Text

Objectives and questions: Synovial inflammation has become an important topic in the pathogenesis and progression of osteoarthritis (OA), highlighting OA as a disease associated with low-grade inflammation rather than one solely caused by wear and tear. The inflammation is characterized by an infiltration of macrophages into the synovial tissue. OA is already known to be associated with an imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages, showing increased levels of M1 macrophages in the OA joint. The cause of this imbalanced polarization is still unknown. Factors like the local microenvironment influence macrophage polarization. With OA synovial fluid (SF) containing pro-inflammatory cytokines potentially polarizing macrophages, this study aimed to investigate its influence on macrophage polarization.

Material and methods: THP-1 cells were differentiated using 1 ng/mL PMA (Phorbol-12-myristat-13-acetat) in RPMI medium with 10% FBS and 1% penicillin/streptomycin. The resulting M0 macrophages were incubated with 20 ng/mL IFN-γ and 10 pg/mL LPS for 72 h to induce M1 polarization, 25 ng/mL IL-4 and 25 ng/mL IL-13 to induce M2 polarization or kept unstimulated (M0). Subsequently, cells were incubated with 20% SF from OA patients (N=43) for 72 h. The SF used varied in OA severity, assessed by the radiological Kellgren-Lawrence (KL) score. Macrophage polarization was analyzed using flow cytometry (M1: CD68, CD80, CD86; M2: CD68, CD163, CD206) and validated using qRT-PCR (M1: IL-1β, IL-8; M2: Fibronectin, CCL18).

Results: After OA SF incubation, macrophage polarization shifted towards M2 in previously M1- and M2-stimulated cells. This was reflected by increased numbers of M2 macrophages in flow cytometry analysis, as well as expression of fibronectin and CCL18. Unstimulated macrophages only exhibited an increase in fibronectin expression. Furthermore, a reduced M1 polarization was observed with reduced IL-1β and IL-8 expression across all conditions. Regarding the radiological severity of OA, incubating unstimulated cells with SF from patients with a KL score of 3 resulted in higher numbers of M1 and M2 macrophages than a score of 4. Similar outcomes were observed in M2-stimulated cells, which showed a higher CCL18 expression with KL3 SF.

Discussion and conclusions: Our data indicate that OA SF alone does not induce M1 polarization in OA. In contrast, it creates an activating and anti-inflammatory environment for macrophages, steering both undifferentiated M0 macrophages and activated M1 and M2 macrophages towards a more anti-inflammatory phenotype. These findings demonstrate the complexity of macrophage polarization in OA, suggesting that M1 macrophages in this condition may be unresponsive to local anti-inflammatory stimuli.