Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-κB-mediated IL-8 gene expression

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Abstract

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1β, TNFα or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IκBα degradation was studied by Western blotting and on NF-κB-DNA binding by EMSA. NF-κB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-κB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1β or TNFα stimulated HT-29 cells (by 75% and 85%, respectively; P < 0.001) and prevented IL-1β-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IκBα degradation, and reduced both NF-κB-DNA binding and NF-κB reporter gene up-regulation in IL-1β or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-κB activation and NF-κB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.

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Cells and reagents

Human transformed monocytic (THP-1), colonic epithelial (HT-29), and gastric epithelial (AGS) cells were obtained from ATCC (American Type Culture Collection, Rockville, MD) and grown in RPMI 1640 media (Gibco-BRL). Recombinant human (rh) IL-1β and TNFα were purchased from R&D Systems (Minneapolis, MN). Purified LPS from E. coli 055:B5 was from Sigma (St. Louis, MO). Anti-human IκBα was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-NF-κB p65 was from Boehringer Mannheim

S. boulardii supernatant inhibits IL-8 secretion by cytokine-stimulated human colonic and gastric epithelial cell lines, the active component being less than 10 kDa in size

HT-29 cells were serum starved overnight and then stimulated with IL-1β (10 ng/ml), TNF-α (10 ng/ml) or LPS (100 ng/ml), in the presence or absence of SbS (1:1, v/v). After 12 h incubation, the cell conditioned media were collected and IL-8 protein levels measured by ELISA. As illustrated in Fig. 1A resting HT-29 monolayers released little IL-8 protein while stimulation with IL-1β or TNFα markedly increased IL-8 protein production. The presence of SbS significantly reduced IL-8 production in both

Discussion

We report here that the yeast Saccharomyces boulardii, a probiotic widely used to treat or prevent a variety of human diarrheal diseases, produces SAIF (Saccharomyces anti-inflammatory factor) a small molecular weight, water soluble molecule that inhibits the activation of NF-κB, a transcription factor that plays a central role in human inflammatory responses. The mechanism of NF-κB inhibition exerted by SAIF involves inhibition of IκBα degradation, thereby resulting in sequestration of NF-κB

Acknowledgments

The authors are grateful to Amy Pan, B.Sc., Michel Warny, M.D., Ph.D., and Samer Aboudola M.D. for their contributions.

Sponsor. This work was supported in part by a grant from Biocodex Laboratories, Montrouge, France and NIH Grant DK 33506.

Competing interests statement. Drs. Sougioultzis, Bhaskar, Pothoulakis, and Kelly are named co-inventors on a patent application on Saccharomyces anti-inflammatory factor (SAIF) filed by Beth Israel Deaconess Medical Center, Boston, MA.

Dr. Pothoulakis has

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    These authors contributed equally to this work.

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