Gastroenterology

Gastroenterology

Volume 126, Issue 2, February 2004, Pages 520-528
Gastroenterology

Clinical-liver, pancreas, and biliary tract
Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis

https://doi.org/10.1053/j.gastro.2003.11.019Get rights and content

Abstract

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 probiotics and Escherichia coli (DH5α) genomic DNA were administered intragastricly (i.g.) or subcutaneously (sc) to mice prior to the induction of colitis. Viable or γ-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: ntragastric 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 γ-irradiated or viable probiotics. Mice deficient in MyD88 did not respond to γ-irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of γ-irradiated probiotics, whereas, in TLR9-deficient mice, γ-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.

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Animals

Balb/c, C57Bl/6 (B6), 129XB6F2, congenic mice bearing the C3H-Tlr4Lps-d mutation (i.e., resistant to LPS) on the Balb/c background, and IL-10-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88 (B6), TLR2 (B6), and TLR9 (129XB6F2)-deficient mice were used as described14, 15 and are currently bred in the UCSD vivarium.

Probiotic preparations

Probiotic bacteria (VSL-3) were purchased from VSL Pharmaceutical Inc. (Gaithersburg, MA). Each packet contains viable lyophilized gram+ bacteria of 4

Probiotic and E. coli DNA have immunostimulatory activities

To evaluate the immunostimulatory properties of probiotic DNA, we assessed the ability of probiotic DNA to activate NF-κB and JNK, 2 major signaling pathways involved in TLR activation.14 Probiotic DNA, but not methylated probiotic DNA or calf thymus DNA, activated NF-κB (EMSA), as did ISS-ODN but not control-ODN (Figure 1A). Similar results were obtained for JNK activation (Figure 1B). The activation of these signaling pathways resulted in the induction of IL-12 (p40) and IL-6, which was

Discussion

Persuasive evidence indicates that intestinal microflora play an important role in the initiation and the perpetuation of murine experimental colitis19 and human IBD.5 This fact initially provided the rationale for the use of probiotics to manipulate the intestinal microenvironment. In fact, recent studies have demonstrated the therapeutic effects of probiotics in various models of experimental colitis and in several clinical trials in patients with IBD or related disorders.10 However, the

Acknowledgements

The authors thank L. Beck and J. Uhle for their helpful editorial assistance.

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  • Cited by (0)

    Supported in part by NIH Grants AI-40682 and DK-35108 (to E.R.) and by the Broad Medical Research Program of the Eli and Edythe L. Broad Foundation (to D.R.).

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