title: Aggregating phenotype in Lactobacillus crispatus determines intestinal colonization and TLR2 and TLR4 modulation in murine colonic mucosa.
abstract: The colonic microbiota is a major modulator of the mucosal immune system; therefore, its manipulation through supplementation with probiotics may significantly affect the host's immune responses. Since different probiotics seem to exert various effects in vivo, we tested the relevance of the autoaggregation phenotype on the intestinal persistence of lactobacilli and their ability to modulate the host's innate immune responses. After 14 days of diet supplementation, the aggregating strain Lactobacillus crispatus M247 but not aggregation-deficient isogenic mutant MU5 was recovered from the feces and colonic mucosa of mice. This observation was confirmed by strain-specific PCR amplification and by Lactobacillus-specific denaturing gradient gel electrophoresis analysis. Indeed, L. crispatus M247 increased Toll-like receptor 2 (TLR2) mRNA levels, while it reduced TLR4 mRNA and protein levels in the colonic mucosa, whereas MU5 was ineffective. In colonic epithelial cells (CMT-93 cells) L. crispatus M247 but not MU5 induced time-dependent extracellular signal-regulated kinase-1 (ERK1) tyrosine phosphorylation and TLR modulation, which were abolished in the presence of PD98059 (an ERK1 inhibitor). To assess the functional relevance of probiotic-induced TLR modulation, we determined the consequences of L. crispatus preexposure on TLR4 (lipopolysaccharide [LPS]) and TLR2 [Pam3Cys-Ser-(Lys)4] ligand-mediated effects in intestinal epithelial cells. Preexposure to L. crispatus M247 blunted LPS-induced interleukin-6 (IL-6) release and inhibition of CMT-93 migration over a wound edge, whereas it enhanced TLR2-mediated IL-10 up-regulation. In summary, the aggregation phenotype is required for L. crispatus persistence in the colon and for modulation of TLR2/TLR4 expression through an ERK-dependent pathway. We speculate that the aggregation phenotype in L. crispatus M247 is required to temper epithelial cell responsiveness to bacterial endotoxins, which thus affects the evolution of intestinal inflammatory processes.
authors.affiliation: University of Padua, Department of Histology, Microbiology, and Medical Biotechnologies, Via A. Gabelli 63, Padua 35121, Italy.
authors.forename: Sandra
authors.initials: S
authors.lastname: Voltan
authors.forename: Ignazio
authors.initials: I
authors.lastname: Castagliuolo
authors.forename: Marina
authors.initials: M
authors.lastname: Elli
authors.forename: Stefano
authors.initials: S
authors.lastname: Longo
authors.forename: Paola
authors.initials: P
authors.lastname: Brun
authors.forename: Renata
authors.initials: R
authors.lastname: D'Incà
authors.forename: Andrea
authors.initials: A
authors.lastname: Porzionato
authors.forename: Veronica
authors.initials: V
authors.lastname: Macchi
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.forename: Giacomo C
authors.initials: GC
authors.lastname: Sturniolo
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Morelli
authors.forename: Diego
authors.initials: D
authors.lastname: Martines
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D016207:Cytokines / Q000235:genetics / Q000276:immunology; D004269:DNA, Bacterial / Q000302:isolation & purification; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D007413:Intestinal Mucosa / Q000276:immunology* / Q000382:microbiology; D007422:Intestines / Q000276:immunology / Q000382:microbiology*; D007778:Lactobacillus / Q000254:growth & development / Q000276:immunology / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D019936:Probiotics / Q000008:administration & dosage; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology; D018418:Th2 Cells / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000276:immunology / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism*
chemical_list: D016207:Cytokines; D004269:DNA, Bacterial; D012333:RNA, Messenger; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D048049:Extracellular Signal-Regulated MAP Kinases