QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gaudier, E. Articles by Hoebler, C. Search for Related Content PubMed PubMed Citation Articles by Gaudier, E. Articles by Hoebler, C.

---

Biochemical and Molecular Actions of Nutrients

The VSL# 3 Probiotic Mixture Modifies Microflora but Does Not Heal Chronic Dextran-Sodium Sulfate–Induced Colitis or Reinforce the Mucus Barrier in Mice^1,2

Human Nutrition and Gut Function Department, INRA Nantes, France

³To whom correspondence should be addressed. E-mail: hoebler{at}nantes.inra.fr.

ABSTRACT

The mucus layer covering the epithelium is one of the main lines of defense of the colonic barrier. Both mucus gel and mucin expressions are altered during colonic inflammation and could be involved in epithelial repair. We postulated that modulating colonic mucus and mucins by probiotic supplementation could contribute to healing inflammatory mucosa. Our aim in this study was to determine whether probiotics could repair dextran-sodium sulfate (DSS)-induced chronic colitis in mice, and whether modifications of the colonic mucins could be involved. For that purpose, the VSL#3 probiotic mixture of 8 lactic acid bacteria probiotic strains was administered daily for 2 wk to mice with a mucosa impaired by a mild DSS treatment, and to mice with a normal mucosa. Probiotic strains survived in the gastrointestinal tract, increased the cecal concentrations of bifidobacteria, and modified cecal microflora metabolic activity in both DSS-treated and healthy mice. However, probiotic supplementation did not reverse the inflammation induced by DSS at either the macroscopic or histological level. Concurrently, probiotics did not modify the colonic mucus barrier, in terms of either mucin gene expression or adherent mucus layer thickness. In conclusion, the modification of microflora by supplementation with the VSL#3 probiotic mixture did not help to repair the colonic barrier breakdown caused by DSS treatment. The potential healing roles of mucins were neither confirmed nor invalidated by this study.

KEY WORDS: • probiotics • Muc gene • colonic barrier • dextran sodium sulfate (DSS) • colitis

Normal colonic mucosa is protected by a barrier, composed of several lines of defense: the luminal commensal microflora, the mucus layer, the epithelium itself and its tight junctions, and the intestinal immune system, which interacts with the intestinal nervous system. During colonic inflammation, immune dysfunction is accompanied by a general breakdown of this colonic barrier. We will focus on one of these levels of the colonic barrier, i.e., the mucus layer covering the epithelium. Colonic mucus and its main components, known as mucins, are also altered in human colitis. Aberrant expression of mucin-encoding MUC genes as well as modifications of mucin protein expression (1–3) are observed in colitis, and the thickness of the mucus layer is reduced in ulcerative colitis (4). Animal models of colitis such as interleukin (IL)⁴ 10–/– mice or mice or rats with dextran sodium sulfate (DSS)-induced colitis, also present mucin alterations. IL10–/– mice have hypotrophic goblet cells in the colon but the gene and protein expression of Muc2 are not altered (5); in contrast, mucin expression is modified in DSS-induced colitis (6,7).

Mucins could be involved not only in the protection of the colonic barrier but also in mucosal repair. Indeed, several arguments support a putative role in colonic repair for mucins and trefoil factor 3 (TFF3), both components of colonic mucus. TFF3 properties of increasing mucosal repair are established in different models of inflammation: IL10–/– mice, DSS-induced (8,9) or methodextrate-induced (10,11). The major colonic mucin secreted, MUC2, is coexpressed with TFF3 in colonic goblet cells (12), and the simultaneous increase in their expression in DSS-colitis in rats has been interpreted, although not proven, as a repair reaction of the mucosa (13). Other mucins could also be involved in epithelial repair. For example, MUC5AC and MUC6 are overexpressed in ulcer-associated cell lineage in Crohn’s disease (1) and these cells are involved in epithelial restitution (14).

Numerous probiotic supplementation trials in animals and humans have been carried out to test their efficacy against colonic inflammation. Most of these studies demonstrated the preventive properties of probiotics against inflammation, but very few studies have established curative properties. Among them, modification of colonic microflora by administration of various lactic acid-producing bacteria [VSL#3 cocktail (15), Lactobacillus plantarum 299v (16), L. salivarius 433118, or Bifidobacterium infantis 35624 (17)] decreases the intensity of well-established colitis in IL10–/– mice. The VSL#3 probiotic mixture prevents DSS-induced colitis in mice but has not been tested as a therapeutic treatment (18). In humans, the efficacy of microflora modulation by probiotic administration in healing colonic inflammation has not been demonstrated but relapse prevention properties were proven effective for several probiotic strains. The VSL#3 probiotic mixture is particularly effective in preventing pouchitis relapse (19). VSL#3 probiotics also have promising properties in maintaining remission in ulcerative colitis (UC) (20,21). A mixture of enterococci, bifidobacteria, and lactobacilli, known as BIFICO, succeeded in maintaining remission in UC (22), and the nonpathogenic Escherichia coli strain Nissle was as effective as mesalazine in preventing UC relapse (23,24). In all of these in vivo studies, the effect of the administration of probiotics on colonic mucins has never been investigated, nor has their involvement in colitis prevention or repair.

Our hypothesis was that the VSL#3 probiotic mixture would attenuate the inflammation preinduced by DSS treatment in mice by inducing modifications in colonic mucins. Indeed, the VSL#3 cocktail was recently shown to increase MUC2, MUC3, and MUC5AC but not MUC1 gene expression in vitro in HT29 colonic cells (25). The VSL#3 probiotic cocktail was shown to prevent DSS-induced colitis (18) but, to our knowledge, its curative properties in the DSS model of colitis have never been investigated. The aim of the present study then was to determine for the first time in vivo whether VSL#3 probiotics could repair DSS-induced chronic colitis in mice, and whether modifications of one of the main lines of the colonic barrier defense, i.e., the mucus layer, could be involved.

