Osteoimmunology of Retarded Bone Regeneration in Periodontitis

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This application for Diversity Administrative Supplement (PA-21-071) is written to request for the one-year support for Hispanic Post Bachelorette Trainee, Ms. Elizabeth Leon, under the parent RO1 grant, titled, “Osteoimmunology of regarded bone regeneration in periodontitis.” She will perform the basic research to elucidate the mechanism underlying the pathogenic bone resorption in periodontitis. Through the proposed training, she will not only become familiar with the operational approach to perform basic research related to the oral diseases, but also master the conceptual approach to find the knowledge gap and the way to solve the existing problem. The fundamentals for increasing the rigor and reproducibility, as well as the principle for responsible research conduct will be also learned. Parallelly, she will be working toward acceptance in the program of dentistry. The goal of this training is to support the career development of a future clinician/scientist in the basic and translational research in the dental sciences. Candidate’s research project background: Our unpublished data demonstrated that P. gingivalis’ vesicles can promote the osteoclastogenesis which was significantly attenuated by the vesicles isolated from PAD-KO P. gingivalis. Furthermore, P. gingivalis’ vesicles up-regulated the expression of OC-STAMP in the RANKL- stimulated RAW264.7 osteoclast precursor cells. Hypothesis: We hypothesized that PAD expressed by vesicles of P. gingivalis can citrullinate the host derived Vimentin which, in turn, acts on OC-STAMP expressed by osteoclasts and promotes osteoclastogenesis Project Objectives for Candidate: Objective 1: To evaluate the role of PAD expressed by vesicles of P. gingivalis (Pg vesicle-PAD) in citrullinating the host proteins, including Vimentin, in the co-culture of vesicles and osteoclasts. Objective 2: To examine whether Pg vesicle-PAD-mediated citrullination of host vimentin can alter Vimentin’s effect on promoting RANKL-induced osteoclastogenesis. The vesicles will be isolated from PAD-KO Pg W83 (gift from Dr. Potempa) and WT Pg W83 using a ultracentrifuge machine. Those isolated vesicles, as well as whole bacterial cells, will be co-cultured with osteoclast precursors. The level of citrullination of host proteins will be determined by W-blot and Mass-Spec. The expression of PAD by vesicles will be monitored using a W-blot. Finally, possible engagement of OC- STAMP expressed by osteoclasts which is the receptor for citrullinated Vimentin in osteoclastogenesis will be determined by blocking with anti-OC-STAMP mAb or anti-citrullinated Vimentin mAb. After completing these two objectives, the candidate will have mastered the methods of cell culture, W- blotting, qPCR, flow cytometry, and osteoclastogenesis assays, as well as basic statistical analysis and interpretation of data which strengthen her credential to be a candidate for graduate program.