Differential Regulation of IL-33 by NOD2 as a Potential Mechanism in the Pathogenesis of Chronic Intestinal Inflammation

IL-33 is important in several chronic inflammatory disorders, including inflammatory bowel disease (IBD). NOD2 (nucleotide oligomerization domain protein 2) is a critical intracellular protein that senses bacteria and helps maintain normal gut homeostasis; it is also the major gene linked to Crohn’s disease (CD). We determined whether NOD2 genetic variants differentially regulate IL-33 and whether genetic deletion of NOD2 may have an impact on an experimental model of CD-like ileitis. RAW264.7 mouse macrophages were stably transduced to express human WT, L469F, and R334Q NOD2 variants. IL-33 mRNA and protein levels were measured by qPCR and Western blots following stimulation with MDP, the bacterial ligand for NOD2.  For in vivo studies, we evaluated the effects of NOD2 genetic deletion in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD. Our results showed that although Il33 was increased after MDP stimulation in both WT (P<0.005) and L469F NOD2 macrophage variants (P<0.0002), no difference in MDP-induced Il33 was measured between these two groups.  However, Il33 was significantly downregulated in R334Q NOD2 macrophages after MDP stimulation compared to both WT (P<0.01) and L469F NOD2 (P<0.003).  In fact, in regard to IL-33, macrophages expressing the R334Q NOD2 variant did not respond at all to MDP. Analysis of IL-33 protein confirmed these trends.  In addition, genetic deletion of NOD2 from SAMP mice resulted in a significant decrease in IL-33 (P<0.001), Th2 cytokines (P<0.01, and ileal inflammation (P<0.01). Our results show that IL-33 regulation by NOD2 may represent an important mechanism in the pathogenesis of chronic intestinal inflammation.