Review article: anti-inflammatory mechanisms of action of Saccharomyces boulardii

Authors


Dr C. Pothoulakis, MRL1240, 675 Charles E. Young Drive South, Inflammatory Bowel Disease Center, Division of Digestive Diseases, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA.
E-mail: cpothoulakis@mednet.ucla.edu

Abstract

Aliment Pharmacol Ther30, 826–833

Summary

Background Saccharomyces boulardii, a well-studied probiotic, can be effective in inflammatory gastrointestinal diseases with diverse pathophysiology, such as inflammatory bowel disease (IBD), and bacterially mediated or enterotoxin-mediated diarrhoea and inflammation.

Aim  To discuss the mechanisms of action involved in the intestinal anti-inflammatory action of S. boulardii.

Methods  Review of the literature related to the anti-inflammatory effects of this probiotic.

Results  Several mechanisms of action have been identified directed against the host and pathogenic microorganisms. S. boulardii and S. boulardii secreted-protein(s) inhibit production of proinflammatory cytokines by interfering with the global mediator of inflammation nuclear factor κB, and modulating the activity of the mitogen-activated protein kinases ERK1/2 and p38. S. boulardii activates expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) that protects from gut inflammation and IBD. S. boulardii also suppresses ‘bacteria overgrowth’ and host cell adherence, releases a protease that cleaves C. difficile toxin A and its intestinal receptor and stimulates antibody production against toxin A. Recent results indicate that S. boulardii may interfere with IBD pathogenesis by trapping T cells in mesenteric lymph nodes.

Conclusions  The multiple anti-inflammatory mechanisms exerted by S. boulardii provide molecular explanations supporting its effectiveness in intestinal inflammatory states.

Introduction

The nonpathogenic yeast Saccharomyces boulardii has been prescribed in the past 30 years for prophylaxis and treatment of diarrhoeal diseases caused by bacteria. More recently, clinical as well as experimental evidence with mouse models of inflammation has suggested that this probiotic yeast may have a therapeutic potential for IBD patients. Importantly, S. boulardii, has demonstrated clinical and experimental effectiveness in gastrointestinal diseases with a predominant inflammatory component, indicating that this probiotic might interfere with cellular signalling pathways common in many inflammatory conditions. This review discusses the mechanisms by which S. boulardii modulates intestinal inflammation. A search was performed in PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) using Saccharomyces boulardii or S. boulardii as key words. All manuscripts identified by this search were examined for their potential to suggest or demonstrate involvement of S. boulardii in signalling pathways associated with intestinal inflammation. Based on the available evidence, particular emphasis on the nuclear factor κB (NF-κB) and the mitogen-activated protein (MAP) kinase ERK and p38 signalling pathways was given. This review, however, also discusses evidence for other mechanisms involved in the beneficial action of this probiotic in intestinal inflammatory disease states.

Expression of many proinflammatory genes is regulated by the transcription factor, nuclear factor κB (NF-κB), which is considered the most important global mediator of inflammation in many cell types, including intestinal epithelial cells.1 In the intestine, NF-κB is a pivotal regulator of inflammation in IBD,2 as well as of innate immune responses to pathogenic bacteria.3 NF-κB exists as a dimeric protein, composed of members of the Rel family of proteins that includes p65, RelB, c-rel, p50 and p52.4 In unstimulated cells, NF-κB resides in the cytoplasm bound to its inhibitor, the inhibitory κB (IκB).5 Following stimulation of cells with proinflammatory cytokines, bacteria or bacterial cell products IκB is phosphorylated on two conserved serines by IκB kinases followed by IκB ubiquitination on two N-terminal conserved lysines, leading to IκB degradation by the proteasome while still being bound to NF-κB, which is then free to translocate into the nucleus and activate transcription of numerous proinflammatory genes,6 including interleukin-8 (IL-8) and tumour necrosis factor-α (TNFα).7 Notably, a majority of the immunosuppressive drugs used to induce remission in IBD patients such as corticosteroids, sulfasalazine and anti-TNF-α antibodies are also well established inhibitors of NF-κB.1 However, development of more specific drugs that can target different parts of the NF-κB inflammatory signalling cascade may represent a better and more specific strategy to treat colitis in IBD patients8, 9 and recent advances in probiotic research, including S. boulardii, clearly point to this possibility.

