All Saccharomyces strains used in this study and listed (Table 1), including baker’s yeast S. boulardii and wine-related strains, were originally isolated from a proprietary germoplasm bank of S. cerevisiae (Societe Industrielle Lesaffre 147 Rue Gabriel Peri, BP 6027, 59700 Marcq-en-Baroeul Cedex, France). They were all provided by Lesaffre as a similar dry form with a cell concentration near to 1 × 10¹⁰ colony-forming units (CFU)/g. The different dried yeast products were processed and enumerated as follows. For both in vitro and in vivo assays, yeast was rehydrated with phosphate buffered saline (PBS) (pH 7.2) 1:10 (w/v) at room temperature, and adjusted to an appropriate concentration in CFU/mL. For this purpose, cellular counts together with viability, expressed in %, were routinely performed using a hematimeter (type Thoma) combined with a Trypan blue exclusion method. More than 95% viability was preserved following rehydration. Enumeration was also confirmed a posteriori by plating on YPD-agar and allowing growth during 2 d at 30°C and 37°C.

Some bacterial strains were used as reference strains for immune cell stimulation as previously described[24], also listed (Table 1). Lactobacillus strains were grown under limited aeration at 37°C in MRS medium (Difco) and a Bifidobacterium strain was grown anaerobically in MRS supplemented with 0.05% L-cysteine-hydrochloride (Sigma). Lactococcus lactis MG1363 was grown at 30°C in M17 medium supplemented with 0.5% glucose. Escherichia coli (E. coli) was grown at 37°C in LB medium (Difco). Bacterial cells were grown until stationary phase, washed and resuspended at 1 × 10⁹ CFU/mL in PBS containing 20% glycerol and stored at -80°C until required for later assays. Some routine analyses were also performed using a portable photometer (Densimat bioMerieux)[25]. Cells were adjusted to McFarland 3 and stored at -80°C.

Groups contained 10 mice. When needed, fecal samples from 5 mice were collected, pooled, weighed and mechanically homogenized in sterile neutral and isotonic buffer at 50 mg/mL of feces. Dilutions were plated onto the “yeast-selective” tetracycline (150 μg/mL) YPD-agar (Yeast extract, Peptone, Dextrose, as described elsewhere[26]. No tetracycline-resistant microorganisms (bacteria or fungi) were detected in non-inoculated mice, indicating no or negligible basal colonization of mice gastrointestinal tract (GIT) by non-pathogenic yeast nor Candida species.

PBMCs were isolated from peripheral blood of healthy donors as previously described[24]. Briefly, after a Ficoll gradient centrifugation (Pharmacia, Uppsala, Sweden), mononuclear cells were collected, washed in RPMI 1640 medium (Live technologies, Paisley, Scotland) and adjusted to 2 × 10⁶ cells/mL supplemented with gentamicin (150 g/mL), L-glutamine (2 mmol/L), and 10% foetal calf serum (Gibco-BRL) supplementation.

PBMC (2 × 10⁶ cells/mL) were seeded in 24-well tissue culture plates (Corning, NY). Twenty microliters of the thawed bacterial or yeast suspensions at 10⁹ CFU/mL in PBS containing 20% glycerol (bacteria:cell or yeast:cell ratio of 10:1) or standardized at homogeneous cell density, were added. PBS containing 20% glycerol was used as a negative (non-stimulated) control. On the basis of preliminary time-course studies, 24 h stimulation corresponded to the best time point for cytokine responses of bacteria stimulated-PBMCs. After 24 h stimulation at 37°C in an atmosphere of air with 5% CO₂, culture supernatants were collected, clarified by centrifugation and stored at -20°C until cytokine analysis. Concerning yeast stimulation, freshly rehydrated powders were used at distinct yeast:cell ratios (from 0.1:1 to 5:1), with or without the presence of fungizone (amphotericin B, 10 μg/mL). Neither medium acidification nor bacterial or fungal proliferation was observed. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) using BD Pharmingen antibody pairs (BD Biosciences, San Jose, Ca, USA) for interleukin (IL)-10, interferon γ (IFNγ) and IL-12p70, and R&D systems (Minneapolis, Mn, USA) for human tumor necrosis factor (TNF), according to the manufacturer’s recommendations.

