Lactobacillus crispatus M247: azioni immuno - modulanti e interazioni molecolari con l' epitelio intestinale

Con il primo lavoro è stato identificato un tratto fenotipico di un ceppo di L.crispatus associato alla capacità di persistere e colonizzare il colon dell’ospite e di modificarene la composizione microbica, tale L.crispatus M247 è in grado di modificare, nell’epitelio del colon, il livello di espressione dei TLR2 dei TLR4 sia in vitro che in vivo. Con il secondo studio si identifica un meccanismo antinfiammatorio, prima sconosciuto, indotto da un ceppo probiotico che coinvolge l’attivazione di PPAR-γ e fornisce una nuova visuale sui meccanismi molecolari coinvolti nel dialogo tra epitelio intestinale e microbiota simbionte.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. Accumulating evidence indicates that the peroxisome proliferator activated receptor (PPAR)- is a major player in maintaining intestinal mucosa homeostasis, but whether PPAR- is directly involved in probiotic-mediated effects and the molecular events involved in its activation are not known. Methods: We investigated the role of PPAR- in the immunomodulatory effects of Lactobacillus crispatus M247 on intestinal epithelial cells (IEC) and the role of probiotic-derived H2O2 on PPAR- activity. Results: L crispatus M247 supplementation in mice significantly increased PPAR- levels and transcriptional activity in the colonic mucosa. L crispatus M247 induced PPAR- nuclear translocation and enhanced transcriptional activity in epithelial (CMT-93) cells, as demonstrated by the increased luciferase activity of a PPAR- –responsive element, PPAR- – responsive gene up-regulation, and reduced activity of an nuclear factor- B–responsive element. Pharmacologic PPAR- inhibition or silencing by small interfering RNA cancelled the L crispatus M247–mediated effects in CMT-93 cells. Because Lactobacillus strains producing little H2O2 failed to activate PPAR- , we investigated the role of L crispatus M247– derived H2O2 in PPAR- activation. L crispatus M247 induced a transient rise in intracellular H2O2 and PPAR- transcriptional activity was cancelled by antioxidant or H2O2 scavenger. Toll-like receptor (TLR)-2 was not required for PPAR- up-regulation mediated by L crispatus M247 in mice, although the protective effects of L crispatus M247 on dextran sodium sulfate-induced colitis were less pronounced in TLR-2 / mice. Conclusions: L crispatus M247 uses H2O2 as a signal transducing molecule to induce PPAR- activation in IEC, directly modulating epithelial cell responsiveness to inflammatory stimuli