2nd Annual BMRP Investigator Meeting - Abstract

Toll R-9 Signaling Mediates the Anti-inflammatory Effect of Probiotics in Experimental Colitis

Daniel Rachmilewitz1,a, Kyoko Katakura2, Fanny Karmeli1, Tomoko Hayashi2, Constantine Reinus1, Jongdae Lee2, Kenji Takabayashi2 and Eyal Raz2

Departments of Medicine, 1Shaare Zedek Medical Center (Jerusalem, Israel) and 2University of California, San Diego (U.S.A.)

Live probiotic (PRO) bacteria attenuate experimental colitis and inflammatory bowel disease (IBD).  We showed that immunostimulatory DNA sequences containing the CpG motif, common in certain bacterial genomes, ameliorate experimental colitis (Gastroenterology 122:1428, 2002).

We tested whether the beneficial effect of PRO is due to the immunostimulatory properties of its DNA, whether TLR-9 signaling is required and if so, whether g-irradiated non-viable PRO are equally as effective as viable PRO.

Methylated and unmethylated genomic DNA (50µg) isolated from VSL#3 packets or DNAse treated PRO (50µg) were administered i.g. to mice 2h before induction of colitis with dextran sodium sulphate (DSS).  Viable or g-irradiated PRO (28x108CFU) were administered i.g. daily to mice ten days prior to administration of DSS to their drinking water and for seven days thereafter.  Mice were sacrificed after seven days of DSS administration; disease activity and histological scores were determined.

PRO DNA, but not methylated PRO DNA, was found to activate NF-kB and to induce IL-6 and IL-12 generation by bone marrow-derived macrophages from wildtype mice, but not in TLR-9 null macrophages.  I.g. administration of PRO DNA ameliorated the severity of DSS induced colitis, whereas methylated PRO DNA, calf thymus DNA and DNAse treated PRO had no effect.  The severity of DSS-induced colitis reflected in the disease activity and histological scores was attenuated to the same extent by i.g. administration of non-viable g-irradiated or viable PRO.  Mice deficient in the adaptor protein MyD88 did not respond to g-irradiated PRO, suggesting the involvement of TLR signaling pathways.  The severity of DSS-induced colitis in TLR-2 and TLR-4 deficient mice was significantly decreased by i.g. administration of g-irradiated PRO, whereas in TLR-9 deficient mice g-irradiated PRO had no effect.

Conclusions:

  • PRO DNA has immunostimulatory activity.
  • The protective effect of PRO is mediated by its own DNA.
  • TLR-9 signaling is essential in mediating the anti-inflammatory effect of PRO.
  • Live microorganisms are not required to attenuate colitis since non-viable PRO are equally effective.

aPrincipal Investigator