Osteoarthritis progression is accompanied by inflammatory CD4+ T-cell polarisation

Abstract

Purpose: While OA has generally been considered a “non-inflammatory” disease, there is an increasing body of evidence about the presence of inflammatory cells and cytokines in OA synovium suggesting they play a role in disease progression. It is still controversial which cell types in OA synovium are responsible for maintaining synovial inflammation, and more importantly how the distribution of inflammatory cells, their activation status and polarisation changes with OA progression. The overarching aim of this project was to quantitatively analyse the presence of inflammatory cells and their main pro-inflammatory cytokines in the affected joints of patients with different OA stages. We aimed to investigate T-cell polarisation and activation with OA disease progression. Methods: Synovial membrane (SM), Synovial fluid (SF) and peripheral blood (PB) were harvested from a total of 120 patients (75 women, 45 men, mean age of 67.8 ± 8 years) with different OA stages (Kellgren-Lawrence I-IV). Patients were scheduled for arthroscopy (group 1, K&L I), partial knee replacement (group 2, K&L II-III) and total knee replacement (group 3, K&L IV) in our clinic. PB samples from healthy controls were included. After enzymatic digestion (SM) and gradient centrifugation (SM, PB), mononuclear cells were isolated and analysed by FACS for cell surface and intracellular markers specific for mononuclear cells, T cell subsets (Th1, Th2, Th17, Treg) and activation markers. T-cell cytokine secretion (IL-2, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF) in SF was measured by cytometric bead array (CBA) analysis. Results: Analysis of PB did not show any significant difference within the OA patient groups and in comparison to controls. In SM a distinct infiltration of CD4+ T-cells was present in all three OA groups, which significantly increased with OA stage (p<0.05). Further, CD4+ T-cell subsets in SM and PB samples were analysed. Whereas in PB only a small proportion of CD4+ T-cells were activated and stained positive for the specific subsets, the majority of SM T-cells were differentiated into the T-cell subsets and this increased with OA stage (Figure 1). The highest increase was measured for the inflammatory T cell subsets Th1 and Th17, which significantly altered the Th1/Th2 and Th17/Treg balance. Further analysis of activation markers displayed an activated phenotype of CD4+ T-cells in the SM (CD69+, CD45RO+, CD45RA-, CD62L-) but not in PB. CBA analysis confirmed a significant increase of all T-cell cytokines in the SF of OA groups 2 and 3 when compared to group 1 (p<0.05). Between group 2 and 3, pro-inflammatory Th1 and Th17 cytokines (TNF and IL-17) were further increased and anti-inflammatory Th2 and Treg cytokines (IL-4, IL-10) decreased in group 3. IL-2, IL-6 and INF-γ were slightly higher in group 2. Conclusions: In contrast to SM, no differences were observed between the PB samples of OA patients and controls, showing that the relevant role of inflammatory cells in OA remains a local process. The infiltration of OA synovium with inflammatory cells is not solely a phenomenon of late stage OA. In fact, CD4+ T-cells are already present and activated in the affected joints of early OA stages. T-cell infiltration and polarisation correlates with OA disease stages and shifts the Th1/Th2 and Th17/Treg balance towards an inflammatory T-cell phenotype. Thus, onset and progression of OA is accompanied by an increasing influx of CD4+ T-cells into the SM, their activation, cytokine production and polarisation. These results underline the relevance of T-cells in the inflammatory process of OA, not only in late-stage disease but also in early OA stages. In “classical” inflammatory joint diseases such as rheumatoid arthritis we know that T-cell derived inflammation contributes to cartilage degeneration and joint destruction. The role of these cells in OA needs to be further studied. Nevertheless our current results suggest that T-cell activation and polarisation may play a role in both the initiation and progression of OA, and as such provide a potential therapeutic target.