The murine pro-IL-1�� expression plasmid was used to monitor secretion of this cytokine independent from altered normally processing of endogenous pro-IL-1��. Expression of full-length CARD8 or FIIND domain of CARD8 diminishes NOD2-induced release of IL-1�� into the culture medium of HEK cells in a dose-dependent manner (Fig. 3, E and F). Because NOD2-induced NF-��B activation has previously been shown to induce the expression and secretion of chemotactic cytokine IL-8 (6), we also tested the influence of transient transfection with NOD2 and/or full-length CARD8 and/or the FIIND domain of CARD8 on IL-8 secretion in MDP-stimulated HEK cells (Fig. 3, G and H). NOD2-dependent IL-8 secretion was significantly reduced in the presence of the full-length CARD8 protein and the FIIND domain of CARD8.

The expression of the respective proteins was confirmed by Western blot analysis (supplemental Fig. S3). The FIIND Domain Inhibits Nodosome Assembly As CARD8 negatively affects NOD2-dependent signaling and NOD2-mediated antibacterial effects, we investigated whether CARD8 may disturb the assembly of the nodosome complex via an inhibition of the oligomerization of NOD2. For this reason we transiently expressed Myc- and FLAG-tagged NOD2 together with increasing amounts of the FIIND domain of CARD8 and subsequently studied the MDP-induced oligomerization of NOD2. Through the use of differently tagged NOD2 proteins self-oligomerization could be detected via co-immunoprecipitation studies. The FIIND domain of CARD8 is able to inhibit the oligomerization of NOD2 upon MDP stimulation (Fig. 4).

These data suggest that binding of CARD8 and NOD2 negatively interferes with the oligomerization of NOD2 thereby diminishing MDP-induced NOD2 signaling. Co-expression of GFP alone, which was used as a tag in the FIIND expression plasmid, did not affect the MDP-induced oligomerization (data not shown). Interestingly CARD8 interacts with NALP3, another member of the NOD-like receptor family (13). According to the molecular mechanisms proposed in this study, CARD8 represents an adaptor of the inflammasome complex by binding to the NBD domain of NALP3 and enabling Entinostat it to recruit a second caspase-1 protein via homotypic CARD-CARD interactions. FIGURE 4. CARD8 interferes with MDP-induced NOD2 oligomerization. FLAG- and Myc-tagged full-length NOD2 and the CARD8-(1�C320) (FIIND domain of CARD8) were expressed in HEK cells and self-association of NOD2 was determined in absence or presence of MDP (10 … This study demonstrates novel aspects on the function of CARD8 in epithelial cells. Our data indicate that CARD8 acts as a negative regulator of NOD2 signaling by inhibiting the oligomerization process of NOD2 itself.