The Possible Roles of CD73 and Adenosine in the Osteoimmunological Bone Resorption of Periradicular Periodontitis

Introduction: Periradicular periodontitis is an inflammatory disease caused by perpetual microbial infection in the root canal system which, if left untreated, results in the destruction of tooth supporting tissues. Tenacious infection of virulent bacteria can lead to continuous activation of host immune response which results in excess production of proinflammatory mediators, including the bone resorption factor RANKL. To date, attempts to minimize the destruction of tooth supporting tissues have included laser therapy and phototherapy, but these approaches have been confined to treating and monitoring the exogenous etiology, i.e., bacterial infection. Therefore, to improve the diagnosis and treatment of periradicular periodontitis, new technologies are needed to control host cell biological responses and evaluate the level of inflammation in the root canal system and periradicular area. Objectives: Intracellular generated adenosine triphosphate (ATP) and its metabolite, adenosine (ADO), act as major energy currencies in the cell system. Only recently has it been reported that extracellular ATP (eATP) released from activated neutrophils elicits proinflammatory responses, while ADO, which is generated via enzymatic conversion of ATP by cell surface-expressed ecto-5′-nucleotidase (CD73) and Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), functions as anti-inflammatory mediator. Accordingly, this study investigated the effects of CD73 and ADO on osteoimmunological bone resorption in periradicular periodontitis. Methods: The possible anti-bone loss role of ADO was examined in vitro by reacting RANKL-stimulated RAW264.7 osteoclast precursors with ADO receptor agonist 5′-(N-Ethylcarboxamido) adenosine (NECA). More specifically, the effects of NECA on RANKL-induced osteoclastogenesis were evaluated by monitoring TRAP+ multinucleated cell number and pit formation. Since activated T cells are the major cellular source of RANKL in periradicular periodontitis, the effects of NECA on proliferation and sRANKL production by TCR/CD28-stimulated T cells were examined in vitro. The therapeutic efficacy of NECA on the pathogenic bone loss induced in periradicular periodontitis was evaluated in a pulp exposure-induced periradicular periodontitis model using C57BL/6 wild-type (WT) mice. On the other hand, the impact of ADO loss-of-function was compared between CD73-/-Foxp3-GFP+/+ mice and WT mice, both with experimentally induced periradicular lesion. Periradicular tissues were isolated from sacrificed mice, and the mRNAs of inflammation-associated and bone remodeling-related genes, amount of ATP and size of periradicular lesions were monitored up to 3 weeks using qPCR and µCT. Expressions of CD73 and other proteins in the periradicular tissues were examined by immunohistochemistry and immunofluorescence staining. Results: As determined from the in vitro experiments, NECA significantly suppressed not only RANKL-mediated osteoclastogenesis from RAW264.7 cells (P<0.05), but also significantly downregulated proliferation and RANKL production from TCR/CD28-activated T cells (P<0.05), suggesting that the elicited signal could downregulate RANKL-induced osteoclastogenesis. As determined from the in vivo experiments, the amount of ATP and expression of CD73 mRNA were significantly increased in the mouse periradicular lesion in a time-dependent manner. NECA-treated wild-type (WT) mice demonstrated significantly elevated mRNAs for CD39, CD73, alkaline phosphatase, and RUNX2, while showing decreased expressions of RANKL and IL1-β mRNAs, accompanied by diminished size of periradicular lesion, compared to nondrug-treated control WT mice experimentally induced with periradicular lesion. Finally, the amount of ATP and mRNAs for RANKL, IL1-β, IL-6, and TNF-α, as well as the size of periradicular lesion, were significantly larger (P