Modulation of Intestinal Inflammation by Yeasts and Cell Wall Extracts: Strain Dependence and Unexpected Anti-Inflammatory Role of Glucan Fractions

Yeasts and their glycan components can have a beneficial or adverse effect on intestinal inflammation. Previous research has shown that the presence of Saccharomyces cerevisiae var. boulardii (Sb) reduces intestinal inflammation and colonization by Candida albicans. The aim of this study was to identify dietary yeasts, which have comparable effects to the anti-C. albicans and anti-inflammatory properties of Sb and to assess the capabilities of yeast cell wall components to modulate intestinal inflammation. Mice received a single oral challenge of C. albicans and were then given 1.5% dextran-sulphate-sodium (DSS) for 2 weeks followed by a 3-day restitution period. S. cerevisiae strains (Sb, Sc1 to Sc4), as well as mannoprotein (MP) and β-glucan crude fractions prepared from Sc2 and highly purified β-glucans prepared from C. albicans were used in this curative model, starting 3 days after C. albicans challenge. Mice were assessed for the clinical, histological and inflammatory responses related to DSS administration. Strain Sc1-1 gave the same level of protection against C. albicans as Sb when assessed by mortality, clinical scores, colonization levels, reduction of TNFα and increase in IL-10 transcription. When Sc1-1 was compared with the other S. cerevisiae strains, the preparation process had a strong influence on biological activity. Interestingly, some S. cerevisiae strains dramatically increased mortality and clinical scores. Strain Sc4 and MP fraction favoured C. albicans colonization and inflammation, whereas β-glucan fraction was protective against both. Surprisingly, purified β-glucans from C. albicans had the same protective effect. Thus, some yeasts appear to be strong modulators of intestinal inflammation. These effects are dependent on the strain, species, preparation process and cell wall fraction. It was striking that β-glucan fractions or pure β-glucans from C. albicans displayed the most potent anti-inflammatory effect in the DSS model.


Introduction
Probiotics are a popular alternative to antibiotics [1]. The positive effects of probiotics on humans and animals result either from a direct nutritional effect or a health effect, with probiotics acting as bioregulators of the intestinal microflora and reinforcing the host's natural defences [2].
Saccharomyces cerevisiae var. boulardii (Sb) is described as a biotherapeutic agent in the clinical literature and is reported to be efficacious in the prevention of antibiotic-associated diarrhoea and colitis in humans [3,4]. Orally administered Sb demonstrated clinical and experimental effectiveness in gastrointestinal diseases through modulation of host cell signalling pathways implicated in the pro-inflammatory response such as IL-1b and TNF-a. Sb exerts a trophic effect that restores intestinal homeostasis and activates expression of peroxisome proliferator-activated receptorgamma which protects against gut inflammation [5].
It has recently been reported that Sb decreases inflammation and intestinal colonization by C. albicans in a BALB/c mouse model of colitis induced by dextran-sulphate-sodium (DSS) [6]. Interestingly, parallel studies indicated that Sb reduces C. albicans adhesion to human intestinal cell lines and decreases proinflammatory cytokine mRNA levels in response to C. albicans infection [7,8].
Sb is generally administered as a lyophilized powder [9] and its use as a food additive has only been reported in a number of cases such as in the fermentation of raw vegetable materials [10] and incorporation into commercial yoghurts [11].Taxonomic studies indicate that Sb should be considered as a Saccharomyces cerevisiae strain [12,13]. This leads to the question ''do other strains of S. cerevisiae also possess probiotic properties?'' [14].
