Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis
Tomohiro Watanabe, … , Atsushi Kitani, Warren Strober
Tomohiro Watanabe, … , Atsushi Kitani, Warren Strober
Published January 10, 2008
Citation Information: J Clin Invest. 2008;118(2):545-559. https://doi.org/10.1172/JCI33145.
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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 7

NOD2 stimulation is associated with upregulation of IRF4 expression.

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NOD2 stimulation is associated with upregulation of IRF4 expression. (A)...
(A) NF-κB activation in human monocyte–derived DCs. DCs were preincubated with MDP or medium for 24 hours and then stimulated with LPS, PGN, or flagellin for 2 hours; nuclear extracts of the cells were then obtained and subjected to gel-shift assays; results shown are representative of those obtained with 2 healthy donors. (B) Upregulation of IRF4 in MDP-stimulated human monocyte–derived DCs. Whole-cell extracts prepared from DCs incubated with MDP, LPS, or medium for 24 hours were immunoblotted with Abs against the indicated components; results shown are representative of those obtained in 3 healthy donors. (C) IRF4 expression in monocyte-derived DCs transfected with IRF4 siRNA. DCs were transfected with 2 μg of IRF4 siRNA, IRAK-M siRNA, or control siRNA using the Amaxa nucleofection method; 16 hours after transfection, DCs were stimulated with MDP, LPS, or medium for 24 hours, at which point whole-cell extracts were prepared and subjected to immunoblotting with Abs against the indicated components. (D) Effects of IRF4 or IRAK-M siRNA transfection on cytokine production by human monocyte–derived DCs. DCs (2 × 107/ml) from 6 healthy donors were transfected with IRF4 siRNA, IRAK-M siRNA, or control siRNA, as described in C. After 24 hours of culture with MDP, LPS, or medium, DCs were stimulated with PGN, Pam3CSK4, or LPS for another 24 hours; cultured supernatants were assayed for IL-12p40 by ELISA. *P < 0.05; **P < 0.01 compared with DCs transfected with control siRNA and preincubated with medium (white bars).