Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis
J. Clin. Invest. Tomohiro Watanabe, et al. 118:545 doi:10.1172/JCI33145 [Go to this article.]

Figure 7
NOD2 stimulation is associated with upregulation of IRF4 expression. (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).