MATERIALS AND METHODS

Mice and treatments

Male BALB/c mice (Janvier), 8 wk old, with an initial mean weight of 20.8 ± 0.1 g, were housed in groups of 6 mice/cage and maintained at 23°C in an animal room with a 12-h light:dark cycle (light: 0700–1900 h). Food and water were consumed ad libitum; food intake and body weight were recorded every other day. The diet was formulated according to the AIN-93G standard (26) (INRA, Jouy-en-Josas). All experiments were carried out in accordance with the recommendations of the local Animal Care and Use Committee of Nantes (France).

    Induction of mild experimental colitis. At d 0, mice (n = 120) were randomly assigned to 2 groups, referred to as DSS+ or DSS–. Colonic inflammation was induced in DSS+ mice who received 1% (wt/v) DSS in drinking water for 5 d; the colitis was then maintained by adding 0.2% DSS to the drinking water for 14 d. We showed in a previous study that these doses are adequate to induce and then maintain moderate chronic colitis in mice (27). DSS –mice received only drinking water during the 19-d experimental period.

    Treatment with the VSL#3 probiotic mixture. At d 5, the 2 mouse groups were again split into 2 groups, leading to the final 4 groups (n = 30 each) referred to as DSS–/VSL–, DSS–/VSL+, DSS+/VSL–, and DSS+/VSL+. A 0.2 mL-probiotic suspension was administered once a day to VSL+ mice by gastric gavage over 14 d. The VSL#3 probiotic mixture combines 8 strains of lactic acid–producing bacteria (L. plantarum, L. delbrueckii subsp. Bulgaricus, L. casei, L. acidophilus, Bifidobacterium breve, B. longum, B. infantis, and Streptococcus salivarius subsp. thermophilus) (20) (provided by Dr. C. De Simone, Department of Microbiology, University of L’Aquila, L’Aquila, Italy). The VSL#3 suspension was prepared daily by diluting 4 x 10⁹ colony-forming units (cfu) of freeze-dried VSL#3 in sterile saline. VSL– mouse groups were administered 0.2 mL sterile saline once a day as a control.

Collection of digestive tissues and contents

At the end of the 19-d experimental period, the mice were killed by cervical dislocation. The colon length was first measured in a relaxed position without stretching. Three segments (cecum, proximal colon, and distal colon) were then removed; cecal contents were aseptically collected and immediately used for bacterial enumeration (n = 10/group) or for microbial metabolic activity assessment (n = 10/group). Cecal, proximal, and distal colonic mucosa were cut longitudinally and cleaned with sterile physiological serum. After the evaluation of macroscopic damage, they were immediately frozen with 1 mL of Trizol (Invitrogen Life Technologies) in liquid nitrogen and stored at –70°C for RNA isolation (n = 10/group), or fixed in 4% neutral buffered formaldehyde, dehydrated, and paraffin-embedded for histological observations (n = 10/group). To measure mucus layer thickness, colonic segments with their contents (n = 10/group) were taken from the distal colon 1 cm proximal to the anus, frozen in liquid nitrogen, and stored at –70°C.

Assessment of colonic damage and inflammation

    Body weight gain, evolution of stool diarrhea score, and bloody stool score. Macroscopic symptoms (body weight, stool consistency, presence of blood in stool) were assessed every other day during the course of the experiment. The following scores were given to stool consistency: 0: formed stool; 1: formed and soft stool; 2: not formed stool; and 3: diarrhea. The presence of fecal blood was assessed with Hemocult II (SKD) and given a score of 4. The total stool score was the sum of the blood presence score and the stool consistency score.

    Measurement of colon length; macroscopic and histological scoring of mucosa inflammation. At the end of the study, macroscopic damage to cecal and colonic mucosa was scored by visual evaluation without knowledge of the treatment group using the scale previously described (27). For histological observations, sections (8 µm) of paraffin-embedded tissues were stained with hematoxylin and eosin, and observed by microscopy for inflammation scoring. Histological crypt scoring for inflammation was adapted from Cooper et al. (28): grade 0: intact crypt (Fig. 1A); grade 1: neutrophil infiltration and loss of the basal part of the crypt (Fig. 1B and C); grade 2: neutrophil infiltration, with loss of entire crypt but surface epithelium remaining intact (Figs. 1B and C); grade 3: neutrophil infiltration with loss of both the entire crypt and surface epithelium (Fig. 1C). The proportion of mucosa length area involved for each grade (0–3) was quantified by image analysis with Lucia software (Laboratory Imaging). The histological score for the cecum, proximal colon, and distal colon was calculated as the sum of the products of each grade by the proportion of the length involved. All slides were reviewed by 2 separate researchers who were unaware of the treatments and a mean of their histological score was calculated for each slide.

View larger version (76K): [in this window] [in a new window]   FIGURE 1 Histological damage and scoring in mice subjected to inflammatory treatments. Example of various grades in mouse proximal colon. 0: intact crypt (A); 1: neutrophil infiltration and loss of the basal part of the crypt (B–C); 2: neutrophil infiltration, with loss of entire crypt but surface epithelium remaining intact (B–C); 3: neutrophil infiltration with loss of both the entire crypt and surface epithelium (C). Bars represent 100 µm.

    Real-time PCR quantification of tumor necrosis factor (TNF)- gene expression.

Analysis of cecal microflora composition and activity

    Bacterial enumeration and PCR identification. Cecal contents were immediately serially diluted 10-fold with anoxic half-strength peptone water supplemented with cysteine (0.5 g/L, Sigma); 100 µL of the appropriate dilutions was spread onto selective media for Lactobacillus spp. (Rogosa Agar, Oxoid, Unipath) and for Bifidobacterium spp. (Beerens Agar, Oxoid). Plates were incubated in an anaerobic chamber (H₂:CO₂:N₂, 5:10:85) at 37°C for 72 h. After incubation, colonies were counted. The detection limit was 10⁴ cfu/g wet content.

