Figure 2.

Membrane-bound CD83 expression protects from excessive osteoclastogenesis in mice and men. (A) Osteoclasts were generated from conditional CD83 knockout mice, where CD83 was specifically depleted on CX₃CR₁-expressing cells. The number of large multinucleated TRAP⁺ cells with more than 15 nuclei was assessed (A; far left-hand side) with phosphate-buffered saline (PBS) at n = 3 and sCD83 at n = 3. RT-PCR analyses regarding the expression of CD83 and osteoclast-related genes normalized to WT value (A; right-hand side; sCD83 at n = 3 and PBS at n = 3). (B) CD83 was knocked down in peripheral blood mononuclear cell (PBMC)-derived cells via CRISPR/Cas9 method, and cells were then used for osteoclastogenesis experiments. Knockdown efficacy was determined by flow cytometry of surface CD83 expression on mature dendritic cells derived from the same targeted PBMC stock, which were differentiated into osteoclasts (B; left-hand side). A representative image from out of three independent experiments is shown on the left-hand side. The number of large multinucleated TRAP⁺ cells with more than 15 nuclei was assessed (B; middle) with PBS at n = 3 and sCD83 at n = 3. The RNA levels of osteoclast-related gene transcripts and CD83 were determined by RT-PCR and normalized to the corresponding mock-nucleofected sample of the same donor in order to account for donor-specific variations (B; right-hand side with n = 3). Student’s t-test analyses were performed.