title: 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.
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 16698T, 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.
authors.affiliation: 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.
authors.forename: Alberto
authors.initials: A
authors.lastname: Finamore
authors.affiliation: 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.
authors.forename: Marianna
authors.initials: M
authors.lastname: Roselli
authors.affiliation: 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.
authors.forename: Ambra
authors.initials: A
authors.lastname: Imbinto
authors.affiliation: INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, Toulouse, France; University of Toulouse, National Polytechnic Institute of Toulouse (INP), UMR 1331 Toxalim, Toulouse, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Seeboth
authors.affiliation: INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, Toulouse, France; University of Toulouse, National Polytechnic Institute of Toulouse (INP), UMR 1331 Toxalim, Toulouse, France.
authors.forename: Isabelle P
authors.initials: IP
authors.lastname: Oswald
authors.affiliation: 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.
authors.forename: Elena
authors.initials: E
authors.lastname: Mengheri
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D002467:Cell Nucleus / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis; D054307:Enterotoxigenic Escherichia coli / Q000502:physiology*; D050884:HSP72 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000473:pathology*; D018836:Inflammation Mediators / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000382:microbiology; D007779:Lactobacillus acidophilus / Q000502:physiology*; D021381:Protein Transport; D015398:Signal Transduction; D034421:Sus scrofa; D051195:Toll-Like Receptor 2 / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D016207:Cytokines; D050884:HSP72 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D018836:Inflammation Mediators; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA
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