For each cecal sample and incubation medium, all different types of colonies were aseptically collected, diluted in 100 µL DNase-free water, and stored at –20°C. Colonies collected on Rogosa Agar were tested to determine whether they belonged to Lactobacillus spp. by PCR amplification using species-specific primers R16-l/LbLMA1-rev (29). Colonies grown on Beerens Agar were tested to determine whether they belonged to Bifidobacterium spp. by PCR amplification using species-selective primers g-Bifid-F and g-Bifid-R (30), and also tested for identification of the VSL#3 mixture strain B infantis using strain-specific primer InfY-BV.L/R (31). Then, 2 µL of the diluted colony was amplified directly using Taq DNA polymerase (Promega) without DNA extraction. Denaturation for 5 min at 95°C was followed by 40 cycles composed of 30 s denaturation at 95°C, 30 s hybridization, and 1 min elongation at 72°C. The last cycle was followed by a 7-min final elongation at 72°C. The hybridization temperature was 55°C for R16-l/LbLMA1-rev and gBif primers, and 64°C for InfY-BV.L/R primers. PCR products were run on a 1.5% agarose gel. Only the colonies whose identity was confirmed by PCR were included in the final calculation, and the results were expressed as the log₁₀ of cfu/g cecal contents.

    Assessment of cecal microflora metabolic activity. A commercially available system for biochemical fingerprinting was used: Pheneplate PhP-48 (BioSysinova), consisting of a microtiter plate with 46 dehydrated biochemical substrates (32–34). Inside the anaerobic chamber, cecal contents (n = 10 mice/group) were diluted in 10 mL dilution medium (BioSysinova), and 150 µL of this solution was added to each of the 48 wells of PhP-48, with 100 µL of mineral oil to preserve anaerobiosis. Plates were incubated at 37°C and absorbance at 630 nm was measured every hour for 13 h. The absorbance slope between 2 and 13 h of incubation made it possible to calculate the degradation activity of each substrate.

Mucin and mucus characterization

    Quantification of mucin and trefoil peptide gene expression by real-time RT-PCR. Mucin and TFF3 gene expressions in cecal and colonic mucosa were quantified following the same procedure used for TNF-, as described above. The sequences of the primers used for Muc1, Muc2, Muc3, Muc4, and TFF3 cDNA amplification were the same as those previously described (27).

    Histological morphometry of mucus thickness in the distal colon. To measure mucus layer thickness, transverse sections (20 µm) of frozen distal colon were cut with a cryostat (Microm HM 500OM, Carl Zeiss) and fixed on SuperFrost Plus Gold slides (Menzel-Glaser) (35). Sections were stained with alcian blue pH 2.5-periodic acid Schiff (36). Microscope images (Nikon) of the stained sections were analyzed with Lucia software (Laboratory Imaging). The adherent mucus layer thickness was measured at 5 points of the circumference of 6 different sections for each mouse. The mean of these 30 measurements was considered to be the adherent mucus thickness for each mouse.

Statistical analysis

Statistical analysis was performed using the Statview 5.0 package (SAS Institute). Data from the bacterial counts, inflammation characteristics, and gene expression levels were analyzed using 2-way ANOVA with DSS treatment and VSL#3 supplementation as the main factors. When differences were detected by ANOVA, differences among groups were determined by Fisher’s Protected Least Significance test, followed by the analysis of interaction curves. Serial assessments of DSS and VSL#3 treatments on total stool scores over time were analyzed by 2-way repeated-measures ANOVA. The effect of VSL#3 and DSS treatments on the microflora activity, assessed using the degradation of 46 substrates present on Pheneplates, was determined using UPGMA clustering (37), based on Pearson-product-moment correlation. Phylograms were created online (38,39). Values from each mouse were considered as the experimental unit of all response variables. Data are expressed as means ± SEM. Differences between means were considered significant at P 0.05.

RESULTS

    Microflora composition and degradation activity. First, most of the VSL#3 probiotics-treated mice harbored positive colonies for B. infantis Y1 DNA amplification, with the count of viable B. infantis Y1 reaching a mean of 10⁶ cfu/g cecal wet content (Table 1). This indicates that at least some strains of the probiotic mixture survived gastrointestinal transit and remained viable in the mouse cecum. Surprisingly, colonies providing positive responses to PCR identification performed with specific primers for B. infantis Y1 strain were obtained in 2 of 8 mice with no probiotic supplementation. More generally, plating on selective medium followed by PCR identification of the bacterial colonies established that VSL#3 probiotic supplementation significantly increased the cecal count of Bifidobacteria spp., and tended to increase the cecal count of Lactobacillus spp. (P = 0.100) (Table 1). DSS treatment had no effect on these bacterial counts, and there was no interaction between DSS and probiotic treatments.

    Assessment of colitis. Both clinical symptoms (Fig. 2) and mucosal characteristics (Table 2) showed that DSS treatment induced and maintained a mild colitis, and that the administration of the VSL#3 probiotics mixture did not heal this colitis. Indeed, stool scores were higher in DSS-treated mice than in nontreated mice from d 6 until the end of the experiment (d 19, P < 0.001), with the peak occurring at d 6 (Fig. 2). The induced colitis remained mild because the body weight gain was not significantly affected by DSS treatment (2.1 ± 0.2 g in 19 d for DSS+ mice vs. 2.3 ± 0.2 g in 19 d for DSS– mice). The alteration of colonic mucosa by DSS treatment was also confirmed by several characteristics measured at the end of the experiment (Table 2): DSS treatment significantly decreased the length of colon, increased the macroscopic inflammatory scores all along the cecocolonic tract and the histological scores in the cecum and the proximal colon. DSS treatment also significantly increased the level of TNF- gene expression in the proximal colon, and tended to increase it in the distal colon (P < 0.100). Probiotic treatment did not change any of these inflammatory characteristics, except for a tendency to decrease the histological score of inflammation in the proximal colon of DSS-treated mice (P < 0.100). The interaction between DSS and probiotic treatments was significant for the histological score in the proximal colon because VSL#3 probiotics tended to decrease histological scores in the proximal colon of DSS+ mice but not in the proximal colon of DSS– mice. The interaction between treatments also tended to be significant in the distal colon (P < 0.100).

