Cellular characterisation of advanced osteoarthritis knee synovium

Abstract

Objectives: Osteoarthritis (OA) is increasingly recognised as a whole joint disease, with an important role for synovium. However, the repertoire of immune cells and fibroblasts that constitute OA synovium remains understudied. This study aims to characterise the cellular composition of advanced OA synovium and to explore potential correlations between different cell types and patient demographics or clinical scores.

Methods: Synovium, collected from 10 patients with advanced OA during total knee replacement surgery, was collagenase-digested, and cells were stained for flow cytometry analysis. Formalin-fixed paraffin-embedded synovium was sectioned, stained with immunofluorescence, and imaged using the multiplex Cell DIVE platform. Patient demographics and clinical scores were also collected.

Results: The proportion of immune cells in OA synovium varied between patients (8-38% of all cells). Macrophages and T cells were the dominant immune cell populations, together representing 76% of immune cells. Age positively correlated with the proportion of macrophages, and negatively correlated with T cells. CCR6+ T cells were found in 6/10 patients; these patients had a higher mean Kellgren-Lawrence grade across the three knee compartments. Immunofluorescence staining showed that macrophages were present in the lining as well as distributed throughout the sublining, while T and B cells were mainly localised near vessels in the sublining. Fibroblast subsets (CD45-PDPN+) based on the expression of CD34/CD90 or FAP/CD90 were identified in all patient samples, and some populations correlate with the percentage of immune cells or clinical scores. Immunofluorescence staining showed that FAP expression was particularly strong in the lining layer, but also present throughout the sublining layer. CD90 expression was exclusively found around vessels in the sublining, while CD34 was mostly found in the sublining but also occasionally in the lining layer.

Conclusions: There are significant differences in the relative proportions and subsets of immune cells in OA synovium; exploratory correlative analyses suggest that these differences might be correlated with age, clinical scores, or fibroblast subsets. Additional studies are required to understand how different cell types affect OA pathobiology, and if the presence or proportion of cell subsets relates to disease phenotypes.

Keywords: B cells; CCR6; Fibroblasts; Flow cytometry; Immunofluorescence; Macrophages; Osteoarthritis; Synovium; T cells.

Fig. 1

CD45 cell subsets in advanced OA synovium. A Representative example of flow cytometry plot gating lymphocytes, myeloid cells, and CD45− cells using SSC-A vs CD45. B Relative frequencies (%) of lymphocyte, myeloid, and CD45− subsets as a representation of all viable cells. n=10. C Representative immunofluorescence imaging of immune cells (CD45, cyan) in advanced OA synovium; DAPI staining (blue) was used to visualise nuclei and CD146 (orange) to visualise vessels

Fig. 2

The relative frequency (%) and localisation of macrophages (CD68+) and T cells (CD3+) cells in advanced OA synovium as a percentage of immune cells (CD45+). A–C Flow cytometry was used to reveal the relative frequencies (%) of macrophages and T cells. D–E The differences in relative frequency (%) of T cell subsets (D) and macrophage subsets (E) between T cell dominant (blue) or macrophage dominant (red) patients. F Relationship between the relative frequency (%) of macrophages and T cells and the age of the patient. G The differences in age, BMI, and mean compartmental Kellgren-Lawrence (KL) grade between T cell dominant (blue) or macrophage dominant (red) patients. n=10. H Representative immunofluorescence staining of T cells (CD3, cyan) and macrophages (CD68, red); DAPI staining (blue) was used to visualise nuclei and CD146 (orange) to visualise vessels

Fig. 3

Lymphocytes in advanced OA synovium. A,B Relative frequency (%) of T cells (CD3+) and B cells (CD19+) as a percentage of lymphocytes. C Relative frequency of T cell subsets, including CCR6+, CD8+, GDTCR+, and CD161+ T cells. D-G Relationship between the relative frequency (%) of CCR6+ T cells and age in years (D), body mass index (E), and mean compartmental Kellgren-Lawrence (KL) grade (F). n=10 (H) Representative immunofluorescence staining of CD3 (cyan), CD8 (yellow), CD19 (pink) in advanced OA synovium; DAPI staining (blue) was used to visualise nuclei and CD146 (orange) to visualise vessels

Fig. 4

Myeloid cells in advanced OA synovium. A Relative frequency (%) of myeloid cells positive for CD11c, CD14, CD15, CD40, CD68, or CD206. B Relative frequency (%) CD40 and/or CD206 positive macrophage populations. C Immunofluorescence staining of macrophage markers CD68 (red), CD206 (green), CD163 (cyan), and MERTK (yellow) in advanced OA synovium; DAPI staining (blue) was used to visualise nuclei

Fig. 5

Fibroblast (CD45−PDPN+) subsets in advanced OA synovium. A, B Relative frequency (%) of fibroblast subsets based on fibroblast activation protein (FAP) and CD90 expression, n=10; C table with relative frequency (%) of fibroblast subsets based on expression of FAP/CD90 and CD34/CD90. D, E Relative frequency of fibroblast subsets based on CD34 and CD90 expression, n=8; F–I Relationship between fibroblast subsets as the percentage of all viable cells and mean compartmental Kellgren-Lawrence (KL) grade or immune (CD45+) cells as the percentage of all viable cells. J Immunofluorescence staining of fibroblast (activation) markers podoplanin (PDPN, green), FAP (red), CD90 (pink), and CD34 (yellow) in advanced OA synovium; DAPI staining (blue) was used to visualise nuclei