Mitogen-activated protein (MAP) kinases represent important molecules involved in signal transduction pathways associated with activation of transcription factors and other cell effectors. MAP kinases represent a family of serine/threonine protein kinases involved in many diverse cellular programmes, including inflammation.10 The best known members of the MAP kinase family are the extracellular signal-regulated kinase 1 and 2 (ERK1/2), which preferentially regulate growth and cell division and the c-Jun N-terminal kinase (JNK) and p38 MAP kinase involved mainly in inflammation and apoptosis. The MAP kinases ERK, c-Jun and p38 have been also implicated in NF-κB activation and proinflammatory gene regulation.11, 12

Mechanisms of Saccharomyces boulardii in Escherichia coli (E. coli )-associated proinflammatory signalling

Saccharomyces boulardii and Enterohemorrhagic Escherichia coli signalling.  Enterohemorrhagic Escherichia coli (EHEC) is an important bacterial pathogen causing gastroenteritis and colitis associated with serious morbidity and mortality. The intestinal responses during infection with EHEC include disruption of tight junctional permeability and intestinal inflammation characterized by release of potent immune mediators.13–15 Several lines of evidence also indicate that the proinflammatory response following exposure of intestinal epithelial cells to this pathogen involves activation of the transcription factor NF-κB.16, 17 Using molecular DNA binding approaches, Dahan et al. showed that infection of colonic adenocarcinoma T84 cells with EHEC stimulates increased NF-κB DNA binding activity and causes phosphorylation and degradation of the NF-κB inhibitor IκBα.15 Moreover, exposure of T84 colonocytes to a proteasome inhibitor reduces IL-8 secretion from EHEC-infected T84 cells, underlying the importance of NF-κB signalling in this proinflammatory response.15 Interestingly, pre-incubation of T84 cells with S. boulardii whole yeast prior to EHEC infection inhibits permeability changes, reduces phosphorylation and degradation of IκBα and NF-κB DNA binding activity and reduces secretion of the potent chemoattractant IL-8,18 indicating that S. boulardii exerts a preventive effect on EHEC infection by reducing activation of NF-κB at the colonocyte level. These results, however, do not exclude the possibility that in vivo, S. boulardii can reduce mucosal inflammation by blocking NF-κB-associated pathways in immune cells of the intestinal lamina propria.19

Infection of T84 colonocyte monolayers with EHEC results in activation of all three MAPK kinases, ERK1/2, p38, at the same time when induction of AP-1 DNA binding activity and phosphorylation and degradation of IκBα are evident.15 Importantly, incubation of colonic epithelial cells with inhibitors directed against either ERK1/2 or p38 results in diminished IL-8 secretion in response to EHEC-infection,15 indicating that in addition to NF-κB, EHEC-associated secretion of the chemoattractant IL-8 involves MAP kinase and AP-1 activation. Moreover, exposure of T84 cells to S. boulardii whole yeast prior to EHEC infection inhibits phosphorylation of all three MAP kinases.18 In contrast, S. boulardii does not have a significant effect on EHEC growth or EHEC adhesion.18 Together, the results demonstrate the ability of S. boulardii to modify important host signalling pathways that are activated by bacterial invasion with EHEC. Along the lines of a significant protective effect mediated by S. boulardii cultures during bacterial infection, addition of this probiotic to T84 colonocyte monolayers also diminishes myosin light chain (MLC) phosphorylation and decreases transepithelial resistance in response EHEC.18 As EHEC-induced MLC phosphorylation and alterations of TER do not involve MAP kinases or the NF-κB pathway,15 other, yet to be identified S. boulardii-associated signalling pathways should play a role in this response.

