Fig 2: Osteoclast stimulating factor in serum. (a) RANKL expression in serum after 1 and 4 weeks. Vertical axis represents p-RANKL concentrations (pg/mL). Horizontal axis represents time visualized by different treatment groups. Average level was 200 pg/mL (range 97–350 pg/mL). No differences were observed between the groups. (b,c) RANKL and bone-volume correlation after 1 week and 4 weeks. Vertical axes represent S-RANKL concentrations (pg/mL). Horizontal axes represent bone volume (mm3). Correlation coefficients were investigated for the clustered groups at each time point. No correlation between bone-graft volume and RANKL concentration in serum was found. Control, n = 12 biological independent samples. IHTs, n = 6 biological independent samples.

Fig 3: PD-L2 decreases TRAP activity in osteoclast cultures and its ligand RGMb is expressed by osteoclasts. Osteoclast cultures were established from healthy controls (HC) (A), and from synovial fluid mononuclear cells from chronic rheumatoid arthritis joints (B) TRAP activity was measured after 21 days. TRAP activity was determined by the measured optical density (OD), and data are presented as the OD of stimulated cultures normalized by the OD of non-treated (NT) cultures. Addition of rhPD-L2 significantly decreased TRAP activity in cultures co-stimulated with rhRANKL and rhM-CSF (n ?= ?3–13 donors). Bars represent mean with SD. Asterix (*) represents p ?< ?0.05. (C) Osteoclasts stained for the presence of PD-L2 (green) and a nuclear DAPI staining (blue) with matching isotype control. (D) Osteoclasts stained for the presence of RGMb (red) and a nuclear DAPI staining (blue) with matching isotype control (n ?= ?3). (E) PD-L2 decreased erosions induced by ACPA. Osteoclast activity evaluated by erosions on synthetic calcium phoshphate coating plates (n ?= ?4, replicates). (F) PAD2 mRNA levels in response to PD-L2 stimulation in PBMCs (n ?= ?3) and SFMCs (n ?= ?3) respectively. Levels were not affected by PD-L2 supplement. (G) Surface expression of PD-L2 on CD90+ FLS, non-treated (NT) and stimulated with TNF-a and IFN-? (All n ?= ?4). Expression of PD-L2 increased by stimulation. (H) Surface expression of PD-L2 on CD90+ ?FLS, gated on RANKL+ and RANKL- FLS. Stimulation especially increased PD-L2 expression on RANKL+ FLS. Bars represent mean with SD. Asterix (*) represents p ?< ?0.05 and astrerix (***) represents p ?< ?0.001.

Fig 4: BMD is decreased in PD-L2-/-mice. (A) Femoral bone mineral density (BMD) evaluated by DXA of WT, PD-L2-/-, and PD-L1/L2-/- mice. BMD was significantly lower in PD-L2-/- mice compared with WT mice. (B) and (C) Representative µCT images through the distal femoral metaphysis from a WT and a PD-L2-/- mouse. (D) RANKL serum levels in WT, PD-L2-/-, and PD-L1-/-PD-L2-/- mice. Serum RANKL levels in WT mice were significantly lower than in knockout mice. (E) OPG serum levels in WT, PD-1-/-, PD-L2-/-, and PD-L1-/-PD-L2-/- mice. Serum OPG levels did not differ between WT and knockout mice. (F) The ratio between serum RANKL and OPG in WT, PD-1-/-, PD-L2-/-, and PD-L1-/-PD-L2-/- mice. The ratio did not differ between the mice. (n ?= ?4 in each group). Differences between the groups are evaluated by one-way ANOVA with subsequent t-tests. Bars represent mean with SD. Asterix (*) represents p ?< ?0.05 and ** represents p ?< ?0.01.

Fig 5: Chondrocytes produce Rankl under inflammation. a MLI surgery was performed on 12-week-old mice. After 4 weeks, articular cartilage was isolated from sham and MLI knee joints and gene expression of Rankl was measured (n = 4, *P = 0.050). Data are mean ± SEM. b Medial and lateral knee articular cartilage was isolated from patients undergoing TKA with medial compartment OA. Gene expression of human RANKL comparing more damaged medial cartilage to lateral cartilage (n = 5, *P = 0.031). Data are mean ± SEM. c Primary chondrocytes were cultured with IL-1ß (10 ng·mL-1) ± SC-514 (10 µmol·L-1) for 24 h. Rankl gene expression was measured (Untreated vs IL-1ß ***P = 0.000 2, IL-1ß vs IL-1ß + IKK2i ***P = 0.000 7), with data representing mean ± SEM of n = 4 independent experiments. d Rankl protein expression in IL-1ß treated (24 h) chondrocytes. Data are mean ± SD for n = 2 representative replicates (***P = 0.003 3). e Two weeks post MLI, knee joints were fixed and sectioned for TRAP staining to identify osteoclasts in the subchondral region (arrows). f TRAP positive cells were counted in anterior and posterior compartments of femur and tibia in MLI and Sham joints (n = 8). (Sham Anterior Femur vs MLI Anterior Femur ***P = 0.002). Bars represent mean ± S.D. g MLI and sham surgeries were performed on right and left mouse knees, respectively. After 2 weeks, mice were sacrificed and joints were harvested and fixed. Bone volume/total volume of subchondral region was measured by uCT in same region of each mouse tibia (n = 5 mice) (*P = 0.036 5). h Representative image of subchondral slice from sham and MLI mouse joints displaying decreased bone volume (M and L indicate medial and lateral condyle, respectively)