OP0273 Transmembrane tnf signalling through tnf-ri induces spa-like inflammation, whereas signallingthrough tnf-rii is crucial for new bone formation
Authors: M. van Tok, D. Pots, I. Blijdorp, M. Armaka, G. Kollias, M. van de Sande, D. Baeten, L. Van Duivenvoorde
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> TNF can drive strictly distinct inflammatory pathologies depending on its expression form. Previously, we have shown that transmembrane (tm) TNF rather than soluble TNF contributes to key pathological features of spondyloarthritis (SpA), including new bone formation.<sup><sup>1</sup></sup> <h3>Objectives</h3> Delineate the cellular and molecular mechanisms by which selective tmTNF overexpression leads to SpA-like pathology. <h3>Methods</h3> tmTNF tg mice (TgA86)<sup><sup>2</sup></sup> were crossed with TNF-RI or TNF-RII knock out mice. Animals were followed for 100 days for clinical symptoms of arthritis and spondylitis development. Histology was performed at the end of the study on both peripheral and axial joints. Calvarial mouse fibroblasts were cultured in osteogenic conditions. Differentiation towards osteoblasts was analysed by alizarin red staining, alkaline phosphatase (ALP) staining as well as by qPCR for collagen type I, II, and X, ALP and RUNX2. <h3>Results</h3> Clinical arthritis, visualised by swelling and deformation of front- and hind paws, was observed in 100% of the tmTNF<sup>+/WT</sup> (&gt;20) as well as in all tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup> mice (7/7) but not in tmTNF<sup>+/wt</sup>xTNF-RI<sup>-/-</sup> mice (0/9). Histologically, peripheral synovitis, osteitis and enthesitis were observed in all tmTNF<sup>+/WT</sup> and tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup> mice, confirming previous findings that tmTNF-mediated synovitis requires the presence of the TNF-RI receptor.<sup><sup>2</sup></sup> Similarly, hunch back formation and crinkled tails were observed in the tmTNF<sup>+/WT</sup> and the tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup> mice but not in tmTNF<sup>+/WT</sup>xTNF-RI<sup>-/-</sup> mice. Histology confirmed the presence of inflammatory cellular infiltrates at the edge of the intervertebral units in all tmTNF<sup>+/WT</sup> tg mice and all tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup> mice, but not in tmTNF<sup>+/wt</sup>xTNF-RI<sup>-/-</sup> mice. Whereas these data indicate that TNF-RI is required for tmTNF-induced inflammation, it was striking that 50% (10/20) of the tmTNF<sup>+/WT</sup>versus none (0/7) of the tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup> mice depicted clear histological signs of endochondral new bone formation. To test whether TNF-RII is involved in pathological new bone formation in this model, calvarial fibroblasts skulls from tmTNF<sup>+/WT</sup>, tmTNF<sup>+/WT</sup>xTNF-RI<sup>-/-</sup>, tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup> or WT were differentiated with osteogenic medium with or without IL-17A. tmTNF overexpressing fibroblasts enhanced the osteogenic differentiation as observed by ALP and alizarin red staining and increased mRNA levels of Collagen type I and ALP compared to WT. This enhancement in osteogenesis was maintained in tmTNF<sup>+/WT</sup>xTNF-RI<sup>-/-</sup>-derived fibroblasts but abolished in tmTNF<sup>+/WT</sup>xTNF-RII<sup>-/-</sup>-derived fibroblasts. <h3>Conclusions</h3> The SpA-like phenotype in tmTNF tg mice is crucially dependent on TNF-RI to drive peripheral and axial inflammation, but TNF-RII signalling is required to drive the pathological new bone formation under inflammatory conditions. <h3>References</h3> [1] Van Duivenvoorde L, van Tok M, Baeten D. Annals of the Rheumatic Diseases2014;73:441–441. [2] Alexopoulou L, Pasparakis M, Kollias G. Eur J Immunol1997;27(10):2588–92. <h3>Disclosure of Interest</h3> M. van Tok: None declared, D. Pots: None declared, I. Blijdorp: None declared, M. Armaka Employee of: Biomedcode, G. Kollias Employee of: Biomedcode, M. van de Sande: None declared, D. Baeten Employee of: UCB Pharma, L. Van Duivenvoorde: None declared