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Experimental Biology and Medicine 230:749-756 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Bifidobacterium breve and Streptococcus thermophilus Secretion Products Enhance T Helper 1 Immune Response and Intestinal Barrier in Mice

Sandrine Ménard*, David Laharie{dagger}, Corinne Asensio{dagger}, Teresita Vidal-Martinez*, Céline Candalh*, Anne Rullier{ddagger}, Frank Zerbib{dagger}, Francis Mégraud{dagger}, Tamara Matysiak-Budnik* and Martine Heyman*,1

* INSERM EMI 0212, Faculté Cochin-Necker, Paris, France; {dagger} Laboratoire de Bactériologie, Université Victor Segalen Bordeaux II, Bordeaux, France; and {ddagger} Service d’Anatomo-Pathologie, Hopital Pellegrin, Bordeaux, France

1 To whom requests for reprints should be addressed at INSERM EMI 0212, Faculté Cochin Necker, 156 rue de Vaugirard, 75730 Paris, France. E-mail: heyman{at}necker.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lactic acid bacteria or their secretion products can modulate immune responses differently in normal and inflammatory conditions. This comparative study analyzes the effect of oral administration of living lactic acid bacteria, or their conditioned media, on the epithelial and immune functions of colitis-prone C57BL/6 IL-10-deficient mice. Mice were untreated (control) or infected with Helicobacter hepaticus with or without oral treatment with living bacteria, Bifidobacterium breve C50 and Streptococcus thermophilus 065 (LB), or their culture-conditioned media (CM). Histology, cytokine mRNA, electrical resistance, and barrier capacity of colonic samples as well as cytokine secretion by mesenteric lymph node (MLN) cells were studied. Helicobacter hepaticus mice developed only mild colitis, which was not modified in LB or CM groups. In the CM (but not the LB) group, the colonic barrier was reinforced as compared to the other groups, as evidenced by decreased horseradish peroxidase (HRP) transcytosis and mannitol fluxes and increased electrical resistance. In MLN, the percentage of CD4+ and CD8+ T cells secreting IFN{gamma} was significantly higher in CM (2.06% and 1.98%, respectively) mice than in H. hepaticus (1.1% and 0.47%, P < 0.05) or control mice. In addition, the nonspecific stimulation of IFN{gamma}, TNF{alpha}, and IL-12 secretion by MLN cells was significantly higher in the CM group as compared to the other groups. In the absence of severe colitis, Bifidobacterium breve C50- and Streptococcus thermophilus 065-conditioned media can reinforce intestinal barrier capacity and stimulate Th1 immune response, highlighting the involvement of lactic acid bacteria–derived components in host defense.

Key Words: probiotics • adjuvants • mucosal immunity • intestinal function


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lactic acid bacteria seem to have different effects on immune responses depending on whether they interact with their host in physiological or pathological conditions. Indeed, although they strengthen specific and innate immune responses in healthy individuals, they seem to down-regulate proinflammatory cytokine secretion in pathological conditions such as inflammatory bowel diseases. In physiological conditions, lactic acid bacteria stimulate the IgA secretory immune response to potentially noxious luminal pathogens, and some studies have shown that they could enhance systemic immune response in case of vaccination (1, 2).

In contrast with these immunostimulatory effects in physiological conditions, in inflammatory conditions, particularly in inflammatory bowel diseases in humans, an attenuation of proinflammatory cytokine secretion and tissue damage has been reported with a selected set of probiotic bacteria (3, 4), although no consensus has yet been reached regarding their efficacy (5). The most convincing effect was observed with the prevention of relapse in chronic pouchitis by oral VSL#3 treatment (6, 7). The beneficial effects of probiotics in inflammation could result from their combined capacity to improve intestinal barrier function and to limit inflammatory responses. Reinforcement of the intestinal barrier capacity has been shown both in vitro, using intestinal epithelial cell lines (8, 9), and in vivo, in animal models of gastroenteritis or colitis (10, 11).

