INCREASED EXPRESSION OF NOTCH RECEPTORS ON OSTEOCLAST PROGENITORS INDUCED BY RHEUMATOID ARTHRITIS

Background: Systemic and periarticular bone loss in rheumatoid arthritis (RA) is mediated by osteoclasts, multinucleated cells originating from the myeloid lineage. Recently, Notch signaling pathway has emerged as a potential regulator of osteoclast progenitor (OCP) differentiation and activation. Objectives: The exact role of Notch signaling in the context of arthritis is still unknown; however, its inhibition has beneficial effects in animal arthritis models. We aimed to determine the expression of Notch receptors and ligands on specific OCP subpopulations and define changes that occur in murine collagen-induced arthritis (CIA) and RA patients. Methods: Peripheral blood, synovial tissue and subchondral bone marrow were collected from RA patients, and periarticular bone marrow (PBM) and spleen (SPL) were harvested from male C57/Bl6 mice immunized with chicken type II collagen. Notch 1 to 4 receptor expression on OCPs was analyzed by flow cytometry. Gene expression of Notch receptors/ligands was determined by qPCR from tissues and sorted OCPs. Sorted OCPs were cultured, with addition of MCSF and RANKL, in control, IgG, Jagged (Jag) 1 or Delta (DLL) 1 coated wells. Immunohistochemistry (IHC) for Notch 1 and 2 was performed on sections of murine hind paws. Research was approved by Ethics committee. Results: We previously identified peripheral and periarticular subpopulations of murine and human OCPs, as CD45+CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ and CD45+CD3-CD19-CD56-CD11b+CD14+CCR2+ respectively, specifically associated with arthritis. Flow cytometry revealed that majority of murine splenic and periarticular OCPs express Notch 2, whereas Notch 1 and 4 were expressed on approximately 10% of cells. In CIA, this highly osteoclastogenic population is expanded as is the expression of Notch 4 in PBM and Notch 3 in SPL. Majority of human peripheral-blood OCPs express Notch 2 and 4, with a specific increase in the expression of Notch 1 and 3 in RA. In contrast, RA synovial-derived OCPs mostly express Notch 1 to 3, whereas subchondral OCPs mostly express Notch 1 and 4. Notch ligands were analyzed at mRNA level and revealed expression of Jag1, Jag2 and DLL4 in murine sorted OCPs and Jag1 and DLL1 in human sorted OCPs. Expression of Notch 1 and 2 was confirmed by IHC on arthritic murine hind paws, with Notch 2 expressed by bone marrow, synovial tissue and chondrocytes and Notch 1 expressed by chondrocytes and synovial tissue. Increased expression of Notch 1, Notch 2 and Jag1 was also confirmed in murine arthritic periarticular tissue by qPCR. During osteoclastogenic culture, murine and human OCPs exhibit a similar gene expression pattern with higher initial expression of Notch 1 and 2, and increase in the expression of Notch 3 and 4 with differentiation. Osteoclasts were also differentiated under Notch-ligand stimulation. Coating with DLL1 results in a greater number of cells expressing osteoclast-specific TRAP, whereas Jag1 seemed to inhibit osteoclastogenesis. Conclusion: Our results indicate that murine and human OCPs express a distinct tissue-specific pattern of Notch receptors. Notch signaling in OCPs is increased in arthritis and may contribute to the osteoclastogenic potential and increased bone resorption. Our next aim would be to determine the effect of Notch inhibition on OCP activity and arthritis severity. Acknowledgments: The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, IP-2014-09-7406 and DOK-2018-09-4276. Disclosure of Interests: None declared