In the present study, low doses of DSS were administered to mice for 2 weeks to induce colonic inflammation and promote the establishment of C. albicans colonization, followed by a 3-day restitution period. Either S. cerevisiae strains or glycan fractions were then administered daily by oral gavage for 2 weeks, starting 3 days after the C. albicans challenge, in order to assess their curative effects on both colonic inflammation and acceleration of colonic epithelial restoration. Using the DSS mouse model each dietary yeast was found to have its own effect on colitis and C. albicans colonization. The impact of orally administered glycan fractions extracted from S. cerevisiae that may reverse the adverse effects of DSS and C. albicans, and the biological activity of soluble b-glucan isolated from the C. albicans cell wall, were then investigated in this DSS mouse model.

Results
Comparison of the probiotic potential of S. cerevisiae var. boulardii and S. cerevisiae 1-1 strain S. cerevisiae 1-1 strain (Sc1-1) was selected from our collection as having previously exhibited a probiotic effect. This yeast is prepared as an active dry yeast so that it can react quickly to its environment ( Table 1). As Sc1-1 was comparable in vitro to Sb in terms of decreasing growth and germ tube formation by C. albicans (data not shown), the aim was to compare the Sc1-1 and Sb strains for their ability to reduce C. albicans colonization and intestinal inflammation.
The model used is summarized in Fig. 1A and can be defined as ''curative''. In this model, a single dose of C. albicans was administered to mice receiving DSS. Either the Sb or Sc1-1 strain was given 3 days later when C. albicans colonization was established. Low mortality was observed in mice that received DSS or DSS+C. albicans whereas none of the mice given the same regimen plus S. cerevisiae died (Fig. 1B).
Concerning C. albicans colonization in mice that received DSS (Fig. 1C), the number of colony-forming units (CFUs) in stools gradually increased from the day of C. albicans administration. From S. cerevisiae administration on day 4 to the endpoint, a dramatic reduction in number of C. albicans CFUs was observed.
No difference was observed between the two S. cerevisiae strains in their activity on C. albicans clearance (Fig. 1C).
The cumulated clinical and histological scores ( Fig. 1D-E) were higher in mice that received DSS or DSS+C. albicans whereas they were reduced significantly by the administration of either Sb or Sc1-1 ( Fig. 1D-E). Both strains were equally effective in reducing intestinal inflammation assessed by these parameters (Fig. 2).
To analyze the possible mediators involved in the reduction of the inflammatory response to DSS and C. albicans in the colon following S. cerevisiae administration, we focused on levels of TNF-a and IL-10 mRNA as representative pro-and anti-inflammatory cytokines. As shown in Fig. 1F-G, administration of either Sb or Sc1-1 was associated with a significant reduction in TNF-a expression and increase in IL-10 production. No difference was observed between the two S. cerevisiae strains in their ability to redirect the inflammatory response.
Analysis of the anti-C. albicans and anti-inflammatory properties of other S. cerevisiae strains with different preparation processes As both Sc1-1 and Sb strains displayed identical beneficial effects in this specific model the Sc1-1 strain was used as the reference strain for probiotic activity. In this part of the study, the influence of yeast preparation procedure and other yeast strains was investigated.
From an initial screening involving 10 strains or preparation procedures, five representative examples of strain activities were selected and compared to Sc1-1. These consisted of strains Sc1-2, Sc2, Sc3 and Sc4.
Sc1-1 showed important differences in mortality (Fig. 3A), ability to reduce C. albicans colonization (Fig. 3B) and a decrease in both histological and clinical scores ( Fig. 3C-D). The Sc1-1 strain gave excellent results for all parameters, as did Sc3, which is used for its probiotic activities ( Fig. 4) Interestingly, some of the beneficial effects induced by Sc1-1 were abolished when this strain was prepared as an instant dry yeast ( Table 1). The most striking results were observed with the Sc4 strain, which did not display any particular effect on C. albicans colonization over 2 days compared to the other S. cerevisiae strains. However, the mice were extremely constipated, clinically inflamed and highly colonized with C. albicans starting from day 7. From this point on, we discontinued collecting faeces as the mice were becoming extremely ill. This yeast, which presents optimal growth at low temperatures, dramatically exacerbated both the clinical and histological scores and was associated with high mortality (around 80%). We also found that both C. albicans and Sc4 increased the staging of colitis in mice (Fig. 4). These high clinical and histological scores were associated with high numbers of C. albicans CFUs in different parts of the gut from moribund mice (data not shown). Finally, Sc2, another industrial strain used for the production of yeast proteins, displayed an ''intermediate'' behaviour with reduced beneficial effects and slight worsening of mortality and clinical scores.