The mechanisms of action of probiotics are not completely understood. In particular, it is not clear whether their effects are related to the presence of the living bacteria or rather to the presence of some soluble bacterial factors.

IL-10–deficient mice are frequently used as a model of colitis (12) with chronic intestinal inflammation from an uncontrolled immune response induced by enteric antigens (13), which contributes to the impaired intestinal barrier function frequently observed in murine models of colitis (9) and in patients with Crohn’s disease (14).

Our aim was to use this model to study the effect of two lactic bacteria, Streptococcus thermophilus 065 and Bifidobacterium breve C50, on inflammatory conditions often observed in IL-10–deficient mice and their capacity to modify the immune and intestinal barrier functions in these mice. Special emphasis was placed on the comparison between the effect of bacterial secretion products and that of living bacteria.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Bacteria-Conditioned Media.
The two lactic acid bacterial strains, Bifidobacterium breve C50 and Streptococcus thermophilus 065, provided by Bledina-SA (Steenvoorde, France), were cultured on brain-heart (BH) medium or lactose-enriched M20 medium, respectively, for 24 hrs in anaerobic conditions. Bacteria were pelleted, extensively washed, and placed for 48 hrs in a simplified medium consisting of RPMI containing 20% fetal calf serum (FCS) and 3% inulin (B. breve) or 3% D-glucose (S. thermophilus). Bacterial conditioned media (CM) corresponding to 5 x 105 and 8 x 105 CFU/ml of S. thermophilus and B. breve, respectively, were ultracentrifuged (100,000 g), filtrated on 0.22-µm membrane, and frozen (–80°C) until use.

Experimental Protocol.
Forty-eight female IL-10–deficient C57BL/6 mice (aged 5 weeks at the beginning of experimental protocol) were used. The animals were housed under specific pathogen-free conditions for 2 weeks before being transferred to a conventional litter and fed a standard nonsterile diet. Our aim was to analyze the effect of a 10-week oral treatment with a mixture of either the two living lactic acid bacteria (B. breve and S. thermophilus) or their corresponding culture-conditioned media on the appearance of colitis and changes in the intestinal barrier and immune response. Experiments were conducted in accordance with the ethical guidelines of the French Veterinary Department.

The control group comprised IL-10–deficient mice (n = 12) that, from the age of 6 weeks, received a daily placebo treatment consisting of 200 µl of phosphate-buffered saline (PBS) administered by oral gavage (using a stainless steel feeding tube) for 10 weeks. The H. hepaticus group comprised IL-10–deficient mice (n = 12) that were infected with H. hepaticus (strain CCUG 33637, ATCC 51448) by two oral gavages with 2 x 109 CFU in 500 µl at the age of 5 weeks. One week later, the treatment protocol commenced, during which the animals received a daily placebo consisting of 200 µl of PBS for 10 weeks. The CM group consisted of H. hepaticus–infected IL-10–deficient mice (n = 12) that were treated daily for 10 weeks by oral gavage with 200 µl of a mixture (1/1) of CM obtained from B. breve and S. thermophilus. These CM were prepared as already described (15). The LB group consisted of 12 H. hepaticus–infected IL-10–deficient mice that received 5 x 105 CFU of a mixture (1/1) of living bacteria (LB), B. breve and S. thermophilus, by oral gavage for 10 weeks. At the end of the treatment protocol, all of the mice (aged 16 weeks) were sacrificed to collect colon samples and intestinal lymph nodes. Distal colon fragments were fixed in 4% formaldehyde for histologic examination, and additional fragments were stored at –80°C before extraction of mRNAs.

