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Clinical and Diagnostic Laboratory Immunology logoLink to Clinical and Diagnostic Laboratory Immunology
. 2003 Sep;10(5):787–792. doi: 10.1128/CDLI.10.5.787-792.2003

Effect of Probiotic Bacteria on Induction and Maintenance of Oral Tolerance to β-Lactoglobulin in Gnotobiotic Mice

Guénolée Prioult 1,2, Ismail Fliss 1,*, Sophie Pecquet 1,2
PMCID: PMC193892  PMID: 12965905

Abstract

In this study, the effect of Lactobacillus paracasei (NCC 2461), Lactobacillus johnsonii (NCC 533) and Bifidobacterium lactis Bb12 (NCC 362) on the induction and maintenance of oral tolerance to bovine β-lactoglobulin (BLG) was investigated in mice. Germfree mice were monocolonized with one of the three strains before oral administration of whey protein to induce tolerance. Mice were then injected with BLG and sacrificed 28 or 50 days after whey protein feeding for humoral and cellular response measurement. Conventional and germfree mice were used as controls. Both humoral and cellular responses were better suppressed in conventional mice than in germfree and monoassociated mice throughout the experiment and better suppressed in L. paracasei-associated mice than in mice colonized with B. lactis or L. johnsonii. The latter two mono-associations suppressed humoral responses only partially and cellular responses not at all. This study provides evidence that probiotics modulate the oral tolerance response to BLG in mice. The mono-colonization effect is strain-dependant, the best result having been obtained with L. paracasei.


The oral administration of a dietary antigen results in a mucosal and systemic antigen-specific immunological unresponsiveness called oral tolerance. The establishment of oral tolerance prevents digestive immunoglobulin E (IgE)-mediated hypersensitivity reactions to food antigens and depends on several factors, such as the age of the host, the nature and doses of the antigen, the frequency of feeding, and the protein content of the diet (3, 11, 21, 27).

Among the environmental factors, the composition of the intestinal microbiota seems to play an important role in the modulation of oral tolerance. It has been shown that oral administration of various antigens induces tolerance in conventional mice but not in germfree mice (18, 31, 35). In addition, the oral tolerance response in germfree mice can be restored either by administration of lipopolysaccharide from gram-negative bacteria (35) or by monocolonization of the gut with Bifidobacterium infantis (18, 31) or Escherichia coli (18) but not with Clostridium perfringens or Staphylococcus aureus (18). Contrasting results show that germfree mice can be made tolerant to ovalbumin but that the tolerance is maintained for a shorter time than in conventional mice (20, 21), for example 2 to 3 months of systemic IgG suppression after a single feeding (30) compared to 21 days in germfree mice (21). These results show that the intestinal microbiota play a major role in the induction and long-term persistence of oral tolerance, but further experiments are needed to identify which bacteria are the most effective.

Probiotics are live microorganisms that, when ingested, might have positive effects on human health (5). Some of the purported health benefits include improving the intestinal microbial balance (24), modulating the immune system (7), and decreasing the prevalence of allergy in susceptible individuals (9). Bifidobacteria and lactobacilli are common anaerobes in the human intestinal microbiota (17), and some of them have been reported to display probiotic properties (24). Differences between the intestinal microbial composition in allergic and healthy infants are plausible indicators of bacterial species eliciting probiotic properties, especially promoting oral tolerance. Allergic infants have been shown to be less colonized with bifidobacteria, especially during the period preceding the development of atopic disease (2, 8, 12, 13), suggesting a potential effect of bifidobacteria in the prevention of allergic diseases. In addition, B. infantis has been shown to restore oral tolerance induction in monoassociated mice (18, 31), but no data are available on the effect of other probiotics on induction or long-term persistence of the oral tolerance response. However, Lactobacillus paracasei NCC 2461 has recently been found to stimulate in vitro regulatory T cells producing transforming growth factor β and interleukin-10 (IL-10) (34), cytokines implicated in oral tolerance induction (23).

In the present study, we investigated the influence of L. paracasei NCC 2461, Bifidobacterium lactis Bb12 NCC 362, and Lactobacillus johnsonii NCC 533 on induction and maintenance of oral tolerance to bovine β-lactoglobulin (BLG) in gnotobiotic mice.

