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 8
Mechanism of NOD2-induced IRF4 inhibition of TLR signaling. (A) Whole-cell extracts were prepared from THP1 cells stimulated with MDP or LPS for 24 hours and then immunoblotted with Abs against IRF4, IRAK-M, and actin. (B) THP1 cells (5 × 10⁵/ml) were prestimulated with MDP, LPS, or medium for 24 hours and stimulated with TLR ligands; cultured supernatants were collected at 24 hours and analyzed for cytokine production by ELISA. *P < 0.05 compared with the concentrations of cytokines by cells preincubated with medium and stimulated with TLR ligands (white bars). (C) Physical interactions between IRF4 and RICK, MyD88, and TRAF6. Whole-cell extracts of HEK293 cells transfected with vectors (2 μg) expressing FLAG-tagged human IRF4 and HA-tagged human MyD88 or with untagged RICK, TRAF6, or TRAF2 were immunoprecipitated with anti-FLAG–conjugated beads and then immunoblotted with anti-HA Abs or with anti-RICK, -TRAF6, or -TRAF2. (D) Negative regulation of NF-κB by IRF4. HT-29 cells (1 × 10⁵/96-well plate) transfected with pNF-κB–Luc (50 ng) and pSV–β-galactosidase (10 ng) were cotransfected with vectors expressing human RICK (200 ng), human MyD88 (200 ng), or human TRAF6 (200 ng) with or without an IRF4-expressing vector (50 ng, 200 ng, 1000 ng). *P < 0.05; **P < 0.01 compared with cells without IRF4 transfection (white bars). (E) Physical interaction of IRF4 and RICK in MDP-prestimulated human DCs. DCs were cultured with MDP or medium for 24 hours and then stimulated with Pam₃CSK4 for an additional hour; whole-cell extracts were prepared and then immunoprecipitated with anti-IRF4 Abs and immunoblotted with anti-RICK Abs.