H. hepaticus infection was confirmed in all infected mice by seminested polymerase chain reaction (PCR) in stool samples. After DNA extraction from the fecal samples using the QIAamp DNA stool minikit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions, a PCR was performed using primers F2 16S and R4 16S, detecting conserved bacterial 16S ribosomal DNA (16). These two oligonucleotides produced an amplified product of 1490 bp; 2.5 µl of the fecal DNA preparation was added to a 25 µl (final volume) reaction mixture containing x1 reaction buffer (with 15 mM MgCl2), 0.5 µM each of the two primers, 200 µM each deoxynucleotide, 0.1 µg/µl of bovine serum albumin, and 0.025 U/µl of Taq polymerase (Promega, Charbonniéres les Bains, France). The following conditions were used for amplification: an initial denaturation at 94°C for 5 mins was followed by 40 cycles (denaturation at 94°C for 30 secs, annealing at 55°C for 30 secs, elongation at 72°C for 1 min 20 secs), and a final elongation at 72°C for 7 mins. After purification with a Sephacryl S-400 high-resolution (Amersham Biosciences, Saclay, France) column, a second set of primers was used for the amplification of a Helicobacter spp specific 16S ribosomal DNA fragment. These two oligonucleotides, 5'-GCTATGACGGGTATCC-3' (C97) and 5'-GATTT-TACCCCTACACCA-3' (C98), produced an amplified product of 398 bp. The reaction mixture was the same as that used for the first amplification but without bovine serum albumin. After a denaturation at 94°C for 5 mins, 40 cycles (denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and elongation at 72°C for 30 secs) and a last elongation at 72°C for 7 mins were performed. PCR was still positive in all infected mice 4 weeks after infection.

Clinical and Histological Examination.
Weight Gain.
Mice were weighed twice a week, and their general behavior was recorded.

Histological Analysis.
Tissues were fixed in 4% formaldehyde, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin. Sections were examined blind by a single pathologist (A.R.). Histological findings were reported using a microscopic score ranging from 0 to 4 as previously described (17). This score is based on the degree of epithelial layer erosion, goblet cell depletion, and inflammatory cell infiltrate; (0, normal; 1, minimal evidence of inflammatory infiltrate; 2, significant evidence of inflammatory infiltrate, e.g., cryptitis, crypt abscesses; 3, significant evidence of inflammatory infiltrate with goblet cell depletion; 4, significant evidence of inflammatory infiltrate with erosion of the mucosa).

Preparation of Soluble H. hepaticus and E. coli Soluble Extracts.
A soluble extract of Escherichia coli was prepared from cultures of E. coli (strain isolated from human microflora). The bacteria were harvested and washed extensively in PBS, then lysed by sonication at 4°C. Cell debris were removed by centrifugation at 8000 g for 30 mins; the supernatant was sterile filtered, and protein concentration was determined to be 1.2 mg/ml. Soluble H. hepaticus was prepared from cultures of H. hepaticus (strain CCUG 33637, ATCC 51448) according to the same protocol, leading to a final protein concentration of 1 mg/ml.

The LPS content of the bacterial supernatants after sonication, as measured using the Limulus amebocyte lysate determination (Charles River, Orléans, France) was 8.6 µg/ml for E. coli and <1 ng/ml for H. hepaticus.

Activation Status of Mesenteric Lymph Node Cells.
Mesenteric lymph nodes (MLNs) were collected at sacrifice, and single-cell suspensions were prepared in Cerottini culture medium (DMEM glutamax supplemented with 8% heat-inactivated fetal calf serum; asparagine, 36 mg/liter; arginine, 116 mg/liter; folic acid, 10 mg/liter; HEPES, 1 g/liter; ß-mercaptoethanol, 0.05 mM; penicillin, 100 U/ml; streptomycin, 100 µg/ml; fungizone, 1 µg/ml). Generally, the total cell count was approximately 20 x 106 cells/mouse, which was then adjusted to 2 x 106 cells/ml in culture medium.

