Impact of rising seawater temperature on a phagocytic cell population during V. parahaemolyticus infection in the sea anemone E. pallida.

Climate change is increasing ocean temperatures and consequently impacts marine life (e.g., bacterial communities). In this context, studying host-pathogen interactions in marine organisms is becoming increasingly important, not only for ecological conservation, but also to reduce economic loss due to mass mortalities in cultured species. In this study, we used Exaiptasia pallida (E. pallida), an anemone, as an emerging marine model to better understand the effect of rising temperatures on the infection induced by the pathogenic marine bacterium Vibrio parahaemolyticus. The effect of temperature on E. pallida was examined at 6, 24, or 30 h after bath inoculation with 108 CFU of V. parahaemolyticus expressing GFP (Vp-GFP) at 27°C (husbandry temperature) or 31°C (heat stress). Morphological observations of E. pallida and their Hsps expression demonstrated heat stress induced increasing damage to anemones. The kinetics of the infections revealed that Vp-GFP were localized on the surface of the ectoderm and in the mucus during the first hours of infection and in the mesenterial filaments thereafter. To better identify the E. pallida cells targeted by Vp-GFP infection, we used spectral flow cytometry. E. pallida cell types were identified based on their autofluorescent properties. corresponding to different cell types (algae and cnidocytes). We identified an AF10 population whose autofluorescent spectrum was identical to that of human monocytes/macrophage, suggesting that this spectral print could be the hallmark of phagocytic cells called "amebocytes''. AF10 autofluorescent cells had a high capacity to phagocytize Vp-GFP, suggesting their possible role in fighting infection. This was confirmed by microscopy using sorted AF10 and GFP-positive cells (AF10+/GFP+). The number of AF10+/GFP+ cells were reduced at 31°C, demonstrating that increased temperature not only damages tissue but also affects the immune response of E. pallida. In conclusion, our study provides a springboard for more comprehensive studies of immune defense in marine organisms and paves the way for future studies of the dynamics, activation patterns, and functional responses of immune cells when encountering pathogens.

Specific targeting of inflammatory osteoclastogenesis by the probiotic yeast S. boulardii CNCM I-745 reduces bone loss in osteoporosis.

Bone destruction is a hallmark of chronic inflammation, and bone-resorbing osteoclasts arising under such a condition differ from steady-state ones. However, osteoclast diversity remains poorly explored. Here, we combined transcriptomic profiling, differentiation assays and in vivo analysis in mouse to decipher specific traits for inflammatory and steady-state osteoclasts. We identified and validated the pattern-recognition receptors (PRR) Tlr2, Dectin-1, and Mincle, all involved in yeast recognition as major regulators of inflammatory osteoclasts. We showed that administration of the yeast probiotic Saccharomyces boulardii CNCM I-745 (Sb) in vivo reduced bone loss in ovariectomized but not sham mice by reducing inflammatory osteoclastogenesis. This beneficial impact of Sb is mediated by the regulation of the inflammatory environment required for the generation of inflammatory osteoclasts. We also showed that Sb derivatives as well as agonists of Tlr2, Dectin-1, and Mincle specifically inhibited directly the differentiation of inflammatory but not steady-state osteoclasts in vitro. These findings demonstrate a preferential use of the PRR-associated costimulatory differentiation pathway by inflammatory osteoclasts, thus enabling their specific inhibition, which opens new therapeutic perspectives for inflammatory bone loss.

Correction: Interaction of Saccharomyces boulardii with Salmonella enterica Serovar Typhimurium Protects Mice and Modifies T84 Cell Response to the Infection.

[This corrects the article DOI: 10.1371/journal.pone.0008925.].

An Increase of Seawater Temperature Upregulates the Expression of Vibrio parahaemolyticus Virulence Factors Implicated in Adhesion and Biofilm Formation.

