OP0305 THE ALARMIN S100A9 HAMPERS OSTEOCLAST DIFFERENTIATION FROM CIRCULATING PRECURSORS BY REDUCING THE EXPRESSION OF RANK
Authors: Martijn van den Bosch, Irene DI Ceglie, Arjen Blom, Robab Davar, Colin Logie, Ehsan Habibi, Johannes Roth, Thomas Vogl, Carl Goodyear, Peter van der Kraan, Peter van Lent
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> High levels of the damage-associated molecular pattern (DAMP) S100A8/A9 are produced in the inflamed synovium during experimental and human rheumatoid arthritis (RA), which have been implicated in sterile inflammation-induced bone resorption. We and others have previously shown that stimulation of mature osteoclasts with S100A8/A9 increases their bone-resorptive capacity. In agreement, reduced bone destruction was observed after induction of experimental RA models in <i>S100a9-/-</i> mice. However, its effects on the differentiation of osteoclasts from their precursors remains elusive. <h3>Objectives</h3> Here, we investigated the effects of S100A9 on osteoclast differentiation from CD14<sup>+</sup> circulating precursors. <h3>Methods</h3> CD14<sup>+</sup> monocytes were isolated from buffy coats of healthy donors and differentiated towards osteoclasts with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B (RANK) ligand in the presence or absence of S100A9. Differentiation state of osteoclasts was determined by tartrate-resistant acid phosphatase (TRAP) staining and resorption capacity was quantified using hydroxyapatite-like-coated plates. RNA expression was analyzed with RNA sequencing and qPCR. RANK expression was assessed using FACS. Underlying epigenetic programming was studied using chromatin immunoprecipitation. Secretion of pro-/anti-inflammatory mediators was analyzed with Luminex analysis. <h3>Results</h3> S100A9 stimulation during monocyte-to-osteoclast differentiation resulted in a strong decrease in the numbers of multinucleated osteoclasts, underlined by a decreased resorptive capacity. The thus differentiated cells showed a high production of pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNFα) after 16h of stimulation. In contrast, at day 4, the cells showed a decreased expression of the osteoclast-promoting factor TNFα. Interestingly, we showed that S100A9 stimulation only during the first 16h of culture was sufficient to reduce osteoclastogenesis. To determine the mechanism of how this short S100A9 stimulation might reduce the osteoclast differentiation, we determined the protein expression of RANK. We observed that within this 16h time frame, S100A9 inhibited the M-CSF-mediated induction of RANK, which we found to be associated with changes in various histone marks at the epigenetic level. This S100A9-induced reduction in RANK could be partially reversed by blocking TNFα using etanercept, but not by blocking interleukin-1 (IL-1) with the IL-1 receptor antagonist. <h3>Conclusion</h3> Whereas S100A8/A9 was previously shown to stimulate the resorptive capacity of mature osteoclasts, we here show that early S100A9 stimulation impedes monocyte-to-osteoclast differentiation via reduction of RANK expression that is partially TNFα-mediated. This suggests that the timing of exposure to S100A8/A9 is an important determinant for monocyte-to-osteoclast differentiation. <h3>Disclosure of Interests</h3> Martijn van den Bosch: None declared, Irene Di Ceglie: None declared, Arjen Blom: None declared, Robab Davar: None declared, Colin Logie: None declared, Ehsan Habibi: None declared, Johannes Roth: None declared, Thomas Vogl: None declared, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie, Peter van der Kraan: None declared, Peter van Lent: None declared&nbsp;