A4.1 Synovial macrophages promote TGF-β signalling after intra-articular injections of oxidised LDL in naÏve murine knee joints, preventing production of pro-inflammatory factors S100A8/9, chemokines and aggrecanase-induced neo-epitopes
Authors: W de Munter, AB Blom, PM van der Kraan, J Roth, T Vogl, WB van den Berg, PL van Lent
Journal: Annals of the Rheumatic Diseases (2015)

<h3>Background and objectives</h3> In previous studies we found that synovial macrophages regulate joint pathology during experimental inflammatory osteoarthritis (OA) and that high systemic levels of LDL aggravate OA joint pathology. LDL in inflamed synovium is oxidised and taken-up by macrophages, leading to an activated macrophage phenotype. In this study, we investigate whether direct injection of oxLDL into a murine knee joint induces pathology and elucidate the role of synovial macrophages in that process. <h3>Materials and methods</h3> Knee joints of C57BL/6 mice were injected five consecutive days with 1.2 mg/mL oxLDL, LDL, or an equal volume of vehicle (PBS). This same procedure was performed in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry and RNA expression and protein production by synovium were determined using RT-PCR and luminex, respectively. TGF-β signalling was measured using a functional CAGA-luciferase assay. Data are depicted as mean ± standard deviation. <h3>Results</h3> LDL and oxLDL injection in naïve knee joints did not increase synovial thickening, or production of pro-inflammatory factors (IL-1β, IL-6 and S100A8/9) compared to vehicle injection. TGF-β signalling in synovial wash-outs was, however, significantly increased by 33% (from 84.7 ng/mL/g synovium ± 14.4 to 113.0 ng/mL/g synovium ± 33.3; p &lt; 0.05). Immunohistochemistry of total knee joints showed that oxLDL injection decreased formation of aggrecanase-induced neo-epitopes (NITEGE) compared with vehicle injections, especially in areas along the bone margins that are prone to develop osteophytes (from arbitrary score 1.19 ± 0.57 to 0.33 ± 0.30; p &lt; 0.05). Repeated injections of oxLDL in macrophage-depleted knee joints led to a 3.1 fold increase of synovial thickening, compared with injection of vehicle (p &lt; 0.01), while LDL injections did not alter synovial thickening. Protein levels of S100A8/A9, markers for inflammation, were significantly increased in synovial wash-outs of oxLDL injected joints, compared with LDL (fold increase 5.6; p &lt; 0.05) or vehicle (fold increase 8.3; p &lt; 0.01) injection. RNA levels of chemokines CCL2 (Mcp-1) and CCL3 (Mip-1α) were also significantly upregulated after oxLDL injections (6.7 fold and 4.6 fold, respectively; p &lt; 0.01). No raise in TGF-β signalling was measured in macrophage-depleted joints. NITEGE expression was markedly increased (fold increase 1.92) in the synovium-cartilage contact areas after oxLDL injection (p &lt; 0.05). <h3>Conclusions</h3> Synovial macrophages promote anabolic effects after oxLDL injections, supporting earlier studies which show increased ectopic bone formation during LDL-rich conditions in experimental osteoarthritis. In absence of synovial macrophages, however, oxLDL induces cell influx, production of pro-inflammatory mediators and aggrecanase activity.