Climate change driven seawater temperature (SWT) increases results in greater abundance and geographical expansion of marine pathogens, among which Vibrio parahaemolyticus (Vp) causes serious economic and health issues. In addition, plastic pollution in the ocean constitutes a vector for harmful pathogens dissemination. We investigate the effect of elevated SWT on the expression of genes implicated in adhesion and biofilm formation on abiotic surfaces in the clinical Vp strain RIMD2210633, which expresses hemolysins. Among the genes studied, the multivalent adhesion molecule-7 and the GlcNAc-binding protein A were involved in the adhesion of Vp to abiotic and biotic surfaces, whereas the type IV pili, the mannose-sensitive hemagglutinin, and the chitin-regulated pilins facilitate attachment and biofilm formation. Data presented here show that at 21°C, Vp is still viable but does not either proliferate or express the virulence factors studied. Interestingly, at 27°C and as early as 1 h of incubation, all factors are transiently expressed in free-living bacteria only and even more upregulated at 31°C. These results clearly show that increased SWT has an important impact on the adhesion properties of free-living Vp to plastic support and thus emphasize the role of climate change in the spread of this pathogenic bacteria.

Statistics of pathogenic bacteria in the search of host cells.

A crucial phase in the infection process, which remains poorly understood, is the localization of suitable host cells by bacteria. It is often assumed that chemotaxis plays a key role during this phase. Here, we report a quantitative study on how Salmonella Typhimurium search for T84 human colonic epithelial cells. Combining time-lapse microscopy and mathematical modeling, we show that bacteria can be described as chiral active particles with strong active speed fluctuations, which are of biological, as opposed to thermal, origin. We observe that there exists a giant range of inter-individual variability of the bacterial exploring capacity. Furthermore, we find Salmonella Typhimurium does not exhibit biased motion towards the cells and show that the search time statistics is consistent with a random search strategy. Our results indicate that in vitro localization of host cells, and also cell infection, are random processes, not involving chemotaxis, that strongly depend on bacterial motility parameters.

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice.

Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.

Human Health and Ocean Pollution.

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood. Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health. Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention. Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths. Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks. Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale. Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries. Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas.

Gene expression kinetics of Exaiptasia pallida innate immune response to Vibrio parahaemolyticus infection.

BACKGROUND: Recent sequencing projects on early-diverging metazoans such as cnidarians, have unveiled a rich innate immunity gene repertoire; however, little is known about immunity gene regulation in the host's early response against marine bacterial pathogens over time. Here, we used RNA-seq on the sea anemone Exaiptasia pallida (Ep) strain CC7 as a model to depict the innate immune response during the onset of infection with the marine pathogenic bacteria Vibrio parahaemolyticus (Vp) clinical strain O3:K6, and lipopolysaccharides (LPS) exposure. Pairwise and time series analyses identified the genes responsive to infection as well as the kinetics of innate immune genes over time. Comparisons between the responses to live Vp and purified LPS was then performed. RESULTS: Gene expression and functional analyses detected hundreds to thousands of genes responsive to the Vp infection after 1, 3, 6 and 12 h, including a few shared with the response to LPS. Our results bring to light the first indications that non-canonical cytoplasmic pattern recognition receptors (PRRs) such as NOD-like and RIG-I-like receptor homologs take part in the immune response of Ep. Over-expression of several members of the lectin-complement pathways in parallel with novel transmembrane and Ig containing ficolins (CniFLs) suggest an active defense against the pathogen. Although lacking typical Toll-like receptors (TLRs), Ep activates a TLR-like pathway including the up-regulation of MyD88, TRAF6, NF-κB and AP-1 genes, which are not induced under LPS treatment and therefore suggest an alternative ligand-to-PRR trigger. Two cytokine-dependent pathways involving Tumor necrosis factor receptors (TNFRs) and several other potential downstream signaling genes likely lead to inflammation and/or apoptosis. Finally, both the extrinsic and intrinsic apoptotic pathways were strongly supported by over-expression of effector and executioner genes. CONCLUSIONS: To our knowledge, this pioneering study is first to follow the kinetics of the innate immune response in a cnidarian during the onset of infection with a bacterial pathogen. Overall, our findings reveal the involvement of both novel immune gene candidates such as NLRs, RLRs and CniFLs, and previously identified TLR-like and apoptotic pathways in anthozoan innate immunity with a large amount of transcript-level evidence.