Identification of the cell wall fractions supporting the beneficial and adverse effects on C. albicans colonization and inflammation For this purpose, Sc2 was used as this strain can be easily induced to undergo autolysis in order to prepare cell wall extracts including mannoprotein (MP) and b-glucan fractions (Fig. 5).
In contrast to living cells, none of the cell wall extracts induced mortality (Fig. 6A). However, the changes in body weight and clinical scores were worsened considerably by MP fractions while these parameters were ameliorated by b-glucan fractions (Fig. 6B,  F). Notably, the MP fraction as well as the Sc2 strain induced an important loss in body weight up to day 9 that was correlated with its incapacity to control C. albicans colonization (Fig. 6C). By contrast, administration of b-glucan fraction maintained normal body weight, reduced inflammation scores and promoted C. albicans clearance (Fig. 6A-F). Although the body weight of mice receiving MP fractions started to increase from day 10, the clinical activity score was higher than that of mice receiving b-glucan fractions (Fig. 6E).
Activity of the homologous C. albicans oligoglucoside fraction in the DSS mouse model As the glucoprotein fraction from S. cerevisiae unexpectedly displayed a protective effect in this model, it was decided to investigate both the structure and biological activity of the bglucan fraction from C. albicans. The harsh whole cell extraction procedure leads to fraction-1, known as yeast ghosts (Fig. 7). Fraction-1 (F1) was analyzed by fluorescence microscopy with various fluorescent probes specific for cell wall glycans in comparison to zymosan, which is widely used for b-glucan immunological studies (Fig. 8). Both zymosan and F1 were labelled with monoclonal antibody (mAb) 2G8 specific for b-1,3 glucans and WGA which binds to chitin (Fig. 8). In contrast to zymosan, which was stained with both Concanavalin A (ConA) and GNL, no mannose residue signals were observed for F1. Immunofluorescent staining with antibodies to b-mannose, liable to be synthesized by C. albicans, was also negative with F1 (data not shown). Thus, yeast ghosts have no mannose residues in their cell wall (Fig. 8).
MALDI-MS analysis of the soluble fraction derived from F1 (F2) established that it consisted of a highly polydisperse hexose polymer consisting of 3-27 hexose residues (Fig. 9A). According to its reactivity with anti b-1,3 glucans, this component was susceptible to zymolyase digestion which produced a set of small water soluble fragments (2)(3)(4)(5), as demonstrated by thin-layer chromatography (data not shown) and MALDI-MS analyses (Fig. 9B). After purification of this fraction by reverse phase and adsorption chromatography, its structure was established by NMR as a mixture of b1,3-substituted glucan oligomers with free reducing ends. Furthermore, these oligomers were shown to be partially substituted by a random single b1,6 glucopyranose residue (Fig. 9C).
Mortality, weight loss, clinical activity, histological score and C. albicans colonization After structure characterization of the b-oligoglucoside fraction (F2) extracted from C. albicans, the biological activity of F2 was tested in the DSS mouse model. After C. albicans challenge, mice were given F2 (1 mg/day) from day3 up to the endpoint (Fig. 1A). F2 administration significantly prevented mouse mortality due to either DSS or DSS+C. albicans (Fig. 10A). In contrast to DSS and DSS+C. albicans mice, which developed severe colitis and lost body weight, F2 administration reversed the adverse effect of colitis and the mice showed a significant amelioration of both the clinical inflammation score and body weight ( Fig. 10B-D). Histological examination of colon sections from mice receiving either DSS or DSS+C. albicans showed important colonic inflammation which was associated with mucosal cell loss, crypt damage, mucosal ulceration and accompanying submucosal oedema ( Fig. 10C-D). F2 administration significantly reduced colonic inflammation due to either DSS or DSS+C. albicans ( Fig. 10C-D). C. albicans colonization in DSS-treated mice showed a steady increase as assessed by the number of CFUs in faeces which was consistent with the high load of C. albicans recovered from the stomach, ileum and colon of this group of mice at the endpoint of the experiments ( Fig. 10E-F).
