Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease that arises after injury and affects millions worldwide. There are currently no disease-modifying treatments. PTOA is a complex, multi-tissue joint disease characterized by pain, cartilage degradation, synovial inflammation and fibrosis, and formation of ectopic bone growths called osteophytes. The inherent complexity of this disease is a barrier to developing effective treatments, as little is known about the intricate tissue crosstalk that underlies PTOA progression. Our long- term goal is to uncover and comprehensively characterize cellular and molecular mechanisms central to key pathological sequalae of PTOA: synovial fibrosis, inflammation, and osteophyte formation. We will focus on canonical Wnt/β-catenin (cWnt) signaling. cWnt overactivation has recently been implicated as a driving factor of arthritis. Our data show that the cWnt signaling agonist R-spondin 2 (Rspo2) is strongly induced in multiple joint tissues during PTOA, and that Rspo2 alone is sufficient to induce pathological features characteristic of PTOA. Using single-cell RNA-seq, we profiled synovium of mice with PTOA and found that Rspo2 is produced by synovial lining fibroblasts. We identified a novel population of pro-fibrotic cells that arise after injury and express Lgr cell surface receptors for Rspo2. We showed that synovial fibroblasts respond to Rspo2 by secreting cytokines that in turn activate pro-inflammatory macrophages (known to drive synovial pathology in PTOA). Single-cell profiling also revealed a novel subset of injury-induced, Lgr-expressing osteochondral progenitors in synovium, which we propose give rise to osteophytes. We hypothesize that Rspo2-driven cWnt signaling mediates pathological crosstalk between joint-resident cell types to potentiate PTOA. To test this, our aims in the K99 phase are to: 1) determine the role of Rspo2-driven cWnt signaling in the emergence and function of pro-fibrotic synovial cells during PTOA using transgenic reporter mice, multi-omic analyses, and in vitro differentiation assays, and 2) characterize crosstalk between cWnt-active synovial fibroblasts and pro- inflammatory macrophages, using knockout mice and crosstalk assays. To extend upon my molecular biology and immunology expertise, I will receive rigorous technical and conceptual training from my diverse mentorship committee during the K99 phase, and valuable career guidance. This expert training in bioinformatics; cWnt signaling; bone, cartilage, and synovial biology; and multi-modal imaging, will be crucial for carrying out my K99 aims and especially critical for successfully launching my independent career. These skills will be utilized in my R00 phase to: 3) determine how Rspo2/Lgr signaling promotes osteophyte formation in PTOA, using tissue-specific deletion and reporter mice, and in vitro differentiation assays. This work will significantly extend our understanding of cellular and molecular mechanisms that underpin synovial fibrosis, inflammation, and osteophyte formation in PTOA. These insights will have meaningful, tangible outcomes for human health, by accelerating development of effective disease-modifying treatments for PTOA sufferers.

Post-traumatic osteoarthritis (PTOA) is a complex degenerative joint disease that arises after injury and is characterized by pain, cartilage degradation, synovial inflammation and fibrosis, and formation of ectopic bone growths called osteophytes. PTOA is a chronic, debilitating condition that affects millions worldwide, yet there are currently no disease-modifying treatments. The overarching objective of this proposal is to comprehensively characterize cells and processes responsible for PTOA progression and to provide insights for the development of effective PTOA treatments that will meaningfully and tangibly improve patient outcomes.