Background/Purpose: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint synovial inflammation and progressive cartilage/bone destruction. Although various kinds of cell types, such as T/B lymphocytes, macrophages and synovial fibroblasts, are involved in the pathogenesis of chronic inflammation in RA, bone destruction is considered to be mainly mediated by enhanced activation of osteoclasts. Recently CD4⁺ T helper 17 (Th17) cells have been reported to express RANKL on the cell surface, which were suggested to be important for osteoclastic bone destruction in arthritic joints. However, the RANKL expressed on the surface of Th17 possesses little ability for inducing differentiation, and the practical function of RANKL and Th17 on bone erosion remained elusive. This study aimed to investigate how the bone-resorptive functions of osteoclasts are regulated in situ and how Th17 cells control the osteoclastic bone resorption in vivo.

Methods: To examine in vivo behaviors of mature osteoclasts and Th17 cells, we utilized advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have originally established. To identify mature osteoclasts in fluorescent microscopy, we utilized the mice in which GFP is expressed under the promoter of a vacuolar type H⁺-ATPase a3 subunit, those are preferentially and abundantly expressed in mature osteoclasts (a3-GFP mice). Polyclonally differentiated Th17 cells were labeled with fluorescent dye and then adoptively transferred into a3-GFP mice. We observed calvaria bone tissues of a3-GFP mice by using intravital multiphoton microscopy.

Results: We succeeded in visualizing live mature osteoclasts on the bone surface in situ. By using this imaging system, we could identify different populations of live mature osteoclasts in terms of their motility and function, i.e., ‘static – bone resorptive (R)’ and ‘moving – non resorptive (N)’. Treatment with recombinant RANKL or bisphosphonate 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 could demonstrate that Th17 cells had potency for inducing rapid N to R conversion of mature osteoclasts by RANKL expressed on their cell surface in situ.

Conclusion: By visualizing in vivo behaviors of mature osteoclasts, we for the first time identified different functional subsets 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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dynamic-in-vivo-imaging-of-th17-mediated-osteoclastic-bone-resorption-in-live-bones-by-using-intravital-multiphoton-microscopy/