In contrast to the higher numbers of C. albicans CFUs recovered from different compartments of the gut in DSS-treated mice, oral administration of F2 decreased the number of C. albicans CFUs recovered from stools and all gut segments of DSS-treated mice ( Fig. 10E-F). Furthermore, the reduction in clinical and histological scores was consistent with these low numbers of C. albicans CFUs in different parts of the gut ( Fig. 11 and Table 2).

Discussion
Excessive use of antifungal agents has been implicated in the emergence of antifungal resistance in C. albicans and constitutes a serious clinical problem in hospitals by affecting the natural balance of the intestinal microflora in these individuals [15]. The non-pathogenic yeast, Sb, which is widely prescribed for the treatment of antibiotic-induced gastrointestinal disorders and Clostridium difficile-associated enteropathies, has been shown to be an alternative approach to counterbalance the equilibrium of the intestinal microflora and modulate the innate immune defence [16,17]. Orally administered Sb was successful in both the treatment of inflammatory bowel disease (IBD) and the elimination of C. albicans colonization [7,8,[18][19][20][21].
Recently, it has been shown that Sb decreases both C. albicans colonization and intestinal inflammation in a mouse model of DSS-induced colitis [6]. Following this study, S. cerevisiae strains, MP and b-glucan fractions were screened in a mouse model of DSS-induced colitis. As Sb is considered taxonomically to be a strain of S. cerevisiae [12,13], strain Sc1-1 was compared to Sb in the DSS model. Sc1-1 is a gastro-resistant strain that reacts rapidly to its environment and is widely used in the food industry. Incidentally, it was observed that both Sc1-1 and Sb strains reduced C. albicans filamentation in vitro and C. albicans adhesion to plastic-plate wells (data not shown). In the present study, we did not chose a prophylactic but a curative model in which the animals develop colitis with histological features that are similar to those seen in patients with IBD before starting their treatment.
In this model low doses of DSS were used in order to establish C. albicans colonization, followed by S. cerevisiae or yeast extracts administration to assess their effects on the inflamed colon and colonic epithelium restitution.
Two weeks of DSS administration were scheduled to induce moderate colonic inflammation in mice, with low mortality rates. A recent study by Samonis et al. showed that mice receiving a high daily oral dose of C. albicans (around 10 8 CFU/day) for 2 weeks did not respond to Sb treatment [22]. In our model, a single inoculum of C. albicans was used and Candida colonization was maintained naturally in the mouse gastrointestinal tract by the DSS-induced colitis since a high C. albicans dose could dramatically hide the beneficial effect of Sb. In the present study, and similar to the Sb strain, Sc1-1 decreased both C. albicans colonization and intestinal inflammation in terms of clinical and histological score and mortality. Another notable finding was the acceleration of colonic epithelium restoration in mice treated with these dietary yeasts leading to the absence of submucosal oedema and epithelial erosion. Mechanistically, a recent report on intestinal inflammation showed that Sb secretes motogenic factors that enhance intestinal epithelial cell restitution [23].
