Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis
Tomohiro Watanabe, Naoki Asano, Peter J. Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J. Fuss, Atsushi Kitani, Warren Strober
Tomohiro Watanabe, Naoki Asano, Peter J. Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J. Fuss, Atsushi Kitani, Warren Strober
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Research Article Gastroenterology

Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis

Abstract

The mechanisms underlying the susceptibility of individuals with caspase recruitment domain 15 (CARD15) mutations and corresponding abnormalities of nucleotide-binding oligomerization domain 2 (NOD2) protein to Crohn disease are still poorly understood. One possibility is based on previous studies showing that muramyl dipeptide (MDP) activation of NOD2 negatively regulates TLR2 responses and that absence of such regulation leads to heightened Th1 responses. We now report that administration of MDP protects mice from the development of experimental colitis by downregulating multiple TLR responses, not just TLR2. The basis of these in vivo findings was suggested by in vitro studies of DCs, in which we showed that prestimulation of cells with MDP reduces cytokine responses to multiple TLR ligands and this reduction is dependent on enhanced IFN regulatory factor 4 (IRF4) activity. Further studies of mouse models of colitis showed that this inhibitory role of IRF4 does in fact apply to MDP-mediated protection from colitis, since neither IRF4-deficient mice nor mice treated with siRNA specific for IRF4 were protected. These findings indicate that MDP activation of NOD2 regulates innate responses to intestinal microflora by downregulating multiple TLR responses and suggest that the absence of such regulation leads to increased susceptibility to Crohn disease.

Authors

Tomohiro Watanabe, Naoki Asano, Peter J. Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J. Fuss, Atsushi Kitani, Warren Strober

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Figure 9

Systemic administration of MDP prevents the development of TNBS colitis by upregulating IRF4 expression.

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Systemic administration of MDP prevents the development of TNBS colitis ...
Mice administered intrarectal TNBS on day 0 were injected with MDP or PBS i.p. on days –3, –2, and –1 and also administered 100 μg of HVJ-encapsulated control siRNA or IRF4 siRNA by intrarectal instillation on days –2, –1, 0, and 1. (A) IRF4 expression in CD11b+ myeloid cells from MLNs and spleens from mice on day 0 (top); IRF4 expression in whole-cell extracts of CD11b+ myeloid cells from MLNs isolated from mice treated with IRF4 siRNA on day 0 (bottom). (B) Changes of body weight in mice treated with MDP and siRNAs. **P < 0.01 compared with body weight of mice treated with PBS. (C) H&E-stained colonic tissue of mice harvested on day 4. Histology of PBS-treated mice and IRF4 siRNA–treated mice showed massive infiltration of mononuclear cells as well as destruction of crypt architecture; histology of control siRNA-treated mice showed almost normal colon tissue with minimal infiltration of mononuclear cells. Original magnification, ×100. (D) MLN cells (1 × 106/ml) isolated from mice on day 4 were stimulated with a broad range of TLR ligands; cultured supernatants were collected at 48 hours and analyzed for cytokine production by ELISA. *P < 0.05; **P < 0.01 compared with the concentrations of cytokines from PBS-treated mice (white bars).