Mechanisms of Saccharomyces boulardii in Enteropathogenic Escherichia coli infection.  Enteropathogenic Escherichia coli (EPEC) is an important causative agent of infant diarrhoea in developing countries.20 Infection with EPEC is characterized by the formation of attaching-and-effacing lesions in the involved intestinal areas20 as well changes in permeability and loss of the integrity of the intestinal barrier associated with changes in specific tight junctional proteins, such as ZO-1.21In vitro experiments indicated that EPEC are able to adhere to the surface of S. boulardii,22 indicating the potential for Sboulardii to modify EPEC infection by direct binding to this pathogen. Important mechanistic studies from Czerucka et al. examined the effects of S. boulardii in barrier function alterations of T8 colonic epithelial cell monolayers following infection with EPEC.23 Exposure of T84 cells to S. boulardii whole yeast cultures blocks transepithelial resistance and permeability changes, reverses impaired ZO-1 distribution and delays apoptosis of epithelial cells in response to EPEC.23 Interestingly, EPEC triggers ERK1/2 MAP kinase activation in T84 colonocytes and this response is also inhibited by S. boulardii.23 This probiotic also significantly reduces the number of intracellular EPEC, an effect similar to the one obtained by exposure of T84 monolayers to a specific ERK1/2 inhibitor prior to EPEC infection, suggesting that modulation of MAP kinase signalling by S. boulardii may account for this protective effect. However, as previous studies suggested that translocation of EPEC from the membrane to the intracellular milieu is not associated with alterations in transepithelial resistance,24 other signalling pathways modulated by S. boulardii might play a role in this response.

Recently, Buts et al. suggested an additional target by which S. boulardii can affect E. coli-mediated infection.25 Lipopolysaccharide (LPS) is a well-studied endotoxin released from pathogenic E. coli strains that by binding to Toll-like receptor 4 stimulates proinflammatory responses in the intestine and other organs. These investigators presented direct evidence indicating that a protein phosphatase released by S. boulardii is able to dephosphorylate LPS from E. coli strain O55B5 at two phosphorylation sites important for expression of its proinflammatory activity.25 As a result, incubation of LPS with S. boulardii protein phosphatase reduced circulating levels of TNF-α following intraperitoneal injection of dephosphorylated LPS injection in rats.25

Mechanisms of Saccharomyces boulardii in Clostridium difficile (C. difficile) infection.  The most prominent causative agent of antibiotic-associated diarrhoea and colitis, C. difficile, mediates intestinal inflammation and mucosal damage by releasing two potent exotoxins, toxin A and toxin B.26 Several pieces of evidence suggest that S. boulardii represents the most effective probiotic that can prevent or, together with other agents, treat antibiotic-associated diarrhoea and recurrent C. difficile infection.27 Early studies with an animal model for C. difficile toxin A-induced enteritis indicated that either cultured suspension or filtered conditioned medium from S. boulardii inhibits toxin A-mediated diarrhoea, intestinal inflammation and histological damage by reducing toxin A-receptor binding.28 This effect is mediated by release of a 54 kDa protease from S. boulardii cultures that digests both toxin A and its receptor binding sites.29 The importance of this protease to inhibit responses to both toxins A and B in native human colon was also directly demonstrated using polyclonal antibodies directed against this specific S. boulardii serine protease.30 Another study in mice also demonstrated that S. boulardii stimulates specific intestinal anti-toxin A immunoglobulin levels, thus enhancing the host’s intestinal immune response against C. difficile toxin A.31 As, in a large clinical trial, the antibody response against toxin A appears to be pivotal for the development of symptoms during C. difficile infection,32 increased anti-toxin antibodies may be an important mechanism for S. boulardii-mediated protection from C. difficile diarrhoea and inflammation and possibly other diarrhoeal illnesses by increasing the overall immune response of the host.

Clostridium difficile toxin A exerts its proinflammatory effects by stimulating transcription of proinflammatory genes, such as IL-8 from human colonocytes or macrophages, via mechanisms involving activation of NF-κB leading to increased transcription of proinflammatory genes.33, 34 Studies from our group previously showed that in human monocytes and colonocytes, C. difficile toxin A activates the MAP kinases ERK and p38 and presented evidence that both these MAP kinases are involved in IL-8 gene expression and cell necrosis in response to toxin A.35, 36 In addition, culture supernatants from S.  boulardii inhibit IL-8 production and activation of the MAP kinases Erk1/2 and JNK/SAPK, but not p38, induced by C. difficile toxin A in human colonic NCM460 cells.37 Moreover, administration of S. boulardii supernatants into mouse ileal loops not only blocks toxin A-associated increased fluid secretion, mucosal inflammation and elevated cytokine release but also inhibits Erk1/2 activation in vivo in response to this toxin. Thus, S. boulardii secretes a factor(s) able to alter inflammatory diarrhoea by modifying important inflammatory signalling pathways relevant not only to C. difficile toxin-induced intestinal inflammation but to other forms of gut inflammation as well. Interestingly, a recent study by Sougioultzis et al. showed that S. boulardii supernatants inhibit increased IL-8 expression in response to IL-1β or TNF-α in colonic epithelial cells at the protein and mRNA level.19Saccharomyces boulardii supernatants also inhibit IL-8 production, prevent IκBα degradation and reduce NF-κB activation in response to IL-1β or LPS stimulation in THP-1 monocytes/macrophages.19 This group of investigators also demonstrated that a low molecular weight heat stable and water soluble factor purified from S. boulardii supernatants is able to block NF-κB activation and NF-κB-mediated IL-8 gene expression in target cells.19 Thus, this S. boulardii anti-inflammatory factor (SAIF) may be responsible, at least in part, for the beneficial effects of S. boulardii in inflammatory diarrhoea of infectious and non-infectious aetiologies. Further studies are needed to identify the specific site of action of SAIF on the NF-κB signalling cascade.

