1D). This is molecularly similar to the interaction of CARD8 with NALP3 where the FIIND (CARD8) domain binds to the NBD domain of NALP3 (13, 31). CARD8 and ARQ197 Sigma NOD2 Co-localize in Intestinal Inflammation To investigate the in vivo relevance of these findings localization of endogenous CARD8 and NOD2 was analyzed in human colonic tissue biopsies of patients with Crohn disease. As shown in Fig. 2A CARD8 expression is elevated in the latter group and strong co-localization of NOD2 and CARD8 can be detected in epithelial cells in the inflamed colonic mucosa of CD patients (Fig. 2B). This may argue for a potential role of the CARD8/NOD2 interaction on NOD2 signaling in intestinal epithelial cells. Moderate overlapping expression can also be detected in mononuclear cells in the lamina propria in both the uninvolved colon of healthy individuals and CD patients.
No signal was detected when omitting the respective primary antibodies. FIGURE 2. CARD8 is up-regulated in intestinal inflammation and co-localizes with NOD2 in the mucosa of Crohn disease patients. A, protein extracts were prepared from biopsies from the colonic mucosa of healthy individuals (HN) and patients with Crohn Disease ( … CARD8 Negatively Influences NOD2-mediated Responses Because NOD2 is involved in the defense of bacterial invasion and intracellular bacterial killing (28, 32,�C35) the impact of CARD8 on NOD2-mediated defense against bacterial infection with Listeria monocytogenes was investigated. Expression of CARD8 and co-expression of CARD8 and NOD2 resulted both in an increased cellular bacterial invasion (Fig.
3A), indicating that CARD8 is able to abolish the protective effect of NOD2 against L. monocytogenes. This is likely due to the inhibition of nodosome assembly by CARD8. However, it is also conceivable that CARD8 is masking binding sites of NOD2 critical for bacterial killing, which have been identified previously (32) and thereby disabling bactericidal action. FIGURE 3. CARD8 increases bacterial cytoinvasion and suppresses NOD2-induced NF-��B activity and secretion of IL-1�� and IL-8. A, CARD8 and/or NOD2 were expressed in HEK Batimastat cells following infection with L. monocytogenes and gentamicin-mediated elimination … To further dissect the influence of CARD8 on NOD2-mediated signaling, MDP-induced NF-��B activation and IL-1�� and IL-8 release were measured. Co-expression of CARD8 and NOD2 in HEK cells significantly reduces MDP-induced NF-��B-activation in a dose-dependent manner (Fig. 3B). Consistently, siRNA-mediated knockdown of endogenous CARD8 (Fig. 3C) resulted in an increased NF-��B promoter transactivation in HeLa cells compared with controls (Fig. 3D).