Cytokine Secretion: Enzyme-Linked Immunosorbent Assay (ELISA) and Flow Cytometry.
ELISA.
MLN cells were seeded on 24-well culture plates in duplicate at 2 x 106 cells/well in 1 ml of Cerottini culture medium with or without stimulation by soluble H. hepaticus (1 µg/ml) or soluble E. coli (1 µg/ml) in duplicate. After 72 hrs, supernatants were collected and frozen at –20°C until assayed. Three proinflammatory cytokines, IFN{gamma}, TNF{alpha}, and IL-12, were tested using duoset ELISA kits (R&D, Abingdon, UK).

Flow Cytometry.
IFN{gamma} secretion by MLN cells in basal conditions without stimulation was assessed by flow cytometry using the mouse IFN{gamma} secretion assay (Miltenyi Biotec, Paris, France). MLN cells were seeded for 16 hrs on 24-well culture plates at 106 cells/well in 1 ml RPMI containing 5% mouse serum. The cells were then washed in cold buffer, and capture antibody against IFN{gamma}/CD45 was added for 5 mins on ice, after which the samples were incubated for 45 mins at 37°C under slow continuous rotation to allow cytokine secretion. Samples were washed and treated with anti-IFN{gamma} APC-conjugated detection antibody for 10 mins on ice. The cells were then counter-stained with monoclonal antibody against CD3-PE, CD4-FITC or CD3-PE, CD8-FITC. A total of ~100,000 mesenteric lymph node cells/mouse were passed in the flow cytometer (FACS Calibur and CELLQuest software, Becton-Dickinson, Pont de Claix, France) to count the IFN{gamma}-secreting cells in the selected CD3+CD4+ and CD3+CD8+ cell populations. Gated cells comprised about 30,000 cells (CD4 or CD8), and the absolute number of IFN{gamma}-secreting cells varied from 20 (control mice) to 1400 (CM mice).

Activation Markers on T Cells.
CD44 and CD62L markers were used to measure T-cell activation. MLN cells were stained with anti-CD3-PE and anti-CD44-FITC antibodies (Becton Dickinson, Pont de Claix, France). CD62L staining was achieved using a biotinylated anti-CD62L antibody, which was detected by a PE-cyanin7-streptavidin conjugate (Becton Dickinson).

Activation Status in Colonic Mucosa: Quantitative PCR Analysis.
Distal colonic fragments (3 to 5 mm) were placed in RNA later (Ambion, Austin, TX) and stored at –80°C until use. The fragments were placed in RLT buffer (Qiagen) and homogenized using a Fast Prep apparatus. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Three micrograms of total RNA were reverse-transcribed for 75 mins at 37°C in a reaction mixture containing 50 µg/ml of random hexamers (Promega), 10 IU/µl of Moloney murine leukemia virus reverse transcriptase (M-MLV RT), x1 RT buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl, 15 mM MgCl2), 0.01 M dithiothreitol, 125 lM of each dNTP, and 2 IU/µl of RNaseOUT (Invitrogen, Cergy Pontoise, France). After denaturation of the reverse transcriptase, cDNAs were stored at –20°C.

IL-12 mRNA was quantified by real-time RT-PCR using the corresponding primers and taqman probes provided by Applied Biosystems (Assay-on-demand) and the TaqMan Universal PCR Master Mix (Applied Biosystems, Courtaboeuf, France). IFN{gamma} expression was quantified using SYBR green PCR Master Mix (Applied Biosystems) and 900 nM of the forward and reverse primers (18). The PCR reaction was carried out using an ABI PRISM 7700 sequence detection system. After an initial step at 95°C for 10 mins, 40 cycles were performed as follows: denaturation at 95°C for 15 secs and annealing and extension at 60°C for 1 min. Data were referred to the expression of TATA box binding protein by calculating 2–{Delta}CT, with {Delta}CT the difference in threshold cycles of target and housekeeping genes. All results are expressed as arbitrary units (AU).