Regarding the RT-PCR results, both Sb and Sc1-1 reduced the expression levels of pro-inflammatory cytokine TNF-a mRNA in the colonic mucosa with subsequent enhancement of IL-10 mRNA expression that inhibits intestinal injury [24]. Additionally, different pro-inflammatory cytokines were investigated in this set of experiments and were consistent with TNF-a expression. Further investigation is required to determine the role of Th17/ Treg responses in different sets of experiments [25,26]. A recent study in patients with IBD showed that Sb reduced TNF-a production and significantly inhibited T-cell proliferation induced by intestinal inflammation [19]. Generally, the biological activities of S. cerevisiae in gastrointestinal inflammatory conditions are mediated through modulation of host pro-inflammatory responses not only by the whole yeast, but also by secreted factors able to interfere with host signalling molecules that control inflammation at different levels such as NF-kB [27,28]. Sb produces a soluble anti-inflammatory factor that inhibits NF-kB activation and attenuates pro-inflammatory signalling in host cells. In addition, Sb stimulates IL-10 secretion from intraepithelial lymphocytes infected by C. albicans and Escherichia coli [29]. As Sc1-1 was shown to be comparable to Sb and presents the same beneficial features against C. albicans and intestinal inflammation, Sc1-1 was considered as the reference strain in the DSS model. To assess if the observed anti-inflammatory properties were strain-dependent, other S. cerevisiae strains were selected deliberately for their high phenotypic diversity. The possible influence of yeast preparation process on anti-C. albicans activity was also studied. Surprisingly, some strains had a dramatic effect in the DSS mouse model and the process of yeast preparation also had an influence on the yeast's biological properties [30]. Each strain selected in this study was well characterized in vitro in terms of cell growth, osmostress, fermentation, viability and metabolites. However, different factors could influence the biological activity of the strains when introduced by gavage in the DSS mouse model: (i) the resistance of the cell wall related to the yeast preparation process [30]; (ii) the viability of the strain in the stomach, ileum and colon; (iii) its interaction with the microflora and intestinal mucosa [31]; and (iv) its ability to produce soluble anti-inflammatory factors in the milieu triggering expression of mediators in the intestinal epithelium and cells of monocyte lineage present in the submucosae [27]. Altogether, each strain has its own unique properties and supports specific activities within the host. The in vitro findings, together with the results for all S. cerevisiae strains analyzed in this study, suggest that Sc1-1 has beneficial biological activities reversing all aspects of colitis, including histological damage, diarrhoea and mucosal levels of the pro-inflammatory mediator TNF-a.
The cell wall is an essential structural component of yeast cells playing a central role in the interaction of yeasts with their environment. Unfortunately, the biological activities of S. cerevisiae cell wall components are still unclear in terms of C. albicans colonization and intestinal inflammation. Two components (MP and b-glucans) produced industrially were explored in our experimental model. With MP fraction administration, C. albicans colonization was not consistent with intestinal inflammation parameters, suggesting that MP fractions have differential effects on C. albicans colonization and intestinal inflammation. In contrast to MP fractions, GP fraction administration decreased the number of C. albicans CFUs concomitantly to all intestinal inflammation parameters.
Both of these components are known to be potent immunological activators, but their mechanisms of action are different and controversial [32][33][34]. As an example, both MP and b-glucans act positively on tumour cells and several microbial infections [34,35]. Conversely, administration of b-glucans derived from C. albicans has been shown to exacerbate arthritis in mice [36]. Structurally, MP have extensive N-and O-linked mannosylation which serve as ligands for galectin-3 (Gal-3), mannose receptor and DC-SIGN on macrophages and dendritic cells [37]. Different MP express b-Man epitopes, which have been identified as the principal ligand for Gal-3 [38]. In a previous study using the DSS model with C. albicans, Gal-3 knock-out mice were less affected by intestinal inflammation and C. albicans colonization than wild-type animals [39]. Recently, it was shown that C. glabrata deficient in b-Man was less virulent in DSS-treated mice as revealed by low clinical and histological scores and reduction of C. glabrata colonization [40]. bglucans have affinities towards different receptors such as CD11b/ CD18 [41], located on neutrophils, or Dectin-1 on macrophages [42]. This results in b-glucan activation of cytokine production and in turn activation of adaptive immunity. Thus, b-glucans attenuate the impact of colitis compared to MP [43].