BALB/c mice (female, 7 to 8 wk old) were obtained from Charles River (St Germain sur l’Arbresle, France). A standardized murine TNBS colitis model was used in which moderate levels of inflammation were induced[27]. Briefly, a 50 μL solution of 100 mg/kg TNBS (Sigma) in 50 % ethanol was slowly administered in the colon via a 3.5 F catheter. Freshly rehydrated yeast suspensions (100 μL), containing 1 × 10⁹ CFU/mL in NaHCO₃ buffer were administered intragastrically to mice each day, starting 4 d before until the day of TNBS administration while control mice received the corresponding buffer. Mice were weighed, bled from the retro-orbital venous plexus and killed 72 h after TNBS administration. Cleared sera were frozen and stored until cytokine assays at -20°C. Colons were removed, washed and opened longitudinally. Inflammation grading was performed by two blinded observers, using the Wallace scoring method[28]. Results are expressed as % protection, corresponding to the reduction of the mean macroscopic inflammation score of treated mice (n = 10) in comparison to the mean score of TNBS-treated control mice (NaHCO₃ buffer-treated mice, n = 10)[27]. Histological analysis was performed on May Grünwald-Giemsa stained 5 μm tissue sections from colon samples fixed in 10% formalin and embedded in paraffin and tissue lesions were scored according to the Ameho criteria[29]. Additionally, the degree of polymorphonuclear neutrophil infiltration of the distal colon was assessed by quantifying myeloperoxidase (MPO) - a neutrophil granule enzyme - as reported earlier[30]. A commercial preparation of prednisone (Cortancyl, Sanofi Aventis, France) was used as a positive control of protection and was orally administered for 2 subsequent days at 10 mg/kg starting at the day before TNBS administration. When needed, heparinized whole blood was collected by retro-orbital puncture and, after separation, mice sera samples were stored at -20°C for subsequent analysis.

Murine IL-6 and serum amyloid A (SAA) protein levels were measured by ELISA using commercial antibodies from Pharmingen antibody pairs (BD Biosciences, San Jose, Ca, USA), and Biosource International (Camarillo, Ca, USA), respectively, with a lower limit of sensitivity of 15 pg/mL for IL-6 and 30 ng/mL for SAA.

All analyses were performed as comparison of experimental groups with respective controls by the non-parametric one-way analysis of variance Mann-Whitney U-testing, or by Student-T testing where appropriate. Differences were judged to be statistically significant when the P value was < 0.05. Data are presented as mean ± SE.

All experiments were performed in an accredited establishment (number 59-35009; Institut Pasteur de Lille, France) and using approved guidelines according to French Ethical Committee and European Union Normatives (number 86/609/CEE).

To determine the optimal dose of yeast that can induce cytokine production in human PBMCs, immuno-competent cells were first stimulated with three different multiplicity levels of infection (0.1:1, 1:1 and 5:1) of three live yeast strains and compared with immuno-stimulatory effects of reference bacteria (ranging from weak to strong inducers). Substantial amounts of IL-10, and to a lesser extent for TNF were dose-dependently induced by yeast cells (Figure 1A and B, respectively) while no or very low levels of the pro-inflammatory indicators IL-12 p70 and IFNγ could be detected following yeast stimulation (Figure 1C and D). Following these results, the yeast:host cell ratio of 1:1 was retained for further comparative analyses between strains in order to mimic physiological interactions between cells but without saturating the stimulatory capacity. In addition, the variable responsiveness due to individual blood donors was highly minimized by expressing data as % of a reference strain instead of in pg/mL, as shown (Figure 2, panel A and B). PBMCs from 5 donors were stimulated with each of the six yeast strains covering distinct subspecies and origins (Table 1).