MATERIALS AND METHODS

Animals.

Germfree female C3H/He mice, 3 to 5 weeks old, were used. The study was done in accordance with Canton de Vaud (Switzerland) veterinary service regulations. The mice were bred in our animal breeding unit and maintained in a light-controlled environment under sterile conditions in plastic isolators until just before sacrifice. A sterile whey protein-free diet was fed in the breeding unit for at least three generations and then throughout the experiment. Three-week-old conventional female BALB/c mice (IFFA Credo, Abresle, France) were used as controls and fed the same diet.

Bacterial strains and monocolonization of mice.

The three probiotic strains used in the present study were from the Nestlé Culture Collection (Nestec, Lausanne, Switzerland) and originally isolated from human feces: L. johnsonii NCC 533, L. paracasei NCC 2461, and B. lactis Bb12 NCC 362. Strains were kept frozen at −80°C before being subcultured twice for 24 h at 37°C under anaerobic conditions in brain heart infusion broth (Oxoid, Ltd., Basingstoke, United Kingdom) supplemented with 0.5 g of l-cysteine/liter. Cell enumerations were done after 24 h of broth culture on nonselective blood agar (BioMerieux, Marcy l'Etoile, France), and plates were incubated at 37°C for 48 h under anaerobic conditions (AnaeroGen; Oxoid). Germfree mice were then inoculated by intragastric intubation with 0.3 ml of a 24-h pure culture containing 5 × 108 CFU of either L. johnsonii, L. paracasei, or B. lactis/ml. Colonization was monitored by bacterial counts in fecal samples of two mice per group every 2 days for the first 2 weeks and then once per week for the next 7 weeks on blood agar. Bacterial counts were expressed as the mean of the two counts per time point.

Oral exposure to BLG and immunizations.

The five groups of 20 mice (germfree, conventional, and L. johnsonii, L. paracasei, or B. lactis associated) were divided into subgroups of 10 for antigen treatment. At 18 days after bacterial feeding, one subgroup from each group was orally given 36 mg of whey protein (lacprodan 80, 62% BLG; MD-Foods, Skanderborgvej, Denmark) by intragastric intubation. The other subgroup received a single feeding of saline solution as negative control. Five days later, all mice were immunized subcutaneously with 100 μg of BLG (Sigma) and 100 μg of ovalbumin (OVA; Sigma) in 2% Al(OH)3. OVA was used as a control to assess the specificity of the response and potential bystander effects. Five mice from each subgroup were sacrificed 28 or 50 days after BLG feeding under isoflurane anesthesia. Mice spared on day 28 received two additional subcutaneous injections of BLG and OVA, 21 and 35 days after antigen feeding. Intestines, blood samples, and spleens were collected for measuring bacterial colonization, antibody titers, and cell proliferation, respectively. Sera were kept frozen at −25°C until used.

Bacterial counts.

The colon and small bowel (divided into jejunum and ileum) were aseptically weighed, homogenized with an Ultra-Turrax (15 s; 13,500 rpm), and diluted in Ringer solution containing 0.5 g of l-cysteine/liter. Lactobacilli were cultured on de Man-Rogosa-Sharpe agar (Oxoid), whereas B. lactis was cultured on Eugon agar (Becton Dickinson, Basel, Switzerland) with 40% (vol/vol) tomato juice. Plates were incubated at 37°C for 48 h under anaerobic conditions (AnaeroGen). The results are expressed as the mean of two counts per group ± the standard error of the mean (SEM).

IgE, IgG1, and IgG2a levels in serum.

The amount of BLG- and OVA-specific IgE in serum was determined by enzyme-linked immunosorbent assay (ELISA) as previously described (25), with some modifications. Briefly, 96-well microplates were coated overnight at 4°C with 0.5 mg of BLG or OVA/ml diluted in carbonate-bicarbonate buffer (pH 9.6). Plates were then washed four times with phosphate-buffered saline-0.05% Tween 20, blocked with 0.5% fish gelatin (Sigma) for 3 h at room temperature, and washed again before the addition of sera. Twofold serial dilutions of sera samples from individual mice were assayed in duplicate at 37°C for 2 h with gentle agitation. Specific IgE was detected by using a peroxidase-labeled rat anti-mouse IgE (Readysysteme AG, Bad Zurzach, Switzerland) and 0.04% O-phenylenediamine dissolved in phosphate citrate buffer (pH 5.0) in the presence of H2O2. The reactions were stopped after 15 min of incubation at room temperature by adding 25 μl of 25% (vol/vol) sulfuric acid to each well, and optical densities were measured at 490 nm.