View larger version (15K): [in this window] [in a new window]   FIGURE 2 Evolution of total stool score in mice that were or were not subjected to probiotic and inflammatory treatments. Values are means ± SEM; n = 10/treatment. DSS treatment significantly increased total stool score over time from d 6 to 9, P < 0.0001.

View larger version (119K): [in this window] [in a new window]   FIGURE 3 Periodic acid Schiff-alcian blue stained sections of mouse distal colon in mice that were (C and D) or were not (A and B) subjected to inflammatory treatments, and administered probiotics (B and D) or saline (A and C) during the last 14 d. The arrows point out the adherent mucus layer. Bars represent 100 µm.

DISCUSSION

The aim of the present study was to determine whether a VSL#3 probiotic cocktail delivered in vivo in mice was able to reduce moderate colitis induced and maintained by DSS treatment, and to modulate one of the colonic barrier components that could be involved in mucosal repair, i.e., mucus. We established that supplementation with the VSL#3 probiotic mixture modified the cecal microflora composition and activity but did not heal DSS-induced colitis and had no effect on colonic mucins and mucus. We will discuss the effect of probiotic supplementation on microflora and mucins, and the possible reasons for the ineffectiveness of the VSL#3 mixture in healing DSS-induced colitis in mice.

First, there was evidence to prove that VSL#3 probiotic bacteria that were delivered by gastric gavage did survive in the upper gastrointestinal tract and remained active (either directly or through interactions with the endogenous bacteria, as discussed below) in the cecal contents, in both normal and DSS-treated mice. We detected the B. infantis Y1 strain from the VSL#3 mixture surviving in the cecal content of most of the supplemented mice. Specific DNA probes for identification of other bifidobacteria strains of the VSL#3 mixture were published in the literature (31,40,41). Nevertheless, under our experimental conditions, none succeeded in detecting their target strain, in either the VSL#3 cocktail or the contents of VSL#3-supplemented mice. As a consequence, we were not able to quantify all VSL#3 strains and thus to establish whether the global increase we observed in both Bifidobacterium spp. and in Lactobacillus spp. stemmed only from the supplementation or was the result of a stimulated development of the commensal Bifidobacterium spp. and Lactobacillus sp. In addition to modifying the intestinal microbiota composition, probiotics are able to affect some microbiotal activities (42,43). Whether these changes are carried out by bacterial enzymes from the probiotics or from the indigenous microbiota as modified by the probiotics is not clear; however, considering the intimate relation between colonic microbiota activity and large gut health (44), such changes could contribute to the mechanisms by which probiotics interact with gut physiology. The effect of VSL#3 on the metabolic activity of the intestinal microbiota has rarely been assessed in previous VSL#3-supplementation assays and only a few activities have considered its effect. Brigidi et al. (45) showed that it increased ß-galactosidase and decreased urease fecal activities in humans. To achieve a more complete view of the VSL#3effect on microbiotal activity, we chose to assess it using the Ph-plate® system. This approach has proven useful in describing the effect of some dietary compounds on microbial activity (32,46). Thus, we demonstrated that supplementation with the VSL#3 cocktail tended to increase the metabolic potential of the cecal microbiota; in particular, there was an increased ability to ferment raffinose and a tendency to increase the utilization rate of galactose, maltose, palatinose, sucrose, lactose, lactulose, melibiose, sorbitol, and amygdalin. Considering that most of the VSL#3 species are able to ferment these sugars (47), and that amygdalin and sorbitol are fermentable substrates for 2 of these species [L. casei and L. plantarum (47)], this observation would sustain the hypothesis of metabolic changes carried out by bacterial enzymes from the probiotic. However, because of the metabolic redundancy of the intestinal microbiota (the utilization of the specific simple substrates studied is not related to a specific bacterial type but could be attributable to more than one bacteria), the real metabolic activity of the VSL#3 strains during their transit through the gastrointestinal tract has yet to be demonstrated.

Second, we hypothesized that VSL#3 probiotics could modulate mucin gene expression or mucus layer thickness, which would help in healing the inflamed mucosa. However, our in vivo supplementation with the VSL#3 probiotic cocktail did not modify Muc gene expression in either normal or altered colonic mucosa in mice. These results contradict an in vitro study showing that the VSL#3 cocktail increased MUC2, MUC3, MUC5AC gene and protein expression in undifferentiated colonic HT29 cell lines (25). Several factors can explain the discrepancy between the 2 models. In vitro cell lines present many characteristics that are very different from physiological conditions. The modulation of MUC gene expression by VSL#3 in HT29 cells could be due to their tumoral characteristics, to their undifferentiated status, or to the fact that a mucus layer does not cover them. Moreover, in vivo epithelial cells are subject to various humoral and neuronal regulations, which make their homeostasis more stable than in vitro. Conditions of probiotic administration also differed between the 2 models: in vitro, probiotics were given alone in the medium, at a considerably high concentration (100 bacteria/cell), whereas in vivo, even if a large amount was given daily and if the microflora was indeed changed by supplementation, the strains administered were still included in a very complex microflora and might be much less in contact with the epithelial cells covered by mucus than in a very simplified in vitro model. Because VSL#3 probiotic supplementation did not change Muc and TFF3 gene expression, their possible role in mucosa healing can neither be confirmed nor invalidated by our results.