As our results also showed a beneficial effect of b-glucans on inflammation/colonization, insoluble ghost yeast cells derived from C. albicans containing b-glucans were prepared and compared to zymosan which is widely used in b-glucan studies where many investigators refer to it as b-glucan [44,45]. Zymosan stimulates the production and activity of pro-inflammatory cytokines [45]. Additionally, when chemically characterized zymosan containing only b-(1-3)-glucans was added to macrophage cells, the production of IL-10, reactive oxygen species (ROS) and TNF-a increased in a dose-dependent way [46]. Bonifazi et al. demonstrated the capacity of zymosan to activate both inflammatory and tolerogenic dendritic cells (DCs) leading to the triggering of both Th17 and Treg cells in vivo [47]. Our observations showed that zymosan contains both mannans and b-glucans exposed together on the cell wall surface in comparison to C. albicans ghosts that contain only b-glucans. This evidence prevented us from further studies on zymosan. Different observations showed that the biological activities of soluble b-glucans differ from those of cellassociated b-glucans [32,48,49]. Ishibashi et al. showed that insoluble cell wall b-glucans induced intensive inflammatory and immunomodulating activities compared to soluble b-glucans [49]. Following the b-glucan analysis, the chemical structure of the soluble b-glucan fraction derived from C. albicans ghosts was characterized and its biological activities were tested in the DSS mouse model. Interestingly, orally administered b-glucans from C. albicans decreased intestinal inflammation and C. albicans colonization.Several reports show that b-glucan enhances the immune response and improves the clearance of pathogenic bacteria in animal models [50][51][52]; this supports our findings that smaller oligoglucosides derived from C. albicans showed beneficial activities against C. albicans and these results are comparable to b-glucans derived from S. cerevisiae. However, it may also be hypothesized that these individual oligoglucosides could block receptors such as dectin-1 and CD11b/CD18 and prevent multivalent binding necessary for strong triggering of the inflammatory responses [53].
Besides the importance of yeast molecules sensing for immune response, a third player may also possibly act in the general interplay. This is the mouse microbiota. Oligosaccharides are well known prebiotics active on the intestinal flora [54,55], and although such a role has not been investigated for C. albicans derived oligoglucosides it cannot be ruled out. Altogether, these results demonstrate that oligoglucosides behave differently from the original C. albicans whole yeast cells in the DSS mouse model.
In summary, Sc1-1 was found to be comparable to Sb and had beneficial biological activities against C. albicans and intestinal inflammation. Clinical trials are currently being conducted with Sc1-1 and promising results have been seen in patients with IBD. In the second part of this study, we focused on cell wall components involved in direct contact with the host and demonstrated that, in contrast to MP, b-glucan fractions from either S. cerevisiae or C. albicans have a more potent antiinflammatory effect against colonic colitis induced by DSS in mice. In conclusion, this study generated some progress in deciphering the nature of the yeast molecular components differentially favouring inflammation and/or C. albicans clearance. Future studies will include experiments on oligosaccharide administration to mice in order to determine how these glycans stimulate the growth of beneficial bacteria in the gut and boost the immune system providing therapeutic perspectives for digestive disorders and life-threatening fungal infections of endogenous origin.

Yeast strains
The yeast strains used in this study are shown in Table 1.