The production of BLG-specific IgG1 and IgG2a was tested by the same method, with plates coated with 30 mg of BLG/ml and blocked with 20% (vol/vol) fetal calf serum (FCS; Bioconcept, Basel, Switzerland) in phosphate-buffered saline containing 0.05% Tween 20. Threefold serial dilutions of sera samples from individual mice were tested. Specific IgG1 and IgG2a were detected by using biotin-conjugated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates, Inc., Birmingham, Ala.), followed by the addition of streptavidin-peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) and tetramethylbenzidine-H2O2 as peroxidase substrate. The reactions were stopped by the addition of 1 M phosphoric acid, and the optical densities were measured at 450 nm.

Serum samples from nonimmunized and immunized BALB/c mice were used as negative and positive controls, respectively, on each plate. The cutoff dilution (Dc) was defined as the dilution yielding twice the absorbance of the negative controls. Titers were calculated as log10(1/Dc) and are expressed as the mean of five mice ± the standard deviations (SD) for each subgroup.

Splenocyte proliferation.

Spleens from five mice were pooled in RPMI 1640 medium (Bioconcept) enriched with 5% FCS, and splenocyte proliferation was performed as described by Pecquet et al. (25). Briefly, splenocytes were obtained by pushing spleens through a cellular sieve prior to eliminating erythrocytes by hypotonic shock. Cells were then rinsed three times with FCS-enriched RPMI and resuspended at 5.0 × 106 cells/ml of RPMI 1640 supplemented with 10% FCS, 2 mM l-glutamine, 100 U of penicillin, and 100 μg of streptomycin. Cells were cocultured with either BLG (2.5 mg/ml) or phytohemagglutinin (PHA) at 125, 25, or 1 μg/ml or no stimulating agent, followed by incubation in 96-well plates at 37°C in a humidified atmosphere of 5% CO2 for 48 h (each condition in triplicate). Antigen concentrations were previously selected as optimal concentrations for cell proliferation (25). [3H]thymidine was added to the wells 6 h before harvest, and incorporated radioactivity was measured by scintillation counting. At 48 h after the initiation of the cultures, supernatants were harvested for assessment of gamma interferon (IFN-γ) production by using ELISA commercial kits (Quantikine Murine R&D Systems, Minneapolis, Minn.).

Statistical analysis.

Antibody titers are expressed as the means ± the SD for the five mice from each subgroup. The statistical significance of comparisons between the BLG-fed subgroups and saline-fed subgroups was assessed by using the Student t test. A P value of <0.05 was considered significant.

RESULTS

Gut colonization by probiotics in monoxenic mice.

The colonization of germfree mice with B. lactis, L. johnsonii, or L. paracasei, as indicated by fecal counts obtained throughout the experiment, was evaluated (Fig. 1A). The three strains increased rapidly in number during the 18 days after inoculation and remained stable thereafter. All three strains colonized the intestine at levels increasing in the distal direction, as measured on day 50 (Fig. 1B). In the jejunum, the L. johnsonii counts were highest, reaching (3.5 ± 0.7) × 108 CFU/g compared to (2.3 ± 1.6) × 107 and (4.0 ± 2.8) × 106 CFU/g for L. paracasei and B. lactis, respectively. In the ileum, L. johnsonii, at (9.5 ± 0.2) × 108 CFU/g, was still more abundant, followed by B. lactis and L. paracasei at (3.5 ± 0.1) × 108 and (4.0 ± 3.2) × 107 CFU/g, respectively. Bacterial counts for L. johnsonii and B. lactis were thus higher in the ileum than in the jejunum. Colonization in the colon (reaching >1010 CFU/g) was higher than in the jejunum or ileum for the three strains, and no statistical difference between strains was found. Intestinal counts done at day 28 (first sacrifice) gave similar results (data not shown).