Epithelial Barrier Function of the Colonic Mucosa.
Distal colon fragments from all mice were opened along the mesenteric border, layered on a Millipore filter (8-µm pores), and mounted as flat sheets in Ussing chambers with an exposed surface area of 0.2 cm2. They were bathed on both sides with 1.5 ml of Ringer solution containing 5 mM mannitol, which was continuously thermostated, circulated, oxygenated, and maintained at pH 7.4 with 5% CO2/95% O2. The mucosal and serosal bathing solutions were connected via agar bridges to calomel electrodes for measurement of the transepithelial potential difference (PD) and to Ag-AgCl electrodes for current ({Delta}I) application. The tissue was kept under open-circuit conditions, and electrical measurements were performed using a DVC 1000 voltage/current clamp (World Precision Instruments, Aston, UK). The tissue was regularly clamped at 1 mV to measure {Delta}I and to calculate the electrical resistance (R) according to Ohm’s law ({Delta}PD = R x {Delta}I).

Horseradish peroxidase (HRP; mol wt, 40 kDa) was used as a soluble protein marker of the transcellular transport pathway (transcytosis). Mannitol was used as a small molecular tracer (mol wt, 182 daltons) of the paracellular pathway. HRP (0.4 mg/ml), [3H]HRP (37 kBq/ml) and [14C]mannitol (12.2 kBq/ml) were simultaneously added to the mucosal compartment bathing intestinal fragments. Samples (800 µl) were taken from the serosal compartment at 10, 30, 50, 70, 90, and 110 mins and replaced by fresh Ringer solution. Intact HRP transport from the mucosal to the serosal compartment was determined by enzymatic assay (19) on 200 µl of serosal samples. Total HRP (intact + degraded = 3H-equivalent HRP) and [14C]mannitol fluxes were assessed on 500-µl serosal samples by counting the 3H and 14C radioactivity using liquid scintillation photometry (Kontron, Betamatic) after double-labeling correction, and are expressed as ng/hr·cm2 and nmol/hr·cm2, respectively. Degraded-HRP fluxes were calculated as total minus intact HRP fluxes. Mean steady-state fluxes obtained from 50 to 110 mins are presented.

Statistical Analysis.
Statistical analysis was performed using the SAS package. The results are expressed as mean ± SE, and the comparison of different parameters among the groups was performed by using analysis of variance and nonparametric tests (Wilcoxon and Kruskal-Wallis). The general linear model procedure was used for multiple group-to-group comparisons. The differences were considered significant for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Weight Gain.
There were slight differences in weight gain expressed as a percentage of initial weight among the different groups of mice during the experimental protocol. Figure 1AGo indicates that LB mice had the lowest growth rate. The treatment of H. hepaticus–infected mice with bacteria-conditioned medium seemed to improve the growth rate as compared to untreated infected mice.



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  		Figure 1. (A) Body weight curves of control IL-10–deficient mice, those infected with H. hepaticus alone (H. hepaticus mice), or those infected with H. hepaticus followed by treatment with either 5 x 105 CFU/day of living B. breve and S. thermophilus (LB mice) or their culture-conditioned media (CM mice). (B) Histological examination of mucosal lesions (grades 0 to 4) observed in distal colon. H. hepaticus–infected mice presented a significantly higher score than control mice. Treatment with CM or LB had no additional effect on histological score. *P < 0.002 as compared with control mice.

 
Colonic Microscopic Score in Mice.
Figure 1BGo shows the histological scores obtained in all of the mice. As compared with control mice (score 0.82 ± 0.18), H. hepaticus–infected mice had a small but significant increase in the inflammatory infiltrate (score 2.10 ± 0.40, P < 0.002), corresponding to mild colitis. This score was not modified in CM- or LB-treated mice (score 2.16 ± 0.29 and 2.28 ± 0.42, respectively).