Preparation of b-glucan fractions from yeasts
The composition in dry matter of spray-dried S. cerevisiae Sc2 cell wall fractions is shown in Table 3 and the preparation procedure for MP and b-glucan fractions from the cell wall of the same strain is shown in Fig. 1. The fractionation and digestion procedure for extraction of the b-glucan fraction from C. albicans is summarized in Fig. 2. Briefly, the cell pellet of C. albicans (50 g wet weight) was incubated twice in 200 ml of 1 M NaOH at 70uC for 30 min. After washing with distilled water, the supernatant was removed and the pellet was oxidized with 100 mM NaIO 4 (Sigma-Aldrich, France) at room temperature for 24 h in the dark [56]. After completion of the reaction, excess periodate was destroyed by adding ethylene glycol. After washing several times with water, the pellet was reduced with 1 M NaBH 4 (Sigma Aldrich, France) at room temperature. The reaction was terminated by lowering the pH to 5 by the addition of acetic acid. After washing several times with water, the insoluble fraction was then lyophilized to produce fraction-1. Fraction-1 was treated with zymolyase 20T (0.2 mg/ mL, Immuno TM ; ICN Biomedicals Inc.) at 37uC for 3 h. Zymolyase inactivation was performed at 70uC for 5 min. After centrifugation, the supernatant was dialyzed against distilled water. The dialyzed solution was loaded onto a Sep-Pak C18 column (Alltech) equilibrated with 0.1% TFA (trifluoroacetic acid). Eluate-1 was evaporated and the resulting oligoglucosaccharides were dissolved in distilled water and further purified on a carbograph column (Alltech carbograph SPE column). Eluate-2 from the carbograph column was lyophilized to produce fraction-2 (F2). Fluorescence microscopy was performed to assess surface oligomannose expression on fraction-1 in comparison to zymosan (Sigma-Aldrich). Fraction-1 and zymosan suspensions deposited on slides were incubated with either monoclonal antibody (mAb) 2G8 specific for b-1,3 glucans [57,58], or wheat germ agglutinin(WGA), which binds to chitin [59], or Concanavalin A (ConA) or Galanthus nivalis lectin (GNL) or DAPI, as described previously [6,40]. For animal experimentation, fraction-2 was suspended in water and divided into 200 mL aliquots (each aliquot of 200 mL contained 1 mg of b-glucans).

Structural analysis of b-glucans
NMR experiments were performed at 300 K using a Bruker AvanceII 900 MHz spectrometer equipped with a 5 mm tripleresonance cryoprobe. Prior to NMR spectroscopic analyses in deuterium, oligosaccharides were repeatedly exchanged in 2   Animals Six-to 8-week-old female BALB/c mice were used. All mice were maintained by Charles River Laboratories (France). Four sets of experiments were performed independently and each experiment was divided into control groups (eight mice/cage), including assessment of the effect of DSS alone, and experimental groups (10 mice/cage).

Ethics statement
All mouse experiments were performed according to protocols approved by the Subcommittee on Research Animal Care of the Regional Hospital Centre of Lille, France, and in accordance with the European legal and institutional guidelines (86/609/CEE) for the care and use of laboratory animals.

Inoculum preparation and induction of colitis
Each animal was inoculated on day 1 by oral gavage with 200 mL of phosphate-buffered saline (PBS) containing 10 7 live C. albicans cells. Mice were given 1.5% DSS (MW 36-50 kDa; MP Biomedicals, LLC, Germany) in drinking water from day 1 to day 14 to induce intestinal inflammation. Three days after C. albicans oral challenge, mice were administered by oral gavage with a single-daily dose of either 10 7 lyophilized S. cerevisiae strains or 1 mg of b-glucan fraction for 2 weeks. Lyophilized S. cerevisiae strains were rehydrated for 30 min in PBS at 37uC before administering to the mice [60]. The presence of yeasts in the intestinal tract was followed daily by performing plate counts of faeces (approximately 0.1 g/sample) collected from each animal [39]. The faecal samples were suspended in 1 mL saline, ground in a glass tissue homogenizer and plated onto Candi-Select medium (Bio-Rad Laboratories, Marnes la Coquette, France). This chromogenic medium is designed for the isolation of yeasts from clinical specimens and is intended to differentiate medically important yeast species depending on the colour of the colonies [61]. Colonies of C. albicans were counted after 48 h incubation at 37uC. The results were noted as colony forming units (CFUs)/mg of faeces.

