A hyperactivating proinflammatory RIPK2 allele associated with early-onset osteoarthritis

Abstract

Osteoarthritis (OA) is a common debilitating disease characterized by abnormal remodeling of the cartilage and bone of the articular joint. Ameliorating therapeutics are lacking due to limited understanding of the molecular pathways affecting disease initiation and progression. Notably, although a link between inflammation and overt OA is well established, the role of inflammation as a driver of disease occurrence is highly disputed. We analyzed a family with dominant inheritance of early-onset OA and found that affected individuals harbored a rare variant allele encoding a significant amino acid change (p.Asn104Asp) in the kinase domain of receptor interacting protein kinase 2 (RIPK2), which transduces signals from activated bacterial peptidoglycan sensors through the NF-κB pathway to generate a proinflammatory immune response. Functional analyses of RIPK2 activity in zebrafish embryos indicated that the variant RIPK2104Asp protein is hyperactive in its signaling capacity, with augmented ability to activate the innate immune response and the NF-κB pathway and to promote upregulation of OA-associated genes. Further we show a second allele of RIPK2 linked to an inflammatory disease associated with arthritis also has enhanced activity stimulating the NF-κB pathway. Our studies reveal for the first time the inflammatory response can function as a gatekeeper risk factor for OA.

Figure 1.

A dominant RIPK2 mutation associates with early-onset OA of the first MTP joint. (A) Family 1 pedigree. Early-onset OA of the first MTP joint segregates as an apparent autosomal dominant trait with age of onset indicated for affected individuals. Arrow marks the proband. (B) Dorsal radiograph of the right foot of the proband. The black arrow indicates the severe loss of cartilage and joint space in the first MTP joint. The white arrow indicates an unaffected second MTP joint. (B′) Lateral view of the first MTP joint demonstrating osteophyte formation. (C) Schematic diagram of the RIPK2 kinase and C-terminal caspase recruitment (CARD) domains and location of the p.Asn104Asp mutation.

Figure 2.

The OA-associated Ripk2^104Asp variant has altered biological activity. (A–G) Wild-type zebrafish 1-cell zygotes were injected with 400–800 pg mRNA encoding the zebrafish Ripk2^104Asn or Ripk2^104Asp protein variant. At 3 dpf, uninjected embryos (A and B) as well as embryos injected with RNA encoding the Asn variant appeared normal (C and D). In contrast 31% (n = 310) of embryos injected with RNA encoding the OA-associated Asp variant were cyclopic with a normal trunk (E and F) or cyclopic with additional axial defects (G). A, C, E and G are lateral views and B, D and F are ventral views with anterior to the left.

Figure 3.

The OA-associated Ripk2^104Asp variant has increased activity supporting survival in response to bacterial infection. 1 dpf wild-type or ripk2^-/- mutant embryos were injected into the yolk cell with PBS or approximately 300 Pseudomonas alcaligenes (PA) bacteria and survival was measured. ripk2^-/- mutant embryos (green) are significantly more susceptible than WT embryos (red) to infection (P < 0.0001). Uninfected controls are indicated in black (WT uninjected) and gray (ripk2^-/- PBS injected). Injection of ripk2^-/- mutant 1-cell embryos with mRNA encoding the WT Ripk2^104Asn protein failed to protect embryos from infection (orange) (P = 0.8498), whereas injection with mRNA encoding the OA-associated Ripk2^104Asp protein provided a significant degree of protection from infection (blue) (P = 0.0412). Data represent six biological replicates with at least 30 animals in each replicate. Statistical significance determined by the Gehan-Breslow-Wilcoxon test. Error bars represent SE and are only in the positive direction for clarity.

Figure 4.

The OA-associated Ripk2^104Asp variant has increased ability to stimulate the NF-κB pathway. Wild-type zebrafish 1-cell zygotes were injected with 400–800 pg of ripk2^104Asn or ripk2^104Asp mRNA and the abundance of gene-specific RNA transcripts was measured 8–9 h later by qRT-PCR. Levels of gene expression relative to uninjected controls are depicted. Embryos with ectopic expression of the OA-associated Ripk2^104Asp variant (red bars) have increased abundance of NF-κB-stimulated, innate immunity pathway and OA-associated gene transcripts compared with wild-type uninjected controls or to embryos that ectopically express the Ripk2^104Asn protein (blue bars). Data represent four biological replicates. Error bars represent ±SEM and statistically significant differences of P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***) were determined by one-way ANOVA with Tukey’s multiple comparisons test.

Figure 5.

The Behçet’s disease-associated Ripk2^269Thr variant and the OA-associated Ripk2^104Asp variant have similar augmented abilities to activate the NF-κB pathway. Wild-type zebrafish 1-cell zygotes were injected with 400–800 pg mRNA encoding zebrafish Ripk2^104Asn, Ripk2^104Asp or Ripk2^269Thr proteins, and gene-specific transcript levels were measured 8–9 h later by qRT-PCR. Levels of gene expression relative to embryos ectopically expressing Ripk2^104Asn protein are depicted. Embryos expressing the Behçet’s disease associated Ripk2^269Thr variant (magenta bars) or the OA-associated Ripk2^269Asp variant (red bars) have similarly increased activation of the transcription of NF-κB-stimulated, innate immunity pathway and OA-associated genes compared with Ripk2^104Asn expressing embryos. Data represent three biological replicates. Error bars represent ±SEM and statistically significant differences of P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***) were determined by one-way ANOVA with Tukey’s multiple comparisons test.