Function of Mesenteric Lymph Node Cells.
Spontaneous and Stimulated Cytokine Secretion as Measured by ELISA.
The TNF{alpha}, IL-12, or IFN{gamma} secretion after stimulation with soluble H. hepaticus extract did not differ from the basal secretion of these cytokines (data not shown). Figure 2Go illustrates the secretion profiles of the three proinflammatory cytokines by MLN cells in basal or soluble E. coli–stimulated conditions. The basal secretion levels of IFN{gamma}, TNF{alpha}, and IL-12 were low (< 70 pg/ml) and similar in all groups of mice. After stimulation with soluble E. coli extracts, these secretion levels were not significantly modified in control mice. However, in H. hepaticus–infected mice, E. coli–stimulated secretion of TNF{alpha} was higher (685 ± 209 pg/ml) than in control mice (184 ± 45 pg/ml, P < 0.02), and E. coli–stimulated secretion of IL-12 tended to be higher (197 ± 39 pg/ml) than in control mice (104 ± 25 pg/ml, P = 0.08), suggesting a higher susceptibility of macrophages/dendritic cells to nonspecific LPS activation in these mice. The most striking effect was an eightfold increase in E. coli–induced IFN{gamma} secretion in the CM group (1594 ± 774 pg/ml) as compared with the control (88 ± 50 pg/ml), H. hepaticus (202 ± 107 pg/ml), and LB (0.6 ± 0.6 pg/ml) groups (P < 0.005), indicating a Th1 skewing of the immune response. Significantly higher TNF{alpha} (x1.6) and IL-12 (x1.5) secretion levels in response to the E. coli extract were also observed in CM mice (1090 ± 333 and 299 ± 74 pg/ml, respectively) as compared to H. hepaticus mice (685 ± 209 and 197 ± 39 pg/ml, P < 0.05), although to a much lesser extent than for IFN{gamma}.



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  		Figure 2. Cytokine secretion by mesenteric lymph node cells under basal conditions or after 72-hr stimulation with soluble E. coli. #Significantly different from E. coli–stimulated control and H. hepaticus mice (P < 0.05). *Significantly different from matched unstimulated cells (P < 0.0001).

 
IFN{gamma} Secretion by MLN Cells as Measured by Flow Cytometry.
Both CD4+ and CD8+ T cells were studied. There was an increase in the number of CD4+ T cells secreting IFN{gamma} in the CM mice (2.06 ± 0.37%) as compared with the control (0.67 ± 0.19%, P < 0.005) or H. hepaticus–infected mice (1.1 ± 0.32%, P < 0.05) (Fig. 3AGo). The same tendency was observed for CD8+ T cells secreting IFN{gamma} (1.98 ± 0.52% in CM mice vs. 0.76 ± 0.26%, P < 0.02; and 0.47 ± 0.09%, P < 0.01; in control and H. hepaticus mice, respectively). In LB mice, the percentage of CD4+ T cells secreting IFN{gamma} (1.95 ± 0.67%) was higher than that observed in control mice (0.67 ± 0.19%, P < 0.03), but not in H. hepaticus mice (1.1 ± 0.32%). This difference was not observed for CD8+ T cells (0.57 ± 0.16%, 0.76 ± 0.26%, and 0.46 ± 0.09% in LB, control, and H. hepaticus mice, respectively).



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  		Figure 3. (A) Spontaneous IFN{gamma} secretion by MLN cells measured by flow cytometry. Results are presented as the percentage of cells secreting IFN{gamma} among the population of CD4+ or CD8+ T cells. (B) CD4+ or CD8+ T cell activation status measured by flow cytometry using the expression of CD44 and CD62L taken as activation markers. *Significantly different from control, H. hepaticus, and LB mice (P < 0.05). #Significantly different from control mice (P < 0.03).

 
It is apparent that, although IFN{gamma} secretion was significantly higher in CD4+ T cells from both CM and LB mice than in H. hepaticus mice (P < 0.03), in CD8+ T cells, such a secretion was significantly increased in CM mice only (P < 0.05) but not in LB mice.

Expression of T Cell Activation Markers CD44 and CD62L by Flow Cytometry.
T cell activation is associated with high expression of CD44 and low expression of CD62L. Figure 3BGo indicates that the expression of CD44 tended to be higher in CM mice, but the difference was statistically significant only for LB mice (31.5 ± 1.9% vs. 23.2 ± 2.4%, P < 0.02). There was a slight decrease in CD62L expression in CM mice as compared with the other groups, but the difference was not statistically significant (P = 0.1).