Modulation of 5-fluorouracil activation of toll-like/MyD88/NF-κB/MAPK pathway by Saccharomyces boulardii CNCM I-745 probiotic.

BACKGROUND AND AIM: Chemotherapy drugs that act via Toll-like receptors (TLRs) can exacerbate mucosal injury through the production of cytokines. Intestinal mucositis can activate TLR2 and TLR4, resulting in the activation of NF-κB. Intestinal mucositis characterized by intense inflammation is the main side effect associated with 5-fluorouracil (5-FU) treatment. Saccharomyces boulardii CNCM I-745 (S.b) is a probiotic yeast used in the treatment of gastrointestinal disorders. The main objective of the study was to evaluate the effect of S.b treatment on the Toll-like/MyD88/NF-κB/MAPK pathway activated during intestinal mucositis and in Caco-2 cells treated with 5-FU. METHODS: The mice were divided into three groups: saline (control), saline + 5-FU, and 5-FU + S.b (1.6 × 1010 colony forming units/kg). After 3 days of S.b administration by gavage, the mice were euthanized and the jejunum and ileum were removed. In vitro, Caco2 cells were treated with 5-FU (1 mM) alone or in the presence of lipopolysaccharide (1 ng/ml). When indicated, cells were exposed to S.b. The jejunum/ileum samples and Caco2 cells were examined for the expression or concentration of the inflammatory components. RESULTS: Treatment with S.b modulated the expressions of TLR2, TLR4, MyD88, NF-κB, ERK1/2, phospho-p38, phospho-JNK, TNF-α, IL-1ß, and CXCL-1 in the jejunum/ileum and Caco2 cells following treatment with 5-FU. CONCLUSION: Toll-like/MyD88/NF-κB/MAPK pathway are activated during intestinal mucositis and their modulation by S.b suggests a novel and valuable therapeutic strategy for intestinal inflammation.

Diversity of Saccharomyces boulardii CNCM I-745 mechanisms of action against intestinal infections.

The yeast Saccharomyces boulardii CNCM I-745 is one of the probiotics recommended for the prevention of antibiotic-associated diarrhea. Studies conducted in vivo and in vitro demonstrated that in the case of infectious diseases there are two potential sites of action of Saccharomyces boulardii CNCM I-745: (1) An action on enteropathogenic microorganisms (adhesion of bacteria and their elimination or an effect on their virulence factors: Toxins, lipopolysaccharide, etc.); and (2) a direct action on the intestinal mucosa (trophic effects, effects on epithelial reconstitution, anti-secretory effects, anti-inflammatory, immunomodulators). Oral administration of Saccharomyces boulardii CNCM I-745 to healthy subjects does not alter their microbiota. However, in the case of diseases associated with the use of antibiotics or chronic diarrhea, Saccharomyces boulardii CNCM I-745 can restore the intestinal microbiota faster. The interaction of Saccharomyces boulardii CNCM I-745 with the innate immune system have been recently demonstrated thus opening up a new therapeutic potential of this yeast in the case of diseases associated with intestinal infections but also other pathologies associated with dysbiosis such as inflammatory diseases.

Saccharomyces boulardii Strain CNCM I-745 Modifies the Mononuclear Phagocytes Response in the Small Intestine of Mice Following Salmonella Typhimurium Infection.

