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.
| Identifer | oai:union.ndltd.org:DocTA/oai:tesionline.unicatt.it:10280/405 |
| Date | 04 February 2009 |
| Creators | LONGO, STEFANO |
| Contributors | MORELLI, LORENZO, MORELLI, LORENZO |
| Publisher | Università Cattolica del Sacro Cuore, PIACENZA |
| Source Sets | Universita Cattolica del Sacro Cuore. DocTA |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis |
| Format | Adobe PDF |
| Rights | reserved |
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