Expression of Cytokines by Quantitative PCR on Distal Colonic Mucosa.
As shown in Figure 4Go, a significant increase in IFN{gamma} mRNA expression was observed in distal colon of CM mice (2.4 ± 0.6 AU, P < 0.001) and LB mice (1.5 ± 0.8 AU, P < 0.05) as compared with H. hepaticus (0.5 ± 0.2 AU) and control mice (0.05 ± 0.02 AU). No significant variation in IL-12 expression was found.



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  		Figure 4. Quantification of cytokine mRNA in the distal colon as measured by real-time PCR and reported as arbitrary units (AU). IFN{gamma} mRNA expression was significantly increased in CM mice (P < 0.001) and LB mice (P < 0.05) as compared with control and H. hepaticus mice. There were no statistically significant differences in IL-12 mRNA expression among the different groups.

 
Epithelial Barrier Function in Distal Colonic Mucosa.
The parameters reflecting paracellular permeability (mannitol fluxes and electrical resistance) and transcellular macromolecular transport (HRP fluxes) in distal colon are reported in Figure 5Go. As compared with control mice, CM mice presented lower HRP transport as attested by the significant decrease in degraded-HRP fluxes (P < 0.02) and, to a lesser extent, intact-HRP fluxes (P = 0.09). In contrast, LB mice presented similar HRP fluxes as control or H. hepaticus mice. Intestinal permeability along the paracellular pathway was also significantly decreased in the CM group as compared with the control and H. hepaticus groups, as attested by a significant decrease in mannitol fluxes (74 ± 11 vs. 116 ± 10 and 109 ± 14 nmol/hr·cm2, P < 0.05) correlating with a significant rise in electrical resistance R (53 ± 5 vs. 41 ± 4 and 40 ± 3 {Omega}·cm2, P < 0.05) in this group of mice. Thus, at the colonic level, both transcellular permeability to macromolecules and paracellular diffusion of small molecules were decreased by treatment with CM (but not with LB), indicating that bacterial secretion products are probably the active components involved in the improvement of intestinal barrier integrity.



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  		Figure 5. Measurement of colonic permeability in IL-10–deficient mice that either did or did not orally receive bacteria-conditioned media or living bacteria. Markers of transcellular (degraded-HRP fluxes) and paracellular (mannitol fluxes) permeability were significantly decreased in CM mice as compared with control and H. hepaticus mice. In parallel, a significant increase of electrical resistance (P < 0.03) and a tendency (P = 0.09) toward a decrease of intact-HRP fluxes in CM mice, as compared with the other groups, confirmed the reinforcement of the colonic barrier by bacteria-conditioned medium. No significant differences were observed in LB mice. *Significantly different from control and H. hepaticus groups (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that, in the absence of important inflammation, lactic acid bacteria–conditioned medium stimulates Th1 immune response and strengthens the colonic barrier, two effects likely to be beneficial in the maintenance of intestinal homeostasis and in immune defense. This study also indicates that both effects are stronger with the bacterial conditioned media than with the living bacteria.

Although IL-10–deficient mice have been widely used as a model of IBD (13), this model is highly conditioned by environmental and genetic factors, and spontaneous colitis is often mild and delayed. Successful induction of colitis in IL-10–deficient mice has been reported (10), but other studies have indicated that these mice do not develop severe colitis even after colonization with H. hepaticus (20). In our experimental setting, C57BL/6 IL-10–deficient mice infected by H. hepaticus developed only mild colitis, with limited histological damage.