Intestinal mononuclear phagocytes (MPs) comprise dendritic cells (DCs) and macrophages (Mφs) that play different roles in response to Salmonella infection. After phagocytosis, DCs expressing CD103 transport Salmonella from the intestinal tract to the mesenteric lymph nodes (MLN) and induce adaptive immune responses whereas resident Mφs expressing CX3CR1 capture bacteria in the lumen and reside in the lamina propria (LP) where they induce a local immune response. CX3CR1+ Mφs are generated from Ly6Chi monocytes that enter the colonic mucosa and differentiate locally. We previously demonstrated that the probiotic yeast Saccharomyces boulardii CNCM I-745 (S.b) prevents infection by Salmonella enterica serovar Typhimurium (ST), decreases ST translocation to the peripheral organs and modifies the pro-and anti-inflammatory cytokine profiles in the gut. In the present study, we investigated the effect of S.b on the migratory CD103+ DCs and the resident CX3CR1+ Mφs. MPs were isolated from the LP of streptomycin-treated mice infected by ST with or without S.b treatment before or during the infection. In S.b-pretreated mice, we observed a decrease of the CD103+ DCs in the LP that was associated with the drop of ST recovery from MLN. Interestingly, S.b induced an infiltration of LP by classical Ly6Chi monocytes, and S.b modified the monocyte-Mφ maturation process in ST-infected mice. Our results showed that S.b treatment induced the expansion of Ly6Chi monocytes in the blood as well as in the bone marrow (BM) of mice, thus contributing to the Mφ replenishment in LP from blood monocytes. In vitro experiments conducted on BM cells confirmed that S.b induced the expansion of CX3CR1+ Mφs and concomitantly ST phagocytosis. Altogether, these data demonstrate that Saccharomyces boulardii CNCM I-745 modulates the innate immune response. Although here, we cannot explicitly delineate direct effects on ST from innate immunity, S. b-amplified innate immunity correlated with partial protection from ST infection. This study shows that S.b can induce the expansion of classical monocytes that are precursors of resident Mφs in the LP.

The Saccharomyces boulardii CNCM I-745 strain shows protective effects against the B. anthracis LT toxin.

The probiotic yeast Saccharomyces boulardii (S. boulardii) has been prescribed for the prophylaxis and treatment of several infectious diarrheal diseases. Gastrointestinal anthrax causes fatal systemic disease. In the present study, we investigated the protective effects conferred by Saccharomyces boulardii CNCM I-745 strain on polarized T84 columnar epithelial cells intoxicated by the lethal toxin (LT) of Bacillus anthracis. Exposure of polarized T84 cells to LT affected cell monolayer integrity, modified the morphology of tight junctions and induced the formation of actin stress fibers. Overnight treatment of cells with S. boulardii before incubation with LT maintained the integrity of the monolayers, prevented morphological modification of tight junctions, restricted the effects of LT on actin remodeling and delayed LT-induced MEK-2 cleavage. Mechanistically, we demonstrated that in the presence of S. boulardii, the medium is depleted of both LF and PA sub-units of LT and the appearance of a cleaved form of PA. Our study highlights the potential of the S. boulardii CNCM I-745 strain as a prophylactic agent against the gastrointestinal form of anthrax.

Saccharomyces boulardii modifies Salmonella typhimurium traffic and host immune responses along the intestinal tract.

Salmonella enterica serovar Typhimurium (ST) is an enteropathogenic Gram-negative bacterium that causes infection following oral ingestion. ST spreads rapidly along the gastrointestinal tract (GIT) and invades the intestinal epithelium to ultimately reach internal body organs. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is prescribed for prophylaxis of diarrheal infectious diseases. We previously showed that S.b-B prevents weight loss in ST-infected mice and significantly decreases bacterial translocation to the spleen and liver. This study was designed to investigate the effect of S.b-B on ST migration along the GIT and the impact of the yeast on the host's early innate immune responses. Bioluminescent imaging (BLI) was used to evaluate the effect of S.b-B on the progression of luminescent Salmonella Typhimurium (ST-lux) in the GIT of mice pretreated with streptomycin. Photonic emission (PE) was measured in GIT extracts (stomach, small intestine, cecum and colon) at various time periods post-infection (PI). PE analysis revealed that, 45 min PI, ST-lux had migrated slightly faster in the mice treated with S.b-B than in the untreated infected animals. At 90 min PI, ST-lux had reached the cecum in both groups of mice. Adhesion of ST to S.b-B was visualized in the intestines of the mice and probably accounts for (1) the faster elimination of ST-lux in the feces, and (2) reduced translocation of ST to the spleen and liver. In the early phase of infection, S.b-B also modifies the host's immune responses by (1) increasing IFN-γ gene expression and decreasing IL-10 gene expression in the small intestine, and (2) elevating both IFN-γ, and IL-10 mRNA levels in the cecum. BLI revealed that S.b-B modifies ST migration and the host immune response along the GIT. Study findings shed new light on the protective mechanisms of S.b-B during the early phase of Salmonella pathogenesis.

