Saccharomyces boulardii Inhibits ERK1/2 Mitogen-activated Protein Kinase Activation Both in Vitro and in Vivo and Protects against Clostridium difficile Toxin A-induced Enteritis

Abstract

Saccharomyces boulardii (Sb), a probiotic yeast, protects against intestinal injury and inflammation caused by a wide variety of enteric pathogens, including Clostridium difficile. Given the broad range of protective effects of Sb in multiple gastrointestinal disorders, we hypothesize that Sb modulates host signaling pathways involved in intestinal inflammatory responses. In this study, we found that Sb culture supernatant (SbS) inhibits interleukin-8 production induced by C. difficile toxin A or IL-1␤ in human colonocyte NCM460 cells in a dosedependent fashion. Furthermore, SbS inhibited IL-1␤ and toxin A induced Erk1/2 and JNK/SAPK but not p38 activation in NCM460 cells. To test whether this inhibition also occurs in vivo, we used a previously established mouse ileal loop model. On its own, SbS had no significant effect on basal fluid secretion or intestinal histology. However, Erk1/2 activation was significantly inhibited by SbS in toxin A exposed mouse ileal mucosa. In control loops, toxin A increased fluid secretion (2.2-fold), histological score (3.3-fold), and levels of the chemokine KC (4.5-fold). SbS pretreatment completely normalized toxin A mediated fluid secretion ( p < 0.01), and histopathologic changes ( p < 0.01) and substantially inhibited toxin A-associated KC increases ( p < 0.001). In summary, the probiotic yeast S. boulardii inhibits C. difficile toxin A-associated enteritis by blocking the activation of Erk1/2 MAP kinases. This study indicates a new mechanism whereby Sb protects against intestinal inflammation and supports the hypothesis that Sb modulates host inflammatory signaling pathways to exert its beneficial effects.

Inhibition of the cytotoxic effect of Clostridium difficile in vitro by Clostridium butyricum MIYAIRI 588 strain

“…Studies on the probiotic yeast S. boulardii have found that it inhibits C. difficile toxin in vitro by secreting a protease that degrades the mature protein (Castagliuolo et al, 1999) and also by modulating the gut immune response (Chen et al, 2006;Kyne et al, 2001). However, data on the recently sequenced genome of CBM588 show that, although it contains protease genes, none of them is confirmed as being secreted (unpublished data).…”

ProbioticSaccharomyces boulardiiCNCM I-745 prevents outbreak-associatedClostridium difficile-associated cecal inflammation in hamsters

“…Studies also indicate that S.b can be used in combination with vancomycin as therapy for relapsing CDI (37, 50). The putative mechanisms involved in the effectiveness of S.b in CDI include effects directed against the microbiome, the host, as well as C. difficile and its toxins (4,6,21,40,43). However, most of the experimental work examining the effects of S.b in CDI models has been limited to nonoutbreak-associated C. difficile strains, mostly with strain VPI10463.…”

The host immune response to Clostridium difficile

“…One explanation for these findings is that C. difficile toxins cause direct injury to the intestinal epithelium, which is associated with a robust host inflammatory response with neutrophil activation and recruitment leading in turn to greatly amplified intestinal injury. In fact, a wide range of anti-inflammatory agents can reduce intestinal injury in animal models of CDI (Anton et al, 2004;Chen et al, 2006;Cottrell et al, 2007;Kim et al, 2005Kim et al, , 2007Kokkotou et al, 2009;Pothoulakis et al, 1993;Warny et al, 2000). The use of combinations of antibiotic and anti-inflammatory agents to treat severe CDI in humans warrants greater attention and investigation, especially given the rising incidence of severe and fatal disease (McDonald et al, 2006;Redelings et al, 2007).…”

“…It has been suggested that differences in the intracellular effects of toxin B variants produced by toxin A-negative strains (compared to toxin B from toxin A-positive strains) may be responsible for the ability of these strains to cause disease in humans. Innate inflammatory responses that are activated by the glucosyltransferase activity of C. difficile toxins A and B include the NF-kB and MAP kinase pathways (Chae et al, 2006;Chen et al, 2006;Jefferson et al, 1999;Kim et al, 2005;Warny et al, 2000). Monocytes and macrophages are activated and a variety of proinflammatory cytokines including IL-1b, TNF-a and IL-8 are released (Jefferson et al, 1999;Linevsky et al, 1997;Sun et al, 2009;Warny et al, 2000).…”