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OP0040 Dynamic in vivo imaging of TH17-mediated control of osteoclast function in live bones by using intravital multiphoton microscopy

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint synovial inflammation and progressive cartilage/bone destruction. Recently it has been reported that Th17, a CD4+ T cell subset expressing RANKL on its cell surface, increases in the synovium of RA patients and enhances bone destruction associated with osteoclasts. However, the mechanism of Th17-mediated control of mature osteoclast function in vivo has remained unclear. Objectives This study aimed to investigate how the bone-resorptive functions of osteoclasts are regulated in situ and how Th17 cells control osteoclastic bone resorption in vivo. Methods To visualize fluorescently-labeled mature osteoclasts, we utilized the mice in which GFP is expressed as a fusion protein with a vacuolar type H+-ATPase “a3 subunit”, preferentially and abundantly expressed in mature osteoclasts (a3-GFP mice) [1]. Polyclonal Th17 cells were differentiated in vitro using anti-IL-4, anti-IFNγ, IL-6, TGFβ and IL-23, labeled with fluorescent dye and then adoptively transferred into a3-GFP mice before imaging. Calvaria bone tissues of a3-GFP mice were observed by using an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have originally established [2,3]. Results We have succeeded in visualization of live mature osteoclasts on the bone surface in situ. Because GFP is expressed as a fusion protein with a3 subunit, GFP fluorescence does not only serve as a marker for mature osteoclasts, but it provides information on the subcellular distribution of V-ATPase in osteoclasts. We identified different populations of live mature osteoclasts, i.e., “static – bone resorptive (R)” and “moving – non resorptive (N)”. Treatment with recombinant RANKL or bisphosphonate (risedronate) changed the composition of these populations as well as total number of mature osteoclasts. We also found that rapid RANKL injection converted the moving (N) osteoclasts to static (R) ones without any changes in total number of osteoclasts, suggesting a novel action of RANKL in controlling mature osteoclast function. Furthermore, we showed that Th17 cells could induce rapid N to R conversion of mature osteoclasts by RANKL expressed on their cell surface. Conclusions By visualizing in vivo behaviors of mature osteoclasts, we for the first time identified different populations of live osteoclasts on the bone surface, from “static – bone resorptive” to “moving – non resorptive”. Furthermore, RANKL turned out not only to promote the differentiation of osteoclasts but also to regulate the bone-resorptive function of fully differentiated mature osteoclasts. RANKL-bearing Th17 cells were shown to control bone resorption of mature osteoclasts, demonstrating novel actions of Th17 that may be a novel therapeutic target in RA. References Sun-Wada et al., J. Cell Sci. 122: 2504, 2009. Ishii et al., Nature, 458: 524, 2009. Ishii et al., J. Exp. Med.,207: 2793, 2010. Disclosure of Interest None Declared