GSE124928
Transcriptomic profiling of osteoarthritis synovial macrophages reveals a tolerized phenotype compounded by a weak corticosteroid response
It is well-known that long-term osteoarthritis prognosis is not improved by corticosteroid treatments. Here we investigate what could underlie this phenomenon by measuring the short term corticosteroid response of osteoarthritic joint synovial macrophages (OA-Mf). Overall design: Monocytes were purified from healthy blood donor buffy coats as described previously. Monocyte-derived Mf were differentiated for 6?days in RPMI 1640 medium with 10% human serum. Training and tolerization involved exposure to lipopolysaccharide (LPS; 5?ng/ml) or ß-glucan (BG; 10?µg/ml) during the first 24?h of monocyte differentiation. Synovial tissue from end-stage OA patients was obtained after joint replacement surgery. Post-operative biopsies from patients with end-stage OA were handed over to the Experimental Rheumatology laboratory of the Radboud Institute for Molecular Life Sciences. The synovial tissue was separated from muscle, fat and ligament tissue and cultured overnight in RPMI 1640 with 10% fetal bovine serum (FBS) at 37°C to let cells from a vascular origin leach out of the synovial tissue. The next day, the synovial tissue was first cut into small pieces and then digested using plain RPMI medium containing a final concentration of 0.1?mg/ml DNase I (Roche, Mannheim, Germany, 11284932001), 1?mg/ml Collagenase D (Roche, 11088858001) and 2.4?mg/ml Dispase II (Sigma, Burlington, MA, USA, D4693-1G) on a roller at 37°C for 1?h. Afterwards, a single-cell suspension was obtained by pipetting up and down to mechanically break down the tissue. Next, the suspension was washed and run through a 70?µm cell strainer, after which the digestion reaction was stopped by adding pure FBS. Erythrocytes were removed using lysis buffer (155?mM NH4Cl, 12?mM KHCO3 and 0.1?mM EDTA) and the reaction was later stopped by phosphate-buffered saline (PBS) addition. To isolate CD14+ cells, magnetic-activated cell sorting (MACS) buffer (PBS, 1.5% acid citrate dextrose-A, 1% FBS) and CD14-coated microbeads were added to the sample, which was then run through a MACS column. After five washing steps, CD14+ cells were eluted following the manufacturer's (Miltenyi Biotec, Bergisch Gladbach, Germany) instructions and 200?000 obtained synovial cells were resuspended per 6?cm dish in RPMI medium supplemented with 10% FBS. Treatment with a final concentration of 1?µM TA (Sigma, T6501) or only dimethyl sulfoxide (DMSO) solvent (0.1% final concentration) lasted 4?h for all cell types. As a positive control for the TA treatment, peripheral blood mononuclear cells from a healthy blood donation buffy coat were exposed to TA under the same culture conditions as the isolated CD14+ OA-Mf using the same set of reagents and equipment.