FIG. 1.

FIG. 1.

(A) Fecal bacterial counts in monoassociated mice expressed as means taken from two mice per time point. (B) Intestinal bacterial counts in monoassociated mice obtained 68 days after oral administration of bacterial suspensions, expressed as the means ± the SEM from two mice per group.

Induction of humoral tolerance to BLG.

Oral administration of whey protein prior to immunization with BLG suppressed the 28-day humoral response to BLG, with the immunoglobulin isotype (IgE, IgG1, and IgG2a) suppressed and the degree of suppression depending on the bacterial flora (Fig. 2A to C). In conventional and L. paracasei-associated mice, BLG-specific IgE, IgG1, and IgG2a were significantly suppressed (P < 0.01), whereas L. johnsonii-associated mice exhibited significantly lower BLG-specific IgE (P < 0.01) and IgG1 (P < 0.05) titers, but similar IgG2a titers. BLG-specific IgG1 and IgG2a titers in the serum of mice associated with B. lactis were at the same level for saline and whey protein treatments, whereas some IgE suppression was observed (P < 0.01) for whey protein-fed mice. The strongest overall suppression of the humoral response to BLG was obtained in conventional mice, showing log reductions of 1.4, 1.6, and 0.7 for IgE, IgG1, and IgG2a, respectively. Among probiotic bacterium-associated mice orally exposed to whey protein, the decrease in specific antibody titers was more apparent in mice colonized with L. paracasei, followed by L. johnsonii and B. lactis, for the three immunoglobulin isotypes tested. Moreover, the humoral suppression reported was specific to BLG, whereas OVA-specific IgE titers measured in whey protein-fed subgroups were similar to those obtained in saline-fed subgroups, regardless of intestinal colonization (Fig. 2D).

FIG. 2.

FIG. 2.

BLG-specific IgE (A to E), IgG1 (B to F), and IgG2a (C to G) and OVA-specific IgE (D to H) titers in the sera of conventional mice (Conv), germfree mice (GF), or mice mono-associated with L. paracasei (L. para.), L. johnsonii (L. john.), or B. lactis at 28 days (A to D) and at 50 days (E to H) after the animals were fed saline (▪) or whey proteins (□). Titers were calculated as the log10(1/Dc) and are expressed as the mean of five mice ± the SD for each subgroup. The statistical significance of comparisons between the BLG-fed subgroups and saline-fed subgroups was assessed by using the Student t test (✽, P < 0.05; ✽✽, P < 0.01).

Maintenance of humoral tolerance to BLG.

The intestinal microbiota influenced the long-term persistence (day 50) of specific humoral unresponsiveness (Fig. 2). As expected, the humoral suppression (BLG-specific IgE, IgG1, and IgG2a) observed in conventional mice at day 28 was maintained at day 50 (Fig. 2E to G). L. paracasei-associated mice retained a significant suppression of anti-BLG IgE (P < 0.05) and IgG1 (P < 0.01) (Fig. 2F), whereas the suppression of IgG2a response was lost (Fig. 2G). In mice colonized with B. lactis, the BLG-specific IgE response suppression was at least maintained (P < 0.01) (Fig. 2E), whereas the anti-BLG IgG1 titer increased in the saline-treated condition and remained at the day 28 level in the BLG-treated subgroup, indicating an apparent suppression (Fig. 2F). In germfree mice, the humoral response pattern observed at day 50 was similar to that observed at day 28, while for L. johnsonii-associated mice the humoral suppression disappeared at day 50. These observations suggest a strain-dependent effect of probiotic bacteria on oral tolerance induction and long-term persistence, the better result having been obtained with L. paracasei. As observed at day 28, the suppression of the humoral response was specific to orally administered BLG, as shown by the lack of any decrease in the OVA-specific IgE titers in any groups of mice (Fig. 2H). Moreover, the humoral immune suppression at day 50 was better maintained in conventional mice, as shown by log reductions of 1.6, 1.4, and 1.6 for IgE, IgG1, and IgG2a, respectively, which were always higher that those obtained for germfree and monoassociated mice.