Saccharomyces boulardii improves intestinal epithelial cell restitution by inhibiting αvß5 integrin activation state.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2ß1 integrin collagen receptors. In the present study, we demonstrated that α2ß1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvß5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvß5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvß5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvß5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Modification of Salmonella Typhimurium motility by the probiotic yeast strain Saccharomyces boulardii.

BACKGROUND: Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain. CONCLUSIONS: This study reveals that S.b-B modifies Salmonella's motility and trajectory which may account for the modification of Salmonella's invasion.

Calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance in colorectal cancer xenografts.

CPT-11 (irinotecan), the first-line chemotherapy for advanced stage colorectal cancer, remains inactive in about half of patients (primary chemoresistance) and almost all initial responders develop secondary resistance after several courses of treatment (8 months on average). Nude mice bearing HT-29 colon cancer xenografts were treated with CPT-11 and/or an NF-κB inhibitor for two courses. We confirm that NF-κB inhibition potentiated CPT-11 anti-tumoural effect after the first course of treatment. However, tumours grew again at the end of the second course of treatment, generating resistant tumours. We observed an increase in the basal NF-κB activation in resistant tumours and in two resistant sublines, either obtained from resistant HT-29 tumours (HT-29R cells) or generated in vitro (RSN cells). The decrease of NF-κB activation in HT-29R and RSN cells by stable transfections with the super-repressor form of IκBα augmented their sensitivity to CPT-11. Comparing gene expression profiles of HT-29 and HT-29R cells, we identified the S100A10/Annexin A2 complex and calpain 2 as over-expressed potential NF-κB inducers. SiRNA silencing of calpain 2 but not of S100A10 and/or annexin A2, resulted in a decrease in NF-κB activation, an increase in cellular levels of IκBα and a partial restoration of the CPT-11 sensitivity in both HT-29R and RSN cells, suggesting that calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance. Thus, targeted therapies directed against calpain 2 may represent a novel strategy to enhance the anti-cancer efficacy of CPT-11.

Saccharomyces boulardii improves intestinal cell restitution through activation of the α2ß1 integrin collagen receptor.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2ß1 integrin activity was assessed using adhesion assays and the analysis of α2ß1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2ß1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2ß1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Interaction of Saccharomyces boulardii with Salmonella enterica serovar Typhimurium protects mice and modifies T84 cell response to the infection.

BACKGROUND: Salmonella pathogenesis engages host cells in two-way biochemical interactions: phagocytosis of bacteria by recruitment of cellular small GTP-binding proteins induced by the bacteria, and by triggering a pro-inflammatory response through activation of MAPKs and nuclear translocation of NF-kappaB. Worldwide interest in the use of functional foods containing probiotic bacteria for health promotion and disease prevention has increased significantly. Saccharomyces boulardii is a non-pathogenic yeast used as a probiotic in infectious diarrhea. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we reported that S. boulardii (Sb) protected mice from Salmonella enterica serovar Typhimurium (ST)-induced death and prevented bacterial translocation to the liver. At a molecular level, using T84 human colorectal cancer cells, we demonstrate that incubation with Sb before infection totally abolished Salmonella invasion. This correlates with a decrease of activation of Rac1. Sb preserved T84 barrier function and decreased ST-induced IL-8 synthesis. This anti-inflammatory effect was correlated with an inhibitory effect of Sb on ST-induced activation of the MAPKs ERK1/2, p38 and JNK as well as on activation of NF-kappaB. Electron and confocal microscopy experiments showed an adhesion of bacteria to yeast cells, which could represent one of the mechanisms by which Sb exerts its protective effects. CONCLUSIONS: Sb shows modulating effects on permeability, inflammation, and signal transduction pathway in T84 cells infected by ST and an in vivo protective effect against ST infection. The present results also demonstrate that Sb modifies invasive properties of Salmonella.