Third, VSL#3 probiotic supplementation, despite altering the cecal microbiota composition and activity, had no therapeutic effect in our model of DSS-induced colitis in rodents. This lack of efficacy could stem from inappropriate colocalization of both phenomena, but as shown in Table 2, and as previously emphasized by Moreau et al. (48), DSS-induced inflammation in rodents not only affects the colon but also the cecum of the animals. Thus, although our observation was limited to the cecum due to the insufficient amounts of colonic contents, it indicates that even though VSL#3 probiotic activity was preserved after transit through the upper digestive tract, it lacked efficacy in healing the cecal mucosa. Such a result is somewhat contradictory to the previous studies that have often demonstrated the beneficial anti-inflammatory effect of the VSL#3 cocktail in both humans and animals. This probiotic cocktail appears to be effective in a range of gut inflammations in humans: it prevents pouchitis onset (49), relapse of pouchitis (19,21), relapse of ulcerative colitis (50), and relapse of Crohn’s disease under antibiotic treatment (51). In animals, the results are not as obvious: VSL#3 probiotic administration succeeded in improving colitis in IL10–/– mice (15) and in preventing iodoacetamide-induced colitis (52). However, it did not prevent dinitrobenzene sodium-induced colitis in mice (52). A recently published study states that VSL#3 probiotic administration before or at the same time as DSS-colitis induction reduced the severity of the inflammation in mice (18). Together, these results suggest that VSL#3 probiotic efficacy would depend more on the colitis-inducing agent and the administration protocol than on the species. Moreover, considering the latter study and the 2 others that previously demonstrated some positive effects of other probiotics (53,54) in preventing the development of DSS-induced colitis in rats and mice, this appears to be a suitable model with which to test the preventive effect of probiotics toward colitis. We therefore assume that it will also be convenient to test the curative effect of VSL#3.

We now address the discrepancies between our results and the study performed by Rachmilewitz et al. (18) because of its close similarity to our study, with respect to the nature of the probiotics, the daily dose, and the colitis-inducing agent. In addition to demonstrating the efficacy of VSL#3 in mice with DSS-induced colitis, these authors showed that the protective effect of VSL#3 is due to its DNA sensing by toll-like receptor 9 (TLR9) (18). Moreover, using a synthetic unmethylated cytosin-phophoryl-guanosin oligodinucleotide (CpG-ODN), they were able to mimic the DNA properties of the VSL#3 cocktail in preventing DSS-induced colitis and in healing established DSS-induced colitis, when administered before or at the same time as the colitis inducer. The major difference between that study and ours is that we used VSL#3 probiotics as a treatment, and thus we administered it after the onset of the colitis.

According to the theory of Rachmilewitz et al. (18), VSL#3 probiotics DNA is responsible for its anti-inflammatory properties. Our results suggest that VSL#3 DNA would have fewer curative than preventive anti-inflammatory properties. This was demonstrated previously for CpG-ODN by Obermeier et al. (55). In the mouse model of colitis, CpG-ODN injections before DSS treatment prevented colitis development, whereas CpG-ODN administration from d 3 after colitis induction exacerbated the colitis. This could be explained by the fact that CpG-ODN and DSS induce the same inflammatory mechanisms. The perpetuation of DSS colitis and CpG-ODN immunostimulation are both characterized by a Th1 phenotype with elevated levels of proinflammatory cytokines (56,57). The protective effect of pretreatment with CpG-ODN was shown to be due to a temporary increase in interferon- production in both colonic cells and mesenteric lymph node cells, which corresponded to a "desensitization" of mice to subsequent DSS-treatment (55). On the contrary, the exacerbation of the inflammation caused by the administration of CpG-ODN after the beginning of DSS treatment was due to the enhancement by CpG-ODN of the already established DSS-induced Th1 response. A similar scenario could explain why, under our conditions, VSL#3 probiotic treatment after the onset of disease did not heal the mucosa, whereas Rachmilewitz et al. (18) found a protective effect of VSL#3 DNA in preventing DSS-induced colitis.

Nevertheless, in contrast to Obermeier et al. (54), Rachmilewitz et al. (18) showed that CpG-ODN given on d 8 after the first DSS administration was able to diminish the colitis. It is regrettable that they did not test VSL#3 DNA efficacy under the same curative conditions. Indeed, their results obtained with CpG-ODN cannot be generalized to VSL#3 DNA. The CpG-ODN used by Rachmilewitz et al. (18) contained a double CpG motif 5'-AACGTTCGAG-3' and, to our knowledge, CpG motifs present in VSL#3 DNA have not been published. Different sequences and lengths of CpG motifs could lead to different affinities for the TLR9 receptor (58) and thus to different immunostimulatory properties of the DNA. The role of repeated vs. single exposure to immunostimulatory DNA should also be explored: single (18) vs. repeated (55) administration of CpG-ODN had opposite consequences in healing DSS-induced colitis. This could also be the case with VSL#3 probiotic DNA.

Finally, it should be recalled that mechanisms distinct from DNA immunostimulation were suggested for the effects of the VSL#3 cocktail on colonic mucosa. On the one hand, VSL#3 effects on epithelial permeability in vitro were reproduced by a heat-inactivated VSL#3 cocktail and by a protein factor contained in the VSL#3 supernatant (15). On the other hand, bacterial debris of the VSL#3 mixture, probably containing bacteria cell walls, membranes, and DNA fragments, was shown to induce IL-10 production by blood and intestinal dendritic cells and to inhibit the generation of proinflammatory Th1 cells (59). The latter effect was the opposite of VSL#3 DNA stimulation of the Th1 reaction. These studies suggest that it might then be too restrictive to limit VSL#3 probiotic effects to their DNA properties. The different compounds of the 8 strains making up the VSL#3 mixture are probably simultaneously recognized by the host, and VSL#3 probiotic effects on colonic mucosa would be the result of more complex mechanisms.

