AB0091 PD-1 AND GAL3 REGULATE OSTEOCLAST DEVELOPMENT IN RHEUMATOID ARTHRITIS

Abstract

Background:Bone erosions in rheumatoid arthritis (RA) is a major complication. Despite improved treatment, erosions still occur and progress. Therefore, a continuous investigation of the interplay between bone regulation and immune activity is needed.Co-inhibitory receptors, like CTLA-4, participate in modulating osteoclast activity1, and blocking these receptors in cancer treatment results in autoimmune disease2. Programmed death 1 (PD-1) is a central co-inhibitory receptor, also present in a soluble (s) form. We have previously shown that sPD-1 is associated with disease activity and reduced bone erosions in RA3. PD-1 and its ligands are glycosylated and the glycosylation affects signaling through the PD-1 pathway4,5. Galectins (GAL) bind to specific glycosylation patterns on glycoproteins by their carbonrecognition domain. Gal-3 can bind to multiple immune receptors shaping the immunological response6.Objectives:We aimed to investigate if PD-1 and GAL-3 modulate osteoclastogenesis in RA.Methods:Plasma and synovial fluid (SF) samples were collected from patients with chronic (c) RA (>8 years of disease, n=14) SF and blood samples were obtained when patients presented with disease flare. Soluble PD-1 and GAL-3 were investigated by ELISA. GAL-3:PD-1 complexes were captured in an optimized ELISA using both GAL-3 and PD-1 antibodies. Surface plasmon resonance was used to evaluate on the binding properties between GAL-3 and PD-1, on a Biacore 3000. Lactose was used to block the potential binding. Cells from the synovial fluid (SFMC) were differentiated into osteoclasts with M-CSF and RANKL, recombinant human (rh) PD-1 and rhGAL-3 were added and osteoclast formation evaluated by TRAP in the supernatant.Results:Soluble PD-1 and GAL-3 were present in both plasma and SF from cRA patient, and the ratio (PD-1/GAL-3) was increased in SF. PD-1:GAL-3 complexes were detected in both plasma and SF, with the highest concentration in SF. The binding between PD-1 and GAL-3 was confirmed by surface plasmon resonance analyses, with an estimated Kd of 5uM. Binding could be blocked by addition of lactose, confirming the binding to be glycan dependent. In SFMC osteoclast cultures, rhPD-1 and rhGAL-3 slightly decreased osteoclast formation evaluated by TRAP. However, combining rhPD-1 and rhGAL3 further potentiated the reduction in osteoclast formation by 37%.Conclusion:We confirm glycan depended binding between the co-inhibitory receptor PD-1 and GAL-3. Both sPD-1 and Gal-3, and the PD-1:GAL-3 complexes, are upregulated in the inflamed joint at site of erosions. In vitroRA culture demonstrates that GAL-3 potentiate the functions of PD-1 and reduces osteoclastogenesis. These findings indicate that the binding between Gal-3 and PD-1 could provide a novel target to control erosions in RA. Futurein vivostudies on this interaction is needed.