Cellular and molecular heterogeneity in the synovial tissue of osteoarthritis patients

Abstract

Osteoarthritis (OA) is the most common form of arthritis and one of the leading causes of disability globally. There are no disease modifying treatments, and for end stage disease, total joint replacement is the only therapeutic option. There is a major unmet clinical need for OA patients. Compared to peripheral blood and inflammatory arthritis (IA), there has been less research into the cellular mechanisms and inflammatory processes involved in the synovial tissue of OA. A barrier to this research has been difficulty in isolating cells from tissues at the site of disease activity. I aimed to identify and compare the cellular phenotype, functional capacity and gene expression of synovial tissue mononuclear cell subsets in OA and IA. After assessing an array of published tissue digestion protocols, I developed a standardised synovial tissue digestion protocol allowing the isolation of multiple cell subsets with high yields, preserved cellular antigens and high cell viability. A multi-colour flow cytometry panel was established to allow the accurate identification and fluorescence-activated cell sorting of cDC2 dendritic cells, CD14+ monocytes, HLA-DR+CD14+ macrophages and CD4+ T cells. Computational analysis of flow cytometry data sets demonstrated greater cellular infiltrate in IA, but an increased proportion of macrophages in OA. However, this proportion was not seen in all OA patients. Owing to their increased but varied proportion, and previously described role in OA pathogenesis, I conducted an in-depth analyses of these cells. OA macrophages had higher expression of surface proteins associated with activation such as CD206, FOLR2 and CD86. Functionality of isolated macrophages was retained, demonstrated by their high phagocytic activity. Next generation RNA-sequencing was performed to better understand their function and heterogeneity in OA synovial tissue. Two distinctive OA endotypes were proposed based on functional gene signatures. One gene signature comprised of cell cycle and proliferation mechanisms, whilst the other consisted of cartilage and tissue development. Despite its central role in arthritic processes, the role of the synovium has remained obscure. This optimised digestion protocol now allows the interrogation of individual cell subsets. Analysis of synovial macrophages using this protocol has demonstrated distinct endotypes within OA patients. Improved investigation into the pathogenesis of OA, especially in the context of OA subtypes, can now be conducted. This could ultimately lead to the modification of treatment strategies for OA patients.