Anti-inflammatory mechanisms of Saccharomyces boulardii during Shigella infection.  Infection with Shigella is characterized by an acute inflammatory response in the colonic mucosa associated with severe, often bloody diarrhoea and intense abdominal pain. In intestinal epithelial cells, Shigella triggers a proinflammatory response characterized by activation of the NF-κB pathway,38 leading to secretion of various cytokines such as IL-838, 39 that attract leucocytes and results in significant epithelial cell damage. Using a gnotobiotic mouse model of Shigella flexneri infection, Rodrigues et al. demonstrated that Sboulardii cultures protect mice from pathogen-associated tissue damage without reducing Shigella flexneri numbers in the intestine.40 In a recent study, Mumy et al. showed that exposure of colonic epithelial cell monolayers to S. boulardii whole yeast cultures or conditioned medium improves barrier integrity and decreases ERK, NF-κB activation, IL-8 secretion and migration of polymorphonuclear leucocytes across cell monolayers in response to Shigella flexneri exposure.41 This S. boulardii anti-inflammatory effect was also confirmed in vivo using a human foetal colon tissue transplanted into scid mice,41 indicating a similar protective mechanism in human infection.

Modes of action of Saccharomyces boulardii in IBD.  Recent clinical trials indicated that S. boulardii might be beneficial as an adjunctive therapy in CD as well as flare-ups in UC patients.42–44 Studies with animal IBD models also underscore the potential for S. boulardii to reduce inflammatory colonic responses and provide potential mechanisms involved in this beneficial effect. Using the chemical model of the CD-like TNBS-induced colitis, Lee and colleagues found that S. boulardii whole yeast administration substantially reduces all aspects of colitis, including histological damage, diarrhoea and mucosal levels of the proinflammatory mediators IL-1β, IL-6, TNF-α and iNOS.45 Peroxisome proliferator activated receptor gamma (PPAR-γ) is a nuclear receptor expressed in the colon and colonic epithelial cells and represents a novel therapeutic target in intestinal inflammation and IBD.46 Interestingly, the same group of investigators showed that cultures of S. boulardii stimulate expression of PPAR-γ in HT-29 colonic epithelial cells47 and elevate transcription of this receptor in the colon of animals exposed to TNBS.45Saccharomyces boulardii inhibited TNFα-mediated regulation of PPAR-gamma and IL-8 by blocking activation of NF-κB. Interestingly, silencing of PPAR-γ expression in colonocytes reverses the inhibitory effect of S. boulardii in IL-8 gene expression, suggesting that activation of PPAR-γ in response to S. boulardii represents another molecular mechanism involved in its anti-inflammatory action.45 Similarly, S. boulardii reduced clinical score and severity of colitis following administration of DSS, a well-established animal model characterized by UC-like colonic pathology. Saccharomyces boulardii whole yeast also decreased colonization of Candida albicans following DSS administration.48 Although the mechanism of these beneficial responses was not determined, the authors of this study provided evidence that Toll-like receptors, bacterial recognition sites involved in IBD pathogenesis,49 might play a role in this response. However, additional studies are needed to clarify the nature and the mechanism participating in this effect.

