Contribution of the NOD/RIPK2 signaling pathway to osteoarthritis susceptibility - SUMMARY Osteoarthritis (OA) is a debilitating disease characterized by loss of joint space, degeneration of cartilage at articular surfaces, remodeling of bone and other joint tissues, and inflammation. Although it is the major cause of disability in the aged population, not a single disease modifying drug is available. The functions and identities of the biological processes that pose vulnerabilities to OA and need to be targeted therapeutically are not known! We propose elevated NOD/RIPK2 signaling in joint tissues confers heightened susceptibility to OA. Our analyses of 151 families with dominantly inherited OA yielded 7 novel OA-susceptibility alleles affecting 6 components of the NOD/RIPK2 signaling pathway. This pathway utilizes intracellular NOD receptors to sense damage-associated molecular patterns and signals via RIPK2 to elicit tissue-specific responses. We studied one disease allele, Ripk2Asn104Asp, in depth. Functional analyses indicate the variant protein drives elevated pathway signaling. Mice carrying the dominant Ripk2104Asp allele, introduced by genome editing, have significantly increased sensitivity to post-traumatic OA, and aged Ripk2104Asp mice have severe spontaneous OA. We hypothesize regulation of Nod/Ripk2 signaling is a central component of the homeostatic signaling networks and cell processes that maintain the synovial joint. The initiating factors for OA are unknown. Although chronic systemic inflammation has been proposed as an OA susceptibility factor, unoperated young Ripk2104Asp mice have elevated expression of OA-associated and inflammatory markers in their knees without coincident elevation of markers of systemic inflammation. In Aim 1 we use a genetically modified mouse that permits conditional expression of the dominant Ripk2104Asp allele in selected cell types to identify cells in which Ripk2 signaling affects OA, thus testing our hypothesis that localized aberrant Nod/Ripk2 signaling restricted to the joint is sufficient to augment susceptibility to OA. Mouse models of aging-dependent OA are rare and are needed to discover the changes that indicate early stages of OA. Using mice that constitutively express the Ripk2104Asp allele, in Aim 2 we test the hypotheses that elevated Ripk2 signaling: i) promotes OA-associated gene expression of multiple joints, and ii) specifically causes accelerated onset of histologically recognizable age-dependent OA in the weight-bearing knee. RNA- seq analyses will be used to uncover changes in gene expression patterns and cell populations in the joint that parallel development of OA. Aim 3 tests the hypothesis that the p.Asn104Asp substitution enhances ubiquitination of Ripk2, which leads to prolonged or enhanced signaling. In Aim 4 we test whether therapeutic attenuation of Ripk2 signaling in Ripk2104Asp or WT mice can reduce sensitivity or severity of acute injury. The work will have direct clinical impact, informing efforts to identify biomarkers of susceptibility or early stages of disease and efforts to design and develop therapies for OA.