Presence of C. albicans colonization in the gastrointestinal tract
To check for C. albicans colonization, the animals were sacrificed and the gastrointestinal tract was removed and separated into the stomach, ileum and colon. The tissues were cut longitudinally. After removal of intestinal contents, the tissues were washed several times in PBS to minimize surface contamination from organisms present in the lumen [62]. Serial dilutions of homogenates were performed. The results were noted as C. albicans CFUs/mg of tissue.

Assessment of clinical parameters
The mortality rate of DSS-treated mice was determined daily and a colon biopsy was taken immediately after death for histological analysis. Total body weight was measured daily. The data are expressed as mean percent change from starting body weight. Daily clinical activity score ranging from 0 to 8 was calculated as described elsewhere [39,63].

Determination of histological score
Rings of the transverse part of the colon were fixed overnight in 4% paraformaldehyde-acid and embedded in paraffin for histo- Figure 11. Summary of the effects of S. cerevisiae strains or glycan fractions on Candida DSS-treated mice. Radar blots showed the effect of each S. cerevisiae strain or glycan fraction on: (1) mortality; (2) body weight loss; (3) clinical score; (4) histological score; and (5) C. albicans colonization. Sb, Sc1-1 and Sc3 strains improved the intestinal damage due to DSS-induced colitis and C. albicans colonization. Sc1-2, Sc2 and Sc4 strains had a dramatic effect on all inflammation signs including Candida colonization. Regarding the glycan fractions, both b-glucans and F2 reduced all colitis parameters and eliminated C. albicans colonization, while MP was less effective at controlling these parameters. doi:10.1371/journal.pone.0040648.g011 Table 2. Effect of different strains and cell wall extracts on C. albicans colonization and inflammation in the curative C. albicans DSS model. logical analysis. Cross-sections (4 mm thick) were stained with haematoxylin-eosin (Sigma-Aldrich, France). Histological scores were evaluated by two independent, blinded investigators who observed two sections per mouse at magnifications of 610 and 6100. The scores were determined in accordance with Siegmund et al. [63] and the sections were evaluated for the following two subscores: (i) a score for the presence and confluence of inflammatory cells, including neutrophils, in the lamina propria and submucosa or transmural extension; and (ii) a score for epithelial damage, focal lymphoepithelial lesions, mucosal erosion and/or ulceration and extension to the bowel wall. The two subscores were added together and the combined histological score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage).

Real-time mRNA quantification
Total RNA was isolated from colon samples using a NucleoSpin RNA II kit (Macherey-Nagel, France) following the manufacturer's instructions, with 20-50 units of DNase I (RNase-free) at 37uC for 30 min to avoid contamination with genomic DNA. RNA quantification was performed by spectrophotometry (Nanodrop; Nyxor Biotech, France). Reverse transcription of mRNA was carried out in a final volume of 26 mL from 1 mg total RNA using 300 U M-MLV reverse transcriptase (Invitrogen, France) according to the manufacturer's instructions with 500 ng oligo(dT) 12-18 and 50 U ribonuclease inhibitor (RNase-Out, Promega). PCR was performed using an ABI 7000 prism sequence detection system (Applied Biosystems, France) with SYBR green (Applied Biosystems, France). Amplification was carried out in a total volume of 25 mL containing 0.5 mL of each primer [6,39] and 1 mL of cDNA prepared as described above. SYBR green dye intensity was analyzed using Abiprism 7000 SDS software (Applera Corp.). All results were normalized to the housekeeping gene b-actin.

Statistical analysis
Data are expressed as the mean 6 SE of five mice in each group. All comparisons were analyzed by the Mann-Whitney U test. Statistical analyses were performed using the StatView TM 4.5 statistical program (SAS Institute Inc., Meylan, France). Differences were considered significant when the P value was ,0.05.