Search

Career situation of first and presenting author Post-doctoral fellow. Introduction Osteocytes derive from bone-forming osteoblasts and are located deep inside the bone matrix. They are encysted in cavities (lacunae) and form dendritic extensions to develop a dense sentinel network inside the bone. Osteocytes are not passive cells. They modulate bone remodelling through regulation of both osteoclast and osteoblast activity. Immature osteocytes express the transmembrane glycoprotein podoplanin (PDPN/gp38)1 which is important for dendrite elongation and osteocyte function.2 Moreover it has been reported that PDPN expression is regulated by inflammatory cytokines including TNFα, IL-6, IL-22, TGF-β1, IFN-γ3 and is highly expressed in synovial tissues from RA patients.4 However the role of PDPN in osteocytes during inflammatory disease has not previously been investigated. Objectives In this study we investigated the effect of osteocyte-specific deletion of PDPN in the KBxN serum transfer (ST) mouse model of rheumatoid arthritis. Methods Dmp1 Cre mice were crossed with PDPNflox/flox mice to generate osteocyte specific conditional knockout mice as well as appropriate PDPNflox/fox controls. KBxN ST arthritis was used to study the effect of PDPN deletion on mouse arthritis progression. PDPN expression was assessed by immunohistochemistry. Osteoclast numbers were calculated by TRAP staining. Loss of cartilage and pannus formation was evaluated by Safranin-O and H and E staining. Standard microCT and synchrotron microCT analysis was used to assess bone density parameters, osteocyte location and bone erosions. Results PDPN is expressed in osteocytes at sites of bone erosions in inflamed joints of KBxN ST mouse model. Osteocyte-specific PDPN deletion has no effect on trabecular and cortical bone density parameters in mouse tibiae under resting conditions, as has been previously reported.2 However, loss of PDPN on osteocytes leads to significantly more bone erosions in the resolving phase of the KBxN ST model compared to non-deleted controls. Deletion of osteocyte PDPN has no effect on pannus formation, cartilage loss and osteoclast numbers. Conclusions Osteocyte-derived PDPN has been suggested as an important sensor for bone damage and this study demonstrates its bone-protective function during inflammatory arthritis. References Zhang KQ, et al. Molecular and Cellular Biology 2006;26:4539–4552. Staines KA, et al. J Cell Physiol 2017;232:3006–3019. Honma M, Minami-Hori M, Takahashi H, Iizuka HP. J Dermatol Sci 2012;65:134–140. Del Rey MJ, et al. Plos One 2014;9:e99607. Disclosure of Interest None declared.

Career situation of first and presenting author Student for a master or a PhD. Introduction Sclerostin is a Wnt inhibitor and has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Sclerostin loss-of-function leads to a higher bone mass and bone strength. Therefore, inhibition of sclerostin is currently considered as a promising treatment for osteoporosis. Surprisingly, in a TNFα-dependent arthritis mouse model (hTNFtg) the genetic deficiency of sclerostin caused a deterioration of disease severity. hTNFtg mice lacking sclerostin displayed enhanced and bone erosion associated with an elevated number of osteoclasts within the joint. Objectives In order to understand the underlying mechanisms, we aimed to investigate the direct and indirect impact of sclerostin on osteoclast differentiation and bone erosion in arthritis. Methods Sclerostin knockout (sost-/-) mice were crossbred with hTNFtg mice to obtain sost-/-/hTNFtg mice, from which synovial fibroblasts (SF) were isolated. Cocultures of synovial fibroblasts and green fluorescent protein (GFP+) bone marrow-derived macrophages (BMM) were performed and osteoclastogenesis was analysed. Receptor activator of NF-kB ligand (RANKL) and macrophage colony stimulating factor (MCSF) expression was measured by ELISA and IL-1a expression by Western Blot. Viability of osteoclasts precursors was measured by MTT Assay. Results In cocultures of SF and GFP+ BMM, osteoclast formation was enhanced by sost-/-/hTNFtg SF compared to hTNFtg SF. Expression of RANKL and MCSF, two crucial factors for osteoclast differentiation, was not different between the genotypes. Interestingly, stimulation of wildtype BMM with conditioned media (CM) from hTNFtg or sost-/-/hTNFtg synovial fibroblasts showed no TRAP+ cells at all. However, CM supplemented with RANKL lead to an elevated number of osteoclasts using sost-/-/hTNFtg CM compared to hTNFtg CM, pointing to a secreted factor promoting osteoclast development. In this regard, the osteogenic factor IL-1α, was higher expressed in sost-/-/hTNFtg SF than in hTNFtg SF. Moreover, the treatment of cocultures with recombinant sclerostin lead to a decreased number of osteoclasts in both genotypes. Accordingly, sclerostin inhibited osteoclastogenesis in monocultures when administered in the pre-differentiation phase, whereas no effect was observed in the differentiation phase, indicating an inhibitory effect of sclerostin mainly on osteoclast precursors. Conclusions Sclerostin deficiency in hTNFtg SF promotes RANKL-mediated osteoclast differentiation, which is likely dependent on the inhibitory effect of sclerostin itself and/or on the promoting effect of higher levels of IL-1α. Disclosure of Interest None declared.