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Figure 1 Dose-dependent cytokine production in human peripheral blood mononuclear cells (PBMCs), stimulated with bacterial reference strains and 3 yeast strains at distinct multiplicity levels of infection [multiplicity levels of infection (MOI) 0. 1, 1 and 5 yeasts/cells] for 24 h. Cytokine levels were analyzed in supernatants by enzyme-linked immunosorbent assay (ELISA). Panels A, B, C and D respectively show results for interleukin (IL)-10, tumor necrosis factor (TNF), IL-12 p70 and interferon γ (IFNγ), respectively. Results are expressed in pg/mL, mean ± SE, n = 3 distinct donors.

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Figure 2 Standardized assessment of cytokine production in human PBMCs from distinct donors. Immuno-competent cells are stimulated with 5 bacterial reference strains for 24 h and cytokine levels were analyzed in supernatants by ELISA. A: IL-10 release for 5 individual donors. Results are expressed in pg/mL; B: Standardized IL-10 release with values expressed as % of Escherichia coli (E. coli) value for each of the 5 donors.

All individual strains of yeast induced relatively high levels of the anti-inflammatory IL-10, reaching the values generally obtained with lactobacilli (mean/max values 535/2300 pg/mL). However, in contrast to bacteria, no significant strain-specificity was observed among different strains of yeasts, which all induced similar levels of cytokines (Figure 3A). Analysis of the cell culture supernatants revealed moderate induction of TNF after yeast stimulation, as compared with bacterial stimulation, again without a clear strain dependency (Figure 3B). Yeasts induced extremely low levels of the pro-inflammatory signals IL-12 and IFNγ as compared to some bacteria such as Lactococcus lactis MG1363 or Lactobacillus acidophilus NCFM (Figure 3C and D). Concerning the intrinsic immuno-stimulatory capacity of yeast cells in vitro, we can conclude that no specific stimulation pattern could be discriminated among the six strains, while all strains favored anti-inflammatory profiles with a high IL-10/IL-12 ratio, low induction of TNF and negligible levels of IFNγ.

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Figure 3 Cytokine production in human PBMCs, stimulated with bacterial reference strains and 6 yeast strains for 24 h. Cytokine levels were analyzed in supernatants by ELISA. A: IL-10; B: TNF; C: IL-12 p70; D: IFNγ. Results are expressed in % of E. coli TG1 value for panel A and in % of Lactococcus lactis MG1363 for B, C and D, including mean ± SE; n = 5.

Next to the effect of yeasts on cytokine induction on PBMCs in vitro, we compared the ability of the 3 strains LV01, LV02 and LV09 to survive the digestive tract of mice. Following 0, 1 and 5 consecutive days of intra-gastric feeding of 10⁸ CFU of each of the yeasts, faecal enumeration showed equivalent survival counts for all the strains with a progressive increase with the duration of the treatment (Figure 4). The systematic increase by 2 log units between day 1 and day 5 suggests that proliferation occurs in the gut, rather than simple transit and shedding. Although we cannot speculate on the long term persistence and colonization of the yeast species, a 5-d-treatment clearly leads to a noticeable presence of living yeasts in the colon lumen (up to 10⁸ CFU/g for the 3 strains). A significant faecal recovery of yeast colonies from healthy mice was still detectable 10 d after the last feeding (more than 10⁵ CFU/g, data not shown). It is noteworthy to mention that neither diarrhoea nor slight changes in faecal character, nor weight disturbance or other deleterious signs were observed in mice treated with those food-grade yeasts.

