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      Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

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          Abstract

          Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698 T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans.

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          Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.

          Toll-like receptors (TLRs) play a crucial role in host defense against microbial infection. The microbial ligands recognized by TLRs are not unique to pathogens, however, and are produced by both pathogenic and commensal microorganisms. It is thought that an inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia. Here, we show that commensal bacteria are recognized by TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, we find that activation of TLRs by commensal microflora is critical for the protection against gut injury and associated mortality. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury-and provide a new perspective on the evolution of host-microbial interactions.
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            Differential Roles of TLR2 and TLR4 in Recognition of Gram-Negative and Gram-Positive Bacterial Cell Wall Components

            Toll-like receptor (TLR) 2 and TLR4 are implicated in the recognition of various bacterial cell wall components, such as lipopolysaccharide (LPS). To investigate in vivo roles of TLR2, we generated TLR2-deficient mice. In contrast to LPS unresponsiveness in TLR4-deficient mice, TLR2-deficient mice responded to LPS to the same extent as wild-type mice. TLR2-deficient macrophages were hyporesponsive to several Gram-positive bacterial cell walls as well as Staphylococcus aureus peptidoglycan. TLR4-deficient macrophages lacked the response to Gram-positive lipoteichoic acids. These results demonstrate that TLR2 and TLR4 recognize different bacterial cell wall components in vivo and TLR2 plays a major role in Gram-positive bacterial recognition.
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              Probiotic Mechanisms of Action

              Probiotics are live microorganisms that provide health benefits to the host when ingested in adequate amounts. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria. Probiotics have demonstrated significant potential as therapeutic options for a variety of diseases, but the mechanisms responsible for these effects have not been fully elucidated yet. Several important mechanisms underlying the antagonistic effects of probiotics on various microorganisms include the following: modification of the gut microbiota, competitive adherence to the mucosa and epithelium, strengthening of the gut epithelial barrier and modulation of the immune system to convey an advantage to the host. Accumulating evidence demonstrates that probiotics communicate with the host by pattern recognition receptors, such as toll-like receptors and nucleotide-binding oligomerization domain-containing protein-like receptors, which modulate key signaling pathways, such as nuclear factor-ĸB and mitogen-activated protein kinase, to enhance or suppress activation and influence downstream pathways. This recognition is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage. A clear understanding of these mechanisms will allow for appropriate probiotic strain selection for specific applications and may uncover novel probiotic functions. The goal of this systematic review was to explore probiotic modes of action focusing on how gut microbes influence the host.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                14 April 2014
                : 9
                : 4
                : e94891
                Affiliations
                [1 ]Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA), Centro di Ricerca per gli Alimenti e la Nutrizione (Research Center on Food and Nutrition, CRA-NUT), Rome, Italy
                [2 ]INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, Toulouse, France
                [3 ]University of Toulouse, National Polytechnic Institute of Toulouse (INP), UMR 1331 Toxalim, Toulouse, France
                Charité, Campus Benjamin Franklin, Germany
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AF MR IPO EM. Performed the experiments: AF MR AI JS. Analyzed the data: AF MR IPO EM. Contributed reagents/materials/analysis tools: AF MR. Wrote the paper: EM.

                Article
                PONE-D-14-03314
                10.1371/journal.pone.0094891
                3986366
                24733511
                892b53ee-4ded-4e5e-a974-126fb1a52ac1
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 29 January 2014
                : 20 March 2014
                Page count
                Pages: 10
                Funding
                The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under the grant agreement n° 227549, and partly from the Italian MiPAAF funds, Qualità Alimentare e Funzionale-QUALIFU programme, (D.M.2087/7303/09-29/1/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                T Cells
                Immune Cells
                Computational Biology
                Population Modeling
                Infectious Disease Modeling
                Immunology
                Clinical Immunology
                Immunomodulation
                Immune Response
                Inflammation
                Immune System
                Innate Immune System
                Immunity
                Immune Activation
                Immunoregulation
                Immunity to Infections
                Medicine and Health Sciences
                Gastroenterology and Hepatology
                Gastrointestinal Infections
                Infectious Diseases
                Infectious Disease Control
                Research and Analysis Methods
                Histochemistry and Cytochemistry Techniques
                Immunohistochemistry Techniques
                Immunohistochemical Analysis
                Immunologic Techniques
                Immunoassays
                Immunofluorescence

                Uncategorized
                Uncategorized

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