Showing papers by "Luis G. Bermúdez-Humarán published in 2016"

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease

Abstract: Background Crohn’s disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusions A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Lactobacillus casei BL23 regulates Treg and Th17 T-cell populations and reduces DMH-associated colorectal cancer.

Abstract: Background Chronic intestinal inflammation alters host physiology and could lead to colorectal cancer (CRC). We have previously reported beneficial effects of the probiotic strain of Lactobacillus casei BL23 in different murine models of intestinal inflammation. In addition, there is an emerging interest on the potential beneficial effects of probiotics to treat CRC. We thus explored whether L. casei BL23 displays protective effects on CRC.

Anti-nociceptive effect of Faecalibacterium prausnitzii in non-inflammatory IBS-like models.

Abstract: Visceral pain and intestinal dysbiosis are associated with Irritable Bowel Syndrome (IBS), a common functional gastrointestinal disorder without available efficient therapies. In this study, a decrease of Faecalibacterium prausnitzii presence has been observed in an IBS-like rodent model induced by a neonatal maternal separation (NMS) stress. Moreover, it was investigated whether F. prausnitzii may have an impact on colonic sensitivity. The A2-165 reference strain, but not its supernatant, significantly decreased colonic hypersensitivity induced by either NMS in mice or partial restraint stress in rats. This effect was associated with a reinforcement of intestinal epithelial barrier. Thus, F. prausnitzii exhibits anti-nociceptive properties, indicating its potential to treat abdominal pain in IBS patients.

Gnotobiotic Rodents: An In Vivo Model for the Study of Microbe-Microbe Interactions.

Abstract: Germ-free rodents have no microorganisms living in or on them, allowing researchers to specifically control an animal’s microbiota through the direct inoculation of bacteria of interest. This strategy has been widely used to decipher host-microbe interactions as well as the role of microorganisms in both i) the development and function of the gut barrier (mainly the intestinal epithelium) and ii) homeostasis and its effects on human health and disease. However, this in vivo model also offers a more realistic environment than an assay tube in which to study microbe-microbe interactions, without most of the confounding interactions present in the intestinal microbiota of conventionally raised mice. This review highlights the usefulness of controlled-microbiota mice in studying microbe-microbe interactions. To this end, we summarize current knowledge on germ-free animals as an experimental model for the study of the ecology and metabolism of intestinal bacteria as well as of microbe-microbe interactions.

Identification of novel anti-inflammatory probiotic strains isolated from pulque

Abstract: Probiotics are live microorganisms which when administered in adequate amounts, confer health benefits on the host. Their use is more and more widespread for both prevention and treatment of diseases, including traveler’s diarrhea and inflammatory bowel diseases (IBDs). In this work, we isolated and characterized novel candidate probiotic strains from pulque (xaxtle), a traditional Mexican alcoholic fermented beverage. A total of 14 strains were obtained from xaxtle samples isolated from three different Mexican regions. Species identification was performed by biochemical methods and 16S rRNA gene targeted PCR. The isolates belonged to the Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus composti phylogenetic groups, with L. brevis being the most dominant group. Bacteria were tested for lysozyme, low pH, and bile acid resistance. Moreover, the strains were tested for adherence to human intestinal epithelial cells and screened for their immunomodulatory properties using a cellular model. Selected bacterial strains with anti-inflammatory properties were then tested in vivo in a dinitro-benzene sulfonic acid (DNBS)-induced chronic colitis mouse model, and weight loss, gut permeability, and cytokine profiles were measured as readouts of inflammation. One of the selected strains, Lactobacillus sanfranciscensis LBH1068, improved mice health as observed by a reduction of weight loss, significant decreases in gut permeability, and cytokine modulation. Altogether, our results highlighted the potential of lactobacilli isolated from pulque and in particular the strain L. sanfranciscensis LBH1068 as a novel probiotic to treat IBD.

Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice.

Abstract: Growing evidence supports the efficacy of many probiotic strains in the management of gastrointestinal disorders associated with deregulated intestinal barrier function and/or structure. In particular, bifidobacteria have been studied for their efficacy to both prevent and treat a broad spectrum of animal and/or human gut disorders. The aim of the current work was thus to evaluate effects on intestinal barrier function of Bifidobacterium animalis ssp. lactis CNCM-I2494, a strain used in fermented dairy products. A chronic dinitrobenzene sulfonic acid (DNBS)-induced low-grade inflammation model causing gut dysfunction in mice was used in order to study markers of inflammation, intestinal permeability, and immune function in the presence of the bacterial strain. In this chronic low-grade inflammation mice model several parameters pointed out the absence of an over active inflammation process. However, gut permeability, lymphocyte populations, and colonic cytokines were found to be altered. B. animalis ssp. lactis CNCM-I2494 was able to protect barrier functions by restoring intestinal permeability, colonic goblet cell populations, and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of several TJ proteins, in particular for claudin-4. Finally, B. lactis strain counterbalanced CD4+ lymphocyte alterations in both spleen and mesenteric lymphoid nodes. It restores the Th1/Th2 ratio altered by the DNBS challenge (which locally augments CD4+ Th1 cells) by increasing the Th2 response as measured by the increase in the production of major representative Th2 cytokines (IL-4, IL-5, and IL-10). Altogether, these data suggest that B. animalis ssp. lactis CNCM-I2494 may efficiently prevent disorders associated with increased barrier permeability.

