NOD2 and CD.

C rohn’s disease (CD) is not a single disease entity, but a group of conditions with similar clinical features, but distinct genetic, serological, and environmental characteristics. Genome-wide association studies (GWAS) of CD patients have identified many disease susceptibility loci, but it must be noted that following identification of gene susceptibility loci there is still a need for further investigation by functional studies in order to determine if there is indeed causality between the loci and the disease. To date, however, there have been only limited studies on the functionality of many of the associated susceptibility genes. The intestinal surface is the most critical barrier in the body between what is internal and the environment. It has the highest exposure to microbial products of any surface as well as to a broad range of environmental antigens. The complex luminal environment plays a central role in the maturation of the mucosal immune system with major implications on the development of tolerance, while immune dysregulation with inflammation is associated with enhanced gut permeability and the failure of systemic tolerance. Inflammation in CD is thought to result from an inappropriate activation of inflammation by intestinal luminal antigens in genetically susceptible individuals. There are two major families of microbial pattern recognition receptors (PRR) that appear central to this. The first are the nucleotide-binding oligomerization domain (NOD) receptors, where polymorphisms in NOD2 were the first, and still the most established, risk loci associated with CD. The second family is the Toll-like receptors (TLRs) that also act as microbial receptors and activators of the innate immune system. The article by Cooney et al links two well-established CD susceptibility loci with defects in the detection and processing of bacteria, which could result in reduced bacterial clearance from the intestinal mucosa and the resultant chronic inflammatory changes observed in CD. NOD2 recognizes numerous luminal antigens and induces autophagy in antigen-presenting dendritic cells (DCs), and the autophagy-related protein (ATG)16L1 is required for the formation of autophagosomes. By measuring autophagosome induction, through assessing the degree of relocalization of light chain 3 (LC3) from diffuse to punctate staining on the cell surface, this article identified that the bacterial ligand, muramyldipeptide, was able to induce autophagosome formation in DCs primarily through the activation of NOD2. The formation of autophagosomes following NOD2 activation was also noted to be at levels similar to those achieved following stimulation of the TLRs 1, 2, and 4. Knocking down NOD2 expression in the DCs reduced autophagosome formation. This decrease also occurred following the reduction of ATG16L1, ATG5, and ATG7 expression through the use of siRNAs, and the inhibition of phosphatidylinositol 3-kinase (PI3K), indicating that these are required for NOD2-mediated autophagy. Antigen presentation was also noted to be enhanced following muramyldipeptide activation of NOD, as the MHC class II expression levels were observed to be increase on the surface of DCs as were the protein expression levels of HLA-DR. Reduction in ATG16L1, ATG5, and ATG7 reduced the HLA-DM colocalization of LC3, indicating that NOD2 induction of autophagy can also influence NOD2-mediated MHC class II antigen presentation in DCs. DCs were then isolated from CD patients with variant NOD2 expression. These cells were noted to induce autophagy to the same extent as wildtype (WT) NOD2expressing DCs following stimulation of the TLRs, where relocalization of LC3 occurred in the same way in both groups. Following specific stimulation of NOD2 with muramyldipeptide, however, relocalization of LC3 did not occur in variant NOD2-expressing DCs. This was also associated with a failure of HLA-DM to colocalize with LC3 and a lack of upregulation of MHC class II surface expression. When DCs were taken from CD patients with the CD-associated susceptibility variant ATG16L1 T300a, but with WT NOD2 expression, similar outcomes were observed. These findings indicate that CD DCs expressing either a NOD2 or ATG16L1 variant were defective in autophagy following specific stimulation of NOD2, with defects in MHC class II processing and T-cell antigen-specific responses. Finally, the article demonstrated that NOD2 variants in DCs could affect bacterial handling. It was noted that fewer bacteria localized to the lysosomes and that there was diminished bacterial killing within the DCs isolated from CD patients with variant NOD2-expression compared DOI 10.1002/ibd.21727 Published online 20 April 2011 in Wiley Online Library (wileyonlinelibrary.com). Received for publication March 6, 2011; Accepted March 16, 2011. Copyright VC 2011 Crohn’s & Colitis Foundation of America, Inc.