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Figure 4 Fecal recovery of the 3 distinct yeast strains in yeast-fed mice, at days 0, 1 and 5. Data were obtained by counting dilutions of stools on appropriate “yeast-selective”-tetracycline agar plates. Results are expressed in Log CFU/g of stools. Results are representative for 2 separate experiments, SD are not represented for clarity reasons. ND: Not detected; CFU: Colony-forming units.

In order to evaluate whether the administration of distinct yeast strains equally impacted on TNBS-induced colitis, we compared the performance of the three strains LV01, LV02 and LV09 with the anti-inflammatory corticoid drug (prednisone) which we and others previously used in this experimental setting[31,32]. Since some anti-inflammatory effects of lactobacilli were observed both after oral and systemic administration[33,34], we assessed whether intraperitoneal administration of the yeast LV01 could also rescue mice from colitis.

Rectal administration of TNBS causes progressive weight loss in vehicle-treated mice, reaching up to 22.5% of the initial weight 72 h after induction of colitis (Figure 5A and B). As expected, prednisone prevented body weight loss associated with experimental colitis (-3.3%, P < 0.001). The oral treatments by the 3 yeast strains also significantly attenuated the deleterious weight changes for the LV01 (-9.9%, P < 0.01), and to a lesser extent for LV02 and LV09 (18%, P < 0.05). In contrast, the systemic administration of the strain LV01 (LV01-IP) had no additional effect on weight.

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Figure 5 Changes in body weight during the preventive interventional study on experimental colitis. A: Time-course changes of weight for control mice and 2-4-6-trinitrobenzene sulfonic acid (TNBS)-treated mice, covering saline vehicle, prednisone and yeast pre-treated animals. Results are expressed as % of initial weight; SDs are not represented for clarity; B: Final weight loss 3 d after colitis. Results are expressed as mean ± SE (n = 10 per group), ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs TNBS-saline.

After the onset of colitis, anti-inflammatory effects were measured macroscopically for the prednisone-treated as well as for the LV01-treated mice (66% and 27% reduction of macroscopic damage, respectively, P < 0.01, Figure 6A). More moderately LV02 reduced the colonic insult (18% of control, P < 0.05) while intra-peritoneal injection of LV01 failed to influence the macroscopic scores.

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Figure 6 Effects of the various treatments 3 d after the TNBS-induced colitis on colonic macroscopic damage (Wallace) (A), histology scores (Ameho) (B) and myeloperoxidase (MPO) activity (C). Mice received intra-rectally either saline or TNBS following oral pre-treatment by prednisone (10 mg/kg, 2 d), by various yeast strains for 5 d or after intra-peritoneal administration (IP) of the yeast at the onset of colitis. Results are expressed as mean ± SE (n = 10 per group), ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs TNBS-saline.

Considering the histological assessment of colitis in various conditions (Figure 6B), both the prophylactic treatment by prednisone and LV01 significantly reduced the microscopic scores of inflammation (by 60.3% and 35%, respectively, P < 0.05), while neither LV02 nor LV09 treament could be associated with a significant reduction of the histopathological features observed during active colitis (mucosal erosions, oedema, ulcerations areas and even necrosis). These observations, including thickening of the submucosa and inflammatory infiltrates, were correlated with the colonic myeloperoxidase activities measured (MPO, Figure 6C) and with similar reductions for the steroid drug and LV01 (P < 0.01). In contrast, no improvement, and even some aggravation, was depicted for strain LV01 provided by the systemic route, as supported by massive neutrophil infiltrates. Representative aspects of the corresponding colon sections are given (Figure 7). Mouse colons with vehicle-treated TNBS-induced colitis exhibited massive goblet cell depletion and showed disorganized mucosal architecture, including muscle layer necrosis (Figure 7B). By contrast, corticoid-treated animals showed a well-conserved appearance, more similar to normal structures despite a slight thickening of the submucosa. Only minor thickening of submucosa and superficial lesions, mainly oedema, were seen in mice treated with the yeast LV01, in complete absence of necrosis. Erosions were more important in LV02-pretreatment, although necrosis was restricted to the upper luminal compartment and the crypt’s architecture was still identifiable. Treatment with strain LV09 given orally and strain LV01 after intraperitoneal administration were characterized by prominent inflammatory infiltrates and transmural inflammation with necrosis reaching the deeper muscle layers.