A large scale in vitro screening of Streptococcus thermophilus strains revealed strains with a high anti-inflammatory potential

Abstract: In spite of its contribution to health benefits of yogurt, probiotic properties of Streptococcus thermophilus remain less explored. Hence, we evaluated the capacities of 30 strains of different origins, to resist the stresses prevailing in digestive tracts, of adhering to the mucus producing HT29-MTX cells, as well as their anti-inflammatory properties. First, on the basis of results obtained by multilocus sequence typing, two very closely related groups were distinguished phylogenetically. However, it appeared that in spite of this phylogenetic proximity, resistance to low pH, bile salts and H2O2 and their capacities of adhesion highly varied from one strain to another. Furthermore, most of the strains reduced the production of the pro-inflammatory interleukin IL-8 after co-incubation with HT-29 cells, while they induced production of the anti-inflammatory interleukin IL-10, when incubated with Peripheral Blood Mononuclear Cells. On the basis of ratio of synthesis of IL-10 and of IL-12, currently used to evaluate the anti-inflammatory potential of a probiotic bacterium, three strains appeared to display a strong and promising in vitro anti-inflammatory potential, suggesting that they could be appropriate for elaborating anti-inflammatory functional fermented foods. Finally, the Principal Component Analysis method enabled us to cluster strains into 6 classes displaying distinct phenotypic properties.

A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces.

Abstract: Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri, leading to a stimulation of the host immune response.

Evaluation of the biosafety of recombinant lactic acid bacteria designed to prevent and treat colitis.

Abstract: Inflammatory bowel diseases (IBDs) affect the gastrointestinal tract and are characterized by recurrent inflammation that requires lifelong therapies. Probiotics such as lactic acid bacteria (LAB) have been proposed to complement current treatment protocols for these patients; however, their characteristics are strain dependent. In this regard, certain novel characteristics are only possible through the genetic modification of these beneficial micro-organisms. Different delivery systems, such as protein delivery of anti-oxidant enzymes and anti-inflammatory cytokines, have been shown to be effective in preventing and treating IBD in animal models. In this study, the safety of the recombinant LAB (recLAB) Streptococcus thermophilus CRL807 : CAT, S. thermophilus CRL807 : SOD, Lactococcus lactis NCDO2118 pXILCYT : IL-10, L. lactis MG1363 pValac : IL-10 and L. lactis MG1363 pGroESL : IL-10 with proven beneficial effects was compared to their progenitor strains S. thermophilus CRL807, L. lactis NCDO2118 or L. lactis MG1363. The prolonged administration of these genetically modified strains showed that they were just as safe as the native strains from which they derive, as demonstrated by normal animal growth and relative organ weights, absence of microbial translocation from the gastrointestinal tract, normal blood parameters and intestinal histology. The results show the potential use of these recLAB in future therapeutic formulations; however, the use of modern bio-containment systems is required for the future acceptance of these recLAB by the medical community and patients with IBD.

Oral administration of Lactococcus lactis expressing recombinant 15-lipoxygenase-1 (15 LOX-1) modulates chemically induced colitis in mice

Abstract: Background: Inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, are characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the oral administration of a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a dextran sodium sulfate (DSS)-induced IBD mouse model. Methods: The anti-inflammatory strain L. lactislacti NCDO 2118 was modified to produce active 15-LOX-1 and tested in in a DSS induced IBD mouse model. Results: This 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model and decreased pro-inflammatory cytokines such as IFN-γ and IL-4 while increasing the anti-inflammatory cytokine IL-10. L. lactis NCDO 2118 acts not just as a tool to delivery 15-LOX-1 in the inflamed gut mucosa but still retains its anti-inflammatory effects. Conclusions: This strain could be used in novel adjunct IBD treatment protocols.

Bifidobacterium animalis subsp lactis CNCM-I2494 restores gut barrier permeability in chronically low-grade inflamed mice

Abstract: Objectives: Growing evidence supports the efficacy of many probiotics in the management of deregulated gut barrier. Bifidobacteria have been studied for their efficacy to treat a broad spectrum of gut disorders. The aim of this work was to study the effects of Bifidobacterium animalis subsp lactis CNCM-I2494, a strain used in fermented dairy products for a long time, on intestinal barrier function. Methodology and results: We first induced gut dysfunction in mice by a chronic low-grade inflammation model. Markers of inflammation, gut permeability and immune function were studied pointing out the absence of an active inflammation process. Nevertheless, gut permeability, lymphocyte populations and colonic cytokines were found to be altered in challenged mice. CNCM-I2494 was able to restore the function of the intestinal barrier improving intestinal permeability and restoring colonic goblet cell populations and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of some TJ proteins, particularly claudin-4 protein. Finally, CNCM-I2494 counterbalanced CD4+ lymphocyte alterations in both spleen and mesenteric lymphoid nodes. Conclusion: Altogether, these data suggest that CNCM-I2494 may be an efficient prevention and therapeutic tool for disorders associated with increased barrier permeability.