In conclusion, we established that VSL#3 probiotic supplementation could modify cecal microbiota by increasing its concentration in bifidobacteria and by modulating its metabolic activity. However, supplementation with the VSL#3 probiotic mixture did not change the mucus barrier, either in terms of Muc gene expression or in terms of mucus gel thickness. The increase in the quantity of bifidobacteria and in the metabolic potential of the microbiota was not sufficient to repair the breakdown of the colonic barrier caused by DSS treatment. Although this chemically induced model of colitis with DSS was previously shown to be relevant for demonstrating the preventive properties of probiotics and among them, the VSL#3 cocktail, our study clearly demonstrated for the first time that VSL#3 probiotics were not able to heal an established chronic inflammation in this DSS-induced animal model of colitis.

ACKNOWLEDGMENTS

The authors thank Professor J.-F. Mosnier (Pathological Anatomy Service, Laennec Hospital, Nantes) for his help with the quantitative analysis of histological scores. The authors are grateful to G. Poupeau, M. Rival, and A. David for their excellent technical assistance. We thank Dr. Claudio De Simone for the critical review of this manuscript.

FOOTNOTES

¹ Supported by the World Cancer Research Fund.

² Supplemental Figure 1 is available as Online Supporting Material with the online posting of this paper at www.nutrition.org.

⁴ Abbreviations used: cfu, colony forming unit; CpG-ODN, cytosin-phophoryl-guanosin oligodinucleotides; DSS, dextran-sodium sulfate; IL, interleukin; MUC, human mucin gene; MUC, human mucin protein; Muc, mice mucin gene; Muc, mice mucin protein; TFF3, trefoil factor 3; TLR, toll-like receptor; UC, ulcerative colitis. Mouse groups: DSS+/VSL+, treated with dextran-sodium sulfate and supplemented with VSL#3 probiotic cocktail; DSS+/VSL–, treated with dextran-sodium sulfate without probiotic supplementation; DSS–/VSL+, supplemented with VSL#3 probiotic cocktail; DSS–/VSL–, control group.

Manuscript received 10 May 2005. Initial review completed 8 June 2005. Revision accepted 29 August 2005.

LITERATURE CITED

1. Buisine MP, Desreumaux P, Leteurtre E, Copin MC, Colombel JF, Porchet N, Aubert JP. Mucin gene expression in intestinal epithelial cells in Crohn’s disease. Gut. 2001;49(4):544-551.[Abstract/Free Full Text]

2. Rhodes JM. Colonic mucus and ulcerative colitis. Gut. 1997;40(6):807-808.[Free Full Text]

3. Tytgat KM, van der Wal JW, Einerhand AW, Buller HA, Dekker J. Quantitative analysis of MUC2 synthesis in ulcerative colitis. Biochem Biophys Res Commun. 1996;224(2):397-405.[Medline]

4. Pullan RD, Thomas GA, Rhodes M, Newcombe RG, Williams GT, Allen A, Rhodes J. Thickness of adherent mucus gel on colonic mucosa in humans and its relevance to colitis. Gut. 1994;35(3):353-359.[Abstract/Free Full Text]

5. Makkink MK, Schwerbrock NM, Mahler M, Boshuizen JA, Renes IB, Cornberg M, Hedrich HJ, Einerhand AW, Buller HA, et al. Fate of goblet cells in experimental colitis. Dig Dis Sci. 2002;47(10):2286-2297.[Medline]

6. Faure M, Moennoz D, Montigon F, Mettraux C, Mercier S, Schiffrin EJ, Obled C, Breuille D, Boza J. Mucin production and composition is altered in dextran sulfate sodium-induced colitis in rats. Dig Dis Sci. 2003;48(7):1366-1373.[Medline]

7. Renes IB, Boshuizen JA, Van Nispen DJ, Bulsing NP, Buller HA, Dekker J, Einerhand AW. Alterations in Muc2 biosynthesis and secretion during dextran sulfate sodium-induced colitis. Am J Physiol Gastrointest Liver Physiol. 2002;282(2):382-389.

8. Itoh H, Beck PL, Inoue N, Xavier R, Podolsky DK. A paradoxical reduction in susceptibility to colonic injury upon targeted transgenic ablation of goblet cells. J Clin Invest. 1999;104(11):1539-1547.[Medline]

9. Mashimo H, Wu DC, Podolsky DK, Fishman MC. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science. 1996;274(5285):262-265.[Abstract/Free Full Text]

10. Vandenbroucke K, Hans W, Van Huysse J, Neirynck S, Demetter P, Remaut E, Rottiers P, Steidler L. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology. 2004;127(2):502-513.[Medline]

11. Xian CJ, Howarth GS, Mardell CE, Cool JC, Familari M, Read LC, Giraud AS. Temporal changes in TFF3 expression and jejunal morphology during methotrexate-induced damage and repair. Am J Physiol Gastrointest Liver Physiol. 1999;277(4 Pt 1):785-795.

12. Longman RJ, Douthwaite J, Sylvester PA, Poulsom R, Corfield AP, Thomas MG, Wright NA. Coordinated localisation of mucins and trefoil peptides in the ulcer associated cell lineage and the gastrointestinal mucosa. Gut. 2000;47(6):792-800.[Abstract/Free Full Text]

13. Renes IB, Verburg M, Van Nispen DJ, Buller HA, Dekker J, Einerhand AW. Distinct epithelial responses in experimental colitis: implications for ion uptake and mucosal protection. Am J Physiol Gastrointest Liver Physiol. 2002;283(1):169-179.

