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.

IntroductionSystemic rheumatic conditions are often accompanied by intraocular inflammatory disease (termed uveitis). Despite the frequent manifestation of uveitis with arthritis, very little is understood of the underlying mechanisms that mediate the eye’s susceptibility to disease. The genetically susceptible SKG mouse strain develops arthritis that arises from an inherent mutation that disrupts T-cell antigen receptor signal transduction and thymic selection. The ensuing T-cell–mediated disease is further modulated through exposure to microbial triggers. The purpose of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T cells that drive arthritis influences uveitis in SKG mice.MethodsSKG mice (BALB/c mice that harbor the W163C point mutation in zeta-chain-associated protein kinase 70 [i.e., ZAP-70]) were housed under arthritis-resistant, specific pathogen–free conditions. Arthritis was induced by intraperitoneal injection with fungal glucans (zymosan or curdlan). Arthritis onset and severity were evaluated by clinical scoring, histopathology and infrared imaging within the joints. Periocular traits involving blepharoconjunctivitis were evaluated by clinical scoring and histology. Eyes were evaluated for signs of anterior uveitis using intravital videomicroscopy to document cell-trafficking responses within the iris vasculature and stroma and by histology to detect inflammatory infiltrate and tissue damage within the anterior and posterior eye segments.ResultsExposure to zymosan resulted in the predicted arthritic, sexually dimorphic phenotype in SKG mice. The eyes of SKG mice exhibited episodic intravascular cellular responses to zymosan or curdlan as indicated by significant increases in leukocyte–endothelium interactions akin to ocular vasculitis. However, despite the significant increase in early cell-trafficking responses, cellular infiltration into the iris stroma was not observed and histopathological signs indicative of a sustained uveitis were absent. Instead, eyes of SKG mice developed blepharoconjunctivitis that coincided with arthritis and exhibited sexual dimorphism.ConclusionsThis study underscores the complexity surrounding the pathogenesis of uveitis and its relationship with arthritis. The findings suggest that distinct mechanisms exist by which pathogenic autoimmune T cells target the eyes versus joints, which likely involves the environmental context but nonetheless should be taken into account in the identification and development of effective therapies for each organ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0725-z) contains supplementary material, which is available to authorized users.

We recently identified the innate receptor Mincle, a C-type lectin receptor (CLR) that signals through Syk and Card9, as essential in induction of ocular autoimmunity, using experimental autoimmune uveitis (EAU). Now, we further delineate molecular and cellular factors involved in orchestrating this Mincle-dependent mechanism. EAU is typically induced by immunization with interphotoreceptor retinoid-binding protein (IRBP) in CFA and then evaluated by fundus imaging and histology. When the synthetic Mincle agonist TDB (trehalose-6-6′-dibehenate) was used in lieu of CFA, TDB-immunized mice developed uveitis (p<0.05; n=8/group). This effect required Mincle and its downstream signaling adaptor, Card9, as EAU was reduced in the absence of either molecule (p<0.05, n=10–15/group). When Syk, a kinase upstream of Card9, was pharmacologically inhibited by piceatannol (given i.p., q4d, d0–16), TDB-induced EAU was abrogated (p<0.05 vs. control; n=15/group). These results demonstrated that the adjuvant effect of innate signaling through Mincle is necessary and sufficient to support EAU. Studies with bone marrow (BM) chimeras, performed to define the cellular source of Card9 and Mincle-activated responses, showed that Card9 expression was required in hematopoietic, rather than non-hematopoietic cells (p<0.05; n=10/group). Since Card9 is primarily expressed by myeloid cells, Cre-Lox recombination was used to generate conditional KO mice with CD11c+ dendritic cells (DC) lacking Syk. Syk-deficiency in DCs resulted in reduced disease early after EAU induction (p<0.05 vs. control; n=6–10/group). These studies uncover an essential role for Mincle and the Syk/Card9 axis in autoimmune uveitis.

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.