Induction and maintenance of cellular tolerance to BLG.

For additional evidence that tolerance is modulated by probiotics, stimulation of splenocyte proliferation in vitro by the antigen was examined. Conventional and L. paracasei-associated mice exhibited strong induction and maintenance of cellular tolerance to BLG, as shown by lower splenocyte proliferation in whey protein-fed subgroups compared to saline-fed subgroups both at day 28 (Fig. 3A) and at day 50 (Fig. 3B). However, the cellular response suppression seemed to be slightly better induced and maintained in conventional mice than in L. paracasei-associated mice. Moreover, the suppression observed was real and not merely apparent due to a lack of cell proliferation during the assay, since the cells responded normally to PHA (25 μg/ml) both at day 28 (Fig. 3E) and at day 50 (Fig. 3F). Finally, the induction and maintenance of cellular tolerance to BLG were confirmed by measurement of IFN-γ produced by BLG-stimulated splenocytes. For conventional and L. paracasei-associated mice, whey protein feeding produced marked suppression of IFN-γ production relative to saline fed controls (Fig. 3C and D). For the other groups of mice, the failure to manifest any cellular tolerance at either day 28 (Fig. 3A) or day 50 (Fig. 3B) obviated confirmation by IFN-γ measurement (Fig. 3C and D).

FIG. 3.

FIG. 3.

Splenocyte proliferation (A and B) and IFN-γ production (C and D) in response to restimulation with BLG in vitro measured 28 days (A and C) and 50 days (B and D) after oral administration of saline or whey protein (WP) in conventional mice (Conv), germfree mice (GF), or mice monoassociated with L. paracasei (L. para.), L. johnsonii (L. john.), or B. lactis. Spleens from five mice were pooled for cell culture; proliferation was determined by measuring [3H]thymidine incorporation and is expressed as the mean counts per minute (± the SEM) of triplicate wells. IFN-γ production in triplicate wells 48 h after the initiation of the culture was determined by ELISA and is shown as the percentage of saline-fed control subgroups (± the SEM). (E and F) The levels of splenocyte proliferation (in counts per minute) of conventional (circles) and L. paracasei-associated (triangles) mice, given either saline (solid symbols) or whey protein (open symbols) orally, in response to PHA (125, 25, and 1 μg/ml) at day 28 (E) and day 50 (F) are shown. Values are means of triplicate determinations.

DISCUSSION

Althought not well documented, the role of intestinal microbiota in the oral tolerance response appears to be important. The most significant studies have reported the effect of bacterial lipopolysaccharides (22, 35) or bacterial strains (C. perfringens or S. aureus) on oral tolerance induction in gnotobiotic mice models (18, 31). Only one probiotic strain (B. infantis) has been tested for induction of tolerance (18, 31), and no study has investigated the effect of probiotics on long-term persistence of tolerance. However, an in vitro study revealed that L. paracasei NCC 2461 seemed to stimulate regulatory T cells (34) and could thus potentially contribute to induce tolerance. To gain insight into the effect of L. paracasei NCC 2461 and other probiotics on the induction and maintenance of orally induced unresponsiveness, we attempted to induce tolerance to BLG in mice colonized with either L. paracasei NCC 2461, B. lactis Bb12 NCC 362, or L. johnsonii NCC 533 relative to two control groups (i.e., conventional and germfree mice).

In our C3H/He germfree mice, oral tolerance to BLG was shown to be partially induced and maintained in the humoral but not in the cellular immune system. These findings are in agreement with those of Moreau and Gaboriau-Routhiau regarding the suppression of peripheral antibody responses in the same strain of mouse (21). However, conflicting results reported for C3H/He (14) and BALB/c (18, 31) germfree mice show a lack of tolerance induction. The significant hypotrophy of Peyer's patches in germfree mice (28) and the resulting absence of T cells (18), which are essential for oral tolerance induction to proteins (4, 18), could explain the failure of tolerance induction under germfree conditions. However, induction of cellular unresponsiveness in the absence of Peyer's patches has been reported by Spahn et al. (29). Although divergent results have been obtained in germfree mice, all studies have concluded that oral tolerance is promoted by intestinal microbiota because it is always greater and maintained longer in conventional mice than in germfree mice (20, 31).

