Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis

Background & Aims: We tested whether the attenuation of experimental colitis by live probiotic bacteria is due to their immunostimulatory DNA, whether toll-like receptor (TLR) signaling is required, and whether nonviable probiotics are effective. Methods: Methylated and unmethylated genomic DNA isolated from probiotics (VSL-3), DNAse-treated problotics and Escherichia coli (DH5alpha) genomic DNA were administered intragastricly (i.g.) or subcutaneously (sc) to mice prior to the induction of colitis. Viable or gamma-irradiated probiotics were administered i.g. to wild-type mice and mice deficient in different TLR or in the adaptor protein MyD88, 10 days prior to administration of dextran sodium sulfate (DSS) to their drinking water and for 7 days thereafter. Results: Intragastric and sc administration of probiotic and E. coli DNA ameliorated the severity of DSS-induced colitis, whereas methylated probiotic DNA, calf thymus DNA, and DNase-treated probiotics had no effect. The colitis severity was attenuated to the same extent by i.g. delivery of nonviable gamma-irradiated or viable probiotics. Mice deficient in MyD88 did not respond to gamma-irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of gamma-irradiated probiotics, whereas, in TLR9-deficient mice, gamma-irradiated probiotics had no effect. Conclusions: The protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon. TLR9 signaling is essential in mediating the anti-inflammatory effect of probiotics, and live microorganisms are not required to attenuate experimental colitis because nonviable probiotics are equally effective.