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Figure 7 Histological features of yeast-mediated protection. The figure shows representative May-Grünwald and Giemsa-strained colon sections of control healthy mice (A) or day 3 after instillation of TNBS in Balb/c mice pre-treated with either vehicle (B), prednisone (C), LV01 (D), LV02 (E), LV09 (F) and LV01-IP (G). Original magnification, × 20.

The levels of the blood markers of inflammation SAA and IL-6, while significantly increased after TNBS administration, were strikingly reduced by prednisone and LV01 treatments, approaching baseline values (P < 0.01). LV02 strain also minimized the pro-inflammatory cytokine IL-6 in the serum (P < 0.05), with a similar trend observed for SAA (P = 0.055, Figure 8).

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Figure 8 Effects of the various treatments 3 d after the TNBS-induced colitis on blood markers. A: Serum amyloid A protein (SAA); B: IL-6. Mice received intra-rectally either saline or TNBS following oral pre-treatment by prednisone (10 mg/kg, 2 d), by various yeast strains for 5 d or after intra-peritoneal administration (IP) of the yeast at the onset of colitis. Results are expressed as mean ± SE (n = 10 per group), ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, 0.05 < ^dP < 0.1 vs TNBS-saline.

In conclusion, TNBS caused increases in mucosal and systemic inflammatory symptoms that can be prevented by an orally administered anti-inflammatory drug (prednisone) as well as the food-grade yeast LV01. This was substantiated on the basis of reduction of body-weight loss, macroscopic damage, histological scores, colonic inflammatory infiltrates and serum markers. The yeast strain LV02 showed an intermediate protection capacity with sometimes partial and borderline effects in the degree of attenuation, depending on the marker considered. LV09 only lowered the weight loss with no improvement of other colitis-related symptoms. These results support the relevance of yeast for colitis protection, but distinct performance of various strains highly suggests strain-dependent mechanisms.

As for probiotic bacteria, probiotic yeast such Saccharomyces boulardii (S. boulardii) can be effective in gastrointestinal pathologies such as inflammatory bowel disease (IBD) and bacterial- or enterotoxin-mediated diarrhea and inflammation. However, very few yeast strains have been studied as possible biotherapeutics agents. The underlying mechanisms of action of probiotics are not fully understood but there is some evidence that they exert their beneficial effect by multiple and specific modes of action depending on the type of the probiotic (bacteria, yeast), the species and strain.

The yeast-mediated capacity to lower inflammation is suggested to mainly involve epithelial cells and barrier function, as demonstrated by (1) the weakness of specific immunomodulatory response in vitro; and (2) the lack of protection by S. boulardii when administered by the systemic but not the oral route. In contrast with the performance of some probiotic bacteria which the authors previously showed to be directly correlated with their in vitro anti-inflammatory potential, the immunoregulatory mechanism of probiotic yeasts fails to directly act on the cytokine level.

As for probiotic bacteria, strain specificity also matters for yeast as for probiotics, but mechanisms, and consequently screening methods, partly differ between yeast and bacteria. A careful and appropriate selection of strains is then required to screen the biodiversity of yeasts for specific clinical studies including applications in IBD as well as for other dedicated therapeutic uses. This will impact on future product composition to enhance or synergize gastrointestinal related disorders by using probiotics.

Probiotics are defined as living microorganisms that may have beneficial effects on health, including mainly food-grade bacteria and non-pathogenic yeasts.

The paper by Benoit Foligné and co-workers investigated the anti-inflammatory effects of various yeasts strains in experimental colitis in mice. The overall design of the study is fair and the results are stimulating for future research.