S1681 Investigation of NOD2 Signaling in Dendritic Cells

Background Nucleotide-binding oligomerization domain containing 2 (NOD2) is an intracellular pattern-recognition receptor activated by muramyldipeptide, a component of peptidoglycan contained in bacterial cell walls. Mutations in the NOD2 gene that occur in the muramyldipeptde-recognition domain (such as 1007fsinsC NOD2) predispose to ileal Crohn's disease (CD). NOD2 is expressed in intestinal epithelial cells including Paneth cells, and also in antigen-presenting cells such as dendritic cells (DCs). DCs process information from innate immune receptors such as NOD2 and translate this into specific signals that govern generation of correctly targeted or aberrant immune responses. Stimulation of NOD2 may induce cytokines, defensins, TH responses and autophagy, these being attenuated in the presence of 1007fsinsC NOD2. It remains unclear how this loss of function leads to the pro-inflammatory changes of CD.We have investigated themolecular basis of NOD2 signaling in DCs focusing on 1) documenting large scale gene expression changes following NOD2 triggering in combination with toll-like receptors (TLRs) and 2) using proteomic analysis to delineate the protein complex in which NOD2 acts. A comparison with effects occurring on expression of 1007fsinsC NOD2 reveals further aspects of NOD2 function in DCs that may have implications for inflammation in CD. Methods Large scale gene expression profiling was undertaken using mRNA and microRNA micro arrays pre and post stimulation through NOD2 +/TLRs. Data was analyzed in Genespring and validated using luminex, FACS, Western blot or qPCR. Biological experiments are being undertaken to investigate the effect of differential regulation of individual genes on NOD2 function. For proteomic studies, green-fluorescence protein (GFP)-tagged WT and 1007fsinsC NOD2 expression vectors were generated for expression in HEK293 cells and DCs. Proteins associating with GFP NOD2 are being identified using LC/MS-MS and Q-TOF analysis. Results NOD2 stimulation results in a transcriptional program in DCs that acts synergistically with that of TLRs to induce maturation of DCs and upregulation of inflammatory mediators. Comparison with signaling through 1007fsinsC NOD2 largely demonstrates loss of this synergy, although there is also loss of expression of some TLR negative regulators, which may accentuate signaling through these particular TLRs in specific contexts. Conclusion NOD2 induces signaling in DCs in a largely synergistic manner with TLRs that is lost following expression of 1007fsinsC NOD2. Definition of the protein complex in which NOD2 acts will help define the mechanism of NOD2 signaling in immune cells.