1956). All samples were aliquoted and kept at –20 °C until use. EPS solutions were passed through a 0.22 µm filter prior to addition to cell cultures.

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 mL-1 50 mM mercaptoethanol , 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin, all from Gibco). Cells were transferred to 96-well round bottom plates at a concentration of 5x105 cells mL-1. Various concentrations of bifidobacterial extracts were added alone or with phytohemagglutinin (PHA) (10 µg mL-1) to the cells. They were incubated for 48 h in a humidified 5 % CO2 atmosphere at 37 °C.

Few studies suggest that EPS produced by certain lactic acid bacteria may have immunostimulatory (Hosono, Amenati, Natsume, Hirayama, Adachi & Kaminogawa, 1997), antitumoral (Oda, Hasegawa, Komatsu, Kambe & Tsuchiya, 1983; Ebina, Ogata & Murata, 1995) activities. Induction of cytokine (IFN-γ and IL-1α) 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, Harata, Uemura, Saito, Kaneko & Itoh, 1998; Kitazawa et al., 2000; Nishimura-Uemura, Kitazawa, Kawai, Itoh, Oda & Saito, 2003). We therefore sought to evaluate the effect of crude and fractioned bifidobacterial EPS as well as cytoplasm and cell wall extracts on splenocyte proliferation and production of IFN-γ and IL-10. Since similar results were obtained with all fractions of the three isolates RBL64, RBL81, and RBL82, we present herein only the comparison of RBL64 and B. lactis Bb12.

The average yields of crude (total) EPS obtained from 24-hour cultures of isolates RBL81, RBL82, RBL64, and B. lactis Bb12 were respectively 0.53, 0.42, 0.32, and 0.33 mg mL-1 of supernatant. Concentrations of EPS were in the same range as those obtained by Roberts et al. (1995) from Bifidobacterium longum BB-79 (0.47 g L-1). EPS yield has been reported to vary widely depending on species and culture conditions. For example, Streptococcus thermophilus has been shown to produce 20 to 100 mg EPS L-1 in fermented milk medium (Vaningelgem et al., 2004), while Lactobacillus rhamnosus RW-9595M yielded 2350 mg L-1 of purified EPS when grown on supplemented whey permeate at 37 °C (Bergmaier, Champagne, & Lacroix, 2003). Lactococcus lactis subsp. cremoris SBT 0495 has been reported to produce 600 mg L-1 in lactose-containing medium (Higashimura, Mulder-Bosman, Reich, Iwasaki & Robin, 2000).

Bifidobacterial EPS were fractionated into F1 (> 100,000 Da), F2 (5,000 to 100,000 Da) or F3 (< 5,000 Da) portions. Proportions of each fraction of the isolate are shown in Figure 2.1. Fraction F1 of all isolates was found to have a higher EPS content (ranging from 63.5 to 79.3%) than fraction F2 (18.1 to 30.9%) or F3 (1.6 to 5.6%).

Isolate RBL82 produced more EPS in the molecular weight range > 100,000 Da than did RBL64 or RBL81. The comparison of the EPS molecular weight profiles of isolate RBL82 and Lactobacillus rhamnosus RW-9595M (Bergmaier et al., 2001) is shown in Figure 2.2. With the aid of Figure 2.3, which shows three peaks corresponding respectively 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 weight. 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. Tone-Shimokawa, Toida and Kawashima (1996), who analysed cell wall polysaccharides of Bifidobacterium 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 weight range of F1 appears consistent with the findings of Roberts et al. (1995), who estimated by chromatography the average weight EPS from Bifidobacterum longum BB-79 to be greater than 200,000 Da.

The crude EPS preparation had only a weak stimulating effect on cell proliferation (Figure 2.4). Tzianabos, Wang and Kaspera (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 (structural alteration induced by heat or trichloroacetic acid treatment), causing losses of their properties to stimulate the cell proliferation.

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, Tzianabos, Bryant, Niemeyer, Ploegh, and Kasper (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, Corthesy, Kusy, Porcelli, Kasper and Tzianabos (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 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 Bifibobacterium longum BB-79 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 B. lactis Bb12 can be clearly observed after 48 h of incubation. Figure 2.4 shows that splenocyte proliferation is dose dependent and the SI is significantly increased at protein concentrations ranging from 20 to 40 µg mL-1. 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 Bifidobacterium 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 β-lactoglobulin in mice. Other studies have reported that Gram-positive 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 Gram-positive 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 bacteria-derived 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 splenocytes (Gobbetti, Corsetti, Smacchi, Zocchetti, and De Angelis, 1998; Samartsev, Astapovich and 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 the 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 of S-proteins may provide additional variation (Beveridge & Graham, 1991; Labischinski & Maidhof, 1994).

IFN-γ 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-γ 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-γ. 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 synergistic effects with PHA. Levels of IFN-γ and IL-10 produced by splenocytes in the presence of bifidobacterial extracts with PHA are shown in Figure 2.5 and Figure 2.6. A significant increase in IFN-γ production was obtained in splenocyte cultures with bacterial cell wall at concentrations of 20 to 40 µg mL-1. More than 4 ng mL-1 of IFN-γ was obtained by adding cell wall from B. lactis Bb12 while little or none was produced by adding EPS (Figure 2.5c). IL-10 was not detected (Figure 2.6c) 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 mL-1) 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 pro- or anti- inflammatory cytokines. Indeed, IFN-γ 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-γ is important in host defence 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 cytokine secretion (IL-12 and TNF-α) 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.

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 to fight against allergic response. Increased lymphocyte proliferation and IFN-γ production in response to lactic acid bacteria in rat spleen has been previously reported (De Simone, Bianchi Salvadori, Jirillo, Baldinelli, Bitonti & Vesely, 1989; De Simone, 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-γ production and Th1 phenotype expression (De Wall Malefyt, Abrams, Bennet, Figdor & De Vries, 1993; D’Andrea, Aste-Amezaga, Valiante, Ma, Kubin, & Trinchieri, 1993). Establishment of a robust Th1-type immunity in the site of infection is a prerequisite for effective defence against most viruses and intracellular bacteria, whereas the coinduction of Th2-type immunity lead to the maintenance of immune homeostasis during Th1-mediated 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).

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

© Tahar Amrouche, 2005