Background/Purpose:

Blau syndrome is an autosomal dominant disorder caused by mutations in nucleotide-binding oligomerization domain 2 (Nod2) and characterized by arthritis, dermatitis and uveitis.  Nod2 binds muramyl dipeptide (MDP), a conserved structure from bacterial peptidoglycan, and activates NF-kB and MAPK signaling cascades.  Prior in vitro studies using reporter assays indicated that Nod2 containing Blau mutations caused enhanced activation of NF-kB, suggesting a gain of function disease mechanism for Blau syndrome.  However, studies with peripheral blood mononuclear cells (PBMCs) from patients with Blau syndrome stimulated with MDP show blunted cytokine responses.  We tested the gain of function 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).  Regulatory elements controlling Nod2 expression were not altered in this model. 

Methods:

Knock in mice were studied for systemic cytokine responses.  Bone marrow derived macrophages (BMDM) from knock in mice were studied by western blot analysis for Nod2 expression and intracellular signaling, and by ELISA assays.  Macrophages derived from patients with Blau syndrome were studied for cytokine responses to MDP and phosphorylation of p38 mitogen activated protein kinase (MAPK).

Results: R314Q heterozygous (+/m) and homozygous (m/m) mice did not spontaneously develop arthritis or dermatitis.  BMDM from R314Q mice showed a reduction in full length (116 kD) Nod2 protein levels compared to wild type (WT) mice and they expressed an 80 kD protein that reacted with anti-Nod2 on western blotting.  WT and mutant mice showed comparable amounts of Nod2 mRNA in BMDM and analysis of mRNA by PCR did not identify a splice variant.  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 correlating with the copy number of the mutation.  Macrophages derived from PBMCs of two patients with classic, familial Blau syndrome (a mother and son carrying an R334W mutation) showed negligible IL-6 and IL-8 production, and significantly reduced activation of p38 MAPK, in response to MDP compared to a healthy control. 

Conclusion:

These data indicate that R314Q-Nod2 mice as well as patients with Blau syndrome studied here have lost the ability to respond to MDP, a pathway that is dependent on Nod2.  Rather than a gain of function as previously thought, the mutations cause a deficiency of Nod2-dependent responses to MDP and raise the possibility that Blau syndrome may fall within the spectrum of an immunodeficiency disease.  This observation may provide insights into why granulomas form in Blau syndrome and it might have important implications for treatment.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/blau-syndrome-associated-nod2-mutations-limit-production-of-il-6-and-kcil-8-in-knock-in-mice-and-in-patients-suggesting-a-loss-of-function-disease-mechanism/