In our gnotobiotic mice colonized with probiotics, the level of oral tolerance response was strain dependent and lower than in conventional mice. Similarly, Maeda et al. (18) recently found that the tolerance response was restored in mice monoassociated with B. infantis or E. coli but not with C. perfringens or S. aureus. In our B. lactis- or L. johnsonii-associated mice, oral tolerance was partially induced humorally but not in the cellular system. In contrast, L. paracasei strongly induced and maintained cellular and, to a lesser degree, humoral unresponsiveness. The mechanisms by which this strain stimulates in vivo the tolerance response requires focused study. It is nevertheless well documented that the development of immunological tolerance to orally fed antigens depends on digestion and subsequent transportation events in the intestinal epithelium, especially in the gut-associated lymphoid tissues and in Peyer's patches (4, 10) and that probiotics participate in antigen degradation in the gut (26). Hence, we had postulated first that the better a probiotic colonizes small intestine, the stronger is the oral tolerance; however, our results here prove the opposite. Indeed, although L. johnsonii was found to thoroughly colonize both the jejunum and the ileum, cellular tolerance was not induced. In contrast, L. paracasei, which colonized less (less than one-tenth the level of L. johnsonii), induced and maintained better tolerance.

A strain-dependent activation of the immune system could explain the differences observed in oral tolerance modulation. L. paracasei has been found to be a strong inducer of IL-12 protein expression in vitro (34), triggering IFN-γ secretion by T cells and IFN-γ production a few days after oral feeding has been correlated with oral tolerance induction (15, 16). Moreover, L. paracasei has been shown to stimulate the production of suppressive cytokines (transforming growth factor β and IL-10) involved in active immune suppression in CD4+-T-cell culture (23) and inhibit their proliferation, as well as to suppress both in vitro (34) and in vivo (6) Th2 cell activity. Similarly, we observed a suppression of splenocyte proliferation and a significant decrease of antigen-specific IgE and IgG1 in L. paracasei-associated mice. Hence, the immunological effects observed in vitro and in vivo in association with L. paracasei could partly explain the tolerance induction reported in the present study. Moreover, recent work on the stimulation of Th2-cell-mediated responses in vivo by L. johnsonii (6) indicated that oral tolerance was poorly induced and not maintained with this strain. Corroborative data are not yet available on the immune deviation produced by B. lactis. In summary, we may speculate that L. paracasei actively participates in the induction of oral tolerance to BLG via a mechanism of immune stimulation, particularly by inducing the active suppression pathway by stimulation of regulatory T cells.

Conserved microbial components have been known to stimulate the immune system through the recognition of several receptors on immune cells such as the Toll-like receptors (TLRs) (19). TLR2 was found to be a receptor for peptidoglycan and lipoprotein by dimerizing with TLR6, whereas TLR4 was reported as a lipopolysaccharide and lipoteichoic acid receptor (19, 32). Lipoteichoic acids are found in most gram-positive bacteria (including lactobacilli), and the induction of oral tolerance to ovalbumin has recently been reported through the stimulation of Th1 cells via TLR4 signaling (33). Hence, L. paracasei could induce and maintain oral tolerance to BLG through the recognition of TLR4 by its lipoteichoic acids, but this hypothesis remains to be elucidated.

The intestinal microbiota play an important role in oral tolerance induction and maintenance. Tolerance is better induced and maintained in mice colonized with a complete and diversified microbiota (conventional mice). An impaired tolerance response is observed when the flora are absent or limited to one bacterial strain. However, oral tolerance is induced and maintained in L. paracasei-associated mice, indicating that this strain is a potential probiotic for the prevention of milk allergy in infants. Further studies are needed to determine whether combinations of probiotics may fully restore induction and maintenance of tolerance in gnotobiotic mice and to understand the mechanism by which L. paracasei NCC 2461 acts.

Acknowledgments

We thank Catherine Schwartz and Robert Beumer for careful breeding of germfree mice; Consolée Aletti, Isabelle Rochat, and José-Louis Sanchez-Garcia for skilled technical assistance; Nabila Ibnou-Zekri for fruitful discussions; and Stephen Davids for critical reading of the manuscript.

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