Citrobacter rodentium (C. rodentium) is a gram-negative bacterium that colonizes the intestine and causes attaching-and-effacing colonic lesions similar to those observed in EPEC and EHEC infections.50 On the basis of intestinal inflammatory phenotype observed in C. rodentium infection and the overall important role bacteria play in the pathogenesis of intestinal inflammation and IBD,51 several groups use C. rodentium mouse infection as an experimental model to study IBD.52 Wu et al. reported that treatment of mice with S. boulardii whole yeast reduces colonic inflammation, attenuates weight loss and inhibits histological damage and intestinal permeability changes in response to infection with C. rodentium.53 These ameliorating effects of S. boulardii are associated with significantly lower numbers of C. rodentium adherent to the mucosa, as well as reductions in Tir protein secretion and translocation into mouse colonocytes and expression and secretion of EspB,53 both important virulent factors for C. rodentium disease pathogenesis.54 Although S. boulardii has no direct bactericidal effect against C. rodentium, this probiotic is able to reduce expression of EspB and Tir proteins in vitro, an effect that can be replicated when S. boulardii supernatants are used.53 Thus, S. boulardii provides protection against colitis associated with C. rodentium infection by releasing soluble factor(s) able to modulate C. rodentium adherence to epithelial cells and inhibit expression of potent virulent factors secreted by this microbe.

A recent exciting study by Dalmasso et al.55 provides evidence for a novel S. boulardii mechanism involved in IBD colitis with possible implications in the entire ‘probiotics’ field.56 These investigators examined the effect of peroral administration of S. boulardii in a T cell transfer model of colitis in severe combined immunodeficient (SCID) mice and found that the probiotic yeast prevents colonic inflammation and clinical signs of colitis.55 These protective responses are associated with diminished colonic NF-κB activity and reduced levels of several pro-inflammatory cytokines. In this study, oral administration of S. boulardii reduced IFN-γ production by CD4+ T cells in the colon, but increased it in the mesenteric lymph nodes, indicating a possible redistribution of IFN-γ–producing T cells. Additional transfer experiments demonstrated that increased accumulation of CD4+ T cells takes place when the receiving, but not the donor mice are provided with S. boulardii, indicating that T-cell retention occurs at the mesenteric lymphatic tissue. In this study, Dalmasso et al. also identified a soluble factor in S. boulardii condition media involved in endothelial cell-mediated rolling of T-cells,55 although the identity of this factor(s) remains to be elucidated. In conclusion, trapping of T-cells in mesenteric lymph nodes represents a unique protective mechanism afforded by S. boulardii against IBD. Although the anti-inflammatory effects of several other probiotics, including E. coli Nissle 1917, VSL # 3 and Lactobacilli appear to involve similar cellular mechanisms,57 T-cell trapping in lymph nodes has never been reported as a probiotic mechanism.

Conclusions

Results from experiments elucidating the mechanisms involved in the anti-inflammatory action of S. boulardii in a diverse and large number of diarrhoeal disease models with a predominant inflammatory component demonstrate the ability of this probiotic to affect key signalling pathways in intestinal host cells participating in the pathogenesis of inflammation. These studies support the notion that the beneficial effects of S. boulardii in gastrointestinal inflammatory conditions are mediated through modulation of host proinflammatory responses not only by the whole yeast, but also by secreted factors able to interfere with host’s signalling molecules controlling inflammation at different levels, such as the NF-κB and the MAP kinase pathways (Figure 1). Future studies on the molecular basis for S. boulardii regulation of the colonocyte and immune cell function will enable us to understand better the mechanisms of the prophylactic and therapeutic effects of this probiotic yeast in infectious inflammatory diarrhoea and IBD, and shed light on the potential new applications of S. boulardii in enteric diseases.

Figure 1.

 Model illustrating the potential cellular mechanism of action of Saccharomyces boulardii (S.b.) in infectious inflammatory states: Clostridium difficile toxins, or pathogenic bacteria such as EPEC and EHEC activate the MAP kinases ERK 1/2 and p38 as well as the NF-kB (p65/p50) system leading to transcription of proinflammatory genes such as IL-8 (Interleukin 8) that promote inflammation. Saccharomyces boulardii produces a factor or different factors that inhibit these signalling pathways thereby blocking secretion of proinflammatory cytokines and reducing inflammatory diarrhoea.

Acknowledgements

Declaration of personal interests: Dr Pothoulakis has served as a Consultant for Biocodex Inc., and Prometheus Laboratories, Inc., and has received research funding from Prometheus Laboratories, Inc. Declaration of funding interests: This work was funded in part by NIH PO-1 DK 33506 grant.

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