14. Wright NA, Poulsom R, Stamp G, Van Norden S, Sarraf C, Elia G, Ahnen D, Jeffery R, Longcroft J, Pike C. Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease. Scand J Gastroenterol Suppl. 1992;193:76-82.[Medline]

15. Madsen K, Cornish A, Soper P, McKaigney C, Jijon H, Yachimec C, Doyle J, Jewell L, De Simone C. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology. 2001;121(3):580-591.[Medline]

16. Schultz M, Veltkamp C, Dieleman LA, Grenther WB, Wyrick PB, Tonkonogy SL, Sartor RB. Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis. 2002;8(2):71-80.[Medline]

17. McCarthy J, O’Mahony L, O’Callaghan L, Sheil B, Vaughan EE, Fitzsimons N, Fitzgibbon J, O’Sullivan GC, Kiely B, et al. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut. 2003;52(7):975-980.[Abstract/Free Full Text]

18. Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, Rudensky B, Akira S, Takeda K, Lee J, et al. Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis. Gastroenterology. 2004;126(2):520-528.[Medline]

19. Gionchetti P, Rizzello F, Venturi A, Brigidi P, Matteuzzi D, Bazzocchi G, Poggioli G, Miglioli M, Campieri M. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology. 2000;119(2):305-309.[Medline]

20. Mimura T, Rizzello F, Helwig U, Poggioli G, Schreiber S, Talbot IC, Nicholls RJ, Gionchetti P, Campieri M, Kamm MA. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis. Gut. 2004;53(1):108-114.[Abstract/Free Full Text]

21. Venturi A, Gionchetti P, Rizzello F, Johansson R, Zucconi E, Brigidi P, Matteuzzi D, Campieri M. Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmacol Ther. 1999;13(8):1103-1108.[Medline]

22. Cui HH, Chen CL, Wang JD, Yang YJ, Cun Y, Wu JB, Liu YH, Dan HL, Jian YT, Chen XQ. Effects of probiotic on intestinal mucosa of patients with ulcerative colitis. World J Gastroenterol. 2004;10(10):1521-1525.[Medline]

23. Kruis W, Schutz E, Fric P, Fixa B, Judmaier G, Stolte M. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther. 1997;11(5):853-858.[Medline]

24. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet. 1999;354(9179):635-639.[Medline]

25. Otte JM, Podolsky DK. Functional modulation of enterocytes by gram-positive and gram-negative microorganisms. Am J Physiol Gastrointest Liver Physiol. 2004;286(4):613-626.

26. Reeves PG, Nielsen FH, Fahey GC, Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939-1951.[Abstract/Free Full Text]

27. Hoebler C, Gaudier E, de Coppet P, Rival M, Cherbut C. Muc gene are differently expressed during onset and maintenance of inflammation in dextran sodium sulfate-treated mice. Dig Dis Sci. 2005; In press.

28. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993;69(2):238-249.[Medline]

29. Dubernet S, Desmasures N, Gueguen M. A PCR-based method for identification of lactobacilli at the genus level. FEMS Microbiol Lett. 2002;214(2):271-275.[Medline]

30. Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, Matsumoto K, Oyaizu H, Tanaka R. Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl Environ Microbiol. 2002;68(11):5445-5451.[Abstract/Free Full Text]

31. Brigidi P, Vitali B, Swennen E, Altomare L, Rossi M, Matteuzzi D. Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction. Syst Appl Microbiol. 2000;23(3):391-399.[Medline]

32. Hojberg O, Canibe N, Knudsen B, Jensen BB. Potential rates of fermentation in digesta from the gastrointestinal tract of pigs: effect of feeding fermented liquid feed. Appl Environ Microbiol. 2003;69(1):408-418.[Abstract/Free Full Text]

33. Lund B, Adamsson I, Edlund C. Gastrointestinal transit survival of an Enterococcus faecium probiotic strain administered with or without vancomycin. Int J Food Microbiol. 2002;77(1-2):109-115.[Medline]

34. Sternberg S, Brandstrom B. Biochemical fingerprinting and ribotyping of isolates of Actinobacillus equuli from healthy and diseased horses. Vet Microbiol. 1999;66(1):53-65.[Medline]

35. Szentkuti L, Lorenz K. The thickness of the mucus layer in different segments of the rat intestine. Histochem J. 1995;27:466-472.[Medline]

36. Jordan N, Newton J, Pearson J, Allen A. A novel method for the visualization of the in situ mucus layer in rat and man. Clin Sci (Lond). 1998;95(1):97-106.[Medline]

37. Sneath , Sokal . A novel method for the visualization of the in situ mucus layer in rat and man. Numerical taxonomy. Freeman San Francisco.

38. Garcia-Vallve S. A novel method for the visualization of the in situ mucus layer in rat and man. DendroUPGMA: a dendrogram construction utility that uses Pearson coefficients and the UPGMA algorithm. Universitat Rovira i Virgili (URV) Tarragona Spain [2002 March]. Available from: URL: http://www.fut.es/debb/UPGMA/Example.html.