As already mentioned, a clear dichotomy is observed between the anti-inflammatory effect of probiotics in inflammatory context and their immune stimulatory effects under physiological conditions. Indeed, lactic acid bacteria have the capacity to stimulate the immune response under basal conditions, both in vitro (15, 21) and in vivo, during vaccination in animals (1) or in humans (2). Lactic acid bacteria also stimulate secretory immune response to luminal pathogens. In addition, various species of Lactobacilli or Bifidobacteria have been shown to enhance the secretion of specific IgA, especially in case of rotavirus infection in children (22, 23). Nonspecific, anti-infectious mechanisms of defense also seem to be enhanced by ingestion of selected strains of lactic acid bacteria. In adults, the consumption of milk fermented with B. bifidum or L. acidophilus LA1 induced phagocytosis of E. coli, as assessed in vitro (24). The activation of the monocytes/macrophages (25) by ingestion of lactic acid bacteria may be responsible for the enhancement of innate immunity, an effect explained by the fact that cell walls or DNA of gram-positive bacteria are potent activators of toll-like receptors.

In contrast, lactic acid bacteria tend to moderate exacerbated cytokine secretion and to improve inflammatory conditions in humans (7) and in experimental models of colitis (10).

The experimental conditions achieved in the present study correspond to a subnormal rather than a pathological state because histological damage is moderate, and spontaneous proinflammatory cytokine secretion in intestinal mucosa and MLN cells is low. In these conditions, B. breve and S. thermophilus stimulated local Th1 immune response and reinforced the colonic epithelial barrier. Surprisingly, bacteria-conditioned media were more efficient in producing these effects than the living bacteria. Although viability of bacteria is considered a major aspect of probiotic activity, especially in lactose intolerance, there is emerging evidence that bacterial components or secretion products are involved in the effects on the immune system (15, 26). The mechanisms by which lactic acid bacteria or their conditioned media could act locally on intestinal and immune function were analyzed. First, the reactivity of intestinal immune cells in MLNs was tested under basal conditions and in response to specific (soluble H. hepaticus extract) and nonspecific (soluble E. coli extract containing LPS) stimuli. In our experimental setting, the basal secretion of cytokines by MLN cells was negligible, confirming the low score of colitis. However, in mice receiving bacteria-conditioned medium, cells did not respond to specific antigens (H. hepaticus) but gained the capacity to respond to a nonspecific stimulus (E. coli), a phenomenon not observed in control or H. hepaticus mice. This was attested to by an eightfold increase in IFN{gamma} secretion and to a lesser extent in TNF{alpha} and IL-12 secretion, by MLN cells of CM mice. A possible interpretation could be that CM mice received bacterial DNA present in bacteria-conditioned medium, a component shown to be immunostimulant and to act as adjuvant in healthy individuals (1, 27). The absence of effect of living bacteria could be because bacterial DNA in this case is not exposed in the intestinal lumen but hidden within the bacterial cells. Although bacterial DNA was shown to reduce inflammation in experimental colitis (26), under our subnormal conditions, this effect could be mainly immunostimulant.

In addition to polarizing T cells toward Th1 response, treatment with bacteria-conditioned medium had a positive effect on epithelial barrier, as evidenced by a reinforcement of the distal colonic barrier, both at the transcellular and paracellular levels. A decrease in HRP fluxes indicated lower transcytosis whereas decreased mannitol fluxes and increased electrical resistance reflected decreased paracellular diffusion and tight junctional complex reinforcement. These results are in agreement with those obtained in guinea pigs fed a dehydrated fermented (B. breve and S. thermophilus) milk (28) or in IL-10–deficient mice treated with VSL#3-conditioned medium (9).

In conclusion, in this context of mild inflammation, the administration of B. breve– and S. thermophilus–conditioned medium, in addition to reinforcing intestinal barrier function, strongly stimulated the Th1 immune system, a feature that may highlight the interest of lactic acid bacteria in immune defense and vaccination.


   Footnotes
 
S. Ménard was recipient of a fellowship from the "Ministére délégué à la Recherche." The work was partly supported by Blédina SA.

Received for publication June 8, 2005. Accepted for publication July 19, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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