NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or DisoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.

Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-B, a transcription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn's disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34؉ hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14 ؉ (monocytes), CD15 ؉ (granulocytes), and CD40؉ /CD86 ؉ (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNF␣ resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-B binding element that was required for transcriptional activity in response to TNF␣. Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative IB␣ blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNF␣ or lipopolysaccharide, both p50 and p65 subunits of NF-B were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-B, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.

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Caspase activating and recruitment domain 8 (CARD8) potently inhibits NF-κB signaling, which plays a key role in inflammation, and may contribute to avoid a pathologic activation of NF-κB; however, the transcriptional mechanisms regulating CARD8 expression and the relevance of this protein in inflammatory diseases are poorly understood. We found a NF-κB-binding element within the human CARD8 promoter that was required for transcriptional activity in response to TNF-α and the p65 subunit of NF-κB. Moreover, TNF-α and overexpression of p65 induced the formation of NF-κB-CARD8 promoter complexes. Thus, CARD8 may control NF-κB activation through a regulatory loop. To study the relevance of CARD8 in chronic inflammatory disorders, we functionally characterized a deleterious polymorphism (p.C10X) and studied its association with rheumatoid arthritis (RA). Transfection of cell lines with the allelic variants of CARD8 revealed that full-length (CARD8-L) but not truncated (CARD8-S) protein inhibits NF-κB transcriptional activity, and abrogates the binding of NF-κB to its consensus site. Furthermore, in contrast to the full-length protein, CARD8-S did not modify the expression of NF-κB target genes (cIAP, A1), in response to TNF-α. We analyzed the p.C10X polymorphism in 200 patients with RA, and found that homozygous carriers of the CARD8-S allele have higher disease activity score (p = 0.014), more extra-articular manifestations (p = 0.03), and a lower probability of clinical remission (p = 0.03) than the CARD8-L allele carriers. Overall, our findings provide molecular insight into the expression of CARD8 by NF-κB, and suggest that a deleterious polymorphism of CARD8 may help predict the severity of RA.

NLRP2 has been shown to inhibit the NF-κB signaling pathway, and thus may contribute to modulate the inflammatory response, where NF-κB plays a major role. In this study, we report that expression of NLRP2 is induced upon differentiation of CD34+ hemopoietic progenitors into granulocyte or monocyte/macrophages. We also found that NLRP2 was up-regulated following differentiation of mesenchymal stem cells toward adipocytes. Notably, stimulation of HEK293T cells with TNF-α or overexpression of the p65 subunit of NF-κB resulted in up-regulation of NLRP2 and the formation of NF-κB-NLRP2 promoter complexes. Moreover, ectopic expression of p65 but not of other transcriptional regulators induced transactivation of the NLRP2 promoter. Thus, NLRP2 may control NF-κB activation through a regulatory loop. Nucleotide changes within the NACHT domain of other NLRP proteins have been associated with hereditary fever syndromes and chronic inflammatory diseases. We identified five single nucleotide polymorphisms present in the NACHT domain of NLRP2 by sequencing genomic DNA from 319 healthy controls. The frequencies of the rare alleles varied between 0.2 and 10%. Of note, one of these variants, I352S was unable to block the transcriptional activity of NF-κB and the formation of NF-κB-DNA-binding complexes following stimulation with TNF-α. Overall, our findings provide molecular insight into the expression of NLRP2 by NF-κB and suggest that a polymorphism within the NACHT domain of NLRP2 may contribute to the amplification of inflammatory responses due to a reduction of inhibitory signals on the NF-κB pathway.