39. Garcia-Vallve S, Palau J, Romeu A. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol Biol Evol. 1999;16(9):1125-1134.[Abstract]

40. Brigidi P, Swennen E, Vitali B, Rossi M, Matteuzzi D. PCR detection of Bifidobacterium strains and Streptococcus thermophilus in feces of human subjects after oral bacteriotherapy and yogurt consumption. Int J Food Microbiol. 2003;81(3):203-209.[Medline]

41. Vitali B, Candela M, Matteuzzi D, Brigidi P. Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR. Syst Appl Microbiol. 2003;26(2):269-276.[Medline]

42. Fujiwara S, Seto Y, Kimura A, Hashiba H. Establishment of orally-administered Lactobacillus gasseri SBT2055SR in the gastrointestinal tract of humans and its influence on intestinal microflora and metabolism. J Appl Microbiol. 2001;90:343-352.[Medline]

43. Lay C, Sutren M, Lepercq P, Juste C, Rigottier-Gois L, Lhoste E, Lemee R, Le Ruyet P, Dore J, Andrieux C. Influence of Camembert consumption on the composition and metabolism of intestinal microbiota: a study in human microbiota-associated rats. Br J Nutr. 2004 Sep;92(3):429-438.[Medline]

44. Guarner F, Malagelada JR. Gut flora in health and disease. Lancet. 2003;361(9356):512-519.[Medline]

45. Brigidi P, Vitali B, Swennen E, Bazzocchi G, Matteuzzi D. Effects of probiotic administration upon the composition and enzymatic activity of human fecal microbiota in patients with irritable bowel syndrome or functional diarrhea. Res Microbiol. 2001;152(8):735-741.[Medline]

46. Smith AH, Mackie I. Effect of condensed tannins on bacterial diversity and metabolic activity in the rat gastrointestinal tract. Appl Environ Microbiol. 2004;70:1104-1115.[Abstract/Free Full Text]

47. Holdeman LV, Cato EP, Moore WEC. Effect of condensed tannins on bacterial diversity and metabolic activity in the rat gastrointestinal tract. Anaerobe laboratory manual. 4th edition ed. V.P.I. Anaerobe Laboratory Blacksburg, Virginia.

48. Moreau NM, Toquet CS, Laboisse CL, Nguyen PG, Siliart BS, Champ MM, Dumon HJ, Martin LJ. Predominance of caecal injury in a new dextran sulphate sodium treatment in rats: histopathological and fermentative characteristics. Eur J Gastroenterol Hepatol. 2002;14(5):535-542.[Medline]

49. Gionchetti P, Rizzello F, Helwig U, Venturi A, Lammers KM, Brigidi P, Vitali B, Poggioli G, Miglioli M, Campieri M. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology. 2003;124(5):1202-1209.[Medline]

50. Ulisse S, Gionchetti P, D’Alo S, Russo FP, Pesce I, Ricci G, Rizzello F, Helwig U, Cifone MG, et al. Expression of cytokines, inducible nitric oxide synthase, and matrix metalloproteinases in pouchitis: effects of probiotic treatment. Am J Gastroenterol. 2001;96(9):2691-2699.[Medline]

51. Campieri M, Rizzello F, Venturi A, Poggioli G, Ugolini F, Helwig U, Amadini C, Romboli E, Gionchetti P. Combination of antibiotic and probiotic treatment is efficacious in prophylaxis of post-operative recurrence of Crohn’s disease: a randomized controlled study vs mesalamine. Gastroenterology. 2000;(118):A4179.

52. Shibolet O, Karmeli F, Eliakim R, Swennen E, Brigidi P, Gionchetti P, Campieri M, Morgenstern S, Rachmilewitz D. Variable response to probiotics in two models of experimental colitis in rats. Inflamm Bowel Dis. 2002;8(6):399-406.[Medline]

53. Okamoto T, Sasaki M, Tsujikawa T, Fujiyama Y, Bamba T, Kusunoki M. Preventive efficacy of butyrate enemas and oral administration of Clostridium butyricum M588 in dextran sodium sulfate-induced colitis in rats. J Gastroenterol. 2000;35(5):341-346.[Medline]

54. Setoyama H, Imaoka A, Ishikawa H, Umesaki Y. Prevention of gut inflammation by Bifidobacterium in dextran sulfate-treated gnotobiotic mice associated with Bacteroides strains isolated from ulcerative colitis patients. Microbes Infect. 2003;5(2):115-122.[Medline]

55. Obermeier F, Dunger N, Strauch UG, Grunwald N, Herfarth H, Scholmerich J, Falk W. Contrasting activity of cytosin-guanosin dinucleotide oligonucleotides in mice with experimental colitis. Clin Exp Immunol. 2003;134(2):217-224.[Medline]

56. Lipford GB, Heeg K, Wagner H. Bacterial DNA as immune cell activator. Trends Microbiol. 1998;6(12):496-500.[Medline]

57. Obermeier F, Kojouharoff G, Hans W, Scholmerich J, Gross V, Falk W. Interferon-gamma (IFN-gamma)- and tumour necrosis factor (TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium (DSS)-induced colitis in mice. Clin Exp Immunol. 1999;116(2):238-245.[Medline]

58. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995;374(6522):546-549.[Medline]

59. Hart AL, Lammers K, Brigidi P, Vitali B, Rizzello F, Gionchetti P, Campieri M, Kamm MA, Knight SC, Stagg AJ. Modulation of human dendritic cell phenotype and function by probiotic bacteria. Gut. 2004;53(11):1602-1609.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Voltan, I. Castagliuolo, M. Elli, S. Longo, P. Brun, R. D'Inca, A. Porzionato, V. Macchi, G. Palu, G. C. Sturniolo, et al. Aggregating Phenotype in Lactobacillus crispatus Determines Intestinal Colonization and TLR2 and TLR4 Modulation in Murine Colonic Mucosa Clin. Vaccine Immunol., September 1, 2007; 14(9): 1138 - 1148. [Abstract] [Full Text] [PDF]

				M. C. Collado, J. Meriluoto, and S. Salminen Development of New Probiotics by Strain Combinations: Is It Possible to Improve the Adhesion to Intestinal Mucus? J Dairy Sci, June 1, 2007; 90(6): 2710 - 2716. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Online Supporting Material Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gaudier, E. Articles by Hoebler, C. Search for Related Content PubMed PubMed Citation Articles by Gaudier, E. Articles by Hoebler, C.