Background/Purpose: Clinical management of localised bone formation and joint ankylosis constitute major challenges in patients with seronegative inflammatory spondyloarthropathies (SpAs).  Whether TNF receptor superfamily members, such as Death Receptor 3 (DR3) and their ligands modulate bone pathology in SpAs is not characterised.  For the first time we evaluated DR3’s role in controlling osteoblast(OB)-dependent new bone formation using the spontaneous ankylosing enthesopathy model in aging DBA/1 mice.

Methods: Osteoprogenitor cells (OPCs) cultured from the bone marrow of male DR3-deficient (DR3^ko) and age-matched wild-type (WT) DBA/1 mice were differentiated into OB using β-glycerophosphate, ascorbic acid and dexamethasone. DR3 and RANKL expression were tested by flow cytometry (fold change over isotype reported).  Functional analyses; OB differentiation (by alkaline phosphatase (ALP) staining) and mineralisation (by alizarin red staining) were performed. Cellular (TNF-like protein 1A (TL1A) mRNA) and soluble cytokines (osteopontin (OPN), osteoprotegerin (OPG) and soluble RANKL) were measured by RT-PCR and ELISA respectively. Finally, a fluorescent optical probe (BoneTag™) was used to visualise and measure in vivo mineralization in 10-month old WT and DR3^komice. Mean±SEM values reported; statistical significance tested by One or 2-way ANOVA as appropriate.

Results: DR3 was expressed on both OPC (1.8±0.2) and OB (1.4±0.8) from WT mice. In DR3^ko levels (1.2±0.1, P<0.05) were comparable in both OPC and OB but significantly lower than WT. For the first time, we showed that TL1A (DR3’s ligand) was constitutively expressed by OPC and OB from WT and DR3^ko. Thus autocrine signalling via the DR3/TL1A pathway functionally translated into a significant elevation in ALP (P<0.05), expression of the Ca2+ binding osteoid protein OPN (P=<0.0001) and mineral apposition (P<0.0001) over the 26 day OB differentiation timecourse (WT versus DR3^ko cultures). OB derived cytokines (RANKL and OPG) control bone formation by regulating osteoclastogenesis. Pro-osteoclastogenic RANKL expression on OPCs and OB was comparable in WT versus DR3^ko whilst soluble RANKL was not detectable in any of the culture supernatants. However, OPG, a soluble inhibitor of osteoclast formation, was significantly elevated across the timecourse in WT versus DR3^ko (P<0.05). When compared against DR3^ko, in vivo incorporation of a fluorescent calcium-chelating probe was striking and significantly higher (P<0.01) in the thoracic vertebrae of WT mice 24 hours after injection. In contrast, the efficiency of probe uptake in WT knee joints and tails was low and comparable with DR3^kolevels in the spine.

Conclusion: These data identify new OB-dependent homeostatic roles for DR3 in bone and potentially explain the atypical pattern of new bone formation in the axial skeleton of mice that spontaneously develop ankylosing enthesopathy. Further studies in human disease are justified to explore the clinical implications of our findings.

F.L. Collins funded by an Arthritis Research UK PhD Studentship.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/death-receptor-3-increases-region-specific-osteoblast-dependent-aberrant-new-bone-formation-in-the-axial-skeleton-of-mice-in-a-spontaneous-model-of-ankylosing-enthesopathy/