Background Inflammatory conditions affect bone turnover, as is seen in rheumatoid arthritis (RA). Inflammatory cytokines in RA stimulate osteoclasts and shift the balance between bone formation and resorption. Programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2, are important for maintaining self-tolerance, and this pathway plays a prominent role in RA. Recently, repulsive guidance molecule b (RGMb) and bone morphogenetic protein (BMP) was identified as a novel receptor complex for PD-L2 (1). We previously showed the PD-1 pathway to be associated with radiographic progression in RA (2). The interaction of PD-L2 with the RGMb/BMP receptor complex could potentially provide a link between bone homeostasis and the PD-1 pathway.
Objectives To investigate the possible association between bone homeostasis and the PD-1 pathway in RA.
Methods We generated osteoclasts from RA synovial fluid mononuclear cells (SFMCs) (3), stimulated them with recombinant (rec) PD-1 and PD-L2, and subsequently evaluated these osteoclasts for TRAP activity, and expression of PD-1, PD-L2 and RGMb. We induced RA-like disease in PD-1-and PD-L2-KO mice by immunization with Freund's adjuvant and chick collagen, and evaluated clinical arthritis. After sacrifice, paws were paraffin embedded and stained for TRAP and osteocalcin. The femur was subjected to ex vivo DXA scanning. In addition, we stimulated normal mouse calvarial cells with the recombinant proteins in a BMP reporter assay to evaluate the potential of PD-1 and PD-L2 to affect BMP signaling.
Results Recombinant PD-1 and PD-L2 increased TRAP activity in osteoclasts generated from RA SFMCs, at day 21 (p=0.046). Pre-osteoclasts in the culture expressed both PD-L2 and RGMb. The KO mice did not develop clinical signs of arthritis, ensuring intact mobility and constant bone load. DXA scan revealed that immunized PD-L2 KO mice had a higher bone mass density than other KO mice, WT and non-immunized PD-L2 KO (p=0.03). These mice did not differ histologically in TRAP and osteocalcin expression. Rec PD-L2 significantly decreased BMP signaling in the BMP reporter assay.
Conclusions The clinical association between the PD-1 pathway and radiographic progression in early RA points to its importance in RA bone homeostasis. The increase in TRAP activity after incubation with recombinant PD-L2 supports this association. The increased bone mass density observed in immunized PD-L2 KO mice further supports a potential role for PD-L2 in bone homeostasis. The expression of PD-L2 in pre-osteoclasts also suggests a role for PD-L2 in bone homeostasis. These data provide a new insight into the close relation between the immune system and bone, as well as suggest a potential therapeutic target in diseases involving inflammatory bone changes.
Xiao Y, et. al Journal of Exp Med. 2014 Apr 21;3(1):81.
Greisen SR, et al. Scand J Rheum. 2013 Nov 1;43(2):101–8.
Greisen SR, et al. APMIS. 2015 Sep;123(9):779–86.
Disclosure of Interest None declared