The biochemical mechanism by which mutations in nucleotide-binding oligomerization domain containing 2 (NOD2) cause Blau syndrome is unknown. Several studies have examined the effect of mutations associated with Blau syndrome in vitro, but none have looked at the implication of the mutations in vivo. To test the hypothesis that mutated NOD2 causes alterations in signaling pathways downstream of NOD2, we created a Nod2 knock-in mouse carrying the most common mutation seen in Blau syndrome, R314Q (corresponding to R334Q in humans). The endogenous regulatory elements of mouse Nod2 were unaltered. R314Q mice showed reduced cytokine production in response to i.p. and intravitreal muramyl dipeptide (MDP). Macrophages from R314Q mice showed reduced NF-κB and IL-6 responses, blunted phosphorylation of MAPKs, and deficient ubiquitination of receptor-interacting protein 2 in response to MDP. R314Q mice expressed a truncated 80 kDa form of NOD2 that was most likely generated by a posttranslational event since there was no evidence for a stop codon or alternative splicing event. Human macrophages from 2 patients with Blau syndrome also showed a reduction of both cytokine production and phosphorylation of p38 in response to MDP, indicating that both R314Q mice and cells from patients with Blau syndrome show reduced responses to MDP. These data indicate that the R314Q mutation when studied with the Nod2 endogenous regulatory elements left intact is associated with marked structural and biochemical changes that are significantly different from those observed from studies of the mutation using over-expression, transient transfection systems.
Blau syndrome is an autosomal dominant disorder caused by mutations in NOD2 and characterized by arthritis, dermatitis and uveitis. NOD2 binds MDP and activates NF-kB and MAPK signaling cascades. Prior in vitro studies reported that NOD2 containing Blau mutations caused enhanced activation of NF-kB, suggesting a gain of function in mutated NOD2 caused Blau syndrome. We tested this hypothesis in vivo by creating a knock-in mouse where a point mutation resulted in a change of arginine [R] to glutamine [Q] at position 314 (R314Q) of NOD2 (position 314 in mice corresponds to 334 in humans). R314Q heterozygous (+/m) and homozygous (m/m) mice did not spontaneously develop arthritis or dermatitis. Bone marrow derived macrophages (BMDM) from R314Q mice showed a reduction in NOD2 protein levels compared to wild type (WT) mice despite comparable amounts of NOD2 mRNA. MDP treatment of BMDM showed reduced activation of NF-kB and p38 MAPK in +/m and m/m compared to WT mice that correlated with the copy number of mutated NOD2, with the greatest reduction in m/m mice. In response to ip MDP, reduced levels of IL-6 and KC were detected in the serum of +/m and m/m mice, also in manner correlating with the copy number of the mutation. These data indicate that R314Q-NOD2 mice do not demonstrate a gain of function of the NOD2 pathway. Rather, R314Q causes a deficiency of NOD2 and raises the possibility that Blau syndrome may fall within the spectrum of an immunodeficiency disease.
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