Saccharomyces boulardii inhibits inflammatory bowel disease by trapping T cells in mesenteric lymph nodes.

BACKGROUND & AIMS: Saccharomyces boulardii is a nonpathogenic yeast used for treatment of diarrhea. We used a mice model of inflammatory bowel disease (IBD) to analyze the effects of S boulardii on inflammation. METHODS: Lymphocyte-transferred SCID mice, displaying IBD, were fed daily with S boulardii. Weight loss and inflammatory status of the colon were monitored. Nuclear factor-kappaB activity was assessed in the colon. The CD4(+) T-cell production of interferon (IFN) gamma was evaluated by enzyme-linked immunosorbent assay, and a comprehensive reverse-transcription polymerase chain reaction (RT-PCR) analysis for both colon and mesenteric lymph nodes was performed. Finally, we analyzed cell migration mechanisms in vitro and in vivo. RESULTS: S boulardii treatment inhibits IBD. S boulardii induces an accumulation of IFN-gamma-producing T-helper 1 cells within the mesenteric lymph nodes correlated with a diminution of CD4(+) T-cell number and IFN-gamma production by CD4+ T cells within the colon. The influence of S boulardii treatment on cell accumulation in mesenteric lymph nodes was also observed in normal BALB/c mice and involves modifications of lymph node endothelial cell adhesiveness by a yeast secretion product. CONCLUSIONS: S boulardii has a unique action on inflammation by a specific alteration of the migratory behavior of T cells, which accumulate in mesenteric lymph nodes. Therefore, S boulardii treatment limits the infiltration of T-helper 1 cells in the inflammed colon and the amplification of inflammation induced by proinflammatory cytokines production. These results suggest that S boulardii administration may have a beneficial effect in the treatment of IBD.

Saccharomyces boulardii prevents TNF-alpha-induced apoptosis in EHEC-infected T84 cells.

Induction of apoptosis and necrosis by enterohemorrhagic Escherichia coli (EHEC) has been reported in vivo and in vitro, but features of cell death were not noted in those reports. Since tumor necrosis factor-alpha (TNF-alpha) has been implicated in the apoptosis of invasive bacteria, we investigated the role of this cytokine in EHEC-induced apoptosis. We hypothesize that the probiotic yeast strain Saccharomyces boulardii that interferes with EHEC-induced pro-inflammatory pathways delays EHEC-induced apoptosis. By 6 h of infection, flow cytometry analysis of T84 cells demonstrated that 40% of cells were FITC-annexin-V-positive and 40% of cells incorporated both annexin and propidium iodide (PI). Simultaneously, western blot analysis demonstrated that procaspases-8 and -3 were cleaved. Fragmentation of internucleosomal DNA revealed evidence of apoptotic leader formation after 8 and 9 h of infection. Procaspase-9 activation and 3',3-dihexyloxacarbocyanine iodide (DiOC(6)) incorporation were observed at 3 h of infection. In cells preincubated with S. boulardii and infected with EHEC in the presence of yeast, the quantities of procaspases-8, -9 and -3 did not vary, and no DNA fragmentation was observed. The TNF-alpha transcript level and the level of secreted TNF-alpha increased considerably (P<0.001vs control cells) at 6 h of infection in EHEC-alone-infected cells, but were significantly reduced in cells infected in the presence of S. boulardii (P<0.001vs EHEC-alone-infected cells). The presence of anti-TNF-alpha antibody during infection reduced by 30% the level of FITC-annexin V-positive cells. Altogether, these findings demonstrated that: (i) EHEC infection stimulated TNF-alpha synthesis that is implicated in apoptosis of T84 cells; and (ii) S. boulardii induced a decrease in TNF-alpha and related apoptosis in EHEC-infected T84 cells.