Osteoarthritis (OA) is a whole joint disease associated with cartilage destruction and chronic inflammation. Catabolic factors, including arthritogenic enzymes are produced by various cell types in the joints, accumulate in the synovium and accelerate tissue damage. Previously, we reported that the synovial lymphatic vessels drain intra-articularly injected dye to draining lymph nodes, which is reduced in joints of post-traumatic OA (PTOA), suggesting that sufficient lymphatic drainage may play a critical role in maintaining joint health. However, the contribution of the synovial lymphatic system (SLS) to the OA pathogenesis is not well understood. The aim of this thesis is to explore:​ 1) how the SLS contributes to the pathology of OA;​ 2) whether targeting the SLS is beneficial for OA;​ 3) what is the cellular and molecular mechanisms underlying targeting SLS for OA treatment, using mouse models of PTOA. Our overall hypothesis is that OA progression is associated with defective SLS caused by pro-inflammatory macrophages (macs), which is induced by the altered ubiquitination status of key proteins.

We demonstrate that synovial lymphatic vessels remove the arthritogenic enzyme MMP13 from synovium tissues to the draining lymph nodes in mice with PTOA and inflammatory arthritis, indicating that dysfunction of the SLS may directly contribute to the progression of PTOA. We show that lymphatic endothelial cells (LECs) isolated from PTOA synovium have an inflammatory phenotype that is induced by pro-inflammatory macs. Bortezomib, a clinically used proteasome inhibitor that blocks the degradation of ubiquitinated proteins, reduces proinflammatory mac-mediated LEC inflammation associated with decreased joint tissue damage. To investigate if the altered protein ubiquitination in macs contributes to PTOA pathogenesis, we used mice with global (Itch-/-) and mac-specific depletion (MΔItch) of ubiquitin E3 ligase Itch based on its known function as a negative regular of inflammation by affecting protein ubiquitination status. Itch-/- and MΔItch mice develop a more severe PTOA compared to control mice, which is associated with increased synovial pro-inflammatory macs, suggesting that Itch depletion in macs exacerbates PTOA progression by promoting the pro-inflammatory polarization of macs. To understand the molecular mechanism by which Itch promotes the proinflammatory polarization of macs, we performed proteomics using purified ubiquitinated proteins from various macs subsets and identified IL-1α specifically being ubiquitinated in proinflammatory macs. Interestingly, IL-1α ubiquitination is further increased in Itch-/- proinflammatory macs, which is associated with increased mature form of IL-1α. Thus, ubiquitin E3 ligase Itch negatively regulates pro-inflammatory polarization of mac by modulating the ubiquitination status of IL-1α.

In summary, we demonstrate that restoring the SLS function by modulating the protein ubiquitination status is protective against PTOA progression.